**Botulinum Toxin A for Sialorrhoea Associated with Neurological Disorders: Evaluation of the Relationship between Effect of Treatment and the Number of Glands Treated**

**Domenico A. Restivo 1,\*, Mariangela Panebianco 2, Antonino Casabona 3, Sara Lanza 4, Rosario Marchese-Ragona 5, Francesco Patti 6, Stefano Masiero 7, Antonio Biondi <sup>8</sup> and Angelo Quartarone <sup>9</sup>**


Received: 21 December 2017; Accepted: 22 January 2018; Published: 27 January 2018

**Abstract:** *Background*: Sialorrhoea and drooling are disabling manifestations of different neurological disorders. The aim of this study was to evaluate the effects of botulinum neurotoxin type A (BoNT/A) injection on hypersalivation in 90 patients with neurological diseases of different aetiologies, and to define the minimum number of injected salivary glands to reduce sialorrhoea. Determining the minimum number of glands that need to be engaged in order to have a significant reduction in drooling may be very useful for establishing the minimum total dosage of BoNT/A that may be considered effective in the treatment of hypersalivation. *Methods*: Twenty-five mouse units (MU) of BoNT/A (*onabotulinumtoxin A,* Botox; Allergan, Irvine, CA, USA; 100 MU/2 mL, 0.9% saline; or *incobotulinumtoxin A*, Xeomin; Merz Pharma, Germany; 100 MU/2 mL, 0.9% saline) were percutaneously injected into the parotid (p) glands and/or submandibular (s) glands under ultrasound control. On this basis, patients were divided into three groups. In group A (30 patients), BoNT/A injections were performed into four glands; in group B (30 patients), into three glands, and in group C (30 patients), into two glands. Patients treated in three glands (group B) were divided into two subgroups based on the treated glands (2 p + 1 s = 15 patients; 2 s + 1 p = 15 patients). Similarly, patients being injected in two glands (group C) were subdivided into three groups (2 p = 10 patients; 1 p + 1 s = 10 patients; 2 s = 10 patients). In patients who were injected in three and two salivary glands, saline solution was injected into the remaining one and two glands, respectively. Assessments were performed at baseline and at 2 weeks after the injections. *Results*: BoNT/A significantly reduced sialorrhoea in 82 out of 90 patients. The effect was more evident in patients who had four glands injected than when three or two glands were injected. The injections into three glands were more effective than injections into two glands. *Conclusions*: Our results have shown that BoNT/A injections induced a significant reduction in sialorrhoea in most patients (91%). In addition, we demonstrated

that sialorrhoea associated with different neurological diseases was better controlled when the number of treated glands was higher.

**Keywords:** sialorrhoea; drooling; salivary glands; swallowing; botulinum toxin; eccrine glands; *onabotulinumtoxin A*; *incobotulinumtoxin A*

**Key contribution:** This study evaluated the effects of BoNT/A injection on hypersalivation in a wide number of neurologic patients and, also, it evaluate whether the number of treated glands might influence the efficacy of the treatment.

#### **1. Introduction**

Sialorrhoea or hypersalivation and drooling are known to be associated with several neurological disorders [1]. Other than a disabling social problem, hypersalivation is often associated with impairment of swallowing coordination. This condition affects about 10% of patients with cerebral palsy and post-traumatic encephalopathy [2] and 20% of patients with amyotrophic lateral sclerosis [3]. In Parkinson's disease, its frequency varies from 10% to 84% [4].

Salivation is controlled by the autonomic nervous system via cholinergic nerve fibres. In healthy adults, the parotid (p) and submandibular (s) glands—the major salivary glands—account for about 95% of the total salivary secretion. The remaining 5% is produced by the lingual and minor glands [5].

Traditional treatment of excessive drooling includes anticholinergic oral drugs, surgical intervention and local irradiation of salivary glands [6,7]. However, these treatments are poorly tolerated, are invasive and are often ineffective in a number of patients. In fact, systemic anticholinergic drugs are often ineffective and produce unacceptable side effects such as blurred vision, urinary retention, and cardiac arrhythmia. Surgical intervention and local irradiation of salivary glands have been performed, but these are invasive procedures that are often unacceptable to patients and their caregivers.

Recently, the percutaneous injection of botulinum neurotoxin type A (BoNT/A) into salivary glands has been shown to be effective in abolishing excessive sialorrhoea associated with several neurological disorders [8–13]. In addition, studies on botulinum neurotoxin type B (BoNT/B) in patients affected with cervical dystonia have shown a relatively high incidence of dry mouth [14]. This suggests that BoNT/B may be more effective in the treatment of hypersalivation than BoNT/A [14]. However, in our country, BoNT/B treatment can be performed only in patients unresponsive to BoNT/A.

The rationale for the use of BoNT/A in this condition is the selective block of presynaptic release of acetylcholine from the cholinergic endings supplying eccrine salivary glands [15,16]. Usually, the effect of this treatment lasts several months. Although encouraging, the results of these studies were obtained from a restricted number of subjects. Moreover, in previous studies, a correlation between the number of glands treated and the amount of salivation reduction was not investigated.

The aim of the present study was thus to evaluate the effects of BoNT/A injection on hypersalivation in a wide number of patients with different neurological disorders and, in addition, to evaluate whether the number of treated glands might influence the efficacy of the treatment. The determination of the minimum number of glands that should be engaged in order to have a significant drooling reduction may be very useful for establishing the minimum total dosage of BoNT/A that may be considered effective in the treatment of hypersalivation.

#### **2. Results**

In evaluating the effects of BoNT/A, we detected four different levels of responsiveness to the treatment. We scored from 3 (good responders with a reduction of 75% in the roll weight) to 0 (no response; when weights of the wet rolls did not change); intermediate and poor responders were scored with 2 or 1, respectively, when the reduction of their roll weights was equal to 50% or 25% (see Table 1).

**Table 1.** The demographic characteristics of the 90 neurological patients with hypersalivation treated with BoNT/A injections into different salivary glands. Abbreviations: PD, Parkinson disease; ALS, amyotrophic lateral sclerosis; BI, brain injury; CP, cerebral palsy; s, submandibular; p, parotids; Inco = incobotulinum toxin A; Ona = onabotulinumtoxin A.



**Table 1.** *Cont.*

Eighty-two out of 90 patients (91%) were responders to the treatment. In almost all patients, sialorrhoea was dramatically reduced at 2 weeks (mean dental roll weight before BoNT/A: 0.8 ± 0.08 g; mean dental roll weight 2 weeks after BoNT/A: 0.25 ± 0.1 g). Only 8 (8.8%) out of 90 patients did not benefit from the treatment. Patients with injections into four, three and two glands showed large differences over the three groups (Figure 1 F2,87 = 48.78; *p* < 0.000001), with the highest average score being observed for the four-gland group (2.63 ± 0.13) and lower scores recorded for the three-gland group (1.73 ± 0.13) and for the two-gland group (0.9 ± 0.09). Significant differences were also observed for each paired comparison (four glands vs. three glands: *p* = 0.000003; four glands vs. two glands: *p* < 0.000001; three glands vs. two glands: *p* < 0.000007).

The narrow bars inserted in Figure 1 represent the scores recorded when injections were unevenly distributed between parotid and submandibular glands. No significant differences were detected and all the scores were similar regardless of which glands were most engaged.

**Figure 1.** Values of scores obtained over the groups with injections into four, three and two glands (large bars) and in the groups where injections were unevenly distributed between parotid (p) and submandibular (s) glands (narrow bars). The error bars represent the standard error, \*\*\* indicates differences with *p* < 0.00001, and ns indicates no statistical differences.

Although the score for *incobotulinum toxin* was higher than the score for *onabotulinumtoxin*, the type of toxin did not produce significant differences when the score was measured over the entire sample of patients (N = 90; F1,84 = 2.47; *p* = 0.12), and no interaction was observed between the number of glands engaged and the type of toxin (F2,84 = 2.76; *p* = 0.069). This is reported in Figure 2.

**Figure 2.** Values of scores observed with respect to the two types of toxins injected (incobotulinum and onabotulinum). Large bars represent the results obtained from the entire sample of patients; the narrow bars show the subdivision over the groups with injections into four, three and two glands. The error bars represent the standard error, \*\*\* indicates differences with *p* < 0.00001, and ns indicates no statistical differences.

When the dataset was separated into two subsamples associated with each toxin, significant differences were observed over the three groups for both the effect of *incobotulinumtoxin* (F2,30 = 22.45; *p* < 0.000001) and *onabotulinumtoxin* (F2,54 = 40.14; *p* < 0.000001). The largest influence was detected in group A and, except for the comparison between group A and group B for *incobotulinumtoxin* and between group B and C for *onabotulinumtoxin*, the other paired comparisons were statistically different.

The overall effect of disease was statistically significant (F4,85 = 6.68; *p* < 0.0001). The Bonferroni multiple comparison showed that the main contribution to this result was provided by the paired differences between ALS and CP (*p* = 0.0033), PD (*p* < 0.0001) and stroke (*p* = 0.0022).

Only one patient complained of dysphagia 7 days after BoNT/A injection, which disappeared within 15 days; another two patients had a hematoma at the site of injection, which needed compressive bondage for 2 h.

#### **3. Discussion**

This is the first randomized, blinded study exploring the relationship between the effect of BoNT/A injections and the number of salivary glands injected.

Our results showed that BoNT/A injections induced a dramatic reduction of sialorrhoea in almost all patients (91%). The reduction of sialorrhoea was evident 2 weeks after the injection. However, the production of saliva was still enough that swallowing of both foods and drink in all patients was not impaired. In fact, parotid and submandibular glands together account for about 95% of the total salivary secretion. The remaining 5% is produced by the lingual and minor glands [5].

The effect of salivation reduction was more significant in patients with four glands treated than patients with three or two glands treated. Patients treated in three glands showed a more significant reduction in hypersalivation than patients treated in two glands. In patients treated in three glands, no significant differences were observed between injections into one parotid and two submandibular glands and injections into one submandibular gland and two parotid glands. In patients treated in two glands, no significant differences were observed among patients who were injected into two parotids, two submandibular glands or one parotid and one submandibular gland, respectively.

Otherwise, we cannot exclude that the beneficial effect was due to the different total doses that were used. Both dose and number of injected glands may have a synergic effect in reducing hypersalivation.

It is noteworthy that the score recorded for the *incobotulinumtoxin A* tended to be higher than the *onabotulinumtoxinA,* and the two toxins showed slightly different effects over the groups. In fact, as both of the toxins influenced the score of patients treated in four and two glands, *incobotulinumtoxin A* differentiated the scores associated with patients treated in three and two glands, while *onabotulinumtoxin A* exhibited an effect between patients treated in four and three glands. Thus, the overall influence of BoNT/A derived from a differentiated effect of the single toxins.

The major number of injected glands did not produce any side effects and the dose of BoNT/A used for each gland was safe in all patients. This treatment has the advantage that it avoids the use of oral anticholinergic medications, which has previously been administered in most patients for the symptomatic therapy of the sialorrhoea.

However, this study did not establish whether BoNT/A parotid injections are superior to BoNT/A submandibular injections in reducing hypersalivation. In fact, when only two parotids or two submandibular glands were injected (Group C, 2p and 2s patients, respectively), no significant differences were observed.

In addition, our findings have shown that injections in patients with sialorrhoea associated with ALS were less effective than in patients with PD, stroke, BI or CP. A possible explanation for these differences may reside in a different saliva composition (more prevalence of mucinoses component in the saliva of patients with ALS) or with a more impaired oral/preparatory phase of swallowing in ALS, with consequent major saliva pooling in the mouth. However, further studies specifically focused on this topic and involving a larger number of ALS patients are needed before drawing definitive conclusions.

Our study demonstrated that sialorrhoea due to different neurological diseases may be successfully managed with injections into four or at least three salivary glands. This treatment is safe, simple and highly effective.

#### **4. Materials and Methods**

#### *4.1. Patients*

A consecutive series of 117 patients with neurological disorders arising from different aetiologies was screened in our hospital from January 2005 to December 2016 for hypersalivation. Out of these 117 patients with sialorrhoea, ninety subjects (59 men and 31 women; mean age: 53.4 ± 17.6 years) who had experienced a high frequency and severity of hypersalivation and drooling in the preceding 6 months satisfied all of the inclusion/exclusion criteria and were enrolled in the study (Figure 3). We included those patients who had wet rolls with a roll-weight at least ten times heavier than the dry rolls. Thirty out of 90 patients were affected by Parkinson disease (PD), 11 were suffering from amyotrophic lateral sclerosis (ALS), 21 were affected by stroke, 8 by brain injury (BI) and 20 by cerebral palsy (CP). Table 1 shows the demographic characteristics of these 90 patients.

**Figure 3.** Study profile.

#### *4.2. Inclusion criteria*

(1) Accepting to participate in the study; (2) age ≥ 18 and ≤ 75 years; (3) the diagnosis of one of the following neurological disorders often associated with hypersalivation and/or drooling: diagnosis of PD, stroke, ALS, CP, or BI; (4) severely disabling sialorrhoea lasting for at least 6 months.

