WO2019243376A1

WO2019243376A1 - Intramuscular injection of botulinum toxin for the treatment of vulvodynia

Info

Publication number: WO2019243376A1
Application number: PCT/EP2019/066108
Authority: WO
Inventors: Philippe Picaut; France CATUS; Andrew Goldstein
Original assignee: Ipsen Biopharm Limited
Priority date: 2018-06-18
Filing date: 2019-06-18
Publication date: 2019-12-26

Abstract

Method of treating one or more symptoms of vulvodynia in a subject, comprising administering to the subject a therapeutically-effective amount of botulinum neurotoxin. Composition for use in treating one or more symptoms of vulvodynia in a subject comprising botulinum neurotoxin. Computer system programmed to receive information related to a subject's response to administration of botulinum neurotoxin, store that response in a database, and transmit the response to a medical practitioner. Non-transitory computer-readable storage medium storing instructions that, when executed by a computer system, causes the computer system to perform the aforementioned steps. Intravaginal device for use in treating one or more symptoms of vulvodynia, the device configured to administer botulinum neurotoxin to a subject in need of such treatment.

Description

INTRAMUSCULAR INJECTION OF BOTULINUM TOXIN FOR THE

TREATMENT OF VULVODYNIA

Field of the Invention

The present invention relates generally to treating one of more symptoms of vulvodynia, and more particularly, to a method of treating one or more symptoms of vulvodynia using botulinum neurotoxin. The invention also relates to a composition comprising botulinum neurotoxin that is useful in treating one or more symptoms of vulvodynia. The invention further relates to a computer system programmed to receive information related to a subject’s response to administration of botulinum neurotoxin, store that response in a database, and transmit the response to a medical practitioner. The invention additionally relates to a non-transitory computer-readable storage medium storing instructions that, when executed by a computer system, causes the computer system to perform the aforementioned steps. The invention also relates to a computer-implemented method of determining a dosage regimen for the administration of botulinum neurotoxin. The invention also relates to a device for use in treating one or more symptoms of vulvodynia, the device configured to administer botulinum neurotoxin to a subject in need of such treatment.

Background

Vulvodynia is chronic vulvar pain in the absence of objective abnormalities such as infection or dermatoses. Harlow et al., Journal of the American Medical Women’s Association, 58:82—88, 82 (2003); Falsetta at 210. As many as 28% of women suffer from vulvodynia at some point in their lives. Pukall et al., J. Sex Med., 13:291—304, 292 (2016); Falsetta et al., British Journal of Obstetrics and Gynaegology, 124:210-218, 210 (2017). Vulvodynia is often characterized as either: localized vulvodynia, which is pain localized at a particular vulvar site; or generalized vulvodynia, which involves unprovoked, diffuse vulvar pain affecting the entire vulvar area (Pukall et al. at pages 291— 292) . Vestibulodynia is localized vulvodynia involving pain at the vaginal opening. In cases wherein such pain occurs only with touch or pressure, the vestibulodynia is termed provoked vestibulodynia (PVD). PVD is the most common form of vulvodynia. Vulvodynia can cause sexual dysfunction and affective disorders such as depression and anxiety.

The cause of vulvodynia is unknown, but it is believed that aberrant nociception, site- specific inflammatory responses, and/or muscle over-activity have roles in the disorder. Yoon et al, International Journal of Impotence Research, 19:84—87, 86 (2007); Falsetta et al, Abstract, 211—212; Reissing et ak, Psychosom. Obstet. Gynaecol. 26:107—113 (2005); Pelletier, Br. J. Oermatolog , 164:617—622

(2011).

It has been found that botulinum neurotoxin may be effective in treating one or more symptoms of vulvodynia (Yoon et al. at page 84). Botulinum neuro toxin acts on the presynaptic motor neurons and inhibits the release of acetyl choline. This leads to a reduction in muscle tone. In addition, it is believed that botulinum neurotoxin acts by suppressing the release of nociceptive agents and inflammatory agents, such as substance P. Yoon at 86; Falsetta at 211.

Botulinum neurotoxin is produced by Clostridium botulinum in the form of a large protein complex comprising botulinum neurotoxin itself complexed to a number of accessory proteins. There are at present at least nine different classes of botulinum neurotoxin, namely: BoNT serotypes A, B, C, D, E, F, G, H and X (known respectively as BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H and BoNT/X), all of which share similar structures and modes of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing anti-sera, with such classification by serotype correlating with percentage sequence identity at the amino acid level. Botulinum neurotoxin proteins of a given serotype are further divided into different subtypes on the basis of amino acid percentage sequence identity.

Different serotypes have different substrate specificities: BoNT/A and BoNT/E cleave SNAP -25, serotypes /B, /D, /F and /G cleave synaptobrevin/VAMP. BoNT/C cleaves SNAP -25 and syntaxin 1, syntaxin2 and syntaxin 3. BoNT/X has been found to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP 5, Ykt6.

BoNT/A is commercially available from, eg., Ipsen (DYSPORT®, Ipsen Limited, Slough, UK), Allergan (BOTOX®, Allergan Inc., Irvine, CA, USA), Merz Pharma (XEOMIN®, Merz Pharma GmbH, Frankfurt am Main, Germany), and Medytox (CORETOX®, Medytox Inc., Seoul, South Korea), whereas botulinum neurotoxin type B is sold by Elan (NYOBLOC®/NEUROBLOC®, Solstice Neurosciences Inc., San Diego, CA, USA).

Topical application of anaesthetic or hormonal creams require daily administration prior to the pain-trigger event. Even then, previous placebo-controlled trials have not demonstrated any benefit to the use of treatments. Orally administered drugs, particularly antidepressants and anticonvulsants have also failed to provide any clinical benefit in placebo-controlled trials. In addition, such drugs have systemic side effects such as somnolence and fatigue which limits there clinical potential. While treatment of the symptoms of vulvodynia with botulinum neurotoxin has been described, with some positive case series and reports (Goldstein et al. J. Sex. Med. (2016) 13:572- 590), the only previous double-blinded controlled trials showed treatment with botulinum neurotoxin to be statistically indistinguishable from the placebo controls (Petersen et al. J. Sex. Med. (2009) 6:2523-2537 and Diomande et al. Arch. Gynecol. Obstet.. (2019) 299(4):993-1000). Accordingly, there remains a need to determine the most efficacious target muscles (or combinations thereof) in order to achieve a clinically relevant result and improve treatment outcomes whilst limiting clinically undesirable side effects.

Summary of the Invention

The inventors have developed a method of treating symptoms of vulvodynia by the administration of a botulinum toxin. In particular, the inventors have developed a comprehensive injection paradigm which targets the specific muscles involved in provoked vestibulodynia (PVD) and which can comprise multiple injections to said muscles to ensure diffusion of the botulinum neurotoxin through said muscles and hence improved treatment efficacy. In addition, by specifically targeting only the muscles involved in PVD, and dyspareunia in particular, this reduces the potential for undesirable side effects such as incontinence of stool, flatus or urine.

Accordingly, a method is provided for treating one or more symptoms of vulvodynia. The method comprises administering a therapeutically-effective amount of a botulinum neurotoxin to a subject determined to be in need of such treatment, wherein the botulinum neurotoxin is administered to the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and/or the right deep transverse perineal muscle. The botulinum toxin may be administered in an amount from about 200 to about 800 units per treatment session.

The subject may suffer from, for example, pain at the vaginal vestibule, chronic vulval pain, sexual dysfunction, affective disorder (eg, depression and/or anxiety), and/or provoked vestibulodynia (PVD). The botulinum neurotoxin may, for example, have at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99%, preferably 80%, 90%, 95%, 97%, 98%, or 99%, sequence identity to the amino acid sequence of a botulinum neurotoxin of serotypes A, B, C, D, E, F, G, H, or X. The use of recombinant, hybrid and chimeric botulinum toxins is also encompassed. In preferred embodiments, the botulinum neurotoxin is botulinum neurotoxin A, even more preferably the botulinum toxin is DYSPORT®.

In the present invention, botulinum neurotoxin may be administered to the bladder and/ or to the pelvic floor.

Typically, botulinum neurotoxin is administered to the muscles of the pelvic floor. Preferably, botulinum neurotoxin is administered to each of the: right and left bulbospongiosus muscles; right and left pubococcygeous muscles; right and left superficial transverse perineal muscles; and the right and left deep transverse perineal muscles. It has surprisingly been found that administration of botulinum neurotoxin to these muscles and/ or combination of muscles provides additional efficacy in treating the symptoms of vulvodynia. In particularly preferred embodiments, the botulinum neurotoxin is administered in the following order: (1) to a first bulbospongiosus muscle, anterior to the hymeneal ring, (2) to a first pubococcygeous muscle, anterior to the hymeneal ring; (3) to the first bulbospongiosus muscle, posterior to the hymeneal ring; (4) to the first pubococcygeous muscle, posterior to the hymeneal ring; (5) to a second bulbospongiosus muscle, anterior to the hymeneal ring, (6) to a second pubococcygeous muscle, anterior to the hymeneal ring; (7) to the second bulbospongiosus muscle, posterior to the hymeneal ring; (8) to the second pubococcygeous muscle, posterior to the hymeneal ring; (9) to the transverse perineal muscles anterior to the hymeneal ring.

The amount of botulinum neurotoxin administered and/ or a dosing regimen for botulinum neurotoxin may be, at least in part, based on the severity of the vulvodynia, the weight of the subject, and/ or the subject’s previous experience with treatment with botulinum neuro toxin.

In an embodiment, the subject’s response to treatment with botulinum neurotoxin is measured following an administration of botulinum neurotoxin. Exemplary methods for assessing the subject’s response to treatment are described herein. Thereafter, the amount of a subsequent administration of botulinum neurotoxin and/or a dosing regimen for botulinum neurotoxin is determined based on the subject’s response. The subject’s response may be recorded, for example, onto a computer device.

Accordingly, the method for treating one or more symptoms of vulvodynia according to the present invention may further comprise determining a dosing regimen for botulinum neurotoxin or the amount and/ or frequency for a subsequent administration of botulinum neurotoxin based on the subject’s response to a previous administration of botulinum neurotoxin.

According to the method for treating one or more symptoms of vulvodynia according to the present invention, the amount of an administration of botulinum neurotoxin or a dosing regimen for botulinum neurotoxin may be determined in part by the level of pain measured following the insertion of a probe into the subject’s vagina. In particular, a probe may be inserted into the subject’s vagina and, if the level of pain measured following insertion of the probe is below a threshold level, a larger sized probe is inserted and the level of pain measured and, if the level of pain measured remains below the threshold level, probe(s) of increasing size are inserted until the level of pain measured following such insertion is at or above the threshold level, and the amount of botulinum neurotoxin to administer or the dosing regimen for botulinum neurotoxin is determined based on the level of pain measured following the insertion of the final probe.

The method of treating one or more symptoms of vulvodynia according to the present invention may comprise a treatment session in which BoNT/A is administered to a female subject diagnosed with vulvodynia, the method comprising the following injection protocol: (a) injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s left bulbospongiosus muscle, anterior to the hymeneal ring, wherein the injection is made at about the 5 o’clock position at about 45° laterally and about 45° posteriorly, towards the hip, and at a depth of about 5 mm from the outer surface of the skin; (b) injecting about 8% of the total dose BoNT/A administered during the treatment session to the subject’s left pubococcygeous muscle, anterior to the hymeneal ring, wherein the injection is made at the same position as the first injection but at a depth of about 10 mm from the outer surface of the skin; (c) injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s left bulbospongiosus muscle, posterior to the hymeneal ring, wherein the injection is made at about the 5 o’clock position at about 45° laterally and about 45° posteriorly, towards the hip, and at a depth of about 5 mm from the outer surface of the skin; (d) injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s left pubococcygeous muscle, posterior to the hymeneal ring, wherein the injection is made at the same position as the previous injection but at a depth of about 10 mm from the outer surface of the skin; (e) injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s right bulbospongiosus muscle, anterior to the hymeneal ring, wherein the injection is made at about the 7 o’clock position at about 45° laterally and about 45° posteriorly, towards the hip, and at a depth of about 5 mm from the outer surface of the skin; (f) injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s right pubococcygeous muscle, anterior to the hymeneal ring, wherein the injection is made at the same position as the previous injection but at a depth of about 10 mm from the outer surface of the skin; (g) injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s right bulbospongiosus muscle, posterior to the hymeneal ring, wherein the injection is made at about the 7 o’clock position at about 45° laterally and about 45° posteriorly, towards the hip, and at a depth of about 5 mm from the outer surface of the skin; (h) injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s right pubococcygeous muscle, posterior to the hymeneal ring, wherein the injection is made at the same position as the previous injection but at a depth of about 10 mm from the outer surface of the skin; and (i) injecting about 36% of the total dose of BoNT/A administered during the treatment session to the subject’s transverse perineal muscles, anterior to the hymeneal ring, wherein: about 18% is administered at about the 6 o’clock position at about 45° laterally and about 45° posteriorly, towards the coccyx, and at a depth of about 5 mm from the outer surface of the skin, about 18% is administered at about the 6 o’clock position at about 45° laterally and about 45° posteriorly, towards the coccyx, and at a depth of about 10 mm from the outer surface of the skin; and wherein, after the foregoing injection protocol, the female subject experiences a lessening or elimination of one or more symptoms of vulvodynia.

The invention also relates to a computer system programmed to perform steps of a computer-implemented method, the method comprising: receiving information related to a subject’s response to treatment with botulinum neurotoxin according to the invention; storing the response information into a database; and transmitting the response information to a medical practitioner. The computer system may comprise: a first computer device that is programmed to receive the response information; a second computer device on which the database resides; and a third computer device which receives the transmitted response information. Said system may be capable of determining a subsequent dosing regimen for the administration of botulinum neurotoxin to the subject.

