**"Appropriate Treatment" and Therapeutic Window in Spasticity Treatment with IncobotulinumtoxinA: From 100 to 1000 Units**

#### **Giancarlo Ianieri \*, Riccardo Marvulli, Giulia Alessia Gallo, Pietro Fiore and Marisa Megna**

Department of Basic Sciences, Neuroscience and Sense Organs, University of Bari "Aldo Moro", G. Cesare Place 11, 70125 Bari, Italy; ricmarv81@hotmail.it (R.M.); giulia.gallo1985@gmail.com (G.A.G.); p\_fiore@hotmail.it (F.P.); marisa.megna@uniba.it (M.M.)

**\*** Correspondence: igc@neurol.uniba.it; Tel.: +39-080-559-5519

Received: 12 February 2018; Accepted: 23 March 2018; Published: 28 March 2018

**Abstract:** Many neurological diseases (ischemic and hemorrhagic stroke, multiple sclerosis, infant cerebral palsy, spinal cord injuries, traumatic brain injury, and other cerebrovascular disorders) may cause muscle spasticity. Different therapeutic strategies have been proposed for the treatment of spasticity. One of the major treatments for tone modulation is botulinum toxin type A (BTX-A), performed in addition to other rehabilitation strategies based on individualized multidisciplinary programs aimed at achieving certain goals for each patient. Therapeutic plans must be precisely defined as they must balance the reduction of spastic hypertonia and retention of residual motor function. To perform and optimize the treatment, an accurate clinical and instrumental evaluation of spasticity is needed to determine how this symptom is invalidating and to choose the best doses, muscles and times of injection in each patient. We introduce an "appropriate treatment" and no "standard or high dosage treatment" concept based on our retrospective observational study on 120 patients lasting two years, according to the larger Therapeutic Index and Therapeutic Window of Incobotulinumtoxin A doses from 100 to 1000 units. We studied the efficiency and safety of this drug considering the clinical spasticity significance for specialist physicians and patients.

**Keywords:** spasticity; botulinum toxin type A; appropriate treatment; Therapeutic Index

**Key Contribution:** To underline larger Therapeutic Index and Therapeutic Window of IncobotulinumtoxinA doses (from 100 to 1000 units) considering clinical spasticity significance for specialist physicians and patients.

#### **1. Introduction**

Spasticity is the most common complication of many neurological diseases followed by ischemic and hemorrhagic stroke, multiple sclerosis, infant cerebral palsy, spinal cord injuries, traumatic brain injury, and other cerebrovascular disorders [1].

Comprehending the various mechanisms of muscle tone alteration and the quantitative evaluation of muscle rheological modifications can lead to the development of more precise and targeted therapeutic interventions for the treatment of spasticity [2,3]. Anatomically, there is a reduction in type II fibers and an increase in type I fibers. Clinically, it is an involuntary motor disorder, characterized by hypertonic muscle tone with increased excitability of the muscle relaxation reflex and increased tendon reflexes. Muscle weakness or paresis in the limbs is associated with spasticity and contributes to loss of motor dexterity and functional capacity [4]. Spasticity, if left untreated, can hinder the functional result by promoting persistent abnormal postures which produce muscle-tendon contractions and bone deformities. Substantial complications resulting from spasticity include

movement impairment, difficulty in managing hygiene and self-care, poor self-esteem, body image alteration, pain, and pressure ulcers. In addition, patients with severe spasticity may suffer poor social interaction drastically worsening quality of life [5,6].

For these clinical problems and their related high social costs, various therapeutic strategies have been proposed for the treatment of spasticity including surgical, medical, and rehabilitation procedures. One of the major treatments for muscle tone modulation is botulinum toxin type A (BTX-A) administered intramuscularly [5,7]. BTX-A, by modulating the release of acetylcholine from synaptic vesicles, brings a reduction in muscle tone and, if associated with appropriate rehabilitation treatment, can stop the cascade of events that causes muscle fibrosis with subsequent retraction and joint blocks. BTX-A is indicated when spasticity is focal or segmental and interferes with active or passive operation. The primary purpose of spastic muscle treatment is to maintain length and to allow normal limb placement to avoid secondary shortening of soft tissues. In general, treatment with BTX-A is performed in addition to other rehabilitation strategies based on individualized multidisciplinary programs aimed at achieving tailored goals for each patient. Therapeutic plans must be precisely defined as they must balance the reduction of spastic hypertonia and retention of residual motor function. Although there is no consensus on when BTX-A therapy should begin or how long it should last, BTX-A intramuscular infiltrations are considered the first line of medical treatment for focal/segmental spasticity [8].

Proper use of botulinum toxin both in terms of dosage and injection requires careful monitoring of spastic hypertonia over time. The most commonly used muscular tone measurement is the Modified Ashworth Scale (MAS), where resistance to the passive muscle extension is rated in five points on an ordinal scale. MAS has been criticized for non-standardization of extension speed in manual trials, it does not quantify an absolute resistance, it is physician-dependent, only applies to distal body segments, and has low sensitivity to small variations in muscle tone [9]. We have also discussed the reliability and validity of this scale. MAS is reliable for measuring the muscle tone of some muscle groups such as the elbow, wrist, and knee flexors [10]. Considering the difficulties in distinguishing between the increase in muscle tone and soft tissue rigidity, as well as lack of correlation with functional changes after each treatment, appropriate clinical and instrumental measurements should be used to obtain reliable and accurate values of their rheological properties (tone, elasticity, and stiffness). A tool with these portable and reliable features is MyotonPRO®, a painless and non-invasive device which can provide quantitative and objective evaluations of muscle properties [11,12].

A proper assessment of muscle properties is important to make appropriate clinical decisions and to monitor therapy results in patients with spasticity.

The aim of this retrospective observational study is to objectify the efficacy and safety of 100 to 1000 units botulinum toxin treatment to modulate spasticity according to the individual patient's needs.

#### **2. Results**

Patients were initially divided into three groups according to the botulinum toxin A dosage, as reported in the Methods section. During the observation period, some patients switched to another group because the dosage used increased (Table 1).

**Table 1.** Number of patients in each group at the beginning of study and after 9 months when there were no other switches. In the first group largest number of patients switched to another group (the second one).


In group A, average doses increased during the study, but there was no statistical significance. At the third injection, dosage was increased in 10 patients (33%) due to poor clinical effects or to treat

more muscles, so they switched to group B. In the following injections, these patients improved their clinical and instrumental measurement of spasticity (Figures 1 and 2).

**Figure 1.** Myometric muscle tone evaluation in group A. This figure shows a statistically significant reduction in muscle tone after 30 days from the first and from the last injection in 20 patients treated with dosage up to 400 UI (**a**); For 10 patients of this group, muscle tone reduction was lower in the first and second cycle of injections; when we increased dosage (up to 700 UI, red line) due to increased units for each muscle or having injected other muscles not evaluated with myotonPRO®, reduction was statistically significant after 30 days for each cycle of injection (**b**). \* *p* < 0.05. BB = Biceps Brachii, FSD = Flexorum Superficial Digitorum, GM = Gastrocnemius Medialis, GL = Gastrocnemius Lateralis.

**Figure 2.** Functional Independence Measure (FIM) value in group A. The dark blue line shows statistical improvement of FIM in 20 patients treated with dosage up to 400 UI. Instead, the green and dashed line shows a statistical improvement of FIM in other 10 patients of this group when we increased dosage (up to 700 UI, red line) due to increased units for each muscle or to having injected other muscles. \* *p* < 0.05. We considered the first three and last cycles of injection because statistical improvement of data occurred after dosage increased in 10 patients and it was constant until the end in both subgroups.

In group B, average increase in dosage was not statistically significant during the study. After the first injection, eight patients (20%) showed no clinical and instrumental improvement, so we increased dosage (we treated muscles with greater dosage rather than treating more muscles) and they switched to group C. In the following injections, these eight patients improved their clinical and instrumental measurement of spasticity (Figures 3 and 4).

**Figure 3.** Myometric muscles tone evaluation in group B. This figure shows a statistically significant reduction in muscle tone after 30 days from the first and from the last injection in 32 patients treated with dosage from 400 UI to 700 UI (**a**); For 8 patients of this group, muscle tone reduction was lower in the first and second cycle of injections; when we increased dosage (up to 1000 UI, red line) due to increased units for each muscle (rather than having injected other muscles), reduction was statistically significant after 30 days for each cycle of injection (**b**). \* *p* < 0.05. BF = Biceps Femoris, AM = Adductor Magnus.

**Figure 4.** FIM value in group B. The dark blue line shows statistical improvement of FIM in 32 patients treated with dosage up to 700 UI. Instead, the green and dashed line shows a statistical improvement of FIM in other 10 patients of this group when we increased dosage (up to 1000 UI, red line) due to increased units for each muscle or having injected other muscles. \* *p* < 0.05.

In the group C, average doses at the end of study were statistically significant compared to the beginning (from 775.65 ± 30.45 to 986.65 ± 13.67, *p* <0.05). During the study all patients improved their clinical and instrumental measurement of spasticity (Figures 5 and 6).

**Figure 5.** Myometric muscle tone evaluation in group C. In each cycle of treatment, a statistically significant reduction in muscle tone after 30 days from injection was found; this figure shows data of first and last cycle of treatment. \* *p* < 0.05. TB = Triceps Brachii, BR = Brachioradialis, RF = Rectus Femoris, TA = Tibialis Anterior.

**Figure 6.** FIM value in group C. FIM value in all 50 patients treated with dosage from 700 UI to 1000 UI showed a statistically significant improvement (\* *p* < 0.05). This figure shows values of first and last cycle of treatment.

#### *Adverse Events*

We found a good safety profile for long-term use of Incobotulinumtoxin A, also in patients treated with dosage up to 1000 UI. The adverse events reported were rare (Table 2).

Considering all injection sessions, only four cases (3.3%) of excessive local muscle weakness were found, two cases (1.6%) of transient generalized weakness lasting 20 and 10 days respectively and only one case of mild dysphagia (0.8%). More specifically:


No patients abandoned treatment over the study.


**Table 2.** Summary of adverse events in each group. AE were rare and transient.

#### **3. Discussion**

This study demonstrated long term treatment efficacy of IncobotulinumtoxinA in the management of muscle spasticity using variable doses.

According to the severity of spasticity, clinically and instrumentally evaluated, and to the number of muscles treated, we injected different botulinum toxin A doses measuring spasticity improvement after each injection cycle. In 10 of 30 patients in group A and 8 of 40 patients in group B, the dose administered was increased after the third and first injection respectively due to a non-significant clinical and instrumental effect; in group A, we increased units for each muscle or we increased number of muscles treated; in group B, we only increased units for each muscle. Different studies demonstrated that improper injection techniques or a denatured toxin resulted in therapeutic failure [13–15]. In the 18 patients of our study, injection technique or toxin A reconstitution were the same as the other patients, so therapeutic failure was due to incorrect dosage or clinical evaluation of the patient. After the total units administered were changed, spasticity improved after each injection in all of the 18 patients.

Changing dosage and using higher doses than approved is possible following a ratio known as Therapeutic Index (TI) or Therapeutic Ratio (TR) [16]. In clinical practice, the TI is the range of doses at which a medication appears to be effective in clinical trials for a median of participants without unacceptable adverse events. For most drugs, this range is wide enough, and the maximum concentration of the drug and the area under the concentration–time curve achieved when the recommended doses of a drug are prescribed lies sufficiently above the minimum therapeutic concentration and sufficiently below the toxic concentration [17]. Thus, it can be expected that at the recommended prescribed doses, drugs present clinical efficacy with an adequate safety margin. A higher TI means a safer drug. A drug is generally considered having a good safety profile if its TI exceeds the value of 10 [16,18]. Patients with multifocal spasticity require higher total doses of botulinum toxin A and several studies have demonstrated IncobotulinumtoxinA having a high TI. With escalating total doses, a higher number of spasticity patterns were successfully treated, leading to increasing improvements in muscle tone, shown by consistent decreases in clinical and instrumental evaluation. In this study, the use of doses from 100 to 1000 UI demonstrate that IncobotulinumtoxinA has a wide therapeutic window, indicating the drug has a good safety profile, since, even at high doses [13] side effects were mild and transient.

In this study we used myometric evaluation (Myoton PRO®) to objectively assess muscle tone. In an objective, simple, repeatable and non-invasive way, we evaluated even minimum changes in muscle tone; we obtained a broader pathophysiological vision of rheological properties of muscle tissue in different neurological, orthopedic, and sports pathologies [19]. In fact, during any pharmacological and/or physiokinesis and/or instrumental spasticity treatment, we studied therapeutic efficacy and we monitored the desired clinical effects without any alterations either in the omolateral antagonist muscles or in the contralateral agonists and antagonist muscles [20]. For example, wrist and finger

control is very compromised after stroke; evaluating properties of the muscles responsible for these functions can help the physiatrist to realize the severity of the disability that the patient is encountering [21]. Instead, evaluation of triceps muscle tone may provide useful indications of load and balance alterations while standing and walking, with consequent repercussions on the entire musculoskeletal system [22,23].

In daily clinical practice, a number of organizational and methodological aspects must always be taken into account when planning a treatment strategy that includes the administration of botulinum toxin A [24]. These aspects include goals and treatment methods, clinical evaluation methods (MAS, myometry, FIM) injection programs (injecting muscles, injection technique, number of injection site for muscle, dose, and dilution), and other therapies (including targeted rehabilitation programs) to be integrated into the therapeutic plan [24,25]. Muscle weakness in the limbs and paresis is associated with spasticity and contributes to loss of motor dexterity and functional capacity. Spasticity, if untreated, can hinder the functional result by promoting persistent abnormal postures which produce muscular-tendon contractions and bone deformities [26]. Moreover, spasticity complications include movement impairment, difficulty in managing hygiene and self-care, poor self-esteem, body image alteration, pain, and pressure ulcers [26,27].

