WO2020234573A1

WO2020234573A1 - Screening method to determine suitability for participation in a clinical trial

Info

Publication number: WO2020234573A1
Application number: PCT/GB2020/051202
Authority: WO
Inventors: Richard Langford; Laurent PONS; Stephen Evans
Original assignee: Ipsen Biopharm Limited
Priority date: 2019-05-17
Filing date: 2020-05-15
Publication date: 2020-11-26
Also published as: GB201907016D0

Abstract

The invention relates to a screening method to determine a subject's suitability for participation in a clinical trial for the treatment of pain. Methods of determining a subject's sensitivity to local anaesthetic and methods of screening a subject for suitability for participation in a clinical trial for the treatment of pain are also provided.

Description

SCREENING METHOD TO DETERMINE SUITABILITY FOR PARTICIPATION IN A CLINICAL TRIAL

FIELD OF THE INVENTION

The invention relates to a screening method to determine a subject's suitability for participation in a clinical trial for the treatment of pain, based on an individual's sensitivity to local anaesthetic.

BACKGROUND OF THE INVENTION

Pain is the most common symptom for which subjects seek medical attention. Clinically, pain can be divided into three categories: (1) Acute pain, usually arising from injury or surgery that is expected to disappear when that injury is healed. (2) Chronic pain arising from malignant disease; the majority of people with metastatic cancer have moderate to severe pain and this is resolved either by successful treatment of the disease or by the death of the patient. (3) Chronic pain not caused by malignant disease; this is a heterogeneous complaint, caused by a variety of illnesses, including arthritis and peripheral neuropathies, that are usually not life-threatening but which may last for decades with increasing levels of pain. Chronic pain in particular affects between one-third and one-half of the population of the United Kingdom (UK), corresponding to just under 28 million adults and is predicted to increase due to an ageing population

Nociceptor sensory neurons mediate the detection of harmful/injurious stimuli, and their aberrant activation is known to result in chronic pain. Some conventional therapies, such as opioids act centrally to inhibit neurotransmission between the primary nociceptive neurons and the projection neurons. Others, such as non-steroidal anti-inflammatory drugs (NSAIDs) have their main action peripherally, by inhibiting prostaglandin release at injury sites.

Conventionally, there are few effective treatments for non-malignant chronic pain, with conventional therapies having limited effectiveness and often significant side effects. For example, opioids are often associated depression of the respiratory system, constipation and particularly addiction. NSAIDS alone are only effective in reducing some types of mild pain to acceptable levels. They are regarded as having a ceiling of activity above which increasing doses do not give increasing pain relief. Furthermore NSAIDs also have side effects with long-term use, such as ulceration of the gastrointestinal tract, that limit their usefulness in chronic complaints.

Chronic pain also has major socioeconomic consequences. In view of the limitations and disadvantages of current therapies, there is, therefore, a significant unmet need for clinically effective, safe treatments for pain, particularly chronic pain. Like all therapeutics, pain medications must undergo clinical trials to determine their safety and efficacy prior to being approved for clinical use. Unfortunately, there is a high placebo response in clinical trials for the management of pain, such that trials frequently fail to distinguish the analgesic effect from placebo, even when the treatment being tested has known efficacy. Consequently, investigational medicinal products for pain treatment often fail to show the necessary efficacy in clinical trials. In order to address the significant unmet need for new effective and safe treatments for pain there is therefore a concomitant need for clinical trial methodology that can accurately determine the clinical efficacy of an investigational medicinal product. One of the biggest problems with clinical trials for pain indications is the high placebo response and identifying suitable subjects for these trials and this is the purpose of this invention. In other words, there is a need to screen subjects prior to inclusion in a clinical trial to investigate a potential new treatment for pain in order to reduce or exclude false positive results from the clinical trial.

SUMMARY OF THE INVENTION

The present inventor has developed a new method for screening a subject for suitability for participation in a clinical trial for the treatment of pain. In particular the method devised by the present inventor uses a cross-over screening methodology in which a subject is treated with a local anaesthetic and a placebo (in either order), and his or her pain perception is determined following either treatment and the subject's pain perception after the placebo and local anaesthetic treatments are compared. This is done in a double-blind manner, such that neither the subject nor the person administering the treatments knows what has been administered. In this way, a subject acts as his or her own control to eliminate confounding factors. Advantageously, the inventor's method allows subjects with high placebo effects to be identified, and therefore eliminated from the pool of subjects for a clinical trial for the treatment of pain, leading to more accurate clinical trials and hence improved identification of valid clinical treatments. Accordingly, the present invention provides a local anaesthetic for use in screening a subject for suitability for participation in a clinical trial for the treatment of pain, comprising: (a) administration of the local anaesthetic or a placebo to said subject; (b) assessing said subject for pain perception; (c) in the case where the local anaesthetic was administered in step a, administration of the placebo, or in the case where the placebo was administered in step a, administration of the local anaesthetic; (d) assessing said subject for pain perception; and (e) determining whether a subject is suitable for participation in the clinical trial on the basis of a comparison of the pain perception assessment of step b and step d; wherein steps a to e are carried out sequentially.

In some embodiments, step c is carried out at least three days, at least five days, or at least seven days after step a, and preferably step c is carried out one week after step a. Pain perception may be assessed prior to step a and/or step c, preferably before both steps a and c, to determine a baseline of pain perception. In some embodiments: (i) step b is carried out up to 24 hours after step a; and/or (ii) step d is carried out up to 24 hours after step c; wherein preferably step b is carried out up to 24 hours after step a, and step d is carried out up to 24 hours after step c. In some embodiments step b and/or step d comprises at least two separate assessments for pain perception, optionally wherein: (i) in step b pain perception is assessed at least one of: 30 minutes after step a; hourly up to 8 hours after step a; and/or hourly between 20-24 hours after step a; and preferably pain perception is assessed at each of these times; and/or (ii) in step d pain perception is assessed at least one of: 30 minutes after step c; hourly up to 8 hours after step c; and/or hourly between 20-24 hours after step c; and preferably pain perception is assessed at each of these times. The local anaesthetic may be lidocaine; and/or the placebo saline; wherein preferably the local anaesthetic is lidocaine and the placebo is saline. Steps a to d may be conducted in a double-blind manner. Pain perception may be assessed using the Numeric Rating Scale (NRS). In some embodiments, determining whether a subject is suitable for participation in a clinical trial on the basis of a comparison of the pain perception assessment of step b and step d comprises determining if the pain perception following administration of the local anaesthetic is at least two points lower on the NRS compared with the pain perception following administration of the placebo. The investigational medicinal product (IMP) for investigation in the clinical trial for the treatment of pain may be a clostridial neurotoxin, preferably a botulinum neurotoxin (BoNT) or tetanus neurotoxin (TeNT). Said botulinum neurotoxin may be a wild-type BoNT, or a modified BoNT, preferably wherein the BoNT is a wild-type BoNT/A, BoNT/B, BoNT/Cl, BoNT/D, BoNT/E, BoNT/F or BoNT/G, more preferably wherein the BoNT is a wild-type BoNT/A. In some embodiments: (i) step a takes place a maximum of 21 days before the first administration of the IMP or the corresponding IMP placebo in a clinical trial for the treatment of pain; and/or (ii) step c takes place a maximum of 14 days before the first administration of the IMP or the corresponding IMP placebo in a clinical trial for the treatment of pain. The pain may be chronic pain, preferably abdominal or thoracic chronic scar pain. The clinical trial may be a double-blind trial for investigating the IMP wild-type BoNT/A compared with a placebo, wherein the wild-type BoNT/A is administered at a dose of 2.5 U/injection site, 10 U/injection site or 20 U/injection site and the subject is monitored for 16 weeks post-administration of the IMP. In some embodiments the clinical trial assesses parameters comprising: (i) NRS; (ii) stimulus-evoked NRS; (iii) temperature of the painful area; (iv) size of the painful area; (v) time to onset of analgesic effect; (vi) peak analgesic effect; (vii) time to peak analgesic effect; (viii) duration of analgesic effect; and/or ix) SF-36 quality of life. The invention also provides a method of determining local anaesthetic sensitivity comprising: (a) administering the local anaesthetic or a placebo; (b) assessing for pain perception; (c) in the case where the local anaesthetic was administered in step a, administering the placebo, or in the case where the placebo was administered in step a, administering the local anaesthetic; and (d) assessing for pain perception; wherein steps a to d are carried out sequentially.

The invention further provides a method for screening a subject for suitability for participation in a clinical trial for the treatment of pain comprising: (a) carrying out the method of determining local anaesthetic sensitivity of the invention; and (i) comparing the pain perception assessed in step b with the pain perception assessed in step d; and (ii) determining whether a subject is suitable for participation in the clinical trial on the basis of the comparison of step ii.

The invention further provides a method for screening a subject for suitability for a particular treatment for pain comprising: (a) carrying out the method of determining local anaesthetic sensitivity of the invention; and (i) comparing the pain perception assessed in step b with the pain perception assessed in step d; and (ii) determining whether a subject is suitable for a particular treatment for pain on the basis of the comparison of step ii.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Injection paradigm exemplifying how the local anaesthetic or IMP may be administered (e.g. by subcutaneous or intradermal injection) to the painful area.

Figure 2: Exemplary study design illustrating the medical eligibility assessment (screening); the screening method of the invention (Part A) and a subsequent clinical trial for the treatment of pain (Part B) for which subjects are selected for participation on the basis of the screening of Part A.

Figure 3: Illustration of the Numerical Rating Scale (NRS) for pain. This is an 11-point scale in which 0 corresponds to no pain and 10 corresponds to the worst possible pain.

Figure 4: Exemplary administration scheme for the administration of an IMP, such as BoNT/A to the painful area.

Figure 5: Box and Whisker Plot of Peak Effect (Randomised Population), Max=maximum; min=minimum; NRS=numerical rating scale. For each treatment group, worst NRS is shown on the left, and average NRS on the right.

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 description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the references herein and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

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.

Screening a Subject for Suitability for Participation in a Clinical Trial for the Treatment of Pain

It is well-established from analysis of clinical trials for analgesics, particularly opioid analgesics, that such clinical trials frequently fail to distinguish the analgesic effect from placebo, despite known efficacy of the drug. This often arises due to large placebo effects being recorded, making it difficult to attribute efficacy to the treatment being investigated. That being the case, despite the unmet need in the art for new effective and safe treatments for pain, it is often difficult to assess whether a potential new pain treatment is clinically efficacious, potentially preventing beneficial new treatments from entering the clinic, or at least slowing down their assessment and ultimate authorisation.

The present inventor is the first to appreciate that by first screening subjects for their response to a local anaesthetic, such as lidocaine (lignocaine), it is possible to filter out subjects with a high placebo response. This enables the selection of subjects to form a patient population for a clinical trial which will more accurately be able to determine the clinical efficacy of a new investigational medicinal product. In particular, the use of local anaesthetics, such as lidocaine (lignocaine) is especially beneficial in screening of subjects for suitability for participation in clinical trials where a locally applied analgesic agent, such as a botulinum neurotoxin, is used to manage the pain from a specified area of the body (e.g. post-surgical neuralgia (scar) pain).

Accordingly, the invention provides a local anaesthetic for use in screening a subject for suitability for participation in a clinical trial for the treatment of pain, comprising: (a) administration of the local anaesthetic or a placebo to said subject; (b) assessing said subject for pain perception; (c) in the case where the local anaesthetic was administered in step a, administration of the placebo, or in the case where the placebo was administered in step a, administration of the local anaesthetic; (d) assessing said subject for pain perception; and (e) determining whether a subject is suitable for participation in the clinical trial on the basis of a comparison of the pain perception assessment of step b and step d; wherein steps a to e are carried out sequentially from a to e.

Thus, the invention provides a cross-over screening method in which a subject is treated either with a local anaesthetic or a placebo (in either order) and their pain perception assessed in response to said administration. In other words, subjects who received an injection of local anaesthetic at the initial administration (step a) are treated with placebo in the subsequent administration (step c), and those subjects who received placebo at the initial administration (step a) are treated with local anaesthetic in the subsequence administration (step c). The subject's pain perception in response to the local anaesthetic is compared with their pain perception in response to the placebo, and this comparison used to determine whether or not said subject is suitable for participation in a clinical trial for the treatment of pain. It is not important whether the subject is first administered with the local anaesthetic and then the placebo, or whether the subject is first administered with the placebo and then the local anaesthetic.

