Biomarker Comparisons Between Adult Menthol and Non-Menthol Smokers

Mohamadi A. Sarkar, M.Pharm., Ph.D., Senior Principal Scientist Client Services

1 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Outline

ƒ Background ƒ Total Exposure Study – Design and Conduct ƒ Comparisons between adult menthol and non-menthol smokers – Demographics – Biomarkers of Exposure – Metabolite Ratios – Biomarkers of Potential Harm – Nicotine Dependence Scores ƒ Summary & Conclusions

2 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Background

ƒ Most of the studies assessing cigarette smoke exposure between adult menthol and non-menthol smokers report no

differences (Ahijevych et al. 1996; Rosenblatt et al. 1998; Patterson et al. 2003; Heck 2009; Wang et al. 2009; Muscat et al. 2009; Ritchie et al. 1997)

ƒ Research on possible effects of menthol on nicotine and a NNK metabolism has yielded inconsistent findings (Muscat et al. 2009; Ritchie et al. 1997; Berg et al. 2009; Ahijevych et al. 2002; Benowitz et al. 2004)

– It has been hypothesized that the metabolic inhibition may impact disease risk and/or smoking behavior

a4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

3 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 PM USA Study Total Exposure Study – TES

ƒ The TES was a stratified, multi-center, cross-sectional study that had 3585 evaluable adult smokers and 1077 evaluable non- smokers from 31 states (39 investigative sites) across the U.S.

States with sites

4 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 TES Study Conduct

ƒ Participants were generally healthy adult males and females, 21 years of age and older ƒ The study was Institutional Review Board approved and conducted in accordance with Good Clinical Practice ƒ Smoking status was defined as consumption of a minimum of one commercially available cigarette per day over the last 12-months ƒ Blood and 24-hour urine samples as well as other measurements were collected from the participants ƒ Adult smokers were asked to return all cigarette butts smoked over the 24-hour period

5 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Objectives of the TES

ƒ Primary objectives: – To estimate the exposure of U.S. adult cigarette smokers to selected cigarette smoke constituents – To investigate the relationship between cigarette smoke exposure of U.S. adult smokers and tar delivery a ƒ Secondary objectives: – To compare selected biomarkers of U.S. adult smokers to adult non-smokers – To evaluate smoking behaviour as it relates to cigarette smoke exposure – To investigate the relationship between selected biomarkers of potential harm and cigarette smoke exposure

a Measured by the filter method

6 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Biomarkers of Exposure

Biomarker Sample Representative matrix smoke constituent Gas Phase Carboxyhemoglobin Blood Carbon monoxide 3-hydroxypropylmercapturic acid (3- 24-hour urine Acrolein HPMA) Monohydroxybutenylmercapturic acid 24-hour urine 1,3-Butadiene (MHBMA) and dihydroxybutylmercapturic acid (DHBMA) Particulate phase 4-Aminobiphenyl adducts Blood Aminobiphenyl Nicotine, cotinine, trans-3’- 24-hour urine Nicotine hydroxycotinine and their glucuronide conjugatesA Total NNALB 24-hour urine NNKC Total 1-hydroxypyrene 24-hour urine Pyrene

AThe molar sum of nicotine and its five major metabolites were expressed as nicotine equivalents (NE) B 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanol; C 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

7 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Biomarkers of Potential Harma

Biomarker of Potential Harm Matrix Oxidative Stress 8-epi-prostaglandin F2α (ng/24h) Urine Inflammation White Blood Cells (x1000/uL) Blood hs C-Reactive Protein (mg/L) Blood Fibrinogen (mg/dL) Blood 11-dehydrothromboxane B (ng/24h) Urine Platelet Activation 2 Endothelial von Willebrand Factor (%) Blood Function Microalbumin (mg/24h) Urine Lipid Metabolism Total Cholesterol (mg/dL) Blood HDL Cholesterol (mg/dL) LDL Cholesterol (mg/dL) Triglycerides (mg/dL) Lung Function FEV1 (% of predicted) Air Volume FVC (% of predicted)

aExamples of some of the BOPHs, other biomarkers not listed include Hematological measures as well as markers of Metabolism, Cardiovascular, Renal and Hepatic function and enzymes

