Methods for enhancing transdermal delivery of botulinum toxins

Abstract

A non-invasive technique for enhancing the delivery of botulinum toxins through the skin, utilizing low-frequency ultrasound as a pretreatment, is provided. The ultrasound- facilitated transdermal delivery approach provides an efficient, comfortable and needle-free alternative to conventional injection-based administration of botulinum toxins, and is applicable to a wide range of therapeutic and cosmetic uses, including, reducing or modulating sweat gland activity, relaxing or inhibiting muscle activity, alleviating pain, minimizing wrinkles or fine lines, and treating or preventing pathologies such as neuromuscular and neurological disorders, glandular dysfunction, urological conditions, ophthalmological disorders, gastrointestinal conditions and / or respiratory disorders

Description

[0001] METHODS FOR ENHANCING TRANSDERMAL DELIVERY OF BOTULINUM TOXINS

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to the use of non-invasive methods for increasing skin permeability, particularly but not exclusively, the use of ultrasound for enhancing the transdermal delivery of botulinum toxins.

[0004] BACKGROUND

[0005] Botulinum toxins are neurotoxins produced by the bacterium Clostridium botulinum. They are among the most potent biological substances known, acting by inhibiting acetylcholine release at neuromuscular junctions, which results in temporary muscle paralysis. Several serotypes have been identified (A-G), but types A and B are the most widely used in medical and cosmetic practice due to their safety and effectiveness when administered in controlled doses.

[0006] Botulinum toxin type A (BTX-A) is the most common form in therapeutic and cosmetic applications. Commercial formulations include Botox®, Dysport®, and Xeomin®. Botulinum toxin type B (BTX-B) is used less frequently, but available in formulations such as Myobloc®, often chosen for patients who develop resistance to BTX-A.

[0007] In medicine, botulinum toxins are employed to manage conditions caused by excessive or inappropriate muscle contractions, such as cervical dystonia, blepharospasm, hemifacial spasm, chronic migraine, spasticity in cerebral palsy or post-stroke patients, and hyperhidrosis (excessive sweating). In cosmetics, BTX-A is widely used to reduce the appearance of dynamic wrinkles - most notably frown lines, forehead lines, and crow's feet - by temporarily relaxing facial muscles responsible for their formation.

[0008] Despite their clinical benefits, botulinum toxin injections are associated with several challenges, among which are local adverse effects such as bruising, swelling, pain, and / or redness at the injection site.

[0009] SUMMARY

[0010] Botulinum toxins have revolutionized therapeutic and cosmetic practice, offering options for muscle overactivity and facial rejuvenation. Though minimally invasive, their current administration, nevertheless, involves painful, multiple subcutaneous injections that may lead to potential side effects, including redness, swelling, and local infection. The present disclosure addresses the yet unmet need for non-invasive means to effectively deliver botulinum toxin transdermally. Disclosed herein is a non-invasive technique for enhancing the delivery of botulinum toxins such as Xeomin® through the skin by utilizing low-frequency ultrasound as a pretreatment. This transdermal delivery method improves the efficiency and comfort of treatment modalities involving botulinum toxins such as, but not limited to, treatment of hyperhidrosis, a condition characterized by excessive sweating.

[0011] In one aspect, the present disclosure relates to a non-invasive method for transdermal delivery of botulinum toxin (BTX), the method comprising the steps of:

[0012] (a) applying ultrasound to a target skin region of a subject in need thereof for a cumulative treatment duration of from about 3 sec to about 10 min; and

[0013] (b) topically administrating to the ultrasound-treated skin region a therapeutically and / or cosmetically effective amount of BTX, thereby transdermally delivering BTX to the subject.

[0014] Steps (a) and (b) may be repeated one or more times in the same or a different skin region, delivering the same or different amounts of BTX.

[0015] The ultrasound may be applied at a frequency of about 20-200 kHz in pulsed mode or in a continuous mode. In some embodiments, the ultrasound is applied for a period of from about 5 sec to about 7 min.

[0016] Embodiments disclosed herein pertain to treating, alleviating or preventing a condition or disorder that may benefit form transdermal delivery of botulinum toxin. The methods disclosed herein exert therapeutic and / or cosmetic effects in multiple indications, including for example, cutaneous manifestations (e.g., skin wrinkles), neuromuscular and neurological disorders, pain syndromes, glandular dysfunction, urological conditions, dermatological and scalp conditions, ophthalmological disorders, gastrointestinal and sphincter-related conditions, respiratory disorders, and emerging therapeutic applications. The effects exerted include, but are not limited to, reducing sweat gland activity, relaxing muscle activity and / or alleviating pain.

[0017] Exemplary diseases disorders and conditions that may be treated, alleviated or prevented by a method disclosed herein include: primary or secondary hyperhidrosis; cosmetic skin conditions such as wrinkles, fine lines, and facial rhytides; neuromuscular disorders such as dystonia, spasticity, tremor, muscle hyperactivity and Tourette syndrome; pain disorders such as chronic migraine, tension-type headaches, chronic neck pain, myofascial pain, neuropathic pain or postoperative pain; (v) glandular conditions such as seborrhea or seborrhea; urological or pelvic conditions such as detrusor overactivity, overactive bladder, urinary incontinence or chronic pelvic pain; dermatological conditions such as acne, rosacea, psoriasis, or pruritus; scalp or hair conditions such as excessive scalp sweating (craniofacial hyperhidrosis) or alopecia; headache disorders such as chronic migraine, severe tension-type headaches or chronic neck pain; ophthalmic disorders such as strabismus (ocular misalignment), blepharospasm (involuntary eyelid spasms), extraocular muscle spasms, or dry eye syndrome; gastrointestinal and sphincter disorders such as achalasia, chronic anal fissures, gastroesophageal reflux disease (GERD) or swallowing disorders; neurological disorders such as involuntary muscle activity; an autonomic or cardiac disorder such as cardiac arrhythmias, atrial fibrillation or myocardial infarction; carpal tunnel syndrome (CTS); wound healing and scar modulation; temporomandibular joint (TMJ) disorders; fibromyalgia; laryngospasm; airway hyperresponsiveness; synovitis and intra-articular inflammation; depression and anxiety; and / or excessive drooling in Parkinson's or ALS patients.

[0018] A method disclosed herein may be applied for therapeutic, prophylactic, or rehabilitative purposes in humans or animals.

[0019] The botulinum toxin may be of type A, B, C, D, E, F or G, optionally formulated as a topical cosmetic or medicinal formulation. In some embodiments, the botulinum toxin employed is type A or B BTX.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Some embodiments are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments may be practiced.

