**5. Animal Evidence of BoNT-A for IC**/**BPS**

Although there is currently no definite animal model for CPPS, several chemical-induced cystitis animal models have been used to investigate the pathophysiology and develop a new treatment strategy for this disease. Lucioni et al. explored the effect of BoNT-A on the sensory neurotransmitters in chronic and acute injury models of the rat bladder by intraperitoneal injection of cyclophosphamide (CYP) and incubation of the bladder preparation with hydrochloric acid (HCl). The study found that a greater release of neuropeptides substance P (SP) and CGRP caused by acute injury with HCl and suggested that there is a potential therapeutic effect of BoNT-A in the treatment of neurogenic inflammation of the bladder [50].

Cayan et al. used 41 female Sprague-Dawley rats with intravesical instillation of HCl monthly to induce chemical cystitis and maintain chronic inflammation. These rats injected with 2-3 units of BoNT-A into the bladder detrusor and saline as the control group. Urodynamic studies showed that BoNT-A treatments increase the maximum bladder capacity and bladder compliance compared to the control group at the beginning and end of the study. The histological examinations reported similar counts of mast cells and leukocyte infiltration in these two groups. In this animal model of chemical cystitis, injected BoNT-A into the bladder detrusor led to improvements in vesical function which may be an alternative, minimally invasive treatment compared to other surgical modalities for a chronic inflammatory condition to improve deteriorated bladder function [51].

Smith et al. demonstrated intraperitoneally injected 150 mg/kg CYP to induce chronic cystitis in female Sprague-Dawley rats. It showed an increase of voiding frequency and hyperactivity of bladder. Treatment with CYP or BoNT-A did not affect the release of ATP in resting urothelium. Injection of CYP led to hypoosmotic stimulation and an increase of ATP release in chronic cystitis. After BoNT-A treatment, hypoosmotic shock-induced ATP decreased significantly. Cystometry revealed that CYP injection increased non-voiding bladder contraction. BoNT-A instillation markedly reduced non-voiding contraction frequency that was induced by CYP injection. However, neither CYP nor BoNT-A nor a combination of CYP + BoNT-A had any effect on the contraction frequency of bladder voiding. Furthermore, intravesical instillation of BoNT-A did not affect the release of ATP from the serosal side, implying that its effects were confined to the urothelial side of the bladder preparation [52].

Vemulakonda et al. studied the inhibitory function of BoNT-A on afferent pathways of chronic inflammation in the bladder. Among four groups of female Sprague-Dawley rats, namely group 1: saline-treated, group 2: BoNT-A treated, group 3: CYP treated, group 4: BoNT-A and CYP treated, all animals received intravesical protamine sulfate (1%), followed by intravesical BoNT-A or saline, and subsequently CYP or saline-injected intraperitoneally. Compared to saline controls, the study showed an increase of L6 and S1 c-fos immunoreactive cells after CYP treatment. BoNT-A/CYP treated group presented with a significant decrease of L6 and S1 c-fos immunoreactive cells compared with the CYP group. There was no significant difference in presentation between these two groups of saline and BoNT-A alone. Cystometrogram revealed that the increase of the non-voiding intercontractile interval in the BoNT-A/CYP group was more than 10-fold in CYP group. Conclusively, in a CYP animal model of chronic bladder inflammation, intravesical BoNT-A significantly inhibits the afferent neural response without impairing efferent bladder function [53]. Table 3 concludes the animal studies of applying BoNT-A for IC/BPS treatment.


#### **Table 3.** Study of BoNT-A treatment for IC/BPS in the animal.

Cystitis of rats was induced by chronic CYP model that reported by Vizzard [54]. Intraperitoneal injection with CYP (150 mg/kg) was administered every third day to a total of three doses to achieve chronic inflammation. CYP: cyclophosphamide. HCl: hydrochloric acid. SP: substance P.

#### **6. Clinical Use of BoNT-A for Sexual Pain Syndrome**

Women with sexual pain disorders experience genital pain during sexual intercourse occurring at the periods including before, in the process, or after the sexual activity that involves the clitoris, vulva, vagina, and/or perineum, thus causing difficulty in sexual intercourse and personal distress. Dyspareunia is painful sexual intercourse, in which pain can occur over the external genitalia, inside the vagina or deeper pelvis due to numerous medical, physical, social, or psychological causes. Generally, the prevalence of dyspareunia was reported to affect between 8%–21.1% of women [55]. More sexual pain disorders were reported in female patients with CPP than women without CPP [1]. Morrissey et al. reported a prospective pilot open-label study of 21 women with CPP and refractory high-tone pelvic floor dysfunction (HTPFD) under needle electromyography (EMG) guidance with BoNT-A injections [30]. They prepared 300 U of BoNT-A with nonpreserved saline in a 10-mL syringe and attached it to a 12.5 cm disposable monopolar EMG needle electrode. BoNT-A was injected into the spastic PFM trigger point (30 U), other deeper PFMs including pubococcygeus, iliococcygeus, and coccygeus (30 U each as needed), and obturator internus muscles (up to 60 U into each side). Of these 21 female patients, 66.7% had vulvodynia. After treatment, the dyspareunia VAS score showed significant improvement at weeks 12 (5.6, *p* = 0.011) and 24 (5.4, *p* = 0.004) than baseline (7.8). The Female Sexual Distress Scale (FSDS) showed significant improvement of sexual function at 8 weeks (27.6, *p* = 0.005), 12 weeks (27.9, *p* = 0.006), and 24 weeks (22.6, *p* < 0.001) compared with baseline

