The weights of (-)-Nutlin-3 loose snus were selected to match the weight of the most frequently used pouched snus (1 g, also included in this study) and the median portion weight reported in the survey (2.5 g). A sensory assessment was performed by questionnaire for the snus products to investigate whether differences in nicotine absorption resulted in variation in reported subjective effects. CYP2A6 genotyping was included because the rate of nicotine metabolism is a potential variable affecting the measured pharmacokinetic endpoints. Methods Study Design This was an open-label, randomized, 6-way, crossover study conducted at the Clinical Research and Trial Centre, Lund University Hospital, Sweden (trial registration number: ISRCTN11703777). The study was conducted in accordance with the ��Declaration of Helsinki,�� ICH GCP and EU Directive 2005/28/EC.

The study was approved by the Central Ethics Review Board, Stockholm, Sweden. Written informed consent was obtained from all subjects before entering the study and undergoing any study-related procedures. The Investigator site ensured that subjects were offered health advice and information on tobacco cessation helplines. The study enrolled 20 healthy subjects. Each subject visited the clinic seven times: once for screening and physical examination and six times for product administration. All 20 subjects tested all the six products; one product per visit was assigned in random order using a validated system. Each product administration visit lasted for 4 hr and there was a gap of at least one whole day between product administration visits.

Subjects were asked not to consume tobacco or nicotine products or foods and drinks that could act as cytochrome P450 enzyme inhibitors (specifically caffeine or herbal teas, cruciferous vegetables) for 12 hr prior to product administration. In addition, no food intake was allowed 1 hr prior to product administration. Subjects were excluded if they had used medications with a known mechanism of action on the cyclooxygenase pathway 14 days prior to first product administration. Screening of urine for drugs of abuse was performed using a standard test kit SYVA? Rapidtest d.a.u? 10. Safety monitoring and reporting, including concomitant medications, was performed throughout the study period and 1 week after completion of last product use.

The subjects were also monitored for cardiovascular changes prior to and at intervals after test product administration (pulse rate, blood pressure at 15, 30, and 60 min; heart rate via ECG records at 30, 60, and 90 min). CYP2A6 has been Entinostat reported to play a major role in the metabolism of nicotine and a number of genetic polymorphisms have been described which affect the rate of nicotine metabolism and inactivation (Haberl et al., 2005; Schoedel, Hoffmann, Rao, Sellers, & Tyndale, 2004).