**2. Biology and Mechanism of BoNT-A**

Botulinum toxin is a neurotoxin produced by Clostridium botulinum [6]. It was first isolated as a crystalline product in 1946 [10] and was found to have a paralyzing effect on hyperactive muscles in the 1950s [11,12]. To date, eight immunologically antigenic distinct subtypes have been identified, i.e., subtypes A, B, C1, C2, D, E, F, and G [13]. Botulinum neurotoxin subtype A is potent, with the longest duration of action among these subtypes, and is commonly used in clinical practice [14]. Currently, several commercial forms of BoNT-A are available, where Botox® (Allergan, Irvine, CA, USA) and Dysport® (Ipsen, Slough, UK) are the two most widely used agents [15]. It should be noted that these products cannot be considered equivalent in terms of dose, efficacy, or safety owing to having different fragments of botulinum toxin and different manufacturing processes [6]. Although a few studies have suggested the efficacy of 1 unit of Botox to be similar to 3–5 units of Dysport [16,17], this simple linear exchange equation has been questioned in different BoNT-A applications [18].

The mechanism by which BoNT-A inhibits target muscle contractions is well-known as the blocking of acetylcholine release from presynaptic efferent nerves at the neuromuscular junctions via cleaving of synaptosome-associated protein 25 (SNAP-25) and preventing the docking of acetylcholine-containing vesicles to the neuronal cell membrane [19]. The toxin also blocks the release of other neurotransmitters, including adenosine triphosphate, substance P, calcitonin gene-related peptide, and downregulates sensory receptors such as vanilloid (TRPV1) and purinergic (P2 × 3) receptors [20,21] known as an afferent nerve desensitizer. The effect of BoNT-A on urethral striated muscle is thought to block the presynaptic release of acetylcholine in the neuromuscular junction and subsequently achieve a chemical sphincterotomy, which is believed to relieve the USD and improve VD [22,23]. An animal study has also shown a reduction in the release of norepinephrine in the urethra after BoNT-A injections at the EUS of rats, supporting its use in the treatment of EUS overactivity [24]. A decrease in maximum urethral pressure (MUP) of an average of 27 cm H2O was noted after BoNT-A injection in DSD patients [5].

Besides the direct effect on the EUS, BoNT-A injections at the EUS may lead to recovery of bladder detrusor contractility in VD patients with detrusor underactivity (DU) and hyperactive EUS or PRES [25]. It has been proposed that the suppression of EUS contraction will deactivate the afferent signals inhibiting the bladder reflex [26,27], however, this neuromodulation effect still awaits further confirmation.

### **3. Urethral Sphincter BoNT-A Injections in Patients with Detrusor Sphincter Dyssynergia**

DSD is defined as involuntary contractions of the EUS during detrusor contractions [28]. DSD is mainly caused by damage to the upper motor neurons lying between the pontine micturition center and the sacral spinal cord, such as in the morbidities associated with spinal cord injury (SCI), multiple sclerosis (MS), and transverse myelitis (TM) [29–33]. DSD usually causes significant VD, leading to chronic urinary retention, recurrent urinary tract infection (UTI), high detrusor voiding pressure, autonomic dysreflexia, vesicoureteral reflux, and possible renal damage [34]. Alpha-blockers have been used to decrease outlet resistance, but the results of urodynamic studies have not been convincing [35]. Clean intermittent catheterization (CIC) is an effective alternative for patients to

empty their bladder, but some patients are not able to tolerate it well due to upper limb impairment or psychological unwillingness [36]. Surgical sphincterotomy is another drastic option for those who fail in the treatments mentioned above, however, many of such patients experience a worsened quality of life due to persistent incontinence and a high long-term failure rate [37]. BoNT-A is thought to be an alternative, as it can block the release of acetylcholine from presynaptic vesicles at the neuromuscular junction, which causes temporary, reversible chemo-denervation of the external EUS [38].

Dykstra et al. [5] first described both transperineal and transurethral injections of BoNT-A to the EUS in 1988 as a new therapeutic approach for 11 male patients with SCI and DSD. In this preliminary study, weekly injections using different dosages of BoNT-A effectively decreased the MUP and post-void residual (PVR) urine volume. Electromyography (EMG) further confirmed sphincter denervation in all patients. The authors then conducted a small sample, randomized, double-blind placebo-control trial in five SCI male patients with DSD to compare low dose BoNT-A with normal saline by weekly injections for three weeks. They concluded that the therapeutic effects were found only in the BoNT-A injection group [39].