8 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Questionnaire and Topography

ƒ Demographics ƒ Diet and Consumption ƒ Physical Activity ƒ Employment ƒ Smoking History ƒ Fagerström Test for Nicotine Dependence and other questions

ƒ Smoking Topography was measured using the CreSS® Micro Device – Puff Count – Puff Volume – Puff Duration – Inter-Puff Interval

9 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Outline

ƒƒƒ BackgroundBackgroundBackground ƒƒƒ TotalTotalTotal ExposureExposureExposure StudyStudyStudy ––– DesignDesignDesign andandand ConductConductConduct ƒ Comparisons between adult menthol and non-menthol smokers – Demographics ––– BiomarkersBiomarkersBiomarkers ofofof ExposureExposureExposure ––– MetaboliteMetaboliteMetabolite RatiosRatiosRatios ––– BiomarkersBiomarkersBiomarkers ofofof PotentialPotentialPotential HarmHarmHarm ––– NicotineNicotineNicotine DependenceDependenceDependence ScoresScoresScores ƒƒƒ SummarySummarySummary &&& ConclusionsConclusionsConclusions

10 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Demographicsa

Menthol Non-Menthol (N=1044) (N=2297) Age (years, mean (SD)) 41.8 (12.1) 42.0 (13.0) Gender, No. (%) Female 664 (63.6%) 1258 (54.8%) Male 380 (36.4%) 1039 (45.2%) Race, No. (%) African-American 448 (42.9%) 166 (7.2%) White 596 (57.1%) 2131 (92.8%) BMI (kg/m2), Mean (SD) 28.57 (7.33) 27.31 (6.29) CPDb , Mean (SD) 15.0 (8.7) 16.8 (9.0)

Tar Yield (mg), Mean (SD) 10.4 (6.2) 8.5 (4.9)

a60 Menthol and 184 Non-Menthol Multiracial, Asian, Native American, Other and Missing values are not included; bNumber of smoked per day, based on number of butts returned

11 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Demographicsa - By Race

Whites African-Americans Characteristics Menthol Non-Menthol Menthol Non-Menthol (N=596) (N=2131) (N=448) (N=166)

Age (years), Mean (SD) 43.7 (12.9) 41.9 (13.2) 39.3 (10.5) 43.3 (10.9) Gender, No. (%) Female 419 (70.3%) 1188 (55.8%) 245 (54.7%) 70 (42.2%) Male 177 (29.7%) 943 (44.2%) 203 (45.3%) 96 (57.8%) CPDb , Mean (SD) 18.1 (9.2) 17.2 (9.1) 10.9 (5.7) 12.1 (7.4)

Tar Yield (mg), Mean (SD) 8.9 (5.4) 8.7 (4.8) 12.8 (6.4) 5.9 (5.4)

Years Smoking, Mean (SD) 23.8 (12.9) 22.4 (13.1) 17.0 (11.4) 21.7 (12.5)

a60 Menthol and 184 Non-Menthol Multiracial, Asian, Native American, Other and Missing values are not included; bNumber of cigarettes smoked per day based on butts returned

12 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Outline

ƒƒƒ BackgroundBackgroundBackground ƒƒƒ TotalTotalTotal ExposureExposureExposure StudyStudyStudy ––– DesignDesignDesign andandand ConductConductConduct ƒ Comparisons between adult menthol and non-menthol smokers ––– DemographicsDemographicsDemographics – Biomarkers of Exposure ––– MetaboliteMetaboliteMetabolite RatiosRatiosRatios ––– BiomarkersBiomarkersBiomarkers ofofof PotentialPotentialPotential HarmHarmHarm ––– NicotineNicotineNicotine DependenceDependenceDependence ScoresScoresScores ƒƒƒ SummarySummarySummary &&& ConclusionsConclusionsConclusions

