**4. Conclusions**

Intratrigonal injection of botulinum toxin A in patients with BPS/IC is an effective and safe long-term treatment option. Moreover, the duration of each treatment seems to be sustained along all of the treatments, even when the number of injections is high. Age, basal pain severity and short duration of the first treatment effect do not seem to predict whether a patient will be a long-term responder to OnaBotA. The main adverse effects are mild, simple UTIs and straining, occurring in a minority of procedures. A specific phenotype for OnaBotA responders is lacking, but given the widely positive effect of the treatment, it should be offered as a third line of therapy.

#### **5. Material & Methods**

This is a retrospective study, analyzing our cohort of patients diagnosed with BPS/IC and treated with intratrigonal botulinum toxin in a tertiary public university hospital in Porto (Porto, Portugal) between 2009 and 2022. The procedure and the OnaBotA preparation, in our institution, are carried out by different surgeons in the urology department and by different nurses in our surgical center.

BPS/IC is diagnosed in compliance with guidelines, following the exclusion of more common conditions through physical examination, patient history, uroculture, urinalysis, cystoscopy, urinary cytology, neurological examination and in some cases pelvic magnetic resonance. All patients performed bladder hydrodistension and urothelium biopsies, before initiating OnaBotA treatment, as part of the diagnostic workup. A total of 47 patients were identified.

The clinical and surgical records of the participants, available in our hospital database, were utilized for the analysis of data related to the interventions—every medical record such as written appointments, date of surgery and surgery details are available in the hospital database. The process of collecting the electronic records into files was conducted following the Ethics Committee's approval for the study. Patients were assessed for pain intensity using a 10-point visual analogue scale (VAS) (results were from 0 to 10; a higher number corresponded to higher pain). For the evaluation of each patient, the authors accessed the number of treatments and the treatment duration (the duration between an injection and the request for a new one). The overall time of disease follow-up was also accessed. Adverse effects of the procedure, such as urinary tract infection, straining and urinary retention were analyzed when recorded.

Three groups of patients were defined to identify possible predictors of outcome. Group A comprised all patients currently in treatment (with the disease controlled after an injection or patients that already requested a reinjection); group B comprised patients who were non-responders to OnaBotA (they could have already shown treatment response but now are non-responders to OnaBotA and abandoned the treatment but not the clinic); and group C are patients lost to follow-up but for non-therapeutic reasons.

A total of 25 patients met the criteria for group A, 17 met the group B criteria, and five met the criteria for group C. For the evaluation of possible predictors of long-term success, we compared the characteristics of group A with group B patients, such as age, the initial pain intensity evaluated by the VAS score, the overall treatment duration, and also the duration of the first treatment (excluding patients that only received one injection). Moreover, we evaluated whether a rapid loss of effect after the first treatment was predictive of a general treatment failure or the need for more frequent injections. The treatment maintenance was evaluated and displayed as a Kaplan-Meier graphic, providing information on the proportion of drop-outs and patients still in OnaBotA. The number

of injections performed before abandoning OnaBotA treatment, as well as the number of injections performed by patients still in treatment, are represented in the graphic.

Most treatments were performed outside of a trial, since from a total of 198 treatments, only 71 (35%) of them were in a clinical trial set EudraCT: 2014-001013-81, "Treatment of Bladder Pain Syndrome with Onabotulinum toxin A" (ProBaBle). This leads us to consider that these data reflect real-world practice.

#### *5.1. Procedure Technique and Follow Up*

In all our patients, the procedure, the drug and the dose administered were the same. The botulinum toxin A used was OnaBotA (Allergan, Irvine, CA, USA), and it was injected under light sedation through a 23-gauge needle (Coloplast A/S, Humlebaek, Denmark) inserted 3 mm into the trigonal wall with a 70◦-lens cystoscopy control. A total of 100 Units were distributed throughout 10 sites (10 U per 1 mL saline)—Figure 3. A preoperative diagnostic workup was performed to guarantee that the individuals had a negative uriculture and no symptoms of cystitis. Patients were evaluated 2 to 3 weeks after the procedure to access early complications.

**Figure 3.** Schematic representation of the 10 bladder sites of OnaBotA injection.

## *5.2. Statistical Analysis*

The statistical program IBM® SPSS® v.28.0 (IBM Corp., Armonk, NY, USA) was used for data analysis. The Kolmogorov-Smirnov test was used to assess the normality of the distribution of continuous variables. Continuous variables with a normal distribution are presented as mean ( ±standard deviation). Non-normally distributed variables are reported as the median (percentile 25; percentile 75) and a Student's t-test was used to compare the variables with normal distribution.

The authors chose to compare groups A and B for predictor analysis. Group C includes five patients that abandoned OnaBotA treatment early for reasons unrelated to the treatment or their clinical situation.

Clearance from the hospital Ethics Committee (Protocol number 337-21: Effect of intratrigonal botulinum toxin in patients with BPS/IC in a single center) was obtained.

**Author Contributions:** Conceptualization, R.P. and P.A.-M.; methodology, F.B.; software, P.A.-M.; validation, F.C., R.P. and F.B.; formal analysis, A.F.-M. and F.B.; investigation, A.F.-M. and P.A.-M.; data curation, A.F.-M.; writing—original draft preparation, A.F.-M.; writing—review and editing, P.A.-M., R.P., F.B. and F.C.; visualization, F.B.; supervision, P.A.-M., R.P., F.B. and F.C.; project administration, R.P.; funding acquisition, All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding. **Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional or Ethics Committee of Centro Hospitalar e Universitário de São João, Porto, Portugal (Protocol number 337-21: Effect of intratrigonal botulinum toxin in patients with BPS/IC in a single-centre, on 8 November 2021).

**Informed Consent Statement:** Patient consent was waived. Since the data is electronically recorded in the hospital system, after the Hospital's Ethics Committee study approval we could use those information without revealing patients' identity (which we did not).

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to Hospitals Ethics Committee policy.

**Conflicts of Interest:** The author Pedro Abreu-Mendes declares that only Francisco Cruz has the current conflict of interest: he is a consultant for Allergan, Astellas, Bayer, and Recordati; has received lecture fees from Allergan and Astellas; and is a trial investigator for Astellas, Bayer, Ipsen and Recordati.
