Urinary Leukotriene E4 and 2,3-Dinor Thromboxane B2 Are Biomarkers of Potential Harm in Short-Term Tobacco Switching Studies

Author Manuscript Published OnlineFirst on September 26, 2019; DOI: 10.1158/1055-9965.EPI-19-0342 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

1 Urinary Leukotriene E4 and 2,3-Dinor Thromboxane B2 are Biomarkers of Potential 2 Harm in Short-Term Tobacco Switching Studies 3 Patrudu Makena1*, Gang Liu1, Peter Chen1, Charles R. Yates2, and G. L. Prasad1 4 5 1RAI Services Company, Winston-Salem, NC, United States 6 2Quinn Pharms, Inc., Knoxville, TN, United States

7 Running title: LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm 8 9 Abbreviations 10 t-PGDM: tetranor-prostaglandin D Metabolite 11 t-PGEM: tetranor-prostaglandin E Metabolite

12 PGF2α: prostaglandin F2α;

13 8-i-PGF2α: 8-iso-PGF2α;

14 2, 3-d-8-i-PGF2α: 2,3-dinor-8-iso PGF2α;

15 11-d-TXB2: 11-dehydro thromboxane B2;

16 2, 3-d-TXB2: 2,3-dinor-thromboxane B2;

17 LTE4: leukotriene E4; 18 12(S)-HETE: 12S-hydroxyeicosatetraenoic acid 19 20 *Corresponding Author: P Makena, RAI Services Company, P.O. Box 1487, Winston-Salem, 21 NC 27102, USA E-mail: [email protected] 22 Keywords: biomarkers of potential harm, tobacco, leukotriene E4, 2,3-dinor thromboxane B2 23 24 Conflict of Interest 25 Patrudu Makena, Gang Liu, Peter Chen and GL Prasad are full-time employees of RAI Services 26 Company. RAI Services Company is a wholly owned subsidiary of Reynolds American Inc., 27 which is a wholly owned subsidiary of British American Tobacco plc. Charles R. Yates is a full- 28 time employee of the National Center for Natural Products Research. 29 30 Word count: 3,846 31 Number of figures: 5 32 Number of tables: 1

Downloaded from cebp.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 26, 2019; DOI: 10.1158/1055-9965.EPI-19-0342 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

33 Abstract 34 Background: Modified risk tobacco products (MRTPs) can reduce harm by decreasing exposure 35 to combustion-related toxicants. In the absence of epidemiological data, biomarkers of potential 36 harm (BoPH) are useful to evaluate the harm-reducing potential of MRTPs. This study evaluated 37 whether arachidonic acid (AA)-derived metabolites serve as short-term BoPH for predicting 38 harm reduction in tobacco product-switching studies. 39 Methods: We used 24-hr urine samples from participants in a series of short-term studies in 40 which smokers switched from combustible to non-combustible tobacco products (oral smokeless 41 tobacco products or electronic nicotine delivery system [ENDS]) or abstinence. Pre- and post- 42 switching samples were analyzed by LC-MS/MS for alterations in select AA metabolites 43 including prostaglandins, isoprostanes, thromboxanes, and leukotrienes. 44 Results: Switching to abstinence, dual-use of combustible and non-combustible products, or

45 exclusive use of non-combustible products resulted in reduced 2,3-d-TXB2 levels. Moreover, 46 switching smokers to either abstinence or exclusive use of oral tobacco products resulted in

47 reduced LTE4, but dual-use of combustible and oral tobacco products or ENDS did not. A two-

48 biomarker classification model comprising 2,3-d-TXB2 and LTE4 demonstrated the highest 49 performance in distinguishing smokers switched to either abstinence, or to ENDS and oral 50 smokeless tobacco products.

51 Conclusions: Urinary 2,3-d-TXB2 and LTE4 can discriminate between combustible tobacco 52 users and combustible tobacco users switched to either abstinence or non-combustible products 53 for five days.

54 Impact: 2,3-d-TXB2 and LTE4, which are linked to platelet activation and inflammation, 55 represent BoPH in short-term tobacco product-switching studies. Thus, from a regulatory

56 perspective, 2,3-d-TXB2 and LTE4 may aid in assessing the harm reduction potential of MRTPs. 57 58

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

59 Introduction 60 Cigarette smoking is an independent risk factor for lung cancer, chronic obstructive

61 pulmonary disease (COPD), and cardiovascular disease (1-4). Cigarette smoke is a dynamic

62 aerosol containing several thousand chemicals, including various toxicants, which are generated

63 during the combustion process. Many of the toxicants cause long-term adverse health outcomes

64 including cancer due to chronic smoking (5). Ninety-three of the toxicants have been designated

65 as harmful and potentially harmful constituents by the United States Food and Drug

66 Administration (US FDA) (6). Smoking abstinence is the best option to reduce harm from

67 cigarette smoking (7).

