**4. Therapeutic Mechanism of Botulinum Toxin A on Bladder Oversensitivity**

BoNT-A has both motor and sensory effects in treating patients with DO or OAB. BoNT-A can inhibit the release of ACh and other neuropeptides from nerve terminals by cleaving the synaptosomal associated protein 25 kDa (SNAP-25), causing paralysis of the affected neuromuscular junctions [45]. In human bladders, Coelho et al. revealed that synaptic vesicle protein 2 (SV2) and SNAP-25 immunoreactive fibers were distributed throughout the suburothelium and muscle layer. Extensive co-localization of both proteins was noted in nerve fibers. SV2 is expressed more in parasympathetic fibers than in sympathetic or sensory fibers [46]. In nerve terminals, synaptic vesicles fuse with the presynaptic membrane where they release the neurotransmitter into the neuromuscular or neuroglandular junction. Intravesical BoNT-A injection relieves OAB symptoms as it enters bladder neurons binding to SV2, causing cleavage of SNAP-25 and preventing exocytosis of the neurotransmitter-containing vesicle at the nerve terminal [47,48]. Further studies revealed that after a single BoNT-A injection, cleaved SNAP-25 immunoreactive fibers were abundant throughout the guinea pig bladder tissue in the mucosa and muscular layer, significantly affecting the parasympathetic fibers. [49] However, because of the tight barrier function of the urothelium, intravesical instillation of BoNT-A cannot cleave SNAP-25 in nerve fibers. The bladder urothelium also expresses intracellular targets SNARE (Soluble *N*-ethylmaleimide-sensitive fusion Attachment protein REceptor) and binding receptor SV2 for BoNT-A uptake. BoNT-A has been shown to suppress the hypotonic-evoked ATP release from the cultured rat urothelial cells. BoNT-A injection can suppress sensory mechanisms and micturition reflexes after affecting the urothelial function of transmitter release [50].

Reduction of the expressions of TRPV1 and P2X3 on the suburothelial sensory afferents had been found in patients with DO treated with detrusor BoNT-A injections. [51] Patients also experience improvement of urinary frequency and reduction of the urgency severity after BoNT-A injection [52,53]. However, not all patients have similar sensory and motor therapeutic effects after BoNT-A injection. The reported success rates are between 60%-80% in patients with OAB [52–59]. Reduction of the urgency severity has been noted to be associated with long-term therapeutic efficacy after BoNT-A injections in patients with idiopathic DO [60]. Patients with both sensory and motor effects after BoNT-A injection showed a significantly better long-term therapeutic effect than the patients with motor or sensory effects alone. [60] Change of bladder capacity and increase of bladder fullness sensation after BoNT-A injections are also noted in these clinical trials. The improvement of bladder oversensitivity is likely to result from reduction of P2X3 and TRPV1 receptor expressions on the suburothelial afferent nerves [51].

In addition, BoNT-A injections for OAB and IC can effectively improve bladder sensory symptoms of frequency urgency and bladder pain in association with reduction of urinary NGF levels [61–64]. Recent studies also demonstrated that BoNT-A injection can inhibit cyclooxygenase-2 (COX-2) and prostaglandin E2 receptor 4 (EP4) expressions in the bladder tissue and block cyclophosphamide-induced bladder inflammation and overactivity. The increase of intercontractile intervals after BoNT-A injection indicates that BoNT-A can effectively improve both sensory and motor functions [65]. BoNT-A might also have a neuromodulatory or anti-inflammatory effect on the bladder wall in patients with OAB or IC, resulting in long-term sensory effect, possibly through CNS desensitization.

#### **5. Clinical E**ff**ects of Botulinum Toxin A on Bladder Oversensitivity in OAB**

The pathophysiology of OAB might be urotheliogenic or myogenic or could be due to neurogenic inflammation or BOO. As the pathophysiology of OAB might not clearly be determined before treatment, antimuscarinic agents or beta-3 adrenoceptor agonists usually cannot effectively treat all patients [66,67]. Recent investigation hypothesized that OAB might be a sensory disorder due to conditions activating the afferent nerve activities and resulting from the mild OAB subtype (hypersensitive bladder, frequency

without urgency) to the moderate subtype (OAB dry, with urgency but no urgency incontinence) or severe subtype (OAB wet, with urgency urinary incontinence). [44] In a long-term, large-scale study, Wennberg et al. found the incidence of urgency incontinence and OAB increased over a 16-year span, with a certain percentage of patients having symptoms progress or regress [68].