The invention further relates to a non-transitory computer-readable storage medium storing instructions that, when executed by a computer system, causes the computer system to perform steps of a computer-implemented method, the method comprising: receiving information related to a subject’s response to treatment with botulinum neurotoxin according to the present invention; storing the response information into a database; and transmitting the response information to a medical practitioner.

The invention further provides a computer-implemented method of determining a dosage regimen for administration of botulinum neurotoxin, the method comprising: receiving information related to a subject’s response to treatment as conducted according to a method for treating one or more symptoms of vulvodynia, the method comprising administering a therapeutically-effective amount of botulinum neuro toxin to a subject determined to be in need thereof, wherein the botulinum neurotoxin is administered to the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and/ or the right deep transverse perineal muscle; receiving the response information; and determining a subsequent dosing regimen for the administration of botulinum neurotoxin to the subject based on the response information. Said method may further comprise sending an instruction to administer botulinum neurotoxin accordingly. The invention also provides a computer-readable medium comprising instructions which, when executed by a computer, cause the computer to carry out said method; and a data processing device comprising a processor configured to perform said method.

The invention further provides a device for use in treating a symptom of vulvodynia, the device configured to perform the method of the invention. Said device may be connected to a computer device which, based on information related to the subject’s response to treatment, determines a subsequent or optimal dosing regimen for the administration of botulinum neurotoxin to the subject and instructs the device to adjust its administration of botulinum neurotoxin accordingly

Additionally, the invention relates to a composition for use in treating one or more symptoms of vulvodynia, the composition comprising a therapeutically-effective amount of botulinum neurotoxin. The botulinum neurotoxin may, for example, have at least about 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to the amino acid sequence of a botulinum neurotoxin of serotypes A, B, C, D, E, F, G, H, or X. In an embodiment, the botulinum neurotoxin is DYSPORT®.

The invention provides a method for treating one or more symptoms of vulvodynia, the method comprising administering a therapeutically-effective amount of botulinum neurotoxin to a subject determined to be in need thereof, wherein the botulinum neurotoxin is administered to the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and/ or the right deep transverse perineal muscle. In some embodiments, the amount of botulinum neurotoxin administered is from about 200 to about 800 units per treatment session. The botulinum neurotoxin may be administered in the following order: (1) to a first bulbospongiosus muscle, anterior to the hymeneal ring, (2) to a first pubococcygeous muscle, anterior to the hymeneal ring; (3) to the first bulbospongiosus muscle, posterior to the hymeneal ring; (4) to the first pubococcygeous muscle, posterior to the hymeneal ring; (5) to a second bulbospongiosus muscle, anterior to the hymeneal ring, (6) to a second pubococcygeous muscle, anterior to the hymeneal ring; (7) to the second bulbospongiosus muscle, posterior to the hymeneal ring; (8) to the second pubococcygeous muscle, posterior to the hymeneal ring; (9) to the transverse perineal muscles anterior to the hymeneal ring. The botulinum neurotoxin may have at least about 80 % sequence identity to the amino acid sequence of a botulinum neurotoxin of serotypes A, B, C, D, E, F, G, or H. In some preferred embodiments, the botulinum neurotoxin is BoNT/A. The subject may suffer from pain at the vaginal vestibule. In some embodiments, the symptom is an affective disorder. In some embodiments, the symptom is depression or anxiety. The amount of botulinum neurotoxin administered may be determined based in part on the level of severity of the vulvodynia. Said method may further comprises determining a dosing regimen for botulinum neurotoxin or the amount and/or frequency for a subsequent administration of botulinum neurotoxin based on the subject’s response to a previous administration of botulinum neurotoxin. The amount of an administration of botulinum neurotoxin or a dosing regimen for botulinum neurotoxin may be determined in part by the level of pain measured following the insertion of a probe into the subject’s vagina. In some embodiments, a probe is inserted into the subject’s vagina and, if the level of pain measured following insertion of the probe is below a threshold level, a larger sized probe is inserted and the level of pain measured and, if the level of pain measured remains below the threshold level, probe(s) of increasing size are inserted until the level of pain measured following such insertion is at or above the threshold level, and the amount of botulinum neurotoxin to administer or the dosing regimen for botulinum neurotoxin is determined based on the level of pain measured following the insertion of the final probe. Said method may further comprise recording the subject’s response to an administration of botulinum neurotoxin. In some embodiments, the method may comprise a treatment session in which BoNT/A is administered to a female subject diagnosed with vulvodynia, the method comprising the following injection protocol: injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s left bulbospongiosus muscle, anterior to the hymeneal ring, wherein the injection is made at about the 5 o’clock position at about 45° laterally and about 45° posteriorly, towards the hip, and at a depth of about 5 mm from the outer surface of the skin; injecting about 8% of the total dose BoNT/A administered during the treatment session to the subject’s left pubococcygeous muscle, anterior to the hymeneal ring, wherein the injection is made at the same position as the first injection but at a depth of about 10 mm from the outer surface of the skin; injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s left bulbospongiosus muscle, posterior to the hymeneal ring, wherein the injection is made at about the 5 o’clock position at about 45° laterally and about 45° posteriorly, towards the hip, and at a depth of about 5 mm from the outer surface of the skin; injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s left pubococcygeous muscle, posterior to the hymeneal ring, wherein the injection is made at the same position as the previous injection but at a depth of about 10 mm from the outer surface of the skin; injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s right bulbospongiosus muscle, anterior to the hymeneal ring, wherein the injection is made at about the 7 o’clock position at about 45° laterally and about 45° posteriorly, towards the hip, and at a depth of about 5 mm from the outer surface of the skin; injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s right pubococcygeous muscle, anterior to the hymeneal ring, wherein the injection is made at the same position as the previous injection but at a depth of about 10 mm from the outer surface of the skin;injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s right bulbospongiosus muscle, posterior to the hymeneal ring, wherein the injection is made at about the 7 o’clock position at about 45° laterally and about 45° posteriorly, towards the hip, and at a depth of about 5 mm from the outer surface of the skin; injecting about 8% of the total dose of BoNT/A administered during the treatment session to the subject’s right pubococcygeous muscle, posterior to the hymeneal ring, wherein the injection is made at the same position as the previous injection but at a depth of about 10 mm from the outer surface of the skin; and injecting about 36% of the total dose of BoNT/A administered during the treatment session to thd subject’s transverse perineal muscles, anterior to the hymeneal ring, wherein: about 18% is administered at about the 6 o’clock position at about 45° laterally and about 45° posteriorly, towards the coccyx, and at a depth of about 5 mm from the outer surface of the skin, about 18% is administered at about the 6 o’clock position at about 45° laterally and about 45° posteriorly, towards the coccyx, and at a depth of about 10 mm from the outer surface of the skin; and wherein, after the foregoing injection protocol, the female subject experiences a lessening or elimination of one or more symptoms of vulvodynia.

The invention further provides a computer system programmed to perform steps of a computer-implemented method, the method comprising: receiving information related to a subject’s response to treatment as conducted according to the method of the invention; storing the response information in a database; and transmitting the response information to a medical practitioner. In some embodiments, the response information is received by the computer system from a first computer device, the database resides on a second computer device, and the response information is transmitted by the computer system to a third computer device. In some embodiments, the system is capable of determining a subsequent dosing regimen for the administration of botulinum neuro toxin to the subject.

The invention further provides a non-transitory computer-readable storage medium storing instructions that, when executed by a computer system, causes the computer system to perform steps of a computer-implemented method, the method comprising: receiving information related to a subject’s response to treatment as conducted according to the method of the invention; storing the response information into a database; and transmitting the response information to a medical practitioner.

The invention also provides a device for use in treating a symptom of vulvodynia, the device configured to perform the method of any one of the invention. In some embodiments, the device is connected to a computer device which, based on information related to the subject’s response to treatment, determines a subsequent or optimal dosing regimen for the administration of botulinum neurotoxin to the subject and instructs the device to adjust its administration of botulinum neurotoxin accordingly.

Description of the Drawings

Figure 1 depicts injection sites for administering botulinum neurotoxin to treat one or more symptoms of vulvodynia: (A) is a site for injecting botulinum neurotoxin to the left bulbospongiosus and left pubococcygeous muscles, anterior to the hymeneal ring; (B) is a site for injecting botulinum neurotoxin to the left bulbospongiosus and left pubococcygeous muscles, posterior to the hymeneal ring; (C) is a site for injecting botulinum neurotoxin to the right bulbospongiosus and right pubococcygeous muscles, anterior to the hymeneal ring; (D) is a site for injecting botulinum neurotoxin to the right bulbospongiosus and right pubococcygeous muscles, posterior to the hymeneal ring; and (E) is a site for injecting botulinum neuro toxin to the left and right superficial transverse perineal muscles and to the left and right deep transverse perineal muscles (all four muscles are administered at this injection site).

Detailed Description of the Invention

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

Described herein is a method for treating one or more symptoms of vulvodynia comprising administering a therapeutically-effective amount of botulinum neurotoxin to a subject determined to be in need of such treatment, such as a female mammal (e.g. human) diagnosed with vulvodynia.

It is to be understood that the present invention is not limited to the embodiments described herein. Indeed, numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein will be employed in practicing the invention. In describing the invention, where a range of values is provided with respect to an embodiment, it is understood that each intervening value is encompassed within the embodiment.

Described herein is a method for treating one or more symptoms of vulvodynia in a subject determined to be in need thereof. The method comprises administering to the subject a therapeutically-effective amount of botulinum neurotoxin. A“subject” as used herein refers to a mammal, e.g. a primate, preferably a human, in need of such treatment. A subject may be adult or juvenile. For example, the subject may be a female human patient.. The terms, "individual," "patient" and "subject" are used interchangeably herein.

A "subject in need" of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

The invention provides a method for treating one or more symptoms of vulvodynia, the method comprising administering a therapeutically-effective amount of botulinum neurotoxin to a subject determined to be in need thereof, wherein the botulinum neurotoxin for administration to the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and/ or the right deep transverse perineal muscle.

The invention also provides a botulinum neurotoxin for use in a method for treating depression and/or anxiety, the method comprising administering a therapeutically-effective amount of botulinum neurotoxin to a subject determined to be in need thereof, wherein the botulinum neurotoxin for administration to the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and/ or the right deep transverse perineal muscle. Said method may optionally comprise a step of assessing said subject for one or more symptom of depression and/or anxiety before treatment, after treatment or before and after treatment. Any appropriate method or technique for assessing one or more symptom of depression and/ or anxiety may be used. Suitable methods/ techniques are known in the art, as non limiting examples, the Patient Health Questionnaire-9 (PHQ-9) or Beck Depression Inventory (BDI) may be used to assess one or more symptom of depression and the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), may be used to assess one or more symptom of anxiety. Any/ ll of the disclosure herein in relation to the treatment of vulvodynia using a botulinum neurotoxin applies equally and unreservedly to the treatment of depression and/or anxiety using botulinum neurotoxin. In other words, any features disclosed in relation to a method of treating vulvodynia should be considered equally individualized/disclosed in the context of treating depression and/or anxiety.

To "treat," as used here, means to deal with medically. It includes, for example, administering a botulinum neurotoxin according to the invention to prevent one or more symptom of vulvodynia or to lessen the severity of said symptom(s). As compared with an equivalent untreated control, “treating” or ameliorating one or more symptom of vulvodynia may reduce the severity of said symptom(s) by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. Examples of vulvodynia and pain perception assessment techniques, methods, parameters and standards are described herein.

The administration of botulinum neurotoxin is typically performed by a medical practitioner. As used herein, the term“medical practitioner” includes a clinician, physician, nurse, medical technician, or the like. However, in some embodiments, the subject may self-administer the botulinum neurotoxin.

In nature, botulinum neurotoxins are synthesised as a single-chain polypeptide that is modified post-translationally by a proteolytic cleavage event to form two polypeptide chains joined together by a disulphide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site that is located between the cysteine residues that provide the inter-chain disulphide bond. It is this di-chain form that is the active form of the toxin. The two chains are termed the heavy chain (H-chain, HC), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain or LC), which has a molecular mass of approximately 50 kDa. The H-chain comprises an N-terminal translocation component (H_N domain) and a C-terminal targeting component (H_c domain). The cleavage site is located between the L-chain and the translocation domain components. Proteolytic activation is of botulinum neuro toxins is crucial because after receptor binding and internalisation by endocytosis, subsequent acidification of the endosome is believed to cause the a conformational change in the protein, leading to insertion of the H_N domain into the endosomal membrane, formation of a translocation pore and delivery of the L-chain into the cytoplasm, where the di-sulphide bond is reduced and the L-chain released. Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin)— see Gerald K (2002) "Cell and Molecular Biology” (4th edition) John Wiley & Sons, Inc. The acronym SNARE derives from the term Soluble NSF Attachment Receptor, where NSF means N-ethylmaleimide -Sensitive Factor. SNARE proteins are integral to intracellular vesicle fusion, and thus to secretion of molecules via vesicle transport from a cell. The protease function is a zinc-dependent endopeptidase activity and exhibits a high substrate specificity for SNARE proteins. The protease is described as non-cytotoxic, as it does not kill the cell in which it acts. Accordingly, once delivered to a desired target cell, the non- cytotoxic protease is capable of inhibiting cellular secretion from the target cell. The L-chain proteases of botulinum neurotoxins are non-cytotoxic proteases that cleave SNARE proteins.

The botulinum neurotoxin-producing strain is preferably Clostridium botulinum, but is not limited thereto, and it will be apparent to those skilled in the art that any strain capable of producing a botulinum neurotoxin may be used in the present invention. As used herein, the term“botulinum neurotoxin” is meant to include not only a neurotoxin produced biologically, but also any modified, recombinant, hybrid, fusion, and chimeric botulinum neurotoxins. A modified or recombinant botulinum neurotoxin may, for example, contain modifications as compared to a wild-type botulinum neurotoxin ( e.g. in its amino acid sequence) so as to have similar or better properties than the wild-type botulinum neurotoxin. In addition, the term“botulinum neurotoxin” as used herein is meant to include any and all known botulinum neurotoxin serotypes, including serotypes A, B, C, D, E, F, G, H and X, as well as botulinum neurotoxin complexes (e.g., 300, 600 and 900 kDa complexes), and high purity botulinum neurotoxins (botulinum neurotoxins that are free from complexes with other proteins), which are all useful in the practice of the present invention.