#### *4.3. Exclusion criteria*

The presence of other neurological diseases or the inability to give informed consent because of cognitive impairment.

All patients gave their written informed consent to the BoNT/A treatment after the approval of the local ethics committee.

#### *4.4. Treatment*

Twenty-five mouse units (MU) of botulinum neurotoxin type A (BoNT/A; Botox, [*onabotulinumtoxin A*], Allergan, Irvine, CA, USA; 100 MU/2 mL, 0.9% saline; or twenty-five mouse units (MU) of BoNT/A, Xeomin [*incobotulinumtoxin A*], Merz Pharma, Frankfurt, Germany; 100 MU/2 mL, 0.9% saline) were injected into each parotid gland using a 27G 3/4 needle. The submandibular glands were also injected with 25 MU each. Out of 90 patients, 57 patients were treated with *onabotulinumtoxin A* and 33 patients with *incobotulinumtoxin A*. The total number of patients treated with *onabotulinumtoxin A* injections was more than the number of patients treated with *incobotulinumtoxin A* because the latter was introduced later on. BoNT/A was percutaneously injected in all patients into the salivary glands under ultrasound control using a linear electronic probe 7.5 MHz (Aloka 650-SSD), which allowed the operator to accurately inject into the targeted salivary gland. Injections were performed once in all patients. Before injection, patients underwent an objective assessment of sialorrhoea and then they were randomised into three groups according to the number of treated glands. The number of glands that were injected was randomly assigned using a computer-generated list. Patients were divided into three groups (group A, B, and C). In group A (N = 30 patients; 10 females and 20 males; age range: 18–73 years), BoNT/A injections were performed into four glands (2p + 2s); in group B (N = 30 patients; 12 females and 18 males; age range: 18–72 years), BoNT/A injections were performed into three glands (2p + 1s, N = 15 patients; 2s + 1p, N = 15 patients) and in group C (30 patients; 10 females and 20 males; age range: 21–73 years), BoNT/A was injected into two glands (1p + 1s, N = 10 patients; 2p, N = 10 patients; 2s, N = 10 patients). In patients with four salivary glands treated, the total dose of botulinum toxin injected was 100 MU (25 UI for each gland). In patients with three and two salivary glands injected, the total dose was 75 MU and 50 MU (25 MU per gland), respectively. In patients injected into three and two salivary glands, an equal amount of saline solution was injected into the remaining one and two glands, respectively.

#### *4.5. Assessment*

Assessments were made at the same time of the day for each visit and patients were seated upright. Patients were inhibited from eating or drinking one hour before each assessment. Assessments were performed by a physician examiner who was blinded to the treatment allocation at baseline, and then at 2 weeks after the injection. All patients were blinded to their treatment allocation. Both treating and physician examiners reported their evaluations in separate case report forms. Hypersalivation was measured with six dental rolls placed in six different areas of the mouth and then retained there for 5 min. The difference in weight between the dry and wet rolls was calculated. The procedure was repeated after 15 min. The mean value of these consecutive assessments was taken as the final value.

#### *4.6. Statistical analysis*

Likert's transformation was obtained to change the qualitative results of BoNT/A injection into salivary glands into a score ranging from 0 (no effect) to 3 (maximum effect = 75% reduction of sialorrhoea). Two-way analysis of variance (ANOVA) was performed to evaluate the effect of the number of glands receiving toxin injection and the type of toxin. One-way ANOVA was applied to the two subgroups with injections into three glands (2p + 1s, 1p + 2s; N = 15 for each group) and the three subgroups with injections into two glands (2p, 2s, 1p + 1s; N = 10 for each group). An additional one-way ANOVA was used to estimate the effect of disease on the score value. For the ANOVA with three levels, the multiple comparisons were adjusted by Bonferroni correction. The data are expressed as means and standard errors, and the results were considered significant when *p* < 0.05. Statistical analyses were performed using SYSTAT version 11 (Systat Inc., Evaston, IL, USA).

**Author Contributions:** D.A.R., R.M.-R., and S.M. conceived and designed the experiments; D.A.R., F.P., S.L., A.Q. performed the experiments; A.C., A.B., and M.P. analyzed the data; A.C. wrote the paper.

**Conflicts of Interest:** The authors declare no conflict of interest. We had no founding sponsor.

#### **References**

1. Banerjee, K.J.; Glasson, C.; O'Flaherty, S.J. Parotid and submandibular botulinum toxin a injections for sialorrhoea in children with cerebral palsy. *Dev. Med. Child. Neurol.* **2006**, *48*, 883–887. [CrossRef] [PubMed]


© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Therapeutic Approaches of Botulinum Toxin in Gynecology**

#### **Marius Alexandru Moga 1, Oana Gabriela Dimienescu 1,\*, Andreea Bălan 1, Ioan Scârneciu 1, Barna Barabas, <sup>2</sup> and Liana Ples, <sup>3</sup>**


Received: 31 March 2018; Accepted: 19 April 2018; Published: 21 April 2018

**Abstract:** Botulinum toxins (BoNTs) are produced by several anaerobic species of the genus *Clostridium* and, although they were originally considered lethal toxins, today they find their usefulness in the treatment of a wide range of pathologies in various medical specialties. Botulinum neurotoxin has been identified in seven different isoforms (BoNT-A, BoNT-B, BoNT-C, BoNT-D, BoNT-E, BoNT-F, and BoNT-G). Neurotoxigenic Clostridia can produce more than 40 different BoNT subtypes and, recently, a new BoNT serotype (BoNT-X) has been reported in some studies. BoNT-X has not been shown to actually be an active neurotoxin despite its catalytically active LC, so it should be described as a putative eighth serotype. The mechanism of action of the serotypes is similar: they inhibit the release of acetylcholine from the nerve endings but their therapeutically potency varies. Botulinum toxin type A (BoNT-A) is the most studied serotype for therapeutic purposes. Regarding the gynecological pathology, a series of studies based on the efficiency of its use in the treatment of refractory myofascial pelvic pain, vaginism, dyspareunia, vulvodynia and overactive bladder or urinary incontinence have been reported. The current study is a review of the literature regarding the efficiency of BoNT-A in the gynecological pathology and on the long and short-term effects of its administration.

**Keywords:** botulinum toxin; chronic pelvic pain; overactive detrusor; vaginism

**Key Contribution:** This review highlights the efficiency of BoNT-A in the gynecological pathology (vaginism, vulvodynia, chronic pelvic pain or urinary tract disorders) pointing out the effects of BoNT-A after the first injection and during the follow up period.

#### **1. Introduction**

The incidence of chronic pelvic pain in women is constantly increasing, and is approximately 15% worldwide [1]. Jarell et al. [2] defined chronic pelvic pain as pain not related to gastrointestinal problems, menstruation or sexual activity, with a complex etiology. Chronic pelvic pain affects both women and men through common mechanisms, involving the central nervous system. The result is a regional pain syndrome that affects the entire pelvis. The triggers may be relatively benign, but individuals predisposed to chronic pelvic pain syndrome develop a series of sensory abnormalities and may perceive normal sensations as increased, until the point of unbearable pain and dysphoria. It is also associated with psychological, sexual, social and behavioral problems [2,3]. Other sequelae

of this pathology include: decreased physical activity, impairment of social and family relationships, depression and accompanying vegetative signs such as sleep and appetite dysfunctions [4]. Potentially beneficial drugs include medroxyprogesterone depot or hormone therapy, but only in association with behavioral therapy [5,6]. Several treatments including conventional or homeopath drugs have been proposed for the management of this pathology. Medicinal plant supplements are therapeutic alternatives, when traditional interventions (surgery, anti-inflammatory or antalgic medication) fail to manage the disease [7]. Medicinal herbs have various effects on the women reproductive system, being used worldwide, in several pathologies including vulvodynia, vaginism, chronic pelvic pain of unknown etiology or urinary tract pathologies [3]. A study conducted in six European countries in 2016 pointed that several plants, such as *Plantago psyllium*, *Prunus Africana* or *Equisetum arvense*, could be used in the treatment of chronic pelvic pain of gynecological or urinary origin. Even if medicinal herbs are frequently used worldwide, new studies are mandatory to propose new drugs [7].

Another alternative to the treatment of several gynecological diseases, more studied nowadays, is Botulinum toxin (BoNT). Botulinum Toxin A (BoNT-A) has been used to treat various gynecological pathologies such as: chronic pelvic pain, vaginism, dyspareunia and urinary incontinence with overactive bladder or sphincter dyssynergia. This article is a review of the current published data regarding the administration of BoNT in gynecological pathology, but, to recommend the wider use of this treatment, it is essential to carry out more research. BoNT/A1 and BoNT/B1 are the only BoNT types used for clinical purposes and BoNT-A is the most studied isoform for therapeutic purposes. Clinical trials on this topic have defined the safety and tolerability profile of BoNT-A [8]. All patients from the clinical studies injected with BoNT-A understood the possible undesirable effects of the treatment, giving their approval through signing the informed consent [9]. The incidence of adverse effects was observed to be approximately 25% in the BoNT-A treated groups compared to 15% in the control group. Among the side effects of the treatment with BoNT-A, the most frequently mentioned was the focal weakness, erythema, edema or hyperesthesia [10]. BoNT-A is used in various fields of medicine: dermatology, motion disorders, ophthalmic disorders, and gastrointestinal disorders, as well as in urogynecology pathologies, having high efficiency with minimal adverse effects. Table 1 summarizes the clinical applications of BoNT in medicine [11].

Regarding the use of other serotypes, such as BoNT-B, it has been used to induce human muscle paralysis but, according to Sloop and coworkers' research, the paralysis resulting from BoNT-B is not as efficient as the one resulting from BoNT-A [12].


**Table 1.** Clinical uses of BoNT-A.

#### **2. Botulinum Toxin**

BoNT are proteic neurotoxins produced by anaerobic sporulated bacteria of the *Clostridium* genus. Pirazzini et al. [13,14] described in a comprehensive review four different clostridial groups (Clostridium *Botulinum* groups I–IV, *Clostridium baratii* and *Clostridium butyricum*) that are known to produce the seven serotypes of BoNTs (BoNT-A to BoNT-G) [15]. Based on the amino-acid sequences, the serotypes are divided into subtypes, being more than 40 BoNT subtypes identified [16].

The first neurotoxin serotypes were identified in 1919 (BoNT-A and BoNT-B) and the last one in the year 1969 (BoNT-G) [16–18]. In 2017, Zhang et al. described a new BoNT-serotype (BoNT-X) [19]. The particularity of this serotype is that can cleave VAMP4 (which mediates vesicle fusion between endosome and TGN) [20,21] and Ykt6—an atypical SNARE without transmembrane domain (an essential protein in yeast, involved in membrane fusion events such as ER-Golgi, intra-Golgi, autophagosome formation) [22]. Zornetta et al. [23] also described in 2016 the first non-Clostridial botulinum like toxin (BoNT-Wo) identified from *Weissella oryzae* (an anaerobe isolated from fermenting rice) [24]. The particularity of this toxin is that it cleaves the VAMP at a single site (a unique Trp-Trp peptide bond, localized within the juxtamembrane segment of VAMP). Recently, Zhang et al. reported another Botulinum Neurotoxin-like toxin in the *Enterococcus faecium* strain isolated from cow feces (BoNT-En) [25]. BoNT-En cleaves two proteins that mediate synaptic vesicle exocytosis in neurons: SNAP-25 and VAMP-2.

BoNT consists of two chains: a heavy chain of 100 kDa and a light chain of 50 kDa linked by a disulfide bond which is extending from the heavy chain and surrounds the light chain like "a belt" [26]. The heavy chain contains a N-terminal HC that mediates the translocation of the LC (which acts as a protease, cleaving various proteins of BoNT-A, -C and -E cleave SNAP-25; BoNT-B, -D, -F and -G cleave VAMP1, -2 and -3; and BoNT C cleaves syntaxin 1) into the endosomal membranes. The cleavage of one of the three SNARE proteins prevents neurotransmitter release from neurons by blocking the fusion of synaptic vesicles to plasma membranes [13,27,28]. Table 2 summarizes the BoNT serotypes, subtypes, the target SNARE proteins, and their intracellular compartments.

After exceeding the intestinal barrier, BONTs spread into the extracellular fluids, entering the lymphatic system, followed by spreading into the blood circulation [29], without crossing the blood barrier. BoNTs can bind to any neurons, but they are distributed primarily to the peripheral nerve terminals [14]. The molecular mechanism of BoNTs inside nerve terminals is described in Figure 1.

**Figure 1.** The five steps of BoNTs' mechanism of action inside nerve terminal.


The first step is binding to the presynaptic vesicle membrane of the nerve terminals, through HC domain, to two independent receptors: a PSG receptor and a protein receptor of the synaptic vesicle (glycosylated SV2 in case of BoNT-A1 and BoNT E1, synaptotagmin I/II for BoNT-B1, BoNT-DC and BoNT-G) [30–34]. The next step involves internalization of BoNT, through dual binding with synaptic vesicle receptors and PSG. Following this process, the strength of BoNT interactions with the membrane increases [13,14,33].