13 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Biomarkers of Exposure

Menthol Non-Menthol (N=1044) (N=2297) CPD 15.0 (8.7) 16.8 (9.0) Tar Yield (mg) 10.6 (6.2) 8.5 (4.9)

NE (mg/24H)a 12.8 (7.8) 13.5 (7.9)

Total NNAL (ng/24H)b 399.9 (294.8) 436.7 (309.5)

Serum Cotinine (ng/mL)a 188.9 (108.4) 183.4 (105.4)

COHb (% Sat.)a 5.2 (2.27) 5.4 (2.23)

Data Shown as unadjusted Mean (SD); Number of evaluable observations varied for each biomarker;a Analysis based on publication by Wang et al 2009; b Unpublished data from additional analysis

Overall, no statistically significant differences (p>0.05) in biomarkers of exposure were observed between menthol and non-menthol smokers

14 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Biomarkers of Exposure – By Race

Whites African-Americans Biomarker Menthol Non-Menthol Menthol Non-Menthol (N=596) (N=2131) (N=448) (N=166)

CPD 18.1 (9.2) 17.2 (9.1) 10.9 (5.7) 12.1 (7.4)

Tar Yield (mg) 8.9 (5.4) 8.7 (4.8) 12.8 (6.4) 5.9 (5.4)

NE (mg/24H)a 14.2 (8.4) 13.8 (7.9) 10.8 (6.4) 10.4 (7.1)

Total NNAL (ng/24H)b 455.4 (318.1) 445.9 (313.8) 326.5 (242.3) 318.2 (215.4)

COHb (% Sat.)a 5.5 (2.4) 5.4 (2.3) 4.7 (2.0) 4.8 (2.3)

Serum Cot. (ng/mL)a 182.6 (100.9) 183.00 (104.1) 197.3 (117.3) 189.1 (120.3)

Data Shown as unadjusted Mean (SD); Number of evaluable observations varied for each biomarker;a Analysis based on publication by Wang et al 2009 b Unpublished data from additional analysis

Overall, no statistically significant differences (p>0.05) in biomarkers of exposure were observed between menthol and non-menthol smokers

15 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Outline

ƒƒƒ BackgroundBackgroundBackground ƒƒƒ TotalTotalTotal ExposureExposureExposure StudyStudyStudy ––– DesignDesignDesign andandand ConductConductConduct ƒ Comparisons between adult menthol and non-menthol smokers ––– DemographicsDemographicsDemographics ––– BiomarkersBiomarkersBiomarkers ofofof ExposureExposureExposure – Metabolite Ratios ––– BiomarkersBiomarkersBiomarkers ofofof PotentialPotentialPotential HarmHarmHarm ––– NicotineNicotineNicotine DependenceDependenceDependence ScoresScoresScores ƒƒƒ SummarySummarySummary &&& ConclusionsConclusionsConclusions

16 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Major Metabolic Pathways of Nicotine in Humans

OH CYP2A6, CYP2B6 H H H CYP2E1 CYP2A6 O O N N N CH CH3 CH N 3 N N 3 Cotinine trans-3'-hydroxycotinine Nicotine

UGT2B10 UGT2B17 UGT2B10 UGT2B17

OGluc H H H N N O O CH CH N N 3 N 3 CH3 Gluc Gluc N

Nicotine-N-Gluc Cotinine-Gluc trans-3'-hydroxycotinine-Gluc

17 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Overview of NNK Metabolism

OH NO HOOC O NO O N HO N HO O CH CH3 NO 3 CYP2A6 OH N CYP2A13 (Lungs) N N UGT1A4 (N-Gluc) CH3 NNAL UGT1A10 (O-Gluc) NNK UGT2B7 (O-Gluc) N UGT2B10(N-Gluc) UGT2B17 (O-Gluc) NNAL-O-Glucuronide α-hydroxylation metabolic activation OH NO N pathways CH3 + N

NNK-N-oxide NNAL-N-oxide O OH 6-hydroxyNNK -OOC

OH OH

NNAL-N-Glucuronide

Adapted from Stepanov et al Cancer Epidemiol Biomarkers Prev. 2008, 17:1764-1773.

