Nicotine is the addictive substance found in various tobacco products. CYP2A13 is an enzyme localized in the lungs, metabolizes tobacco-specific nitrosamine carcinogens that contribute to lung cancer. Therefore, pinpointing CYP2A13 inhibitors is an approach to lower tobacco-based lung cancer risk. Cinnamaldehyde is a common flavoring agent in the fluids of electronic nicotine vaping devices. Cinnamaldehyde was found to be a potent inhibitor of CYP2A6, another enzyme that metabolizes nicotine. Because CYP2A13 and CYP2A6 exhibit overlap in substrate/inhibitor selectivity, the goal here was to evaluate the inhibition of CYP2A13 by cinnamaldehyde. A time-dependent inhibition coumarin assay was performed to determine the kinetic parameters for cinnamaldehyde in recombinant CYP2A13. Primary incubations contained cinnamaldehyde, CYP2A13 Supersomes, and potassium phosphate buffer. Incubations were initiated with NADPH. Secondary incubations contained coumarin, NADPH, and potassium phosphate buffer. At selected time points, an aliquot of the primary incubation mixture was transferred to the secondary incubation tubes, which were terminated with trichloroacetic acid after heating at 37°C for 5.5 minutes. A linearity study was conducted to determine the appropriate termination time. CYP2A13 activity was measured by the detection of hydroxycoumarin using high-performance liquid chromatography (HPLC) and a fluorescence detector. Hydroxycoumarin formation decreased with time and inhibitor concentrations. Maximal inhibition following an 18-minute incubation was 38.3 ± 1.6 and 4.0 ± 0.6%. The maximal rate of inhibition was 0.109 per minute. The results provide evidence that cinnamaldehyde is a time-dependent inhibitor of CYP2A13. Furthermore, cinnamaldehyde appears to be a more potent inhibitor of CYP2A13 than CYP2A6, based on the maximal rate of inhibition. The results imply that cinnamaldehyde could interfere with the bioactivation of nitrosamine lung carcinogens. Additional kinetic studies are needed to confirm the results of this study and to evaluate the safety and toxicity profiles of cinnamaldehyde in complex physiological models.

 Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant pathogen responsible for ~1/3 of antimicrobial resistance-associated mortality in the USA. The glycopeptide, vancomycin, remains the primary treatment for invasive MRSA infections while lipopeptides (e.g. daptomycin) and lipoglycopeptides (e.g. dalbavancin) are common alternatives. All MRSA are resistant to beta-lactams (e.g. nafcillin) but synergistic antimicrobial activity between vancomycin or daptomycin and beta-lactams is commonly observed. Some investigators attribute this synergy to the “seesaw effect”, a phenomenon where the susceptibility to beta-lactams increases with declining vancomycin or daptomycin susceptibility. However, the association between synergy and the seesaw effect hasn’t been rigorously tested. The objective of this study was to determine whether the seesaw effect was necessary for synergy. We used standard time-kill methods to test for synergy between nafcillin and vancomycin, daptomycin or dalbavancin in a series of isogenic strains with reduced susceptibility to vancomycin, daptomycin, and dalbavancin. Two strains exhibited the seesaw effect (N315-VAN8, N315-D1) with nafcillin while 1 didn’t (N315-DAL0.5). All time-kills were performed using half the minimum inhibitory concentration(MIC) for each drug. Nafcillin concentrations were capped at peak physiological concentrations if the MIC was above this value and all experiments were performed in duplicate. Bacterial survival was counted at 0, 4, 8, and 24-hours. Synergy was defined as ≥2 log10 colony forming units per milliliter increase in bacterial killing of the combination compared to the most active single agent. All strains tested exhibited synergy between nafcillin and vancomycin, daptomycin, and dalbavancin, independent of the seesaw effect, suggesting that these two phenomena are distinct. This is important because the emergence of the seesaw effect cannot be detected clinically. This suggests that the seesaw effect is not a therapeutically relevant phenomenon. Further work is warranted to characterize strains that don’t exhibit beta-lactam synergy to identify which strains we should target with combination therapy.

 Over 30% of patients with epilepsy remain resistant to antiseizure drugs (ASD), despite over 20 therapies available. Individuals that do not achieve seizure control with two or more ASDs are considered “pharmacoresistant”. Limited preclinical studies have suggested that the early chronic administration of ASDs that selectively inactivate sodium channels may dramatically alter subsequent pharmacoresistance in chronic seizure models of epilepsy. However, it is unknown whether inhibition of slow inactivation state of sodium channels specifically leads to pharmacoresistance. Therefore, we sought to determine whether the chronic administration of lacosamide (LCM), which inhibits the slow-inactivation state of sodium channels, would lead to a similarly pharmacoresistant chronic seizure model. Male CF-1 mice were treated with an anticonvulsant dose of LCM (4.5 mg/kg, ip) or vehicle one hour prior to each corneal kindling session twice/day for three weeks until animals achieved the fully kindled state of five consecutive Racine stage 5 seizures. Once mice had achieved kindling criterion, they were given a two week stimulation-free period. We then tested the sensitivity of kindled seizures of both treatment groups to 9 mg/kg (ip) lacosamide. There were 7/9 vehicle-kindled mice (77.7%) versus 3/10 LCM-kindled mice that achieved kindling criterion (p=0.0698). There was also a significant time x treatment effect on kindled seizure severity (F (23, 391) = 2.169, p<0.002), demonstrating that chronic LCM administration significantly delayed acquisition of the fully kindled state. Further, administration of 9 mg/kg LCM reduced seizure severity in both LCM- and VEH-kindled mice, indicating that LCM-kindled mice are not resistant to escalating doses of LCM. To assess the mechanism of pharmacoresistance, immunohistochemistry data will be further discussed, specifically the differences in sodium channel subunit expression and neuroinflammation in LCM- versus VEH-kindled mice. This study provides insight into how ASD monotherapy early in the epileptogenesis process may influence pharmacoresistance and the development of the epilepsy.