68 Epidemiological outcomes require the availability of potentially reduced harm products in the

69 marketplace and sustained exclusive use of these products over many years. Hence, interim

70 measures, such as short-term biomarkers, are useful to evaluate the effect of alternate, novel,

71 potentially reduced harm tobacco products on consumers. In the context of tobacco products,

72 such biomarkers have been described as biomarkers of potential harm (BoPH) (8). Several BoPH

73 have been suggested and extensively investigated in smokers (9-11). These markers serve as

74 early indicators of physiological changes due to product use, which could potentially inform of

75 perturbations in biological processes leading to smoking-related diseases.

76 Smoking-induced oxidative stress and inflammation are important drivers of underlying

77 disease mechanisms. However, there are few well-established and validated functional BoPH

78 that serve as predictive biomarkers of smoking-related diseases. For example, forced expiratory

79 volume 1 (FEV1) is the most widely used BoPH of respiratory function and is used as a

80 quantitative measure to characterize COPD. Two potential BoPH related to these mechanisms

81 that have consistently distinguished smokers from non-smokers include F2 isoprostane iP2FIII

82 and white blood cell counts (12-14). 3

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

83 Combustion-related toxicants drive the adverse health effects associated with cigarette

84 smoking (5,8,15). In addition to cigarettes, non-combustible tobacco products including

85 smokeless tobacco products (e.g., chewing tobacco, moist snuff, snus), electronic nicotine

86 delivery systems (ENDS), and tobacco heating products (THPs), which do not generate

87 combustion-related toxicants, exist in the current market place (16,17). Although, there are

88 differences in the product constituents and consumer population (18) existing US and Swedish

89 epidemiological data demonstrate that both products, may present less risk than cigarette

90 smoking (19). For example, the risks of lung cancer are much lower for US (11.7 fold) and

91 Swedish (12.8 fold) smokeless tobacco users as well as for switchers from cigarettes to

92 smokeless tobacco than for cigarette smokers (19,20). Therefore, smokeless tobacco products

93 provide an alternative for those smokers who cannot or are unwilling to quit tobacco product use

94 (19,21). However, there is limited information on the effects of the use of other non-combustible

95 tobacco products (i.e., ENDS and THPs), which produce an aerosol that is chemically far less

96 complex than cigarette smoke.

97 To better understand the biological and pathophysiological effects of combustible and non-

98 combustible tobacco products, several cross-sectional biomarker discovery studies, which

99 included cigarette smokers (SMK), moist snuff consumers (MSC), and non-tobacco consumers

100 (NTC) have been conducted to identify BoPH for product evaluation (22,23). Further, SMK

101 exhibit enhanced arachidonic acid (AA) metabolism compared to MSC and NTC, as evidenced

102 by increased AA production (22). Increased production of AA metabolites, isoprostanes and

103 leukotriene E4 (LTE4), which are markers of oxidative stress and inflammation, were observed.

104 Thus, cigarette smoking evokes a pro-inflammatory phenotype, highlighted by increased

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

105 synthesis of AA and its metabolites that include prostaglandins, prostacyclins, thromboxanes,

106 leukotrienes, and hydroxyeicosatetraenoic acids (12,13,24).

107 The biological effects of smoking have been known to persist for a long time after complete

108 cessation, and the evaluation of certain effects of switching to modified risk tobacco products

109 (MRTPs) also could require several months. For example, six or more months of smoking

110 abstinence is necessary to detect changes in WBC levels (25,26). A significant change in FEV1

111 is detectable more than 6-12 months after smoking abstinence (27), suggesting that FEV1 is a

112 long-term biomarker of lung function. Therefore, evaluation of the health effects of potential

113 MRTPs in clinical trials becomes challenging, as confining study volunteers for extended periods

114 to ensure study compliance in residential settings is not practical. Hence, we sought to identify

115 BoPH that rapidly change after a few days of smoking abstinence and/or switching to alternate

116 tobacco products. Based on previous studies, which reported that select AA metabolites rapidly

117 change upon smoking abstinence (28), and their established role as markers of oxidative stress

118 and inflammation, we set out to determine whether the AA metabolites would serve as short-

119 term reversible BoPH.

120 In this study, we assessed the levels of a panel of urinary AA-derived metabolites, including

121 prostaglandins, isoprostanes, thromboxanes, and leukotrienes, as BoPH in smokers who either

122 abstained from smoking or switched to an alternate tobacco product for five days in a residential

123 setting. Urine samples were obtained from three separate short-term product switching studies in

124 which SMK were switched from their usual brand (UB) combustible cigarette to non-

125 combustible products (i.e., oral smokeless tobacco products or ENDS products) or abstinence.