[0022] In the drawings:

[0023] Fig. 1 is a bar graph presenting the fluorescence-based quantification of transdermal delivery of Xeomin® fluorescently labeled with Hyalite™ Fluor 488 ("XeominHF488"). The concentration (mg / mL) of fluorescently labeled XeominHF488 detected in the receiver compartment of Franz diffusion cells after 24 hours is shown for three test groups: PBS (control), XeominHF488 applied without pre-application of US, and XeominHF488 applied following US pre-treatment (US + XeominHF488). Data are presented as mean ± SD (n = 3); Figs. 2A-2C are confocal fluorescence microscopy images of cross-sectional pig skin obtained after 24 hours of ex vivo exposure to XeominHF488. The spatial pattern of fluorescence demonstrates treatment-dependent differences in skin penetration for three test groups: skin treated with PBS only (2A; control); skin treated with XeominHF488 without ultrasound (US) preapplication (2B); and skin treated with XeominHF488 following US application (2C). The green fluorescence signal (emission at 523 nm) represents the distribution of XeominHF488 within the stratum corneum (SC), epidermis, and dermis, while background tissue autofluorescence is also visible. Scale bars = 100 pm;

[0024] Figs. 3A-3B are graphs showing the depth-dependent distribution of XeominHF488 in pig skin. 3A: a graph depicting average fluorescence intensity (RFU) plotted as a function of tissue depth (pm) for three treatment groups: PBS only, XeominHF488 only (Xeomin) and ultrasound + XeominHF488 (US + Xeomin). 3B: a bar graph presenting quantitative analysis of fluorescence intensity across successive 20 pm depth intervals. Data are presented as mean ± SD, derived from 27 measurements per group (three measurements per image, three images per sample, n = 3 independent biological replicates). Statistical significance was determined using two-way ANOVA, with thresholds of p < 0.05, p < 0.01, p < 0.001, and p < 0.0001;

[0025] Figs. 4A-4B are collections of representative images of mouse hind paws following iodinestarch staining and ImageJ™ analysis. 4A: stained paws from five experimental groups: untreated control; 6-hour saline application; 6-hour Xeomin® application; ultrasound (US) pre-treatment followed by 6-hour saline application; and US pre-treatment followed by 6-hour Xeomin® application. Dark regions correspond to areas of active sweat secretion. 4B: binary images generated using ImageJ™ software, in which black areas denote sweat-secreting regions and white areas indicate absence of sweat activity; and

[0026] Fig. 5 is a bar graph showing reduction in sweating area (%) across experimental groups of mouse hind paw model of induced sweating: saline application (Saline); Xeomin® application (Xeomin); US pre-treatment followed by saline application (US); and US pre-treatment followed by Xeomin® application (US + Xeomin). Sweating reduction was quantified using the iodinestarch assay combined with ImageJ™ analysis. For each animal, the sweating area of the treated paw was compared to that of the untreated contralateral paw, which also underwent iodinestarch staining. Data are expressed as mean ± SD. Statistical analysis was performed using oneway ANOVA; p < 0.001, p < 0.0001. DETAILED DESCRIPTION

[0027] The present disclosure relates to a non-invasive method that relies on ultrasound as a pre-treatment to enhance the transdermal delivery of botulinum toxins through the skin. This method dramatically reduces the pain and side effects associated with the conventional multiple injections treatment and facilitates toxin delivery through the skin layers to the subcutaneous tissue.

[0028] The present disclosure is based on the inventors' discovery that pre-application of low- frequency ultrasound (US) enabled the transdermal delivery of botulinum toxin (BTX) type A (Xeomin®). Ex vivo studies using pig ear skin in a Franz diffusion cell demonstrated that US pretreatment facilitated penetration of fluorescently labeled Xeomin® across all skin layers, with 6.0 ± 0.9% of the applied dose detected in the receptor compartment, as confirmed by fluorescence quantification and histological analysis. In vivo experiments conducted on the hind paws of mice to assess the biological effect of Xeomin® on sweat reduction, showed that only the group receiving both US pretreatment and Xeomin® exhibited a statistically significant reduction in sweat secretion compared to saline, Xeomin®-only, and US-only controls (p < 0.001), as determined by an iodine-starch assay and ImageJ analysis. The procedure was well tolerated, with no observable tissue damage or functional impairment. These findings demonstrate that low-frequency ultrasound effectively enhances transdermal delivery of Xeomin®, providing a needle-free method for reducing sweat secretion and offering a promising alternative to botulinum toxin injections for hyperhidrosis treatment, while also holding potential for broader non-invasive cosmetic and therapeutic applications.

[0029] In one aspect, the present disclosure relates to a non-invasive method for transdermal delivery of botulinum toxin, the method comprising the steps of:

[0030] (a) applying ultrasound to a target skin region of a subject in need thereof for a cumulative treatment duration of from about 3 sec to about 10 min; and

[0031] (b) topically administrating to the ultrasound-treated skin region a therapeutically and / or cosmetically effective amount of BTX, thereby transdermally delivering BTX to the subject.

[0032] Optionally, steps (a) and (b) of a disclosed method may be repeated at least one more time (e.g., once, twice, three times or more) as needed in order to achieve efficient penetration of therapeutically or cosmetically effective amounts of BTX into deep layers of the skin. For example, after the first ultrasound application, a further ultrasound treatment may be applied to the skin target region, optionally followed by administration of BTX. The one or more further US applications may target same skin region and / or different skin regions, for example, nearby, or partially overlapping skin regions so as to expose larger skin area to BTX application. Each time the US is applied it can be applied withing the same or different time frames of from about 3 sec to about 10 min. In some embodiments, US is applied for a cumulative treatment duration of 5 sec to about 7 min, either once, twice or more times, as needed.

[0033] When steps (a) and (b) of a claimed method are repeated two or more times, the amount of BTX provided to the skin may be the same or different.

[0034] Ultrasound refers to acoustic waves with frequencies above the audible range of human hearing, generally greater than about 20 kilohertz (20 kHz). Ultrasound requires a physical medium (solid, liquid, or gas) to propagate, relying on mechanical waves generated by the vibrations of an elastic substance. Ultrasound frequencies are categorized into three main ranges: low-frequency ultrasound (LFUS; 20-200 kHz), therapeutic ultrasound (0.7-3 MHz), and high-frequency sonophoresis (>3 MHz). Low frequencies are commonly used for applications like drug delivery, leveraging enhanced skin permeability. Higher frequencies provide detailed imaging of superficial structures such as skin. Medical applications of ultrasound include tumor therapy, kidney stone fragmentation, and wound healing.

[0035] In the context of the present disclosure, ultrasound encompasses the application of such acoustic energy to biological tissue, particularly skin, to facilitate the transdermal delivery of therapeutic or diagnostic agents.

[0036] Embodiments disclosed herein LFUS, typically within the range of about 20 kHz to about 200 kHz, which has been demonstrated to significantly enhance skin permeability. Low- frequency ultrasound can induce both cavitational and microstreaming effects within the skin's outermost layer, the stratum corneum, resulting in temporary disruption of lipid bilayers and the formation of localized transport pathways or "microchannels." These mechanisms allow for the enhanced penetration of small molecules, macromolecules, nanoparticles, and other agents across the skin barrier.

[0037] The application of LFUS may be continuous or pulsed, and can be modulated in intensity, duty cycle, or exposure time to optimize delivery efficiency while minimizing adverse effects such as tissue heating or irreversible skin damage.

[0038] As used herein, the term "intensity" of ultrasound refers to the average power delivered per unit area of the ultrasound transducer surface, expressed in watts per square centimeter (W / cm2). When it is stated that US is delivered at an intensity of about 1.5-20 W / cm2, this encompasses the range of acoustic power densities that may be used to induce enhanced skin permeability while minimizing irreversible tissue effects. In certain embodiments, the intensity may be more narrowly selected within a sub-range of about 5-12 W / cm2to balance efficacy in promoting transdermal delivery with safety and user comfort.