As used herein, the term "screening a subject for suitability for participation in a clinical trial for the treatment of pain" means determining, using the methods and techniques described herein whether a subject responds sufficiently to the local anaesthetic as administered, such that the subject may usefully be included as a subject in the intended clinical trial. A subject may be determined to be suitable for participation in a clinical trial for the treatment of pain if they do not demonstrate a high response to the administration of a placebo, i.e. do not demonstrate a high placebo effect or response (these terms may be used interchangeably), as defined herein. Instead, a subject may be determined to be suitable for participation in a clinical trial for the treatment of pain if they demonstrate a desirable response to the local anaesthetic as administered herein, i.e. they demonstrate a high local anaesthetic effect or response (these terms may be used interchangeably), as defined herein. Subjects suitable for participation in a clinical trial may be considered "responders" to the local anaesthetic, and^' "non-responders" to the placebo. The placebo response and/or the local anaesthetic response may be independently determined using any appropriate method. In particular, the placebo response and/or the local anaesthetic response may be determined by pain perception assessment as described herein.

In accordance with the present invention both a local anaesthetic and a placebo shall be administered to a subject undergoing screening for suitability for participation in a clinical trial for the treatment of pain. In a first step (step a), the subject is administered local anaesthetic or placebo. In the case where the local anaesthetic was administered in the first step, administration of the placebo shall be carried out in the subsequent administration step (step c). In contrast in the case where the placebo was administered in the first step (step a), administration of the local anaesthetic shall be carried out in the subsequent administration step (step c). In accordance with the invention, the subject's pain perception is assessed following administration of the local anaesthetic and following administration of the placebo (steps b and d). This allows the subject's suitability for participation in a clinical trial for the treatment of pain to be determined based on a comparison of the assessed pain perception (step e).

Steps a to e of the method are conducted at defined time intervals. In particular, the skilled person will be aware that the length of time between the initial administration step (step a) and subsequent administration step (step c) may be determined by a number of factors including but not limited to the choice of local anaesthetic, the dose and route of administration of the local anaesthetic the pharmacokinetics of the local anaesthetic, and the time needed for the subjects pain perception to return to baseline. Typically the subsequent administration (step c) is carried out at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least ten days, at least 11 days, at least 12 days, at least 13 days or at least 14 days after the initial administration (step a). Preferably the subsequent administration (step c) is carried out at least five days to at least eight days after the initial administration (step a), and more preferably seven days (one week) after the initial administration (step a). The time interval between steps a and c is intended to allow a subject's response to return to baseline following the initial administration (step a) and prior to the subsequent administration (step c).

In step b, the subject is assessed for pain perception following the initial administration (step a). Pain perception may be assessed using any method or criteria defined herein. Pain perception following the initial administration (step b) may be carried out up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours following said initial administration (step a). Preferably, step b is carried out up to 24 hours following step a. Step b may involve one or more separate assessments of pain perception, typically at least two, at least five, at least ten, at least 12, at least 15, at least 20 or more separate assessments of pain perception. The one or more separate assessments of pain perception may be conducted every 30 minutes, hourly, every two hours, every four hours, every 8 hours, every 12 hours or more. By way of non-limiting example, in step b the assessment of pain perception in the subject may be carried out up to 30 minutes, 45 minutes, one hours, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, 12 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 48 hours following step a.

Preferably, in step b pain perception is assessed at least one of: 30 minutes after step a; hourly up to 8 hours after step a; and/or hourly between 20-24 hours after step a; and preferably pain perception is assessed at each of these times.

In step d the subject is assessed for pain perception following the subsequent administration (step c). Pain perception following the subsequent administration (step d) may be assessed using any method or criteria defined herein. Step d may be carried out up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours following the subsequent administration (step c). Preferably, step d is carried out up to 24 hours following step c. Step d may involve one or more separate assessments of pain perception, typically at least two, at least five, at least ten, at least 12, at least 15, at least 20 or more separate assessments of pain perception. The one or more separate assessments of pain perception may be conducted every 30 minutes, hourly, every two hours, every four hours, every 8 hours, every 12 hours or more. By way of non-limiting example, in step d the assessment of pain perception in the subject may be carried out up to 30 minutes, 45 minutes, one hours, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, 12 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 48 hours following step c.

Preferably, in step d pain perception is assessed at least one of: 30 minutes after step c; hourly up to 8 hours after step c; and/or hourly between 20-24 hours after step c; and preferably pain perception is assessed at each of these times.

Step b may be carried out up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours following step a and step d may be carried out up to 12 hours, up to 24 hours, up to 36 hours, up to 48 hours following step c. Preferably, step b is carried out up to 24 hours following step a and step d is carried out up to 24 hours following step c. Steps b and d may each involve one or more separate assessments of pain perception, typically at least two, at least five, at least ten, at least 12, at least 15, at least 20 or more separate assessments of pain perception. The one or more separate assessments of pain perception may be conducted every 30 minutes, hourly, every two hours, every four hours, every 8 hours, every 12 hours or more. By way of non-limiting example, in both step b and step d the assessment of pain perception in the subject may be carried out up to 30 minutes, 45 minutes, one hours, two hours, three hours, four hours, five hours, six hours, seven hours, eight hours, 12 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or 48 hours following step a or c respectively.

Preferably, in step b pain perception is assessed at least one of: 30 minutes after step a; hourly up to 8 hours after step a; and/or hourly between 20-24 hours after step a and in step d pain perception is assessed at least one of: 30 minutes after step c; hourly up to 8 hours after step c. More preferably pain perception is assessed at all of these times for both steps b and d.

Thus, in a particularly preferred embodiment of the invention, if the day of the initial administration (step a) is considered as day 0: pain perception step b will comprise assessing pain perception 30 minutes after step a, hourly up to 8 hours after step a, and hourly between 20-24 hours after step a; the subsequent administration (step c) is carried out on day 7; pain perception step d will comprise assessing pain perception 30 minutes after step c, hourly up to 8 hours after step c, and hourly between 20-24 hours after step c; and subsequently a comparison of the pain perception assessment of step b and step d will be conducted to determine whether a subject is suitable for participation in the clinical trial. The clinical trial will begin a maximum of 14 days after the subsequent administration (step c), as described below.

The time points/periods for assessing pain perception, particularly in steps (b) and/or (d) may be modified depending on the local anaesthetic to be used in the screening method of the invention. By way of non-limiting example, if the local anaesthetic gives rise to a delayed response (e.g. relative to lidocaine), then the time points/periods for assessing pain perception in steps (b) and/or (d) may be extended.

In order to allow a baseline pain perception to be determined, the assessment of pain perception may additionally be carried out prior to administration of the local anaesthetic and/or prior to the administration of the placebo, i.e. prior to step a and/or step c. The assessment of pain perception in the subject may be carried out at any time prior to administration of the local anaesthetic and/or prior to the administration of the placebo, i.e. prior to step a and/or step c. By way of non-limiting example, the baseline assessment of pain perception in the subject may be carried out up to 30 min, 45 min, 1 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours or up to 48 hours prior to the administration of the local anaesthetic and/or placebo, i.e. prior to step a and/or step c. Typically the baseline assessment is carried out up to 30 minutes or up to one hour prior to step a and/or step c.

In a preferred embodiment of the invention, the assessment of pain perception is carried prior to the administration of the local anaesthetic and placebo and following administration of the local anaesthetic and placebo, i.e. prior to both step a and step c. Thus, in a particularly preferred embodiment of the invention, if the day of the initial administration (step a) is considered as day 0: a baseline pain perception assessment will be carried out on the subject (typically within 30 minutes or an hour) of the initial administration of step a; pain perception step b will comprise assessing pain perception 30 minutes after step a, hourly up to 8 hours after step a, and hourly between 20-24 hours after step a; on day 7, a baseline pain perception assessment will be carried out on the subject (typically within 30 minutes or an hour) of the subsequent administration of step c; the subsequent administration (step c) is carried out on day 7; pain perception step d will comprise assessing pain perception 30 minutes after step c, hourly up to 8 hours after step c, and hourly between 20-24 hours after step c; and subsequently a comparison of the pain perception assessment of step b and step d will be conducted to determine whether a subject is suitable for participation in the clinical trial. The clinical trial will begin a maximum of 14 days after the subsequent administration (step c), as described below. An exemplary study protocol according to the invention is shown in Figure 2 herein.

In step e, the subject's pain perception following administration of the local anaesthetic and the subject's pain perception following administration of the placebo are compared. This comparison allows the determination of whether a subject is suitable for participation in a clinical trial for the treatment of pain. In particular, if there is a sufficient different between the subject's pain perception following administration of the local anaesthetic and the subject's pain perception following administration of the placebo, i.e. if the subject has a sufficiently decreased pain perception assessment following administration with the local anaesthetic as compared with the placebo, this indicates that the subject does not elicit an unfavourably high placebo effect and is suitable for participation in a clinical trial for the treatment of pain. A sufficiently decreased pain perception assessment following administration with the local anaesthetic as compared with the placebo, may be determined based on any clinically appropriate criteria, as assessed by the attending physician, for example a sufficient reduction in the pain perception assessments using the methodology described herein.

When NRS scores are used to assess pain perception, a sufficiently decreased pain perception assessment following administration with the local anaesthetic as compared with the placebo may be a decrease in NRS score of at least two, at least three, at least four or more NRS points following administration of the local anaesthetic compared with administration of the placebo. In a preferred embodiment, there is a decrease in NRS score of at least two NRS points following administration of the local anaesthetic compared with administration of the placebo. In other words:

[NRS predose NRS 1 hour post-injection test] local anaesthetic [NRS predose NRS 1 hour post-injection test] placebo— 2 When assessing pain perception, it will also be determined whether the pattern of pain relief (i.e. a decreased pain perception as assessed as described herein) is clinically plausible. The assessment for determining the plausibility of the pattern of pain relief may depend on a number of factors, including the local anaesthetic used. Any appropriate means may be used to determine the clinical plausibility of the pain relief, and such means are well known in the art. A clinician will readily be able to determine whether the reported pain relief is clinically plausible using standard techniques known in the art and without undue burden. By way of non-limiting example, in the case of lidocaine, the pattern of pain relief may be considered plausible if the onset of pain relief is reported by 30 minutes from time of completing the injections and/or pain perception scores after the local anaesthetic administration rise again by 2-8 hours post-administration.

When determining whether a subject is suitable for participation in a clinical trial, other factors may also be considered, as clinically appropriate. These include, but are not limited to, whether the investigation has concerns regarding compliance with safety procedures or other concerns regarding the subject's suitability. One of skill in the art will readily be able to identify and assess appropriate factors, which are known in the art.

Typically a subject may be determined to be suitable for participation in a clinical trial f they are consistent in their assessment of pain perception. By way of non-limiting example, a subject may be considered consistent in their assessment of pain perception if: (i) they report variability of less than two points for the average spontaneous NRS score before screening according to the present invention; (ii) they report variability of less than two points they report variability of less than two points for the average spontaneous NRS score before pre -quantitative sensory testing (QST); (iii) they report variability of less than two points they report variability of less than two points for the average spontaneous NRS score before pre-dosing in the clinical trial; and (iv) the subject's pattern of pain relief is clinically plausible; preferably all of (i) to (iv).

The screening method of the invention may correspond to part A of Figure 2 as shown herein.

The term "double-blind", as used herein, refers to an approach in which neither the subject receiving an agent, nor the person administering an agent knows which the identity of the agent which they are receiving/administering. A double-blind procedure helps to minimise the effects of bias, whether that be from the subject receiving a particular agent (and their beliefs about the effectiveness/safety of the agent) or from the researcher/clinician, whose knowledge of the identity of the agent may influence the results of the trial. In an embodiment of the present invention, the steps of administration of the local anaesthetic and placebo, and of the assessment of pain perception in the subject are conducted in a double-blind manner. The screening of the invention is typically carried out in a double-blind manner. The clinical trial may also be double-blinded. The randomisation of subjects to allow for a double-blind screening method and/or a double blind clinical trial may be carried out using any standard randomisation method known in the art.

The cross-over screening method of the present invention may also be used in the context of screening subjects for their suitability for a particular therapy or treatment of interest, preferably a therapy or treatment for pain. Any and all disclosure herein in relation to the screening methods of the invention for use in screening subjects for suitability for participation in clinical trials for the treatment of pain is generally applicable, and applies equally and without restriction to the use of these screening methods for determining the suitability of a subject for a particular therapy or treatment, particularly a therapy or treatment for pain. Said therapy or treatment for pain may be any as described herein in the context of clinical trials. A particularly preferred treatment for pain is a clostridial neurotoxin, e.g. a botulinum neurotoxin, as described herein. Briefly, a subject is treated either with a local anaesthetic or a placebo (in either order) and their pain perception assessed in response to said administration. In other words, subjects who received an injection of local anaesthetic at the initial administration (step a) are treated with placebo in the subsequent administration (step c), and those subjects who received placebo at the initial administration (step a) are treated with local anaesthetic in the subsequence administration (step c). The subject's pain perception in response to the local anaesthetic is compared with their pain perception in response to the placebo, and this comparison used to determine whether or not said subject is suitable for a particular therapy or treatment, particularly a therapy or treatment for pain. It is not important whether the subject is first administered with the local anaesthetic and then the placebo, or whether the subject is first administered with the placebo and then the local anaesthetic.