126 127 Materials and Methods

128 Ethical conduct of clinical studies

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

129 Clinical studies were performed in accordance with the US Code of Federal Regulations

130 (CFR) governing Protection of Human Subjects (21 CFR Part 50), Financial Disclosure by

131 Clinical Investigators (21 CFR Part 54), and Institutional Review Board (IRB) (21 CFR Part 56).

132 In addition to these federal regulations, these studies followed the 1996 guidelines of the

133 International Conference on Harmonization, commonly known as Good Clinical Practice (GCP),

134 which are consistent with the Declaration of Helsinki as adopted in 2008.

135 Study design 136 A brief description of three sponsored clinical studies (from RJ Reynolds Tobacco Company

137 [RJRT] and RJR Vapor [RJRV] company) in which smokers who completely switched to either

138 abstinence or to test products in a confinement setting for a period of 5 days is provided below

139 (Supplementary Figure 1). The demographics of enrolled subjects in the three studies are

140 summarized in Table 1. The three studies included generally healthy adult male and female

141 smokers who were primarily Caucasian and African American. The mean subject age ranged

142 from 38 to 43 years. The representation of Hispanics was very limited in the study groups.

143 Study I (Cigarette Per Day Reduction [CPDR] study) was a single-center, randomized,

144 controlled, open-label, parallel group study, designed to evaluate the dose-effect relationships

145 between cigarette per day reduction and biomarkers of exposure (BoE) (29). For the current

146 analysis, 24-hr urine samples from smokers switched from 20 cigarettes per day (CPD) to

147 abstinence (0 CPD cohort) were used to evaluate AA metabolites.

148 Study II (Modern Smokeless Tobacco Product (STP) study) was a multi-center, open-label,

149 randomized, forced-switching, parallel cohort study designed to evaluate changes in tobacco

150 product use behavior and levels of selected BoE (30). Smokers were randomized into one of six

151 different test product groups: Group 1: dual use of UB cigarettes and Camel Snus; Group 2:

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

152 exclusive Camel Snus use; Group 3: exclusive Camel Sticks use; Group 4: exclusive Camel

153 Strips use; Group 5: exclusive Camel Orbs use; and Group 6: tobacco product abstinence. In this

154 trial, two Snus variants were available for participant selection: Frost (mint) or Mellow (non-

155 mint). The Sticks, Strips, and Orbs tested were generally similar in composition, consisting of

156 finely-milled tobacco and other food-grade ingredients, and contained a total alkaloid content of

157 approximately 3.2, 1.0, and 1.1 mg, respectively. These products are used orally and allowed to

158 dissolve in the mouth.

159 Study III (ENDS exposure study) was a single-center, randomized, controlled, switching,

160 open-label, parallel cohort study (31). Smokers were enrolled and randomized to one of two

161 cohorts switching to either Vuse (VS) Original or Menthol variant, for five days after baseline ad

162 libitum smoking of their UB cigarettes. Smoking status at enrollment was defined as self-

163 reported smoking of at least 10 CPD for at least six months with an expired carbon monoxide

164 (ECO) level of >15 ppm.

165 The VS products are composed of a battery, heating element, microchip, sensor, and a cartridge

166 containing propylene glycol, glycerin, nicotine, flavorings, and water. Drawing on the mouthpiece

167 causes the heating element to aerosolize the liquid in the cartridge and delivers a puff of aerosol to

168 the user.

169 Urine biomarker measurement 170 24-hour urine samples were collected beginning on study days -3 and 4 (29-31). LC-MS/MS

171 was used to measure urinary AA metabolites including PGF2α, 8-i-PGF2α, 2,3-d-8-i-PGF2α, t-

172 PGDM, t-PGEM, 2,3-d-TXB2, 11-d-TXB2, LTE4, and 12(S)-HETE at Analytisch-Biologisches

173 Forschungslabor GmbH (ABF GmbH, München, Germany) (32).

174 Classification Models

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

175 A biomarker-based gradient boosting model was constructed to classify smoker status (i.e.,

176 pre- and post-switching). The model performance, defined as the classifier’s ability to identify

177 the tobacco use groups correctly, was computed as the area under the curve (AUC) from the

178 receiver operator characteristic (ROC) curve. The ROC curve was plotted as a true positive rate

179 (sensitivity) as a function of false positive rate (1-specificity). To build biomarker-based

180 classifiers for evaluation of VS products, the datasets from Study III (i.e., VS Original and VS

181 Menthol) (75 subjects and 2 time points) were used to build, train, and cross-validate the model.

182 During model fitting, the datasets were randomly split into five folds, four of which were used to

183 train the model and one of which was used for cross-validation. This process (five-fold cross-

184 validation) was repeated ten times and the average of the model performance metrics were

185 computed.