This study highlights the safety of high dose treatment with IncobotulinumtoxinA [28]. Few (less than 5%) and of no clinical relevance were the adverse events found. With different and high doses particularly, we obtained a safe and effective treatment for patients with chronic upper and lower limb spasticity following brain injury; using doses above 400 U enables treating a greater number of muscles and clinical spasticity patterns, resulting in increased improvements of muscle tone, goal attainment, and global efficacy, without compromising patients' safety or tolerability [13,28]. High IncobotulinumtoxinA dosage offers the potential for comprehensive, well-tolerated and effective spasticity treatment of more clinical patterns, which allows greater focus on patients' needs and goals with respect to lower doses in chronic spasticity [14,15,29].

Finally, our study also shows that repeated and long-term treatment (two years) with IncobotulinumtoxinA does not lead to any reduction in clinical efficacy due to antibodies forming against the active substance and/or excipients of the pharmacological preparation [30]. In fact, the possible development of botulinum toxin A antibodies related to injection frequency and dosage is a source of variability for possible adverse events [24]. In this study, several sets of high dose toxin exclude the adverse events and the development of antibodies against the toxin stimulated by higher doses. Certainly, using a highly purified botulinum toxin A formulation (IncobotulinumtoxinA), free from complexing proteins, is associated with a relatively low risk of immunogenicity and represents a therapeutic advantage for a long-term treatment with higher doses [13–15,29,30].

#### **4. Conclusions**

Botulinum toxin chemo-denervation has become popular because it is effective due to its local selectivity and its effects are repeatable and safe without important adverse events or the development of antibodies. To perform and optimize the treatment, an accurate clinical and instrumental evaluation of spasticity is needed to determine how this symptom is invalidating and to choose the best doses, muscles, and times of injection in each patient. Considering larger Therapeutic Index and Therapeutic Window (from 100 to 1000 UI) of IncobotulinumtoxinA, we can better modulate spasticity by considering its clinical significance for each patient. Therefore, we could introduce the concept of an "appropriate treatment" instead of a "standard or high dosage treatment"; this allows us to underline the actual clinical needs of each patient.

#### **5. Materials and Methods**

This was a retrospective observational study lasting two years. One hundred and twenty adult patients (Table 1) with spasticity due to ischemic/hemorrhagic stroke, multiple sclerosis, spinal cord injuries, traumatic brain injury, and other cerebrovascular disorders were recruited. All subjects gave their informed consent for inclusion before participating in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol number of the Ethics Committee was 5590. All patients signed the informed consent form. Exclusion criteria were: age above 80 or below 18, muscle fibrosis/tendon retraction detected by ultrasound, concomitant treatment with other muscle relaxants, peripheral myopathy/neuropathy, cognitive deterioration (Mini-Mental State Examination ≥ 24) and positive history of allergy to the drug.

#### *5.1. Study Design*

Patients were initially divided into three groups (data homogeneity are in Table 3) according to the botulinum toxin A dosage used at the beginning of study (IncobotulinumtoxinA, Xeomin®, Merz Pharma, 100 U/mL in normal saline):


The doses were chosen depending on the severity of spasticity clinically evaluated and on the number of muscles treated.

**Table 3.** Patient demographics and baseline characteristics in the three groups; data demonstrate homogeneity of the samples.


Treated muscles were Biceps Brachii, Brachioradialis, Triceps Brachii, Superficial FlexorumDigitorum, Ulnar FlexorumCarpis and Opponenspollicis for upper limb, Rectus Femoris, Biceps FemorisAdductor Magnus, Tibialis Anterior, Flexor Hallucis Longus, Gastrocnemius Medialis and Lateralis, Soleus, Tibialis Posterior and Flexor Digitorum for lower limb, with different average dosage (see Tables 4 and 5).

**Table 4.** This table shows how many patients of each group had a specific muscle treated and its percentage. In the first group, soleus was injected in 100% of patients; in the second group, the most treated muscle was biceps brachii, while in the third one they were triceps surae muscles.



**Table 4.** *Cont.*

**Table 5.** Minimum, Maximum, Average ± SD dosage of each muscle in the three groups at the beginning of the study. In the first group, soleus received the highest dose compared to the other muscles (average 73.3 ± 2.5 UI). In the second group, muscles with highest doses were biceps brachii, superficial flexorum digitorum, gastrocnemius medialis, gastrocnemius lateralis, soleus, and tibialis posterior. In the third group, muscles with high doses were adductor magnus and those listed in the previous group.


During the study, patients received rehabilitation (stretching of injected muscles, active and passive limb mobilization, walking training, and global muscle strengthening) daily for the first 30 days after injection, then followed by three days a week until the next injection.

#### *5.2. Outcome Measures*

The evaluation method applied included the Functional Independence Measure (FIM, an international standard of disability measurement that differentiates motor from cognitive impairment; in our study we considered only motor impairment [31,32]) and myometric measurement (MyotonPRO®, tool that determines an objective value of muscle tone, elasticity and stiffness); furthermore, we took into consideration (muscle) tone values of superficial muscles [33,34]).

All assessments for each patient were performed at recruitment (during the 1st injection session), at every injection session, and during follow ups (one month after each session).

#### *5.3. Statistical Analysis*

Statistical analysis was carried out using the IBM SPSS Statistics program for Windows. Myometric measurements were analyzed with TWO WAY ANOVA method while FIM measurements with the t-student test. The alpha level for significance was set at *p* < 0.05. Data are expressed as average. In group A and group B we considered two patient subgroups according to dosage used at the beginning and the end of study.

**Acknowledgments:** We did not receive sources of funding, grants or funds for covering the costs to publish in open access.

**Author Contributions:** Giancarlo Ianieri and Riccardo Marvulli conceived and designed the experiments; Riccardo Marvulli and Giulia Alessia Gallo performed the experiments; Giancarlo Ianieri, Marisa Megna, Pietro Fiore analyzed the data; all the authors wrote the paper.

**Conflicts of Interest:** The authors declare no conflict 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* **Botulinum Toxin in the Field of Dermatology: Novel Indications**

#### **Yoon Seob Kim, Eun Sun Hong and Hei Sung Kim \***

Department of Dermatology, Incheon St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea; kysbbubbu@hotmail.com (Y.S.K.); dmsun99@gmail.com (E.S.H.)

**\*** Correspondence: hazelkimhoho@gmail.com; Tel.: +82-32-280-5105; Fax: +82-32-506-9514

Academic Editor: Siro Luvisetto Received: 20 November 2017; Accepted: 14 December 2017; Published: 16 December 2017

**Abstract:** Since its approval by the US Food and Drug Administration in 2002 for glabellar wrinkles, botulinum toxin (BTX) has been widely used to correct facial wrinkles. As a result, many consider BTX synonymous with cosmetic dermatology. Recent studies indicate that BTX elicits biological effects on various skin cell types via the modulation of neurotransmitter release, and it seems that BTX has a wider zone of dermatologic influence than originally understood. Clinicians and researchers are now beginning to explore the potential of BTX beyond the amelioration of facial lines and encouraging results are seen with BTX in a variety of skin conditions. In this paper, we review novel dermatological indications of BTX which includes (but not limited to) scar prevention, facial flushing, post-herpetic neuralgia and itch. These areas show great promise, but there is definite need for larger, double-blinded, randomized control trials against established treatments before BTX becomes a clinical reality.

**Keywords:** botulinum toxin; biological effect; various cell types; neurotransmitter; dermatology; novel indication

#### **1. Introduction**

Botulinum toxin (BTX) is a potent neurotoxin produced by the bacterium *Clostridium botulinum.* Seven distinct isoforms (BTX-A, B, C, D, E, F, and G) have been described, with BTX-A and BTX-B being commercially available. BTX blocks the release of acetylcholine and a number of other neurotransmitters from presynaptic vesicles by deactivating SNARE proteins and has a long history of therapeutic application in neurological conditions with a strong efficacy and safety profile. As widely known, the skin interacts with the nervous system and there is increasing evidence that the neurological system directly participates in cutaneous inflammation and wound healing [1,2]. With that said, BTX has been used experimentally in a number of dermatological conditions which include scar prevention, facial flushing, post-herpetic neuralgia and itch with good results. The general mechanism which underlies these novel indications includes suppression of mast cell activity, and the inhibition of substance P, calcitonin gene-related peptide (CGRP) and glutamate release. In this review, we analyze the possible off-label applications of BTX based on published data.

#### **2. Off-Label Use of BTX in Dermatology**

#### *2.1. BTX in Hypertrophic Scar Treatment*

Scars are defined as marks that remain after the healing of a wound. They cause significant cosmetic concern, especially when located on conspicuous areas such as the head and neck. Hypertrophic scars and keloids represent an aberrant response to the wound healing process and are characterized by dysregulated growth and excessive collagen formation [3].

BTX has been reported as a treatment measure for hypertrophic scars and keloids in a number of studies [4–7] (Table 1). In one study [4], BTX injection (2.5 IU/cm3) was performed once a month for three months, leading to a significant decrease in erythema, itching, and pliability of the scar. In another study [7], 12 keloid patients received BTX injection (70–140 IU per session, every 3 months for a maximum of 9 months) and achieved more than 50% improvement in symptoms, size, height, and induration of the scar. In a randomized controlled trial (RCT) [5], the efficacy of BTX (5 IU/cm3, 3 sessions, repeated every 8 weeks) was compared with that of steroid injection (triamcinolone, kenacort 10 mg/cc, 6 sessions, repeated every 4 weeks) in keloids, where BTX led to a more significant reduction of subjective complaints (itch and pain of the scar).

The molecular mechanism of BTX on hypertrophic scars and keloids is not yet perfectly explained, but BTX has been shown to inhibit the proliferation of fibroblasts derived from hypertrophic scar tissues. In addition, BTX is reported to suppress the expression of transforming growth factor (TGF)-β1, collagen I and III, α-smooth muscle actin and myosin II protein in keloid fibroblasts [8–11].

One particularly favorable aspect of BTX is its ability to control the subjective symptoms of hypertrophic scars. BTX can immobilize the local muscles of a scar and reduce skin tension caused by the muscle pull [12]. This relieves trapped nerve fibers in keloids, neutralizing the itch and pain associated with small-fiber neuropathy [13]. Another advantage of BTX is the absence of skin atrophy and telangiectasia which is often seen after steroid injection.

The limitations of BTX on hypertrophic scars and keloids would be the high cost of the drug (with the dosages mentioned in prior studies) and its potential effect on the surrounding muscles. Due to these limitations, many suggest the use of BTX as an adjuvant rather than first line treatment for hypertrophic scars.

#### *2.2. BTX in Scar Prevention*

Nowadays, many acknowledge the role of active scar prevention important in post-operative scar management. A key factor that determines the final cosmetic appearance of a surgical scar is the tension that acts on the wound edges during the healing phase [14,15]. By blocking acetylcholine neurotransmitter release from peripheral nerves, BTX allows near-complete elimination of dynamic muscle tension on the healing wound. The tension relieving properties, together with the direct inhibitory effects of BTX on fibroblasts and TGF-β1 expression support its usage in surgical scar prevention [16–18]. The anti-inflammatory effect of BTX and its action of the cutaneous vasculature calms down the inflammatory phase (immediate to 2–5 days) of the wound healing process which may also contribute to scar prevention.

A number of studies have reported the effectiveness of BTX in scar prevention [19–22] (Table 2). In a split-scar RCT [19], the safety and efficacy of early postoperative BTX injection was assessed in 15 thyroidectomy scar patients. A single treatment with either BTX (20–65 IU) or 0.9% saline (control) was applied to fresh scars (within 10 days of thyroidectomy), where the BTX-treated halves showed a significantly better outcome in terms of scar scales and patient satisfaction compared to the saline treated sides. In 2006, Gassner [21] tested whether postoperative injection of BTX improved facial scars following forehead lacerations and excisions. BTX (15–45 IU) was injected to post-op scars within 24 h after wound closure to produce enhanced wound healing and improved cosmesis compared to placebo (normal saline) injection.

BTX is best used for op scars. It would be optimal to inject BTX intraoperatively or shortly (preferably within days) after the surgery. To note, BTX should be avoided in open wounds as it delays wound closure.





#### *2.3. BTX in Rosacea and Facial Flushing*

Rosacea is a common inflammatory dermatosis characterized by persistent erythema, telangiectasia, papules, pustules, and facial flush. Oral medication, topicals, and laser therapy are routinely performed but often fail to relieve the facial flush. Persistent facial flushing is also a troublesome menopausal symptom.

A number of reports demonstrate the possible action of BTX on rosacea and menopausal hot flashes [23–26] (Table 3). In a prospective pilot study [23], the effect of BTX on the Dermatology Life Quality Index (DLQI) of patients with facial flushing was examined. BTX was injected once up to a total dose of 30 units on the cheeks which led to a significant decrease in DLQI at 2 months follow-up. Odo et al. [26] reported BTX (6.2 IU of abo-BTX per injection point, 40 points over the face, chest, neck, and scalp) to significantly reduce the mean number of menopausal hot flashes at day 60. The effect of abo-BTX was also investigated in 15 patients with rosacea. 15–45 IU of BTX was injected to the face which resulted in a statistically significant improvement in erythema at 3 months follow-up [24]. Adverse effects were rarely reported in the studies.

One possible mechanism by which BTX improves flushing is the potent blockade of acetylcholine release from autonomic peripheral nerves of the cutaneous vasodilatory system [27,28]. It is also well-known that BTX inhibits the release of inflammatory mediators such as substance P and calcitonin gene-related peptide (CGRP) [29]. The reduction and control of local skin inflammation may allow the erythema to fade out.