[0039] The term "pulsed mode" refers to the intermittent delivery of ultrasound energy in cycles of "on" and "off" periods rather than continuous application. The "duty cycle" is defined as the percentage of time during which ultrasound is actively applied within a given cycle. For example, a duty cycle of about 10-70% indicates that ultrasound is applied for 10-70% of the total cycle duration, with the remainder being an "off" period. In some embodiments, a duty cycle of about 50% is conducted, meaning that ultrasound is applied for half of each cycle and withheld for the other half. Pulsed operation can reduce thermal buildup, allow for tissue recovery, and enhance the efficiency of cavitational effects, thereby improving the overall safety and efficacy of ultrasound-assisted transdermal delivery.

[0040] When steps (a) and (b) of a disclosed method are repeated one or more times, each time the US is applied it can be applied in the same or a different mode and / or with the same or a different intensity.

[0041] In accordance with the present disclosure, low-frequency ultrasound serves as a preparatory step to temporarily disrupt the skin barrier, promoting the effective and targeted delivery of botulinum toxins.

[0042] The skin comprises three principal layers: the epidermis, dermis, and hypodermis. The epidermis includes the stratum corneum (SC), a barrier of corneocytes embedded in a lipid matrix that regulates water loss and provides protection against mechanical, chemical, and microbial insults, and the underlying viable epidermis, primarily composed of keratinocytes involved in barrier and immune functions. Corneocytes are derived from keratinocytes and are the terminally differentiated cells that make up the SC. As keratinocytes move outward through the epidermis, they undergo a process called cornification (keratinization), during which they lose their nucleus and organelles.

[0043] The dermis is a connective tissue layer containing collagen and elastic fibers that provide strength, flexibility, and resilience, and is organized into papillary and reticular regions. The papillary dermis is the uppermost, thin layer of the dermis, directly beneath the epidermis. It is a loose connective tissue rich in capillaries, lymphatic vessels, and sensory nerve endings. Contains dermal papillae, which project upward toward the epidermis. The papillary dermis provides nutrients and oxygen to the avascular epidermis. It contributes to sensory perception (touch, pain, temperature). The reticular dermis is the deeper, thicker portion of the dermis, beneath the papillary region. It is a dense irregular connective tissue, rich in collagen and elastin fibers, with larger blood vessels, lymphatics, nerves, sweat glands, sebaceous glands, and hair follicles. The reticular dermis provides tensile strength and elasticity to the skin and plays a major role in thermoregulation and mechanical resilience.

[0044] Beneath the dermis lies the hypodermis also known as the subcutaneous layer, consisting mainly of adipose tissue interlaced with connective tissue, providing cushioning, metabolic exchange, and structural support. The hypodermis is situated between the dermis and the underlying muscular structures. Its thickness is variable, influenced by factors such as body location, age, gender, ethnicity, and the individual's overall health and nutritional state. This layer is predominantly composed of white adipocytes (fat cells containing large lipid droplets that occupy most of the cell's volume), which are embedded in a network of collagen fibers. The hypodermis is structured into fat lobules, each supplied by its own arteriole and encased in connective tissue. Every adipocyte maintains contact with at least one capillary, facilitating efficient metabolic exchange and cellular function.

[0045] The skin serves as a route for transdermal drug delivery, offering advantages such as bypassing gastrointestinal degradation and first-pass metabolism while minimizing systemic toxicity associated with oral dosing. It reduces the risk of gastrointestinal discomfort, stomach ulcers, and potential liver or kidney damage associated with prolonged oral medication use. However, permeability is restricted by the SC. This highly organized and compact structure limits passive delivery to small, lipophilic molecules (<500 Da) that can achieve therapeutic effects at low systemic concentrations. Alternatives such as hypodermic injection overcome this barrier but are invasive, whereas transdermal approaches provide a non-invasive, patient-friendly option.

[0046] In some embodiments, a non-invasive method for transdermal delivery of botulinum toxin disclosed herein is applied for treating, alleviating or preventing a disease, condition or disorder that may benefit form transdermal delivery of botulinum toxin.

[0047] In some embodiments, a disclosed method exerts a therapeutic effect such as, but not limited to, reducing sweat gland activity, relaxing muscle activity, alleviating pain and / or treating a glandular, dermatological, or neurological disorder. In some embodiments, a disclosed method is applied for treating hyperhidrosis, a condition characterized by excessive and uncontrollable sweating that exceeds physiological thermoregulatory needs.

[0048] The skin plays a central role in thermoregulation through sweating, mediated by eccrine glands located in the dermis. Eccrine glands, numbering 2-4 million across the body, secrete sweat in response to cholinergic stimulation, enabling evaporative cooling. Their density varies with body size and region, being highest on the palms, soles, and forehead. A key player in the sweating process is acetylcholine, a neurotransmitter released by sympathetic nerve fibers that innervate the eccrine sweat glands. When acetylcholine binds to muscarinic receptors on the sweat gland, it triggers an increase in intracellular calcium concentrations, leading to the secretion of sweat.

[0049] Primary hyperhidrosis is idiopathic and focal, typically beginning during adolescence, affecting specific areas such as the axillae, palms, soles, and craniofacial region, while secondary hyperhidrosis is often linked to underlying medical conditions or medication use. The pathophysiology of primary hyperhidrosis involves hyperactivation of the sympathetic nervous system, which leads to excessive stimulation of eccrine sweat glands. This dysregulation results in an abnormally high baseline level of sweat production and heightened responses to emotional or physical stimuli.

[0050] Hyperhidrosis has profound impacts on patients' quality of life, contributing to emotional distress, social withdrawal, and professional challenges. Despite the availability of treatments, many existing options, including topical antiperspirants, systemic medications, iontophoresis, and botulinum toxin injections, are associated with significant limitations such as temporary effects, discomfort, or adverse outcomes like compensatory sweating. Surgical approaches, such as thoracic sympathectomy, are reserved for severe cases and carry substantial risks.

[0051] In some embodiments, hyperhidrosis is treated with botulinum toxin type A (BTX-A).

[0052] Derived from Clostridium botulinum, BTX-A acts by blocking acetylcholine release at the neuromuscular junction, thereby inhibiting eccrine sweat gland activity and reducing excessive sweating in targeted regions with high concentration of eccrine sweat glands areas such as the underarms, palms, and forehead. The treatment effect typically lasts between three to four months, after which normal gland function gradually resumes. BTX-A injections have demonstrated remarkable efficacy in treating hyperhidrosis, outperforming many alternative therapies. Clinical studies have revealed impressive outcomes, with 93.8% of participants experiencing a 50% reduction in axillary sweating within four weeks of treatment. This high success rate and safety profile similar to placebo, positions botulinum toxin as a leading option for hyperhidrosis management. The treatment offers longer-lasting relief compared to topical solutions, without resorting to more invasive surgical interventions.

[0053] Despite its high efficacy, BTX-A requires localized intradermal injections for clinical application in hyperhidrosis, which are often associated with significant pain - particularly in sensitive regions such as the palms. To mitigate discomfort, various anesthesia techniques have been used, including topical anesthetics, cryoanalgesia, and regional nerve blocks. However, these approaches can lead to side effects such as temporary hand dexterity impairment or mild neuropathies resulting from repeated nerve injury. Additional adverse effects may include transient numbness, paresthesia, and minor hematomas at injection sites.