The botulinum neurotoxin may be of serotypes A, B, C, D, E, F, G, H, or X. The botulinum neurotoxin may be a protein containing modifications as compared to wild-type botulinum neurotoxins. For example, the botulinum neurotoxin may be a protein having an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity, preferably at least 80%, 90%, 95%, 97%, 98%, or 99% sequence identity, to the amino acid sequence of a wild-type botulinum neurotoxin of serotypes A, B, C, D, E, F, G, H, or X.

In one embodiment the botulinum neuro toxin may be BoNT/A. A reference BoNT/A sequence is shown as SEQ ID NO: 1. In another embodiment the botulinum neurotoxin may be BoNT/B. A reference BoNT/B sequence is shown as SEQ ID NO: 2.

In another embodiment the botulinum neurotoxin may be BoNT/C. A reference BoNT/ sequence is shown as SEQ ID NO: 3.

In another embodiment the botulinum neurotoxin may be BoNT/D. A reference BoNT/D sequence is shown as SEQ ID NO: 4.

In another embodiment the botulinum neurotoxin may be BoNT/E. A reference BoNT/E sequence is shown as SEQ ID NO: 5.

In another embodiment the botulinum neurotoxin may be BoNT/F. A reference BoNT/F sequence is shown as SEQ ID NO: 6.

In another embodiment the botulinum neuro toxin may be BoNT/G. A reference BoNT/ G sequence is shown as SEQ ID NO: 7.

In one embodiment the botulinum neurotoxin may be BoNT/X. A reference BoNT/X sequence is shown as SEQ ID NO: 8.

As well as encompassing wild-type botulinum neuro toxins (also called native botulinum neurotoxins, unmodified botulinum neurotoxins and holotoxins), the term“botulinum neurotoxin” is also intended to embrace modified botulinum neurotoxins and derivatives thereof, including but not limited to those described below. A modified botulinum neurotoxin or derivative may contain one or more amino acids that has been modified as compared to the native (unmodified) form of the botulinum neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the botulinum neurotoxin. By way of example, a modified botulinum neurotoxin may have modified amino acid sequences in one or more domains relative to the native (unmodified) botulinum neurotoxin sequence. Such modifications may modify functional aspects of the toxin, for example biological activity or persistence. Thus, in one embodiment, the botulinum neurotoxin of the invention is a modified botulinum neurotoxin, or a modified botulinum neurotoxin derivative, or a botulinum neurotoxin derivative.

A modified botulinum neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (such as a modified H_c domain), wherein said modified heavy chain binds to target nerve cells with a higher or lower affinity than the native (unmodified) botulinum neurotoxin. Such modifications in the H_c domain can include modifying residues in the ganglioside binding site of the H_c domain or in the protein (SV2 or synaptotagmin) binding site that alter binding to the ganglioside receptor and/ or the protein receptor of the target nerve cell. Examples of such modified botulinum neurotoxins are described in WO 2006/027207 and WO 2006/114308, both of which are hereby incorporated by reference in their entirety.

A modified botulinum neurotoxin may have one or more modifications in the amino acid sequence of the light chain, for example modifications in the substrate binding or catalytic domain which may alter or modify the SNARE protein specificity of the modified L-chain. Examples of such modified botulinum neurotoxins are described in WO 2010/120766 and US 2011/0318385, both of which are hereby incorporated by reference in their entirety.

A modified botulinum neurotoxin may comprise one or more modifications that increases or decreases the biological activity and/or the biological persistence of the modified botulinum neurotoxin. For example, a modified botulinum neurotoxin may comprise a leucine- or tyrosine- based motif, wherein said motif increases or decreases the biological activity and/ or the biological persistence of the modified botulinum neurotoxin. Suitable leucine-based motifs include xDxxxLL, xExxxLL, xExxxIL, and xExxxLM (wherein x is any amino acid). Suitable tyrosine-based motifs include Y-x-x-Hy (wherein Ely is a hydrophobic amino acid). Examples of modified botulinum neurotoxins comprising leucine- and tyrosine-based motifs are described in WO 2002/ 08268, which is hereby incorporated by reference in its entirety.

The term“botulinum neurotoxin” is intended to embrace hybrid and chimeric botulinum neurotoxins. A hybrid botulinum neurotoxin comprises at least a portion of a light chain from one botulinum neurotoxin or subtype thereof, and at least a portion of a heavy chain from another botulinum neurotoxin or botulinum neurotoxin subtype. In one embodiment the hybrid botulinum neurotoxin may contain the entire light chain of a light chain from one botulinum neurotoxin subtype and the heavy chain from another botulinum neurotoxin subtype. In another embodiment, a botulinum neurotoxin may contain a portion (e.g. the binding domain) of the heavy chain of one botulinum neurotoxin subtype, with another portion of the heavy chain being from another botulinum neurotoxin subtype. Similarly or alternatively, the therapeutic element may comprise light chain portions from different botulinum neurotoxins. Such hybrid or chimeric botulinum neurotoxins are useful, for example, as a means of delivering the therapeutic benefits of such botulinum neurotoxins to patients who are immunologically resistant to a given botulinum neurotoxin subtype, to patients who may have a lower than average concentration of receptors to a given botulinum neurotoxin heavy chain binding domain, or to patients who may have a protease- resistant variant of the membrane or vesicle toxin substrate (e.g., SNAP-25, VAMP and syntaxin). Hybrid and chimeric botulinum neurotoxins are described in US 8,071,110, which publication is hereby incorporated by reference in its entirety. Thus, in one embodiment, the botulinum neurotoxin of the invention is a hybrid botulinum neurotoxin, or a chimeric botulinum neurotoxin.

The term “botulinum neurotoxin” is also intended to embrace retargeted botulinum neurotoxins. The three domains of BoNT (LC, H_N, H_<-) are functionally and structurally distinct and the boundaries of each domain for each sub-serotype has been defined previously by the Applicant. This has been exploited in the Applicant’s Targeted Secretion Inhibitor (TSI) platform where the host cell receptor binding domain (H_c) is replaced by other binding domains (e.g., EGFR) to retarget the rest of the BoNT molecule, LH_N (LC + H_N) to a different cell type. Note that the H_N domain has a“belt” region that wraps around the LC— this is believed to behave as a pseudo inhibitor and have a chaperone function during LC translocation.

The term“botulinum neurotoxin” is also intended to encompass variants and fragments of botulinum neurotoxin, provided they retain the analgesic effect of the corresponding native botulinum neurotoxin.

The term “botulinum neurotoxin” may also embrace newly discovered botulinum neurotoxin protein family members expressed by non-clostridial microorganisms, such as the Enterococcus encoded toxin which has closest sequence identity to BoNT/X, the Weissella ory^ae encoded toxin called BoNT/Wo (NCBI Ref Seq: WP_027699549.1), which cleaves VAMP2 at W89- W90, the Enterococcus faecium encoded toxin (GenBank: 0T022244.1), which cleaves VAMP2 and SNAP25, and the Chryseobacterium pipero encoded toxin (NCBI Ref.Seq: WP_034687872.1).

Without being bound by theory, it is believed that botulinum neurotoxins are particularly suited for the treatment of vulvodynia as they can both decrease muscular overactivity (one of the causes of vulvodynia, with most patients with PVD having overactive pelvic floor muscles), and also have a direct effect on pain.

Botulinum neurotoxin may be obtained commercially by establishing and growing cultures of C. botulinum in a fermenter, and harvesting and purifying the fermented mixture in accordance with known techniques. The“A” form of botulinum neurotoxin is currently available commercially from several sources, including Ipsen Biopharmaceuticals Limited under the tradename DYSPORT®, from Merz Pharma under the tradename XEOMIN®, from Medytox Inc. under the tradename CORETOX®, and from Allergan Inc. under the tradename BOTOX®. In an embodiment of the present invention, the botulinum neurotoxin is one of these commercially- available forms.

In a particularly preferred embodiment, the botulinum neurotoxin is abobotulinumtoxinA, even more preferably the botulinum toxin is DYSPORT®. DYSPORT® is an injectable form of BoNT/A, which is isolated and purified from Clostridium bacteria producing BoNT/A. Dysport® is formulated as a complex of BoNT/A with haemagglutinin (BoNT/A-HAC), a large therapeutically inert protein used to stabilise the toxin. Dysport® is formulated with lactose (bulking agent) and human serum albumin, and is supplied as a lyophilised powder. It is supplied as a lyophilized powder. DYSPORT® has approved therapeutic indications in the United States for the treatment of adults with Cervical Dystonia (CD), the treatment of upper and lower limb spasticity in adult patients, and the treatment of lower limb spasticity in children to improve tone and spasticity. The medicine was first registered in the United Kingdom in 1990 for other uses and is licensed in more than 80 countries in eight different indications, with over 1,300 peer-reviewed publications.

The botulinum neurotoxin may be administered by any means known in the art, including parenterally, intra-muscularly, intradermally, transdermally and by instillation. Typically botulinum neurotoxin is administered parenterally, for example subcutaneously or intramuscularly. Preferably, botulinum neurotoxin is administered intramuscularly.

Botulinum neurotoxin may be administered to a subject’s bladder and/or pelvic floor. This has the advantage of allowing the treatment of concomitant disease (e.g. interstitial cystitis).

Preferably, administration may be to the muscles around the vulvar vestibule (also referred to as the vaginal vestibule or vaginal opening). For example, botulinum neurotoxin may be administered to the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and/ or the right deep transverse perineal muscle, or any combination thereof. While treatment of vulvodynia with botulinum neurotoxin has been described in the art, the results have been inconsistent, with the only randomized control studies showing no effect compared with the placebo. Further, the prior art does not describe a method of administering botulinum neurotoxin to the combination of all of the aforementioned muscles. It has surprisingly been found by the inventors that administration of botulinum neurotoxin to these muscles in combination provides additional efficacy in treating symptoms of vulvodynia, whilst reducing the risk of undesirable clinical side effects. Accordingly, a method of treating one or more symptom of vulvodynia according to the invention preferably comprises the administration of a botulinum toxin to each of the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and the right deep transverse perineal muscle.

The injection to the transverse perineal muscles may be at about 5 mm below the skin surface and/ or at about 10 mm below the skin surface.

To assist the physician in ensuring that all the relevant muscles are treated, botulinum neurotoxin may be administered to a subject in the following order: (1) to a first bulbospongiosus muscle, anterior to the hymeneal ring, (2) to a first pubococcygeous muscle, anterior to the hymeneal ring; (3) to the first bulbospongiosus muscle, posterior to the hymeneal ring; (4) to the first pubococcygeous muscle, posterior to the hymeneal ring; (5) to a second bulbospongiosus muscle, anterior to the hymeneal ring, (6) to a second pubococcygeous muscle, anterior to the hymeneal ring; (7) to the second bulbospongiosus muscle, posterior to the hymeneal ring; (8) to the second pubococcygeous muscle, posterior to the hymeneal ring; and (9) to the superficial and deep transverse perineal muscles (left and right) anterior to the hymeneal ring. The first bulbospongiosus muscle is the left or right bulbospongiosus, with the second bulbospongiosus muscle being the left bulbospongiosus if the first bulbospongiosus muscle is the right bulbospongiosus and the right bulbospongiosus if the first bulbospongiosus muscle is the left bulbospongiosus. The first pubococcygeous muscle is the left or right pubococcygeous, with the second pubococcygeous muscle being the left pubococcygeous if the first pubococcygeous muscle is the right pubococcygeous and the right pubococcygeous if the first pubococcygeous muscle is the left pubococcygeous.

The administration may be at about the 5 o’clock position when administered to the left bulbospongiosus and pubococcygeous, at about the 7 o’clock position when administered to the right bulbospongiosus and pubococcygeous, and/or at about the 6 o’clock position when administered to the superficial and deep transverse perineal muscles (each of the left and right, superficial and deep transverse perineal muscles are administered at this position).

The botulinum neurotoxin can be presented as a sterile aqueous solution, for instance a pyrogen-free aqueous solution or dispersion, or as a sterile powder for reconstitution into a sterile solution or dispersion. If desired, tonicity adjusting agents, such as sodium chloride, glycerol and/ or various sugars can be added. Stabilizers may be included if desired. The formulation may be preserved by means of any suitable pharmaceutically acceptable preservative, such as a paraben.

In some embodiments, the botulinum neurotoxin is formulated in unit dosage form, for example, as a sterile solution in a vial, or as a vial or sachet containing a lyophilized powder for reconstituting in a suitable carrier, such as saline, for injection. In one aspect, the botulinum neurotoxin is formulated in a solution containing saline and pasteurized human serum albumin, which stabilizes the neurotoxin. The solution may be sterile filtered, filled into individual vials, and then vacuum dried to give a sterile lyophilized powder. In use, the powder can be reconstituted by the addition of sterile unpreserved normal saline (sodium chloride 0.9% for injection).

The potency of the neurotoxin may be expressed as a multiple of the LD_S0 value. One LD_S0 unit is the equivalent amount of neurotoxin which causes the death of 50% (one-half) of a group of test animals, such as laboratory mice. Alternative methods of determining the potency of the neurotoxin may also be employed, including, for example, any method included in the European Pharmacopoeia monograph 01/2005:2113, which is hereby incorporated by reference.