Histological study revealed that chronic inflammation exists in half of the OAB bladders [38]. OAB had also been postulated as a subtype of neurogenic inflammation with vascular and non-vascular inflammatory responses through activation of bladder sensory afferents and release of sensory neuropeptides such as substance P and CGRP [69]. The inflammation might involve locally over-expressed suburothelial receptors such as TRPV1 and P2X3, or central inflammatory responses in the dorsal root ganglia [23,70]. Bladder inflammation leads to afferent nerve activation and results in long-term neuroplasticity which lowers the threshold of nociceptive and mechanoceptive afferent fibers, causing bladder oversensitivity. [71] The dynamic changes of OAB presentation with time might be due to different bladder conditions affecting afferent nerve activities [72].

Although many factors could cause OAB, the downstream pathophysiology of bladder oversensitivity and DO is similar. Through BoNT-A injection, the release of neuropeptides and neurotransmitters such ACh, ATP, substance P, or CGRP can be effectively reduced, causing impairment of sensory afferents and paralysis of the detrusor muscles. Treatment of OAB by intravesical BoNT-A injection has achieved satisfactory results in recent decades [52,58,73–77]. Currently, BoNT-A has been widely used in treatment of urinary urgency and incontinence in OAB patients' refractory to oral medical treatment, and relief of frequency and bladder pain in patients with IC. The application of BoNT-A in OAB has been approved by the U.S. and European FDA and is listed as the third-line treatment in the AUA and EAU guidelines for OAB and urinary incontinence [78,79].

The dose of BoNT-A for OAB has been changed from 200 U or 300 U initially to the currently used 100 U [73]. A recent phase 3 clinical trial confirmed that the therapeutic results are similar between doses of 100 U and greater than 100 U [74,75]. BoNT-A at the dose of greater than 100 U increased the risk of adverse events of acute urinary retention and large post-void residual (PVR), as well as the incidence of urinary tract infection (UTI) [77,80]. Injecting BoNT-A at the trigone or bladder base for patients with OAB could effectively decrease the risk of acute urinary retention or large PVR without affecting the therapeutic effects of reduction of urgency and urgency incontinence [81]. The increased bladder sensation in OAB patients can effectively be decreased and bladder capacity increased after effective BoNT-A injection. Table 1 shows some representative study results on the improvement of bladder oversensitivity, reduction of frequency and urgency episodes, and increase of bladder capacity in OAB patients after different doses of BoNT-A injections.


**Table 1.** The therapeutic effects of botulinum toxin A on the increase of bladder capacity in patients with overactive bladder syndrome.


**Table 1.** *Cont*.

OAB: overactive bladder, IDO: idiopathic detrusor overactivity, NDO: neurogenic detrusor overactivty, DHIC: detrusor overactivity and inadequate contractility, SU: suburethelial injection, D: detrusor injection, MCC: maximal cystometric capacity, FDV: first desire volume, MBC: maximal bladder capacity, Vol: voided volume, PVR: post-void residual.

#### **6. Clinical E**ff**ects of Botulinum Toxin A on Bladder Oversensitivity in IC Patients**

As BoNT-A injections can inhibit the release of ACh at the presynaptic neuromuscular junction and reduce the expressions of TRPV1, P2X3, CGRP and substance P in the urothelial cells and sensory fibers, this treatment has been enthusiastically applied in the treatment of IC [95]. Intravesical BoNTA injection can reduce bladder pain response and inhibit CGRP release from bladder afferent nerves in rat models [96]. The effect of BoNT-A on pain response to irritants might not only reduce bladder oversensitivity but also result in desensitization of the CNS in dorsal horn ganglia after long-term neuroplasticity [97].