[0054] These limitations underscore the need for non-invasive alternatives. The use of low- frequency ultrasound to enhance skin permeability and facilitate transdermal delivery of BTX-A, as disclosed herein, is a promising alternative. This approach overcomes the barrier properties of the stratum corneum and enables effective delivery of large biomolecules such as BTX-A.

[0055] The non-invasive method for transdermal delivery of botulinum toxin disclosed herein may be further utilized for cosmetic, therapeutic, or prophylactic purposes across a wide range of indications. Without limitation, such indications include cutaneous manifestations (e.g., skin wrinkles), neuromuscular and neurological disorders, pain syndromes, glandular dysfunction, urological conditions, dermatological and scalp conditions, ophthalmological disorders, gastrointestinal and sphincter-related conditions, respiratory disorders, and emerging therapeutic applications.

[0056] In some embodiments, the disclosed method is directed to the treatment of cosmetic skin conditions, including but not limited to wrinkles, fine lines, and facial rhytides, wherein BTX attenuates neuromuscular activity underlying repetitive facial muscle contractions, thereby reducing the appearance of visible skin creasing and improving dermal smoothness.

[0057] In some embodiments, a disclosed method is applied for treating, alleviating, or preventing neurological disorders which encompass conditions characterized by abnormal nerve and muscle signaling, such as dystonia (involuntary muscle contractions), spasticity (persistent muscle stiffness, frequently following stroke, spinal cord injury, or other neurological insult), tremors (including head and neck tremors), muscle hyperactivity, myoclonus, myokymia, neuromyotonia, and selected presentations of Tourette syndrome. In such cases, BTX functions by blocking presynaptic release of acetylcholine at the neuromuscular junction, thereby reducing involuntary or excessive muscle contractions and restoring functional muscle tone.

[0058] In some embodiments, the disclosed method affords treatment for pain disorders, including chronic migraine, wherein BTX is typically administered to specific anatomical points in the head and neck, tension-type headaches, chronic neck pain of neurological or musculoskeletal origin, myofascial pain, neuropathic pain, fibromyalgia, and postoperative pain. The therapeutic effect of BTX in such indications is mediated through modulation of nociceptive pathways, including inhibition of neurotransmitters and neuropeptides involved in pain transmission (e.g., substance P, calcitonin gene-related peptide), thereby reducing both peripheral sensitization and central pain processing.

[0059] The methods disclosed herein further extend to additional glandular conditions besides hyperhidrosis, including sialorrhea (excessive salivation) and seborrhea (excessive sebaceous gland activity). In these embodiments, BTX decreases hypersecretion by inhibiting cholinergic stimulation of glandular tissues, thereby normalizing secretion rates. BTX may also be applied to excessive drooling associated with neurodegenerative disorders such as Parkinson's disease or amyotrophic lateral sclerosis (ALS).

[0060] In some embodiments, a disclosed method is applied in the treatment of urological and pelvic conditions, including detrusor overactivity, overactive bladder, urinary incontinence secondary to neurological injury, and chronic pelvic pain. In such contexts, BTX reduces detrusor muscle hyperactivity by inhibiting acetylcholine-mediated contraction, thereby alleviating urinary urgency, frequency, and incontinence.

[0061] Utilization of the method disclosed herein further encompasses dermatological conditions, including acne, rosacea, psoriasis, and pruritus. BTX exerts therapeutic benefit in such disorders through modulation of sebum production, suppression of neurogenic inflammation, and regulation of aberrant cutaneous nerve signaling. In related embodiments, BTX delivered by the method disclosed herein may also be applied for the treatment of scalp and hair conditions, including craniofacial hyperhidrosis and alopecia, wherein BTX reduces sweat gland activity and may enhance hair follicle function through neuromuscular and neurovascular pathway modulation.

[0062] In further embodiments, the disclosed method is directed to treatment, alleviation or prevention of ophthalmological disorders, including strabismus (ocular misalignment), blepharospasm (involuntary eyelid spasms), extraocular muscle spasms, and disorders such as dry eye syndrome associated with abnormal neuromuscular activity. BTX, by inducing temporary chemodenervation of overactive ocular muscles, restores ocular alignment, reduces eyelid spasm frequency, and mitigates ocular surface damage.

[0063] In certain gastrointestinal contexts, BTX delivered transdermally according to the non- invasive US-based method disclosed herein may provide benefit in achalasia (a disorder of lower esophageal sphincter relaxation) through reduction of lower esophageal sphincter tone, chronic anal fissures (via relaxation of the internal anal sphincter), gastroesophageal reflux disease (GERD), and selected swallowing disorders. In each case, BTX mediates localized smooth muscle relaxation and reduces aberrant contractility.

[0064] In some embodiments, the disclosed method is applied in treatment, alleviation or prevention of temporomandibular joint (TMJ) disorders, wherein BTX reduces muscle hyperactivity contributing to jaw pain and bruxism, and to laryngospasm and related respiratory conditions, wherein BTX reduces involuntary spasms of the laryngeal musculature and airway hyperreactivity.

[0065] Further, in accordance with the present disclosure, a method disclosed herein may be directed to treatment or alleviation carpal tunnel syndrome (CTS). Carpal tunnel syndrome is caused by compression of the median nerve within the carpal tunnel, leading to pain, numbness, and weakness in the hand. Botulinum toxin type A blocks the presynaptic release of acetylcholine at neuromuscular junctions. When transdermally delivered into muscles or around nerves in the carpal tunnel region, it can reduce local muscle overactivity (e.g., flexor retinaculum-associated tension) that may contribute to nerve compression. BTX-A can alleviate neuropathic pain by modulating neurotransmitter release (e.g., substance P, glutamate) from sensory neurons and improve microcirculation within the tunnel by reducing vasoconstrictive tone, potentially decreasing perineural edema. This multifaceted action could provide both symptom relief and functional improvement in CTS patients. Current treatment of CTS with BTX-A is via direct injection into the carpal tunnel, under ultrasound or electromyographic (EMG) guidance, to ensure precision and minimize risk of nerve injury.

[0066] In some embodiments, a disclosed method is applied for modulating wound healing or scar formation. BTX reduces dynamic tension around wounds (e.g., facial or joint regions), minimizing stress that can widen scars. It can downregulate fibroblast activity and reduce collagen deposition, leading to finer, less hypertrophic scars. BTX improves microcirculation and decreases ischemia at wound edges by reducing vasospasm and modulate anti-inflammatory effects by inhibiting release of neuropeptides (e.g., substance P, CGRP) and cytokines, potentially reducing local inflammation that contributes to poor wound healing and excessive scarring.

[0067] Clinical applications of BTX in preventing, reducing, or mitigating scarring include, for example, post-surgical scars: BTX injections around surgical incisions (especially in plastic and reconstructive surgery) can improve cosmetic outcomes, producing flatter and less noticeable scars; and hypertrophic scars and keloids: BTX may reduce scar thickness and symptoms (e.g., pain, itching). Clinical applications of BTX in wound healing include, for example, treatment of chronic wounds by enhancing perfusion and healing in ischemic ulcers or pressure sores, and treatment of facial wounds, particularly in high-tension areas such as the forehead or around the eyes, where dynamic muscle pull worsens scar appearance.