(34.5). Resting pressures and maximum contraction pressures of the vagina as measured by vaginal manometry significantly decreased during all follow-up examinations (*p* < 0.05).

Vulvodynia is characterized as genital pain without clear etiology that may have resulted from sexual intercourse and causes sexual pain disorder. In the general population, the estimated prevalence of vulvodynia ranges from 10% to 28% in reproductive-aged women [56]. Aberrant increase in the number of nociceptors, which causes peripheral hypersensitivity, leads to intraepithelial neural hyperplasia and strong pain in the vestibule, which may be the cause of vulvodynia [12]. Approximately 7–8% of women have experienced vulvodynia by age 40 that limited sexual intercourse [57]. BoNT-A can inhibit the release of ACh from sympathetic neurons and parasympathetic neurons to relieve vulvodynia and improve dyspareunia. A retrospective study recruited seven women aged 28–61 years with intractable genital pain that was refractory to conventional treatment [58]. Twenty units of BoNT-A was injected into the pain sites including the vestibule, levator ani muscle and the perineal body. If the symptoms had not subsided totally, 40 U of BoNT-A was injected repeatedly every two weeks. After BoNT-A injections, pain decreased or disappeared in all patients. The mean VAS score decreased to 1.4 from 8.3 before the treatment, with no recurrence. The study showed improvement of sexual life without significant pain or discomfort during or after sexual activity.

Hedebo et al. used BoNT-A to treat vulvodynia refractory to conventional treatment for at least six months [59]. The cohort consisted of 79 women and each received 100 U of BoNT-A injections. The results showed significant improvements in dyspareunia (7.82 to 5.82, *p* < 0.01), Negative Interference in Quality of Life (NIQL) (7.88 to 6.19, *p* < 0.01) and the cotton swab test (6.81 to 5.50, *p* < 0.01).

High doses of BoNT-A injection seem to have effectiveness in the treatment of vulvodynia related to sexual pain syndrome. A randomized, double-blind, three-arm, placebo-controlled study from June 2008 to September 2014 included 32 women aged 23–35 years with provoked vestibulodynia [60]. They subcutaneously injected BoNT-A 50 U (arm A), 100 U (arm B) or saline (arm C) into the dorsal vulvar vestibulum and evaluated pain scores after three months. They injected 100 U of BoNT-A for persistently symptomatic women. At the 6-month visit, symptomatic patients received a second injection of BoNT-A 100 U in arm C. The results showed no significant differences in pain between these three groups after three months from the initial injection. However, significant improvements were observed among all three arms using the von Frey filaments test. Exploratory analyses reported that repeat injections with 100 U of BoNT-A over six months had a significant reduction of pain including VAS and von Frey filaments. Fifty-eight percent of patients assessable after repeat BoNT-A injections with 100 U had symptom-free or ≥ 2 points improvement of VAS score.

In 2016 Pelletier et al. evaluated in a prospective cohort study the long-term effectiveness of BoNT-A injection for more than two years in 19 women with provoked vestibulodynia [61]. Fifty units of BoNT-A were diluted in 1.0 mL saline solution followed by injection into bilateral bulbospongiosus muscles for a total dose of 100 U. After 24 months, 37% of participants had no pain. After treatment, they showed significant improvements in the VAS, Dermatology Life Quality Index (DLQI) and Female Sexual Function Index (FSFI) scores at 24 months compared to baseline (*p* < 0.0001). Eighteen women (95%) were able to have sexual intercourse after 24 months.

BoNT-A was successfully used in sexual pain syndrome and appeared to have long-term beneficial effects for sexual activity (Table 4). It is important to continue further research for investigating the novel treatments in the sexual pain syndrome of women.


**4.**StudiesofBoNT-Ainjectionforsexualpainsyndromeinwomen.

NRS: Numerical rating scale. NIQL: Negative interference in quality of life. FSFI: Female Sexual Function Index. DLQI: Dermatology Life Quality Index.