18 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Estimation of Metabolite Ratios

Phase I Oxidation Metabolite Ratio

3OH Cot./Cot. Ratio = trans-3’-hydroxycotinine Cotinine

Phase II Glucuronidation Metabolite Ratio Glucuronide Ratios = Glucuronide Conjugate Unconjugated

Note: Higher ratio is indicative of rapid detoxification

19 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Comparison of Metabolite Ratio

Menthol Non-Menthol Metabolite Ratio (N = 980) (N = 2189) PHASE I Oxidation Metabolite Ratio 3OHC/Cot. Ratio 2.67 2.52

Data Shown as LSMeans

Overall, no statistically significant difference (p>0.05) in the metabolite ratio was observed between menthol and non-menthol smokers

20 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Comparison of Metabolite Ratios

Metabolite Ratios Menthol Non-Menthol (N = 980) (N = 2189) PHASE I Oxidation Metabolite Ratio

3OHC/Cot. Ratio 2.67 2.52 PHASE II – Glucuronidation Metabolite Ratios Nicotine Gluc. Ratio 1.12 1.09 Cotinine Gluc. Ratio 2.85 2.59 3OHC Gluc. Ratio 1.07 1.09 NNAL Gluc. Ratio 3.15 3.06

Data Shown as LSMeans; Number of evaluable observations varied for each ratio

Overall, no statistically significant differences (p>0.05) in metabolite ratios were observed between menthol and non-menthol smokers

21 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Comparisons of Metabolite Ratio - By Race

Whites African-Americans Menthol Non-Menthol Menthol Non-Menthol (N = 566) (N = 2028) (N = 414) (N = 161) PHASE I – Oxidation Metabolite Ratio 3OHC/Cot. Ratio 2.99 2.85 2.38 2.22

Data Shown as LSMeans

Overall, no statistically significant difference (p>0.05) in the metabolite ratio was observed between menthol and non-menthol smokers

22 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Comparisons of Metabolite Ratios – By Race

Whites African-Americans Menthol Non-Menthol Menthol Non-Menthol (N = 566) (N = 2028) (N = 414) (N = 161) PHASE I – Oxidation Metabolite Ratio 3OHC/Cot. Ratio 2.99 2.85 2.38 2.22 PHASE II – Glucuronidation Metabolite Ratios Nicotine Gluc. Ratio 1.34 1.26 0.93 0.95 Cotinine Gluc. Ratio 3.86* 3.33 2.06 1.97 3OHC Gluc. Ratio 0.96 0.95 1.19 1.24 NNAL Gluc. Ratio 3.38 3.35 2.93 2.80

Data Shown as LSMeans; * p = 0.0363 for cotinine glucuronide ratio between White menthol and non-menthol cigarette smokers; Number of evaluable observations varied for each ratio

Overall, no statistically significant differences (p>0.05) in all but one metabolite ratios were observed between menthol and non-menthol smokers

23 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Statistical Model for Serum Cotinine

Parameter F-ratio p-value Number of CPD 415.8 <.0001 Tar Yield 56.7 <.0001 1) CPD was the most BMI 39.4 <.0001 important statistically 3OHC/Cot Ratio 27.4 <.0001 significant parameter Cotinine Gluc. Ratio 22.2 <.0001 (p<.0001) for Serum Puff duration 20.2 <.0001 Cotinine. Gender 15.0 0.0001 2) Menthol did not have Race 14.7 0.0001 a statistically Age 8.3 0.0003 significant (p>0.05) Alcohol consumption 8.2 <.0001 contribution on Total puff volume 4.3 0.0381 Serum Cotinine. Menthol × Race 3.7 0.0552 Menthol 0.8 0.3678