[0068] Additionally, the non-invasive method for transdermal delivery of BTX may be directed to emerging applications of BTX therapy, including psychiatric indications such as depression and anxiety, as well as additional chronic pain states. Without being bound by theory, such applications may be mediated through BTX's neuromodulatory effects on peripheral nerve terminals and its downstream influence on central nervous system signaling.

[0069] In all embodiments, the conditions described herein may be addressed individually or in combination with BTX delivered transdermally in a controlled, non-invasive manner. The methods disclosed herein thus provide therapeutic efficacy across a broad range of indications while obviating the risks and limitations associated with injectable administration.

[0070] In some embodiments, the non-invasive method of transdermal delivery of BTX, facilitated by ultrasound as disclosed herein, is applied in the field of veterinary medicine for therapeutic, prophylactic, or rehabilitative purposes. The following embodiments are provided by way of non-limiting examples:

[0071] (i) Neuromuscular and musculoskeletal disorders. The disclosed methods may be utilized for the treatment of dystonias, spasticity, or muscle contractures in companion or performance animals, including cats, dogs, and horses. For example, in horses and cats, BTX can be employed to relieve contracted limbs, thereby enabling improved mobility and facilitating rehabilitative therapy.

[0072] (ii) Synovitis and intra-articular inflammation, wherein administration reduces joint pain and lameness. In dogs, for example, BTX may be applied, in accordance with the present disclosure, for osteoarthritis of the hip to thereby facilitate improvement in pain and mobility in certain cases. (iii) Analgesic applications. In other embodiments, BTX is applied as an adjunct for pain management, wherein it functions by reducing the release of pain-related neuropeptides and modulating nociceptive pathways. For example, in canine subjects undergoing mastectomy, BTX administration in accordance with the present disclosure may result in postoperative analgesia and decreased reliance on systemic pain medications.

[0073] (iv) Autonomic and cardiac disorders. In some embodiments, BTX is used to treat cardiac arrhythmias through modulation of autonomic ganglia. For example, BTX administered to atrial ganglionated plexi of dogs can significantly reduce the incidence of atrial fibrillation for periods lasting weeks to months. In further embodiments, BTX is delivered to sympathetic ganglia post- myocardial infarction, wherein it provides cardioprotective effects, including improved cardiac remodeling and reduced arrhythmogenic risk.

[0074] (v) Movement-related disorders. In some embodiments, ultrasound-facilitated transdermal delivery of BTX is applied to conditions characterized by involuntary muscle activity, including myoclonus, myokymia, and neuromyotonia. In canine subjects, BTX treatment can result in improved motor function and suppression of abnormal muscle activity, with therapeutic benefit persisting for several months.

[0075] (vi) Ophthalmic disorders. In some embodiments, BTX is utilized in ophthalmic indications. For example, ultrasound-assisted transdermal delivery may be applied for temporary ptosis induction in dogs, thereby protecting corneal healing following ocular injury or surgery. In other embodiments, BTX may be administered for blepharospasm and other extraocular muscle spasms, wherein repeated applications reduce involuntary eyelid closure and improve visual comfort over extended durations.

[0076] (vii) Respiratory disorders. In some embodiments, BTX is applied for airway hyperresponsiveness. For example, in canine, submucosal BTX administration to bronchial tissue may decrease chemically induced airway hyperreactivity by approximately 60%, with effects persisting up to six months.

[0077] (viii) Gastrointestinal and sphincter disorders. In some embodiments, BTX is applied according to a method disclosed herein to lower esophageal sphincter hypertonicity, such as in achalasia, wherein the BTX reduces smooth muscle tone to improve esophageal relaxation and swallowing. In porcine, for example, BTX may reduce internal anal sphincter tone, demonstrating potential utility for chronic anal fissures and related anorectal dysfunctions. (ix) Urological disorders. In further embodiments, BTX is employed, according to the present disclosure, in urological applications, wherein it modulates detrusor muscle and urethral sphincter activity. In porcine, for example, BTX alters neurochemical patterns of the bladder wall and intramural ganglia, leading to reduced muscle tone and improved bladder function. Additionally, periureteral BTX application may facilitate urolith passage by relaxing smooth muscle and reducing ureteral spasms.

[0078] (x) Reconstructive and surgical assistance. In some embodiments, BTX is applied to enhance outcomes in reconstructive surgery. For example, in minipig, BTX administration may improve myocutaneous flap expansion and reduced contraction, thereby increasing the viability and predictability of reconstructive tissue flaps.

[0079] In all the foregoing embodiments, transdermal delivery of BTX provides therapeutic benefit while obviating the need for direct injections. By combining BTX with ultrasound-assisted permeabilization, the methods disclosed herein allow for controlled, localized, and non-invasive treatment of a diverse range of veterinary conditions, improving animal welfare and expanding clinical utility across multiple species.

[0080] A method disclosed herein may employ BTX formulated in the form of a topical cosmetic formulation or a medicinal formulation.

[0081] As used herein, the term "formulation" refers to a composition comprising one or more active agents in combination with pharmaceutically or cosmetically acceptable excipients, carriers, diluents, stabilizers, or adjuvants, designed to facilitate delivery, stability, bioavailability, and / or efficacy of the active agent when administered to a subject. The term is intended to encompass both cosmetic formulations, which are primarily directed to improving or maintaining the appearance of the skin, hair, or other external tissues, and medicinal formulations, which are intended for therapeutic or prophylactic treatment of diseases or medical conditions.

[0082] Topical cosmetic formulations are compositions of cosmetically active agents designed for application to the skin, scalp, hair, or nails for cosmetic purposes. The cosmetic formulation may further comprise excipients and carriers such as water, oils, emulsifiers, surfactants, stabilizers, preservatives, fragrances, and penetration enhancers suitable for cosmetic use.

[0083] Topical medicinal formulations are compositions of pharmaceutically active agents designed for topical application and intended for diagnosis, treatment, alleviation, or prevention of a disease, disorder, or pathological condition in a subject. Such formulations are subject to regulatory standards for pharmaceutical products.

[0084] A medicinal formulation may include active pharmaceutical ingredients (APIs) such as botulinum toxin, small molecules, antibiotics, antivirals, anti-inflammatories, analgesics, or other therapeutic agents, formulated together with excipients and carriers, including, but not limited to, buffers, isotonic agents, stabilizers, solubilizers, preservatives, viscosity modifiers, and delivery enhancers compatible with pharmaceutical administration.

[0085] The term "therapeutically or cosmetically effective amount", as used herein, refers to an amount of BTX that, when administered to a subject in need thereof, produces a desired therapeutic and / or cosmetic effect.

[0086] A therapeutically effective amount of BTX is an amount sufficient to prevent, reduce, delay, mitigate, stabilize, or eliminate one or more symptoms, signs, or causes of a disease, disorder, syndrome, or condition, or to otherwise provide a clinically meaningful improvement. Such improvement may be subjective (e.g., patient-reported symptom relief) or objective (e.g., measurable physiological or functional change).

[0087] A cosmetically effective amount of BTX is an amount sufficient to produce a perceptible or measurable improvement in a subject's appearance, including but not limited to reduction of wrinkles, fine lines, scars, hyperpigmentation, or other aesthetic imperfections, or enhancement of skin, hair, or tissue quality.