The third step of the process, namely translocation has been extensively studied. The vesicular ATPase proton pump generates a transmembrane pH gradient, to translocate the L-chain from the synaptic vesicle into the cytosol. ATPase inhibitors are an important component, because they block completely the nerve termination intoxications by BONTs [35–40]. After translocation, the L chain is released on the cytosolic side of the membrane. However, this process requires the inter-change disulfide bond to be reduced, because the BONTs that possess reduced inter-chain disulfide bonds do not form channels. Fisher et al. described in their paper the importance of reduction in the inter-chain disulfide bon that needs to take place at any stage before the exposure to the cytosol. It is necessary because it prevents the L-chain translocation. Several enzymatic systems (thioredoxins and glutaredoxins) are involved in the reduction of protein disulfide bond, having a major role in the release of L chain into the neuronal cytosol [41]. After the enzymatic system reduction of the disulfide bond, the toxin can interact with the target proteins [40,42–45].

The final step of the BoNTs mechanism is the cleavage of SNARE proteins with ensuing blockade of neurotransmitter release. The L chains of all BoNTs are specific metalloproteases for one of the SNARE proteins: VAMP, SNAP25 or syntaxin. BONT-A and -E cleave SNAP25; BONT-B, -D, -F, and -G cleave VAMP; and BONT-C targets syntaxin and SNAP25. The result of the proteolytic actions is the prolonged inhibition of the neurotransmitter release, followed by neuroparalysis [13,46,47].

BoNT-A is used in medicine in a wide range of muscular dysfunctions because it acts on nerve endings and inhibits the release of acetylcholine in synaptic spaces, preventing muscle spasm [48]. BoNTs acts on chronic pain, spasm and dystonia and could be successfully used to relieve these symptoms [49,50]. Of all BoNT serotypes, BoNT-A specifically cleaves SNAP-25 and thus prevents the release of acetylcholine in the synaptic space. As the synapses are blocked by the action of the toxin, the neuron will form new ones, a process known as sprouting [26,51]. A systematized description of the mechanism of BoNT-A in pain inhibition can be observed in Figure 2.

**Figure 2.** Mechanism of action of BoNT-A in pain. Inhibition of acetylcholine and neurotransmitter released from motor neuron and nociceptor by BoNT-A reduces pain by inhibiting the pain signal transmission.

#### **3. Review of the Literature Regarding the Gynecologic Indications for the Use of BoNT-A**

#### *3.1. Use of BoNT-A in the Treatment of Vaginism*

The term "vaginism" describes the involuntary, recurrent or persistent contraction of the perineal muscles that surround the outer third of the vagina. It occurs during sexual intercourse and/or penetration with a swab or vaginal speculum during a gynecological examination. This involuntary contraction of the perineal muscles can aggravate or even make sexual life impossible [52].

The severity of vaginism could be classified according to the Lamont Scale, depending on the presenting symptoms and pain during the gynecology exams. Vaginismus was first described in 1978 by Lamont, who divided the pathology into four degrees [53]:

	- o Levator and perineal spasm relieved with reassurance
	- o Able to tolerate vaginal exam
	- o the perineal spasm is maintained through the gynecology exam
	- o Unable to relax for the pelvic exam
	- o Spasm of the levator muscle
	- o Elevation of buttocks to avoid the gynecology exam
	- o Perineal and levator spasm
	- o Adduction of thighs, elevation of buttocks, unable to tolerate the pelvic exam.

Depending on the severity degree, there are cases when penetration with vaginal swabs or vaginal speculum is allowed, but, in severe cases, penetration with any gynecological instrument or sexual intercourse is impossible [54]. Kegel exercises, relaxation and physical examination are considered the first line treatment in this pathology. In addition, voluntary control of perineal muscle contraction is a key factor in the successful treatment of vaginism [53]. Anxiolytic therapeutic agents and local treatments (lubricants and anesthetic creams) have been used as pharmacological treatments of this pathology, but approximately 10% of patients do not find amelioration in the symptoms [54].

Because of the numerous unresponsive cases to the conventional treatment, several authors investigated the effect of BoNT in this pathology [54–57]. The first case of vaginismus treated with BoNT-A was described in 1977 [58] and since then it had been carried out multiple studies.

A retrospective study from 2004 included 24 women aged 19–34 years with 3rd- or 4th-degree vaginism, without previous treatment [54]. Prior to injection, 500 U BoNT-A was diluted with 1.5 mL of saline solution and a total dose of 150–400 U was injected equally into levator ani muscles, in three points on each side, under sedation with Midazolam and with Oxygen administration. For the first cases, 150–200 U BoNT-A was used, with the dose gradually increased for the following patients, up to 400 U. Patients were monitored on average for 12 months and the conclusions showed that: 95.8% of the patients did not show any resistance or showed reduced resistance to post-injection vaginal examinations, 75% achieved satisfactory sexual intercourse after the first injection and 16.7% had mild pain at penetration after the first injection. In addition, recurrent vaginism in patients treated with BoNT-A was not detected.

In a case-control study, the efficiency of BoNT use in the treatment of vaginism was investigated [55]. The cohort consisted of thirteen cases of women diagnosed with vaginism, with an average age of 26.6 years. The cohort was divided into two groups: eight patients suffering from vaginism and five patients diagnosed with vaginism prior to the treatment, considered the control group. The first group was injected with 25 U of BoNT diluted in 1 mL of saline in each bulbospongiosus muscle. The controllers were injected with saline solution. After the injection, the patients were followed for an average of 3.3 months. The results obtained were encouraging: improvements were observed, and, in all cases, sexual life became possible or satisfactory. However, recurrences of vaginism have also been reported. There were no improvements in the control group.

To point out the utility of BoNT-A in the treatment of vaginismus secondary to vulvar vestibulitis syndrome, Bertolasi et al. recruited 39 women whose electromyography (EMG) recordings in the levator ani muscle had showed reduced resting and reduced inhibition during exercise [56]. The patients were injected with BoNT-A into repeated cycles under the guidance of EMG and were followed for an average of 105 (±50) weeks. Four weeks after each cycle, the women underwent EMG evaluations, vaginal examinations, evaluation of bowel and bladder symptoms completed VAS and the female sexual function index scale (FSFI). The results of the questionnaires were satisfactory at the first follow up (at four weeks after the first injection of BoNT-A) and the results maintained, with the increase in the number of subsequent injections. At the end of the follow-up period, 63.2% of the patients were completely cured, 15.4% requested re-injections and 15.4% dropped out the study before finishing it.

Another retrospective study that pointed out the use of BoNT-A in the treatment of vaginism was conducted on a cohort of 20 patients that have been treated with BoNT-A injections during 2005–2009 [57]. The patients were divided according to the severity of vaginism: 12 women with primary vaginism, 5 women with secondary vaginism and 3 women with severe dyspareunia. Initially, low doses of BoNT-A were used, and then the doses increased from 100 U to 150 U, diluted in 2 mL saline solution, injected under sedation (15–20 mL of bupivacaine 0.25% with epinephrine 1: 200.000) in several points along each side of the vagina (into the bulbocavernosus, pubococcygeus and puborectalis muscles). At the time of the study, 16 patients managed to have sexual intercourse in two weeks to three months after the injection and a patient was considered a failure because not even the smallest penetration dilator could ever be used. Patients continued to have a low degree of discomfort and vaginal burns during early sexual intercourse attempts, but this problem disappeared within a few weeks of completing the treatment with BoNT-A.

Table 3 summarizes the clinical studies regarding the use of BoNT-A in the treatment of vaginism.


 **3.** Studies of BoNT-A in the treatment of vaginism.

**Table**

#### *3.2. Use of BoNT in the Treatment of Vulvodynia*

Vulvodynia is a sexual dysfunction manifested by vulvar pain and orgasmic difficulties that cause a difficult sexual life. Women affected by this pathology receive only symptomatic treatment, anti-inflammatory and analgesic drugs, while psychotherapy can treat the fear of pain [60]. In modern medicine, BoNT could be used to treat this pathology when other treatments fail. It acts through a permanent neuromuscular blockage and muscle function recovery is achieved by forming new neuromuscular junctions [61]. The etiology of vulvodynia is not fully known, although it has been extensively researched. The factors involved in this pathology could be: inflammatory, genetic, infectious, hormonal or mechanical [62,63]. These factors induce modifications through three different pathways: sexual function, nervous system pain and pelvic floor muscles [64].

Yoon et al. performed a study regarding the use of BoNT-A in the treatment of vulvodynia [60]. The cohort consisted of seven women with genital pain that were injected with 20–40 U BoNT-A. All patients reported that the pain decreased after injections and the subjective pain score improved from 8.3 to 1.4, with no recurrences (the follow-up period was 4–24 months) Patients have also reported that, after treatment, no significant pain or discomfort occurred during or after sexual intercourse. In 2009, Petersen et al. evaluated in a randomized, double blinded, placebo-controlled study, the efficacy of BoNT-A injection in 32 women with vulvodynia and compared the results with a control group of 32 women [63]. Twenty units of BoNT were diluted in 0.5 mL saline solution and injected into bulbospongious muscles, while, for control cases, 0.5 mL saline solution was used. Both groups achieved a significant reduction in pain and the conclusion was that BoNT-A did not reduce the pain, does not improve sexual function and does not influence the quality of life compared to the placebo group.

Vulvodynia is a syndrome defined by a sharp pain in the vulva that does not have a well-defined cause which makes it very difficult to treat. The most common clinical form of vulvodynia is the provoked vestibulodynia, also named vulvar vestibulitis [65]. BoNT-A administered in high doses appears to have found utility in the treatment of this pathology that does not have a known organic substrate. Pelletier et al. administered BoNT-A to a group of 20 women aged 18–60 with provoked vulvodynia [66]. Each patient was injected with 50 U of BoNT-A into the bulbospongious muscles under EMG guidance. After three months, 80% of patients confirmed a decrease in pain intensity, and quality of life and sexual life improved significantly in the first six months. A retrospective study compared the effects of different doses of BoNT-A and Gabapentin in patients with vulvodynia also concluded that the symptoms of the patients injected with BoNT-A, measured through VAS scale were significantly improved in the group treated with Gabapentin (an anti-epileptic drug, that is also used to treat neuropathic pain) [67,68]. In Table 4 are summarized the clinical studies regarding the use of BoNT-A in the treatment of vulvodynia.

One of the causes of the occurrence of vulvodynia is the aberrant increase in the number of nociceptors. Intraepithelial neural hyperplasia associated with hypersensitivity of peripheral nociceptors generates a strong pain in the vestibule. BoNT was successfully used in this pathology. The pain is released through blocking the release of acetylcholine from parasympathetic neurons and from sympathetic post-ganglionar neurons. BoNT has been also used, with a beneficial effect on dyspareunia. These effects could be explained by two theories: the first refers to the decrease of the pelvic muscular hypertonicity and implicitly of the pain, by paralyzing the musculature. The second mechanism is the blockade of neurotransmission at nociceptive receptors in the submucosal layers of the vestibule [69].


**Table 4.** Studies of BoNT-A in treatment of vulvodynia.

#### *3.3. Use of BoNT-A in the Treatment of Chronic Pelvic Pain*

A persistent pelvic pain, lasting more than six months, which can be conceptualized as a syndrome of somatic functional pain or as a regional pain syndrome defines chronic pelvic pain [5].

One of the causes of chronic pelvic pain syndrome is the spasm of the pelvic muscles, especially the spasm of the levator ani. Myofascial pain and spasm are defined as regional muscular pain characterized by the presence of trigger points. These are hypersensitive points distributed on the levator ani surface, which once touched, cause pain. The pain resulting from reaching these trigger points appears to result from the excessive release of acetylcholine and other neurogenic inflammatory substances from the neuromuscular junction. The management of pelvic floor muscle spasm requires a multidisciplinary approach and treatment strategies including the use of steroids, non-steroidal anti-inflammatory drugs, muscle relaxants, antidepressants, neuromodulators, selective norepinephrine reuptake inhibitors and injection of various substances into the triggering points such as local anesthetics, steroids and BoNT [70–72].

Adelowo et al. designed in 2013 a retrospective study on a cohort of 31 patients to evaluate the role of injections with BoNT-A in the levator ani muscle in women with refractory pelvic myofascial pain [73]. The pain was assessed during palpation of the pelvic floor muscles using a scale of 0 to 10, 10 being the most severe pain possible. Patient reassessment occurred before six weeks after injection and again after ≥6 weeks post injection. Thirty-one patients met the eligibility criteria but two were lost during follow-up. Overall, 79.3% of the patients reported on the re-evaluation a pain relief, while 20.7% reported an improvement in symptoms. The conclusion of this study was that BoNT-A injection into the levator ani proved to be effective for women with refractory myofascial pelvic pain, with only a few limited side effects. In 2006 a double-blind randomized study was conducted to estimate the utility of BoNT versus placebo in women that have reported pelvic spasms and chronic pelvic pain lasting more than two years [74]. Thirty women were injected with 80 U of BoNT while 30 with saline solution in the pelvic floor muscles. Their subjective symptoms (dysmenorrhea, dyspareunia and pelvic pain of non-menstrual origin) were quantified with VAS scores (Visual analog scales). VAS is a measurement instrument used to document the symptoms severity in different patients and to assess the effectiveness of therapy in those patients [75]. The pain of the pelvic floor was measured by vaginal manometry. After six months, the patients were re-evaluated and the conclusion was that the reduction of pelvic spasm could reduce some types of pelvic pain. BoNT-A reduces pelvic floor hypertonia more than placebo, so it could be used in women with refractory pain.