Larger, controlled, randomized studies are warranted to determine the optimal dose and duration of BTX activity on rosacea and facial flushing. BTX injection for facial flushing has additional benefits as it also improve the fine lines and wrinkles by diminishing the pull of the facial depressors.

#### *2.4. BTX in Postherpetic Neuralgia*

Postherpetic neuralgia (PHN) is the most frequent chronic complication of herpes zoster and the most common neuropathic pain resulting from infection. It is conventionally defined as dermatomal pain (usually a score of 40 or higher on a Likert scale ranging from 0-no pain to 100-worst possible pain), persisting at least 90 days after the appearance of the acute herpes zoster rash. PHN causes considerable suffering and results in a health care burden at both the individual and societal levels [30].

Treatment approaches include nonsteroidal anti-inflammatory drugs, gabapentin, opioids, and tricyclic antidepressants as well as topical anesthetics and capsaicin cream, but pain can be resistant to all of these drugs.

A number of reports have been made on the efficacy of BTX in PHN [31–33] (Table 4). Xiao et al. [33] performed a randomized, double-blind, placebo-controlled study on 60 PHN patients with the following arms: the BTX group, the 0.5% lidocaine group, and the 0.9% saline group. All patients were treated once (as for BTX, a dose of 200 IU at maximum), and were followed-up for 3 months. The BTX treated patients were found to have the most significant improvement in Visual Analog Scale (VAS) and sleep quality compared to those of the other two groups. Apalla et al. [32] also performed a RCT where 30 PHN patients received either BTX (200 IU in total) or placebo. BTX patients showed a significant reduction in VAS pain scores as well as the sleep scores which lasted for approximately 16 weeks. In a prospective study, Ding et al. [31] treated 58 PHN with BTX (50 to 100 IU in total) to find promising results (reduced frequency of pain attacks, lower pain severity, reduction in the quantity of painkillers consumed by patients) with very few adverse reactions.

The mechanism involved in the pain-relieving effect of BTX is still unclear, but it is thought that both the peripheral and central mechanism play a role [33]. The peripheral effects of BTX injection come through the inhibition of neuropeptide release from the peripheral nociceptive nerves [34,35]. In addition, BTX has been suggested to exert central nervous system (CNS) effects through axonal transport to the CNS after peripheral application [36,37].

Although promising, cost would be one of the main considerations to BTX use in PHN. Also, unlike other therapeutic modalities, BTX induces antitoxin antibodies which can limit the clinical effectiveness of the drug after repetitive, long-term use.


**Table 3.** Representative studies of BTX in rosacea and facial flushing.

Onabotulinumtoxinunlessotherwise stated,Abo-BTX:Abobotulinum



#### *2.5. BTX in Pruritus*

Pruritus (also known as itch) is an unpleasant sensation of the skin leading to the desire to scratch. Among the 4 subtypes (pruriceptive, neurogenic, neuropathic, and psychogenic itch), the pruriceptive itch is a peripherally induced pruritus arising from the skin and mucosa and is often seen in dermatological disease.

A number of reports have been made on the efficacy of BTX in pruriceptive pruritus [38–40] (Table 5). Recalcitrant pruritus is a hallmark of lichen simplex, a localized variant of atopic dermatitis. In an open pilot study [40], BTX (abo-BTX, 20–80 IU) was injected intradermally into 5 circumscribed lichenoid lesions with recalcitrant pruritus. Within a week, all patients reported to have noticeable alleviation of itching and at 12 weeks, all were still free from the uncontrollable urge to scratch. Itch is also a common and well-recognized problem in burns [38]. Nine patients with recalcitrant itching secondary to burns were treated with BTX (dosage not specified) where the burn itch fell to 0 out of 10 in 4 weeks. The average duration of symptom free period was reported as nine months.

As clinical evidence has revealed the antipruritic effect of BTX, Arendt-Nielsen et al. [39], investigated the effect of subcutaneous administration of BTX on experimentally histamine-induced itch in human skin. In this double-blind, placebo-controlled study, 14 healthy men received BTX and isotonic saline on the volar surface of either forearm. Histamine prick tests were performed four times at the treatment sites (before treatment, and days 1, 3, and 7 after treatment) where BTX reduced the histamine-induced itch intensity, and itch area compared with saline at all time points.

Several possible mechanisms can be responsible for the reduction of pruriceptive itch. Acetylcholine mediates itch in pruritic skin conditions such as atopic dermatitis [41] and BTX inhibits the release of acetylcholine from presynaptic vesicles [42]. BTX is also known to interact with molecules associated with itch and flare such as substance P (releases histamine via the activation of mast cells, promotes vasodilation) and CGRP (a potent vasodilator) [36,43–45]. BTX inhibits the release of such mediators, thus reducing the sensation of itch. Lastly, BTX has also been shown to stabilize mast cells and inhibit their degranulation [46].

Pruritogenic pruritus is usually accompanied by skin inflammation. Since BTX is capable of reducing neurogenic inflammation [29], it is natural to expect improvement of the primary skin disease (e.g., atopic dermatitis, psoriasis) as well, which has in fact been reported through animal studies [47,48], human studies [49] and case reports [50,51]. Although promising in pruritogenic itch (and also in inflammatory dermatoses), we feel that BTX would be best used as an adjunct to conventional therapy. It should be applied focally, considering that the product can induce muscle weakening.

#### *2.6. BTX in Dermatological Conditions Associated with Hyperhidrosis*

A number of skin disease are caused by and/or have symptoms that are exacerbated by hyperhidrosis, a condition that can be treated successfully with BTX.

Pompholyx or dyshidrotic eczema is a common vesiculo-bullous disease of the palms and/or soles. A hallmark of this disease is its tendency to relapse in response to various provoking factors which includes wet work, occlusion and hyperhidrosis. An intra-individual study of 10 patients [52] (Table 6) investigated the use of BTX (mean dose of 162 IU per palm) for pompholyx, using the using the untreated site as control. 70% of patients reported a marked improvement of both sweating and itching on the treated site after 6 weeks. In another side-by-side trial [53], dyshidrotic hand eczema was treated with BTX (100 IU per palm) as an adjunct to topical steroids. Six patients who completed the study were found to have improved symptoms of pompholyx and reduced number of relapses by BTX injection.

The anhidrotic effect of BTX in pompholyx can be explained by its action on smooth muscles surrounding the sweat glands and through the inhibition of acetylcholine release. Inhibition of substance P release also explains the reduction in pruritus [54,55].

Hidradenitis suppurativa (HS) is a chronic inflammatory dermatosis of the apocrine glands which typically affects the axillae and groin. Patients afflicted by HS have severe discomfort and treatment is extremely challenging. It is well-known that a moist environment in folds, especially in the axilla and groin, provides ideal conditions for the flourishing of bacteria and is a precipitating factor of HS.

In 2005, HS on the axillae was first reported to be successfully treated with BTX (abo-BTX, 250 IU) with 10 months of complete remission [56] (Table 6). Khoo et al. [57] also confirmed the efficacy of BTX in HS where a 46-year-old woman with Hurley stage 2 HS responded well to axillary BTX treatment (50 IU per side) with a remission period of 12 months. The patient had been recalcitrant to conventional treatments and also underwent surgical drainage.

The exact mechanism by which BTX affects the disease process in HS is unclear but it is likely that the effect of BTX on sweat production reduces the population of skin flora and its potential inflammatory effect [56,57]. A second hypothesis is that by inhibiting apocrine secretion, BTX prevents the rupture and spread of follicular material from the pilosebaceous unit [57].

BTX has been studied in inverse psoriasis (Table 6) which is also thought to be exacerbated by excessive sweating. A pilot study of 15 patients with flexural psoriasis [49] showed that 50–100 IU of BTX improved subjective symptoms and objective photographic evidence of disease in 87% of patients at 2, 4, and 12 weeks follow-up. It is hypothesized that the beneficial effects of BTX in inverse psoriasis is largely due to the reduction of local sweating in folds [49]. Patients with psoriasis are also known to have a higher concentration of substance P receptors in their skin [58,59], meaning that BTX can reduce pruritus and vasodilation by inhibiting neuropeptide liberation (and preventing substance P binding to multiple receptors).

Hailey–Hailey disease is an autosomal dominant acantholytic disorder with mutation of the *ATP2C1* gene, clinically manifesting as macerated flexural erythema. Heat and sweat aggravate the disease, worsening the discomfort and pruritic symptoms.

Several case reports [60,61] (Table 6) have evidenced improvement of Hailey–Hailey disease with the use of BTX (50–125 IU per side). In one study, the effect of BTX was found to be comparable to that of laser ablation and dermabrasion [61].

BTX can rationally ameliorate the symptoms of Hailey–Hailey disease via its inhibition of acetylcholine and substance P release from the nerve endings [54,55] (Table 6). Although more clinical evidence is needed to prove effectiveness, BTX may be considered as a possible treatment modality for Hailey–Hailey disease recalcitrant to conventional treatment.

#### *2.7. BTX in Oily Skin*

Sebum contributes to the delivery of fat-soluble antioxidants to the skin surface and has antimicrobial activity, thereby functioning as a skin barrier. However, excess sebum blocks the pores, provides nourishment to bacteria, and can result in skin inflammation (e.g., acne, seborrheic dermatitis).

Recently, insights into the effect of BTX on sebum production have been published [62,63] (Table 7). Min et al. [62] randomly assigned 42 volunteers with forehead wrinkles to receive 10 or 20 units of BTX, which was administered in five standard injection sites. Treatment with BTX exhibited significant sebum reduction at the injection site of both groups, with a sebum gradient surrounding the injection point. The efficacy did not improve significantly with higher injection doses and the sebum production recovered to normal levels at 16-week follow-up for both treatment groups. Rose and Goldberg [63] also evaluated the safety and efficacy of BTX on the oily skin of 25 subjects. A 10-point injection was made with BTX (abo-BTX, total amount of 30-45 IU) on the forehead to find significantly lower sebum production and high patient satisfaction.

The mechanism by which intradermal BTX injection results in decreased sebum production is not entirely clear because the role of the nervous system and acetylcholine on sebaceous glands is not well defined. However, it is most likely that the arrector pili muscles and the local muscarinic receptors in the sebaceous glands are targets for the neuro-modulatory effects of BTX. Li et al. [64] demonstrated that nicotinic acetylcholine receptor α7 (nAchRα7) is expressed in human sebaceous glands in vivo, and acetylcholine signal increased lipid synthesis in vitro in a dose-dependent manner. Further study is needed to determine the best candidates, optimal injection techniques and doses.



 Onabotulinum toxin unless otherwise stated, Abo-BTX: abobotulinumtoxin, VAS: Visual Analogue Scale.


**Table 6.** BTX in dermatologic disease associated with hyperhidrosis.


**Table 6.** *Cont.* BTX: Onabotulinum toxin unless otherwise stated, Abo-BTX: Abobotulinumtoxin, VAS: Visual Analogue Scale.

Final cosmetic results were comparable.



BTX: Onabotulinum toxin unless otherwise stated, Abo-BTX: abobotulinumtoxin.

#### **3. Conclusions**

In this review, we highlighted the promising outcomes of BTX in several off-label indications of interest for dermatologists. There is overwhelming evidence that BTX exhibits biological effects on many human cell types, but much is yet to be learned about the drug and its mechanism of action. Knowing that the skin closely interacts with the nervous system, future studies should investigate the link between BTX and the cutaneous neuroimmune system to better understand its therapeutic potential in dermatology. A consensus on the dose regimen and injection technique is also desirable for standardized treatment. Generally, high doses of BTX were applied, with an average total of 300 IU for hypertrophic scars, 50 IU for scar prevention, 50–100 IU for facial flush/rosacea, 100 IU for PHN, 150 IU for pompholyx, 100 IU for HS, 75 IU for inverse psoriasis and 250 IU for Hailey-Hailey disease. Lastly, with the limitations of BTX treatment (high cost, muscle weakening, risk of tachyphylaxis and production of antibodies), BTX may be optimally used as an adjunct in recalcitrant cases to conventional therapy.

**Acknowledgments:** This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (Grant No.: 2017R1C1B5016144) and the 2016 Amore-Pacific grant.

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

#### **References**


© 2017 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* **Antipruritic Effects of Botulinum Neurotoxins**

#### **Parisa Gazerani**

Department of Health Science and Technology, Aalborg University, Frederik Bajers Vej 7A2, A2-208, 9220 Aalborg East, Denmark; gazerani@hst.aau.dk; Tel.: +45-9940-2412

Received: 5 March 2018; Accepted: 27 March 2018; Published: 29 March 2018

**Abstract:** This review explores current evidence to demonstrate that botulinum neurotoxins (BoNTs) exert antipruritic effects. Both experimental and clinical conditions in which botulinum neurotoxins have been applied for pruritus relief will be presented and significant findings will be highlighted. Potential mechanisms underlying antipruritic effects will also be discussed and ongoing challenges and unmet needs will be addressed.

**Keywords:** botulinum neurotoxins; itch; pruritus; antipruritic; clinical; experimental

**Key Contribution:** This review highlights the potential utility of BoNTs to relieve pruritus. Presented information; discussion of limitations and promising results are valuable for both researchers and clinicians in the field.