F-ratio = Effect Mean Square/Residual Mean Square; The parameters were prioritized based on F-ratio; R2=0.27

24 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 The clinical relevance of the differences in metabolism is uncertain

ƒ “Exposure and detoxification of NNK are unlikely to explain, by themselves, the differences in lung cancer risk

among the three populations studied.” (Derby, et al. 2009)

ƒ “Mentholated cigarette smoking inhibits the metabolism of nicotine, but the consequences of this with respect to

addiction or health effects is unclear.” (Benowitz, et al. 2008)

ƒ Inhibition of a single pathway may not have a significant impact on overall exposure since multiple isoforms are involved and alternate pathways are available e.g. Only 14-

17% of NNK is converted to NNAL (Hecht, et al. 2008)

25 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Summary

ƒ Based on the selected biomarkers of exposure investigated, cigarette smoke exposure in adult menthol smokers was not statistically significantly different compared to that observed in non-menthol smokers

ƒ Menthol does not appear to inhibit the metabolism of nicotine or NNK

26 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Outline

ƒƒƒ BackgroundBackgroundBackground ƒƒƒ TotalTotalTotal ExposureExposureExposure StudyStudyStudy ––– DesignDesignDesign andandand ConductConductConduct ƒ Comparisons between adult menthol and non-menthol smokers ––– DemographicsDemographicsDemographics ––– BiomarkersBiomarkersBiomarkers ofofof ExposureExposureExposure ––– MetaboliteMetaboliteMetabolite RatiosRatiosRatios – Biomarkers of Potential Harm ––– NicotineNicotineNicotine DependenceDependenceDependence ScoresScoresScores ƒƒƒ SummarySummarySummary &&& ConclusionsConclusionsConclusions

27 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Biomarkers of Potential Harm

ƒ Biomarkers of oxidative stress, inflammation, endothelial function, coagulation, lipid metabolism and metabolic function were investigated.

28 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Biomarkers of Potential Harma

Biomarker Menthol Non-Menthol Percent Mean (SD) Mean (SD) Difference HDL Cholesterol (mg/dl) 52.6 (15.3) 51.0 (16.3) 3.01 LDL Cholesterol (mg/dl) 114 (36.4) 116 (36.3) -1.62 Oxidized LDL (U/L) 73.5 (21.4) 74.5 (21.0) -1.33 Total Cholesterol (mg/dl) 194 (42.3) 198 (41.6) -1.69 Triglycerides (mg/dl) 142 (106) 164 (141) -13.6 hs C-Reactive Protein (mg/L) 2.91 (2.69) 2.69 (2.55) 8.14 FEV1 (% predicted) 83.5 (19.0) 84.1 (18.0 ) -0.78 8-epi-Prostaglandin-F2α (ng/24h) 1882 (1082) 1893 (1040) -0.61 11-dehydrothromboxane-B2 (ng/24h) 1397 (1016) 1310 (1045) 6.64 White Blood Cells (x1000/uL) 7.74 (2.33) 8.05 (2.27) -3.85

a Selected biomarkers shown in this table

29 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Biomarkers of Potential Harm (BOPH)

Menthol had no statistically significant effect (p>0.05) on the biomarkers of potential harm investigated.

ƒ The highest ranking statistically significant factors were:

– BMI, CPD, Age, Gender, Number of years smoked

ƒ All the statistically significant factors accounted for 4- 26%a of the variability in BOPHs investigated

aexcept for Leptin (R2=0.6) which was primarily driven by BMI

30 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Outline

ƒƒƒ BackgroundBackgroundBackground ƒƒƒ TotalTotalTotal ExposureExposureExposure StudyStudyStudy ––– DesignDesignDesign andandand ConductConductConduct ƒ Comparisons between adult menthol and non-menthol smokers ––– DemographicsDemographicsDemographics ––– BiomarkersBiomarkersBiomarkers ofofof ExposureExposureExposure ––– MetaboliteMetaboliteMetabolite RatiosRatiosRatios ––– BiomarkersBiomarkersBiomarkers ofofof PotentialPotentialPotential HarmHarmHarm – Nicotine Dependence Scores