As used herein, the term“unit” refers to a unit dose of DYSPORT®, which refers to the median intraperitoneal LD_S0 dose of DYSPORT® in mice. It is noted that the median intraperitoneal LD_S0 dose of DYSPORT® in mice is not necessarily the same as the median intraperitoneal LD_S0 dose of another botulinum neurotoxin-containing product, such as BOTOX®, as different pharmaceutical preparations are produced differendy. For example, units of BOTOX®, as described in the art, are not the same as the units of the present invention, which are units of DYSPORT®.

The botulinum neurotoxin is administered to the subject is a“therapeutically effective amount,” that is, an amount sufficient to reduce, ameliorate, or eliminate one or more primary or secondary symptoms of vulvodynia. Primary symptoms include pain in the genital area, which can be characterized as burning, soreness, stinging, rawness, painful intercourse (dyspareunia), aching, throbbing, or itching. The pain may be constant, occasional, or only when pressure is applied to the sensitive area. The pain may be felt in the entire genital area (generalized) or it may be localized to a certain area, such as the opening of the vagina. Genital tissue may appear normal or slighdy inflamed or swollen. In some preferred embodiments of the invention, the subject suffers from pain at the vaginal vestibule. Accordingly, in some preferred embodiments of the invention, the subject suffers from a localized form of vulvodynia, known as vestibulodynia. In some embodiments, the vestibulodynia is general; in other embodiments, the vestibulodynia is caused by touching (known as provoked vestibulodynia or PVD), such as when the subject inserts a tampon, has a pelvic exam, or engages in sexual intercourse. Treatment of PVD is particularly preferred. Secondary symptoms of vulvodynia which may be experienced by a subject to be treated by the present invention include affective disorders such as emotional distress, stress, anxiety, and depression. In some preferred embodiments, the subject to be treated by the present invention suffers from an affective disorder, preferably anxiety or depression.

The amount of botulinum toxin to be administered can be determined, at least in part, by the severity of the one or more symptoms of vulvodynia experienced by the subject. Any appropriate symptom, examples of which are described herein, may be used in this regard. Suitable symptoms, and methods for assessing/ quantifying their severity are known in the art and could be selected and determined by one of ordinary skill in the art, such as the attending physician.

By way of non-limiting example, severity of the symptoms of vulvodynia can be assessed, for example, by the pain felt by a subject during intercourse and/or during a dilator test. One measure of the severity of the vulvodynia is the Vulvar Pain Assessment Questionnaire (VPAQ), which assesses pain as well as emotional response, cognitive response, interference with life, sexual function and self-stimulation/penetration (see, for example, Dargie et al. Pain (2016) 157:2672-2686). The VPAQ includes 55 Likert-type questions and designed to capture 6 domains: pain severity, emotional response, cognitive response and interference with life, sexual function, self stimulation/penetration. The score for each question ranges from 0 to 4 and a mean score is computed for each domain.

Another example of the severity of vulvodynia pain is the Numerical Rating Scale (NRS). The NRS is an 11 -point scale to assess subject pain perception. Subjects are asked to give a number between 0 and 10 that fits best to their pain intensity. Zero represents‘no pain at all’ whereas the upper limit, 10, represents‘the worst pain possible’. Other measures of pain are known in the art (see, for example, Hawker et al. Arthritis Care Res. (2011) 63(S11):S240-S252). Non-limiting examples include: the McGill Pain Rating Index, which measures the subject’s pain over the previous month on a scale from 0 (no pain) to 3 (severe pain) (Hawker et al),· the McGill Visual Analog Scale which measures the subject’s pain intensity for the previous month, ranging from 0 (no pain) to 5 (excruciating pain) (Hawker et al); the Brief Pain Inventory (Cleeland and Ryan Ann. Acad. Med. Singapore (1994) 23:129-138); the Short Form McGill Pain Questionnaire (SF-MPQ) (Melzack Pain (1990) 30:191-197.); the Speculum Rating, which measures pain provoked by the use of a speculum from 0 (no pain) to 3 (severe pain); the Cotton Swab Rating, which measures pain provoked by cotton-swab palpation at six sites within the vulvar vestibule from 0 (no pain) to 3 (severe pain) (Goldstein et al. J. Sex. Med. (2016) 13:575-590); the Pain Severity Subscale of the West Haven-Yale Multidimensional Pain Inventory (MPI); and Likert scales adapted to measure pain. In some preferred embodiments, the Numerical Rating Scale (NRS) can be used to assess the severity of vulvodynia.

Non-limiting examples of vulvodynia severity assessments/pain assessment are detailed below (also see Examples 3 to 8). Any one or more of these outcomes may be used to assess a subject’s response to treatment according to the present invention, or any combination thereof. By way of non-limiting example, a subject may be assessed by the mean change in NRS score from baseline (prior to administration of botulinum neurotoxin) to a suitable time point (e.g. six weeks or more) post-treatment. By way of a further non-limiting example, a therapeutic effect may be quantified as a significant reduction in vaginal pain, e.g. at least a 20%, at least a 30% decrease, at least a 40% decrease, at least a 50% decrease or more in vaginal pain, preferably at least a 30% decrease or greater in vaginal pain. This decrease may be measured using any appropriate scale or index, including those disclosed herein (e.g. the NRS). By way of a further non-limiting example, a therapeutic effect may be quantified as at least a two-point, at least a three-point, at least a four-point or more decrease in vaginal pain. This decrease may be measured using any appropriate scale or index, including those disclosed herein (e.g. the NRS). By way of a further non-limiting example, a therapeutic effect may be quantified as the mean change from baseline to post-treatment in maximum tolerated vaginal pain. Again, this may be measured using any appropriate scale or index, including those disclosed herein (e.g. the NRS).

Any of the assessment techniques described herein may assess pain following insertion of a probe (e.g. a dilator). Pain may be assessed at the dilator size that provokes the maximum tolerated pain (dilator maximum testedsize, DMTS). By way of a further non-limiting example, a therapeutic effect may be quantified as the mean change from baseline to post-treatment in the composite score for vaginal pain. Such a composite score may be arrived as by a combination of dilator-induced pain and the tolerability of the dilator size. This composite score may be measured using any appropriate scale or index, including those disclosed herein (e.g. the NRS). By way of a further non limiting example, a therapeutic effect may be quantified as the mean change from baseline to post treatment in the pain suffered during intercourse. Again, this may be measured using any appropriate scale or index, including those disclosed herein (e.g. the NRS). By way of a further non limiting example, a therapeutic effect may be quantified as the mean change from baseline to post treatment in the number of instances of intercourse. By way of a further non-limiting example, a therapeutic effect may be quantified as the mean change from baseline to post-treatment in the Clinical Global Impression (CGI). By way of a further non-limiting example, a therapeutic effect may be quantified as the mean change from baseline to post-treatment in the Patient Global Impression of change in pain (PGI-C) and/or Patient Global Impression of severity of pain (PGI- S). PGI-C may be assessed by asking a patient to assess how their pain has changed compared with prior to treatment using a 7-point Likert scale (from -3: very much worse to +3: very much improved). PGI-S may be conducted using a 4-point Likert scale (from 0: no pain to 3: severe pain) over a recall period of the past 1 week.The severity of secondary symptoms of vulvodynia can be measured by various means, including: the Female Sexual Function Index (FSFI) (Rosen et al. J. Sex Marital Ther. (2000) 26(2):191-208); the Hamilton Depression Rating Scale (HAM-D); the Hamilton Anxiety Rating Scale (HAM-A); the Profile of Mood States; the Beck Depression Inventory; and the Pain Anxiety Symptoms Scale (PASS), which measures anxiety associated with the fear of pain. Other suitable means for assessment include the Patient-Health Questionnaire 9 (PHQ-9), which is a validated depression module, in which scores each of nine Diagnostic and Statistical Manual of Mental Disorders (DSM) criteria as "0" (not at all) to "3" (nearly every day). The Quality of Life questionnaire Short Form-36 (SF-36) may be used to assess a subject’s pain perception. The SF-36 is a 36-item, subject-reported survey of subject health. The SF-36 consists of eight scaled scores (vitality, physical functioning, bodily pain, general health perceptions, physical role functioning, emotional role functioning, social role functioning and mental health). The higher the score recorded in the SF-36, the less disability. Pelvic floor pressure (resting vaginal pressure and/or maximal sequeeze pressure) may also be assessed. There are also multi-dimensional measures of the symptoms of vulvodynia, such as the Female Sexual Function Index (FSFI) (Rosen et al.), which measures sexual desire or interest, pain associated with vaginal penetration, and other factors. In more detail, the FSFI is a validated self- reported multidimensional 19-item questionnaire for assessing the sexual function in women by evaluating six key domains of sexual function: desire, arousal, lubrication, orgasm, sexual satisfaction, and pain. A total score and domain scores are calculated. Every item is assessed with a score from 0 or 1 to 5, the total score being 2-36. FSFI questions 17 (relating to how often pain or discomfort is experienced during vaginal penetration), 18 (relating to how often pain or discomfort is experienced after vaginal penetration) and 19 (relating to rating the level/degree of pain or discomfort during or following vaginal penetration) are particularly relevant.

If appropriate, modified forms of one or more of these assessments may be used. As a non limiting example, a modified form of the VPAQ (mVPAQ) or the FSFI (mFSFI) may be used. In particular, the standard VPAQ assessment as described in the art may be altered by: (i) modifying the recall period for the VPAQ subscale to the past 1 week; (ii) modifying the recall period for the VPAQ life interference subscale to over the past 1 week; (iii) altering the recall periods for the other VPAQ subscales to over the past 1 month; and/or (iv) reserving the sequence of questions for reporting distress (average/worst) on the pain severity subscale. Preferably all of these modifications are made when the VPAQ is used to assess pain (and hence treatment efficacy) according to the present invention. As a further non-limiting example, the standard FSFI assessment as described in the art may be altered by: (i) modifying the FSFI pain domain (questions 17 to 19) recall period to over the past 1 week; and/ or (ii) altering the other FSFI domains to over the past 4 weeks.

It will be appreciated that a therapeutically effective amount of the botulinum neurotoxin may depend upon a variety of factors, including the method of administration, the weight of the subject, the age of the subject, the size of the muscles, the location of the muscles, the severity of the subject’s vulvodynia, the severity of the subject’s symptoms of vulvodynia, the type and potency of the particular botulinum neurotoxin, the status of the subject’s recovery, the subject’s history with botulinum neurotoxin, the subject’s body mass index, contraindications, if any, and/or the judgement of the attending physician. Therefore, it is not generally practicable to specify an exact therapeutically effective amount, although these may be readily determined by one of ordinary skill. By way of non-limiting example, a physician may increase or decrease the dose based on the efficacy and safety responses. By way of further non-limiting example, in some embodiments and where several doses are approved, a physician may start with low dose in low weight patients, with the potential to increase the dose depending on the observed clinical response.

Typically, the dosage is the lowest amount of botulinum neurotoxin that is therapeutically effective. The administrations may be repeated as necessary, though subsequent administrations may vary in dosage amount, administration route, frequency, muscle type, location, and/or toxin type. The treatment may be continued for as long as the symptoms of vulvodynia persist. In the event treatment is terminated but the symptoms reappear, treatment can recommence. By way of non limiting example, the botulinum neurotoxin may be administered about every three to six months. For more severe vulvodynia, the administration interval may be decreased (i.e. the frequency of administration increased), e.g. botulinum neurotoxin may be administered every month, or every two months, or at another more frequent interval as determined by the subject’s physician using routine skill. For more mild vulvodynia, the administration interval may be increased (i.e. the frequency of administration decreased), e.g. botulinum neurotoxin may be administered every six months, every eight months, or annually, or at another less frequent interval as determined by the subject’s physician using routine skill.

The treatment typically involves administering about 1,500 units or less of the botulinum neurotoxin DYSPORT®, for example from about 1 to about 1,500 units, about 1 to about 1,000 units, about 200 to about 800 units, about 350 to about 800 units, about 450 to about 800 units, or about 500 units per treatment session. Preferably the treatment involves administering about 350 to about 600 units per treatment session, for example about 350 units, about 500 units or about 600 units per treatment session.

In some embodiments, about 5 to 25% of the total dose of botulinum neurotoxin administered during a treatment session is administered to one or more of the following muscles: the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and the right deep transverse perineal muscle. In some embodiments about 8% of the total dose of botulinum neurotoxin administered during a treatment session is administered to one or more of the following muscles: a first bulbospongiosus muscle, anterior to the hymeneal ring; a first pubococcygeous muscle, anterior to the hymeneal ring; a first bulbospongiosus muscle, posterior to the hymeneal ring; a first pubococcygeous muscle, posterior to the hymeneal ring; a second bulbospongiosus muscle, anterior to the hymeneal ring; a second pubococcygeous muscle, anterior to the hymeneal ring; a second bulbospongiosus muscle, posterior to the hymeneal ring; a second pubococcygeous muscle, posterior to the hymeneal ring. In some embodiments about 8% of the total dose of botulinum neurotoxin administered during a treatment session is administered to one or more of the following muscles: the left bulbospongiosus muscle, anterior to the hymeneal ring; the left pubococcygeous muscle, anterior to the hymeneal ring; the left bulbospongiosus muscle, posterior to the hymeneal ring; the left pubococcygeous muscle, posterior to the hymeneal ring; the right bulbospongiosus muscle, anterior to the hymeneal ring; the right pubococcygeous muscle, anterior to the hymeneal ring; the right bulbospongiosus muscle, posterior to the hymeneal ring; the right pubococcygeous muscle, posterior to the hymeneal ring. In some preferred embodiments, about 8% of the total dose of botulinum neurotoxin administered during a treatment session is administered to each of the following muscles: the left bulbospongiosus muscle, anterior to the hymeneal ring; the left pubococcygeous muscle, anterior to the hymeneal ring; the left bulbospongiosus muscle, posterior to the hymeneal ring; the left pubococcygeous muscle, posterior to the hymeneal ring; the right bulbospongiosus muscle, anterior to the hymeneal ring; the right pubococcygeous muscle, anterior to the hymeneal ring; the right bulbospongiosus muscle, posterior to the hymeneal ring; the right pubococcygeous muscle, posterior to the hymeneal ring.