[0088] The therapeutically or cosmetically effective amount may vary depending on factors such as the specific active agent, formulation, route of administration, dosing regimen, severity of the condition, treatment objective, and individual subject characteristics (e.g., age, weight, health status). The term encompasses amounts sufficient to provide partial, temporary, or complete benefit, including prophylaxis, maintenance, or enhancement of the treated condition or cosmetic feature.

[0089] Various embodiments and aspects as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0090] EXAMPLES

[0091] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments in a non-limiting fashion. Materials and Methods

[0092] (1) Preparation of pig skin samples

[0093] Fresh pig ear skin was utilized as a model for the ex vivo experiments. The skin was carefully dissected using a scalpel to obtain a uniform layer, which was then cut into 2x2 cm sections. Samples were used on the day of collection or stored at 2-8°C for no more than 24 hours to ensure integrity. Prior to experimentation, the skin was allowed to reach room temperature for approximately 10 minutes.

[0094] (2) Ex vivo experimental set up

[0095] The permeability of pig skin was assessed using Franz diffusion cells. A Franz diffusion cell comprises two compartments: donor chamber that contains a test formulation (cream, gel, solution, etc.) comprising the active substance / compound(s) to be transdermally delivered, and a receptor chamber filled with a receptor medium (like buffer solution) that collects the permeated compound.

[0096] For testing skin permeability, the stratum corneum of the pig skin was positioned facing the donor compartment, which contained 6 mL of a 1% sodium lauryl sulfate (SLS) solution, and the receiving compartment held 9 mL of phosphate-buffered saline (PBS). The compartments were securely sealed using clamps to prevent leakage.

[0097] Skin conductance measurements were employed to ensure the integrity of the tested pig's skin and to exclude samples with compromised permeability. Ag / AgCI disc electrodes (4 mm diameter) were positioned in both the donor and receptor compartments of a Franz diffusion cell to enable electrical measurements across the mounted skin sample. An AC voltage of 200 mV at 10 Hz was applied using a 33120A Function Generator (Agilent Technologies®), and the resulting current was recorded with a Fluke® 45 Digital Multimeter. From these measurements, skin conductivity was determined as an indicator of barrier integrity. Samples exhibiting conductivity values higher than 0.7 (kO-cm2)-1, which reflect excessive permeability and therefore defective or damaged skin, were excluded from subsequent experiments.

[0098] (3) Ultrasound pre-treatment

[0099] Ultrasound was applied using a QSonica® Q700 Sonicator operating at a frequency of 20 kHz and an acoustic intensity of 8.2 W / cm2. A 1.3 cm diameter probe was positioned 8 mm above the surface of the excised skin mounted in the donor compartment of a Franz diffusion cell. Sonication was delivered in pulsed mode with a 50% duty cycle (1 second on, 1 second off) for a total treatment time of 5 minutes. A 1% sodium lauryl sulfate (SLS) solution served as the coupling medium to enhance acoustic energy transfer. To minimize undesired thermal effects during ultrasound exposure, both the phosphate-buffered saline (PBS) receptor solution and the SLS coupling medium were replaced every 30 seconds.

[0100] Following ultrasound treatment, fluorescently labeled Xeomin® was introduced into the donor compartment. Labeling of Xeomin® was performed using N-succinimidyl ester of 5-(and- 6)-carboxyfluorescein known by the trade name Hyalite™ Fluor 488 (for brevity, the labeled BTX is termed herein "XeominHF488"). The Hyalite™ Fluor 488 Protein Labeling Kit was used in accordance with the manufacturer's instructions, and the final protein concentration was determined to be 0.44 mg / mL by Bradford assay.

[0101] Skin barrier integrity was assessed before and after ultrasound exposure by conductivity measurements across the mounted tissue as described above. Samples exhibiting conductivity values greater than 0.7 (kO-cm2)-1, indicative of compromised or defective skin, were excluded from subsequent analyses.

[0102] After treatment, the skin samples were thoroughly rinsed, embedded in OCT™ compound (Optimal Cutting Temperature compound, Sakura Finetek®), and cryosectioned into 10 pm-thick slices. The sections were analyzed by confocal fluorescence microscopy using a LSM 880 (ZEISS®). Fluorescence images were acquired with an excitation wavelength of 488 nm, and emission was collected within the range of 490-597 nm.

[0103] (4) In vivo studies

[0104] A comprehensive in vivo experimental model was established to evaluate the effect of ultrasound pre-treatment on the transdermal delivery of Xeomin® (Merz Pharmaceuticals) and its subsequent impact on sweat gland activity.

[0105] (a) Device design and application of treatment solutions. A custom-designed fluidretention device was engineered to ensure continuous contact between the treatment solution and the hind paw skin of the mice.

[0106] Initially, a small water balloon was fitted around the ankle joint, extending slightly above it, and filled with 200 pL of solution to completely immerse the paw. The balloon was affixed with a biocompatible biological adhesive, gently secured to prevent leakage while avoiding vascular restriction and tissue necrosis. A waterproof plaster was applied to seal the balloon, and a specialized external device was positioned over the assembly to maintain the natural alignment of the joint, enabling the mice to move freely following recovery from anesthesia. The animals were observed to confirm normal ambulation and ability to perform basic functions prior to the initiation of further treatments.

[0107] (b) Experimental workflow. The experimental workflow consisted of several key stages:

[0108] (i) selection and acclimatization of animals; (ii) ultrasound pre-treatment of the hind paw; (iii) topical application of treatment formulations using a custom-designed fluid-retention device; and (iv) assessment of sweat gland activity via the iodine-starch test.

[0109] (i) Selection of animals

[0110] Female C57BL / 6 mice (8-12 weeks old; 20-25 g body weight) were chosen for their reproducible physiological responses in dermatological and sweat gland studies. Animals were maintained under standard laboratory conditions, including a 12-hour light / dark cycle, controlled temperature (22 ± 2°C), and relative humidity (55 ± 10%). Food and water were provided ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC), and mice were acclimatized for at least 7 days before experimentation to reduce stress and variability.

[0111] (ii) Ultrasound pre-treatment

[0112] For designated groups, ultrasound was applied to enhance skin permeability. The hind paw was placed within a cylindrical treatment chamber filled with 1% sodium lauryl sulfate (SLS) in PBS, which served as a coupling medium. The chamber was sealed with waterproof plaster to prevent leakage.

[0113] Ultrasound was delivered using a QSonica® Q700 Sonicator with a 1.3 cm probe positioned 16 mm from the paw. Parameters included a frequency of 20 kHz, intensity of 10.3 W / cm2, and pulsed operation at a 50% duty cycle (1 second on, 1 second off). Each treatment consisted of three 20-second cycles (1-minute cumulative exposure). To minimize thermal effects, the coupling solution was replaced with freshly prepared SLS / PBS between cycles.