ƒƒƒ SummarySummarySummary &&& ConclusionsConclusionsConclusions

31 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Fagerström Test for Nicotine Dependence (FTND)

450 Non-Menthol Smokers Menthol Smokers 400 380 386 363

350

300 272

242 250

203 190 200 181 173 154 142 150

Number of participants 122 114 93 100 81 72 59 50 26 12 19 4 5 0 012345678910 FTND Score Menthol smokers do not appear to have higher FTND scores

32 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Fagerström Test for Nicotine Dependence (FTND)

Categorization Menthol Adjusteda 95% CI of FTND Scores Status Odds Ratio Five Categories Menthol 1.05 0.91 – 1.22 (Very Low: 0-2 points, Low: 3-4 points, Non-Menthol Medium: 5 points, High: 6-7 points, Very 1.00 Reference High: 8-10 points) Three Categories Menthol 0.97 0.83 – 1.13 (Low (0-3), Medium (4-5) and High (6-10) Non-Menthol scores) 1.00 Reference

Two Categories (Low/Medium Menthol 0.94 0.79 – 1.13 (0-5) and High (6-10) scores) Non-Menthol 1.00 Reference aAdjusted for age, race, gender, education and tar yield category.

Overall, no statistically significant differences (p>0.05) in FTND scores

aAdjustedwere for age, race,observed gender, education between and tar yield category. menthol and non-menthol smokers

33 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Time to First Cigarette (TTFC)

Categorization Menthol Adjusteda 95% CI of TTFC Status Odds Ratio Four Categories Menthol 1.10 0.94 – 1.28 (< 5 mins, 6-30 mins, 30-60 mins Non-Menthol and > 60 mins.) 1.00 Reference Two Categories Menthol 0.88 0.72 – 1.05 (< 30 mins and > 30 mins) Non-Menthol 1.00 Reference

aAdjusted for age, race, gender, education and tar yield category.

Overall, no statistically significant differences (p>0.05) in time to first cigarette were observed between menthol and non-menthol smokers

34 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Outline

ƒƒƒ BackgroundBackgroundBackground ƒƒƒ TotalTotalTotal ExposureExposureExposure StudyStudyStudy ––– DesignDesignDesign andandand ConductConductConduct ƒƒƒ ComparisonsComparisonsComparisons betweenbetweenbetween adultadultadult mentholmentholmenthol andandand nonnonnon---mentholmentholmenthol smokerssmokerssmokers ––– DemographicsDemographicsDemographics ––– BiomarkersBiomarkersBiomarkers ofofof ExposureExposureExposure ––– MetaboliteMetaboliteMetabolite RatiosRatiosRatios ––– BiomarkersBiomarkersBiomarkers ofofof PotentialPotentialPotential HarmHarmHarm ––– NicotineNicotineNicotine DependenceDependenceDependence ScoresScoresScores ƒ Summary & Conclusions

35 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Summary

ƒ Based on the selected biomarkers of exposure investigated, cigarette smoke exposure in adult menthol smokers was not statistically significantly different compared to that observed in non-menthol smokers

ƒ Menthol does not appear to inhibit the metabolism of nicotine or NNK

36 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 Summary

ƒ The biomarkers of potential harm investigated were not statistically significantly different in adult menthol cigarette smokers compared to non-menthol cigarette smokers

ƒ Menthol cigarette smokers did not have higher Fagerström Test for Nicotine Dependence scores compared to non-menthol cigarette smokers

The collective evidence from the TES is in agreement with the epidemiological reports that adult menthol cigarette smokers are not at a greater risk of smoking-related diseases compared to adult non- menthol smokers