The invention also provides a method of determining local anaesthetic sensitivity comprising: (a) administering the local anaesthetic or a placebo; (b) assessing for pain perception; (c) in the case where the local anaesthetic was administered in step a, administering the placebo, or in the case where the placebo was administered in step a, administering the local anaesthetic; and (d) assessing for pain perception; wherein steps a to d are carried out sequentially. The invention further provides a method for screening a subject for suitability for participation in a clinical trial for the treatment of pain comprising carrying out said method of determining local anaesthetic sensitivity; (ii) comparing the pain perception assessed in step b with the pain perception assessed in step d; and (iii) determining whether a subject is suitable for participation in the clinical trial on the basis of the comparison of step (ii). The invention further provides a method for screening a subject for suitability for a particular treatment for pain comprising: (a) carrying out the method of determining local anaesthetic sensitivity of the invention; and (i) comparing the pain perception assessed in step b with the pain perception assessed in step d; and (ii) determining whether a subject is suitable for a particular treatment for pain on the basis of the comparison of step ii.

As used herein, the term "local anaesthetic sensitivity" means that a subject has a plausible clinical, measurable response to a local anaesthetic, particularly the anaesthetic being tested. This can be determined by assessing a subject's pain perception, as described in detail herein.

As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments of all the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms, "individual," "patient" and "subject" are used interchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of pain. A subject can be male or female, adult or juvenile.

A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. pain) or one or more complications related to such a condition, and optionally, have already undergone treatment for pain or the one or more complications related to pain. Alternatively, a subject can also be one who has not been previously diagnosed as having pain or one or more complications related to pain. For example, a subject can be one who exhibits one or more risk factors for pain or one or more complications related to pain or a subject who does not exhibit risk factors.

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 screening method of the invention may be carried out to identify subjects suitable for inclusion in any clinical trial for the investigation of any IMP for the treatment of pain.

As described herein, the administration of the placebo and the local anaesthetic in order to screen patients for suitability for participation in a clinical trial according to the invention are separated temporally (i.e. first one is given, then at a subsequent time-point, the other). Typically when the placebo and local anaesthetic are administered, they are administered at the same site on the body, or in the same locality. For example, in the locality of the pain that is to be treated in the clinical trial. Thus, the administration of the placebo and the local anaesthetic is typically not spatially separated.

Local anaesthetic

As used herein, the term "local anaesthetic", refers to an agent that reduces or eliminates pain sensation when applied locally (i.e. to a restricted area). Local anaesthetics reversibly inhibit nerve transmission by binding voltage-gated sodium channels in the nerve plasma membrane. Voltage-gated sodium channels are integral membrane proteins, anchored in the plasma membrane. When local anaesthetics bind these sodium channels, they render them impermeable to sodium ions and prevent action potential initiation and propagation in neurons. Although voltage-gated sodium channels are present in all nerves, never fibres differ markedly in their susceptibility to conduction blockage by local anaesthetics. In general, neurons having more rapid firing rates are more susceptible to local anaesthetic action. For example, small, non-myelinated neurons (like those which detect and transmit pain stimuli) are much more susceptible to local anaesthetics than large, myelinated neurons (like motor neurons).

In general, local anaesthetics fall into two structural categories, amino esters and amino amides. The designations refer to the linkage between the lipophilic aromatic ring (or, in the case of articaine, the thiophene ring) and the hydrophilic tertiary amine components of local anaesthetics.

A variety of local anaesthetics suitable for use according the present invention are known to those skilled in the art. By way of non-limiting examples, suitable local anaesthetics include lidocaine (also known as lignocaine), articaine, bupivacaine, cinchocaine (dibucaine), etidocaine, levobupivacaine, mepivacaine, prilocaine, ropivacaine, trimecaine, benzocaine, chloroprocaine, cocaine, clyclomethycaine, dimethocaine (larocaine), piperocaine, propoxycaine, procaine (novocaine), proparacaine, tetracaine (amethocaine), menthol, eugenol, spilanthol, saxitoxin, neosaitoxin and tetrodotoxin. In a preferred embodiment of the invention, the local anaesthetic is lidocaine (lignocaine).

Local anaesthetics may be administered with other pharmacologically active agents. By way of non-limiting example, local anaesthetics may be administered with vasoconstrictors to reduce the dispersion of the local anaesthetic from the site of administration and/or to increase duration of action. As a further non-limiting example, local anaesthetics may be administered in combination with opioids, NSAIDS and/or anticonvulsants. Such combinations are also encompassed by the present invention. In a preferred embodiment, the local anaesthetic is administered as a sole pharmaceutically active agent. Placebo

The term "placebo" refers to a dummy medication or treatment. In particular, in relation to the present invention a placebo is a substance or composition with no specific pharmaceutical activity or therapeutic effect against pain. Placebos are typically used as a control in clinical investigations. The administration of a placebo in a screening method of the present invention allows pain perception to be compared following administration of the placebo against pain perception following the administration of a local anaesthetic in the same subject. The administration of a placebo in a clinical trial for investigating an investigational medicinal product (IMP) allows for the effect of the IMP to be determined in isolation from any effects of other components of the administered composition, the act of administration or other confounding factors.

Appropriate placebos for use according to the invention can be selected using standard methodology. Indeed, a variety of placebos suitable for administration according the present invention are known in the art.

By way of non-limiting examples, suitable placebos for use when screening a subject for suitability for participation in a clinical trial for the treatment of pain include saline and components of the composition (e.g. excipients, diluents, etc. as described herein) used to administer the local anaesthetic, but lacking said local anaesthetic. In other words, the placebo may be identical to the composition containing the local anaesthetic, with the sole difference that the local anaesthetic is omitted. In a preferred embodiment, when screening a subject for suitability for participation in a clinical trial for the treatment of pain, saline is used as the placebo. In a particularly preferred embodiment, when screening a subject for suitability for participation in a clinical trial for the treatment of pain, the local anaesthetic is lidocaine (lignocaine) and the placebo is saline.

Typically, when investigating an IM P in a clinical trial for the treatment of pain, the placebo is an identical composition to that comprising the IMP, but wherein the IMP is omitted.

Investigational Medicinal Product (IMP)

The screening method provided by the present invention enables the identification of subjects suitable for participation in a clinical trial for the treatment of pain. Typically, the purpose of said clinical trial is to determine whether an investigational medicinal product (IMP) has therapeutic efficacy in the treatment of pain.

An IMP is a pharmaceutical form of an active substance being tested, or to be tested in a clinical trial. An IM P may not have an existing marketing authorisation. Alternatively, the term IMP includes a medicinal product which has a marketing authorisation but is, for the purposes of the trial: is a) used or assembled in a way different from the form of the product authorised under the authorisation; b) used for an indication not included in the summary of product characteristics under the authorisation for that product; or c) used to gain further information about the form of that product as authorised under the authorisation.

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

According to the invention, an IMP is any pharmaceutical form of any active substance which has potential therapeutic utility in the treatment of pain. One of ordinary skill of the art will readily be able to determine whether an IMP is suitable for investigation according to the methods of the invention.

By way of non-limiting example, the IMP may be a clostridial neurotoxin, such as a botulinum neurotoxin, as defined herein. In such instances, the botulinum toxin is preferably a BoNT/A, such as abobotulinumtoxinA (Dysport^®).

Compositions and Formulations comprising a Local Anaesthetic or IMP

The local anaesthetic and/or IMP can be administered in any pharmaceutically acceptable composition or formulation. One of ordinary skill in the art can design and produce such a suitable formulation or composition based on standard knowledge and routine methods.

A pharmaceutically acceptable composition or formulation typically comprises one or more pharmaceutically acceptable carrier, diluent and/or excipient.

Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; ( 4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminium hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C 12 alcohols, such as ethanol; and (23) other non toxic compatible substances employed in pharmaceutical formulations. Wetting agents, colouring agents, release agents, coating agents, sweetening agents, flavouring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "diluent", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments of all the aspects described herein, the carrier inhibits the degradation of the active agent, e.g. a composition as described herein.

Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use.

Fluid unit dosage forms are typically prepared utilising a pyrogen-free sterile vehicle. The active ingredients, depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle.

In preparing administrable solutions, the local anaesthetic or IMP can be dissolved or suspended in a vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving.

Advantageously additives such as buffering, solubilising, stabilising, preservative or bactericidal, suspending or emulsifying agents may be dissolved in the vehicle.

Dry powders which are dissolved or suspended in a suitable vehicle prior to use may be prepared by filling pre-sterilised drug substance and other ingredients into a sterile container using aseptic technique in a sterile area.

Alternatively the polypeptides and other ingredients may be dissolved in an aqueous vehicle; the solution is sterilized by filtration and distributed into suitable containers using aseptic technique in a sterile area. The product is then freeze dried and the containers are sealed aseptically.

Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration. The components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation. Advantageously, a suspending agent for example polyvinylpyrrolidone is included in the composition/s to facilitate uniform distribution of the components.

Administration

The term "administering", as used here, means to deliver an agent, typically a local anaesthetic, a placebo and/or an IMP to a subject. A variety of means for administering local anaesthetic, placebo and/or IMP as described herein to subjects are known to those skilled in the art. Such methods can include, but are not limited to oral, subcutaneous, intradermal, intramuscular, parenteral, intravenous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, transdermal, injection, or intratumoral administration. Typically administration is local, rather than systemic. By way of non-limiting examples, the local anaesthetic, placebo and/or IMP as described herein may be administered by subcutaneous injection, intradermal injection, local infiltration (e.g. via injection into a skin bleb at the point of injection, or via an ELMA cream) intramuscular injection, intrathecal infusion, intra-cerebroventricular infusion, epidural injection into the central nervous system, intraneural injection, intra-articular injection, intratumoral injection, topical application or transdermal application. Preferably, the local anaesthetic and/or placebo as described herein is administered by intradermal injection, subcutaneous injection or intramuscular injection. Intradermal injection is particularly preferred. The means of administration may depend on the local anaesthetic being used. By way of non-limiting example, when the local anaesthetic is lidocaine, the means of administration may be preferably selected from intradermal injection, subcutaneous injection or local infiltration.

As described herein, the local anaesthetic is typically administered at an effective amount. Additionally or alternatively, the clinical trial for the treatment of pain may be assessing effective amount of the IMP being investigated.

The term "effective amount" as used herein refers to the amount of a composition needed to alleviate at least one or more symptom of the disease or disorder (such as pain), and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of the composition that is sufficient to provide a particular anti-pain effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the active ingredient which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

The dosage ranges for administration of the local anaesthetic or IMP according to the present invention are those which produce the desired reduction or elimination of pain, as may be assessed by pain perception methods as described herein. It will be appreciated that the dosage range required depends on the precise nature of the components, the route of administration, the nature of the formulation, the age of the patient, the weight of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician.

The administration of the local anaesthetic, placebo and/or IMP may comprise multiple separate administrations to achieve the desired "effective amount". For example, the administration may require a single administration, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or more separate administrations. Typically, a maximum of ten separate administrations is used to administer the local anaesthetic (particularly lidocaine (lignocaine)) and/or the IMP. In a preferred embodiment, a maximum of ten separate administrations is used to administer the local anaesthetic (particularly lidocaine (lignocaine)) and a maximum of ten separate administrations is used to administer the IMP. Preferably said administrations are by subcutaneous or intradermal injection.

For the local anaesthetic, the appropriate effective amount and number of separate administrations will depend on numerous factors, as described herein, particularly the particular local anaesthetic to be used. Therefore, it is not generally practicable to specify an exact "effective amount", the means of administration or the number of separate administrations, although these may be readily determined by one of ordinary skill.

By way of non-limiting example, wherein the local anaesthetic is lidocaine (lignocaine), the administration is by subcutaneous or intradermal injection and the dose is selected from 0.5 mg, 1.0 mg, 2.0 mg 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 6.0 mg, 7.0 mg, 8.0 mg, 9.0 mg or 10.0 mg lidocaine per injection. Preferably 2.5 mg lidocaine (lignocaine) per injection is used. A maximum of ten subcutaneous or intradermal injections is preferably used. Thus, in a most preferred embodiment, when lidocaine (lignocaine) is the local anaesthetic, a maximum of ten subcutaneous or intradermal injections each of 2.5 mg is used. The lidocaine (lignocaine) can be administered in any appropriate volume, for example, about 0.1 mL, about 0.2 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL or about 1.0 mL, preferably about 0.3 mL to about 0.7 mL, more preferably about 0.5 mL per administration. Thus, in a particularly preferred embodiment, lidocaine (lignocaine) is administered by subcutaneous or intradermal injections at a dose of 2.5 mg in a volume of 0.5 mL, to a maximum of ten injection points.