Gajraj et al. presented a case of a 60-year-old woman presenting a four-year-long pelvic pain with leg irradiation and irradiation in the vagina and rectum, aggravated by clinostats [76]. The pain was assessed at a level of 4–8 out of 10 on the VAS pain scale. Physical examinations did not reveal any focal neurological signs. The vaginal examination showed sensitivity and tenderness in the right anterior and right posterior lateral regions. The patient was injected in the internal obturator muscle with 0.25% bupivacaine, resulting in a 90% reduction in pain for 12 h. Progressive and postprocedural mean scores on the VAS scale were 7 out of 10 and 1 out of 10, respectively. After a subsequent BoNT-A injection, the patient again reported a 90% decrease in pain for more than three months. In addition, after BoNT injection, there were no adverse effects such as motor weakness, and intestinal or bladder disorders. Therefore, BoNT-A has also shown its efficiency in this case.

Twelve women aged 18–55 years old, with objective hypertonia of the pelvic floor muscles for at least two years and chronic pelvic pain were recruited for testing the utility of BoNT [77]. Forty units of BoNT-A in three different dilutions were administered bilaterally in the puborectal and pubococcygeal muscles under conscious sedation. The results were favorable and uninfluenced by dilution. VAS pain scores improved for the cases with dyspareunia, but non-menstrual pelvic pain experienced insignificant reductions. At four weeks after treatment, it was observed a decrease with 37% in resting pressure measured through pelvic muscle manometer, reduction that decreased until 25% at 12 weeks. In addition, the quality of life scores improved.

A prospective study on women with refractory chronic pelvic pain and pelvic muscle spasm was conducted in 2015 to demonstrate the role of BoNT-A in the treatment of these dysfunctions [78]. BoNT-A injections in spastic pelvic muscles (up to 300 U) were performed through needle electromyographic guidance. Of the 28 women enrolled in the study, 21 qualified for analysis. The average age of the cases was 22–50 years and the comorbidities included interstitial cystitis/bladder pain syndrome in 42.9% of cases and vulvodynia in 66.7% of cases. Overall, 61.9% of subjects have reported improvement in the overall response assessment at four weeks and 80.9% at 8, 12 and 24 weeks post injection compared to baseline. Post-injection adverse reactions were also reported, including worsening of the following pre-existing conditions: constipation (28.6%), stress urinary incontinence (4.8%), fecal incontinence (4.8%) and urinary incontinence (4.8%). The conclusion of this study is consistent with the findings of other studies on this topic and suggests that BoNT could be useful in the treatment of pelvic floor muscle spasm and chronic pelvic pain, refractory to other therapies.

Levator ani syndrome is defined by chronic or recurrent episodes of rectal pain and affects approximately 6.6% of adults. There is no consensus on the pathophysiology of this painful syndrome, although the chronic hypertonia of pelvic floor muscles is the most common explanation [79]. Therefore, through the muscle spasm, the use of BoNT-A was attempted in the treatment of levator ani syndrome, however, without favorable results. Rao et al. conducted a randomized, placebo-controlled study on 12 cases with levator ani syndrome [80]. After BoNT-Administration into the anal sphincter, the duration and intensity of pain and the mean frequency remained unchanged compared to the baseline. The conclusion of the authors was that BoNT-A injections into the sphincter ani is secure but is not effective in relieving anorectal pain associated with this syndrome. Table 5 summarizes the clinical studies regarding the use of BoNT-A in the treatment of chronic pelvic pain and pelvic floor muscle spasm treatment.

Jhang J-F et al. [81] described in their research from 2015 a possible mechanism of BoNT-A on chronic pelvic pain. In several experimental studies involving both rats and humans, membrane receptors TRVP-1 and P2X3 have been observed to be up-regulated in the neuropathic pain. Xiao L. et al. concluded that a possible mechanism of BoNT-A is the reduction of TRPV-1 expression in spinal neurons of rats with hyperalgesia [82]. Muscular spasm seems to be usually associated with chronic pelvic pain [83,84] and the reduction of pain may also reduce the spasm. Myelinated and unmyelinated fibers (group III and IV, respectively) are nociceptors found in muscles, which can be sensitized by bradykinins, prostaglandin E, substance P, calcitonin gene-related peptide (CGRP) [85] and ATP [86]. BoNT-A is supposed to inhibit their release [85] and the activation of spinal cord neurons that are responsible for the pain transmission [87–89]. BoNT-A could also inhibit the spinal motor neurons alpha and gamma, mechanism through which pelvic muscle spasm could be stopped [85]. It is important to continue the research in this area and to investigate the mechanism of action and the effects of BoNT-A in the treatment of chronic pelvic pain.


*Toxins* **2018**, *10*, 169

#### *3.4. Inferior Urinary System Dysfunctions*

#### 3.4.1. Use of BoNT in the Treatment of Interstitial Cystitis (the Painful Bladder Syndrome)

BoNT-A injections into the urethral sphincter have been used in the treatment of lower urinary tract dysfunctions in the last 20 years to reduce bladder emptying, urethral and residual urine pressures [90,91]. The pathogenesis of interstitial cystitis is still uncertain, but there is a hypothesis that asserts that this pathology arises from neurogenic inflammation that activates bladder afferent nerves and causes bladder hypersensitivity [92]. Painful bladder syndrome is a clinical syndrome characterized by pain with supra-pubian localization caused by bladder filling associated with increased urinary frequency both day and night in the absence of proven urinary infection. There is currently no standard treatment [93], but there are studies in the literature that confirm the efficiency of BoNT-A in the management of interstitial cystitis, which has an antinociceptive effect on the visceral afferent nervous fibers [94].

Giannantoni A. et al. selected a group of seven women and instilled them with 200 U BoNT-A diluted in 100 mL of saline, without any anesthesia, to analyze the utility of this treatment in bladder painful syndrome [94]. The exclusion criterion for the cohort was detrusor overactivity. The solution was injected intravesical and maintained for 40 min and the results were evaluated after one week, one month and three months. However, the results were discouraging because short-term benefits were found for four of seven patients. The explanation consists in the molecular weight of BoNT-A that does not allow it to pass the epithelial barrier to the bladder to reach sub-urothelium and to act on nerve endings. Therefore, the intravesical instillation of diluted BoNTs is not an effective treatment in this pathology.

As the intravesical instillation of BoNT-A has not been found to be convenient, studies on this subject have further been conducted and it has been found that increased amounts of sensory fibers are located into the bladder trigonum. To evaluate the tolerability and efficiency of BoNT-A injections in patients with painful bladder syndrome that are refractory to standard treatment, Pinto et al. conducted a study on 17 patients (16 women and one man), which were investigated before treatment and one, three six, and nine months later [93]. All patients received 10 intra-trigonal injections with 10 U BoNT-A, diluted in 1 mL saline (a total of 100 U). The results were favorable: the pain level decreased, the urinary frequency decreased and O'Leary-Sant score increased. Therefore, the authors concluded that intra-trigonal injections with BoNT-A are useful in treating interstitial cystitis, also called painful bladder syndrome.

Satisfactory results were also obtained in the treatment of vesical dysfunctions, by injecting BoNT-A into the bladder wall [95]. Fifteen patients received 200 U BoNT-A diluted in 20 mL saline under general anesthesia and cystoscopic guidance. To evaluate the results, VAS pain scale and drainage charts were recorded, while urodynamic studies were realized before injections and at 1, 3, 5 and 12 months after the treatment. Overall, 86.6% of patients have reported an improvement after one and three months. In 26.6% of cases, at the five months follow up visit, it was observed that the effects persisted; nevertheless, the urinary frequency during day and night was increased. Twelve months after the treatment, the pain reappeared in all patients. Nine patients claimed dysuria one month after treatment.

Kuo et al. conducted a study on 10 patients to demonstrate the efficiency of suburothelial injection of BoNT-A in the management of chronic interstitial cystitis [96]. They injected 100 U of BoNT-A suburothelial in 20 places on five patients.Five other patients were injected with another 100 U of BoNT-A at the trigonal area of the bladder. However, the therapeutic results were disappointing because no favorable evolution was observed after three months of treatment. Carl et al. conducted a pilot study involving 29 patients with painful bladder syndrome and injected them with BoNT-A, to demonstrate that it is useful in the treatment of this pathology [97]. The toxin was injected submucosally, into the trigonal area, and the results contrary to those obtained by Kuo et al. [96], suggested that BoNT-A has an antinociceptive effect on the bladder afferent nerves in patients

with chronic interstitial cystitis. The authors did not experience systemic side effects during and after treatment.

A single center, prospective, non-randomized study was conducted in 2007 by Ramsay et al. to evaluate the efficiency, tolerability and safety of BoNT-A when injected intravesical at patients with interstitial cystitis [98]. Eleven women with average age of 56 years were injected with BoNT-A. The conclusion of the study was that a significant symptoms reduction had been observed at about 10–14 weeks after injection. Another prospective study based on the safety and effects of BoNT-A repeatedly administered to patients with bladder painful syndrome, was conducted on a cohort of 16 patients [99]. They were exposed to four cycles of intratrigonal injections with BoNT-A. A second injection was administered at the three-month follow up visit. Complications such as urinary tract infections or bladder hypersensitivity were evaluated at different intervals. Improvement occurred after approximately 9.9 months (±2.4 months). No total remission of symptoms was seen in any of the patients and 5 of the 16 patients had uncomplicated urinary tract infections. The studies that are pointing out the usefulness of BoNT-A in interstitial cystitis treatment are summarized is Table 6.

Apostolidis et al. proposed a possible mechanism of action of BoNT in the treatment of detrusor activity, but further research is needed to determine the significant effects of BoNT-A in this pathology [100,101]. Karsenty et al. [102] conducted a study in 2008 that pointed out that BoNT-A could also action through inhibition of other neurotransmitters, receptors or neuropeptides. A review from 2016 [103] identified a possible mechanism for the effects of BoNT-A in the treatment of inferior urinary system dysfunctions. Several studies in vivo and vitro included in this research, highlighted that BoNT-A injections into detrusor could decrease the levels of capsaicin receptor TRPV1 and purinergic receptor P2X3 (whose expression is increased) in the suburothelial nerve fibers [104–107]. Patients with detrusor overactivity have shown increased densities of substance P and CGRP, according to Smet et al. [108]. Following those observations, several studies on animals identified a possible mechanism of action of BoNT-A, which consists of the reduced release of CGRP in rat model [109] and an inhibition of SP with reduced activation of P2X3 and TRPV1 receptors in suburothelium and detrusor muscle in guinea pig model [110], leading to peripheral denervation. However, future research must be conducted to clarify the proposed mechanism and the role of BoNT-A therapy in case of inferior urinary system dysfunctions.





3.4.2. Use of BoNT-A in Urinary Incontinence through Neurogenic Overactive Bladder and Idiopathic Overactive Bladder

Urinary incontinence through both neurogenic (NOB) and idiopathic bladder hyperactivity (IOB) is a lower urinary tract dysfunction that affects many women with a prevalence of approximately 25% of the cases worldwide [111]. Bladder overactivity is defined by increased urinary frequency, feeling of urge and nycturia and could be associated with urinary incontinence. Anticholinergic therapy is essential in the treatment of overactive bladder but it also requires lifestyle and behavioral changes. If symptoms do not improve with anticholinergic therapy and lifestyle changes, urodynamic studies and cystourethroscopy are required [112]. Studies confirm the usefulness of intravesical injections with BoNT-A in women with overactive bladder (OB). After the injection of BoNT-A to the patients with neurogenic or idiopathic detrusor overactivity, there was observed a reduction in bladder detrusor pressure during both involuntary and voluntary contractions, which confirmed the BoNT-A influences the motor detrusor innervation. BoNT-A prevents the release of neurotransmitters such as Ach and in addition it acts on adenosine triphosphate (ATP), substance P and glutamate, decreasing the number of sensory receptors and nerves growth factor (NGF) in the bladder wall. These mechanisms of action may explain the utility of BoNT-A in the treatment of urinary incontinence through bladder overactivity [86].

During the period 2005–2009, 99 patients with OB were enrolled in a prospective, randomized, double-blind, placebo-controlled comparative trial [113]. Patients received a single injection with BoNT-A (50 U, 100 U or 150 U) into the vesical muscle. Three months after administration, a 50% improvement from baseline in urge and urinary incontinence was noticed. The 100 U and 150 U doses of BoNT were well tolerated and, in both cases, improvement was recorded. However, injections of 100 U showed reasonable efficiency with a lower post-voiding residual volume risk.

