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Novel ileocolonic release peppermint oil 127 6 Pharmacokinetic analysis The pharmacokinetic profile of small-intestinal and ileocolonic release peppermint oil capsules was determined by menthol-glucuronide analysis in the blood. In total, 14 venous blood samples (+/-8 mL per time point) were collected in heparinized tubes at the time points mentioned above. Within two hours after collection, samples were centrifuged at 3120 rpm for 10 min at 4°C and plasma supernatants were stored at - 80°C until assayed. Samples were analyzed for menthol-glucuronide concentration, the primary metabolite of L-menthol, by a 15 h incubation at 37°C with beta-glucuronidase and using Gas Chromatography Mass Spectrometry (GC/MS); the method has been described in detail elsewhere. 35,36 A detection limit of 5 μg/L applied. Menthol- glucuronide concentrations are expressed in micrograms per liter. Statistical analysis A power calculation was performed (two-sided=0.05; power=0.80; Sd=109; minimal detectable difference in means=150min); at least 7 subjects needed to complete the study to reliably demonstrate a significant difference in Time to reach peak menthol- glucuronide concentration in the plasma (T max ), between the small-intestinal versus the novel ileocolonic release peppermint oil capsules. Anticipating 1 dropout, the aim was to include 8 participants. Statistical analyses were carried out using IBM SPSS statistics 23.0 (Chicago IL, USA) and GraphPad Prism 6.0 (La Jolla, CA, USA) for Macintosh. As the sample size was small ( N =8), data were analyzed using non-parametric tests. T max , T lag , Cmax values were determined directly from the plasma concentration-time profiles for each subject and were analyzed for comparison by the Wilcoxon-signed rank test. AUCs and T 1/2 were calculated by pharmacokinetic software MWPharm 3.80 (Mediware) using the log-linear trapezoidal rule (non-compartmental analysis). AUCs and C max were logarithmically transformed and a 90% CI interval was calculated of the log-transformed parameter ratios (small-intestinal/ileocolonic release) to assess bioequivalence. A P value of less than 0.05 was considered statistically significant. There were no missing data. Safety and tolerability analysis Subjects were monitored for adverse events by direct observation during the first 14 h after peppermint oil administration and again for 1 h after 24 h. During both test periods, participants completed a questionnaire regarding adverse events and tolerability at the 14 time points mentioned above. In addition, vital signs were reviewed