186 After model cross-validation, the datasets from Study I (0 CPD cohort) and Study II (Group 6:

187 abstinence cohort) were used as inputs to test the model’s accuracy in distinguishing between

188 smokers pre- and post-abstinence. The performance of the classification model in the smokeless

189 tobacco product-switching studies was evaluated using the datasets from Groups 2-5 (snus,

190 Sticks, Strips and Orbs, respectively) in Study II. The “caret” package written in R

191 (http://topepo.github.io/caret/index.html) was used for model training, cross-validation, and

192 prediction.

193 Statistical analyses 194 The BoPH levels are presented as total mass (ng/24h). Mean and standard deviation of the

195 biomarkers were calculated for baseline and post-switching in each tobacco use group. To

196 compare the differences between baseline and post-switching in each group, a paired t-test was

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

197 conducted to determine statistical significance (p < 0.05). JMP 10 (SAS Institute, Cary, NC,

198 USA) was used for data analysis.

199 Results

200 Smoking abstinence alters urinary levels of AA-derived metabolites 201 Clinical trials that evaluated the impact of reduced CPD on cigarette smoke toxicant exposure

202 have previously reported reductions in tobacco-related BoE (33,34). Using well-established BoE,

203 Theophilus et al. expanded the tobacco exposure-response relationship by evaluating multiple

204 CPDR levels, including abstinence, in a short-term (5-day) switching study (29). In our effort to

205 identify short-term BoPH, we determined the levels of a panel of AA metabolites in the urine

206 sample collected in the CPDR study.

207 When compared to baseline, urinary LTE4 levels five days after smoking abstinence were

208 reduced approximately 40% (154 ± 128 vs. 93.1 ± 49.0 ng/24 hr, p = 0.004) (Fig. 1). Conversely,

209 following five days of abstinence in Study 1, urinary levels of PGF2α (2262 ± 975.4 vs. 2780 ±

210 1186 ng/24 hr, p = 0.004), 2,3-d-8-i-PGF2α (4084 ± 2394 vs. 4709 ± 1380 ng/24 hr, p = 0.02), t-

211 PGEM (15,282 ± 10,927 vs. 16,043 ± 7415.0 ng/24 hr, p = 0.023), and t-PGDM (3100 ± 1274

212 vs. 4565 ± 1646 ng/24 hr, p = 0.0001) increased (Fig. 1). Urinary levels of 11-dehydro-TXB2, 8-

213 i-PGF2α, and 2,3-d-TXB2 were unaltered five days after smoking abstinence.

214 Leukotriene and thromboxane metabolite levels are reduced after switching from 215 combustible cigarettes to noncombustible oral products 216 To determine whether AA metabolism is altered following short-term switching, we measured

217 AA-derived metabolites in SMK switching from UB cigarettes to abstinence, to modern

218 smokeless tobacco products (snus, Sticks, Strips, or Orbs), or to dual use (UB cigarettes and

219 snus) for five days.

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

220 Urinary LTE4 levels were reduced approximately 26-43% in all cohorts who switched from

221 combustible cigarettes to snus (122 ± 61.2 vs. 68.9 ± 27.9 ng/24 hr, p < 0.001), Sticks (129 ± 101

222 vs. 95.1 ± 72.4 ng/24 hr, p = < 0.001), Strips (105 ± 37.4 vs. 68.7 ± 34.0 ng/24 hr, p < 0.001),

223 Orbs (125 ± 61.4 vs. 77.0 ± 28.7 ng/24 hr, p < 0.001), or abstinence (155 ± 96.3 vs. 94.0 ± 57.5

224 ng/24 hr, p < 0.001) (Fig. 2). However, urinary LTE4 levels remained unchanged in subjects

225 switched to dual use of cigarettes and snus (134 ± 78.5 vs. 127 ± 106 ng/24 hr, p > 0.05). Urinary

226 levels of PGF-2α, 8-iPGF-2α, 2,3-d-8-iPGF-2α, t-PGEM, and t-PGDM were unchanged by any

227 switching or forced abstinence.

228 Urinary levels of 2,3-d-TXB2 were reduced approximately 24-48% across all cohorts

229 randomized to short-term switching, including dual use (2290 ± 1606 vs. 1500 ± 1119 ng/24 hr,

230 p = 0.003), Orbs (1451 ± 899.7 vs. 832.6 ± 439.7 ng/24 hr, p < 0.001), snus (2120 ± 2039 vs.