In some embodiments, about 9% of the total dose of botulinum neurotoxin administered during a treatment session is administered to one or more of the following muscles: the left superficial transverse perineal muscle anterior to the hymeneal ring; the right superficial transverse perineal muscle anterior to the hymeneal ring; the left deep transverse perineal muscle anterior to the hymeneal ring; and the right deep transverse perineal muscle anterior to the hymeneal ring.

Using the exemplary dosage administrations described above, the total dose of botulinum toxin is split equally (as far as practicable) between the muscles to be treated. Other administration protocols which allow for equal dosing of the muscles to be treated can be readily determined by one of ordinary skill in the art.

By way of further non-limiting example, in some embodiments, about 36% of the total dose of botulinum neurotoxin is administered to the transverse perineal muscles anterior to the hymeneal ring. In such embodiments, all four of the following muscles are administered by such a dose: the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and the right deep transverse perineal muscle. Accordingly, in certain such embodiments, each of these muscles receives about 9% of the total dose of botulinum neurotoxin.

The amount of botulinum neurotoxin administered to the subject may be direcdy proportional to the severity of the vulvodynia and/or the symptom(s) of vulvodynia. As a non limiting example, a dosage of from about 1 to about 200 units may be administered to a subject having mild vulvodynia or mild symptom(s) of vulvodynia. A dosage of from about 200 to about 800, preferably from about 350 to about 800, more preferably from about 450 to about 800 or about 350 to 600, units may be administered to a subject having moderate vulvodynia or moderate symptom(s) of vulvodynia. A dosage of from about 800 to about 1,500 units may be administered to a subject having severe vulvodynia or severe symptom(s) of vulvodynia.

The amount of botulinum neurotoxin administered to the subject may be directly proportional to the muscle tone of the subject.

In addition, a lower dose of botulinum neurotoxin may be administered if the subject has exhibited adverse effects following previous treatment with botulinum neurotoxin. Examples of adverse effects include: dysphagia, pneumonia, significant debility, muscle weakness, pneumopathy, urinary incontinence, and/or fecal incontinence. Examples of such a lower dose are from about 50 units to about 500 units. Thus, the method of treating one or more symptom of vulvodynia according to the invention may further comprise; (i) determining a dosing regimen or botulinum neurotoxin; (ii) determining the amount of botulinum neurotoxin for a subsequent administration; and / or (iii) determining the frequency for a subsequent administration of a botulinum toxin; based on the subject’s response to a previous administration of botulinum neurotoxin.

The subject’s response may be recorded into a software program that is configured to receive such information. The program may operate on a computer device. Based on the subject’s response as measured, the program may determine an appropriate dosing regimen for a botulinum neurotoxin.

Also described herein is a method involving determining an appropriate dosage amount and/or an appropriate dosing regimen for botulinum neurotoxin based on an assessment of one or more of the following criteria: the subject’s weight, the severity of the vulvodynia, the severity of the symptoms of the vulvodynia, the status of the subject’s recovery following vulvodynia, the subject’s history with treatment with botulinum neurotoxin, whether the subject has previously experienced vulvodynia, the subject’s body mass index, and/or the subject’s response to a previous treatment with botulinum neurotoxin.

As used herein, the term“dosing regimen” encompasses one or more of the amount of botulinum neurotoxin to be administered (both in total and per site or per time of administration), administration interval, frequency of administration and/or time of administration, or any combination thereof. By way of non-limiting example, if a subject has previously exhibited a therapeutic response to a total dose of 500 units DYSPORT ®, then for subsequent administrations: (a) a dose of about 500 units of DYSPORT ® may be used to treat this subject; or (b) a dose less than 500 units of DYSPORT ® may be used, with the dose being increased following the subsequent administration if the lower dose does not elicit a therapeutic response. By way of a further non-limiting example, if a subject has previously reported a therapeutic response with a treatment interval/ frequency of about three months, then for subsequent administrations: (a) a treatment interval of three months may be used; or (b) a treatment interval/ frequency of more than three months may be used, with the treatment interval being decreased/treatment frequency increased following the subsequent administration if the longer treatment interval/reduced treatment frequency does not elicit or maintain the desired therapeutic response. A new dosing regimen, amount of botulinum neurotoxin to be administered, administration interval, frequency of administration and/ or time of administration may be readily determined by one of ordinary skill in the art based on a subject’s response to a previous treatment with botulinum neurotoxin.

Similarly, one of ordinary skill in the art will readily be able to determine a new dosing regimen, amount of botulinum neurotoxin to be administered, administration interval, frequency of administration and/or time of administration based on the status of the subject’s history with treatment with botulinum neurotoxin and/or whether the subject has previously experienced vulvodynia, or their recovery therefrom. It is also within the routine capabilities of one of ordinary skill in the art to determine an appropriate therapeutically effective amount of botulinum neurotoxin based on a subject’s weight and/or body mass index (BMI).

In some embodiments, the amount of botulinum neurotoxin administered and/or the dosing regimen for botulinum neurotoxin is determined in part by the level of pain measured following the insertion of a probe ( ^'e.g. , a vaginal dilator, speculum) into the subject’s vagina (use of such a probe, or a series of probes of different sizes is known in the art. By way of non-limiting example, Pacik, P. Female Pelvic Medicine & Reconstructive Surgery (2013) 19:S45-S195) describes the use of a set of two dilators of different sizes. According to the invention, sets of five, six, seven, eight or more probes of different sizes may be used. For example, if such insertion provokes severe pain, a higher dosage and/or more frequent dosing regimen (as described herein) may be desired. If only a moderate pain results, a moderate dosage and/or moderately frequent dosing regimen (as described herein) may be used. Pain may be measured using any of the means known in the art, including (but not limited to) those disclosed herein.

In some embodiments, the method involves adjusting future doses or the dosing regimen based on the subject’s response to a previous treatment.

The quantity of neurotoxin administered and the frequency of its administration will be at the discretion of the individual(s) responsible for the treatment (eg., the medical practitioner(s) and/ or the subject), and should be commensurate with questions of safety and the effects produced by the neurotoxin.

In some embodiments, following an initial treatment with botulinum neurotoxin, the amount of a subsequent administration of botulinum neurotoxin and/or a dosing regimen for botulinum neurotoxin may be determined. The amount of such subsequent administrations (s) and/or a dosing regimen may be determined based, at least in part, on the subject’s recovery following a previous treatment with botulinum neurotoxin. For example, a lower dose and/ or lower frequency of administration may be desired if the patient has exhibited significant recovery and a higher dose and/ or higher frequency of administration may be desired if the patient has exhibited a low level of recovery.

The status of a subject’s recovery can be measured using routine methods known to persons of ordinary skill in the art. For example, a subject’s recovery status may be measured using the Physician’s Global Assessment (PGA) of response Busner and Targum Psychiatry (2007) 4(7)28-37). The PGA measures response on a scale from 1 to 7. Preferably, a modified PGA may be used which measures response on a scale from -4 to +4, with -4 indicating that response is markedly worse, to 0, which indicates no change, to +4 indicating that response has markedly improved. For example, a desired response may be a PGA score following administration of 1 or above, 2 or above, 3 or above, or 4. As such, the amount of a dose or dosing frequency may, for example, be increased for a subject exhibiting a PGA score of below 0, the amount of a dose or dosing frequency may, for example, be maintained for a subject exhibiting a PGA score of from 0 to 2, the amount of a dose or dosing frequency may, for example, be reduced for a subject exhibiting a level subject exhibiting a PGA score of between 2 and 4, and further treatment with botulinum neurotoxin may be terminated for a subject exhibiting a PGA score of 4 or above.

Another measure of the status of a subject’s response to treatment involves insertion of a vaginal dilator. Different size dilators may be used with measurement based on the dilator size that provokes the maximum tolerated pain.

Other methods of measuring a subject’s response include the Global Response Index (GRI), the Female Sexual Function Index (FSFI), the Female Sexual Distress Scale (FSDS), the Dermatology Life Quality Index (DLQI), the Pelvic Floor Distress Inventory (PFDI) index, the Visual Analog Scale (VAS), the Pain Numeric Rating Scale (NRS), the Clinical Global Impression scale (CGI), the Patient Global Impression of Improvement scale (PGI-I), the Patient Global Impression of Change scale (PGI-C), the Patient Global Impression of Severity scale (PGI-S), and the Quality Of Life Scale (QOLS). In preferred embodiments the NRS is used. In some embodiments, the information relating to a subject’s response to treatment with botulinum neurotoxin is recorded into a log or diary. In some embodiments, the response information is recorded into or by a software program that operates on a computer device and the response information is stored in a database associated with the computer device. In some embodiments, the response information is recorded into or by a software program that operates on a first computer device and the information is stored into a database on the first computer device or a second computer device that is in communication with first computer device.

In some embodiments, the method may additionally comprise administering a subsequent administration of botulinum neurotoxin, wherein the subsequent administration is determined, at least in part, by the response information. In particular embodiments, the method further comprises: reviewing the subject’s response information; designing a dosing regimen based, at least in part, on the subject’s response information; and administering a subsequent botulinum neurotoxin administration to the subject in accordance with the dosing regimen, wherein the botulinum neurotoxin administration is sufficient to treat or reduce the symptoms of vulvodynia.

In some embodiments, the amount and/or frequency of the subsequent administration of botulinum neurotoxin and/ or a dosing regimen for botulinum neurotoxin may be determined based in part on the level of pain measured following the insertion of a probe (eg., a vaginal dilator, speculum) into the subject’s vagina following an initial administration of botulinum neurotoxin. If the level of pain measured following insertion of the probe is below a threshold level (eg. moderate pain as measured by a pain scale known in the art and as described herein), a larger sized probe (eg., a vagina dilator having a larger diameter) is inserted and the level of pain measured and, if the level of pain measured remains below the threshold level, probe(s) of increasing size (eg., vaginal dilators having larger diameters) are inserted until the level of pain measured following such insertion is at or above the threshold level. The amount of botulinum neurotoxin to administer or the dosing regimen for botulinum neurotoxin is then determined based on the level of pain measured following the insertion of the final probe. The aforementioned steps may be repeated as necessary until treatment of the subject is considered satisfactory. For example, following the aforementioned subsequent administration, a probe may be inserted and the subject’s level of pain determined, with probes of increasing size inserted as necessary until the level of pain reaches a certain threshold. The amount of the next administration of botulinum neurotoxin or a subsequent dosing regimen for botulinum neurotoxin is then determined based on the level of pain measured following insertion of the final probe.

Also described herein is a botulinum neurotoxin for use in the treatment of one or more symptoms of vulvodynia, wherein said botulinum neurotoxin is for administration to the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and/ or the right deep transverse perineal muscle.

Also described herein is the use of a botulinum neurotoxin in the manufacture of a medicament for use in the treatment of one or more symptoms of vulvodynia, wherein said botulinum neurotoxin-containing medicament is for administration to the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and/ or the right deep transverse perineal muscle.

Any and all disclosure herein in relation to methods of treating one or more symptom of vulvodynia applies equally and without reservation to the disclosure of botulinum neurotoxins for use in the treatment of one or more symptoms of vulvodynia. Similarly, any and all disclosure herein in relation to methods of treating one or more symptom of vulvodynia applies equally and without reservation to the use of a botulinum neurotoxin in the manufacture of a medicament for use in the treatment of one or more symptoms of vulvodynia.

In some embodiments, the clinical management of the subject’s recovery is enhanced by virtue of the fact that information about the subject’s recovery is being recorded into a log or diary, or into a software program that is able to receive and communicate information about the subject’s recovery.

The software program useful in the methods described herein is capable of running on any suitable computer device. Various information or data may be received by the software program, including: the time that has elapsed since the previous administration, frequency of administration, symptoms relating to vulvodynia, the effect (if any) of the administration, and/or any other comments about the administration and recovery process. After the software program receives the information that is associated with the subject’s treatment and/or recovery it may store that information into a database. The database can reside on the same computer device used by the practitioner or on a different computer device that is able to communicate with the computer device used by the practitioner.

In some embodiments, the medical practitioner reviews the response information. Using a computer device that is able to communicate with the database, a medical practitioner can access the response information. In one aspect, after reviewing the information, the medical practitioner (eg., clinician) may use this information to determine an appropriate dosing regimen, or to make or suggest an adjustment to an existing dosing regimen. In another aspect, the medical practitioner may use the response information to determine or vary the botulinum neurotoxin dosing regimen, i.e., the mode, amount, or frequency of botulinum neurotoxin administered to the subject. This determination may take into account various factors, including the response information, to arrive at an optimal dosing regimen for that subject.

As used herein, the term“optimal dosing regimen” means the dosing regimen determined by the medical practitioner to be optimal for a particular subject based on a variety of factors, including: the subject’s age, the type of vulvodynia, the potency of the neurotoxin, and/or the subject’s symptoms relating to vulvodynia.

As used herein, the term“computer device” refers to any electronic device for storing and processing data, typically in binary form, according to instructions given to it in a software program, and includes, for example, a desktop, laptop, or tablet personal computers;“netbooks”; mobile communication devices, such as smartphones; personal digital assistants; portable audio or video file players; portable game players; portable electronic readers; or equivalent devices. The computer device can be in communication with another computer device by any suitable type of network (such as internet), and can use any suitable protocol, medium (eg., fiber optic, coaxial cable, wireless broadband, etc.), network interface, or bandwidth.

The computer device used by the subject can be the same as or different from the computer device used by the medical practitioner to access the response information. In some embodiments, the computer device used by the subject is the same computer device that the medical practitioner uses to access the response information. For example, the subject may enter the response information into his/her smartphone and bring that smartphone to the clinic visit to have the information viewed by the medical practitioner directly from the smartphone. In other embodiments, the computer device used to access the response information is different from the computer device used to enter the response information. For example, the response information may be entered into a website portal that is specially designed to collect this type of information and the medical practitioner uses his/her own desktop computer to access the website portal and view the information entered.