[0114] (iii) Topical application of formulations using a custom-designed fluid-retention device. The customized device described above was utilized for fluid (formulation) application and retention around the mouse foot. In treatment groups, the donor solution contained Xeomin® at 0.125 Units / pL (50 Units per 200 pL). Control groups received either isotonic saline or PBS. Solutions remained in contact with the paw for 6 hours. Device integrity was inspected periodically to confirm proper retention and absence of leakage. (iv) Assessment of sweat secretion: iodine-starch test

[0115] Sweat gland activity was assessed using the iodine-starch test, a standard diagnostic method for visualizing active sweat secretion. In this assay, an iodine solution is applied to the skin surface, followed by starch powder; areas of sweat production are identified by the formation of characteristic dark blue-black complexes upon contact with sweat droplets. This allowed for qualitative visualization and subsequent quantitative analysis of sweat reduction following treatment.

[0116] Sweat secretion was evaluated using a standard heating pad of the iodine-starch test, after which an iodine-ethanol solution (0.1 g / mL) was applied to the paw and allowed to air dry for 1 minute. The paw was then coated with a uniform layer of starch-castor oil suspension (1 g / mL). Subsequent exposure to a heating lamp for 5 minutes further enhanced sweating and facilitated the iodine-starch reaction.

[0117] Sweat-producing sites appeared as characteristic dark blue-black spots on the skin surface, resulting from iodine dissolution by sweat and its subsequent reaction with starch. Paws were photographed under standardized conditions, and images were analyzed with ImageJ™ software (NIH) to quantify the sweating area. Percentage reduction in sweat production was calculated by comparing treated paws with the contralateral untreated control paws.

[0118] (5) Statistics

[0119] Data were analyzed for both ex vivo Xeomin® penetration and in vivo sweat inhibition. Quantitative values are presented as mean ± standard deviation (SD). Group comparisons were performed using one-way or two-way ANOVA in GraphPad Prism® software (version 8, GraphPad Software, LLC). Statistical significance was set at p < 0.05. Levels of significance are denoted as follows: p<0.05 (*), p<0.01 (**), p<0.001 (***), and p<0.0001 (****).

[0120] EXAMPLE 1

[0121] Ex vivo transdermal penetration of XeominHF488

[0122] To assess the transdermal penetration of botulinum toxin, Xeomin® was first fluorescently labeled using the Hyalite™ Fluor 488 Protein Labeling Kit.

[0123] The concentration of the labeled XeominHF488 solution was determined to be 0.44 mg / mL, as measured by the Bradford protein assay. Labeling efficiency was estimated at approximately 70%, ensuring sufficient yield and consistent fluorescence intensity for ex vivo and in vivo studies. The successful preparation of XeominHF488 provided a robust analytical tool to visualize and quantify Xeomin® delivery efficiency, tissue distribution, and penetration depth under different treatment conditions.

[0124] Ex vivo experiments were conducted using Franz diffusion cells with excised porcine skin mounted between the donor and receptor chambers, as described in Materials and Methods. Treatment solutions for three tests groups included PBS (control; group 1) and XeominHF488 applied either without pre-application of US (group 2) or after US pre-treatment (group 3). In each group, 3 skin samples were assessed (n=3).

[0125] After 24 hours, receptor chamber samples were collected and analyzed for fluorescence intensity using a microplate reader (excitation at 488 nm; emission at 523 nm). Calibration curves generated from XeominHF488 dilutions in PBS allowed calculation of absolute concentrations. The results are shown on Fig. 1. The following concentrations were obtained:

[0126] US + XeominHF488 (group 3): 0.021 ± 0.004 mg / mL;

[0127] XeominHF488-only (group 2): 0.007 ± 0.001 mg / mL; and

[0128] PBS control (group 1): 0.006 ± 0.001 mg / mL.

[0129] Clearly, group 3 exhibited a significantly higher transdermal permeation compared to the XeominHF488-only and PBS control groups. Statistical analysis (one-way ANOVA) confirmed a significantly greater permeation in the ultrasound-pretreated group compared to both control groups (**p < 0.01; ***p < 0.001).

[0130] Histological examination of skin cross-sections (10 pm, OCT-embedded) was performed by confocal fluorescence microscopy using LSM 880 (ZEISS®). The results are shown in Figs. 2A- 2C.

[0131] As shown in Fig. 2C, XeominHF488 fluorescence was most prominent in the stratum corneum (SC) and epidermis, with penetration extending into the upper dermis in the ultrasound-pretreated group.

[0132] To complement the qualitative visualization, quantitative fluorescence analysis was performed along defined transects spanning the full depth of the skin tissue. Cryosections (10 pm thickness) were prepared from three independent biological replicates per treatment group (n = 3). For each section, three non-overlapping confocal fluorescence images were acquired, and three transects were measured per image. This approach yielded a total of 27 independent fluorescence measurements for each treatment condition, ensuring robust statistical power. Fluorescence corresponding to XeominHF488 (emission at 523 nm) was recorded at multiple depth points from the stratum corneum through the dermis. Data were averaged within successive 20 pm intervals to generate a continuous depth-dependent distribution profile. The resulting graphs shown in Figs. 3A-3B illustrate the spatial penetration and relative concentration of XeominHF488 across distinct skin layers, enabling direct comparison of delivery efficiency between treatment groups.

[0133] Quantitative fluorescence profiling across the skin depth revealed clear differences in distribution between test groups. As illustrated in Figs. 3A-3B, the US + XeominHF488 group demonstrated substantially higher fluorescence intensity compared to both the XeominHF488- only and PBS control groups. The enhancement was most pronounced in the superficial layers, particularly within the stratum corneum and upper epidermis (0-60 pm), confirming effective permeation across the primary skin barrier.

[0134] Statistical analysis showed highly significant differences (p < 0.0001) throughout all epidermal segments, with penetration extending into the upper dermis (up to ~120 pm). Although fluorescence intensity declined progressively with depth, the US + XeominHF488 group retained significantly elevated values even at 180-200 pm (p < 0.05), indicating that ultrasound facilitated not only barrier disruption but also sustained molecular diffusion into deeper layers.

[0135] These results provide strong evidence that ultrasound pre-treatment enhances transdermal delivery by transiently perturbing the stratum corneum, thereby enabling passive diffusion of large biomolecules. The detection of XeominHF488 fluorescence well beyond the epidermis supports the feasibility of this approach for the non-invasive delivery of macromolecular therapeutics across the full thickness of the skin to physiologically relevant target sites such as sweat glands.

[0136] EXAMPLE 2

[0137] In vivo evaluation of sweat reduction following US-assisted Xeomin® delivery

[0138] The functional efficacy of ultrasound-assisted Xeomin® delivery was assessed in a mouse hind paw model of induced sweating. Treatment solutions were applied for 6 hours using a custom-designed fluid-retention device as described in the Materials and Methods section. Four experimental groups were established to determine the effects of ultrasound pre-treatment and Xeomin® application on sweat gland activity. In all cases, the right hind paw was treated, while the left served as untreated control: (1) Saline group (no ultrasound): 200 pL saline (n = 3);

[0139] (2) Xeomin® group (without US): 200 pL Xeomin® (50 Units), (n = 5);

[0140] (3) Saline + US group: US pre-treatment followed by 200 pL saline (n = 6); and

[0141] (4) Xeomin® + US group: US pre-treatment followed by 200 pL Xeomin® (50 Units) (n = 5).

[0142] The percentage reduction in sweat secretion was evaluated using the iodine-starch assay, followed by image analysis with ImageJ software. The results are shown in Figs. 4A-4B.

