PROJECT SUMMARY – PROJECT 2 Historically, the tobacco industry manipulated nicotine concentration and form to offer a range of smokeless tobacco (ST) products to appeal to both new and established users. Termed by the industry as the “graduation” program, they provided new users with “starter products” with low nicotine concentration and low freebase nicotine and “mature” users with higher concentrations of nicotine, mainly in the freebase form, to maintain dependence. They are now following the same playbook for oral nicotine pouches (ONPs), manipulating the nicotine dimensions of concentration and form, and more recently have begun manipulating isomer composition through the use of synthetic nicotine. While ONPs may have promise as a less harmful alternative for smokers and ST users who completely switch, the potential risk for ONP uptake among young new users is concerning. The overall integrative theme of The Ohio State University Tobacco Center for Regulatory Science is: “Flipping the Script”: Using the Industry's Nicotine Playbook to Maximize Public Health. Just as the tobacco industry manipulates nicotine to create ONPs that appeal to young people, with the evidence produced by Project 2 (P2), we posit that the FDA can regulate the nicotine dimensions in ONPs, making them unappealing to new users while still providing smokers and ST users a less harmful but appealing and satisfying alternative. P2 will inform regulations that position ONPs as a public health benefit rather than a threat by identifying the characteristics of nicotine that best support switching to ONPs and evaluating corresponding changes to the oral microbiome among adult smokers and ST users. In Phase 1, using a double-blind, cross- over design, exclusive smokers (n=80) and ST users (n=80) will be randomized to high or low nicotine concentration, and then engage in 5 single-product laboratory use sessions of their usual brand and 4 ONPs (random order) that range in freebase fraction (low, high) and isomer (racemic, >99% S-nicotine). Outcomes include plasma nicotine pharmacokinetics, subjective effects, and behavioral economic demand indicies. In Phase 2, the same participants, after a 1-week usual brand control period, will substitute a randomly assigned ONP for their usual brand for 4 weeks. Study outcomes include use behaviors, including complete switching, measured via ecological momentary assessment and biochemically confirmed (anatabine and anabasine), count of unused ONPs, subjective effects, behavioral economic demand indices, and oral microbiome assessment. P2 is significant and innovative because it uses a mix of tightly controlled in-lab procedures and ad libitum, naturalistic use of commercial tobacco products to assess how nicotine concentration, form, and isomer affect the addiction potential and health effects of ONPs. This is the first study of the combined nicotine dimensions with the intent of facilitating switching from cigarettes and ST to ONPs without ...