The computer device on which the database resides may be the same as or different from the computer device used to receive the response information and/ or the computer device used to access that information. In some embodiments, the computer device on which the database resides is different from both the computer device used to receive the response information and the computer device used by the medical practitioner to access that information. For example, the information may be entered into a website portal via a laptop computer. The information is stored on a web server computer, which is then accessed by the medical practitioner on his/her own computer to view the response information.

The computer system may be capable of determining a subsequent or optimal dosing regimen for the administration of botulinum neurotoxin to the subject. Such a determination may be made based on the response information with the appropriate adjustments in dosing amounts and frequency as discussed above. The invention also provides a software product. As used herein, the term “software product” refers to a non-transitory computer-readable storage medium storing instructions that, when executed by a computer system, causes the computer system to perform the recited steps. The software product may reside on any suitable computer-readable storage medium, such as CD-ROM, DVD, memory, hard disk, flash drive, RAM, ROM, cache, and the like. The software platform for implementing the present invention can vary depending on design considerations such as user preference, cost, implementation, ease of use, machine capabilities, network limitations, etc.

The invention further provides a computer system. The computer system is programmed to perform steps of a computer-implemented method, the method comprising: receiving information associated with the subject’s recovery; storing the information into a database; and transmitting the information to a medical practitioner.

The invention also provides a computer system comprising one or more computer devices that are programmed to perform the methods of the present invention. The hardware platforms used by the subject, the medical practitioner, and/or any other third parties may be different, but operate together as a system. For example, the response information may be entered in one computer device, that information may be stored on a different computer device located remotely (eg., a third party web server), and the medical practitioner may use his/her own computer device to access the information. In this scenario, these three computer devices may be considered to operate together as a system. The physical and/ or functional components of the computer system may be distributed, centralized, or arranged in any suitable manner. Communications between the different physical and/or functional components may be performed in any suitable way. Moreover, the present invention encompasses all the various ways in which the operating work may be divided among different physical and/or functional components.

The response information may be received by the computer system from a first computer device, the database resides on a second computer device, and the response information is transmitted by the computer system to a third computer device. The computer devices may be separate. The first computer device may be in communication with the second computer device and the second computer device may be in communication with the third computer device. In one aspect, the first computer device is programmed to receive the response information from the subject, the database resides on a second computer device, and the third computer device receives the transmitted response information. A non-transitory computer-readable storage medium storing instructions is also provided. The non-transitory computer-readable medium, when executed by a computer system, causes the computer system to perform steps of a computer-implemented method, the method comprising: receiving information associated with the subject’s recovery; storing the response information into a database; and transmitting the response information to a medical practitioner.

A computer-implemented method of determining a dosage regimen for administration of botulinum neurotoxin is also provided, the method comprising: (a) receiving information related to a subject’s response to treatment as conducted according to a method for treating one or more symptoms of vulvodynia, the method comprising administering a therapeutically-effective amount of botulinum neuro toxin to a subject determined to be in need thereof, wherein the botulinum neurotoxin is administered to the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and/ or the right deep transverse perineal muscle; (b) receiving the response information; and (c) determining a subsequent dosing regimen for the administration of botulinum neurotoxin to the subject based on the response information. Said method may further comprise sending an instruction to administer botulinum neurotoxin accordingly. The invention also provides a computer-readable medium comprising instructions which, when executed by a computer, cause the computer to carry out said method. The invention further provides a data processing device comprising a processor configured to perform said method.

Also described herein is a composition useful in treating one of more symptoms of vulvodynia. The composition comprises a therapeutically-effective amount of botulinum neurotoxin. In some embodiments, the composition comprises a diluent, for example a sodium chloride solution. The sodium chloride solution may, for example, be a 0.9% sodium chloride solution. In other embodiments, the composition comprises botulinum neurotoxin as a lyophilized powder which may be reconstituted, for example using a sodium chloride solution.

In an embodiment, the botulinum neurotoxin of the composition is of serotypes A, B, C, D, E, F, G, H, or X. In an embodiment, the botulinum neurotoxin of the composition is a protein containing modifications as compared to wild-type botulinum neurotoxins. In an embodiment, the botulinum neurotoxin of the composition is a protein having an amino acid sequence that has at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% sequence identity to the amino acid sequence of a botulinum neurotoxin of serotypes A, B, C, D, E, F, G, H, or X.

In an embodiment, the botulinum neurotoxin of the composition is naturally-occurring. In another embodiment, the botulinum neurotoxin of the composition is produced via recombinant means. Hybrid and chimeric botulinum toxins are also encompassed within the term“botulinum neurotoxin” according to the present invention.

Preferably, the botulinum neurotoxin of the composition is BoNT/A, also known as botulinum neurotoxin A. In a particularly preferred embodiment, the botulinum neurotoxin is DYSPORT®.

Wherein the invention related to DYSPORT ® and its uses, the composition of the composition typically comprises about 50 to 700 units of DYSPORT®, for example, about 500 units of DYSPORT®.

The composition may be formulated for the administration of botulinum neurotoxin parenterally, intra-muscularly, intradermally, or transdermally. In some embodiments, the composition is formulated for parenteral administration, for example subcutaneous administration. In some preferred embodiments, the composition is formulated for intramuscular administration.

The composition may be in the form of a liquid, a gel, or a powder.

The composition may comprise a lyophilized powder comprising botulinum neurotoxin, or the composition is formed by reconstituting a lyophilized powder comprising botulinum neurotoxin in a solution, for example the aforementioned sodium chloride solution. Following reconstitution, the composition may be stored under refrigeration at about 2 °C to about 8 °C and protected from light.

The composition may be administered to a subject in need thereof in amount determined to be appropriate as described above.

The composition may further comprise an excipient. The excipient may, for example, be a filler, a binder, a disintegrant, an anti-adherent, a solvent, a buffering agent, a preservative, or a humectant. Examples of fillers include cellulose, lactose, sucrose, glucose, mannose, and sorbitol. The composition may comprise, for example, 10 mg/ml lactose. Examples of binders include gelatin, cellulose, polyvinyl pyrrolidone, starch, and sucrose. Examples of disintegrants include polyvinyl pyrrolidone and carboxymethyl cellulose. Examples of preservatives include parabens. Examples of solvents include water, oils, glycerol, propylene glycol, and ethanol. Examples of buffering agents include phosphates, carbonates, citrates, and lactates. Examples of humectants include glycerol, ethylene glycol, and polyethylene glycol (PEG).

Also described herein is a device useful in treating one or more symptoms of vulvodynia. The device is configured to administer botulinum neuro toxin to a subject determined to be in need thereof. The botulinum neurotoxin may be distributed throughout the material of the device. Alternatively, the botulinum neurotoxin may be applied to the surface of the device. As another alternative, the botulinum neurotoxin may be present in a reservoir within the device. Examples of such devices are described in US 2017/0105784 and WO 2018/023027, both of which are incorporated herein by reference.

The device may take any appropriate format. By way of non-limiting example, the device may be an intravaginal device, which may be substantially ring-shaped with an outer edge configured to contact the vaginal wall. By way of a further non-limiting example,

the device may be a pre-filled syringe.

The device may be connected to a computer device. The computer device may be capable of determining a subsequent or optimal dosing regimen for the administration of botulinum neurotoxin to the subject. Such a determination may be made based on the response information with the appropriate adjustments in dosing amounts and frequency as discussed above. The computer device may then instruct the device to adjust its administration of botulinum neurotoxin accordingly.

Also described herein is a method of treating a symptom of vulvodynia, the method comprising inserting the aforementioned intravaginal device into the vagina of a subject determined to be in need of such treatment. The device may remain within the vagina of the subject for as long as necessary to be effective in the treatment. The subject can also have the device removed and reinserted as necessary.

It is to be understood that the present invention is not limited to the embodiments, aspects, or features described above, but encompasses any and all embodiments, aspects, and features within the scope of the following claims.

All references cited in this disclosure are incorporated herein by reference in their entireties. DEFINITIONS

The terms "decrease", "reduced", "reduction", or "inhibit" are all used herein to mean a decrease by a statistically significant amount. In some embodiments, "reduce," "reduction" or "decrease" or "inhibit" typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about

10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about

40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about

65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about

90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, "reduction" or "inhibition" does not encompass a complete inhibition or reduction as compared to a reference level. "Complete inhibition" is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

The terms "increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statically significant amount. In some embodiments of all the aspects described wherein, the terms "increased", "increase", "enhance", or "activate" can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a I 00% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

A "variant" botulinum neurotoxin as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains the relevant biological activity relative to the reference protein, e.g., at least 50% of the wildtype reference protein. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3% or fewer, or 1 % or fewer) of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. It is contemplated that some changes can potentially improve the relevant activity, such that a variant, whether conservative or not, has more than 100% of the activity of wild-type, e.g. 110%, 125%, 150%, 175%, 200%, 500%, 1000% or more.

A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as lie, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity of a native or reference polypeptide is retained. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure. Typically conservative substitutions for one another include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (L), Leucine (L ), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

The term "fragment", when used in relation to a protein, particularly a botulinum neurotoxin, means a peptide having at least 10, at least 20, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700 or more, amino acid residues of the protein in question, but not containing the full-length protein sequence, and which fragment thereof retains the relevant biological activity relative to the reference protein.

As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not. The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

The term“equivalent” as used herein may mean that the two or more values being compared are not statistically significantly different. Preferably the term“equivalent” as used herein means that the two or more values are identical.

Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation. The term“protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term“amino acid sequence” is synonymous with the term“polypeptide” and/ or the term“protein”. In some instances, the term“amino acid sequence” is synonymous with the term“peptide”. In some instances, the term“amino acid sequence” is synonymous with the term “enzyme”. The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three- letter codes for amino acid residues may be used. The 3 -letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be defined only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

Sequence homology

Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subde Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131 ) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

Thus, percent sequence identity is determined by conventional methods. See, for example,

Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Flenikoff and Flenikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes) .

Alignment score for determining sequence identity

BLOSUM62 table

A R N D C Q E G H I L K M F P S T W Y V

A 4

R -1 5

N-2 0 6

D-2-2 1 6

C 0 -3 -3 -3 9

Q4 1 0 0-3 5

E -1 0 0 2 -4 2 5

G 0-2 0-1 -3 -2 -2 6

H -2 0 1 -1 -3 0 0 -2 8

I _i _3 _3 _3 _i _3 _3 _4 _3 4

L -1 -2 -3 -4 -1 -2 -3 -4-3 2 4

K -1 2 0-1 -3 1 1 -2 -1 -3 -2 5

M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2-1 5

F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0-3 06

P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7

S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4

T 0 -1 0-1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2-1 1 5

W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4-3-211

Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7

V 0-3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4 The percent identity is then calculated as:

Total number of identical matches

_ x 100

[length of the longer sequence plus the number of gaps

Introduced into the longer sequence in order to align the two sequences]

Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

Conservative amino acid substitutions

Basic: arginine

lysine

histidine

Acidic: glutamic acid

aspartic acid

Polar: glutamine

asparagine

Hydrophobic: leucine

isoleucine

valine

Aromatic: phenylalanine

tryptophan

tyrosine

Small: glycine

alanine

serine

threonine methionine

In addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and a -methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for botulinum polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4- methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allothreonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et ak, J. Am. Chem. Soc. 113:2722, 1991; Ellman et ak, Methods Enzymol. 202:301, 1991; Chung et ah, Science 259:806-9, 1993; and Chung et ak, Proc. Nad. Acad. Sci. USA 90: 10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et ah, J. Biol. Chem. 271 :19991-8, 1996). Within a third method, E.coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine ). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et ah, Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site -directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993). A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.

Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081 -5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labelling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306- 12, 1992; Smith et al, J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241 :53-7, 1988) or Bowie and Sauer (Proc. Nad. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30: 10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

SEQUENCE INFORMATION

Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue/codon may be optional.