[0143] Representative images of the hind paws after iodine-starch staining shown in Fig. 4A demonstrated sweat-secreting regions as dark areas. The corresponding processed images, in which sweat-secreting regions are displayed in black and non-sweating areas in white, as generated by ImageJ analysis (binary conversion with ImageJ™ software (NIH)) are shown in Fig. 4B. This visual representation enabled quantitative analysis of sweating areas.

[0144] As shown in Fig. 5, a statistically significant reduction in sweating area was observed only in the group that received ultrasound pre-treatment followed by six hours of topical application of Xeomin®. The reduction was highly significant compared to the saline group (p < 0.0001), the US-only group (p < 0.001), and the Xeomin®-only group (p < 0.001). Neither ultrasound nor Xeomin® alone produced a comparable effect, demonstrating that the combined approach is necessary to achieve a meaningful biological outcome.

[0145] Throughout the experiment, no burns, swelling, or visible tissue damage were detected in the US-pretreated groups. Moreover, the custom-designed fluid-retention device maintained solution contact without impairing paw mobility or normal function during the six-hour application period.

[0146] These in vivo results are consistent with the ex vivo findings, where transdermal penetration of Xeomin® was confirmed by fluorescence labeling and histological imaging. The observed reduction in sweat secretion indicates that the neurotoxin successfully reached the upper dermis or superficial subcutaneous layer, where eccrine sweat glands are located and carry out their secretory function. This observation is consistent with the known mechanism of botulinum toxin type A, which inhibits acetylcholine release at the neuromuscular junction of sweat glands. Collectively, these findings support the hypothesis that ultrasound pre-treatment transiently disrupts the stratum corneum - most likely through acoustic cavitation - thereby enabling enhanced non-invasive transdermal delivery of macromolecules such as Xeomin®. The ability to achieve sweat gland inhibition without injections demonstrates the therapeutic potential of this approach for conditions such as hyperhidrosis.

Claims

1. WHAT IS CLAIMED IS:

1. A non-invasive method for transdermal delivery of botulinum toxin (BTX), the method comprising the steps of:(a) applying ultrasound to a target skin region of a subject in need thereof for a cumulative treatment duration of from about 3 sec to about 10 min; and(b) topically administrating to the ultrasound-treated skin region a therapeutically and / or cosmetically effective amount of BTX, thereby transdermally delivering BTX to the subject.

2. A method for treating, alleviating or preventing a condition or disorder that may benefit form transdermal delivery of botulinum toxin (BTX), the method comprising the steps of:(a) applying ultrasound to a target skin region of a subject in need thereof for a cumulative treatment duration of from about 3 sec to about 10 min; and(b) topically administrating to the ultrasound-treated skin region a therapeutically effective amount of BTX, thereby treating, alleviating or preventing a condition or disorder that may benefit form transdermal delivery of botulinum toxin in the subject.

3. The method of claim 2, wherein the condition or disorder that may benefit form transdermal delivery of botulinum toxin is selected from cutaneous manifestations, neuromuscular and neurological disorders, pain syndromes, glandular dysfunction, urological conditions, dermatological and scalp conditions, ophthalmological disorders, gastrointestinal and sphincter-related conditions and / or respiratory disorders.

4. A method for exerting a therapeutic effect selected from reducing sweat gland activity, relaxing muscle activity and / or alleviating pain, the method comprising the steps of:(a) applying ultrasound to a target skin region of a subject in need thereof for a cumulative treatment duration of from about 3 sec to about 10 min; and(b) topically administrating to the ultrasound-treated skin region a therapeutically effective amount of BTX,thereby exerting said therapeutic effect in the subject.

5. The method of any one of claims 2 to 4, for treating, alleviating or preventing one or more of:(i) primary or secondary hyperhidrosis;(ii) cosmetic skin conditions selected from wrinkles, fine lines, and facial rhytides;(iii) a neuromuscular disorder selected from dystonia (involuntary muscle contractions), spasticity (persistent muscle stiffness), tremor, muscle hyperactivity and Tourette syndrome;(iv) a pain disorder selected from chronic migraine, tension-type headaches, chronic neck pain of neurological or musculoskeletal origin, myofascial pain, neuropathic pain or postoperative pain;(v) a glandular condition selected from seborrhea (excessive salivation) or seborrhea (excessive sebaceous gland activity);(vi) an urological or pelvic condition selected from detrusor overactivity, overactive bladder, urinary incontinence due to neurological damage or chronic pelvic pain;(vii) a dermatological condition selected from acne, rosacea, psoriasis, or pruritus;(viii) a scalp or hair condition selected from excessive scalp sweating (craniofacial hyperhidrosis) or alopecia;(ix) a headache disorder selected from chronic migraine, severe tension-type headaches or chronic neck pain;(x) an ophthalmic disorder selected from strabismus (ocular misalignment), blepharospasm (involuntary eyelid spasms), extraocular muscle spasms, or dry eye syndrome;(xi) gastrointestinal and sphincter disorders selected from achalasia (affecting lower esophageal muscles), chronic anal fissures, gastroesophageal reflux disease (GERD) or swallowing disorders;(xii) a neurological disorder selected from myoclonus, myokymia, or neuromyotonia;(xiii) an autonomic or cardiac disorder selected from cardiac arrhythmias, atrial fibrillation or myocardial infarction;(xiv) carpal tunnel syndrome (CTS);(xv) wound healing and scar modulation; and / or(xvi) other condition selected from temporomandibular joint (TMJ) disorders (jaw pain and teeth grinding), fibromyalgia, laryngospasm (vocal cord spasms), airwayhyperresponsiveness, synovitis and intra-articular inflammation, depression and anxiety, or excessive drooling in Parkinson's or ALS patients.

6. The method of claim 5, applied for therapeutic, prophylactic, or rehabilitative purposes in humans or animals.

7. The method of any one of claims 1, 2 and 4, wherein steps (a) and (b) are repeated one or more times in the same or a different target skin region, delivering the same or different amounts of BTX.

8. The method of any one of claims 1 to 7, wherein the ultrasound is applied at a frequency of about 20-200 kHz.

9. The method of any one of claims 1 to 8, wherein the ultrasound is delivered at an intensity of about 1.5-20 W / cm2, optionally about 5-12 W / cm2.

10. The method of any one of claims 1 to 9, wherein the ultrasound is applied in pulsed mode with a duty cycle of about 10-70%, optionally about 50%.

11. The method of any one of claims 1 to 9, wherein the ultrasound is applied in a continuous mode.

12. The method of any one of claims 1 to 11, wherein the ultrasound is applied for a period of from about 5 sec to about 7 min.

13. The method of any one of claims 1 to 12, wherein the botulinum toxin is of one or more of BTX type A, B, C, D, E, F or G.

14. The method of claim 13, wherein the botulinum toxin is BTX type A or type B.

15. The method of any one of claims 1 to 14, wherein the botulinum toxin is fluorescently labeled to monitor transdermal penetration.

16. The method of claim 15, wherein the botulinum toxin is BTX type A labeled with N- succinimidyl ester of 5-(and-6)-carboxyfluorescein.

17. The method of any one of claims 1 to 16, wherein the botulinum toxin is formulated as a topical cosmetic or medicinal formulation.

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