For an IMP, suitable doses, dose intervals and number of separate administrations will depend on numerous factors, as described herein, particularly the particular IMP to be used.

For example, suitable daily doses may be in the range 0.0001-1 mg/kg, preferably 0.0001-0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and particularly preferably 0.004-0.5 mg/kg. The unit dosage can vary from less than 1 mg to 30 mg, but typically will be in the region of 0.01 to 1 mg per dose, which may be administered daily or preferably less frequently, such as weekly or six monthly. When the IMP is a clostridial neurotoxin as described herein, dosing may be based on 2.5 ng of neurotoxin as the IX dose, including dosages in the range 1X-100X (i.e. 2.5-250 ng). Botulinum neurotoxin doses can also be quantified in terms of potency units (U). Potency units are typically specific to the preparation and assay method utilised. For example, potency of abobotulinumtoxinA (Dysport^®) is calculated using a cell-based potency assay to determine the potency relative to a reference standard. One unit of abobotulinumtoxinA corresponds to the calculated median lethal intraperitoneal dose (LD50) in mice. By way of non-limiting example, when the IMP is a clostridial neurotoxin (e.g. BoNT/A, such as abobotulinumtoxinA), the administration is by intradermal injection and the dose is selected from 1U, 2.5U, 5U, 10U, 20U or 25U per injection. Typically 2.5U, 10U and/or 25U are used, and preferably all of 2.5U, 10U and 20U are tested. A maximum of ten intradermal injections is preferably used. A minimum of four intradermal injections is preferably used. Thus, in a most preferred embodiment, when a clostridial neurotoxin (e.g. BoNT/A, such as abobotulinumtoxinA) is the IMP, a minimum of four intradermal injections each of 2.5 U, 10U or 20U (i.e. to a minimum total of 10U) is used and a maximum of ten intradermal injections each of 2.5 U, 10U or 20U (i.e. to a maximum total dose of 200U) is used. The clostridial neurotoxin (e.g. BoNT/A, such as abobotulinumtoxinA) can be administered in any appropriate volume, for example, about 0.05 mL, about 1 mL, about 0.15 mL, about 0.2 mL, about 0.25 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL or about 1.0 mL, preferably about 0.1 mL to about 0.3 mL, more preferably about 0.2 mL per administration.

Wide variations in the required local anaesthetic and/or IMP dosage, however, are to be expected depending on the precise nature of the components, and the differing efficiencies of various routes of administration. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation, as is well understood in the art.

For the placebo, again, the appropriate dose, volume and/or number of separate administrations will depend on numerous factors, as described herein, and will be matched to either the local anaesthetic to be administered (in the case where the placebo is being used to screen a subject for suitability for participation in a clinical trial) or the IMP being investigated (in the case where the placebo is being used in a clinical trial to investigate an IMP).

By way of non-limiting example, when the placebo is being used to screen a subject for suitability for participation in a clinical trial and the local anaesthetic is lidocaine (lignocaine) being administered as a single administration (preferably subcutaneous or intradermal injection), then a single administration of placebo at the same volume as the volume of lidocaine (lignocaine) and using the same means of administration is administered. By way of a further non-limiting example, if the placebo is being used to screen a subject for suitability for participation in a clinical trial and the local anaesthetic is lidocaine (lignocaine) being administered by a maximum of ten separate administrations (preferably subcutaneous or intradermal injections), then the same number of administrations (i.e. to a maximum of ten) of placebo at the same volume as the volume of lidocaine (lignocaine) and using the same means of administration is administered. Preferably said placebo is saline.

By way of non-limiting example, when the placebo is being used in a clinical trial to investigate an IMP and the IMP is a clostridial neurotoxin (preferably BoNT/A, e.g. abobotulinumtoxinA) being administered as a single administration (preferably intradermal injection), then a single administration of placebo at the same volume as the volume of clostridial neurotoxin (preferably BoNT/A, e.g. abobotulinumtoxinA) and using the same means of administration is administered. By way of a further non-limiting example, if the placebo is being used in a clinical trial to investigate an IMP and the IMP is a clostridial neurotoxin (preferably BoNT/A, e.g. abobotulinumtoxinA) being administered by a maximum of ten separate administrations (preferably intradermal injections), then the same number of administrations (i.e. to a maximum of ten) of placebo at the same volume as the volume of clostridial neurotoxin (preferably BoNT/A, e.g. abobotulinumtoxinA) and using the same means of administration is administered. Preferably said placebo is an identical composition to that containing the clostridial neurotoxin (preferably BoNT/A, e.g. abobotulinumtoxinA), but lacking said clostridial neurotoxin.

Pain

The term "pain," as used here, means any unpleasant sensory experience, usually associated with a physical disorder. The physical disorder may or may not be apparent to a clinician. Pain is of two types: chronic and acute. An "acute pain" is a pain of short duration having a sudden onset. One type of acute pain, for example, is cutaneous pain felt on injury to the skin or other superficial tissues, such as caused by a cut or a burn. Cutaneous nociceptors terminate just below the skin, and due to the high concentration of nerve endings, produce a well-defined, localized pain of short duration. "Chronic pain" is a pain other than an acute pain. Chronic pain includes neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain and referred pain.

Typically the pain of the invention is chronic and localised. The term "localised" as used herein means that the pain is perceived within a discrete part or area of the body, rather than being perceived in a diffuse or systemic manner.

Non-limiting examples of pain for which a clinical trial may be used to assess a potential treatment option include: chronic pain; abdominal or thoracic chronic scar pain; post-surgical neuralgia, chronic neuropathic pain; painful diabetic neuropathy (PDN), post-herpetic neuropathy (PHN); trigeminal neuralgia (TN); inflammatory pain; neuropathic pain; a channelopathy; primary erythermalgia (PE); paroxysmal extreme pain disorder (PEPD); spinal cord injury pain; multiple sclerosis pain; phantom limb pain; post-stroke pain; chronic back pain; osteoarthritis pain; cancer- associated pain; HIV-associated pain; chronic inflammatory pain; central neuropathy; peripheral neuropathy; anaesthesia dolorosa; hyperalgesia; hyperpathia; paresthesia; psychogenic pain; back pain; breakthrough pain; erythromelalgia; nerve compression and/or entrapment [e.g., carpal tunnel syndrome, tarsal tunnel syndrome, ulnar nerve entrapment, compression radiculopathy, radicular low back pain, spinal root lesions, spinal root compression, lumbar spinal stenosis, sciatic nerve compression, and/or intercostal neuralgia]; neuritis; pain from chemotherapy; chronic alcoholism (alcoholic polyneuropathy); rheumatoid arthritis pain; pain associated with bums; encephalitis pain; bone fracture pain; neuritis pain; autoimmune disease pain; postoperative pain; dental pain; pain associated with bacterial infection, e.g. a bacterial infection or viral infection; pain associated with radiotherapy; pain associated with gout and irritable bowel syndrome; pain from trauma (such as from lacerations, incisions, burns, foreign bodies or bullet and/or shrapnel injuries, spinal cord injury, brachial plexus avulsion, nerve crush and/or entrapment; nerve transection; visceral pain (such as renal or ureteral colic, irritable bowel syndrome, angina or cardiac pain, cardiac arrhythmia, period pain, interstitial cystitis, rectal pain, pain associated with diarrhoea, appendicitis, cholecystitis and pancreatitis); uremia pain; pain associated with hypothyroidism; pain associated with vitamin deficiency; headache pain (e.g., tension headache, migraine and cluster headache); idiopathic pain (e.g., trigeminal neuralgia, a complex regional pain syndrome [e.g. complex regional pain syndrome I and /or complex regional pain syndrome II], allodynia or fibromyalgia); respiratory pain (e.g., pain associated with asthma, airway hyper-reactivity in asthma, chronic cough, e.g. in asthma and/or chronic obstructive pulmonary disorder); fibromyalgia; hormonal therapy pain; hypothyroidism pain; epileptic pain; ataxia; periodic paralysis; acute itch and/or chronic itch pain.

In a preferred embodiment of the invention, the pain is chronic pain, preferably abdominal or thoracic chronic scar pain.

Pain Perception

Pain is defined by the International Association for the Study of Pain as: "An unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage" (www.iasp-pain.org). Pain is subjective and this definition links emotion and past experience to the sensory event thus also highlighting that pain is a complex experience that includes multiple dimensions. This is because each individual learns the meaning of the word "pain" through experiences related to injury in early life. Stimuli or illnesses that cause pain are likely to damage tissue and hence pain is an experience associated with actual or potential tissue damage. Pain is almost always unpleasant and therefore an emotional experience. Pain is subjective and there are no satisfactory objective measures of pain, hence the subject's self-report of pain is the most reliable indicator of pain.

As used herein, the assessment for pain perception refers to methods of determining a subject's sense of pain. Assessing a subject for pain perception according to the present invention encompasses any method by which the subject's sense of pain may be determined (qualitatively or quantitatively). As described herein, by assessing a subject's pain perception at specific time points pre- and post-treatment with a local anaesthetic, placebo and/or IMP, it is possible to screen a patient for suitability for participation in a clinical trial, and also to assess the clinical efficacy of the IMP. A variety of means for assessing pain perception are known to those skilled in the art. By way of non-limiting example, suitable methods for assessing pain perception in a subject include the following: Numerical Rating Scale (NRS) score; sensory threshold; pain perception threshold; static mechanical allodynia; dynamic mechanical allodynia; temporal summation; pressure pain threshold; conditioned pain modulation; and temperature threshold. Exemplary methods of determining some of these pain perception means as described in more detail herein.

Other non-limiting examples of pain perception measures include: time to onset (e.g. time to decrease from baseline of two points in the spontaneous NRS score), peak-effect ( i.e. maximal decrease from baseline in the spontaneous NRS score); time to peak-effect (i.e. time to reach the peak-effect); duration of effect (i.e. duration between time to onset and last time point for which change from baseline in the spontaneous NRS score is > two points). These may be considered "primary" endpoints or pain perception assessment measures.

Other non-limiting examples of pain perception measures include: change from baseline in the spontaneous NRS score to each scheduled time point; change from baseline in the stimulus- evoked NRS score to each scheduled time point; safety of three intradermal doses of the IMP (e.g. BoNT/A), where safety assessments may include monitoring of adverse effects, concomitant medications and changes from baseline in physical examinations findings including the examination of the scar at the injection/administration sites and vital signs recordings at each scheduled time point. These may be considered "secondary" endpoints or pain perception assessment measures.

Other non-limiting examples of pain perception measures include: change from baseline in SF-36 scores at each scheduled time point; amount of rescue medication taken during the study and time to first intake of rescue medication. These may be considered "exploratory" endpoints or pain perception assessment measures.

Numerical Rating Scale (NRS)

Typically pain perception according to the present invention uses 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'.

The NRS can be used to assess numerous facets of pain, including spontaneous average pain, spontaneous worst pain and spontaneous current pain.

Spontaneous average pain is assessed by asking a subject to select a number that best describes the subject's average pain over a period of time, for example at least 6 hours, 12 hours, 24 hours, or at least 48 hours. Spontaneous worst pain is assessed by asking a subject to select a number that best describes the subject's pain at its worst during a specified period, e.g. at least the previous 6 hours, 12 hours, 24 hours or previous 48 hours. Spontaneous current pain is assessed by asking a subject to select a number that best describes how much pain the subject is in at the time of assessment.

When screening a subject for suitability for participation in a clinical trial, spontaneous current pain NRS scoring may be preferred (either with or without stimulus as described below). When carrying out a clinical trial to investigate an IMP for the treatment of pain, spontaneous average and worst pain NRS scoring may be preferred (either with or without stimulus as described below).

NRS in Response to Stimuli

The NRS can also be used to assess a subject's pain perception in response to a variety of different stimuli. To assess pain perception in response to a stimulus, the subject will be submitted to stimuli of various nature applied to the painful area. Subjects will be asked what are their current NRS scores pre-dose (of the local anaesthetic or IMP) and post-stimulus.

Examples of stimuli used include: (i) light touch (which can be assessed by measuring pain on the surface of the painful area on radial spokes following application of a von Frey filament as described herein); (ii) pressure (pressure pain threshold), which can be assessed by asking the subject to give a NRS score as increasing pressure is applied using a pressure algometer as described herein; and (iii) temperature (which can be assessed by asking the subject for an NRS score for warm, cold and hot stimulation using a thermode applied to the painful area, as described herein).

Quality of Life questionnaire Short Form-36 (SF-36)

Alternatively or in addition, the SF-36 quality of life questionnaire 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.