A comparative study between the response of patients with neurogenic detrusor overactivity and idiopathic detrusor overactivity to the first of administration of BoNT-A into the vesical detrusor was performed in 2005 by Popat et al. [114]. The study included 44 patients with neurogenic bladder overactivity and 31 with idiopathic bladder overactivity. The first group received 300 U BoNT-A and the second group received 200 U BoNT-A. The results were compared 4 and 16 weeks after injection: patients with idiopathic bladder overactivity responded to BoNT-A as well as those with neurogenic bladder overactivity and, despite the lower dose of toxin used, the results were similar.

Schmid et al. conducted a prospective study on 180 cases (135 women) to report the efficiency of a reduced dose (100 U) of BoNT-A injected into the bladder detrusor in case of patients with IOB [115]. Eighty-seven percent of patients experienced an improvement in urodynamic parameters: the urge completely disappeared in 75% of cases and the urinary incontinence disappeared in 84% of patients within two weeks. The frequency of urination decreased from 15 to 7 mictions and no more than 5 to 2 mictions per night were reported.

Brubaker et al. compared a group of 28 women with refractory urge through idiopathic bladder overactivity (200 U BoNT-A administered in the detrusor) with a placebo group of 15 women with the same pathology [116]. Sixty percent of the patients treated with BoNT-A have reported improved symptoms, effects that lasted approximately 373 days, respectively 62 days or less in the placebo cases. In the BoNT-A group, many patients with increased post-voiding residual volume (43%) and urinary tract infection were found in those with an increased post-voiding residual volume (75%).

Khanlow et al. conducted a study based on the effects of intravesical administration of BoNT-A in the management of refractory idiopathic bladder overactivity. Patients were properly informed about the improvement of life quality, the duration of re-injection and the risks for intermittent auto-catheterization. A cohort of 81 patients were injected with 200 U BoNT-A intravesically [117]. After BoNT-A injections, it was observed a significant improvement in the quality of life sustained by repeated injections. In 43% of cases treated with BoNT-A, auto-catheterization was required.

To describe the mid-term outcomes and adaptation of patients to BoNT-A therapy as a management strategy for women with refractory idiopathic bladder overactivity, Dowson et al. developed a cohort of 100 women who received BoNT-A injections as follows: all patients received an injection, 53 received 2, 20 received 3, 13 received 4, 10 received 5, 5 received 6, 3 received 7, 1 received 8, 1 received 9 and 1 received 10 injections [118]. Thirty-seven percent of patients completed the study after the first two injections and 11% of patients required intermittent auto-catheterization. As a possible complication, in 35% of patients, catheterization was required after the first administration of BoNT-A and in 21% of cases bacteriuria was detected. A review paper from 2016 investigated the use of BoNT in adults with urgency urinary incontinence and idiopathic overactive bladder. The conclusions of the study were that 22.9% to 55% cases regained complete continence and showed a significant improvement in the quality of life after treatment [119]. Detailed and comparative studies are found in Table 7.


**7.**StudiesofBoNTinoveractive


**Table7.***Cont*.

#### **4. Conclusions and Future Perspectives**

In this paper, we reveal several pathologies from the gynecological field for which BoNT treatment can be used. Most of these dysfunctions have been shown to be refractory to conventional treatments, but the results after single or repeated cycles of BoNT-A were favorable and the symptoms improved.

The dosage used ranged from 40 U to 400 U in single administration. In some cases, repeated injection cycles were necessary, depending on the symptomatology and the scores obtained after the patients completed standardized questionnaires. The improvement of the symptomatology was objectively certified Susing standardized questionnaires before and after a certain period post-injection.

When compared to BoNT-B, we observed that BoNT-A is used more often with good results, especially because the paralysis resulting from BoNT-B is not as efficient as the one of BoNT-A. It is also desirable to carry out further studies to reach consensus on the optimal BoNT-A dose and administration protocol to create a standardized treatment.

In conclusion, this review highlights that BoNT could be successfully used in treating symptoms of gynecological dysfunctions refractory to conventional treatments, having few side effects and high efficacy.

**Author Contributions:** M.A.M., O.G.D., A.B. and P.L. together initiated, designed, and drafted the manuscript. O.G.D., A.B., I.S. and B.B contributed to the literature collection. I.S, B.B. and P.L.drew the figures. All authors revised the manuscript. All authors read and approved the final manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

The following abbreviations are used in the manuscript:


#### **References**


© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Novel Applications of OnabotulinumtoxinA in Lower Urinary Tract Dysfunction**

#### **Jia-Fong Jhang and Hann-Chorng Kuo \***

Department of Urology, Buddhist Tzu Chi General Hospital, and Tzu Chi University, Hualien 970, Taiwan; alur1984@hotmail.com

**\*** Correspondence: hck@tzuchi.com.tw; Tel./Fax: +886-3865-1825 (ext. 2113)

Received: 25 April 2018; Accepted: 22 June 2018; Published: 26 June 2018

**Abstract:** OnabotulinumtoxinA (BoNT-A) was first used to treat neurogenic lower urinary tract dysfunction (LUTD) 30 years ago. Recently, application of BoNT-A in LUTD have become more common since the approval of intravesical BoNT-A injection for patients with both overactive bladders (OAB) and neurogenic detrusor overactivity (NDO) by regulatory agencies in many countries. Although unlicensed, BoNT-A has been recommended to treat patients with interstitial cystitis/bladder pain syndrome (IC/BPS) under different guidelines. BoNT-A delivery with liposome-encapsulation and gelation hydrogel intravesical instillation provided a potentially less invasive and more convenient form of application for patients with OAB or IC/BPS. BoNT-A injections into the urethral sphincter for spinal cord injury patients with detrusor-sphincter dyssynergia have been used for a long time. New evidence revealed that it could also be applied to patients with non-neurogenic dysfunctional voiding. Previous studies and meta-analyses suggest that BoNT-A injections for patients with benign prostate hyperplasia do not have a better therapeutic effect than placebo. However, new randomized and placebo-controlled trials revealed intraprostatic BoNT-A injection is superior to placebo in specific patients. A recent trial also showed intraprostatic BoNT-A injection could significantly reduce pain in patients with chronic prostatitis. Both careful selection of patients and prudent use of urodynamic evaluation results to confirm diagnoses are essential for successful outcomes of BoNT-A treatment for LUTD.

**Keywords:** botulinum toxin; clinical trial; human; urodynamics

**Key Contribution:** This article summarized recent novel applications of BoNT-A for LUTD, and suggested the possible further works to improve the therapeutic efficacy.

#### **1. Introduction**

Lower urinary tract dysfunction (LUTD) is a condition presented by patients suffering from LUTD linked to one or more structures and/or functions of the lower urinary tract [1]. LUTD is common in both men and women, and the incidence and prevalence increase as people age [2]. A recent large cross-sectional study in China revealed 61.1% of women and 61.2% of men reported LUTD [2]. In general, the pathophysiology of LUTD could be classified into bladder or bladder outlet dysfunction, and treatments of LUTD should focus on etiology. However, not all LUTDs could be treated effectively, even if the etiology is definite. Treatment of functional LUTD, such as interstitial cystitis/bladder pain syndrome (IC/BPS), overactive bladder (OAB), and dysfunctional voiding (DV), remains a challenge to the urologist.

Botulinum toxin (BoNT) is a potent poisonous neurotoxin, which is produced by the bacterium *Clostridium botulinum* and related species [3]. Ingestion of BoNT-poisoned food causes intoxication by inhibiting the release of the neurotransmitter acetylcholine from nerve fibers, thereby inhibiting muscle contractions, which was first described in the early 17th century [4]. BoNT was first isolated in 1895, and now could be classified antigenically and serologically into eight distinguishable exotoxins (A, B, C1, C2, D, E, F, and G) [5]. In 1981, Scott first used onabotulinumtoxinA (BoNT-A) by injecting it into human eye muscles to correct strabismus successfully [6]. Since then, BoNT-A has been widely used to treat many neuropathic pain syndromes and dystonic diseases. In LUTD, the first application of BoNT-A targeted the urethral sphincter. Dykstra used transperineal or cystoscopic injection of BoNT-A into the urethral sphincter in patients with spinal cord injury (SCI) and detrusor-sphincter dyssynergia (DSD) in 1988 [7]. Currently, BoNT-A has been widely used in different kinds of LUTDs, especially in diseases that could not be easily treated with oral medications. In the American Urology Association (AUA) guidelines, BoNT-A injection into the urinary bladder is now a standard treatment for patients with refractory OAB and IC/BPS [8,9]. Newly published clinical trials also revealed novel applications of BoNT-A in different LUTDs and exhibited promising results. The aim of the current article is to review important new applications of BoNT-A in LUTDs and the associated evidence supporting its use.

#### **2. Mechanisms of BoNT-A in LUTDs**

BoNT-A, a potent neurotoxic protein, is well known for its ability to inhibit the release of the neurotransmitter acetylcholine from presynaptic efferent nerves at neuromuscular junctions [5]. BoNT-A consists of a 50-kDa light chain and a 100-kDa heavy endocytosis [10]. Subsequently, the light chain and heavy chain separate in the endosomal vesicle [11,12]. The light chain is the biologically active moiety of BoNT-A. The light chain of BoNT-A cleaves synaptosome-associated protein 25 in the presynaptic nerve terminal, and inhibits the release of acetylcholine by disrupting the fusion of vesicles with the neuron's cell membrane, finally causing flaccid paralysis of muscles [13,14]. Traditionally, the effects of BoNT-A in treating LUTDs, such as OAB and DSD, were believed to be attributed to the inhibition of detrusor or urethral sphincter contractions. Recently, evidence also revealed BoNT-A injection into the bladder also could regulate sensory nerve function by blocking the release of various noxious neurotransmitters, including adenosine triphosphate, calcitonin gene-related peptide, calcitonin gene-related peptide, and substance P [15,16]. Modulation of sensory nerve function might be the mechanism of BoNT-A in some sensory problems predominantly LUTD, such as IC/BPS. In addition, studies showed an anti-inflammatory effect for BoNT-A. Immunohistochemical evidence revealed decreased tryptase expression in the bladder after BoNT-A injections, which suggests a reduction in active mast cells in bladders of IC/BPS patients [17].

#### **3. Intravesical BoNT-A Injection in OAB and Neurogenic Detrusor Overactivity**

OAB is a clinical syndrome, which is characterized by urinary urgency, usually accompanied by frequency and nocturia, with or without urgency, urinary incontinence, in the absence of urinary tract infection (UTI), or other obvious pathology [18]. According to a recent large cross-sectional study in Asia, the prevalence of OAB was 20.8% overall (men 19.5%, women 22.1%) and increased significantly with age [19]. Treatments of OAB are usually started with behavioral therapy and then oral medications such as antimuscarinics or beta-agonists [8]. Although oral medications might be effective, a large-scale study showed that 46.2% of OAB patients discontinued antimuscarinics and stated the reason for treatment discontinuation was "did not work as expected" [20]. In patients with neurogenic detrusor overactivity (NDO) due to SCI, oral antimuscarinics have been reported to increase bladder capacity and decrease intravesical pressure [21]. However, the effect of antimuscarinics is usually poor in NDO patients with severe urgency and incontinence symptoms. In a large series of NDO patients with urinary incontinence, only 32% of patients could become continent after using oxybutynin and trospium [22]. Thus, treatment for patients with OAB and NDO, who were refractory to oral medications, is a daily common challenging problem in the urology clinic.

Intravesical BoNT-A injection for treating patients with SCI and NDO have been reported since 2000 [23]. Schurch first injected 200 to 300 units (U) of BoNT-A into the detrusor muscle of NDO patients [23]. At six weeks of follow-up, complete continence was observed in 17 of 19 (89.4%) cases in which anticholinergic medication was markedly decreased or withdrawn. Evidence from basic and clinical researchers revealed BoNT-A injection could block acetylcholine release from efferent nerves ending by cleaving Synaptosomal-associated protein 25, thereby temporarily inhibiting detrusor muscle contraction and improve bladder storage symptoms [24]. Further investigation also revealed that BoNT-A injection could also inhibit both noradrenaline and adenosine triphosphate release, which have a powerful influence on bladder sensation [25,26]. After years of work by many researchers and clinicians, intravesical BoNT-A injection has provided evidence demonstrating its utility as standard therapy in patients with both OAB and NDO [8,27]. The application of BoNT-A in OAB and NDO has also been approved by regulatory agencies in most countries.

Patients with NDO and OAB who respond to BoNT-A usually need repeat injections every 6 to 12 months [28]. The long-term efficacy of repeat BoNT-A injections was doubtful before, but recent long-term follow-up studies (>5 years) revealed that BoNT-A could decrease urinary incontinence rate and improve quality of life in patients with both NDO and OAB [29,30]. However, treatment compliance might be not satisfactory. Rahnama'i reported that only 25% of patients continued treatment during the six years of follow-up [29]. Most of these patients could not tolerate voiding urinary tract symptoms, urine retention, or urethral catheterization [29].