SEP ID NO: 1 fBoNT/A - UniProt PI 0845.5)

MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDL NPPPE AKQVPVSYYDSTYT STDNEKDNYT KGVTKT FERTYSTDT GRMT J TSTVRGTPFWG GSTTDTET KVTDTN CTNVT QPDGSYR SEET .NT ATT GPS A PTTQFECK SFGHEVT .NT TRNGY GSTQYTR FSPD FTFGFEEST EVDTNPT J G AGKFATPPA VTT . A HET JH A GHR T YGT A TNPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNK AKSIVGTTASLQYMKNVFKEKYLLSEDTSGI<GFSVDKLI<GFDKLYKMLTEIYTEDNFVI<GFF KVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKL KNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLN K GEETTSDTNTE A AEENTST DT .TQQYYT TFNFDNEPENT STENT SSPTTGQT ET .MPNTERFPN GKKYET PKYTMFHYT .R AQEFEHGKSRT AT TNSVNE AT J .NPSR VYTFFSSPYVKKVNK AT E A AMFT GWVEQT VYDFTDETSEV STTDKT A DTTTTTPYT GP A T NT GNMT YKPPFVGALTFS GAVTT J EFTPET A TPVT GTF A T .VS YT A NKVT TVQTTDN A T SKP NEKWDEVYKYTVTNWT .A K VNTQIDLIRKKMKEALEN QAEATKAIINY QYN QYTEEEKNNINFNIDDLSSKLNESINKA MININI<GFLNQCSVSYLMNSMIPYGVKRLEDFDASLI<GDALLKYIYDNRGTLIGQVDRLI<GD KVNNTT STDTPFQT SKYVDNQRT T .STFTEYTKNTTNTSTT .NT .R YESNHT TPT .SR Y A SKTNT GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNN EYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRWFKYSQMINISDYINRWIFVTIT NNRT NNSKTYTNGRT .TDQKPTSNT GNTFf A SNNTMFKT DGCRDTHR YTWTKYFNT FDKET N EI<GEII<GDLYDNQSNSGILI<GDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLK GPRGSVMTTNIYLNSSLYRGTI<GFIIKKYASGNI<GDNIVRNNDRVYINVWKNI<GEYRLATNA SO A GVEKTT .S AT ETPDVGNT SQVWMKSKNDQGTTNKCKMNT .QDNNGNDTGFTGFHQF NNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

SEP ID NO: 2 (BoNT/B - UniProt P10844.3)

MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN KSSGTFNRDVCEYYDPDYT .NTNDKKNTFT QTMTKT FNRTKSKPT GEKT T EMTTNGTPYT G DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH F A SREGFGGTMQMKFCPEYVSVFNNVQENKG A STFNR R GYFSDP A T IT MFfET JFfVT FfGT .Y GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRLNKVLVCISDPNININIYKNI<GFI<GDKYI<GFVEDSEGKYSIDVESFDI<GLYKSLMFGFTETN T A ENYKTKTR A SYFSDST PPVKTKNT T DNETYTTE E GFNT SD KDMEKEYR GQNK ATNKQA YEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSN YTENDFPTNET .TT DTDT .TSKTET PSENTEST TPFNVPVPVYEK QP A TKKTFTDENTTFQY T .YSQTFPT DTRDTST TSSFDD AT T ESNKVYSFFSMDYTKT ANKWE, AGT E AGWVKQTVND FVTE A NKSNTMPKT ADTST TVPYTGT .AT .NVGNET A KGNFEN A FET A G A STT T EFTPET T J PWG A FT T ESYTDNKNKTTKTTDN A T TKRNEKWSPMYGT .TV A QWT .STVNTQFYTTKEGM YI<GALNYQAQALEEIIKYRYNIYSEI<GEKSNINIDFNDINSI<GLNEGINQAIDNINNFINGCSV SYLMKKMIPLAVEI<GLLDFDNTLI<GKNLLNYIDENI<GLYLIGSAEYEKSKVNKYLKTIMPFDL STYTNDTTT .TEMFNKYNSETT ,NNTTT .NT RYKDNNT ,TDT .SGYG A KVEVYDGVET NDKNQFK LTSSAN SKIRVTQN QNIIFN SVFLDFSVSFWIRIPKYKNDGIQNYIF1NEYTIIN CMKNN S GWKISIRGNRIIWTLIDIN GKTKS VFFEYNIREDISEYINRWFFVTITNNLNNAKIYIN G

I<GLESNTDII<GDIREVIANGEIIFI<GLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSY SEYLI<GDFWGNPLMYNKEYYMFNAGNKNSYII<GLKI<GDSPVGEILTRSKYNQNSKYINYRD LYIGEI<GFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKI<GEEEI<GLFLAPISD SDEFYNTIQII<GEYDEQPTYSCQLLFKI<GDEESTDEIGLIGIHRFYESGIVFEEYI<GDYFCIS KWYLI<GEVKRKPYNLI<GLGCNWQFIPI<GDEGWTE

SEP ID NO: 3 ίBoNT/C - UniProt PI 8640.3)

MPITINNFNY SDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNK PPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNN

NTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTF

AAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYG

IAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSI

AKRLNSITTANPSSFNKYIGEYKQKLIRKYRFWESSGEVTVNRNKFVELYNELTQIFTE

FNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNP

ALRKVNPENMLYLFTKFCHKAIDGRSLYNKTLDCRELLVKNTDLPFIGDISDVKTDIFLR

KDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQN

VD YT XSYYYI ESQKT .SPNVEPFTFTR STEE A T .DNS A K VYTYFPTT ,ANKUN A GVQGGT ET .

MWANDWEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILL

EAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQF

NNIS Y QMYDSLNY QAGAIKAKIDLEYKKYSGSDKENIKSQVENLKN SLD VKISEAMNNI

NKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLINLIDSF1NIILVGEVDKLKAKVNNS

FQNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQ

LNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIID

SVKNNSGWSIGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGN

MKIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKEL

DGKDINILFNSLQYTNWKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRN

NNDFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTE

DIYAIGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGICSIGTYRFRLGGD

WYR I l\ YI VPTVKQGNY A ST T ESTSTHWGFVPVSE

SEP ID NO: 4 ZBoNT/D - UniProt P19321.1)

MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSK

PPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLWGSPFMGDS

STPEDTFDFTRF1TTNIAVEKFEN GSWKVTNIITPSVLIF GPLPNILDYTASLTLQGQQSN

PSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYG

INIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDI

AKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFWNIDKFNSLYSDLTNVMSE

WYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPA

LQKLSSESWDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIITDE

TNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYL

NSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANE

WEPFTTNTMKKPTT .PKTSPVS VTTPYTGP A T \ IG\ S A T .R GNFNQ A F AT A GV A FT T EGFP

E FTTP A T GVFTFYSSTQER EKTTKTTEN GT EQR VKRWKPSYQWMVSNWT .SRTTTQFNHTN

Y QMYDSLSY QAD AIKAKIDLEYKKYSGSDKENIKSQVENLKN SLDVKISEAMNNINKFIR

ECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTM

PFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTI

YTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNS

GWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYIN

GELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQI

LRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSV

SDKNPYSRILNGDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNY

GIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLS

TSSFWKFISRDPGWVE SEP ID NO: 5 iBoNT/E - UniProt 000496.2)

MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTS

LKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTP

PNQFHTGP A S A VETKFSNGSQPTT T PNVTTMG AEPPT FETNSSNTST .RNNYMPSNHR FGS

IAIVTFSPEYSFRFNDNCMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPL

ITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYK

D VFEAKY GLDKDASGIY SVNINKFNDIFKKLY SFTEFDLRTKF QVKCRQTYIGQYKYFKL

SNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKG

IRKSICIEINN GELFFVASEN SYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFN SES A

PGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTS

STPT AT T EQPKTYTFFSSEFTNNVNKPVQ A AT FVSWTQQVT .VPFTTE ANQKSTVPKT APTS

IWPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNK

NKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTI

IESKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKIINEVKIN

KLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYF

NKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVN

ISQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEII

WTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNL

GNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYL

LYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDN

LVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVWMNSVGNCTMN

FKNNNGNNIGLLGFKADTWASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK

SEP ID NO: 6 TBoNT/F - UniProt A7GBG3.E

MPWINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFD

PPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGN

EHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVY

DPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIADPAISLAHELIHALHGLYGAR

GVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATR

LSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANK

FKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDK

GLVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNLN

NNYRNNLDEVILD YN SETIPQISN QTLNTLV QDD S YVPRYD SN GTSEIEEHN WDLN VFF

YLHAQKVPEGETNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIR

DFTTE ATPKSTFDKT APTST .WPYVGT .AT .NTGNEVOKENFKE AFET T GAGTT T FVPET T .

IPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRK

EQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIER

FITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNN

SIPFELSSYTNDKILILYFNKLYKKIKDNSILDMRYENNKFIDISGYGSNISINGDVYIY

STNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDC

IRNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGN

SRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETL

Y SDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQN SNFLNINQQRGVY QKPNIFSN

TRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINWDRDVEYRLYADISIAKPEKIIKL

IRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNT

SSNGCFWSFISKEHGWQEN SEP ID NO: 7 ZBoNT/G - UniProt 060393.2)

MPVNIKXFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFN

ASTGVFSKDVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLG

NASTPPDKFAANVANVSINKKIIQPGAEDQIKGLMTNLIIFGPGPVLSDNFTDSMIMNGE1

SPISEGFGARMMIRFCPSCLNVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLY

GIKISNLPITPNTKEFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIA

NRLNIVSSAQGSGIDISLYKQIYKNKYDFVEDPNGKYSVDKDKFDKLYKALMFGFTETN

LAGEY GIKTRY S YFSEYLPPIKTEKLLDNTIYTQNEGFNIASKNLKTEFN GQNKAVNKEA

YEEISLEHLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAETIAYN

TQNNTIENNFSIDQLILDNDLSSGIDLPNENTEPFTNFDDIDIPVYIKQSALKKIFVDGD

SLFEYLHAQTFPSNIENLQLTNSLNDALRNNNKVYTFFSTNLVEKANTWGASLFVNWV

K GVTPP FTSEST OK STTP K V SP VSTTTP YT GP A T .NVGNET A KENFKN A FETGG A A TT .MEET

PET JVPTVGFFTT ESYVGNKGHTTMTTSN AT .KKRPQKWTPMYGT .TVSQWT .STVNTPFYTT

KERMYNALNNQSQAIEKIIEDQYNRYSEEDKMNINIDFNDIDFKLNQSINLAINNIDDFI

NQCSISYLMNRMIPLAVKKLKDFDDNLKRDLLEYIDTNELYLLDEVNILKSKVNRHLKDS

IPFDLSLYTKDTILIQVFNNYISNISSNAILSLSYRGGRLIDSSGYGATMNVGSDVIFND

IGNGQFKLNNSENSNITAHQSKFWYDSMFDNFSINFWVRTPKYNNNDIQTYLQNEYTII

SCIKNDSGWKVSIKGNRIIWTLIDVNAKSKSIFFEYSIKDNISDYINKWFSITITNDRLG

NANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIFGRELNATEVS

SLYWIQSSTNTLKDFWGNPLRYDTQYYLFNQGMQNIYIKYFSKASMGETAPRTNFNNAA

INYQNLYLGLRFIIKKASNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQTQ

LFLAPINDDPTFYDVLQIKKYYEKTTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTY

DNYFCISQWYLRRISENINKLRLGCNWQFIPVDEGWTE

SEP ID NO: 8 (Polypeptide Sequence of BoNT/X)

MKLEINKFNYNDPIDGINVITMRPPRE1SDKINKGKGPFKAFQVIKNIWIVPERYNFTNNT

NDLNIPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIP

LPLV SN GALTLSDNETIAY QENNNIV SNLQANLVIY GPGPDIANNATY GLY STPISN GEG

TLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGIS

NRNFYYNFDTGKIETSRQQNSLIFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTLISE

RLNTVTVENDLLKYIKNKIPVQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVR

KHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNALRAFI

KICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFLISN

KDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELY

EPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPF

KNMSNTINSIETGITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPYIGPLLNI

GNDTRHGDFVGATET .AGTT AT T YVPEFTTPTT VGT EVTGGET .AREQVE ATVNN AT .DKRD

QKWAEVYNITKAQWWGTIHLQINTRLAHTYKALSRQANAIKMNMEFQLANYKGNIDD

KAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKVQDNLKNFDLETKKT

LDKFIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDLINQYKNEIEDYEVLN

LGAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNF

SISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYI

AFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLL

DQFSIYRKELNQNEWKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREY

WSSFGYDYVILSDSKTITFPNNIRY GALYN GSKVLIKN SKKLDGLVRNKDFIQLEIDGYN MGISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTE

TSKPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED

The present invention will now be described with reference to the following non-limiting Examples.

Examples

Example 1— Preparation of DYSPQRT ®

2.5 mL of preservative-free 0.9% sodium chloride solution is drawn and then injected into a vial containing 500 units of DYSPORT®. The vial is tilted side-to-side (and not shaken) for about one minute to ensure solution homogeneity.

Next, 0.5 mL of the reconstituted DYSPORT® is drawn up without inverting the vial. This 0.5 mL solution is then combined with 2.0 mL of preservative-free 0.9% sodium chloride solution. This can be accomplished, for example, by drawing 2.0 mL of preservative-free 0.9% sodium chloride solution into a syringe, connecting that syringe to the syringe containing the 0.5 mL solution of reconstituted DYSPORT®, and using the plunger of the latter syringe to pull 2.0 mL of preservative-free 0.9% sodium chloride solution. This will result in a syringe having 2.5 mL of preservative-free 0.9% sodium chloride solution containing 100 units of DYSPORT®.

Next, a“first” 1 mL syringe is connected to the syringe containing the DYSPORT® solution. The plunger of the first 1 mL syringe is pulled to transfer 0.8 mL of the DYSPORT® solution into the first 1 mL syringe. The syringe is removed and capped. This first 1 mL syringe contains about 32 units of DYSPORT®.

Next, a“second” 1 mL syringe is connected to the syringe containing the DYSPORT® solution. The plunger of the second 1 mL syringe is pulled to transfer 0.8 mL of the DYSPORT® solution into the second 1 mL syringe. The syringe is removed and capped. This second 1 mL syringe contains about 32 units of DYSPORT®.

Next, a“third” 1 mL syringe is connected to the syringe containing the DYSPORT® solution. The plunger of the third 1 mL syringe is pulled to transfer 0.9 mL of the DYSPORT® solution into the third 1 mL syringe. The syringe is removed and capped. This third 1 mL syringe contains about 36 units of DYSPORT®.

The three 1 mL syringes are stored in a refrigerator at 2 to 8° C and protected from light. The solutions contained therein are used within 24 hours and, if not used, are discarded. Before use in injection, the cap is removed and a needle attached. Example 2 - Administration of DYSPQRT ® to subject

Botulinum neurotoxin is administered by intramuscular injection to a subject determined to have vulvodynia on day 1 of each testing cycle. The administration paradigm is as follows:

The first 1 mL syringe containing 0.8 mL of DYSPORT® as reconstituted according to Example 1 is used to inject 0.2 mL of DYSPORT® (about 8 units) to the subject’s left bulbospongiosus, anterior to the hymeneal ring. This is accomplished by injecting at about the 5 o’clock position (with the front of the patient facing the 12 o’clock position) at about 45° laterally and about 45° posteriorly (towards the hip) and at a depth of about 5 mm from the outer the surface of the skin. The same syringe is then used to inject a further 0.2 mL of DYSPORT® (about 8 units) to the subject’s left pubococcygeous, anterior to the hymeneal ring, by injecting at the same position but at a depth of about 10 mm from the outer surface of the skin. The same syringe is then used to inject 0.2 mL of DYSPORT® (about 8 units) to the subject’s left bulbospongiosus muscle, posterior to the hymeneal ring. This is accomplished by injecting at about the 5 o’clock position at about 45° laterally and about 45° posteriorly (towards the hip) and at a depth of about 5 mm from the outer the surface of the skin. The same syringe is then used to inject the final 0.2 mL of DYSPORT® (about 8 units) to the subject’s left pubococcygeous muscle, posterior to the hymeneal ring, by injecting at the same position but at a depth of about 10 mm from the outer surface of the skin.