Clinically Plausible Pain Relief

When assessing pain perception according to the invention, it will also be determined whether the pattern of pain relief (i.e. a decreased pain perception as assessed as described herein) is clinically plausible. The assessment for determining the plausibility of the pattern of pain relief may depend on a number of factors, including the local anaesthetic used. Any appropriate means may be used to determine the clinical plausibility of the pain relief, and such means are well known in the art. A clinician will readily be able to determine whether the reported pain relief is clinically plausible using standard techniques known in the art and without undue burden By way of non limiting example, in the case of lidocaine, the pattern of pain relief may be considered plausible if the onset of pain relief is reported by 30 minutes from time of completing the injections and/or pain perception scores after the local anaesthetic administration rise again by 2-8 hours post administration.

Quantitative Sensory Testing

Quantitative Sensory Testing (QST) measuring changes in sensitivity to different types of sensations, including temperature, touch and pressure. It can form part of the overall assessment of pain perception. Exemplary tests that may be included in QST are described in the examples herein. For example, QST may comprise light touch testing, pressure pain threshold testing, temperature testing, area of pain testing, or any combination thereof, preferably all of these. QST may assess allodynia, hyperalgesia and conditioned pain modulation. A Quality of Life (QoL) questionnaire may also be included as part of QST.

Quantitative Sensory Testing (QST) may be carried out before and/or as part of the screening of the invention. QTS may also be carried out before a subject identified as suitable for participation for a clinical trial is enrolled and/or dosed in said clinical trial. Typically QST is carried out as a baseline assessment or part of a baseline assessment prior to participation in screening and/or a clinical trial.

Area of pain

When screening a subject for suitability for participation in a clinical trial for the treatment of pain according to the present invention, or when determining the therapeutic efficacy of the IMP in said clinical trial, the subject is typically assessed for pain perception in relation to one or more discrete areas of pain.

The longitudinal axis of the painful area and the total painful area will be measured. The boundary of the spontaneous pain felt by the subject as well as the sites for injection at intervals, typically 0.5cm, 1.0cm, 1.5cm or 2.0cm and preferably 2cm and starting 1 cm from the edge of the longitudinal axis of the pain area will be determined as represented in Figure 1. A photograph may be recorded with a ruler for scale. Flaving determined and recorded the area of pain, this information can be used to precisely locate the sites of administration (as defined herein), and also used when assessing the subject's pain perception. By way of non-limiting example, in the case of post-surgical neuralgia, the area of pain is typically the scar tissue and the surrounding area. Clinical Trials for the Treatment of Pain

The use of a local anaesthetic in screening a subject for suitability for participation in a clinical trial for the treatment of pain as described herein can be used as an initial pre-screening phase or part of a clinical trial programme. Accordingly, the present invention provides a clinical trial programme, framework or schedule (all terms which may be used interchangeably) in which the screening method of the invention is carried out prior to initiation of the clinical trial to investigate an IMP for the treatment of pain.

Typically step a of the screening method as described herein (i.e. the initial administration of the screening method) takes place a maximum of 14 days, a maximum of 15 days, a maximum of 16 days, a maximum of 17 days, a maximum of 18 days, a maximum of 19 days, a maximum of 20 days, a maximum of 21 days, a maximum of 22 days, a maximum of 23 days, a maximum of 24 days, a maximum of 25 days, a maximum of 26 days, a maximum of 27 days, a maximum of 28 days prior to the first administration of an IM P or corresponding IMP placebo in a clinical trial for the treatment of pain. Preferably step a of the screening method as described herein takes place a maximum of 21 days prior to the first administration of an IMP or corresponding IMP placebo in a clinical trial for the treatment of pain.

Typically step c of the screening method as described herein (i.e. the subsequent administration of the screening method) takes place a maximum of 7 days, a maximum of 8 days, a maximum of 9 days, a maximum of 10 days, a maximum of 11 days, a maximum of 12 days, a maximum of 13 days, a maximum of 14 days, a maximum of 15 days, a maximum of 16 days, a maximum of 17 days, a maximum of 18 days, a maximum of 19 days, a maximum of 20 days, a maximum of 21 days prior to the first administration of an IMP or corresponding IMP placebo in a clinical trial for the treatment of pain. Preferably step c of the screening method as described herein takes place a maximum of 14 days prior to the first administration of an IMP or corresponding IMP placebo in a clinical trial for the treatment of pain.

More preferably, step a of the screening method as described herein takes place a maximum of 21 days prior to the first administration of an IMP or corresponding IM P placebo in a clinical trial for the treatment of pain and step c of the screening method as described herein takes place a maximum of 14 days prior to the first administration of an IMP or corresponding IMP placebo in a clinical trial for the treatment of pain.

The time interval between the screening method of the invention (particularly steps a and c of the screening method) is intended to allow a subject's response to return to baseline following the screening method and prior to the administration of the IMP. The clinical trial can assess multiple pharmacokinetic and/or pharmacodynamic parameters. Relevant parameters commonly tested in clinical trials for the treatment of pain are known in the art and could be readily selected by one of ordinary skill in the art. Examples of such parameters include, but are not limited to NRS; stimulus-evoked NRS; temperature of the painful area; size of the painful area; time to onset of analgesic effect; peak analgesic effect; time to peak analgesic effect; duration of analgesic effect; and/or SF-36 quality of life as described herein. Methods for assessing these parameters are also known in the art and can be carried out by one of ordinary skill using routine methods and procedures. Examples of assessment methodology are also described herein.

The clinical trial can assess any desired treatment schedule for the IMP of interest. The treatment schedule may vary depending on multiple factors, including the nature of the IMP, the intended dose and/or route of administration. Thus, it is not generally practicable to specify a precise treatment schedule for the clinical trial. However, for any given case, an appropriate treatment schedule can be readily determined by one of ordinary skill in the art.

Similarly, the post-treatment assessment may also vary depending on multiple factors, including the nature of the IMP, the intended dose and/or route of administration. Thus, it is not generally practicable to specify a precise schedule of post-treatment assessment for the clinical trial. However, for any given case, an appropriate of post-treatment assessment schedule, typically including pain perception assessment as described herein, can be readily determined by one of ordinary skill in the art.

By way of non-limiting example, when the IMP is a clostridial neurotoxin, the neurotoxin may be administered at a single time point over one or more sites of administration (preferably intradermal injection), and the subject monitored for up to eight weeks, up to ten weeks, up to 12 weeks, up to 14 weeks, up to 16 weeks, up to 18 weeks, up to 20 weeks, up to 30 weeks, up to 40 weeks, up to 52 weeks following the administration of the neurotoxin. Preferably monitoring occurs for up to 16 weeks post-administration. Pain perception assessment following administration of the clostridial neurotoxin can occur hourly, every two hours, every four hours, every eight hours, every 12 hours, daily, every two days, every three days, weekly, fortnightly or longer. Preferably, pain perception assessment takes place every 12 hours following administration of the clostridial neurotoxin. A non-limiting example of a treatment schedule for an IMP, particularly a clostridial neurotoxin, is shown in part B of Figure 2.

The clinical trial may be for the treatment of any pain as defined herein. Typically the clinical trial is for the treatment of chronic pain (e.g. persisting for more than six months). The clinical trial may be for the treatment of moderate to severe pain, i.e. spontaneous NRS score of 4-8. Typically the pain has been stable for at least the previous month before screening.

Clostridial neurotoxins

According to the invention, the screening method may be used to screen a subject for suitability in participation in a clinical trial for the treatment of pain, wherein the IMP to be investigated is a clostridial neurotoxin.

The invention relates to both single-chain clostridial neurotoxins and di-chain neurotoxins. For the avoidance of doubt, all references herein to "clostridial neurotoxins" as IMPs of the invention relates to both single-chain and di-chain forms of clostridial neurotoxins, unless explicitly excluded by a specific disclosure herein, or if this is clear from the technical context.

Bacteria in the genus Clostridia produce highly potent and specific protein toxins, which can poison neurons and other cells to which they are delivered. Examples of such clostridial neurotoxins include the neurotoxins produced by C. tetani (TeNT) and by C. botulinum (BoNT) serotypes A-G, and X ( see WO 2018/009903 A2), as well as those produced by C. baratii and C. butyricum.

Among the clostridial neurotoxins are some of the most potent toxins known. By way of example, botulinum neurotoxins have median lethal dose (LD₅o) values for mice ranging from 0.5 to 5 ng/kg, depending on the serotype. Both tetanus and botulinum neurotoxins act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. While botulinum neurotoxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, tetanus neurotoxin acts in the central nervous system.

In nature, clostridial 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 (He domain). The cleavage site is located between the L-chain and the translocation domain components. Proteolytic activation is of clostridial neurotoxins 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 clostridial neurotoxins are non-cytotoxic proteases that cleave SNARE proteins.

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

BoNTs are absorbed in the gastrointestinal tract, and, after entering the general circulation, bind to the presynaptic membrane of cholinergic nerve terminals and prevent the release of their neurotransmitter acetylcholine.

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, VAMP5, Ykt6.

Together with BoNT, tetanus neurotoxin (TeNT) produced by Clostridium tetani, make up the clostridial neurotoxin (CNT) family. TeNT exhibits a high degree of sequence and structural homology to BoNT, in particular to BoNT/B, and is the causative agent of tetanus, which is characterized by spastic paralysis. Overall, BoNTs and TeNT share approximately 35% sequence identity. The BoNT catalytic L-chain domains share up to 36% sequence identity and the L-chain domains of BoNT/B and TeNT have over 50% identity. Although differing in clinical manifestation, the fundamental mode of action - inhibition of neurotransmission - is common to all CNTs. The present invention may be used to investigate clostridial neurotoxins as IMPs in clinical trials for the treatment of pain. The invention is suitable for application to many different varieties of clostridial neurotoxin as IMPs. Thus, in the context of the present invention, the term "clostridial neurotoxin" embraces toxins produced by C. botulinum (botulinum neurotoxin serotypes A, B, Cl, D, E, F, G, H, and X), C. tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E), and C. baratii (botulinum neurotoxin serotype F), as well as modified clostridial neurotoxins or derivatives derived from any of the foregoing. The term "clostridial neurotoxin" also embraces botulinum neurotoxin serotype H.

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

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

A modified clostridial 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 clostridial neurotoxins are described in WO 2010/120766 and US 2011/0318385, both of which are hereby incorporated by reference in their entirety. A modified clostridial neurotoxin may comprise one or more modifications that increases or decreases the biological activity and/or the biological persistence of the modified clostridial neurotoxin. For example, a modified clostridial 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 clostridial 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 Hy is a hydrophobic amino acid). Examples of modified clostridial neurotoxins comprising leucine- and tyrosine-based motifs are described in WO 2002/08268, which is hereby incorporated by reference in its entirety.

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

The term "clostridial neurotoxin" is also intended to embrace retargeted clostridial neurotoxins. The three domains of BoNT (LC, H_N, He) 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 (He) 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 "clostridial neurotoxin" is also intended to encompass variants and fragments of clostridial neurotoxin, provided they retain the analgesic effect of the corresponding native clostridial neurotoxin.

The term "clostridial 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 oryzae 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).

In a preferred embodiment a clostridial neurotoxin is a botulinum neurotoxin, more preferably BoNT/A, and more preferably abobotulinumtoxinA, particularly Dysport^® being even more preferred. 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.

In one embodiment the clostridial neurotoxin may be BoNT/A. A reference BoNT/A sequence is shown as SEQ ID NO: 1.

In another embodiment the clostridial neurotoxin may be BoNT/B. A reference BoNT/B sequence is shown as SEQ ID NO: 2.

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

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

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

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

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

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

In another embodiment the clostridial neurotoxin may be TeNT. A reference TeNT sequence is shown as SEQ ID NO: 9. 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" clostridial 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 (I), 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 clostridial 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 lUPACIUB 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 Subtle 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 Henikoff and Henikoff (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-l 5

N -2 06

D-2-2 1 6

C 0-3 -3 -3 9

Q-l 10 0-3 5

E -1 00 2 -4 2 5

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

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

I -1 -3 -3 -3 -1 -3 -3 -4 -3 4

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

K-l 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 0 6

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 clostridial 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 al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. 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 al., 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 al., 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. Natl. 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.