231 1286 ± 1125 ng/24 hr, p = 0.014), Sticks (1691 ± 1000 vs. 1279 ± 840.9 ng/24 hr, p = 0.037),

232 Strips (1663 ± 913.8 vs. 923.0 ± 607.2 ng/24 hr, p < 0.001), and abstinence (1834 ± 921.5 vs.

233 947.9 ± 577.2 ng/24 hr, p < 0.001) (Fig. 3). Urinary 11-dh-TXB2 levels were reduced in smokers

234 who switched to snus (787 ± 276 vs. 638 ± 207 ng/24 hr, p = 0.009). However, urinary 11-dh-

235 TXB2 levels were unaltered when subjects switched to Orbs, Sticks, Strips, or abstinence.

236 Leukotriene and thromboxane metabolite levels are reduced after switching from 237 combustible cigarettes to noncombustible Vuse Solo products 238 Studies have demonstrated BoE reductions in smokers switched to ENDS (35,36); however,

239 to date, none of these studies evaluated the impact of switching on candidate BoPH. Thus, we

240 sought to identify potential urinary BoPH in smokers switched to either VS Original or VS

241 Menthol. Five days after switching to VS Menthol, urinary levels of PGF-2α (2696 ± 1293 vs.

242 3185 ± 1502 ng/24 hr, p = 0.0009), 8-iPGF-2α (669 ± 276 vs. 744 ± 268 ng/24 hr, p = 0.0205),

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

243 and t-PGDM (4243 ± 2034 vs. 4966 ± 1956 ng/24 hr, p = 0.01413) increased. 2,3-d-TXB2

244 urinary levels decreased in smokers switched to either VS Menthol (3604 ± 1917 vs. 2011 ±

245 1762 ng/24 hr, p < 0.001) or VS Original (2561 ± 1618 vs. 1439 ± 1001 ng/24 hr, p < 0.001)

246 (Fig. 4). None of the remaining urinary AA metabolites were altered upon switching to either VS

247 Original or VS Menthol.

248 Classification Model 249 To assess the clinical validity of the biomarkers, a two-biomarker-based classification model

250 was constructed, and its performance quantified using the AUC from ROC (Fig. 5). In this

251 model, only LTE4 and 2,3-d-TXB2 were included, since their urinary levels were significantly

252 decreased across all the various short-term product-switching study cohorts for at least one of the

253 metabolites. The highest AUC was for VS Original (0.88), followed by VS Menthol (0.82) and

254 smoking abstinence (0.72). Furthermore, application of our model using the smokeless tobacco

255 switching cohorts as input data revealed AUCs of 0.76 (Orbs); 0.61 (Sticks); and 0.72 (snus and

256 Strips) (Supplementary Figure 2).

257 258 Discussion 259 Chronic smoking is associated with elevated oxidative stress and inflammation, which are key

260 drivers of smoking-induced pathophysiology (13). While several potential BoPH for smoking-

261 related diseases have been identified (11), it usually requires several months of smoking

262 abstinence to detect meaningful changes in their levels. Our goal was to identify and qualify

263 BoPH following short-term smoking abstinence or switching to potential MRTPs. In this

264 manuscript, we investigated whether the AA metabolites are responsive to short-term smoking

265 abstention and whether they can be used as potential BoPH for potential evaluation of tobacco

266 products.

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

267 The current work evaluated a panel of known biomarkers of oxidative stress, inflammation,

268 and platelet activation in the urine samples collected from three RJRT and RJRV sponsored

269 studies. Key findings from this study are: 1) switching to either abstinence, dual use of

270 combustible and non-combustible products, or exclusive use of non-combustible products

271 resulted in reduced 2,3-d-TXB2; and 2) switching smokers to either abstinence or non-inhaled

272 oral tobacco products culminated in reduced LTE4. Together, these data show 2,3-d-TXB2 and

273 LTE4, as potential BoPH. Further, we constructed a classification model based on these BoPH to

274 evaluate their combined performance in distinguishing smokers pre- and post-switching.

275 The two-biomarker-based model demonstrated the highest performance in distinguishing

276 smokers pre- and post-switching to non-combustible inhaled products (i.e., VS Original and VS

277 Menthol). Moreover, the model performed well (AUC > 0.70) in differentiating smokers

278 switched to either abstinence or smokeless oral tobacco products. Together, these data suggest

279 that a classification model comprising both LTE4 and 2,3-d-TXB2 has the potential to categorize

280 smokers subjected to short-term product switching.