#### **1. Introduction**

Botulinum neurotoxins (BoNTs) are protein neurotoxins that are produced by anaerobic, spore-forming bacteria of the *Clostridium* genus, including *Clostridium botulinum*, *Clostridium butyrricum*, *Clostridium barati,* and *Clostridium argentinensis* [1]. An updated review of biology, pharmacology, and toxicology of BoNTs can be found in M. Pirazzini et al.'s excellent recent review [2]. Since its first therapeutic use in humans in the 1980s [3], clinical use of BoNTs has significantly increased [4]. BoNTs have been used for therapeutic purposes in a diverse range of medical conditions, such as ophthalmology, neurology, gastroenterology, urology, and psychiatry [2,4–6]. Advanced understanding of the mechanism of action of BoNTs has led to increasing use of these molecules with novel, unique, and desirable pharmacological properties [2]. BoNTs have also been tested in many dermatological conditions, several of which with off-label uses [7,8]. This review focuses on the antipruritic effects of BoNTs as presented in clinical and experimental conditions which specifically addressed potential itch-relieving mechanisms. The aim is to highlight the value of BoNTs in an expanded evaluation as potential antipruritic agents in future practice. It is worth mentioning that one case [9] in the literature presented that an application of OnabotulinumtoxinA (100 U) provoked itchiness in a patient when it was used for neuromuscular pain. In that instance, pruritus was treated with oral hydroxyzine and camphor-menthol topical lotion [9].

#### **2. Pruritus**

Itchiness (pruritus) is a common unpleasant sensation that elicits a desire to scratch [10]. Acute itch [11] serves as a warning and self-protective mechanism to prevent potentially harmful irritations. However, chronic itch is a challenging and significant clinical problem [12] which is often associated with skin diseases, systemic diseases, metabolic disorders, and psychiatric disorders [13]. Although scratching temporarily relieves acute itch, persistent itch-scratch cycles often exacerbate skin problems, disrupt sleep, and reduce the quality of life in chronic itch patients [14]. Recent studies have documented a high prevalence of chronic pruritus (13–17%) with a lifetime prevalence of 22–26% [15]. On the basis of international consensus, a threshold of six weeks has been set for the definition of

chronic pruritus. Chronic pruritus is a major challenge to overcome [16] and requires interdisciplinary cooperation despite many interfering factors. Successful treatment usually involves dermatologists, internists, general practitioners, neurologists, gynecologists, and psychiatrists [16]. An understanding of the molecular mechanisms underlying itch has advanced the identification of itch-specific pathways and transmitters for selective targeting [17].

Itch can broadly be categorized as either histaminergic and non-histaminergic [18]. Histamine is released by mast and epithelial cells and binds to H1–H4 receptors, leading to the activation of downstream target molecules within sensory neurons [17,19,20]. Histamine has been identified as the main mediator of itch in several pruritic conditions, such as urticaria and allergic diseases. Antipruritic treatment strategies [12] have been successfully used to target histaminergic pathways [21].

Non-histaminergic itch has attracted more attention in recent years because many chronic itch conditions are resistant to antihistamines, necessitating the need to consider alternative treatments. Cowhage (*Mucuna pruriens*) is a tropical legume known to cause itch, pricking, stinging, and burning sensations that otherwise do not respond to antihistamines. This characteristic has made cowhage a useful tool for studying non-histaminergic mechanisms underlying itch. When cowhage spicules are inserted, the cysteine protease mucunain is released. When the mucanin reaches the nerve endings of primary sensory neurons in the epidermis, it activates protease-activated receptors (PAR) 2 and 4, which are members of the G protein-coupled receptor family [22]. Interestingly, PAR2 and tryptase (an endogenous PAR2 agonist, the most abundant secretory granule-derived serine proteinase released by mast cells causing itch), have been found highly elevated in patients with atopic dermatitis (AD) [23]. Mas-related G protein-coupled receptors (Mrgprs) are involved in the response to non-histaminergic pruritogens [24]. MrgprA3 is expressed in a sub-population of sensory neurons known as peptidergic C-fibers and encodes pruritic effects of chloroquine, which is an antimalarial drug [25,26]. These receptors [27,28] are also responsive to histamine, bovine adrenal medulla 8–22 (BAM8–22), cowhage spicules, and capsaicin. Interestingly, mice lacking MrgprA3 neurons are resistant to pruritogens such as histamine, BAM8–22, SLIGRL, α-methyl-5HT, ET-1, and chloroquine [26]. However, MrgprA3-ablated mice scratch in response to β-alanine [29]. Therefore, MrgrpA3 positive sensory neurons are different from those neurons responding to β-alanine for itch. MrgprD is the receptor activated by β-alanine [30], and mice lacking MrgprD do not scratch following an intradermal injection of β-alanine [29]. Transient receptor potential (TRP) channels are also involved in itch. Histaminergic itch transmission through TRPV1 has been reported [31]. TRPA1 is a downstream target of MrgprA3 and MrgprC11. Ablation of TRPA1 blocks itch in a dry skin mouse model of chronic itch. Mice lacking TRPA1 exhibit no scratch following subcutaneous injections of chloroquine and BAM8–22, but do scratch in response to α-methyl-5HT [17,32]. Voltage-gated sodium channel (NaV) 1.7 has also been found to mediate itch. A monoclonal antibody targeting NaV 1.7 could abolish both acute and chronic itch in mice [33].

Immune cells of skin interact with nerve endings and play an important role in pathological itch. Cytokines released from T helper 2 cells are found elevated in several pruritus conditions [34]. Interleukin-31 (IL-31) is known to play a role in AD [35]. Intradermal injection of IL-31 in mice provokes scratching [36] that is also correlated with elevated expression of IL-31RA in the DRG (dorsal root ganglion) [37].

Taken together, novel findings on peripheral receptors and mediators [38] present that itch is mediated by several different subpopulations of primary sensory neurons. Some itch-provoking substances activate overlapping populations of neurons, while others only activate distinct populations (e.g., chloroquine vs. β-alanine; MrgprA3 vs. MrgprD). Depending on pruritogen, method of delivery, and species, itch-related responses are variable [17]. One must also consider that a cross-talk exists between the neurons and immune cells in the skin [38].

In addition to advancements in peripheral mechanisms of itch, several hypotheses have been proposed for central mechanisms of itch [17,38]. Gastrin-releasing peptide (GRP) and GRP receptors were the first central components of itch identified in the spinal cord [39]. Ablation of inhibitory interneurons (B5-I) in mice resulted in the development of skin lesions and scratching in mice [40]. B5-I interneurons are activated following certain transient receptor potential (TRP) channels signaling and release dynorphin that can block itch signaling [41]. Spinal interneurons that express neuropeptide Y also exist which mediate mechanical itch [42]. Cross-talk between neurons and central glia has also been suggested in modulating itch [43]. For example, in a mouse model of contact dermatitis and AD, spinal reactive astrogliosis has been reported [44]. Toll-like receptor 4 has been found to contribute to this type of astrogliosis in a dry skin mouse model of itch [45]. Apart from astrocytes activation, spinal microglial activation has also been found in mouse models; for example, after intradermal injection of compound 48/80 (histamine-dependent) and 5 -guanidinonaltrindole [46]. Microglial activation can be subsided by intrathecal minocycline (a microglial modulator) which can reduce scratching and symptoms of dermatitis in a mouse model of AD [47].

In short, identification of peripheral and central components of itch [17,38] has pushed the field forward for novel and effective targeting. Several established models of itch exist which are applicable in both animals and humans. These models are useful in understanding itch pathways and also in testing novel antipruritics [48]. Many compounds are in early stages of development and several are going through final phases of antipruritic pipelines.

BoNTs were initially used for muscle hyperactivity [49]. Soon after the identification of broader biological effects (e.g., neuronal and non-neuronal effects in dermal fibroblasts, sebocytes and vascular endothelial cells), additional indications garnered attention and further mechanisms underlying BoNTs effects were proposed [50,51]. The antipruritic effect of Botulinum Toxin Type A (BoNTA) was identified in an open-label pilot study of lichen simplex in 2002 [52]. Its antipruritic effect in dyshidrotic hand dermatitis was also reported in the same year [53,54]. Since then [6], BoNTs have been subjected to investigation for many other pruritic conditions, such as Hailey-Hailey disease and inversed psoriasis. First, one must understand how BoNTs can exert potential effects against itch. An acceptable rationale for application of BoNTs in itch is that acetylcholine mediates itch and BoNTA inhibits the release of acetylcholine from presynaptic vesicles. However, other mechanisms also play a role in antipruritic effects of BoNTs. In the section below, proposed underlying mechanisms of BoNTs in reducing itch are described. Since botulinum toxin type A (BoNTA) is the most used in the current literature, the rest of this manuscript focuses on this neurotoxin unless otherwise stated.

#### **3. Botulinum Toxin Type A (BoNTA)**

BoNTA inhibits vesicular release of neurotransmitters by interfering with exocytotic release. BoNTA is composed of a heavy chain with a receptor-binding site and a translocation domain as well as a light chain with endopeptidase activity. This permits cleavage of synaptosomal-associated protein 25 (SNAP-25) which is an essential molecule for membrane fusion [55]. BoNTA was first known to block acetylcholine release at the neuromuscular junction [56]. It has been used for disorders with abnormal muscle contraction because of its ability to relax spastic muscles [49]. However, it became evident that BoNTA also inhibits the release of other transmitters, such as glutamate, substance P (SP), and calcitonin gene-related peptide (CGRP) [57]. The anti-itch effect of BoNTA is also a result of inhibition of acetylcholine release and other mediators involved in itch [8,9]. As such, evidence from analgesic properties of BoNTs in pain and nociception have also been inspirational for scientists [58–64].

#### **4. Experimental Evidence for Antipruritic Effects of BoNTA in Healthy Humans**

Our group was first to investigate the effect of subcutaneous administration of BoNTA on experimentally induced itch (histamine) in healthy subjects. Fourteen healthy men received BoNTA (5U, BOTOX®, Allergan, NJ, USA) on the volar forearm. Saline was used as control. Histamine prick tests were performed at the application sites before, one, three days, and a week after treatments. Itch intensity and neurogenic inflammation were evaluated. BoNTA significantly reduced histamine-evoked itch intensity, flare size, and vasomotor reactions to histamine [65].

Another study in healthy volunteers looked into the antipruritic effects of BoNTA in a non-histaminergic model where cowhage was used (clinicaltrials.gov; identifier: NCT02639052). In this study, 35 healthy subjects (16 men and 19 women; age 26.8 ± 6.8 years) were enrolled and intradermal BoNTA (10U, BOTOX®, Allergan, NJ, USA) was injected in a 4 × 4 cm area on volar arms. Saline was used as control. Itch intensity following application of cowhage was recorded before treatment and one week, one month, and three months post-treatment. BoNTA reduced cowhage-evoked itch at all time points, suggesting a long-lasting effect. This study was presented at the 9th World Congress on Itch [66].

#### **5. Experimental Evidence for Antipruritic Effects of BoNTA in Animal Models**

Animal studies have also been conducted to look deeper into the cellular-molecular mechanism(s) of antipruritic effects of BoNTA.

It is generally accepted that TRPV1 (transient receptor potential cation channel subfamily V member 1) is essential for histamine-dependent itch [31], whereas TRPA1 (transient receptor potential ankyrin 1) is required for histamine-independent itch, e.g., chloroquine-evoked itch, bile acids-induced cholestatic itch [67], and oxidative stress-induced itch [68,69]. A mice study [70] has investigated the effects of BoNTA on acute and chronic itch and the possible association of TRP channels to antipruritic mechanisms of BoNTA. Findings from this study demonstrated that BoNTA inhibited chloroquine-evoked itch which is considered an acute non-histaminergic model similar to that of compound 48/80-induced itch. Compound 48/80 is a potent histamine-releasing agent, primarily from mast cells, with a subsequent depletion of tissue histamine [71]. It was also presented that, following a single intradermal injection of BoNTA (0.1 U) into the nape of the neck, mRNA expression of TRPV1 and TRPA1 notably decreased in DRG and lasted for seven days. Protein expression of TRPA1 was highly elevated following AEW (acetone–diethylether–water) treatment—a dry skin itch model—and pretreatment with BoNTA could significantly abolish upregulation of TRPA1 expression in this model. Authors proposed that TRPV1 and TRPA1 play an important role in both acute and chronic itch and that BoNTA might exert its anti-itch effects through downregulated expression of TPRV1 and TPRA1 in DRG [70]. This study confirmed that antipruritic effects of BoNTA present independently of mice models and can be used both for histamine-dependent and histamine-independent itch and dry skin-induced chronic itch [70].

Another study studied AD in mice models [72]. AD is accompanied by debilitating itch and a complex interaction is believed to exist between immune cells and nerve fibers [73]. NC/Nga mouse is a relevant animal model to study AD [74] because these animals spontaneously develop AD-like skin lesions under conventional conditions. In this study [72], the authors examined the protective effect of BoNTA (intradermal injection on the rostral back) on AD lesions in NC/Nga mouse. The primary outcome was skin thickness and transepidermal water loss. Authors assessed skin thickness, water loss, skin severity scores, histological alterations of skin, e.g., mast cell count, skin interleukin (IL)-4 mRNA and protein expression, and total serum IgE levels [72].This study showed that BoNTA could significantly suppress AD severity, IL-4 expression level, and the number of infiltrating mast cells [72]. Study period was limited to 14 days and long-term effects were not investigated.

The effects of BoNTA on mast cell activity has also been studied in animal models [75,76]. In a study by Park [75], 10 Sprague Dawley rats were randomly divided into two groups receiving BoNT A and vehicle (control). A distally based 3 × 9 cm random pattern flap including the panniculus carnosus muscle was elevated. BoNTA was administered five days prior to flap elevation. Seven days after flap elevation, tissue samples (1 × 1 cm) were taken from the center of each flap. Findings [75] demonstrated that BoNTA decreased mast cell activity.