#### **4. Intravesical Liposome-Encapsulated BoNT-A Instillation in OAB**

In our prospective pilot randomized controlled study, liposome-encapsulated BoNT-A (Lipotoxin) bladder installation was used to treat patients with OAB [31]. At one month after treatment, the change in urinary frequency and urgency significantly improved in the Lipotoxin group but not in the normal saline instillation group. More importantly, no adverse event such as post-voiding residual volume (PVR), urinary retention, or UTI significantly increased or was reported by patients during the follow-up period. Bladder instillation of Lipotoxin in patients with OAB seems to be an effective treatment without significant adverse effects, but the long-term efficacy still needs to be proved in the future. Although the use of Lipotoxin for treating OAB patients is promising, it has not been used in patients with NDO until now. The therapeutic effect of Lipotoxin in NDO might be not adequate in this case.

Management is usually difficult in some patients who are characterized by urinary urgency, incontinence with incomplete bladder emptying, and with a urodynamic diagnosis of detrusor hyperactivity with impaired contractile function (DHIC) [32]. Recently, we reported our experience with suburothelial injection of 100 U of BoNT-A in patients with DHIC [33]. At six months of follow-up, the subjective urgency symptom scores improved significantly, but urgency episodes did not significantly improve in 21 patients. Acute urinary retention developed in 7 (33.3%) and UTI was noted in eight patients with DHIC (38.1%). The incidence of adverse effects of BoNT-A injection in patients with DHIC was relatively higher than that in patients with OAB. We concluded that the efficacy of intravesical BoNT-A injection for DHIC patients was limited and short-term. Physicians should inform patients of both potential benefits and risks of BoNT-A injection for the treatment of DHIC. A comprehensive urodynamic study before decision-making is recommended to rule out the coexistence of LUTD such as bladder neck dysfunction.

#### **5. Intravesical BoNT-A Injection in Interstitial Cystitis/Bladder Pain Syndrome**

IC/BPS is a clinical syndrome and includes a large group of patients who are defined by the AUA as having "an unpleasant sensation (pain, pressure, discomfort) perceived to be related to the urinary bladder, associated with LUTD of more than six weeks duration, in the absence of infection or other identifiable causes" [9]. The estimated prevalence of IC/BPS among adult females in the US ranges from 2.7% to 6.53% [34]. Until now, the etiology of IC/BPS is still uncertain, and its management is both frustrating and difficult [35]. Smith et al. first treated female IC/BPS patients with intravesical 100 to 200 U BoNT-A injection plus cystoscopic hydrodistention at the same time [36]. We also conducted a prospective, randomized, double-blind clinical trial to investigate the clinical efficacy of

BoNT-A intravesical injection in patients with IC/BPS [37]. Our results showed a significantly greater reduction in pain and increase in cystometric bladder capacity in the BoNT-A group compared with the normal saline injection group at eight weeks of follow-up. Experimental studies conducted in both animals and humans provided laboratory evidence to support BoNT-A injections in the IC/BPS. BoNT-A injections into the bladder have been shown to block the release of noxious neurotransmitters including calcitonin, calcitonin gene-related peptide, glutamate, adenosine triphosphate, and substance P from neurons [38]. Now, intravesical BoNT-A injection is the fourth-line standard treatment in the AUA treatment guideline of IC/BPS [9].

A recent network meta-analysis compared BoNT-A injection with different intravesical therapy for IC/BPS, including bacillus Calmette-Guerin, resiniferatoxin, lidocaine, chondroitin sulfate, oxybutynin, and pentosan polysulfate [39]. The results indicate that in patients with IC/BPS BoNT-A injection has the highest probability of being the best therapy according to the global response assessment, and significantly improves bladder capacity. Recently, we conducted a double-blind, randomized trial to investigate the efficacy of intravesical Lipotoxin instillation in patients with IC/BPS [40]. Patients who received Lipotoxin therapy demonstrated a statistically significant decrease in O'Leary-Sant symptom scores and on the visual analog scale for pain. However, there was no significant difference in improvement between the Lipotoxin and normal saline instillation group. Nevertheless, no significant adverse effect developed in either group, the efficacy of Lipotxoin instillation in IC/BPS might be masked by the placebo effect, and further study is necessary to validate the actual effect. Rappaport et al. recently used TC-3 gel, a novel reverse-thermal gelation hydrogel to deliver BoNT-A into IC/BPS bladders [41]. A single intravesical instillation of 200 U of BoNT-A mixed with 40 mL TC-3 gel could significantly reduce both pain and O'Leary-Sant symptom scores at 12 weeks of follow-up. Preliminary results of instillation of a TC-3 gel-BoNT-A mixture are promising, but further prospective and randomized trials are necessary to prove its efficacy.

#### **6. Urethral Sphincter BoNT-A Injection in Detrusor-Sphincter Dyssynergia**

DSD is characterized by involuntary contractions of the external urethral sphincter during a detrusor contraction, which is caused by central nervous system injury between the pontine micturition center and the sacral spinal cord [42]. Patients with SCI and DSD usually suffer from incomplete bladder emptying and DO. Application of BoNT-A in DSD started as early as 1988 [7]. At that time, it was believed that the effect of BoNT-A could block acetylcholine release from presynaptic vesicles at the neuromuscular junction into the urethral sphincter [43]. However, clinical evidence to support its efficacy remains limited and randomized placebo-controlled trial data was limited until now. Our prospective study showed that 100 U of BoNT-A injection into the urethral sphincter could significantly decrease voiding detrusor pressure and increase maximum flow rate [44]. However, some patients might complain of an increase in incontinence grade and were dissatisfied with the BoNT-A injection. A recent prospective trial enrolled 59 SCI patients with both NDO and DSD. All these patients received both 200 U intravesical and 100 U urethral sphincter injections of BoNT-A at the same time [45]. Patients could experience a significant reduction of detrusor voiding pressure, urinary incontinence episode, and increased voiding volume at 12 weeks of follow-up. Twenty-five patients (42.4%) even reported complete dryness at follow-up. Patients with DSD may become incontinent after urethral sphincter BoNT-A injection, and it might adversely affect the quality of life in these patients. Simultaneously, BoNT-A injections in the detrusor and urethral sphincters are a reasonable treatment for SCI patients with both NDO and DSD. Using urodynamic study results to evaluate both urethral and bladder function in these patients and presenting a thorough explanation of all possible adverse effects as well as expectations is key to increase patient satisfaction.

#### **7. Urethral Sphincter BoNT-A Injection in Dysfunctional Voiding**

As urethral BoNT-A injection had been successfully used in the treatment of DSD in SCI patients, this treatment was further applied to adults with non-neurogenic voiding dysfunction due to bladder

outlet obstruction and urethral sphincter overactivity. Fowler's syndrome consists of difficulty in passing urine or urinary retention due to failure to relax the urethral sphincter in patients without neurological or anatomical abnormality [46]. Treatment of Fowler's syndrome is complicated and patients usually need intermittent self-catheterization [46]. In 2016, an open-label, prospective study enrolled 10 women with difficult urination due to Fowler's syndrome and treated these patients with urethral injection of 100 U of BoNT-A [47]. At 10 weeks of follow-up, the maximal urinary flow rate was significantly increased and the residual volume was decreased. Even four of the five women, who initially had complete retention, could void spontaneously after the treatment. Recently, we also conducted a randomized, double-blind, and placebo-controlled study using BoNT-A injection into the urethral sphincter to treat patients with refractory DV (open bladder neck but a poorly relaxed urethral sphincter, and a normal-to-high voiding pressure with a low urinary flow) [48]. Our results revealed that patients who received BoNT-A injection had a significantly improved international prostate symptom score (IPSS), quality-of-life index, maximum flow rate, voided volume, and decreased detrusor voiding pressure at one month of follow-up. When compared with the normal saline injection group, however, only the total IPSS and voided volume improvement were significantly greater in the BoNT-A injection group. Improvement in other clinical parameters was not significantly different between the BoNT-A and normal saline injection groups. We concluded that urethral sphincter injection with either BoNT-A or placebo could safely and effectively ameliorate clinical symptoms and improve quality of life in patients with DV. Although the exact pathogenetic mechanism remains unknown, local injection itself might have a therapeutic effect on the relaxation of the urethral sphincter, regardless of pharmacologic effects of BoNT-A. Additional studies enrolling more patients with DV are necessary to elucidate the efficacy of BoNT-A urethral injection.

#### **8. Intraprostatic BoNT-A Injection in Benign Prostate Hyperplasia**

Benign prostate hyperplasia (BPH) resulting in bladder outlet obstruction is one of the most common conditions presented in the urology clinic. An epidemiological meta-analysis revealed the occurrence of BPH in age groups 40–49 years, 50–59 years, 60–69 years, 70–79 years, and 80 years and older was 2.9%, 29.0%, 44.7%, 58.1%, and 69.2%, respectively [49]. Mainstream treatment of BPH is usually started with oral medication including an α-adrenergic blocker and a 5-α-reductase inhibitor [50]. Surgical intervention such as transurethral prostate resection is indicated if BPH patients are refractory to treatment with oral medication [51]. The prostate is an organ composed of glandular tissue and fibromuscular stroma. The prostate may cause bladder outlet obstruction not only because of glandular hyperplasia, but also because it may be associated with the dysregulation of smooth muscle contractility in the stroma [52]. Relaxation of smooth muscle in the prostate stroma has been considered a potential target to treat patients with BPH in many pharmacological studies [52]. Given its inhibitory effect on neurotransmitter release from the neuromuscular junction, studies using 100 to 200 U intraprostatic injection of BoNT-A to treat patients with BPH started as early as 2003, and initial results showed promising therapeutic effects [53,54]. BPH patients who received intraprostatic BoNT-A injection experienced significant improvement in both symptom score and quality-of-life index at short-term follow-up (one month) [53,54]. In addition, evidence from both human and animal studies showed prostate apoptosis activity increased after BoNT-A injection [53,55]. However, two large, randomized, double-blind, placebo-controlled trials showed no significant difference between patients who received intraprostatic BoNT-A injection and placebo [56,57]. A meta-analysis including randomized, placebo-controlled trials also suggested that BoNT-A injection for patients with BPH does not have a significantly better therapeutic effect than does placebo [58]. On the other hand, a recent randomized placebo-controlled study, which enrolled only BPH patients with moderate to severe symptoms (IPSS ≥ 19) and pressure-flow study, indicated bladder outlet obstruction showed different results [59]. Patients received BoNT-A injection reported significantly greater improvement of IPSS, maximum flow rate, and PVR when compared with the placebo group at three months of follow-up. The follow-up urodynamic study in the BoNT-A injection group also showed a significant reduction

in bladder outlet obstruction index (54%), which was not significantly changed in the placebo group. This study enrolled only BPH patients with evidence of bladder outlet obstruction and might be the reason why these results are different from previous studies. We suggested intraprostatic BoNT-A should be a reasonable treatment option for moderate to severe BPH patients who are refractory to oral medications and not willing to undergo surgical intervention. Careful selection of patients according to symptom severity and urodynamic study evaluation before treatment might be essential for successful outcome of this treatment.

#### **9. Intraprostatic BoNT-A Injection in Chronic Prostatitis**

Prostatitis is another common problem among many young and middle-aged male patients in the urology clinic. Male patients with chronic prostatitis usually present with pelvic pain/discomfort (perineal, testicular, penis, or pubic area) and voiding symptoms [60]. Treatments of chronic prostatitis should be treated first with oral antibiotics and an α-adrenergic blocker [61]. If there is no obvious symptomatic benefit, medications targeting neuropathic pain or neuromodulation procedures should be considered in these patients [61]. Giorgio et al. first used BoNT-A injection to treat voiding dysfunction in male patients with chronic prostatitis [62]. An animal study revealed the anti-inflammatory and analgesic effects of BoNT-A in prostatitis [63]. In rats with capsaicin-induced prostatitis, Chuang et al. showed that intraprostatic BoNT-A injection could both reduce pain and decrease infiltration of inflammatory cells in the prostate. Falahatkar et al. conducted a prospective, randomized, double-blind, placebo-controlled study to evaluate transurethral intraprostatic injection of 100 U of BoNT-A for patients with chronic prostatitis [64]. Significant improvement in pain score, National Institutes of Health chronic prostatitis symptom index, and quality of life were observed in the BoNT-A injection group at six months of follow-up. When compared with the placebo group, symptom improvements were significantly greater in the BoNT-A group. Another study compared the efficacy of transurethral and transrectal intraprostatic BoNT-A for chronic prostatitis [65]. Both groups demonstrated significant improvement in pain at six months of follow-up, but only patients in the transrectal injection group had significant improvement in the chronic prostatitis symptom index. Both anti-inflammatory and anti-nociceptive effects should be key factors for treating chronic prostatitis with BoNT-A. Although these pilot studies showed promising results of BoNT-A injection for treating chronic prostatitis, additional randomized placebo-controlled trials that enroll more patients are necessary to prove its efficacy.