SEQ ID NO: 1 (BoNT/A - UniProt P10845.5)

MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHEUHAGHRLYGIAINPN

RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTUGQVDRLKDK

VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINI

GSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNN

EYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTIT

NNRLNNSKIYINGRUDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELN

EKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPR

GSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA

GVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAK

LVASNWYN RQI E RSSRTLG CSWEF I PVD DG WG E RP L

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

MPVTINNFNYNDPIDNNNIIMMEPPFARGTG RYYKAFKITDRIWIIPERYTFGYKPEDFN

KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG

DRRVPLEEFNTNIASVTVNKUSNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPAULMHELIHVLHGLY

GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV

DRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN

IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA

YEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSN

YIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQY

LYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVND

FVIEANKSNTMDKIADISUVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELU

PVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMY

KALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSV

SYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYUGSAEYEKSKVNKYLKTIMPFDL

SIYTNDTIUEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFK

LTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNS

GWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYING

KLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSY

SEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLY

IGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISD

SDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCIS

KWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

SEQ ID NO: 3 (BoNT/C - UniProt P18640.3)

MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNK

PPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEEUYRLSTDIPFPGNN

NTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTF

AAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYG

IAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSI

AKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTE

FNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPA

LRKVNPENMLYLFTKFCHKAIDGRSLYNKTLDCRELLVKNTDLPFIGDISDVKTDIFLRK

DINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYPSIDSESEILPGENQVFYDNRTQN

VDYLNSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLM

WANDVVEDFTTNILRKDTLDKISDVSAIIPYIGPALNISNSVRRGNFTEAFAVTGVTILL

EAFPEFTIPALGAFVIYSKVQERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQF

NNISYQMYDSLNYQAGAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNIN

KFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKUNUDSHNIILVGEVDKLKAKVNNSF

QNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLVDTSGYNAEVSEEGDVQ

LNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSNLPGYTIID

SVKNNSGWSIGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNM

KIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKEL

DGKDINILFNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRNNN

DFNEGYKIIIKRIRGNTNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYA

IGLREQTKDINDNIIFQIQPMNNTYYYASQIFKSNFNGENISGICSIGTYRFRLGGDWYR

HNYLVPTVKQGNYASLLESTSTHWGFVPVSE

SEQ ID NO: 4 (BoNT/D - UniProt P19321.1)

MTWPVKDFNYSDPVNDNDILYLRIPQNKUTTPVKAFMITQNIWVIPERFSSDTNPSLSK

PPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDS

STPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSN

PSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYG

INIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDI

AKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSE

VVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPA

LQKLSSESVVDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKIITDE

TNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYL NSYYYLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANE

VVEDFTTNIMKKDTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFP

EFTIPALGVFTFYSSIQEREKIIKTIENCLEQRVKRWKDSYQWMVSNWLSRITTQFNHIN

YQMYDSLSYQADAIKAKIDLEYKKYSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIR

ECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDSHNIILVGEVDRLKAKVNESFENTM

PFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSGYNAEVRVGDNVQLNTI

YTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTIINSIEQNS

GWKLCIRNGNIEWILQDVNRKYKSUFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYIN

GELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQI

LRNVIKDYWGNPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSV

SDKNPYSRILNGDNIILHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNY

GIGIFSIKNIVSKNKYCSQIFSSFRENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLS

TSSFWKFISRDPGWVE

SEQ ID NO: 5 (BoNT/E - UniProt Q00496.2)

MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTS

LKNGDSSYYDPNYLQSDEEKDRFLKIVTKIFNRINNNLSGGILLEELSKANPYLGNDNTP

DNQFHIGDASAVEIKFSNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHRFGS

lAIVTFSPEYSFRFNDNCMNEFIQDPALTLMHEUHSLHGLYGAKGITTKYTITQKQNPL

ITNIRGTNIEEFLTFGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYK

DVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLRTKFQVKCRQTYIGQYKYFKL

SNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKG

IRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFNSESA

PGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSS

IDTALLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADIS

IVVPYIGLALNIGNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNK

NKVIKAINNALKERDEKWKEVYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIE

SKYNSYTLEEKNELTNKYDIKQIENELNQKVSIAMNNIDRFLTESSISYLMKIINEVKIN

KLREYDENVKTYLLNYIIQHGSILGESQQELNSMVTDTLNNSIPFKLSSYTDDKILISYF

NKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTNKNQFGIYNDKLSEVNI

SQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKVSLNHNEII

WTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNL

GNIHVSDNILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYL

LYDKEYYLLNVLKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDN

LVRKNDQVYINFVASKTHLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTMNF

KNNNGNNIGLLGFKADTVVASTWYYTHMRDHTNSNGCFWNFISEEHGWQEK

SEQ ID NO: 6 (BoNT/F - UniProt A7GBG3.1)

MPVVINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFD

PPASLENGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGN

EHTPINEFHPVTRTTSVNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVY

DPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIADPAISLAHEUHALHGLYGAR

GVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATR

LSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANKF

KVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKG

LVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFFVASESSYNENDINTPKEIDDTTNLN

NNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFF

YLHAQKVPEGETNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIR

DFTTEATQKSTFDKIADISLVVPYVGLALNIGNEVQKENFKEAFELLGAGILLEFVPELL

IPTILVFTIKSFIGSSENKNKIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRK

EQMYQALQNQVDAIKTVIEYKYNNYTSDERNRLESEYNINNIREELNKKVSLAMENIERF

ITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYISEHRSILGNSVQELNDLVTSTLNN

SIPFELSSYTNDKIULYFNKLYKKIKDNSILDMRYENNKFIDISGYGSNISINGDVYIY

STNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLNNEYTIIDC IRNNNSGWKISLNYNKIIWTLQDTAGNNQKLVFNYTQMISISDYINKWIFVTITNNRLGN

SRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETL

YSDEPDPSILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSN

TRLYTGVEVIIRKNGSTDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKL

IRTSNSNNSLGQIIVMDSIGNNCTMNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTS

SNGCFWSFISKEHGWQEN

SEQ ID NO: 7 (BoNT/G - UniProt Q60393.2)

MPVNIKXFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFN

ASTGVFSKDVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLG

NASTPPDKFAANVANVSINKKIIQPGAEDQIKGLMTNUIFGPGPVLSDNFTDSMIMNGH

SPISEGFGARMMIRFCPSCLNVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLY

GIKISNLPITPNTKEFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIA

NRLNIVSSAQGSGIDISLYKQIYKNKYDFVEDPNGKYSVDKDKFDKLYKALMFGFTETNL

AGEYGIKTRYSYFSEYLPPIKTEKLLDNTIYTQNEGFNIASKNLKTEFNGQNKAVNKEAY

EEISLEHLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAETIAYN

TQNNTIENNFSIDQLILDNDLSSGIDLPNENTEPFTNFDDIDIPVYIKQSALKKIFVDGD

SLFEYLHAQTFPSNIENLQLTNSLNDALRNNNKVYTFFSTNLVEKANTWGASLFVNWVK

GVIDDFTSESTQKSTIDKVSDVSIIIPYIGPALNVGNETAKENFKNAFEIGGAAILMEFI

PELIVPIVGFFTLESYVGNKGHIIMTISNALKKRDQKWTDMYGLIVSQWLSTVNTQFYTI

KERMYNALNNQSQAIEKIIEDQYNRYSEEDKMNINIDFNDIDFKLNQSINLAINNIDDFI

NQCSISYLMNRMIPLAVKKLKDFDDNLKRDLLEYIDTNELYLLDEVNILKSKVNRHLKDS

IPFDLSLYTKDTILIQVFNNYISNISSNAILSLSYRGGRLIDSSGYGATMNVGSDVIFND

IGNGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTYLQNEYTII

SCIKNDSGWKVSIKGNRIIWTUDVNAKSKSIFFEYSIKDNISDYINKWFSITITNDRLG

NANIYINGSLKKSEKILNLDRINSSNDIDFKUNCTDTTKFVWIKDFNIFGRELNATEVS

SLYWIQSSTNTLKDFWGNPLRYDTQYYLFNQGMQNIYIKYFSKASMGETAPRTNFNNAAI

NYQNLYLGLRFIIKKASNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQTQ

LFLAPINDDPTFYDVLQIKKYYEKTTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTYD

NYFCISQWYLRRISENINKLRLGCNWQFIPVDEGWTE

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

MKLEINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNT

NDLNIPSEPIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLEUSSSIP

LPLVSNGALTLSDNETIAYQENNNIVSNLQANLVIYGPGPDIANNATYG LYSTPISNGEG

TLSEVSFSPFYLKPFDESYGNYRSLVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGIS

NRNFYYNFDTGKIETSRQQNSUFEELLTFGGIDSKAISSLIIKKIIETAKNNYTTUSE

RLNTVTVENDLLKYIKNKIPVQGRLGNFKLDTAEFEKKLNTILFVLNESNLAQRFSILVR

KHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYFEKIESNALRAFI

KICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLFUSN

KDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELY

EPIRNSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPF

KNMSNTINSI ETG ITSTYI FYQWLRSI VKDFSDETG Kl DVI DKSSDTLAIVPYIG PLLN I

GNDIRHGDFVGAIELAGITALLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNALDKRD

QKWAEVYN ITKAQWWGTI H LQI NTRLAHTYKALSRQANAI KM N M E FQLANYKG NIDDKAK

IKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLTKEMIPKVQDNLKNFDLETKKTLDK

FIKEKEDILGTNLSSSLRRKVSIRLNKNIAFDINDIPFSEFDDUNQYKNEIEDYEVLNL

GAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYLRFSATDNF

SISFWIKHPKPTNLLNNGIEYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYI

AFNGWNUTITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLL

DQFSIYRKELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWS

SFGYDYVILSDSKTITFPNNIRYGALYNGSKVUKNSKKLDGLVRNKDFIQLEIDGYNMG

ISADRFNEDTNYIGTTYGTTHDLTTDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETS

KPTFHDYRDWVYSSAWYFQNYENLNLRKHTKTNWYFIPKDEGWDED SEQ ID NO: 9 (TeNT- UniProt P04958.2)

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

EXAMPLES

Example 1 - Pharmacodynamic Assessments

The following pharmacodynamic parameters will be collected and reviewed during the study at specific time points as described in the study schedule of assessments in Table 3. The pharmacodynamics parameters described are generally applicable to the assessment of pain perception in a subject. Numerical Rating Scale

The NRS is a 11-point scale to assess subject pain perception as described in Figure 3. Subjects will be asked for their score.

Spontaneous Average and Worst Pain NRS Score during the Medical Eligibility Assessment Visit

Subjects will be asked to record their average scar-related pain intensity for the previous 24 hours. The subjects will be asked the following question:

"Please rate your pain by selecting the one number that best describes your pain on average during the last 24 hours." Subjects will be asked to record their worst scar-related pain intensity for the previous 24 hours. The subjects will be asked the following question:

"Please rate your pain by selecting the one number that best describes your pain at its worst during the last 24 hours."

Spontaneous Current Pain NRS Score in the Pre-randomisation Run-in Period (Part A)

On the day of the injection test, before the injection test (predose), subjects will be asked what are their current NRS scores, the pain felt by the subject at the time of the assessment. The subjects will be asked to answer the following question:

"Please rate your pain by selecting the one number that best describes how much pain you have right now."

Subjects will then be injected and will report their pain relief within 30 minutes after the injection test if any. Subjects will be asked for their current NRS score at 30 minutes and one hour post-injection test, according to the question just described previously.

Subjects will be provided with a paper diary after injection tests 1 and 2. They will be invited to record at home after discharge their current NRS scores hourly up to eight hours and between 20 and 24 hours post-injection test in this diary.

Spontaneous Average and Worst Pain NRS Score in the Randomised Double-blind Period (Part B)

If subjects are responders, they will be provided with an Actiwatch^® during an Actiwatch^® training visit to record their spontaneous NRS score at home. They will start NRS score recording from the training visit on Actiwatch^® use.

The Actiwatch^® will alarm and alert the subjects twice a day to record their average and maximal NRS scores over the preceding 12 hours. The questions will be asked of the subject by the Actiwatch^®:

"Please rate your pain by selecting the one number that best describes your pain on average during the last 12 hours."

"Please rate your pain by selecting the one number that best describes your pain at its worst during the last 12 hours.

The Actiwatch^® will allow electronic NRS score collection and storage from the Actiwatch^® training to end of study (EoS). Data will be extracted from the Actiwatch^® at each visit. A paper diary will also be provided to subjects in the unlikely event of an Actiwatch^® malfunction during the collection period. One week before post-dosing visit to the CRU, the subjects might be requested to complete the paper diary as a quality control check for the data in the Actiwatch^® and as a backup in case there is a problem with downloading information from the Actiwatch^®.

Stimulus-evoked NRS Score during Quantitative Sensory Testing

Subjects will be submitted to stimuli of various nature applied to the painful area. Stimulus- evoked NRS score will be collected during the assessments.

Subjects will be asked what are their current NRS scores predose and post-stimulus.

At baseline and at the post-dose visits light touch, pressure and temperature will be used as stimuli for Quantitative Sensory Testing (QST). The QST battery of tests is described in full below.

Light Touch

The surface of the painful area will be measured on radial spokes starting from the previously mapped painful area applying a von Frey filament. The filament will be applied until it slightly bends and be left in place for 4-5 seconds. Subjects will be asked to report when the von Frey filament first begins to cause any pain sensation and the distance of that point from the initial mapped painful area will be recorded.