Another animal study looked into the mechanisms of BoNTA in targeting psoriasis. In a KC-Tie2 mouse model of psoriasis [77], researchers showed that intradermal injection of BoNTA improved psoriasiform skin inflammation and epidermal hyperplasia. It also decreased the number of infiltrating CD4<sup>+</sup> T cells and CD11c<sup>+</sup> dendritic cells (DCs) in parallel with reducing the number of blood vessels and their adjacent nerves [77]. The decreased number of blood vessels within the affected skin of the treated mice illustrates the role of nerves and blood vessels in an inflammatory skin disease such as psoriasis. This study illustrates the role of blood vessel and nerve communication in psoriasis and the potential role of BoNTA in blocking this communication. Authors proposed that the persistence of some plaques in psoriasis patients might be explained by local microenvironments within the tissue, including nerve-derived SP and CGRP [77]. BoNTA, a known inhibitor of CGRP and SP release, can help with the interruption of this cascade and may present significant improvement in disease severity as early as two weeks after treatment. Therefore, they proposed that BoNTA may serve as a supplemental agent to topical or biologic therapeutic regimens [77].

#### **6. Clinical Evidence for Antipruritic Effects of BoNTs**

BoNTs have been used in clinics for many dermatological conditions that can present with or without itch. For a review, see A. Campanati et al., Y.S. Kim et al., and A.S. Al-Ghamdi et al. [7,8,78]. A recent review has summarized the use of intradermal BoNTA in treating chronic refractory pruritus based on 11 studies between 1996 and 2016 [79].

Many applications are still off-label [7] and the cases presented below both summarize current clinical evidence and encourage additional well-designed studies to reach a consensus on safe applicability, optimal dose, and delivery route for the standardization of BoNTs use for antipruritic effects.

#### *6.1. Post Herpetic Itch*

Post herpetic itch (PHI) is considered a type of neuropathic itch and has been investigated less than postherpetic neuralgia (PHN) [80]. PHN is a long-term neuropathic pain that remains after the rash from shingles (also known as herpes zoster) has healed. Varicella-zoster virus (VZV) is the cause of herpes zoster. Besides shingles, degenerative nerve root compression (notalgia paresthetica), and sensory polyneuropathy can cause neuropathic itch [81]. Almost half of PHN patients report PHI. This finding suggests that mechanisms underlying PHI and PHN are most likely independent [82]. PHI is a common disorder that equally affects men and women. PHI is age-independent and occurs in both young and old patients. PHI often appears on the head and neck (V1 dermatome) [82].

BoNTA treatment has been successful to reverse pain in PHN [83]. Accordingly, the usefulness of BoNTA in PHI has been considered [84] and the effectiveness of BoNTA for a neuropathic itch caused by dermatomal damage to the thoracic nerves has been presented [84]. In this study, BoNTA injections (dose range 16–25 U) were given in several points within the involved dermatome. Double-blind, randomized, control trials are required in a larger sample size before the use of BoNTA in different types of neuropathic itch such as PHI can be considered. BoNTs could be considered in severe cases of intractable PHI, which are not responsive to other options [80].

#### *6.2. Brachioradial Pruritus*

Brachioradial pruritus (BRP) was first described in Florida in 1968 by Waisman [85] and is classified as a deep itch of the forearms and upper trunk which can worsen with either scratching or sunlight [86]. Brachioradial pruritus is considered another neurogenic itch which often occurs in the upper extremities, usually localized on the dorsolateral forearm overlying the proximal head of the brachioradialis muscle; however, upper arms and shoulders may also be affected [87,88]. BRP might be unilateral or bilateral and it is still considered a common "tropical" dermopathy [89]. It is still not known if BRP is a symptom of neuropathy, similar to chronic cervical radiculopathy, or a condition that occurs secondary to chronic ultraviolet damage. Larger studies for better understanding of BRP are warranted. BRP responds to ice packs but efficacy is only temporary [90]. Lamotrigine and gabapentin have also been found useful for BRP. Intradermal injections of BoNTA (100 IU) was reported in a 59-year-old Caucasian female with BRP for 12 years [86]. This patient had disabling itch and a burning sensation on the upper posterior arms, scapular regions, and neck. A diverse range of topical and systemic treatments, hypnotherapy, and Chinese herbal medicine did not improve the patient's condition. Application of icepacks was not beneficial in this case. However, this patient reported dramatic itch relief, lasting for up to six months, after four rounds of BoNTA injections. It was proposed that the effect of BoNTs for this condition and those similar to it, may have been due in part to its ability to block the release of neurotransmitters involved in itch, e.g., acetylcholine.

#### *6.3. Notalgia Paresthetica*

Notalgia paresthetica (NP) [91,92] is a sensory neuropathic syndrome with pruritus, pain, paresthesia, hypo-hyper-esthesia, and burning as common symptoms. NP is characterized by a brownish itchy patch in the affected area. This condition mainly occurs in the elderly or in association with musculoskeletal disorders driven by spinal nerve compression, particularly at the C4–C6 level [92]. NP is a difficult condition to treat and quality of life is rather low in these patients. Less efficient treatments for NP are partially attributable to its unidentified underlying mechanisms or pathogenesis.

In 2007, two NP patients were treated with BoNTA [84]. Later in 2010, Wallengren and Bartosik [93] reported limited effectiveness of BoNTA treatment in six NP patients. One double-blind randomized clinical trial for NP was reported in 2014 [94], in which the effectiveness of BoNTA was tested in 20 NP patients who were resistant to topical therapies. The study investigated pruritus, effects on hyperpigmentation, and global effectiveness as rated by both patients and investigators. Pruritus rated on VAS (visual analogue scale) did not show any itch reduction when it was compared between patients and controls (receiving saline) [94]. BoNTA treatment also did not improve hyperpigmentation or global efficacy indicators. In this study, injections of 0.1 mL (50 U/mL) for every 1–2 cm2 of hyperpigmented area were given. Maximum dose reached to 200 U [94].

Injection of BoNTA is an option, but further research is required to confirm safety and efficacy of BoNTA for NP. Patient selection and dose also need to be determined.

#### *6.4. Lichen Simplex Chronicus (LSC)*

Lichen simplex chronicus (LSC) is also known as neurodermatitis circumscripta. LSC is an eczematous dermatosis, characterized by intense localized pruritus and thickening of the skin with variable scaling arising secondarily from repetitive scratching or rubbing. This condition can be intense or recurrent and often disrupts sleep, sexual function, and quality of life in affected individuals. Breaking the itch-scratch cycle is challenging. Exact incidence in general population is unknown, but one study demonstrated that 12% of aging patients with pruritic skin presented with LSC [95]. This disorder is observed more commonly in females than in males. BoNTA has been considered an option for LSC [52]. One pilot study investigated the effect of intradermal injection of Abobotulinumtoxin A in five lesions in three patients and found that pruritus diminished within three to seven days in all patients. By four weeks, all lesions had cleared completely with no recurrences [96]. Another case study reported a successful result with BoNTA in a 55-year-old woman with a six-year history of intense facial pruritus at the right side of face [97]. Despite the small sizes of these studies, the antipruritic effects of BoNTA in LSC is promising [78]; however, additional large studies are required to confirm its efficacy in LSC.

#### *6.5. Vulvodynia*

Vulvodynia is a complex disorder [98] affecting 16% of women in the general population. It is described by burning, stinging, itching, irritation, or rawness. The International Society for the Study of Vulvovaginal Disease (ISSVD) has defined vulvodynia as "vulvar pain occurring in the absence of an underlying recognizable disease." There are no clinical or histopathologic criteria for the diagnosis other than consideration and careful evaluation to exclude other causes of pain. Successful therapy often requires a multidisciplinary approach with more than one type of therapeutic intervention. BoNTA has been shown to be effective treatment for vulvodynia [99]. While several small open-label studies have shown improvement in symptoms with botulinum toxin at doses of 20–100

units, the only randomized double-blind, placebo-controlled trial demonstrated no improvement with 20 units over placebo in 64 women with vulvodynia [100].

#### *6.6. Keloids and Hypertrophic Scars*

Keloids and hypertrophic scars are structures formed during the wound healing process and present with dysregulated growth and a high level of collagen formation. To prevent these scars, silicone dressings, laser therapy, and immune response modulators are applied [101]. Intralesional (IL) corticosteroid therapy with triamcinolone acetonide is a common therapy in keloids treatment [102]. In 2000, Gassner and colleagues [103] suggested that BoNTA injections can paralyze muscles close to wounds and subsequently reduce pressure on wound edges. This first study was conducted in a primate model and confirmed the hypothesis. Another study used optical 3D profilometry as an objective evaluation of keloids following treatment by BoNTA [104]. Only four patients were included in this study and no changes was evident on fibroblast proliferation. In a rabbit ear hypertrophic scar model [105], BoNTA also was found to have less effect on hypertrophic index, fibroblast density, and collagen density when it was compared with IL triamcinolone acetonide and 5-fluorouracil [106]. However, other in vitro and experimental animal models support BoNTA as treatment of keloids and scars [107]. BoNTA delays fibroblast growth through the inhibition of the cell cycle which subsequently reduces hypertrophic scar development. BoNTA also decreases the expression of connective tissue growth factor and inhibits the growth of fibroblasts and scar expansion. BoNTA reduces the concentration of TGF-β1 in fibroblasts and decreases the infiltration of inflammatory cells during wound healing; it also reduces fibrosis [107].

In 2015, a large randomized double-blind study tested the effect of BoNTA compared with IL corticosteroid therapy in 24 patients with keloids [108]. In this study, patients were allocated to receive IL steroid every four weeks for six sessions and IL BoNTA 5 IU/cm every eight weeks for three sessions. Hardness, elevation, and redness, together with itching, pain, and tenderness were evaluated and patients were asked for their subjective satisfaction. No significant difference was observed between groups in most of the measured parameters. However, patients receiving BoNTA reported higher satisfaction with their therapy. Authors proposed that BoNTA might have reduced small-fiber neuropathy causing itching, pain, and allodynia [108].

A potential use for BoNTA in keloids and hypertrophic scars is predicted. But additional randomized double-blind controlled trials are needed to compare with current treatments to evaluate efficacy and safety profile. Efficacy in the prevention and treatment of hypertrophic scars might vary according to the scar's location on the body; hence, testing both facial and other body parts such as chest or back is proposed. Surgical and trauma wounds must also be differentiated. In addition, stratification according to ethnicity and age is essential as both elements affect wound healing.

#### *6.7. Psoriasis*

Psoriasis is a skin disorder strongly linked to both genetic and environmental factors. An immunological reaction mediated by T lymphocytes is thought to be the main player in the pathogenesis of psoriasis. Cutaneous inflammation and keratinocyte hyperproliferation are featured characteristics of such a response.

Inverse or flexural psoriasis is a specific form of psoriasis with red, dry, smooth, and shiny skin. Clinically, inverse psoriasis manifests with sharply demarcated erythematous plaques with infiltration that accompany sensations of itching and burning. Common locations of inverse psoriasis include armpits, groin, under the breasts, and in other flexion skin folds, such as around the genitals and buttocks. It is particularly troublesome for patients with deep skin folds and/or those who are overweight. Treatment of inverse psoriasis can be difficult. Steroid creams and ointments are considered effective; however, overuse of steroids can result in side effects, especially thinning of the skin and stretch marks. Skin folds, where inverse psoriasis is common, are susceptible to yeast

and fungal infections. Topical immunomodulators, such as tacrolimus and pimecrolimus, have also been effective.

Administration of BoNTA has been proposed as a novel therapy in inverse psoriasis [109,110] in consideration of its mechanism of action in the neuroglandular junction, which reduces sweating. However, a link between high nerve fiber density in psoriatic skin and elevated CGRP and SP release has been reported. It has been demonstrated that psoriasis undergoes remission phases as a result of innervation loss or lack of nerve function, such as following a nerve injury. This can explain how BoNTA inhibits CGRP and SP release from nerve endings and can lead to subjective reports of improvement after administration of BoNTA.

Zanchi and colleagues [109] reported that in 15 patients with inverse psoriasis, BoNTA presented effectiveness; however, the effect was mainly evaluated by self-assessment in patients rating itch and pain on a visual analogue scale (VAS). In this study, psoriasis that was located in the armpits, submammary sulcus, intergluteal folds, inguinal folds, and umbilicus in patients was treated with BoNTA injections with a total dose of 50–100 U per patient relative to psoriasis extent and severity. Evaluations were performed before and after treatment in weeks 2, 4, and 12. The erythematous area was defined using objective photographic evidence and subjective patient assessment of pain and itch was assessed using a 10-point VAS. BoNTA therapy resulted in improvements in subjective patient symptomatology and objective reductions in erythema and maceration in the treated areas according to photographic evidence. However, findings from this study were questioned [111], pointing to the lack of quantitative assessment for improvement; for example, using psoriasis area and severity index (PASI) or obtaining histological evident before and after the treatment.

Overall, current evidence demonstrates that BoNTA is capable of reducing pain, itch, and inflammation in psoriasis-affected skin. Dermal and epidermal cytokines and peptides produced by keratinocytes, fibroblasts, lymphocytes, and macrophages are involved in the pathogenesis of psoriasis. Interleukin-1 (IL-1) stimulates the proliferation of keratinocytes and the production of cellular adhesion molecules which then stimulate the release of other cytokines (e.g., IL-6, IL-8). IL-6 stimulates the proliferation of B and T lymphocytes, which is an important factor in stimulating keratinocyte growth, and IL-8 exerts a powerful chemotaxis action towards leucocytes. In future randomized clinical trials evaluating the potential role of BoNTA in the treatment of psoriasis, special attention needs to be given to psoriasis as a variable pathology with several spontaneous relapses and remissions over time which can cause difficulties in evaluation of effectiveness. Amount and depth of injection are yet to be determined. Assessment of effectiveness must include both subjective and objective parameters including cutaneous sensory, vasomotor, and autonomic function. Safety, tolerability, and cost effectiveness should also be carefully evaluated before considering BoNTA as a routine clinical practice.