Instead of a once-weekly injection, a once-monthly dose has also been confirmed to effectively improve sphincter function as well as to decrease PVR [40]. In addition, a single transurethral dose of BoNT-A was further evaluated by Petit et al. [41] in 17 male patients with SCI and DSD. Their results showed similar clinical improvements in approximately 70% of the cases with an average therapeutic effect of two to six months. Phelan et al. [42] first confirmed the effectiveness of sphincteric BoNT-A injections in both male and female patients with various etiologies of DSD, including SCI, TM, and MS. De Sèze et al. [43] carried out a randomized, double-blind control trial to compare the efficacy of and tolerability to BoNT-A with a lidocaine single transperineal injection in patients with DSD. Higher patient satisfaction scores and significant decreases in PVR, MUP, and EMG activity were found only in the BoNT-A group. Most of the patients tolerated the treatment well without major complications.

Recently, more data describing the clinical experiences related to sphincteric BoNT-A injections in patients with DSD have been reported. Both transperineal injections [5,40,43–47] and transurethral injections using cystoscopy [5,40–42,48–53] showed promising outcomes in terms of reducing sphincteric activity. Although transurethral injections have been reported to be more effective in terms of decreasing the MUP than the use of a transperineal route [40], no further trials have ever directly compared these two injection methods. Most of the recent publications on this topic involve the use of a sphincteric injection of 100 units of Botox in DSD patients since a more prolonged therapeutic effect was found, as compared to 50 units, in a previous randomized control trial [54].

Urodynamic parameters were commonly used as measurements of objective outcomes after sphincteric BoNT-A injections in patients with DSD. As expected, a significant reduction in EMG activity [5,39,43] and a decrease in MUP [5,40,41,43,44] during the voiding phase have been found. Improvements in PVR [5,40–43,49], detrusor contraction pressure [5,44,48,49], and maximal flow rate (Qmax) [42,48] were also reported as a result of decreased sphincteric resistance. However, unlike SCI patients, a randomized, double-blind, placebo-control trial in 86 patients with MS and DSD showed that a single injection of BoNT-A did not decrease the PVR [44]. The authors posited that the unchanged PVR could be attributed to the lower baseline detrusor contraction pressure. In addition to the outcome measurement, urodynamic parameters have also been used to predict therapeutic outcomes. Several pre-treatment urodynamic parameters, including higher baseline detrusor contraction pressure [41,53], lower baseline sphincteric tone [48,50,55], and a synergic bladder neck [53,55] have been confirmed as predictors of a favorable outcome.

Although objective urodynamic results have been improved after EUS BoNT-A injection, Kuo et al. [52] reported inconsistencies between urodynamic outcomes and patient satisfaction. Even though the PVR and detrusor contraction pressure were improved, patients were not satisfied with the outcomes mainly because of the increased incontinence grade [51]. Notably, for patients with SCI and DSD, a detrusor BoNT-A injection provided a much better quality of life than the case with an EUS injection [52] This result emphasized the importance of continence in a patient's quality of life. Other causes of patient dissatisfaction included persistent difficult urination [51], increases in urgency episodes [51], and the need for repeated injections [45]. For therapeutic outcomes, patient satisfaction was mainly due to improved voiding conditions and fewer autonomic dysreflexia (AD) episodes [52,53]. To summarize the subjective outcomes of sphincteric BoNT-A injections for patients with DSD, 61–88% experienced clinical improvement [5,40–42,48,51] for a therapeutic duration lasting two to six months [40–42,44], more than 80% could regain spontaneous voiding and successfully remove the indwelling catheter or stop CIC [42,48,49], and episodes of autonomic dysreflexia were reduced in more than 50% of the cases [5,43,50,51]. Table 1 summarizes the clinical studies on efficacy and adverse events related to sphincteric BoNT-A injections in the treatment of patients with DSD.

In spite of the benefits of sphincteric BoNT-A injections in patients with DSD based on the literature, the use of different injection protocols among various etiologies makes intergroup comparisons difficult. Further randomized control trials with large case numbers focusing on a single etiology of DSD are necessary to evaluate the therapeutic impacts on both objective and subjective parameters, quality of life, duration of effect, and long-term durability after repetitive injections.





*Toxins* **2019**, *11*, 728

injury; TM =

volume. Sphincteric injections used with preparation other than the typical BoNT-A commercial form, including Botox or Disport, were denoted as "BoNT-A". a UDS

were found in the urethral Botox group. b

Transverse myelitis; UDI-6 =

Urogenital Distress

Individual results in specific disease groups were not available.

Inventory–Short

 Form; UDS =

Urodynamic study; UTI = Urinary tract infection; VAS = Visual analog scale; VV = Voided

improvements