In summary, both researchers and clinicians are determined to develop novel BoNT-A applications for treating LUTDs, improve the convenience of drug delivery, and decrease adverse effects. However, some patients continued to be dissatisfied with the outcome and opted to discontinue injections. For example, in a prospective study, only 68% of refractory OAB patients would like to continue to receive BoNT-A treatment after the first injection [66]. A study also detected neutralizing antibodies to BoNT-A in patients who had received bladder or urethral BoNT-A injections and suggested a possible cause of therapy failure [67]. Physicians should comprehensively evaluate voiding problems in patients and make diagnoses precisely before using BoNT-A to treat LUTDs. Further research that focuses on ways to improve BoNT-A with stronger effects and long-lasting formulations are necessary. Adjustment of BoNT-A dose according to urodynamics, study findings, or in combination with oral medications might improve the efficacy or prolong the duration of the therapeutic effect. A summary of novel applications of BoNT-A in LUTDs is provided in Table 1.



 quality of life; Qmax: maximal urinary flow rate; IPSS: international prostate symptom score; VV: voided volume; PVR: post-voiding residual volume. IC/BPS: interstitial cystitis/bladder pain syndrome; OAB: overactive bladder; DHIC: detrusor hyperactivity with impaired contractile function; DV: dysfunctional voiding; BPH: benign prostate hyperplasia.

#### **10. Conclusions**

Since BoNT-A was first used to treat LUTD 30 years ago, applications have become increasingly prevalent and popular. Intravesical BoNT-A injection for patients with OAB or NDO has been widely used in daily urologic practice and proved by DHIC in the United States and many countries. For IC/BPS patients who have an inadequate response to initial treatment, intravesical BoNT-A injections also have been considered as standard treatment according to different clinical guidelines. BoNT-A delivery with liposome-encapsulation and gelation hydrogel intravesical installation provided a new class of less invasive and convenient application for patients with OAB or IC/BPS. Clinical trials revealed promising therapeutic results of novel BoNT-A applications, including DV, BPH, and chronic prostatitis. However, further randomized and placebo-controlled studies that enroll patients with accurate diagnoses are necessary to prove the efficacy of BoNT-A treatment. Both careful patient selection and prudent use of urodynamic study evaluation to confirm diagnoses are essential to achieve successful treatment outcomes.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Exploiting Botulinum Neurotoxins for the Study of Brain Physiology and Pathology**

#### **Matteo Caleo and Laura Restani \***

CNR Neuroscience Institute, via G. Moruzzi 1, 56124 Pisa, Italy; caleo@in.cnr.it **\*** Correspondence: restani@in.cnr.it; Tel.: +39-050-315-3199

Received: 31 March 2018; Accepted: 23 April 2018; Published: 25 April 2018

**Abstract:** Botulinum neurotoxins are metalloproteases that specifically cleave *N*-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in synaptic terminals, resulting in a potent inhibition of vesicle fusion and transmitter release. The family comprises different serotypes (BoNT/A to BoNT/G). The natural target of these toxins is represented by the neuromuscular junction, where BoNTs block acetylcholine release. In this review, we describe the actions of botulinum toxins after direct delivery to the central nervous system (CNS), where BoNTs block exocytosis of several transmitters, with near-complete silencing of neural networks. The use of clostridial neurotoxins in the CNS has allowed us to investigate specifically the role of synaptic activity in different physiological and pathological processes. The silencing properties of BoNTs can be exploited for therapeutic purposes, for example to counteract pathological hyperactivity and seizures in epileptogenic brain foci, or to investigate the role of activity in degenerative diseases like prion disease. Altogether, clostridial neurotoxins and their derivatives hold promise as powerful tools for both the basic understanding of brain function and the dissection and treatment of activity-dependent pathogenic pathways.

**Keywords:** synaptic transmission; SNAP-25; epilepsy; Parkinson's disease; neurotransmission blockade; electrical activity; prion disease

**Key Contribution:** This review describes the experimental use of botulinum neurotoxins as tools to block synaptic function in specific brain modules and dissect activity-dependent pathways in CNS pathologies.

#### **1. Introduction**

Botulinum neurotoxins (BoNTs) are the pathogenic agents responsible for the manifestation of botulism. The typical flaccid paralysis of botulism induced by BoNTs is due to blockade of cholinergic neurotransmission at the neuromuscular junction and autonomic terminals [1–3].

These toxins are produce by anaerobic bacteria of the genus Clostridium and are among the most potent naturally-occurring substances. The family of BoNTs comprises seven antigenically distinct botulinum neurotoxins (BoNT/A–BoNT/G). For serotypes A, B, E, and F, several subtypes have been described based on differences in amino-acid sequences. For BoNT/A, at least eight subtypes (named A1 to A8) are currently known with different enzymatic activity and toxicological properties [4–6].

BoNTs share a common molecular structure and are composed of a disulphide-linked, ~100-kDa heavy chain and ~50-kDa light chain. They are metalloproteases that bind to presynaptic terminals, enter the cytosol and block neurotransmitter release by specific cleavage of proteins of the soluble *N*-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. The SNARE complex is necessary for synaptic vesicles fusion, thus the net effect is blockade of neurotransmitter release [3,7,8]. The target protein differs according to BoNTs serotype. BoNT/A and E cleave

synaptosomal associated protein of 25 kDa (SNAP-25); BoNT/C acts on both SNAP-25 and syntaxin; BoNT/B, D, F and G cleave vesicle-associated membrane proteins (VAMPs, also known as synaptobrevins).

Despite their toxicity, they produce a prolonged but reversible action at the synapses. Thus, it has been speculated, already decades ago, that small amount of BoNTs could be used therapeutically to treat disorders characterized by hyperexcitability. Historically, the first to make therapeutic use of BoNT/s was Alan B. Scott in the 1970s, for the treatment of strabismus [9]. Subsequently, the Food and Drug Administration has continuously increased the approved uses for botulinum neurotoxin A1 (BoNT/A1). BoNT/A1 is indeed the most used serotype in clinical practice, because the protease has a persistent activity and this allows long lasting duration of the therapeutic effects (months).

To date, approved indications include focal dystonias, spasticity, cosmetic treatments and migraine, and several other applications are emerging. In all of these cases, minute amounts of BoNT are administered in peripheral muscles to locally inhibit transmitter release.

However, BoNTs are also effective in blocking transmitter release at central synapses when directly delivered into the brain [10].

Here we will review literature data reporting BoNTs effects following direct injection into the central nervous system. Specifically, we will describe how these potent and selective synaptic blockers may be exploited to gain insight into mechanisms of brain physiology and dysfunction.

#### **2. Action of BoNTs on Central Synaptic Terminals**

BoNTs enter central neurons mainly via activity-dependent synaptic endocytosis, indeed depolarization increases toxins uptake [11–14]. At least for BoNT/A, neuronal entry also occurs via an alternative pathway independent of synaptic vesicle endocytosis [15,16], which may direct the toxin to the retroaxonal transport pathway [17,18].

Analyses on brain synaptosomes have demonstrated that BoNTs (mainly studies on BoNT/A) interfere with neurotransmitter release of acetylcholine, glutamate, noradrenaline, serotonin and dopamine from central synases ([10]). It is interesting to note that GABAergic terminals are more resistant to BoNT/A intoxication compared to excitatory (glutamatergic) terminals [19,20]. One reason could be that SNAP-25, the synaptic target of BoNT/A, is less expressed in inhibitory than in glutamatergic terminals [20,21]. For example, SNAP-25 is almost absent in perisomatic inhibitory terminals impinging onto principal neurons in the pyramidal layer of hippocampal CA1 [22]. However, recent electrophysiological recordings in embryonic stem cell-derived neurons (ESNs), showed that miniature Inhibitory Post Synaptic Currents (mIPSC) frequencies were already reduced more than 70% 30 min after BoNT/A intoxication, while decrease in miniature Excitatory Post Synaptic Currents (mEPSC) frequencies was detectable only after 70 min [23]. This finding supports the initial increase in frequency of mPSCs in the first hour after BoNT/A treatment, followed by basically a complete silencing of activity around 15 h [23].

Silencing of spontaneous and evoked excitatory postsynaptic potentials was already demonstrated in hippocampal neurons [24,25]. Accordingly, in vivo delivery of BoNT/A or BoNT/E in rodent hippocampus prevents neuronal spiking activity in hippocampal CA1 [26,27].

It is worth noting that BoNT/A produces an efficient blockade of neurotransmitter release by cleaving a small percentage (about 10%) of the SNAP-25. This seems to be due to the dominant negative effect of BoNT/A-truncated SNAP-25 [28]. However, it possible to rescue BoNT/A-induced blockade of neurotransmission by increasing extracellular calcium concentration. Although BoNT/A and BoNT/E share the same synaptic target (SNAP-25), this rescue with calcium is not possible with BoNT/E, probably because serotype E cleaves a larger fragment at the C-terminus of SNAP-25 [24,29].

At the ultrastructural level, our group investigated the morphological changes induced by local delivery of BoNT/A into the hippocampus [30,31]. Hippocampal samples were analyzed at different times following BoNT/A injection (2, 4, 8 weeks). Observation of electron microscope images, focused on the CA1 stratum radiatum, revealed that BoNT/A induced an accumulation of synaptic

vesicles. This accumulation triggered an enlargement of presynaptic terminals which was maximal at 4 weeks [30]. It is noteworthy that these changes were detectable basically only in asymmetric, excitatory synapses, and not in symmetric, GABAergic synapses, confirming a preferential effect of BoNT/A on excitatory terminals [20–22,30]. Axonal enlargements were also observed within the striatum injected with BoNT/A. These enlargements result positive for choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) in rats, but positive only for ChAT in mice [32,33].

#### **3. BoNTs for the Study of Brain Physiology**

A typical feature of BoNTs is that their action is prolonged but reversible. These characteristics make BoNTs, in particular BoNT/E which produces a short-lived blockade, ideal tools to study brain physiology. BoNTs allow a transient "silencing" of specific brain regions after a single administration, which is experimentally more convenient compared to other drugs that need to be continuously infused (e.g., tetrodotoxin or muscimol) [34].

Luvisetto, Pavone and collaborators were among the first to test the impact of direct brain injections of BoNTs in mice. They performed intracerebroventricular (icv) injections of sub-lethal doses of BoNT/A or BoNT/B and assessed various behavioral responses [35], such as active avoidance and object recognition. They also analyzed BoNTs effects on pharmacologically induced locomotor activity. The results indicated no effect on active avoidance acquisition, while there were impairments in the novel object recognition task, and amplified effects of drugs which induce locomotor activity [35]. The same group also tested the effects of central administration of BoNT/A on pain mechanisms [36]. They used a mouse model of formalin-induced pain (injection of formalin into the hindpaw) and the licking response as an index of pain. The data showed that intracerebral BoNT/A affected the licking response in the second phase of formalin test, similar to the effects obtained with peripheral administration [36,37]. Anti-nociceptive effects of central administrations of BoNT/A were later confirmed by other groups in various models of pain [38,39].

Our group has exploited BoNT/E to obtain a sustained but reversible blockade of neurotransmission for about 2 weeks in specific brain regions [27,40]. In particular, to investigate the role of cortical activity in the maturation of visual function, we unilaterally injected BoNT/E into the visual cortex (V1) in rat pups, at the time of eye opening [40]. BoNT/E injection produced a unilateral silencing of V1 for about 2 weeks, completely abolishing visual responses during the so called "critical period" for development of cortical function [41]. We performed electrophysiological recordings 3 weeks following BoNT/E injection (when cleaved SNAP-25 was no longer detectable), in order to assess visual system development when electrical activity was recovered, i.e., at the completion of the normal critical period. We found that BoNT/E-induced silencing of cortical activity did not allow normal maturation of visual function, keeping visual acuity low and extending the duration of the critical period [40]. We also evaluated if these deficits were persistent, or if they reflected only a delay in visual function maturation. Thus we performed behavioral and electrophysiological analyses at a longer time point (more than 2 months following toxin injection), and we confirmed a persistent impairment in visual performance. In conclusion, exploiting BoNT/E delivery to induce a transient silencing of cortical activity during the critical period allowed us to demonstrate that intrinsic cortical activity is necessary for a correct development of visual function [40].

Long-lasting serotypes such as BoNT/A and BoNT/B could be useful to create animal models of pathologies (e.g., dementia, [42]) or to treat hyperexcitability [26] (see below). However, these models could also offer basic knowledge about the role of specific brain regions in behavioral performance. For example, BoNT/B injection into the entorhinal cortex in adult rats produce learning and memory impairments as assessed by maze tests [42].

Similarly, BoNT/E hippocampal injection in adult rats induces deficits in spatial learning during the Morris water maze task, but since BoNT/E action is short-lived, the impairments are completely reversible and confirm a key role of hippocampus in spatial learning [26].

Mapping of the spread of BoNT/E via immunostaining for intact and cleaved SNAP-25 [40,43] demonstrates that toxin action remains confined to the cortical areas close to the injection site, thus allowing regional specificity of the synaptic blockade. Toxin diffusion can be further limited via the use of convection-enhanced delivery (CED), which provides a more homogeneous distribution than conventional bolus injection and does not damage the surrounding tissue [44–47].