281 Smoking abstinence is the accepted “Gold Standard” strategy for reducing harm from

282 cigarette smoking. Consequently, the biological effects of switching to a MRTP must be

283 considered in the context of both smoking and abstinence. In Study I, which was designed to

284 evaluate the dose-effect relationships between CPDR and BoE, we found that abstinence resulted

285 in a significant decrease in urinary LTE4 levels. Leukotrienes (LTs) are lipid signaling mediators

286 produced by mast cells, eosinophils, neutrophils, basophils, and macrophages. LT synthesis is

287 initiated by phospholipase A-mediated cleavage of AA, which then undergoes rapid conversion

288 to LTA4 via 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) (37,38). LTA4

289 is subsequently converted to LTC4, LTD4, and LTE4, collectively known as cysteinyl

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

290 leukotrienes (CysLTs), and LTB4 (Supplementary Figure. 6). Urinary LTE4, the most stable

291 CysLT, is frequently used as a marker of leukotriene synthesis (39,40).

292 Urinary LTE4 levels are closely correlated with number of cigarettes smoked (41,42) and

293 cotinine levels (43). Additionally, adult smokers had up to five-fold higher levels of urinary

294 LTE4 compared with non-smokers (44). Thus, our data demonstrating increased LTE4 levels in

295 smokers is consistent with studies examining the link between cigarette smoking and increased

296 LTE4 levels. Following forced abstinence or switching to smokeless tobacco products, urinary

297 LTE4 levels declined rapidly and to a similar degree to the decrease seen in smokers reducing the

298 number of cigarettes smoked per day. Our findings align with studies which demonstrate that

299 urinary LTE4 levels return to baseline within two weeks of tobacco product abstention (42).

300 There was a similar trend toward reduction of urinary LTE4 levels in smokers switched to either

301 VS Original or VS Menthol, however, the reductions were not statistically significant.

302 Thromboxane A2 (TXA2), the major product of prostaglandin endoperoxides in platelets,

303 induces irreversible platelet aggregation (45). TXA2 is rapidly converted to TXB2 followed by

304 catabolism to 2,3-d-TXB2 (46). 2,3-d-TXB2 is the most abundant metabolite recovered in the

305 urine following intravenous infusion of TXB2 to humans (47). Thromboxane urinary metabolites

306 are frequently used as a marker for platelet activation in smokers (13,48-50). For example, 2,3-d-

307 TXB2 is significantly elevated in healthy chronic smokers when compared to non-smokers (50).

308 Consistent with the literature, we observed that 2,3-d-TXB2 urinary levels were reduced

309 following smoking abstinence. Similar results were obtained in smokers switched to dual use,

310 and in smokers switched to exclusive use of non-combustible products (i.e., oral tobacco

311 products and VS products). Together, these data suggest diminished TXA2 synthesis and platelet

312 activation when smokers switch to non-combustible products.

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

313 The lung is a major site of LT synthesis (51) and elevated LT levels in the lung are a hallmark

314 of airway hypersensitivity-related diseases such as asthma and allergic rhinitis. A decrease in

315 LTE4 levels in smokers who stop smoking or switch to smokeless tobacco products such as snus

316 indicates a decrease in airway hypersensitivity. In smokers who switched to VS Original or VS

317 Menthol, LTE4 levels were lower (17% and 20% respectively), although not statistically

318 significant. However, a boosted-tree-based classification model based on LTE4 and 2,3-d-TXB2

319 data from all three clinical studies revealed that these two BoPH clearly differentiate smokers

320 who switched to non-combustible products in several days from their baseline levels (Fig. 5).

321 Thus, the levels of LTE4 and 2,3-d-TXB2 decline in smokers who switch to non-combustible

322 tobacco products, suggesting improved airway responsiveness and platelet function.

323 Few clinical studies have compared thromboxane synthesis in combustible versus non-

324 combustible tobacco product users. For example, smokeless tobacco users, despite having

325 urinary cotinine levels similar to that of smokers, have urinary levels of 2,3-d-TXB2 (52) and

326 TXB2 (53) similar to those of non-tobacco users. Our study examined the effect of short-term

327 switching on AA metabolites associated with platelet activation across a spectrum of non-

328 combustible tobacco products including Orbs, snus, Sticks, Strips, and VS Original and VS

329 Menthol. Consistently, use of noncombustible tobacco, including vapor products, was associated

330 with reduced platelet activation, as indicated by reduced urinary excretion of 2,3-d-TXB2.

331 Platelet activation was also diminished in smokers switching to either dual use or strict tobacco

332 abstinence. It is unclear why urinary 2,3-d-TXB2 levels were reduced in the abstinence cohort

333 (Study II) and not in smokers abstaining from tobacco use in the CPDR study (Study I). One

334 possible explanation is that smokers in the CPDR study had 2,3-d-TXB2 levels approximately

335 50% lower than smokers in the abstinence cohort (Study II).

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

336 Platelet activation represents a well-established physiologic response elicited by exposure to

337 combustible tobacco toxicants and is known to contribute to the etiology and progression of

338 cardiovascular disease (54,55). Cigarette smoke toxicants induce an inflammatory airway

339 phenotype in which activated platelets serve as a major cellular source of TXA2 (50). For

340 example, acrolein, a toxicant found in cigarette smoke, potentiates platelet aggregation and

341 TXA2 synthesis in response to thrombin by increasing the availability of AA, a substrate for

342 TXA2 formation (56). TXA2, in turn, acts on TXA2 receptors to induce the second-phase

343 response to cigarette smoke (57). Our finding that short-term product-switching yields reduced

344 2,3-d-TXB2, the most abundant urinary metabolite derived from TXA2, suggests that 2,3-d-TXB2

345 may serve as a BoPH of platelet activation.