37 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010 References

ƒ Ahijevych, K., Gillespier, J., Demirci, M., Jagadeesh, J. (1996). Menthol and nonmenthol cigarettes and smoke exposure in black and white women. Pharmacology Biochemistry and Behavior 53(2):355-360. ƒ Ahijevych, K., Dal, G., & Chan, K. (2002). Menthol pharmacokinetics in African American women following menthol cigarette smoking [abstract]. American Thoracic Society International Conference, May 17-22, 2002. Atlanta, GA. ƒ Benowitz, N.L., Herrera, B., & Jacob III, P. (2004). Mentholated cigarette smoking inhibits nicotine metabolism. Journal of Pharmacology and Experimental Therapeutics 310(3):1208-1215. ƒ Benowitz, N.L., Lessov-Schlaggar, C.N., Swan, G.E. (2008). Genetic influences in the variation in renal clearance of nicotine and cotinine. Clinical Pharmacology & Theraputics 84(2):243-247. ƒ Berg, J.Z., Mason, J., Boettcher, A.J., Hatsukami, D.K., & Murphy, E.W. (2010). Nicotine metabolism in African Americans and European Americans: Variation in glucuronidation by ethnicity and UGT2B10 haplotype. Journal of Pharmacology and Experimental Therapeutics 332(1):202-209. ƒ Derby, K.S., Cuthrell, K., Caberto, C., Carmella, S., Murphy, S.E., Hecht, S.S., & Le Marchand, L. (2009). Exposure to the carcinogen 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in smokers from 3 populations with different risks of lung cancer. International Journal of Cancer 125(10):2418-2424. ƒ Hecht, S.S., Carmella, S.G., Stepanov, I., Jensen, J., Anderson, A., Hatsukami, D.K. (2008). Metabolism of the tobacco-specific carcinogen 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone to its biomaker total NNAL in smokeless tobacco users. Cancer Epidemiology Biomarkers and Prevention. 17(3):732-735. ƒ Heck, J.D. (2009). Smokers of menthol and nonmenthol cigarettes exhibit similar levels of biomarkers of smoke exposure. Cancer Epidemiology Biomarkers and Prevention 18(2):622-629. ƒ Muscat, J.E. Chen, G., Knipe, A., Stellman, S.D., Lazarus, P., & Richie, J. (2009). Effects of menthol on tobacco smoke exposure nicotine dependence, and NNAL glucuronidation. Cancer Epidemiology Biomarkers and Prevention 18(1):35-41. ƒ Patterson, F., Benowitz, N., Shields, P., Kaufmann, V., Jepson, C., Wileyto, P., Kucharski, S., Lerman, C. (2003). Individual differences in nicotine intake per cigarette. Cancer Epidemiology Biomarkers and Prevention. 12(5):468-471. ƒ Richie, J.P., Jr., Carmella, S.G., Muscat, J.E., Scott, D.G., Akerkar, S.A. & Hecht, S.S. (1997). Differences in the urinary metabolites of the tobacco- specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in black and white smokers. Cancer Epidemiology Biomarkers and Prevention 6(10):783-790. ƒ Rosenblatt, M.R., Olmstead, R.E., Iwamoto-Schaap, P.N. & Jarvik, M.E. (1998). Olfactory thresholds for nicotine and menthol in smokers (abstinent and nonabstinent) and nonsmokers. Physiology and Behavior 65(3):575-579. ƒ Stepanov, I., Upadhyaya,P., Carmella, S.G., Feuer, R., Jensen, J., Hatsukami, D.K., Hecht, S.S. (2008). Extensive metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in smokers. Cancer Epidemiology Biomarkers and Prevention. 17(7):1764-73. ƒ Wang, J., Roethig, H., Appleton, S., Werley, M.S., Muhammad-Kah, R., & Mendes, P. (2009). The effect of menthol containing cigarettes on adult smokers’ exposure to nicotine and carbon monoxide. Regulatory Toxicology and Pharmacology 57(1):24-30.

38 l Altria Client Services (on behalf of Philip Morris USA) l Presentation to the Tobacco Products Scientific Advisory Committee l July 15-16, 2010