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Of Mice and Men

Posted by amukhin on 19 Feb 2015 at 19:33 GMT

We read this article with great interest, as it addresses the important topic of developing a model for the evaluation of the safety of electronic cigarette (e-cig) use. The authors report that e-cig vapor at some level of exposure may not only cause lung inflammation but reduces anti-bacterial and anti-viral defenses.
One of the critical problems in the development of any animal model for human pharmacology and toxicology is determining the animal doses or the level of exposure that will adequately reflect human conditions. In the described model the authors used the level of e-cig vapor exposure in mice resulting in a blood cotinine level that matches the level observed in regular e-cig users. The use of such a level of exposure in the animal model would be valid only if the intensity of nicotine and cotinine metabolism in mice and in humans were comparable. In reality the metabolism of both compounds in mice is much faster than that in humans. For example the half-life value of nicotine in mice is about 8% of that in humans ( 9 min vs. 120 min) and the half-life value of cotinine in mice is only about 4% of that in humans, 0.6h vs. 16 h (Siu & Tyndale, 2006; Benowitz et al., 2009). Therefore the nicotine exposure of mice will be much higher than that in humans to produce the same level of blood cotinine.
Since the proposed model may be used in further studies and the results of the study could be interpreted by people who are not familiar with the peculiarity of nicotine/cotinine pharmacokinetics in mice, we attempted to estimate the level of nicotine (and e-liquid vapor) exposure used in the discussed study. Since in this study the animals were continuously exposed to nicotine vapor over 90 min and the T1/2 of cotinine in C57BL/6 mice is 37.5 min, the cotinine level (267ng/mL) measured after the termination of nicotine did not reach steady-state (CotSS). Nonetheless, using equation (1) CSS= Ct/(1-exp(-ln(2)/T1/2*t)), CotSS for this study can be calculated as 329 ng/mL. Assuming that the bioavailability of nicotine = 1 and all nicotine is converted to cotinine, CotSS can be calculated using equation (2) CotSS = (Ko nic/CLcot)*R, were Ko nic is the level of nicotine exposure in ng/min, CLcot is the total clearance of cotinine and R is ratio of the molecular weight of cotinine (176) to that of nicotine (162). The general form of equation (2) can be found in Gillette (2012). The ¬¬equation (2) can be rearranged as equation (3) Ko nic, = CotSS * CLcot/R. Using this equation, CotSS=328 ng/mL (see above), CLcot=0.52 mL/min, measured in 25 g C57BL/6 mice (Siu & Tyndale, 2006), R = 1.09, and assuming that the body weight of C57BL/6 mice used in the discussed study was 25g (we were unable to find the body weight in the paper), Ko nic can be calculated as 583 ng/25g/min (2.1 mg/kg/90min). Since animals were exposed to vapor twice per day the daily dose was 4.2 mg/kg nicotine, which corresponds to a daily nicotine exposure dose for a 70 kg human as high as 294 mg/day, which roughly corresponds to smoking 200 cigarettes.
As we stated above, in our calculation two assumptions have been made: 1) the bioavailability of nicotine=1.0; and 2) all nicotine is converted to cotinine. In case the bioavailability is < 1 and/or not all nicotine is converted to cotinine, then in order to reach the measured level of blood cotinine, the level of nicotine exposure would be even higher than the value we calculated.
To support our calculations we also analyzed the data from a published study where C57BL/6 mice received a continuous intravenous nicotine infusion over 10 days (Marks et al., 2004). In this study, using an infusion of nicotine at a rate of 4 mg/kg/h, the observed steady-state level of cotinine was 750 ng/mL. Therefore to obtain the target 267 ng/mL cotinine level reported in the discussed study, the rate of nicotine infusion in these mice would have been 1.76 mg/kg/h (2.64 mg/kg/1.5h). For twice per day exposure the daily dose would be 5.3 mg/kg/day or 370 mg/day for a 70 kg human, more than ten times the nicotine exposure of a typical cigarette smoker.
Taken together our calculations suggest that animals in the discussed study were exposed to nicotine vapor at an intensity which corresponds to a daily exposure in humans on the order of 300-370 mg nicotine. With 1.6% nicotine concentration in e-liquid, to achieve such a dose of nicotine it would be necessary to utilize 18-23 mL e-liquid per day. Using the average value of e-liquid consumption per puff of 0.005mL (Farsalinos et al., 2013) the number of e-cig puffs per day can be calculated as 3600-4600.
It should be noted that in our calculations we postulated that the blood for cotinine measurement was taken immediately after the end of 90 min of exposure. In the results section the authors stated that “Blood was collected … within 1 h of the final exposure” but in the methods section they stated that “exposure was assessed by measuring serum cotinine at 1 h after exposure.” If the last statement is correct, because of the fast elimination of cotinine in mice the level of exposure in the study was 3 times higher (2^(60/37.5) ≈ 3) than the above-calculated values. In other words, to obtain the same exposure in humans the e-cig user should take 11000 – 13000 puffs per day. Assuming 8 hours of sleep per day, in order to acquire such a high number of puffs e-cig users would need to take 11-13 puffs per minute and thus practically take an e-cig puff with each breath.
In conclusion we recommend that the results of the discussed study should be interpreted with caution and that more studies with more realistic levels of e-liquid exposure should be conducted.

Alexey G. Mukhin M.D., Ph.D. and Jed E. Rose, Ph.D.
Center for Smoking Cessation, Duke University Medical Center

Benowitz et al., Handb Exp Pharmacol. 2009; (192):29-60
Farsalinos et al., Int J Environ Res Public Health. 2013; 10(6):2500-14
Gillette J.R. (2012) Kinetic Aspects of Metabolism and Elimination of Foreign Compounds in Animals.
In WB Jakoby (Ed.), Enzymatic Basis of Detoxication, Vol. 1, Chapter 2, Academic Press
Marks et al., Neuropharmacology. 2004; 46(8):1141-57
Siu and Tyndale, Mol Pharmacol. 2007; 71(3):826-34

Competing interests declared: AGM: No competing interests to declare.
JER: Consultancy and patent purchase agreement with Philip Morris International.