346 Some limitations of this BoPH study include: 1) the relatively small sample sizes in all the

347 three studies; 2) the lack of a proportional representation of Hispanics in the studies; 3) need for

348 refinement of the classification model; and 4) the need for consideration of the influence of diet

349 on AA metabolism. Enrollment of the subjects was open to all those who met the

350 inclusion/exclusion criteria and inadequate representation of Hispanics could be due to the

351 location of where the studies were conducted. Although the sample sizes for each of the three

352 clinical studies were relatively small, the two BoPH show consistent changes in the direction and

353 the magnitude. The two-biomarker-based classification model described above was built based

354 on limited size of biomarker data and further refinement using independent larger sample size of

355 clinical datasets may be necessary. Since diet and genetic variation are potential determinants of

356 AA metabolite levels, these factors also might require further consideration (58).

357 In conclusion, enhanced thromboxane and leukotriene biosynthesis are linked to platelet

358 activation, chemotaxis, and airway hypersensitivity, which are early pathophysiological events in

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

359 cardiovascular and pulmonary disease (Supplementary Figure 6). 2,3-d-TXB2 and LTE4,

360 relatively stable metabolites of TXA2 and LTA4, are used as proxies for thromboxane and

361 leukotriene synthesis. In this study, we demonstrated that urinary 2,3-d-TXB2 and LTE4 levels

362 are lower following reduced toxicant exposure resulting from product switching. 2,3-d-TXB2 and

363 LTE4 can discriminate between smokers and smokers switched to either abstinence or non-

364 combustible products over a period of approximately one week. Moreover, these two metabolites

365 reflect altered AA metabolism, which is linked to enhanced platelet activation, leukocyte

366 recruitment, and inflammation. Because 2,3-d-TXB2 and LTE4 possess these key attributes, they

367 represent potential BoPH designed to gauge the reversibility of toxicant exposure response in

368 short-term tobacco product-switching studies.

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

369 Acknowledgements 370 The studies and the manuscript preparation were funded by RAI Services Company. RAI

371 Services Company is a wholly owned subsidiary of Reynolds American Inc., which is a wholly

372 owned subsidiary of British American Tobacco plc. The authors sincerely thank Herman Krebs

373 (RAI Services Company) for sample management and ABF GmbH for bioanalysis of this

374 biomarker discovery study. In addition, we would like to thank Nasrin Nouri who was an intern

375 of RAI Services Company and Quynh Tran, a former employee of RAI Services Company.

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

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LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

560 Table 1. Summary of demographic information

561 Study I (CPDR) Study II (STP study) Study III (ENDS) study Dual Usage of Subject Smoking Tobacco Vuse Solo Vuse Solo Orbs Snus Sticks Strips UB Cigarettes Demographics Cessation Abstinence Original Menthol n=28 n=27 n=28 n=24 and Snus n=30 n=24 n=37 n=38 n=25 39.14 ± 41.07 ± Age, y 42.8 ± 9.17 42.83 ± 11.16 38.03 ± 11.42 40.16 ± 11.76 42 ± 12.49 41.43  11.31 42.71  11.06 12.90 12.29 Gender, n (%) Male 17 (57) 14 (58) 12 (43) 16 (59) 16 (57) 14 (58) 11 (44) 11 (30) 14 (37) Female 13 (43) 10 (42) 16 (57) 11 (41) 12 (43) 10 (42) 14 (56) 26 (70) 24 (63)

Ethnicity, n (%)

Hispanic or Latino 1 (2.6) 0 5 (17) 0 (0) 0 0 1 (4) 3 (12) 3 (12) Non-Hispanic or 37 (97.4) 40 (100) 25 (83) 24 (100) 28 (100) 27 (100) 27 (96) 25 (88) 22 (88) Latino

Race, n (%) Black 7 (23) 3 (13) 6 (21) 5 (19) 6 (21) 1 (4) 2 (8) 14 (38) 24 (63) White 23 (77) 20 (83) 18 (64) 22 (81) 20 (71) 21 (88) 23 (92) 20 (54) 10 (26) Other 0 1 (4) 4 (15) 0 2 (8) 2 (8) 0 3 (8) 4 (11)

BMI, kg/m 27.96 ± 5.27 26.30 ± 6.46 26.67 ± 5.87 27.70 ± 5.98 27.07 ± 4.78 25.24 ± 5.34 29.16 ± 7.23 27.99 ± 7.25 29.31 ± 5.39

