**4. Conclusions**

This long-follow-up pilot study shows that repeated intravesical instillations of Sapylin after BoNT/A injection can produce significantly better clinical outcomes than BoNT/A alone in IC/PBS patients. Further research comparing BoNT/A injection plus Sapylin instillation, BoNT/A alone, or Sapylin alone in the treatment of IC/BPS is warranted with a large, multicenter, randomized, placebo-controlled trial.

## **5. Materials and Methods**

## *5.1. Participants and Ethics*

From March 2015 to April 2020 in our hospital, 70 IC/BPS patients were selected according to the following: (1) diagnosed according to National Institute of Diabetes and Digestive and Kidney (NIDDK) guidelines [29]; (2) an O'Leary-Sant Interstitial Cystitis Symptom Index score (ICSI) of more than 9 and disease duration of more than 6 months; (3) previous adequate unsuccessful treatments in accordance with guidelines including oral anti-cholinergic agents, pain-killers, amitriptyline, intravesical hyaluronic acid, heparinoids and so on. Those patients with urinary cancers, bacterial genitourinary infections, bladder tuberculosis, radiation-induced or chemical cystitis, sexually transmitted diseases, pelvic inflammatory disease and endometriosis were excluded [30]. All patients were required to provide their informed consent to receive BoNT/A injection alone or BoNT/A plus Sapylin and they made their own decision about which treatment to receive. Furthermore, 8 cases were removed because of missing data. Finally, this study investigated 34 patients who received repeated intravesical instillations of Sapylin after BoNT/A injection (Mix group) and 28 patients who accepted a submucosal injection of BoNT/A alone (Control group).

After treatment, to evaluate its efficacy, we performed a questionnaire investigation of symptoms via telephone and outpatient interviews. The primary outcome was the global response assessment (GRA) at 3 and 6 months after treatment: "As compared to when you started the current study, how would you rate your overall pelvic symptoms now?", with seven response categories: 1 = Markedly worse, 2 = Moderately worse, 3 = Slightly worse, 4 = No change, 5 = Slightly improved, 6 = Moderately improved and 7 = Markedly improved. Participants who reported "moderately or markedly improved" were considered as the responders to the treatment. Others were defined as non-responders. The secondary outcomes were pelvic pain and urgency/frequency patient symptom score (PUF), visual analogue scale (VAS), O'Leary-Sant ICSI and Interstitial Cystitis Problem

Index (ICPI). Further, a 3-day urinary diary was used to evaluate the mean voided volume per micturition, maximal voided volume per micturition and mean urinary frequency per 24 h. Cystoscopy was performed under local anesthesia before treatment. Saline was delivered into the bladder until the patients' bladder pain or discomfort became intolerable. Then, the total saline delivery volume was calculated as the functional maximal bladder capacity.