An initial pilot trial was performed by Smith et al. who treated 13 IC patients with 100 U to 200 U of Dysport or BoNT-A into the trigone and bladder base and found subjective improvement in 69% of patients after treatment. They concluded that BoNT-A might have an antinociceptive effect on bladder afferent nerves [98]. Kuo et al. injected BoNT-A suburothelially to treat 10 women with IC and seven had symptom improvement. The cystometric bladder capacity significantly increased from 210 ± 63.8 mL to 287 ± 115 mL (*p* = 0.05), however, patients who responded to treatment also had dysuria after BoNT-A injection [95]. The therapeutic effect of BoNT-A on IC patients was further confirmed by Giannantoni et al., who treated 14 patients with 200 U of BoNT-A at 20 sites of the trigone and bladder base. After treatment, 12 patients (85.7%) had subjective improvement at one and three months, where urinary frequency and bladder pain decreased and functional capacity increased significantly [99]. However, the therapeutic efficacy decreased to none at the one-year follow-up [100]. Nevertheless, intravesical BoNT-A injection could reduce bladder pain, urinary frequency, and improve psychosocial functioning. Table 2 shows some representative study results of the changes of bladder capacity and improvement of bladder oversensitivity that are significant in IC bladders after different doses of BoNT-A injections. Due to significant therapeutic efficacy the application of BoNT-A on IC, it has also been recommended as the third-line for IC refractory to lifestyle modulation and medication for pain and urothelial glycosaminoglycan replenishment [101].


**Table 2.** The therapeutic effects of botulinum toxin A on the increase of bladder capacity and decrease of bladder oversensitivity in patients with interstitial cystitis.

SU: suburothelial injection, D: detrusor injection, T: trigonal injection, MBC: maximal bladder capacity, IC: interstitial cystitis, BoNT-A botulinum toxin A.

The dose of BoNT-A for IC has not been well determined. Pinto et al. used 100 U of BoNT-A to treat IC women by 10 trigonal injections and found subjective improvement in all patients at oneand three-month follow-up and the therapeutic efficacy remained for nine months in more than 50% of patients [104]. The therapeutic efficacy of 100 U of BoNT-A on IC patients was demonstrated in a large cohort. Significant improvement of interstitial cystitis symptom index (ICSI) and interstitial cystitis problem index (ICPI, 23.6 ± 5.9 versus 15.2 ± 8.5, *p* = 0.000), VAS (5.3 ± 2.2 versus 3.3 ± 2.4, *p* = 0.000), functional bladder capacity (136 ± 77.6 versus 180 ± 78.2, *p* = 0.000) and global response assessment (0.3 ± 0.8 versus 1.4 ± 1.0, *p* = 0.000) were shown at six months after 100 U of BoNT-A injection. BoNT-A injection has been proven to be a safe and effective procedure for relief of bladder pain and increase of bladder capacity in IC patients [105].

Kuo et al. conducted a randomized controlled trial (RCT) to compare the clinical effectiveness of 100 U or 200 U of BoNT-A intravesical injections followed by cystoscopic hydrodistention and hydrodistention alone in 67 IC patients. Among three groups, IC symptom score significantly decreased in all, but bladder pain VAS reduction and functional and cystometric bladder capacity increases were only noted in the BoNT-A groups at three months [102]. Recently, Pinto et al. compared the efficacy and safety of trigonal injections of onabotulinumtoxinA and saline in patients with IC. They found significant reduction of bladder pain in the BoNT-A group than the saline group at week 12. BoNT-A significantly improved IC symptom score and quality of life at all timepoints and the PVR did not increase at the endpoint, indicating that BoNT-A trigonal injection had safe and effective treatment outcomes without PVR increase [112].

In the histopathological investigations, Kuo et al. also investigated bladder tissue NGF mRNA at baseline and after BoNT-A treatment and found the NGF levels significantly increased in IC patients at baseline and decreased to normal in responders after BoNT-A treatment [61]. Shie et al. found that mast cell activity and apoptotic cell count did not decrease significantly; Bax and p-p38 but not tryptase content decreased significantly after a single BoNT-A injection [106]. After three repeated BoNT-A injections every six months, significant decrease of tryptase, Bax, p-p38 contents and apoptotic cell counts were noted, and SNAP-25 content in the bladder also decreased after BoNT-A injections. The immunohistochemistric improvements are also associated with clinical symptomatic improvements. This evidence further confirms that chronic inflammation and apoptotic signaling molecules in the IC bladder wall can be reduced significantly after repeated BoNT-A injections in IC bladders [42]. Repeat BoNT-A injections are needed to achieve a better and more durable success in the

treatment of IC [107]. Repeated BoNT-A injections plus hydrodistention provides bladder pain relief and bladder capacity increase in responders. Through reduction of bladder suburothelial inflammation, the defective urothelial repair and improvement of cell differentiation ensue, leading to a healthy urothelium and thereby improving the clinical symptoms of IC [36].