562 563

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

564 Figure 1: Differentiating urinary arachidonic acid metabolites in smokers switched to 565 abstinence. Twenty-four-hour urine samples obtained at baseline and five days post-switching 566 from smokers switched from 20 cigarettes per day to abstinence (0 cigarettes per day) were used 567 to measure arachidonic acid metabolites by LC-MS/MS. PGF2α, 2,3-d-8-i-PGF2α, t-PGEM, and 568 t-PGDM increased following 5 days of abstinence, while LTE4 decreased. The metabolite 569 measurements were presented in box plots. In each box plot, the upper, middle and lower edges 570 represent 25%, 50%, and 75% quantiles respectively and individual data points represent subject- 571 level measurements. *P < 0.05, **P < 0.01, ****P <0.0001 when comparing pre- and post- 572 switching metabolite levels. 573

574 Figure 2: Urinary LTE4 levels in smokers switched to smokeless tobacco products. Twenty- 575 four-hour urine samples obtained at baseline and five days post-switching from smokers 576 switched from usual brand (UB) cigarettes to smokeless tobacco products were used to measure 577 arachidonic acid metabolites by LC-MS/MS. Urinary LTE4 levels were reduced in all cohorts in 578 which combustible tobacco products were prohibited (Orbs, snus, Sticks, Strips, and abstinence). 579 Urinary LTE4 levels remained unchanged in subjects switching to Dual Use. The metabolite 580 measurements were presented in box plots. In each box plot, the upper, middle and lower edges 581 represent 25%, 50%, and 75% quantiles respectively and individual data points represent subject- 582 level measurements. ***P < 0.001, ****P <0.0001 when comparing pre- and post-switching 583 metabolite levels. 584

585 Figure 3: Urinary 2,3-d-TXB2 levels in smokers switched to smokeless tobacco products. 586 Twenty-four-hour urine samples obtained at baseline and five days post-switching from smokers 587 switched from usual brand (UB) cigarettes to smokeless tobacco products were used to measure 588 arachidonic acid metabolites by LC-MS/MS. Urinary 2,3-d-TXB2 levels were reduced in all 589 cohorts in which combustible tobacco products were prohibited (Orbs, snus, Sticks, Strips, and 590 abstinence) and in smokers switched to Dual Use. The metabolite measurements were presented 591 in box plots. In each box plot, the upper, middle and lower edges represent 25%, 50%, and 75% 592 quantiles respectively and individual data points represent subject-level measurements. *P < 593 0.05, **P < 0.01, ***P < 0.001, ****P <0.0001 when comparing pre- and post-switching 594 metabolite levels. 595 596 Figure 4: Urinary arachidonic acid metabolites in smokers switched to Vuse Solo. Twenty- 597 four-hour urine samples obtained at baseline and five days post-switching from smokers 598 switched from usual brand (UB) cigarettes to Vuse Solo products were used to measure 599 arachidonic acid metabolites by LC-MS/MS. Five days after switching, urinary levels of PGF-2α, 600 8-iPGF-2α, and t-PGDM were increased in smokers switched to Vuse Solo Menthol, while 2,3-d- 601 TXB2 urinary levels were decreased in smokers switched to either Vuse Solo Menthol or Vuse 602 Solo Original. The metabolite measurements were presented in box plots. In each box plot, the 603 upper, middle and lower edges represent 25%, 50%, and 75% quantiles respectively and 604 individual data points represent subject-level measurements. *P < 0.05, ** LTE4 is statistically 605 significant (p < 0.001) upon log transformation of the data in both cohorts (Vuse Solo Menthol 606 or Vuse Solo Original), ***P < 0.001, ****P <0.0001 when comparing pre- and post-switching 607 metabolite levels. 25

LTE4 and 2,3-d-TXB2 as Biomarkers of Potential Harm

608 609 Figure 5: Arachidonic acid metabolite-based classification model predicts short-term 610 product switching. The tree-based binary classification model built on LTE4 and 2,3-d-TXB2 611 data was evaluated using validation datasets from Vuse Solo Original (Vuse O), Vuse Solo 612 Menthol (Vuse M), and Smoking Abstinence cohorts. Their corresponding ROC curves are 613 shown in blue (Vuse Solo Original), red (Vuse Solo Menthol) and brown (Smoking Abstinence 614 from Study I and II), respectively. 615 616

Urinary Leukotriene E4 and 2,3-Dinor Thromboxane B2 are Biomarkers of Potential Harm in Short-Term Tobacco Switching Studies

Patrudu Makena, Gang Liu, Peter Chen, et al.

Cancer Epidemiol Biomarkers Prev Published OnlineFirst September 26, 2019.

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