The first painful position will be located on the skin and this process will be done for each radial spoke. The resulting points will then be transferred onto a tracing paper sheet and connected to define the outline of the painful area submitted to light touch. The area of pain will be calculated by using 1 cm² squares on the tracing paper (see area of pain below).

Sensory Threshold (ST): Seventeen, progressively rigid, monofilament, von Frey fibres (filaments represent stimuli from 0.039 - 4386mN) will be used for this test. Test the area with von Frey's filament, starting from the lowest/ thinnest monofilament. Each filament should be applied to the skin at a 90° angle with sufficient force to bend or bow the filament. The filament is held in place for 1.5 seconds and then removed. The exact threshold is found by repetitive testing ascending fibre sizes. The patient is instructed to respond "Yes" when a stimulus was felt. Each filament is applied up to 3 times in increasing filament thickness and the patient should say 'Yes' at least twice for the threshold filament.

Pain Perception Threshold (PPrT): This test is performed similar to ST but the response is the monofilament producing discomfort/ pain. Static Mechanical Allodynia (SMA): This test is performed by applying the plastic base of a von Frey filament for 10 seconds with sufficient pressure, indenting the skin-testing area. This is recorded in a 11-point Numerical Rating Scale (NRS).

Dynamic (Brush evoked) Mechanical Allodynia (DMA): DMA is evoked by gently stroking the test area with a foam brush. This is recorded in a 11-point Numerical Rating Scale (NRS).

Temporal summation: This test is done only on the painful site. A baseline NRS score will be obtained from the patient with the vFF as part of the PPrT measurement. This fibre will be used for the test. The patient is given a repetitive stimulation consisting of 30 repetitions of a pressure stimulus applied for 1 second duration for 30 seconds. The magnitude of the stimulus is set at the level of the subjects' pressure pain threshold. Patients rate the pain intensity on a NRS at the end of the 30 seconds and this is repeated for up to 10 sets or it is necessary to stop due to the level of discomfort.

Pressure (Pressure Pain Threshold)

An evoked current NRS score will be recorded for each pressure intensity. The pressure pain threshold (PPT) assesses the level of pressure causing pain. In post-surgical neuralgia subjects, light, normally non-painful pressure applied at the most painful point causes/may cause intense pain. The PPT is assessed by the investigator by using a pressure algometer (contact area 1 cm² ), documented in the source data, and transferred to the eCRF. The threshold for pressure induced pain is measured and then repeated in three series of slowly increasing stimulus intensities (at a rate of about 50 kPa/s). The subject must not be able to look at the readings during the measurement. For the final PPT, the arithmetic mean of all three consecutive measurements will be calculated.

Pressure Pain Threshold (PPT): A hand-held pressure algometer (Algometer type II, Somedic Production AB, Sweden, diameter contact tip 10mm; cover 2mm thick rubber; standardised and constant speed of pressure increase of 0.3kg/s) is used to measure the PPT's in KPa (Kilo Pascal). The probe is placed perpendicular to the skin and standard incremental pressure is applied until the subject perceived the pressure as pain when the procedure is immediately terminated. At each site, a set of 3 measurements are taken at 4 different nearby points and an average PPT value is calculated from the 12 measurements. If the patient feels too uncomfortable then restrict to fewest number of consistent readings (3 to 4). Conditioned Pain Modulation (CPM), also known as Diffuse Noxious Inhibitory Control (DNIC): CPM refers to an endogenous pain modulatory pathway. This is described as "pain inhibits pain". CPM occurs when a response to a second, often spatially distant, noxious stimuli inhibits the response from a painful stimulus.

Ischemic arm test to measure DNIC: DNIC is measured by inducing a heterotopic noxious conditioning stimulation. This is evoked by an inflated blood pressure cuff and thereby creating an ischaemic compression of the arm. Blood pressure cuff positioned on the arm was inflated above systolic pressure (200 mmHg) for 10 minutes, or until a Numerical Rating Scale of 6 was achieved. The point on the painful site with the lowest PPT value (average of 3 measurements) is chosen to measure the CPM response. A set of 3 PPT measurements are then taken at this point following which the cuff is deflated. The average of the 3 PPT measurements are taken.

Temperature

A thermal stimulation will be provided from a thermode placed on the painful area. The temperature of the thermode is controlled by a Medoc TSA II NeuroSensory Analyser (Medoc Ramat Yishai, Israel). The TSA-II - NeuroSensory Analyser is a precise, computer controlled device capable of generating and documenting response to highly repeatable thermal and stimuli, such as warmth, cold, heat-induced pain and cold-induced pain. The thermal sensory testing element measures the thresholds for four sensory sub-modalities:

Warm sensation, for normal subjects, usually at 1- 2 °C above adaptation temperature (C fibre mediated sensation),

Cold sensation, for normal subjects, usually at 1- 2 °C below adaptation temperature (A- delta fibres mediated sensation),

Heat induced pain, threshold around 45 °C (mostly C fibre mediated sensation, with some involvement of A-delta fibres).

Cold induced pain, the most variable and difficult to assess of all previous modalities, at about 10 °C (combination of both C and A-delta fibre mediated sensation).Warm detection and heat pain thresholds will be determined. The start of the temperature will be 32 °C and gradually ramped by 1 °C/sec. The heat pain threshold will be obtained when the subject will experience a painful feeling. The thermode will automatically shut down at a preset temperature of 53 °C in order to avoid any tissue damage. Cold detection and cold pain thresholds will also be determined, as programmed by the TSA II.

Temperature Thresholds: This test measures the cold detection threshold (CDT), warm detection threshold (WDT), cold pain threshold (CPT) and heat pain threshold (HPT) and paradoxical heat sensation. Four sensory sub-modalities are measured using a computer-controlled thermode with surface area of 9 cm² which is connected to a patient-activated push-button (TSA-II Quantitative NeuroSensory Analyzer; Medoc, Ramat Yishai, Israel). The system contains a pre-loaded software which dictates the temperature changes. The baseline temperature is automatically set at 32 °C. The temperature then decreases at a constant rate of 1 °C /s until the subject perceived the thermode as cold and immediately pressed a push-button. Four consecutive measurements are taken with the thermode returning to baseline temperature each time. Similarly, HDT, is measured but with the temperature increased from 32 °C. Subsequently, CPT and HPT are determined in a similar manner, and in that order but by taking an average of 3 consecutive measurements. To avoid thermal injury the thermode with automatically cut off at 0 °C at the lower end and at 50 °C at the higher end.

Area of Pain

The scar pain area reported by the subject will be mapped and drawn on the skin with a skin marker pen. The longitudinal axis of the painful area and the total painful area will be measured. The boundary of the spontaneous pain felt by the subject as well as the sites for injection at 2-cm intervals and starting 1 cm from the edge of the longitudinal axis of the pain area will be marked as represented in Figure 4. A photograph will be recorded with a ruler for scale and used to reproduce the administration scheme on injection test 1, injection test 2 and IMP dosing on Day 1. Mapping of the painful area was also performed post dose, as specified in Table 3.

Quality of Life questionnaire Short Form-36

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 the less disability. Example 2 - Study Outline

The study will be conducted in compliance with lECs/IRBs, informed consent regulations, the Declaration of Helsinki and ICH Guidelines related to GCP. Any episode of noncompliance will be documented. The electronic data capture (EDC) system will comply with the FDA, 21 CFR Part 11, Electronic Records, Electronic Signatures, and FDA, Guidance for Industry: Computerized Systems Used in Clinical Trials.

In addition, the study will adhere to all applicable international and local regulatory requirements.

All or some of the obligations of the sponsor will be assigned to a CRU or a CRO.

As shown in Figure 2, the study will consist of:

• A medical eligibility assessment visit: 28 days maximum before IMP dosing,

• A Part A, pre-randomisation run-in period (also referred to as the screening stage) (as per

Example 3):

o First injection test: 21 days maximum before IMP dosing

o Second injection test: one week after first injection test, 14 days maximum before IMP dosing,

o A Part B, randomised double-blind period (as per Example 4):

o A baseline period including a QST and Actiwatch^® training, safety assessments and a QoL: within 7 days before IMP dosing,

o A one-day IMP dosing,

o A 16-week randomised double-blind period. Subjects are expected to participate in this study for a maximum of 20 weeks.

The study will be considered to have started when the first subject has provided signed informed consent and will be considered to have ended after the last subject has completed his EoS visit. The overall study is anticipated to last approximately one year.

Example 3 - Pre-randomisation Run-in Period (Part A)

Prior to the pre-randomistion run-in period (i.e. the screening stage), potential subjects will be assessed for medical eligibility using relevant criteria as per Tables 1 and 2 within 28 days prior to IMP dosing on Day 1 (see Example 4). Table 1: Inclusion criteria

Table 2: Exclusion criteria

Upon consent and after successful assessment of medical eligibility and within 21 days maximum before IMP dosing on Day 1, subjects will attend the clinical research unit (CRU) for an injection test 1 visit, they will receive a pre-randomisation run-in number and allocated to one of the two treatment sequence groups in a chronological order, i.e. first eligible subject after the screening visit will be given the first run-in number. Subjects will be randomised with a ratio 2:2 (blocks of eight subjects) to receive either: (1) lidocaine on injection test 1 then placebo (saline) on injection test 2; or (2) placebo (saline) on injection test 1 then lidocaine on injection test 2.

Following randomisation, each subject will be injected in a double blind fashion with either local anaesthetic (lidocaine) or placebo (saline). Lidocaine will be prepared extemporaneously as a 0.5% v/w solution and 0.5 mL will be injected per injection site, providing 2.5 mg of lidocaine per injection site. Placebo doses are similarly prepared extemporaneously as a 0.9% v/w preservative- free sodium chloride solution (saline). 0.5 mL of lidocaine or placebo will be administered per injection site.

On the day of injection test 1, before the injection test (predose), subjects will be asked what are their current NRS score, the pain felt by the subject at the time of the assessment. The subjects will be asked to answer the following question: "Please rate your pain by selecting the one number that best describes how much pain you have right now.", i.e. their spontaneous current pain NRS score. Pain intensity will be scored using a 11-point NRS (from score 0 for "no pain" up to 10 for "worst possible pain"), as per Figure 3.

Lidocaine or saline will then be injected subcutaneously at a maximum of 10 injection points in the scar pain area.

Following administration of either lidocaine (local anaesthetic) or saline (placebo), subjects will be assessed for pain perception on site within 30 minutes after the injection test 1 and then be discharged from the CRU from 1 hour post-injection test 1 and continue to record their NRS score. Subjects will be invited to record at home after discharge their current NRS scores hourly up to eight hours and between 20 and 24 hours post-injection test 1.

Subjects will be contacted by telephone by the site to check their safety and collect their NRS score post-injection test 1 at the end of the injection test 1 day and on the following day.

Approximately one week later and within 14 days maximum before IMP dosing on Day 1, subjects will come back to the CRU for injection test 2. They will be crossed over and injected with the other agent. They will be assessed for pain perception on site and then be discharged from the CRU from 1 hour post-injection test 2 and continue to record their NRS score at home in an identical manner to that described above in relation to injection test 1. They will be contacted by telephone by the site to check their safety and collect their NRS score post-injection test 2 at the end of the injection test 2 day and on the following day.

After completion of a subject's injection test 2, the blind will be broken by an investigator to check if the subject is a responder. Subjects will be considered as responders and randomised to Part B if:

• The pattern of pain relief is sufficiently greater with lidocaine than saline using the formula below:

[NRS predose - NRS 1 hour post-injection test] lidocaine - [NRS predose NRS 1 hour post-injection test] placebo— 2

• The pattern of pain relief is clinically plausible in the investigator's opinion, e.g. onset of pain relief reported by 30 minutes from time of completing the injections, pain scores after the lidocaine injection test rising again by 2-8 hours post-injection;

• The investigator has no concern regarding compliance with study procedures and safety.

Albeit considered unlikely, should the results be equivocal, or if an unexpected but plausible pattern of response emerges, the decision as to whether a subject should proceed in the study, will be adjudicated by mutual agreement of the investigator and the sponsor. Subjects will be contacted by telephone by an investigator to inform them of the result. If subjects are assessed as responders and there are no concerns over subject safety, compliance with study assessments, they will enter in Part B. The scheduled dates should be with sufficient time between study visits test 1, test 2 and baseline. This is to permit the scar area to return to baseline after examination, test dosing or QST procedures.

This screening stage may be carried out to identify subjects prior to any clinical trial for the investigation of an IMP for the treatment of pain. Example 4 - Randomised Double-blind Period (Part B)

Subjects deemed to be responders based on the outcome of Example 3/Part A will be enrolled in the double-blind, randomised, placebo controlled, proof-of-concept study in subjects with abdominal or thoracic chronic scar pain to assess the analgesic properties of intradermal doses of Dysport^® (Clostridium botulinum toxin A-haemagluttinin complex).