#### *6.8. Pompholyx*

Dyshidrotic eczema, also called pompholyx, is a common relapsing vesicular-bullous disease found on the palm or soles of feet [112]. The pathogenesis of this condition is still unresolved; however, one study examined the roles of aquaporin 3 and aquaporin 10, which are water channel proteins located in the epidermis, and concluded that overexpression of these channels may play a role [113]. Wet works, sweating, and occlusion are among the provoking factors. Pain, itch, and burning sensations together with discomfort in wearing gloves or shoes, bacterial infection, or mycosis are among the common symptoms.

An improvement in hand eczema was observed in patients with palmar hyperhidrosis following intradermal BoNTA [53]. This study was conducted in 10 patients with bilateral vesicular hand dermatitis where BoNTA injections (100 U BOTOX®) or saline (control) were given in either hand. Seven out of the 10 patients reported a good or very good effect of the treatment. Another study [54] applied topical corticosteroids on both hands in combination with intracutaneous injections of BoNTA (100 U BOTOX®) in six patients with more severely affected hands. A rapid improvement in pruritus

and vesiculation was observed in the treated hand with combination therapy. A case study [114] has also demonstrated BoNTA effects in palmar pompholyx. No placebo-controlled trial is available. Therefore, effective and safe application of BoNTA for dyshidrotic eczema requires further validation.

#### *6.9. Postburn Itch*

Itching is a common secondary symptom related to burn injuries [115]. Research has proposed multiple mechanisms underlying itch as secondary to burn conditions. Several medications have been identified and used to manage this condition. BoNTA has also been considered as an option. In 2012, a study was conducted [116] to investigate the effectiveness of BoNTA and found that 87.5% of patients rated their postburn itch as severe (>7). Following the administration of BoNTA, itch intensity dropped to zero within four weeks. The average duration of the symptom-free period was nine months (range 3–18 months). BoNTA might be an option for burn-associated itch which are resistant to conventional therapies. This study [116] only included a small sample size and larger studies are warranted before establishment of this treatment at clinic.

#### *6.10. Fox–Fordyce Disease*

Fox–Fordyce disease (FFD), characterized by intensely pruritic papules in apocrine gland-bearing regions, is a rare disorder for which there is currently no definitive treatment or known cure [117]. FFD is a chronic, pruritic disorder caused by keratin plugging of the follicular infundibulum at the distal portion of the apocrine sweat duct and less often by plugging of apoeccrine ducts. This obstruction causes apocrine sweat retention and, over time, rupture of glands with secondary inflammatory dermal alterations. The etiology remains unclear although epidemiological data support a hormonal component, as women between 15 and 35 years of age are more commonly affected and this condition may remit after menopause.

The condition is often intensely pruritic and is usually associated with hypohidrosis. Pruritus is aggravated by emotional, physical, or pharmacological stimulations that enhance sweating. Therapeutic knowledge of FFD is derived from case reports but no large case series has been carried out. Topical and intralesional corticosteroids are often a first-line therapy. In medication-refractory cases, surgical interventions have proven to be successful. A case has been presented in which BoNTA injections resulted in the disappearance of pruritus and a partial clinical response after one session [117]. This response was sustained over time. The study's authors suggested that chemodenervation of cholinergic nerve terminals to the eccrine and apoeccrine glands, inhibiting their sweat secretion, might be considered as underlying mechanism for the effects seen in this case. Other cases of hyperhidrotic pruritic axillary granular parakeratosis responders to BoNTA have also been reported [118]. Clinical trials to evaluate optimal treatment regimen with BoNTA for FFD are required.

#### *6.11. Hailey-Hailey Disease*

Hailey-Hailey disease (familial benign pemphigus) is a rare genetic skin disease, often presented with blisters or vesicles and erythematous plaques in skin folds. Axilla, groin, neck, and inframammary folds are amongst the most common sites of disease manifestation. The disease's associated red scaly areas can be itchy. The topical and oral corticosteroids, oral retinoid, cyclosporine, and methotrexate used for treatment are often linked to side effects. There are cases of Hailey-Hailey disease treated with BoNTA with successful outcome [119,120]. Reduced sweating and local irritation help to improve lesions conditions as well as a reduction in itch. BoNTA can potentially be considered as a treatment option, in particular for those patients with limited response or intolerance to other treatments [120].

#### *6.12. Rhinitis*

Rhinitis is an inflammation of the nasal mucous membranes, presented with nasal discharge, nasal obstruction, sneezing, and itching [121,122]. It is a common disease affecting around 20% of general population and can be divided into infectious, allergic, occupational, drug-induced, hormonal, and idiopathic rhinitis (IR). The latter is also known as non-allergic, noninfectious perennial rhinitis, intrinsic, or vasomotor rhinitis. BoNTs have been used in both allergic [123] and idiopathic rhinitis (IR) [124,125].

Allergic rhinitis (AR) is a noninfectious inflammatory disorder in nasal mucosa provoked by an allergen exposure and an IgE-mediated immune response. The major mediator of nasal inflammation in AR is histamine which causes symptoms such as vascular permeability, mucus secretion, and stimulation of the sensory nerve fibers. Other mediators involved are neurokinin A, SP, CGRP, VIP, and neurotrophins. Current treatments include intranasal corticosteroids, antihistamines, mast cell stabilizers, and leukotriene receptor antagonists. AR can benefit from BoNTA injection, when it is given intranasally [124]. No serious adverse or systemic effects have been noted but burning after injection, nasal dryness, and epistaxis have been recorded. Nasal injection of BoNTA has shown comparable therapeutic effect to cetirizine in AR [126]. Arguments for the use of BoNTA in rhinitis are grounded in its modulatory effect on the secretory tone, which is related to the action of autonomic nervous system. A similar rationale has been used for the positive effects of BoNTA in Frey syndrome, hyperhidrosis, and sialorrhea. AR's symptoms result from the activation of inflammatory mediators and an imbalance in the autonomic nervous system. Histamine, prostaglandin, and leukotrienes enhance vascular permeability and produce edema, in addition to altering the balance of the autonomic nervous system [127]. Underlying mechanisms of IR remain to be elucidated; however, autonomic nervous system imbalance with a dominant parasympathetic tone in the nasal mucosa has been proposed. Nasal blockage and rhinorrhea are more common in IR, while itching and sneezing are mostly present in AR. All symptoms can be prevented by application of BoNTA.

Several other mechanisms for BoNTA effect in nasal mucosa have been proposed [124]. For example, BoNTA can induce apoptosis in the nasal glands, inhibit acetylcholine release from nasal mucosa nerve endings, decrease the release of neuropeptides (e.g., VIP and SP) from the trigeminal and parasympathetic nerve endings, and inhibit acetylcholine release from preganglionic cholinergic nerve endings in the sphenopalatine ganglion. As such, it has been suggested that targeting an upstream source of parasympathetic innervation at the sphenopalatine ganglion can potentially affect both nasal mucosa and nasal glands to yield an additive effect from the intraganglionic injection. A study proposed a technique to inject BoNTA into the posterior lateral nasal wall, which is located adjacent to the sphenopalatine ganglion. They conducted a pilot study with this technique applying a low dose of 25 units, resulting in only moderate discomfort to participants but yielding safe and effective results. Improved effects on congestion and itch had already been seen with dosages above 12 units. Accordingly, the authors suggested that low dose administration of BoNTA can be advantageous from a safety perspective.

BoNTA also may be considered in patients resistant to other treatments or intolerant to current treatments, e.g., nasal corticosteroids or systemic antihistamines. This treatment also allows for longer lasting effects, beneficial for patients. However, larger studies are required to identify the long-term effects and safety profile of BoNTA [126]. Further investigation is needed to identify whether BoNTA is to be used in clinic for rhinitis and which technique would yield the optimal outcome (e.g., posterior injection, turbinate injection, septal injection, or topical) [128]. RT001 is a novel topical gel formulation which contains a purified 150kDa BoNTA protein that has been used in a rat model [129]. The gel formula includes a prietary peptide to enhance transcutaneous and transmucosal flux of BoNTA. In the model, after a single intranasal administration of RT001, associated clinical signs of rhinitis, including inflammation, were significantly resolved within 5 days after treatment.

The optimal dose and patient selection also need to be determined. In addition, it is still unclear whether and how repetitive administration of BoNTA would influence the outcome. Desensitization following repeated application is still an open question. Another important point is that some outcome measures are difficult to be objectified, for instance, nasal pruritus. Hence, analyzing effectiveness calls for the development of some objective methods to complement existing subjective instruments.

#### **7. Concluding Remarks and Future Perspectives**

This review highlighted the potential for BoNTs with a major focus on BoNTA to relieve pruritus. A lack of a sufficient number of randomized controlled trials, limited sample size in the current literature, diverse range of outcome measures, and a lack of knowledge about placebo effects make it difficult to draw a firm conclusion on the antipruritic effects of BoNTs. In addition, for each condition, several critical components remain unidentified, including safe and effective dosage, route of delivery (also considering new formulations of BoNTs), single versus repeated application with optimal interval, and standardization of techniques used in outcome measures. In addition, a strategy for patient selection and precise identification of responders in terms of gender, age, and ethnic background would substantially aid in targeting the right group for optimal effect. The long-lasting effects of BoNTs make it desirable in terms of patients' compliance. However, measured use must also be considered in terms of cost and comparable effectiveness with other agents available for each pruritus condition. Most of the studies presented in the literature have suggested BoNTA as an option; however, not as a first-line therapy and predominantly for those patients either who are having recurrent problems or who are non-responders to other treatment options. It is likely only a matter of time before the full potential of BoNTs for pruritus is elucidated. However, for the time being, focus should be on more common conditions or those for which stronger evidence exists for successful use of BoNTs can be on those conditions that are more common and stronger evidence exist in the literature for successful use of BoNTs.

**Conflicts of Interest:** The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in this manuscript. No writing assistance was utilized in the production of this manuscript.

#### **References**


© 2018 by the author. 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/).

### *Article* **A Study and Review of Effects of Botulinum Toxins on Mast Cell Dependent and Independent Pruritus**

**Roshni Ramachandran 1, Marc J. Marino 1, Snighdha Paul 1, Zhenping Wang 2, Nicholas L. Mascarenhas 2, Sabine Pellett 3, Eric A. Johnson 3, Anna DiNardo <sup>2</sup> and Tony L. Yaksh 1,\***


Received: 7 March 2018; Accepted: 21 March 2018; Published: 23 March 2018

**Abstract:** Pruriceptive itch originates following activation of peripheral sensory nerve terminals when pruritogens come in contact with the skin. The ability of botulinum neurotoxins (BoNTs) to attenuate transmitter release from afferent terminals provides a rationale for studying its effect on pruritus. This study investigated the effects of BoNT/A1 and BoNT/B1 on mast cell dependent (Compound 48/80:48/80) and independent (Chloroquine:CQ) scratching. C57Bl/6 male mice received intradermal injection of 1.5 U of BoNT/A1, BoNT/B1 or saline 2, 7, 14 and 21 days prior to ipsilateral 48/80 or CQ at the nape of the neck. Ipsilateral hind paw scratching was determined using an automated recording device. The effect of BoNTs on 48/80 mediated mast cell degranulation was analyzed in human and murine mast cells and the presence of SNAREs was determined using qPCR, immunostaining and Western blot. Pre-treatment with BoNT/A1 and BoNT/B1 reduced 48/80 and CQ induced scratching behavior starting on day 2 with reversal by day 21. Both serotypes inhibited 48/80 induced mast cell degranulation. qPCR and immunostaining detected SNAP-25 mRNA and protein, respectively, in mast cells, however, Western blots did not. This study demonstrates the long-lasting anti-pruritic effects of two BoNT serotypes, in a murine pruritus model using two different mechanistically driven pruritogens. These data also indicate that BoNTs may have a direct effect upon mast cell degranulation.

**Keywords:** botulinum toxin; itch; SNARE; VAMP; mast cells; compound 48/80; chloroquine

**Key Contribution:** BoNT serotypes show long lasting anti-pruritic effects and may have a direct effect on mast cells.

#### **1. Introduction**

Pruritus or itch is an unpleasant sensation that promotes scratching as a primary response. Chronic itch is a debilitating and dominating symptom accompanying several disorders including skin conditions such as atopic dermatitis (AD) as well as in systemic (renal and liver failure) [1–3] and neurological disorders (diabetic neuropathy and shingles) [4,5]. Pruriceptive itch, as seen in AD, originates following the activation of peripheral sensory nerve terminals associated with allergic reactions induced by insect bites or when pruritogens come in contact with the skin. Among the several subtypes of primary afferent nerve fibers, a role for C-fibers has been demonstrated in detecting and transmitting pruriceptive signals to the neuraxis [6,7].

Many forms of itch are mediated by histamine released from mast cells that activate a subset of neurons expressing TRPV1 receptors as evidenced by the effects of TRPV1 antagonism in histamine evoked activation of dorsal root ganglion (DRG) neurons [8] and reduced histamine evoked scratching behavior [9]. Pruritogens, such as chloroquine (CQ), induce itch via mast cell-independent pathways [10]. Mas-related G protein coupled receptor (Mrgpr) has emerged as a novel class of receptors in histamine independent itch pathways and MrgprA3 is the receptor for CQ. In contrast to histamine dependent pathways, where TRPV1 functions downstream of histamine receptors to promote itch, the histamine-independent pathway utilizes TRPA1 as a key transduction channel downstream of the MrgprA3 receptor [11,12].