The second 1 mL syringe containing 0.8 mL of DYSPORT® as reconstituted according to Example 1 is used to inject 0.2 mL of DYSPORT® (about 8 units) to the subject’s right bulbospongiosus, anterior to the hymeneal ring. This is accomplished by injecting at about the 7 o’clock position (with the front of the patient facing the 12 o’clock position) at about 45° laterally and about 45° posteriorly (towards the hip) and at a depth of about 5 mm from the outer the surface of the skin. The same syringe is then used to inject a further 0.2 mL of DYSPORT® (about 8 units) to the subject’s right pubococcygeous, anterior to the hymeneal ring, by injecting at the same position but at a depth of about 10 mm from the outer surface of the skin. The same syringe is then used to inject 0.2 mL of DYSPORT® (about 8 units) to the subject’s right bulbospongiosus muscle, posterior to the hymeneal ring. This is accomplished by injecting at about the 5 o’clock position at about 45° laterally and about 45° posteriorly (towards the hip) and at a depth of about 5 mm from the outer the surface of the skin. The same syringe is then used to inject the final 0.2 mL of DYSPORT® (about 8 units) to the subject’s right pubococcygeous muscle, posterior to the hymeneal ring, by injecting at the same position but at a depth of about 10 mm from the outer surface of the skin. The third syringe containing 0.9 mL of DYSPORT® (as reconstituted according to Example 1) is used to inject 0.45 mL of DYSPORT® (about 18 units) to the subject’s superficial and deep transverse perineal muscles anterior to the hymeneal ring. This is accomplished by injecting at about the 6 o’clock position (with the front of the patient facing the 12 o’clock position) at about 45° laterally and about 45° posteriorly (towards the coccyx) and at a depth of about 5 mm from the outer the surface of the skin (both left and right, superficial and deep transverse perineal muscles are administered to with this injection). The same syringe is then used to inject the final 0.45 mL of DYSPORT® (about 18 units) to the superficial and deep transverse perineal muscles, anterior to the hymeneal ring, by injecting at the same position but at a depth of about 10 mm from the outer surface of the skin (both left and right, superficial and deep transverse perineal muscles are administered to with this injection).

The injection sites are depicted in Figure 1. A total of approximately 500 units of DYSPORT ® is administered to the subject.

Example 3 - Use of DYSPORT ® to treat PVD

A 36 year old woman with PVD, presents with symptom duration of 14 months and a baseline score of 6 on the NRS scale when tested with the 0.75 inch dilator - the size that provokes the maximum tolerated pain (dilator maximum tested size, DMTS). She receives a course of DYSPORT ® injections according to Example 2. Within 6 weeks her pain perception as recorded on the NRS scale drops to 4 using the baseline DMTS (0.75 inches).

The treatment is repeated 12 weeks later and three months later the patient reports no return of PVD symptoms.

Example 4— Use of DYSPORT ® to treat vulvodynia

A 23 year old woman with vuvlodynia, presents with symptom duration of 8 months and a baseline score of 9 on the NRS scale when tested with the 0.5 inch dilator (DMTS). She receives a course of DYSPORT ® injections according to Example 2. Within 6 weeks her pain perception as recorded on the NRS scale drops to 6 using the baseline DMTS (0.5 inches).

The treatment is repeated twice at 12 week intervals and four months after the last treatment the patient reports no return of vuvlodynia symptoms. Example 5— Use of DYSPQRT ® to treat vestibulodynia

A 42 year old woman with vestibulodynia, presents with symptom duration of 3 years and a baseline score of 6 on the NRS scale when tested with the 0.5 inch dilator (DMTS) and has bilateral provoked pain at the posterior vestibule on a cotton-bud test, with pain at positions 5, 6 and 7 o’clock. She receives a course of DYSPORT ® injections according to Example 2. Within 6 weeks the DMTS increases from the 0.5 inch dilator to the 0.75 inch dilator and her pain perception as recorded on the NRS scale drops to 4 using the baseline DMTS (0.5 inches). Thus, her compositie score (combination of pain perception measured by NRS and the DMTS) significantly improves.

The treatment is repeated 24 weeks later and four months later the patient reports no return of vestibulodynia symptoms.

Example 6— Use of DYSPQRT ® to treat PVD

A 30 year old woman with PVD, presents with symptom duration of 2 years and a baseline score of 8 on the NRS scale when tested with the 0.75 inch dilator (DMTS). She receives a course of DYSPORT ® injections according to Example 2. Within 6 weeks her mean CGI (as assessed by her attending physician) significandy improves.

The treatment is repeated twice at 12 week intervals and four months after the last treatment the patient reports no return of PVD symptoms.

Example 7— Use of DYSPORT ® to treat PVD

A 38 year old woman with PVD, presents with symptom duration of 10 months and a baseline score of 5 on the NRS scale when tested with the 0.5 inch dilator (DMTS). She receives a course of DYSPORT ® injections according to Example 2. Within 6 weeks her mean PGG-C (as assessed by the patient) significantly improves.

The treatment is repeated 12 weeks later and three months later the patient reports no return of PVD symptoms. Example 8— Use of DYSPQRT ® to treat vestibulodynia

A 19 year old woman with vestibulodynia, presents with symptom duration of 11 months and a baseline score of 10 on the NRS scale when tested with the 0.75 inch dilator (DMTS). She receives a course of DYSPORT ® injections according to Example 2. Within 6 weeks her mean PGG-S (as assessed by the patient) significantly improves.

The treatment is repeated twice at 12 week intervals and four months after the last treatment the patient reports no return of vestibulodynia symptoms.

Claims

1. A botulinum neurotoxin for use in a method for treating one or more symptoms of vulvodynia, the method comprising administering a therapeutically-effective amount of botulinum neurotoxin to a subject determined to be in need thereof, wherein the botulinum neurotoxin for administration to the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and/or the right deep transverse perineal muscle.

2. The botulinum neurotoxin for use of claim 1, wherein the botulinum neurotoxin is for administration in an amount from about 200 to about 800 units per treatment session.

3. The botulinum neurotoxin for use of claim 1 or 2, wherein the botulinum neurotoxin is for administration in the following order: (1) to a first bulbospongiosus muscle, anterior to the hymeneal ring, (2) to a first pubococcygeous muscle, anterior to the hymeneal ring; (3) to the first bulbospongiosus muscle, posterior to the hymeneal ring; (4) to the first pubococcygeous muscle, posterior to the hymeneal ring; (5) to a second bulbospongiosus muscle, anterior to the hymeneal ring, (6) to a second pubococcygeous muscle, anterior to the hymeneal ring; (7) to the second bulbospongiosus muscle, posterior to the hymeneal ring; (8) to the second pubococcygeous muscle, posterior to the hymeneal ring; (9) to the transverse perineal muscles anterior to the hymeneal ring.

4. The botulinum neurotoxin for use of any one of claims 1—3, wherein the botulinum neurotoxin has at least about 80 % sequence identity to the amino acid sequence of a botulinum neurotoxin of serotypes A, B, C, D, E, F, G, H, or X.

5. The botulinum neurotoxin for use of any one of claims 1—4, wherein the botulinum neurotoxin is BoNT/A.

6. The botulinum neurotoxin for use of any one of claims 1—5, wherein the subject suffers from pain at the vaginal vestibule, preferably wherein the subject suffers from vestibulodynia.

7. The botulinum neurotoxin for use of any one of claims 1—6, wherein the symptom is an affective disorder.

8. The botulinum neurotoxin for use of any one of claims 1—7, wherein the symptom is depression or anxiety.

9. The botulinum neurotoxin for use of any one of claims 1—8, wherein the amount of botulinum neurotoxin for administration is determined based in part on the level of severity of the vulvodynia.

10. The botulinum neurotoxin for use of any one of claims 1—9, wherein said method of treating further comprises determining a dosing regimen for botulinum neurotoxin or the amount and/or frequency for a subsequent administration of botulinum neurotoxin based on the subject’s response to a previous administration of botulinum neurotoxin.

11. The botulinum neurotoxin for use of any one of claims 1—10, wherein the amount of an administration of botulinum neurotoxin or a dosing regimen for botulinum neurotoxin is determined in part by the level of pain measured following the insertion of a probe into the subject’s vagina.

12. The botulinum neurotoxin for use of any one of claims 1—11, wherein the amount of botulinum neurotoxin for administration or the dosing regimen for the botulinum neurotoxin is pre determined by a method comprising: inserting a probe into the subject’s vagina and, if the level of pain measured following insertion of the probe is below a threshold level, a larger sized probe is inserted and the level of pain measured and, if the level of pain measured remains below the threshold level, probe(s) of increasing size are inserted until the level of pain measured following such insertion is at or above the threshold level; and the amount of botulinum neuro toxin for administration or the dosing regimen for botulinum neurotoxin is based on the level of pain measured following the insertion of the final probe.

13. The botulinum neurotoxin for use of claim 11 or 12, wherein the probe is a vaginal dilator.

14. The botulinum neurotoxin for use of any one of claims 1—13, further comprising recording the subject’s response to an administration of botulinum neurotoxin.

15. The botulinum neurotoxin for use of any one of claims 1—14, wherein said method of treating comprises a treatment session in which BoNT/A for administration to a female subject diagnosed with vulvodynia using the following injection protocol:

(a) about 8% of the total dose of BoNT/A administered during the treatment session is for injection to the subject’s left bulbospongiosus muscle, anterior to the hymeneal ring, wherein the injection is made at about the 5 o’clock position at about 45° laterally and about 45° posteriorly, towards the hip, and at a depth of about 5 mm from the outer surface of the skin;

(b) about 8% of the total dose BoNT/A administered during the treatment session is for injection to the subject’s left pubococcygeous muscle, anterior to the hymeneal ring, wherein the injection is made at the same position as the first injection but at a depth of about 10 mm from the outer surface of the skin;

(c) about 8% of the total dose of BoNT/A administered during the treatment session is for injection to the subject’s left bulbospongiosus muscle, posterior to the hymeneal ring, wherein the injection is made at about the 5 o’clock position at about 45° laterally and about 45° posteriorly, towards the hip, and at a depth of about 5 mm from the outer surface of the skin;

(d) about 8% of the total dose of BoNT/A administered during the treatment session is for injection to the subject’s left pubococcygeous muscle, posterior to the hymeneal ring, wherein the injection is made at the same position as the previous injection but at a depth of about 10 mm from the outer surface of the skin;

(e) about 8% of the total dose of BoNT/A administered during the treatment session is for injection to the subject’s right bulbospongiosus muscle, anterior to the hymeneal ring, wherein the injection is made at about the 7 o’clock position at about 45° laterally and about 45° posteriorly, towards the hip, and at a depth of about 5 mm from the outer surface of the skin;

(f) about 8% of the total dose of BoNT/A administered during the treatment session is for injection to the subject’s right pubococcygeous muscle, anterior to the hymeneal ring, wherein the injection is made at the same position as the previous injection but at a depth of about 10 mm from the outer surface of the skin; (g) about 8% of the total dose of BoNT/A administered during the treatment session is for injection to the subject’s right bulbospongiosus muscle, posterior to the hymeneal ring, wherein the injection is made at about the 7 o’clock position at about 45° laterally and about 45° posteriorly, towards the hip, and at a depth of about 5 mm from the outer surface of the skin;

(h) about 8% of the total dose of BoNT/A administered during the treatment session is for injection to the subject’s right pubococcygeous muscle, posterior to the hymeneal ring, wherein the injection is made at the same position as the previous injection but at a depth of about 10 mm from the outer surface of the skin; and

(i) about 36% of the total dose of BoNT/A administered during the treatment session is for injection to the subject’s transverse perineal muscles, anterior to the hymeneal ring, wherein:

about 18% is for administration at about the 6 o’clock position at about 45° laterally and about 45° posteriorly, towards the coccyx, and at a depth of about 5 mm from the outer surface of the skin,

about 18% is for administration at about the 6 o’clock position at about 45° laterally and about 45° posteriorly, towards the coccyx, and at a depth of about 10 mm from the outer surface of the skin; and

wherein, after the foregoing injection protocol, the female subject experiences a lessening or elimination of one or more symptoms of vulvodynia.

16. A computer-implemented method of determining a dosage regimen for administration of botulinum neurotoxin, the method comprising:

receiving information related to a subject’s response to treatment as conducted according to a method for treating one or more symptoms of vulvodynia, the method comprising administering a therapeutically-effective amount of botulinum neuro toxin to a subject determined to be in need thereof, wherein the botulinum neurotoxin is administered to the left bulbospongiosus, the right bulbospongiosus, the left pubococcygeous, the right pubococcygeous, the left superficial transverse perineal muscle, the right superficial transverse perineal muscle, the left deep transverse perineal muscle, and/ or the right deep transverse perineal muscle;

receiving the response information; and

determining a subsequent dosing regimen for the administration of botulinum neurotoxin to the subject based on the response information.

17. The method of claim 16 further comprising sending an instruction to administer botulinum neurotoxin accordingly.

18. A computer-readable medium comprising instructions which, when executed by a computer, cause the computer to carry out the method of claim 16 or 17.

19. A data processing device comprising a processor configured to perform the method of claim 16 or 17.