In this study conducted at Zelo Phase 1 Unit at Copenhagen University Hospital - Bispebjerg and Frederiksberg, Copenhagen, Denmark, we investigated the effect of a single dose of imipramine (50 mg) on urethral and anal pressure in healthy women. The findings from the urethral pressure measurements and a thorough description of the study design, detailed eligibility criteria, and methodologies have been published by Kornholt et al. [15].

The study was approved by the Regional Ethics Committee of the Capital Region of Denmark (approval number H‐17007330) and the Danish Medicines Agency (EudraCT no. 2017‐000119‐18), and was registered at www.ClinicalTrials.org (identifier NCT03102645). Further, the study adhered to the Declaration of Helsinki and Good Clinical Practice guidelines. All participants provided written informed consent upon enrolment.

This study was designed as a double-blind, randomized, placebo-controlled crossover study in healthy women. We recruited 16 healthy female volunteers by advertisement and randomized them to one of two treatment sequences (imipramine to placebo or placebo to imipramine). The Capital Region Pharmacy performed the balanced randomization (50% of the participants receiving imipramine at the first study visit) and prepared the numbered blinded dosing kits containing 50 mg imipramine and a visually identical placebo according to the sequence allocation list. The participants were consecutively assigned randomization numbers specifying their dosing kit. Imipramine was selected as the TCA study drug for this combined UPR and AAR study owing to its notable off-label use in managing stress urinary incontinence [16].

On each of the two study visits, separated by at least 7 days of washout, baseline measurements of the urethral and anal pressure were performed using UPR and AAR respectively. Subsequently, a single dose of 50 mg imipramine or placebo was administered to the participants. After 1 h of rest, corresponding to the mean time to peak plasma concentration for imipramine, 1-h post-dose UPR and AAR measurements were performed. The participants were interviewed about adverse events at the end of study visits 1 and 2, the start of study visit 2, and via telephone 5 days following the final study visit.

Healthy women aged 18 to 55 with a body mass index (BMI) between 18.5 and 30.0 kg/m2, who were nonsmokers and consented not to breastfeed, become pregnant, and participate in other clinical trials during the study period, were eligible for this study. Primary exclusion criteria were pregnancy, any clinically relevant history or evidence of disease evaluated by the investigator, history of clinically significant urinary incontinence, and the use of any prescription or over-the-counter drugs in the 2 weeks before study drug administration (except for paracetamol and hormonal contraceptives) [15].

The AAR assessments and the parameters obtained from these have previously been described [17,18,19]. The AAR measurement catheter consists of a 45-cm polyvinyl chloride (PVC) tube with a thin, fully collapsible, polyurethane bag, 70 mm long, mounted on the distal end, which can be inflated to a maximum diameter of 5 mm. At its proximal end, the PVC tube is connected to a probe with a loudspeaker, a microphone, and another PVC tube to which a handheld syringe, used to inflate and deflate the bag, is connected. Sound waves initiated by a digital signal processor are amplified by the loudspeaker, reflected from the bag, and recorded by the microphone. By applying the Ware–Aki algorithm to the reflected sound waves, computer software (Oticon A/S Rhinometrics 5.6.1.1, Copenhagen, Denmark) calculates the cross-sectional area of every millimeter along the 70-mm-long bag and, in this way, corresponding pressure measurements and cross-sectional areas are obtained. In the further analysis, only the measurements from the point of minimal measurable cross-sectional area, representing the high-pressure zone of the anal canal, are used.

With the participant in the lithotomy position, the polyurethane bag is placed in the anal canal using a baby feeding tube as a guidewire. AAR measurements are recorded during rest and during voluntary contractions of the pelvic floor (squeeze). During the resting condition, the pressure is increased from 0 to 200 cmH2O over 7 s and decreased again to 0 cmH2O over 7 s. This cycle (inflation/deflation) is then repeated ten times, and the mean value of these ten measurements is used for analysis. Subsequently, the participant is asked to contract the pelvic floor (“as trying not to pass gas”). When familiar with the correct squeezing technique, the pressure is increased from 0 to 200 cmH2O in 7 s, and while the participant relaxes the pelvic floor, the pressure is decreased again. This squeezing procedure is repeated five times, and the mean value of five measurements is used for analysis. The total time to obtain a complete set of AAR measurements is approximately 4 min.

All UPR and AAR examinations were performed by a single operator (TR), whereas the AOP was calculated and agreed upon by two of the authors (TR and NK). All involved were blinded to the participants’ sequence allocation until statistical analyses were completed, implicating analysis of the treatment effect as A versus B before we requested the complete unblinding of the study from the hospital pharmacy.

Based on a prior AAR study, we anticipated a within-subject standard deviation (SD) of 21 cmH2O for the squeezing opening pressure [11]. With a sample size of 16 subjects, our study had a power of 80% to detect a 15 cmH2O difference in squeezing opening pressure between imipramine and placebo using a two-sided significance level of 0.05.

The baseline demographics of the subjects and the within-subject standard deviation (SD) were summarized descriptively. Changes in mean AOP (imipramine versus placebo) from baseline to the 1-h outcome were analyzed using cros analyses, which adjust for period effects. As generally recommended, we ensured a long washout between the study visits to minimize the risk of carry-over effects and omitted formal tests for carry-over [20, 21]. To investigate potential placebo effects, changes in mean AOP from baseline to the 1-h outcome on placebo days were analyzed using paired t tests. Safety data were summarized descriptively.

The statistical analyses for this paper were performed using RStudio version 1.0.136 (RStudio, Boston, MA, USA). Graphical plots were generated using GraphPad Prism version 9.4.1 for Windows (GraphPad Software, San Diego, CA, USA).