Botulinum neurotoxins (BoNTs), including the A1 (Botox) and B1 (Myobloc) serotypes, attenuate neurotransmitter release in neurons by the cleavage of terminal soluble N-ethylmaleimidesensitive-factor attachment protein receptors (SNAREs) [13–15]. Data indicate that when BoNT/A1 and B1 are given subcutaneously in the paw, the toxin is taken up in the peripheral terminal and transported back to the central terminal of the primary afferent [16,17]. Studies from our lab as well as from other groups have shown that both subcutaneous (sc) BoNT/A1 and BoNT/B1 reduce local intradermal capsaicin evoked flares in animal [17,18] and human models [19–22], reflecting the local inhibitory effect upon release of vasodilatory peptides (substance P (sP)/ calcitonin gene-related peptide (CGRP)) from the peripheral terminal evoked by TRPV1 receptor. In addition, following peripheral delivery of BoNTs, cleaved SNAREs are detected in the dorsal root ganglia and dorsal horn along with an associated block of sP release [17]. While BoNTs primarily seem to affect motor neurons in botulism, it is well known that BoNTs can efficiently enter and block neurotransmission in other neuronal subpopulations as well. However, entry and effects on non-neuronal secreting cells, such as mast cells, are less explored, in part because the vesicle release machinery utilizes different (non-neuronal) SNARE proteins that based on the literature are not the targets of medically employed BoNTs. However, an anti-pruritic effect of BoNT/A1 has been demonstrated clinically in several skin disorders, including dermatitis [23], burn induced itch [24], and lichen simplex [25], a localized variant of AD in which acetylcholine appears to be a dominant pruritic mediator. BoNT/A1 also reduced the itch intensity, blood flow and neurogenic inflammation in response to the histamine prick test in human skin [19]. These results jointly suggest the use of BoNTs in treating pruritus, although the mechanism of action remains unknown including whether observed effects are a result of direct action of the BoNT on mast cells or an indirect effect via neurons. The present study demonstrates anti-pruritic effects of BoNT/A1 and BoNT/B1 on histamine dependent compound 48/80 and histamine-independent CQ-induced scratching behavior in mice, and for the first time shows an effect of the BoNTs on cultured murine and human mast cells.

#### **2. Results**

#### *2.1. BoNT/A1 and BoNT/B1 Injection Reduced 48/80 and CQ Induced Scratching*

Behavioral responses were recorded for 40 min in the C57Bl/6 mice following intradermal injection of 48/80 and CQ at the nape of the neck. Both pruritogens injected unilaterally induced ipsilateral scratching behavior. The total number of scratches in the 40 min period increased significantly following intradermal injection of mast cell-dependent 48/80 and mast cell-independent CQ (Figure 1).

Bouts of scratching induced by 48/80 and CQ were reduced by 1.5 U of ipsilateral BoNT/A1 and BoNT/B1 given locally (intradermal) two days prior to the intradermal injections of pruritogens. Analysis of total scratching in the 40 min period showed that this reduction was statistically significant. Importantly, the 1.5 U of intradermal BoNT-A1 or BoNT-B1 did not produce detectable alterations in motor function or strength. Animals displayed normal grasping behavior as measured by a suspension test where the animals were required to grip onto the wire mesh for at least 1 min and showed normal hind limb placing and stepping reflexes [17].

**Figure 1.** Compound 48/80 and chloroquine induced scratches: (**A**) time course of scratching induced by intradermal injection of compound 48/80 (50 μL of 1 mg/kg) (*N* = 8) or Chloroquine (50 μL of 2mg/mL) (*N* = 8) over a period of 40 min (CQ); (**B**) histogram showing cumulative scratch count following compound 48/80 and CQ in 40 min. All data are expressed as Mean ± SEM. \*\*\* *p* < 0.001 as compared to the saline treated group (*N* = 7).

#### *2.2. BoNT/A1 and BoNT/B1 Have a Long Duration of Effect in Reducing Compound 48/80 and CQ Induced Scratching*

One of the hallmarks of pharmaceutical BoNTs is their long duration of action, lasting 2–6 months in humans after intramuscular injection. Local intramuscular injection of BoNT/A1 in mice results in local paralysis that peaks at day 2 after injection and slowly decreases in effect over the following 2–3 weeks [26]. To determine whether effects of BoNT/A1 and B1 on 48/80 and CQ induced scratching have a similarly long-lasting duration, 1.5 U BoNT/A1 or BoNT/B1 or saline were given on days 2, 7, 14 and 21 days prior to administration of 48/80 and CQ treatment on the same side of the neck. BoNT/A1 and BoNT/B1 significantly reduced 48/80 induced scratching behavior on days 2, 7 and 14, but not on day 21 as compared to the saline treated group, suggesting a reversal of effect of BoNT by day 21 (Figure 2). A similar long-lasting effect of BoNT/A1 and B1 was observed on CQ induced scratching as well, where pretreatment with unilateral BoNT/A1 and B1 significantly reduced CQ induced scratching behavior on days 2, 7 and 14 with a complete reversal by day 21 (Figure 2). In both cases, a slow recovery to normal scratching behavior was observed over time, similarly as is seen with muscle paralysis after BoNT treatment. Interestingly, even though BoNT/A1 has a significantly longer duration of action than BoNT/B1 in causing muscle paralysis, in the pruritus assay, both toxins had a similar duration of action in suppressing 48/80 or CQ induced scratching behavior.

**Figure 2.** Duration of action of BoNT/A1 and BoNT/B1 in reducing compound 48/80 and chloroquine induced scratching: Mice were treated with intradermal saline (control), BoNT/A1 (1.5 U), or BoNT/B1 (1.5 U) at 2, 7, 14, or 21 days prior to intradermal administration of compound 48/80 (**A**,**B**) or Chloroquine (**C**,**D**). The total scratches per minute were observed over a 40 min time interval after administration of compound 48/80 or chloroquine. Plots indicate mean ± SEM for cumulative flinches observed at days 2, 7, 14 and 21. \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. saline; # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 as compared to saline, *N* = 8 animals per group.

#### *2.3. BoNT/A1 and BoNT/B1 Reduce Compound 48/80 Induced Murine and Human Mast Cell Degranulation*

The inhibitory effects of BoNT/A1 and B1 on 48/80 induced scratching observed in the in vivo studies could be due to a direct effect of the BoNTs on mast cells or secondary to the inhibition of mediator release from primary afferents inhibited by BoNTs. In order to determine whether BoNT/A1 and B1 directly affected the functioning of mast cells, an in vitro assay using isolated murine and human iPSC derived mast cells was performed. The primary murine mast cells were treated with 48/80 for 20 min at 37 ◦C, leading to degranulation as evidenced by β-hexosaminidase release compared to untreated control cells. As expected, treatment with CQ did not induce mast cell degranulation. Interestingly, pre-treatment of both murine and human mast cells with 0.5 U of BoNT/A1 and /B1 for 24 h significantly reduced 48/80 mast cell degranulation (Figure 3). This indicates a direct effect of BoNT/A1 and B1 on mast cells.

**Figure 3.** Effect of BoNT on compound 48/80 induced mast cell degranulation in human (hMC) and mouse mast cell culture (mMC): Mast cell β-hexosaminidase release (as index of mast cell degranulation) following compound 48/80 (48/80, 10 μg/mL) and chloroquine (CQ) (**A**); mast cell β-hexosaminidase release induced by 48/80 (1μg/mL) 24 h after treatment with 0.5 U of BoNT/A1 or BoNT/B1 in murine mast cell culture (**B**) and in human mast cell culture (**C**). \*\*\* *p* < 0.001 compared to other groups.

#### *2.4. Western Blot Analysis of Expression and Effect of BoNT/A1 and BoNT/B1 on SNAP-25 and VAMP 1/2/3 in Human and Murine Mast Cells*

The cellular target of BoNT/A1 is the neuronal SNAP-25 and for BoNT/B1 is VAMP-1 and 2, which are essential components in the neuronal vesicle release machinery. The BoNTs cleave their respective target SNARE proteins, which is the mode of action by which BoNTs block neurotransmitter release. While the degranulation machinery identified in mast cells utilizes SNAP-23 and VAMP-7/8, which are not cleaved by BoNT/A1 and /B1, the observed inhibition of degranulation of mast cells by BoNTs indicates a mechanism other than SNARE cleavage. In order to confirm this in our model, cultured murine and human mast cells were treated with BoNT/A1 and BoNT/B1 for 24 h and SNAP-25 and VAMP isoforms were analyzed using Western blot. Consistent with previous reports, Western blot analysis did not detect expression of SNAP-25 (Figure 4). It should be noted that spinal cord samples loaded as a positive control on the same immunoblot membrane clearly showed SNAP-25 expression, suggesting that mast cells do not express SNAP-25 at detectable levels. Though expression of VAMP-1/2/3 was observed in mast cells, BoNT/B1 surprisingly did not reduce full-length VAMP 1/2/3 levels in these BoNT treated mast cells, suggesting no cleavage of BoNT-B1 specific VAMP proteins.

**Figure 4.** Expression of SNARE proteins in mast cells: Representative image of Western blots showing expression of SNAP-25 or VAMP-1/2/3 in the mast cells with or without BoNT/A1 or B1 treatment (0.5 U for 24 h). Spinal cord (SC) tissue was used as a positive control for SNAP-25 expression. Mast cells did not express SNAP-25 and hence no effect of BoNT/A1 on SNAP-25 was observed. VAMP 1/2/3 were expressed in mast cells; however, they were not affected by pre-treatment with BoNT/B1; this was repeated three times.

#### *2.5. Expression of SNAP-25 and VAMP 1/2/3 Cleavage with or without BoNT/A1 or B1 Treatment, Respectively, in Human and Murine Mast Cells*

Although Western blot did not detect any expression of SNAP-25 in human and mast cell culture, immunostaining was able to detect cleaved products of SNAP-25 following BoNT/A1 treatment on both human and murine mast cells. The SNAP-25 antibody used in the present study detects only the cleaved products (cSNAP-25). The control groups of murine and human MC did not show cSNAP-25 staining; however pre-treatment of the mast cells with BoNT/A1 for 4 h showed a dose-dependent increase in cSNAP-25 staining. DAPI was used to stain the nuclei of the mast cells (Figure 5A). RT-qPCR analysis on human and mouse mast cells showed expression of SNAP-25 mRNA, suggesting the presence of at least low levels of SNAP-25 in mast cells (Figure 5B). The VAMP antibody used recognizes the intact molecule. Therefore, reduction of VAMP protein expression was used as a measure of VAMP cleavage. In control animals, VAMP expression was observed in the control group along with the DAPI stained nuclei. Following pre-treatment with BoNT/B1, the cells showed a reduction in VAMP expression. Thus, VAMP cleavage was significantly greater in the BoNT/B1 treated group as compared to PBS control (Figure 5A).

**Figure 5.** Detection of cSNAP-25 and VAMP-2, respectively, in mouse and human mast cell culture: (**A**) representative images of BoNT/A1-cleaved SNAP-25 and VAMP-2 immunostaining following treatment with BoNT/A1 and BoNT/B1 (10 pM); (**B**) RT-qPCR of SNAP-25 expression in mouse and human mast cell culture. *N* = 3.

#### **3. Discussion**

The present study demonstrated the anti-pruritic effects of two well characterized and clinically employed BoNT serotypes, Botulinum Toxin A1 (Botox©) and Botulinum Toxin B1 (MyoBloc©) over time in a murine pruritus model using two different mechanistically driven pruritogens. While involvement of SNARE proteins in release of pruritus stimulating mediators from mast cells has been demonstrated [27,28], effects of BoNT/A1 and BoNT/B1 on mast cell release has not been explored. The present study showed that BoNTs may have a direct effect on mast cells in altering its local degranulation, and that this effect may be independent of SNARE cleavage.

#### *3.1. Mast Cell Dependent and Independent Pruritogens*

Pruriceptive itch is induced peripherally due to the activation of nerve fibers located in the epidermis and is the type of itch observed in several dermatological conditions such as atopic dermatitis, psoriasis, etc. [29]. Pruriceptive itch can be experimentally triggered by several exogenous and endogenous substances. In the present study, we utilized two pruritogens, mast cell dependent, 48/80 and mast cell independent, CQ. Compound 48/80 degranulates mast cells to release histamine [30], which induces itch by binding to and activating C-fibers via gating the TRPV1 channel [8,9]. Other mediators released from mast cells such as serotonin, bradykinin, and prostaglandin can potentiate the effects of histamine induced itch sensation [31]. CQ, on the other hand, elicits itch in a mast cell independent pathway, presumably by activating Mrgpr /TRPA1 receptors [11,12]. A recent study shows that 48/80 may have a direct action on neurons in addition to degranulating mast cells [32] and the interpretation of the results in this study does consider this possibility. However, it should be noted that a previous study has reported mast cell mediated neuronal activity of 48/80 [33].

#### *3.2. Anti-Pruritic Effect of Botulinum Toxin*

BoNT/A1 and BoNT/B1 have been extensively used in clinical and pre-clinical studies for elucidating the mechanisms by which they can inhibit pain transduction in several pain related disorders. Ample evidence now suggests that BoNTs influence release of several neurotransmitters such as acetylcholine, glutamate, CGRP, sP, and serotonin [34–36]. The release of these neurotransmitters may play a contributing role in induced itch [37,38]. Patients with AD show an increase in density of nerve fibers containing CGRP and sP [39]. Furthermore, pre-clinical studies in pain models have shown that peripheral BoNT can block the release of neurotransmitters from the local afferents as well as from the central nerve terminals [17,18,40–43], suggesting a possible pathway in which BoNTs may influence the transmission of itch signals to higher brain centers. Our data from the present study suggests that both BoNT/A1 and BoNT/B1 significantly inhibited the scratching behavior induced by two mechanistically different pruritogens. These findings are in accordance with a clinical study showing that BoNT/A1 could reduce the histamine pin prick induced itch intensity in human skin along with diminished blood flow and neurogenic inflammation [19]. Further clinical studies have suggested anti-pruritic effects of BoNT/A1 on conditions accompanied with itch such as lichen simplex, rhinitis, inverse psoriasis, burn induced itch and dermatitis [23–25]. However, no studies have determined the effect of BoNT/B1 in pruritus so far.