#### **4. Exploiting BoNTs in Pathological Brain Conditions**

We have already reported examples of how BoNTs could be exploited to study the role of electrical activity in physiological, developmental brain processes [40]. In addition, BoNTs delivery could be useful to address the impact of electrical activity in neurodegenerative pathologies. Indeed, while it is known that synaptic degeneration precedes cell loss (e.g., [48]), little is known about mechanisms that tag synapses for degeneration. In this context, our group hypothesized that in a hippocampal mouse model of prion disease (a neurodegenerative disease associated with aggregates of misfolded proteins), synaptic degeneration was activity-dependent. To verify this hypothesis, we injected BoNT/A into the hippocampus of mice with prion disease and we analyzed synaptic degeneration at the ultrastructural level by electron microscopy [30]. Contrary to our expectations, we failed to find differences in the density of degenerating synapses between BoNT/A- and vehicle-injected prion mice. The morphology of the degenerating synapses was also indistinguishable between the two groups. These experiments challenge the idea that dysfunctions in synaptic vesicle release trigger the elimination of synaptic boutons, at least in prion-induced neurodegeneration [30,31].

Recently, Spalletti et al. (2017) used BoNT/E to produce a transient silencing of the contralesional hemisphere in a mouse model of focal stroke in the motor cortex. One of the main hypothesis in the stroke field is the "inter-hemispheric competition model", which posits an enhanced transcallosal inhibition from the healthy to the lesioned side. To reduce this interhemispheric inhibition, the authors applied BoNT/E to block activity in the contralesional motor cortex immediately after the stroke. They found a significant recovery of motor function in the treated animals. Importantly, functional recovery was further enhanced when the silencing of the healthy side was coupled with physical rehabilitation of the affected arm [43].

Since BoNTs block neurotransmitter release, it is not unexpected that they have been exploited to treat, similarly to the peripheral nervous system, pathologies characterized by hyperexcitability. The most frequent category of central pathologies associated with hyperexcitability is epilepsy. About six millions of persons in Europe develop epilepsy, and around 30% of these are pharmaco-resistant [49]. This means that there are no drugs available to suppress or decrease their seizures. In the worst cases, the only clinical solution is a surgical intervention which physically removes the main epileptic focus. Consequently, efforts for discovering new therapeutic treatments are warranted. Our group and others have investigated whether central, local BoNTs delivery could suppress seizures in animal models of epilepsy [4,26,27,45,50]. The first serotype used was BoNT/E, tested in animal models of acute seizures, triggered by hippocampal administration of pro-convulsant agent kainic acid (KA) [26]. To measure BoNT/E effects, authors performed behavioral and electrographic analyses, demonstrating that BoNT/E delivery is effective in decreasing number and duration of seizures triggered by KA. BoNT/E effects were not limited to the electrophysiological level, but the toxin had an impact also on hippocampal histopathological changes, such as neuronal loss. This neuroprotection likely depends on blockade of excitoxicity phenomena occurring during prolonged electrical activity [26]. The neuroprotective action elicited by BoNT/E has been demonstrated also in a model of focal ischemia [51]. The potent vaso-constricting peptide endothelin-1 (ET-1) was delivered intrahippocampally in adult rats, followed 20 min later by BoNT/E injection in CA1. To evaluate BoNT/E action on excitoxicity, that is, on glutamate release, the authors performed in vivo microdialysis. Data showed that BoNT/E-injected rats had a decreased glutamate release. This synaptic effect was matched with a decrease in CA1 neuronal loss, as measured by immunohistochemistry [51]. Thus, the neuroprotective action by BoNT/E depends on the inhibition of the release of glutamate and occurs via downregulation of proapoptotic proteins, such as caspase-3 [52].

Based on these initial, encouraging data on acute seizures, BoNT/E was tested also in a mouse model of chronic seizures that resembles mesial temporal lobe epilepsy (MTLE), one of the most common pharmacoresistant forms of epilepsy in humans, obtained by intrahippocampal injection of KA [27,50]. The authors initially tested the impact of BoNT/E delivery on epileptogenesis (i.e., the development of spontaneous ictal events) following an episode of status epilepticus triggered by KA. The findings indicated that BoNT/E-mediated synaptic blockade during epileptogenesis was not effective in blocking the occurrence of spontaneous seizures. However, BoNT/E treatment was associated with histopatological protection; there was less neuronal loss in CA1 and the dispersion of granule cells in the dentate gyrus was potently prevented [27]. In a second work, the authors investigated if BoNT/E delivery was sufficient to reduce seizures during the chronic phase of epilepsy [50]. Mice injected with KA were implanted with bipolar electrodes, and after a period of baseline recording sessions, BoNT/E was infused directly into the epileptic hippocampus. Subsequent electrophysiological recordings clearly proved that BoNT/E delivery produces a reduction in total seizure duration and frequency [50].

One may argue that to be practically useful in the treatment of epilepsy, focal treatments require a long duration of action. Other serotypes of BoNTs with a prolonged proteolytic activity, like BoNT/A or BoNT/B, are ideal tools. Indeed, a couple of studies have used these serotypes to block seizures for longer periods in the amygdala kindling model, an experimental paradigm that allows to follow seizures for weeks to months. Gasior and colleagues (2013) directly infused BoNT/A or BoNT/B into the amygdala, via convection-enhanced delivery (CED) [45]. Therapeutic effects of both toxins were assessed by measuring after-discharge threshold and other parameters of the amygdala-kindled seizures at different times (3, 7, 10, 15, 21, 35, 50, and 64 days) after the administration. Results pointed to the anti-convulsant effects of both toxins, as assessed with EEG measures (i.e., elevation in after-discharge threshold of stimulation and seizures duration). The anti-convulsant action persisted until day 50. It interesting to note that, whilst BoNT/B was also effective in reduction of behavioral seizures, BoNT/A did not reach significance values in this parameter [45].

Another manuscript exploited infusion of BoNT/A (specifically serotype A2) to reduce seizures in kindled mice [53]. In half of the animals, BoNT/A2 was able to completely block the appearance of seizures. In addition, the toxin decreases the level of seizures, at least until 18 days following injection.

Taken together, these results suggest that BoNTs are quite effective in amelioration of epileptic activity, and they could be potentially used as focal antiepileptic treatments.

One might envision another possible "diagnostic" use of BoNTs in epilepsy, especially for BoNT/E, which has the shorter duration of action. In patients eligible for resection surgery, it is fundamental to precisely map brain epileptic foci, to remove all the hyperexcitable areas and render the patient seizure-free after surgery. The mapping is usually performed by non-invasive imaging techniques (such as magnetoencephalography (MEG) and functional MRI (fMRI)), or by EEG with chronically implanted electrodes [54], however the results are not always satisfactory, and patient could suffer of residual seizures also after surgery. In this context, local delivery of botulinum toxins could represent a strategy to functionally map the epileptogenic areas, and check whether the silencing of the presumptive focus is effective in abolishing seizures.

Another promising application of local delivery of BoNT/A is the therapeutic treatment of movement disorders and neurotransmission dysfunction typical of Parkinson's disease (PD). PD is characterized by an imbalanced cholinergic hyperactivity in the striatum, due to the loss of dopaminergic neurons of the substantia nigra. Since BoNT/A blocks neurotransmitter release, including acetylcholine (ACh), the toxin was injected directly into the striatum, in animal models of PD [32,33,55–57]. In particular, the rodent model of 6-hydroxydopamine (6-OHDA) produces a hemi-parkinsonism. Wree and colleagues (2011) tested effects of BoNT/A injected 6 weeks following lesion with 6-OHDA. BoNT/A action was evaluated using the apomorphine-induced contralateral

rotation test. Apomorphine is a dopamine (DA) receptor agonist and stimulates the supersensitive dopamine receptor D2 (DRD2) in the lesioned hemisphere, causing a net rotation away from the side of the lesion, that is, anti-clockwise [58]. Infusion of BoNT/A into the ipsilateral, lesioned striatum is able to reverse this rotation movement until 3 months [32]. Authors observed also enlarged axonal varicosities in BoNT/A (BiVs) injected-animals (possibly due to synaptic vesicles accumulation as seen in hippocampus by Caleo and co-authors [30]). Immunohistochemical analysis revealed that these axonal varicosities were cholinergic, but some of the BiVs were found to be positive for tyrosine hydroxylase (TH) [32,55]. In a subsequent work, these cholinergic varicosities induced by BoNT/A were investigated in detail [55]. They evaluated the number of ChAT-positive interneurons as well as the density and the volumetric size of the BiVs. In the ipsilateral side of BoNT/A-injected rats, with 6-OHDA lesion, the numeric density of BiVs reached a maximum 3 months after BoNT/A, while their volume increased during the whole time course of the experiment. However, no differences were detectable in the number of ChAT-positive neurons, up to 1 year following BoNT/A injection. This last result is important because it speaks in favor of a lack of cytotoxic effects of BoNT/A [55].

A similar study has been performed in mice, to extend possible therapeutic BoNT/A applications to genetics mouse models of PD [33]. Authors injected increasing doses of BoNT/A, finding no differences in the number of ChAT-positive interneurons. Increasing BoNT/A doses (from 25 pg to 200 pg), led to an increased BiV volume, and a decreased number of small BiVs. It is noteworthy that, in contrast to rats, TH-immunoreactive BiVs were not found in BoNT/A-infused mice [33].

Intrastriatally injected BoNT/A appears also to induce changes in receptor expression, likely due to activity silencing. For example, BoNT/A reduced density of dopamine receptor D2/D3, whereas other key receptors (such as dopamine 1 (D1), noradrenergic (a1 and a2) and serotonergic (5HT2A) receptors) remained basically unaltered in rats [57]. Since authors found few weeks after unilateral 6-hydroxydopamine (6-OHDA) lesion a significant increase of D2/D3 receptor ratio, the therapeutic effects of BoNT/A probably resides in reducing the interhemispheric imbalance in D2/D3 receptor density in lesioned rats.

Altogether, these results indicate how intracerebrally injected BoNTs could induce synaptic silencing and long-lasting changes in neurotransmitter-related proteins, that ultimately produce therapeutic benefits (see Table 1 for a summary).


**Table 1.** Exploiting botulinum neurotoxins (BoNTs) in pathological brain conditions. The table summarizes the main studies that have exploited central delivery of botulinum neurotoxins to treat pathological brain conditions.

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#### **Table 1.** *Cont*.

#### **5. Intracerebral BoNTs: Future Directions**

BoNT clinical indications are continuously increasing, thanks to advantages such as very long duration, high potency, and complete reversibility of action [3].

There is currently considerable interest in developing novel forms of BoNTs with optimized therapeutic properties and neuronal selectivity (i.e., neuromuscular junction vs. sensory endings), which could offer new treatment opportunities. On one hand, the natural repertoire of BoNTs offers a wide variety of molecules with specific actions in neuronal cells and in vivo mouse models [59]. Second, an engineering approach has been taken to modify the pharmacological properties of native toxins by specific mutations. For example, a mutated BoNT/A1 has been created with faster onset and a shorter duration of action than BoNT/A1 wild type [60], opening the way to design BoNT variants with novel and useful properties.

The group of Bazbek Davletov has quite recently developed a new technology, named "protein-stapling", by which it is possible to re-assemble chimeric clostridial neurotoxins starting from two separate modules, that is, the light chain/translocation domain and the receptor-binding domain [61,62]. This technology is not only useful to safely produce active toxins, but also allows engineering of toxins. The first engineered toxin was an analogue of the botulinum neurotoxin type A, called BiTox. The structural evaluation of BiTox suggests that the re-assembled BoNT/A could be substantially longer than the native molecule. However, BiTox demonstrated similar efficiency to that of native BoNT/A in proteolytic cleavage of SNAP-25 in vitro and in vivo, and thus in neurotransmitter silencing [61]. Interestingly, and clinically relevant, potency of BiTox at the neuromuscular junction is reduced, probably because of the bigger size of the molecule. Thus, systemic toxicity is reduced in BiTox injected subjects, and this represents a considerable advantage for clinical applications [61].

Engineered neurotoxins could also be exploited to enhance the selectivity for selected neuronal populations, combining the receptor-binding domain with different catalytic chains. For example, the same group has combined BoNT/A protease with the TeNT binding domain, allowing intoxication of different neuron populations compared to the native BoNT/A [62]. This chimera has a nociceptive action at central level, but has no action on motoneurons (as it caused neither flaccid nor spastic paralysis), resulting safer and potentially relevant for medical applications. On the other side, engineered toxins are interesting also for basic neuroscience research. Indeed, this chimera, following direct delivery into the rat visual cortex, was able to modulate sensory function [62].

**Author Contributions:** M.C. and L.R. wrote and discussed the manuscript.

**Acknowledgments:** We acknowledge financial support from AIRC (Italian Association for Cancer Research) grant #IG18925, Regione Toscana (RONDA Project, "Programma Attuativo Regionale" financed by FAS—now FSC), CNR InterOmics project, and CNR NanoMax project.

**Conflicts of Interest:** The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

#### **References**


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