#### *3.3. Duration of Action of BoNT*

Activity of BoNT is attributed to neuronal cell entry by the toxin, release of the light chain (LC) into the cells cytosol, and cleavage of terminal SNAREs by the LC, blocking vesicular transmitter release [13]. The duration of action of BoNT depends on the persistence of the catalytically active intracellular LC [44,45]. While in humans the duration of pharmaceutical BoNTs varies from two to six months, depending on the dose, mice usually recover from paralytic effects after local intramuscular injection within three weeks [26,46]. Similarly, the BoNT/A1 and B1 induced reduction in itch behavior in the murine model used in this study lasted for about two weeks, with mice showing gradual reversal

that was complete by day 21 (Figure 3). This similarity in duration and gradual reversal indicates a possibly similar mechanism of action of BoNTs in mast cells as in neurons, or an indirect effect of BoNTs in pruritus due to neuronal release inhibition.

#### *3.4. Possible Mechanism of Action of BoNT/A1 and BoNT/B1 in Reducing Induced Itch*

Effects of both the pruritogens, 48/80 and CQ are believed to be mediated by the activation of C-fiber terminals in the epidermis. Compelling evidence suggests the role of TRPV1 and TRPA1 ion channels in these subsets of neurons downstream to histamine and CQ [11,12] to mediate calcium induced activation of SNAREs that mobilize synaptic vesicle release, thereby promoting itch sensation. Patients with AD show intense staining of CGRP and substance P-immunoreactive fibers, and uptake of BoNT has previously shown to inhibit the release of these neurotransmitters. Therefore, the reduction in induced itch behavior in mice by BoNT/A1 and B1 could be at least in part due to an effect of the toxins on the C-fibers, rather than a direct effect on mast cells.

Interestingly, we observed that pre-treatment with both BoNT/A1 and BoNT/B1 impaired 48/80 induced mast cell degranulation in cultured murine and human mast cells, indicating that BoNTs may also have a direct effect on mast cells. This is in agreement with previous experiments conducted by Park and colleagues that showed a decrease in mast cell activity seven days following BoNT/A1 treatment in rat skin tissue [47]. While the mast cell release machinery involves SNAREs, which are the target of BoNTs in neurons, the SNARE isoforms considered to be required for mast cell degranulation (SNAP-23 and VAMP-7 and 8) are insensitive to BoNT/A1 and /B1 [28,48,49]. In our study, very low levels of SNAP-25 mRNA expression were observed in both murine and human mast cells, with immunohistochemistry studies confirming the findings for both SNAP-25 and VAMP-1/2/3 and indicating cleavage of these SNARE isoforms by BoNT/A1 and B1. However, western blot data suggested absence or very low levels of SNAP-25 in mast cells, and levels of the BoNT/B1 sensitive VAMP-1/2/3 in mast cells appeared to be unaffected by BoNT/B1. Similar discrepancy in the SNAP-25 expression in mast cells using various detection methods has been previously reported [50]. This result, although confounding, is intriguing and leads to the speculation that BoNTs may utilize a non-canonical mechanism other than SNARE cleavage to inhibit release of secretory granules from mast cells. For example, one possibility could be hindrance in the trafficking of membrane proteins such as TRP receptor subunits to the plasma membrane of mast cells. The role of BoNT/A1 in inhibiting TRPV1 receptor function by affecting regulated endocytosis and reduction in TRPV1 receptor expression has been previously demonstrated in the trigeminal as well as in suburothelial nerve fibers [51,52] (Shimizu et al., Apostolidis et al.). Furthermore, studies have shown that 48/80 degranulation of mast cells employ calcium induced exocytosis in mast cells [53] and BoNTs primarily inhibit the normal depolarization- evoked calcium currents [54]. More studies are required to elucidate the inhibitory mechanism of botulinum toxins on mast cell degranulation and whether the observed in vivo effects are due to direct or indirect action of BoNTs on mast cells.

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

#### *4.1. Animals*

Adult male C57Bl/6 mice, 25–30 g (Harlan Sprague Dawley Inc., Indianapolis, IN, USA), were housed in the vivarium for a minimum of 2 days before use, maintained on a 12/12-h day-night cycle and given free access to food and water. All studies were carried out according to protocols approved by the Institutional Animal Care and Use Committee of the University of California, San Diego, CA, USA. Ethical approval code and date: S00137M and 26 March 2015 (IACUC).

#### *4.2. Drugs*

Drugs employed were compound 48/80 (48/80) (1 mg/mL) or CQ (2 mg/mL) (Sigma Aldrich, St Louis, MO, USA). BoNT/A1 (Botox©, onabotulinumtoxin A, Allergan Inc., Carlsbad, CA, USA) and

BoNT/B1 (Myobloc©, Rimabotulinumtoxin B, Solstice Neurosciences, Louisville, KY, USA) solutions were prepared from stock solutions of 50 U/mL and 5000 U/mL, respectively. These products were then serially diluted to the final concentration in 0.9% saline. All solutions were stored at 4 ◦C and brought to room temperature prior to use.

#### *4.3. Drug Delivery*

Mice were anesthetized (2.5% isoflurane, with 80% oxygen and 20% room air) and were shaven on the dorsolateral aspect of the neck and upper shoulder. Using a 29 G needle (insulin syringe) intradermal injection of 50 μL of BoNT/A1 or BoNT/B1 (1.5 U) or saline was administered. Intradermal injections of 48/80 or CQ (50 μL) were administered on the day of behavioral testing.

#### *4.4. Behavior*

On the day of testing, animals were placed in a plexiglass cylindrical chamber and a detection band was placed around the hind paw ipsilateral to the shaven area. To initiate scratching behavior, intradermal (ID) injection of 48/80 or CQ was administered in the middle of the shaven area of skin using a 29 G needle. The itch behavior is recorded over the period of 40 min using a paw motion detector (PMD). The PMD detects the movement of a non-ferrous metal band placed around one hind paw of the rodent (band weight = 0.1 g). The testing apparatus consists of cylindrical chambers (mouse: 8.5 cm diameter/22.5 cm tall). Under each cylinder is a pair of circular concentric electromagnetic coils, which serve respectively as antennae for transmission and reception. The transmitter coil assembly emits a 5–8 mW, 6–8 kHz, sinusoidal electromagnetic field. The detection principal is that Eddy currents created by the movements of the ferrous and nonferrous metals perturb the EM field. Such perturbations are detected and produce an output waveform [55,56]. Data were acquired electronically.

#### *4.5. Mast Cell Culture*

Primary murine MCs were generated from C57BL/6 mouse bone marrow and cultured in RPMI 1640 medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 25 mM HEPES (pH 7.4), 4 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Recombinant murine IL-3 (1 ng/mL, R&D Systems, Minneapolis, MN, USA) and recombinant murine stem cell factor (20 ng/mL, R&D Systems, Minneapolis, MN, USA) were also included to allow for in vitro differentiation. After 4 weeks, the MCs were fully differentiated, as confirmed by the expression of CD117 (c-Kit) and FcεRI. Cell maturation was confirmed by metachromatic staining with toluidine blue. The purity of MCs was greater than 98%. For the detailed procedure, see [57].

Primary human MCs were derived from human cord blood CD34+CD45+ cells from healthy donors (STEMCELL Technologies, Seattle, WA, USA) according to Kirshenbaum and Metcalfe [58]. They were cultured in Stemline II hematopoietic stem cell medium (Sigma Aldrich, St. Louis, MO, USA) with recombinant human SCF and IL-6 (100 ng/mL, Peprotech, Rock Hill, NJ, USA) for 9 weeks. MC differentiation was confirmed by CD117 (c-Kit) and FcεRI expression, and maturation was confirmed by metachromatic staining with toluidine blue. The purity of MCs was greater than 98%.

#### *4.6. Mast Cell Degranulation Assay*

Degranulation was assessed by measuring the activity of β-hexosaminidase in the supernatants of 1 × 105 MCs in 200 <sup>μ</sup>L Tyrode's buffer (0.1% BSA, 0.1% glucose, 2 mmol/L MgCl2, 137.5 mmol/L NaCl, 12 mmol/L NaHCO3, 2.6 mmol/L KCl, pH 7.4) incubated for 24 h with 0.5 U of BoNT-A1 or -B1 before the addition of 1 μg/mL 48/80 (Sigma Aldrich, St Louis, MO, USA). For comparison of mast cell degranulation by 48/80 to CQ, 10 μg/mL 48/80 was used. For each sample assayed, supernatant aliquots (20 μL) were mixed with substrate solution (100 μL) which consisted of 10 mM

4-methylumbelliferyl-2-acetamide-2-deoxy-b-D-glucopyranoside (EMD Millipore, Billerica, MA, USA) in 0.1 M sodium citrate buffer (pH 4.5) and were incubated for 2 h at 37 ◦C in the dark. The reaction mixtures were excited at 365 nm and measured at 460 nm in a fluorescence plate reader (Gemini EM microplate spectrofluorometer; Molecular Devices, Sunnyvale, CA, USA). To determine the total cellular content of this enzyme, an equivalent number of cells were lysed with 1% Triton X-100 (Sigma Aldrich, St Louis, MO, USA). Release of β-hexosaminidase was calculated as the percentage of the total enzyme content.

#### *4.7. Immunohistochemistry on Mast Cells*

Mast cells were attached to a glass slide by using Shandon Cytospin 2 cytocentrifuge (Thermo Fisher Scientific, Waltham, MC, USA). The cells were stained with 1 mg/mL anti BoNT/A1-cleaved-SNAP-25 Ab, which recognizes only the BoNT/A1cleavage product of SNAP-25 and not the full-length SNAP-25, and anti-VAMP-2 Ab (Synaptic Systems, Goettingen, Germany) according to the manufacturer's instructions. Slides were mounted in ProLong Anti-Fade reagent with DAPI (Molecular Probes, Eugene, OR, USA). We imaged the cells using the Bx51 research microscope (Olympus, Center Valley, PA, USA) and X-Cite 120 fluorescence illumination systems (EXFO Photonic Solutions, Mississauga, ON, Canada).

#### *4.8. mRNA Isolation and Real-Time Quantitative PCR*

Total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) and 1 μg of total RNA was used for cDNA synthesis by using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions. cDNA was amplified using Real time-PCR in an ABI 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). RNA analysis reagents (SYBR Green Master Mix) were from Bio-Rad, Hercules, CA, USA. We used the comparative ΔΔ cycle threshold method to quantify gene expression. Target gene expression levels in the test samples were normalized to the endogenous reference glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F: 5 -CCA ACC GCG AGA AGA TGA CC-3 and R: 5 -GAT CTT CAT GAG GTA GTC AGT-3 ) levels and reported as the fold difference relative to GAPDH gene expression in untreated baseline control. All assays were performed in triplicate and the experiments were repeated at least three times.

#### *4.9. Western Blot Analysis*

Following 24 h treatment with BoNT-A1 or BoNT-B1 on murine and human mast cells, cell lysates were prepared by solubilizing cells in RIPA buffer (Life Technologies, Carlsbad, CA, USA) with protease inhibitor cocktail (Sigma Aldrich, St Louis, MO, USA), at 1 × 107 cells/mL. Cells were incubated for 1 h on ice for complete lysis, and the lysates clarified by centrifugation at 4 ◦C, for 10 min at 12,000 RPM. Supernatants were collected and stored on ice for immediate use, or at –80 ◦C until needed. Total protein concentration of the clarified cell lysates was determined by BCA protein assay (Life Technologies, Carlsbad, CA, USA) prior to loading on a gel.

For the Western blot analysis, five micrograms of total cell lysate of each sample were separated on a 12% Bis-Tris NuPAGE gel with MES running buffer (all from Life Technologies). For the mouse spinal cord cell lysate (SC) controls, primary mouse spinal cord cell lysates were prepared as previously described [45], and 8 μl of untreated primary mouse spinal cord cell lysates were used. Proteins were transferred to a PVDF membrane (Millipore 0.45 micron for the SNAP-25 blots, and GE Healthcare (Little Chalfont, UK) 0.2 micron for the VAMP blots) by semi-dry transfer. The membranes were probed with antibodies to beta-actin (Abcam, Cambridge, UK) and VAMP-1/2/3 (Synaptic Systems) (top gel), a polyclonal anti-SNAP-25 antibody (Synaptic Systems) (middle gel), or a monoclonal anti-SNAP-25 antibody (Synaptic Systems) (bottom gel). Images were obtained using PhosphaGlo reagent (KPL, Gaithersburg, MD, USA) and a Fotodyne/FOTO/Analyst FX imaging system (Fotodyne, Hartland, WI, USA).

#### *4.10. Statistical Analysis*

The data for each variable was put in tabular form (i.e., Excel worksheet). Summary statistics were computed and include group means and standard deviations and numbers of animals per group. Statistical analysis was performed using GraphPad Prism 6, v6.0c (GraphPad Software, San Diego, CA, USA). For comparison of 48/80 and CQ induced scratching, results were compared using a one-way ANOVA across doses or time. Bonferroni post hoc tests were used to compare groups at similar doses or times. For all post hoc comparisons, multiplicity adjusted *p*-values were calculated. In each case, Bonferroni post hoc tests (e.g., *t*-tests with Bonferroni corrections) were undertaken and presented in the graphics and figure legends for values between *p* < 0.01 and *p* < 0.0001.

**Acknowledgments:** This work was supported by DA15353 (TY), 5R21AI113580-02 (RR) and DA02110 (SP).

**Author Contributions:** R.R. and T.L.Y. conceived and designed the experiments; R.R., M.J.M., S.P., Z.W., N.L.M. and S.P. performed the experiments; R.R., S.P. and Z.W. analyzed the data; A.D. and E.A.J. contributed to reagents/materials/analysis tools; R.R. wrote the paper.

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

#### **References**


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