The rationale of this study is to test the hypothesis that Dysport^® is effective in relieving chronic scar pain when administered intradermally. This study will also aim at characterising the pharmacodynamic profile of any analgesic effect of a predefined dose range (time to onset, peak- effect, time to peak-effect and duration of effect).

Baseline visit

Following confirmation of eligibility after injection test 2 of the screening method of Example 3, subjects will be assigned to a randomisation number and allocated to one of the treatment groups in a chronological order, i.e. first eligible subject will be given the first randomisation number. Mirror lists of randomisation numbers will also be produced to allow the randomisation of replacement subjects (e.g. the subjects who withdraw within six first weeks after IMP dosing) or to be used for the replacement of kits which could be damaged during the IMP reconstitution.

An initial visit to the CRU will be conducted within 7 days prior to Day 1 in order to carry out a QST (including light touch, pressure, temperature as stimuli) [stimulus-evoked NRS] and completion of QoL questionnaire SF-36. Safety assessments (physical examination, vital signs measurements, collection of adverse events (AEs) and recording of concomitant medication) will also be carried out. Subjects will also receive training on how to use an Actiwatch^® (device to be used for collection of the spontaneous NRS score). They will start to enter their NRS scores in their Actiwatch^®, and will be provided with a paper diary as a back-up solution for NRS score collection.

Blinding

Only the pharmacist or delegate in charge of drug preparation at the clinical research unit (CRU) and the monitor responsible for pharmacy monitoring will be partially unblinded. These personnel will be fully trained on the importance of their role in maintaining the blind for the subject, the investigator and the remainder of the study team. The unblinded staff at the CRU will not carry out any study assessments other than those they are assigned to. All other staff at the CRU, the sponsor and the subjects will be blinded to treatment allocations during the study.

Code-break Two types of code-break envelopes will be set up for the pre-randomisation run-in period (Part A, Example 3) and for the randomised double-blind period (Part B, Example 4).

Two sets of individual sealed code-break envelopes of each type will be prepared by the sponsor's randomisation manager to enable emergency code-break procedures for individual subjects without compromising the blind of the study. One set will be provided to the CRU and one set provided to the sponsor department of global patient safety (GPS).

In an emergency situation, which requires the identification of the study treatment group, the investigator may break the treatment code. If so, the investigator is requested to: inform the monitoring office at the earliest opportunity that the blind has been broken for an emergency situation; reseal the code-break envelope; sign, date and provide reason for the code-break on the emergency code-break form, and on the sealed envelope.

The date and reason for code-breaking should also be recorded in the electronic case report form (eCRF).

Specific case, to check if the subject is a responder at the end of the run-in period, the investigator will unblind the subject by (re-)opening the code-break-envelope associated to his(her) assigned pre-randomisation run-in number. The investigator will sign, date and specify "End of run-in period" as the reason for the code-break on the envelope.

Monitors should routinely check the integrity of the envelopes that are stored at the study site. They must collect envelopes from the study site prior to study close-out and ensure that they are all intact. If envelope(s) have been opened at the site or by the sponsor's representative, the monitor must ensure a written explanation is clearly documented (opener's name, dated signature and reason for opening) on the visit status page of the eCRF.

Confirmation of the integrity of all code-break envelopes at study completion must be documented in the trial master file (TMF). All sets of the sealed individual subject envelopes must be kept in the TMF in the co-ordinating office at study completion for proof of integrity.

Stopping Rules and Discontinuation Criteria

The complete study can be terminated prematurely at any time if the sponsor judges it necessary for any reason. In that case, all scheduled procedures and assessment for subjects who are still in the study will be performed. Some possible reasons for the closure of a study site may include: failure of the investigator staff to comply with the protocol or with the GCP guidelines; new and significant safety concerns; inadequate subject recruitment.

During the conduct of the study, all adverse effects (AEs) including serious AEs (SAEs) will be reviewed as they are reported from the study site to identify new and significant safety concerns. Withdrawal of Subjects

In accordance with the declaration of Helsinki and International Council for Harmonisation (ICH) GCP, each subject is free to withdraw from the study at any time, for any reason (e.g. withdrawal of consent, AE).

The investigator can withdraw a subject from the study at any time for any reason (e.g. protocol deviation, non compliance with the protocol conditions, lack of cooperation, in the event of concurrent illness, AE, or other reasons concerning the health or well-being of the subject). The reason for and date of withdrawal from the study must be recorded in the eCRF. If withdrawal is based on subject's decision every attempt will be made to determine: the reason for withdrawal; whether the subject also decides to withdraw his/her consent for the sponsor to collect and use the data collected up to the withdrawal point.

Data collected prior to subject withdrawal may be kept in study records and shared for further analyses unless the subject formally specifies his/her decision to withdraw consent for using data already collected. Should a subject be withdrawn from the study after IMP dosing and before normal study completion, all efforts will be made to complete the end of study assessments and report the observations up to the time of withdrawal as thoroughly as possible. A complete final evaluation at the time of the subject's withdrawal should be made whenever possible.

Subjects who withdraw within the first six weeks after IMP dosing for reasons other than lack of efficacy will be replaced. Replacement subjects will receive the same schedule and treatment as the subject they have replaced.

IMP for the Randomised Double-blind Period

The IMP is a solution for single intradermal injection (0.2 mL per injection site) of either Dysport^® or placebo. Active doses are prepared extemporaneously at the CRU pharmacy with marketed Dysport^® 500 U powder for solution for injection reconstituted and further diluted with saline to reach the targeted dose as indicated on the Dysport^®/placebo vial. Placebo doses are prepared extemporaneously at the CRU pharmacy with a placebo powder for solution for injection reconstituted and further diluted with saline using the same procedure as for the corresponding Dysport^® dose. They are indistinguishable from the active (Dysport^®) formulation after reconstitution. The pharmacist will prepare 2 mL of Dysport^®/placebo solution to inject 0.2 mL per injection site with a maximum of 10 injections in the scar pain area. Dosage Selection

The anhidrotic properties of a range of single intradermal 0.2 mL doses of Dysport^® (2.5, 10 and 20 U) were studied in healthy volunteers (Ipsen Study Y-52-52120-207). These doses were well tolerated and showed a dose-related increase in the anhidrotic area using the Minor's starch-iodine test. The maximal mean areas were 1.1, 2.7 and 3.7 cm² (corresponding radius of 0.6, 0.9 and 1.1 cm) for 2.5, 10 and 20 U respectively. Those same dose levels of 2.5, 10 and 20 U and route of administration have been selected for the individual injection points in the present study. The minimal dose will be 2.5 U injected in four sites (i.e. total dose of 10 U).

The maximal diffusion radius (1.1 cm for Dysport^® 20 U), together with the specification related to the maximal length of the pain area (i.e. no more than 10 cm) have been taken into consideration for the injection paradigm where individual injection points will be given intradermally 2 cm apart and the maximal number of injection points limited to 10 (see Figure 1). The maximal allowed total dose in the present study will thus be 200 U in the Dysport^® maximal dose group (20 U), which is consistent with the maximal recommended intradermal dose in the product label.

Randomised Period

The subjects will be required to attend the CRU on several occasions during the study:

• On Day 1 in the morning, based on investigator judgement, some of the screening/run-in assessments may be redone to confirm that subjects are still compliant with the inclusion/exclusion criteria and study restrictions. If the subject is considered as stable in pain perception from the screening visit, subjects will undergo pre-dose safety assessments and then receive the dose of IMP. After IMP dosing, subjects will undergo post-dose assessments as described in Table 3. They will be instructed to continue entering their NRS score in the Actiwatch^® and will be provided with a paper rescue medication diary to note their post-dose rescue medication intake (i.e. for scar pain) and NRS score immediately before intake at home (details on rescue medication are provided below). They will be discharged after a minimum of two hours post-dosing.

• Out-patient visits: Subjects will be required to come back to the CRU for out-patient visits on Week 6 and Week 12.

During the randomised double-blind period (Part B), subjects will also be contacted by telephone by the CRU every day up to Day 7 (subjects might visit the CRU if required for safety purpose) and every two weeks after IMP dosing for collection of adverse effects (AEs), concomitant medications and compliance with pain assessments and other study requirements. The outpatient visits and calls from Week 2 post-dosing can occur +/- three days around the scheduled date.

Early Discontinuation or End of Study Visit

Subjects will be discharged from the study after completion of an EoS visit for follow-up assessments which will happen 16 weeks after the IMP dosing +/- three days or earlier in case of early discontinuation (ED). The principal investigator will inform the family doctor and pain clinic (if applicable) with details of participation in the clinical study and any relevant information which may help the treating physician in the management of their patient.

Lifestyle Restrictions

Diet

As poppy seeds can cause a positive result on the drug of abuse (DOA) test, subjects will be advised to avoid eating poppy seeds/food containing poppy seeds for at least 24 hours before attending any DOA test. Subjects diet is expected to remain stable for the duration of the study.

Alcohol

Subjects should abstain from alcohol for 24 hours prior to each visit to the CRU. Excessive alcohol consumption (>21 units per week) should be avoided for the duration of the study.

Physical Activity

Subjects should not change their exercise regime from screening until completion of the study.

Prohibited Concomitant Medications and non Drugs Therapies

The following medications and therapies should not be started during the study:

• Physiotherapy,

• Transcutaneous electrical nerve stimulation therapy,

• Antidepressants and anxiolytics,

• Anticonvulsant medication used to treat pain,

• Opioids

• Non steroidal anti-inflammatory drugs except for brief periods of treatment (up to two

• days) for other pain or fever, • Acetylsalicylic acid (aspirin) except for low doses taken for cardioprotection or brief

• periods of treatment (up to two days) e.g. for episodes of fever,

• Corticosteroids (injected to treat the scar pain),

• Local anaesthetics (injected to treat the scar pain, unless for the pre-randomisation run-in

• period),

• Medications listed in the exclusion criteria.

Rescue medication (as detailed below, i.e. paracetamol up to 4000 mg per day in divided doses which can be substituted by co-codamol 30 mg/500 mg for the first week post-IMP administration) is allowed except within 12 hours before QST assessments. Subjects must not take medication containing paracetamol and rescue medication without having discussed this with the investigator.

Subject will also be cautioned on the use of any other medication (over-the-counter or prescription containing paracetamol e.g. Lemsip^®).

Rescue Medication

Paracetamol (acetaminophen), taken orally as required, is the rescue pain medication during the randomised double-blind period (Part B) and will be provided by the CRU to the subject. It can be taken up to four times a day and should not exceed a daily dose of 4000 mg in divided doses. A single dose of rescue medication is defined as 1000 mg (two tablets). The single paracetamol dose may be lowered to 500 mg (one tablet) if the investigator/subject feels that the dose is higher than what may be required to provide adequate analgesic effect. For the first week, in Part B post-dose, the investigator may substitute paracetamol with co-codamol (30 mg/500 mg, maximum 4000 mg of paracetamol per day).

If the subject takes the maximum rescue medication dose of 4000 mg paracetamol per day for three consecutive days and still reports uncontrolled scar pain, the investigator should consider discontinuing the subject from the study. If uncontrolled pain elsewhere in the body were to develop, the management of this should be handled on a case-by-case basis. The rescue medication should be swallowed whole with water and must not be broken or crushed. The subject will be cautioned on the use of any other medication (over-the-counter or prescription containing paracetamol e.g. Lemsip®). Rescue medication should not be taken in conjunction with other analgesic medication unless discussed with the investigator.

If rescue medication is taken, an AE and the corresponding rescue medication should be recorded in the electronic case report form (eCRF). Table 3: Study Schedule of Assessments

AE=adverse event; CRU=clinical research unit; D=study day; ECG=electrocardiogram; ED=early discontinuation; EoS=end of study; IMP=investigational medicinal product; NRS=numerical rating scale; QoL=quality of life; QST=quantitative sensory testing; W=study week.

[a] Visit to be performed ± 3 days around the scheduled date.

[b] In the evening and on the following morning to check safety and collect spontaneous NRS scores.

[C] Once the NRS scores from injection tests 1 and 2 are known and blind has been broken, the subject's responder status is determined. Subjects will be contacted by telephone by an investigator to inform of the result, and in the case of responders, they will enter in Part B.

[d] Subjects will be contacted by telephone by the CRU every day up to Day 7 and every 2 weeks after IMP dosing for collection of AEs, concomitant medications and compliance with pain assessments and other study requirements.

[e] Vital signs in both standing and supine position.

[f] Includes an examination of the scar at injection sites.

[g] Vital signs in supine position only h Just before the IMP dosing.

[i] Subjects will be provided with a paper diary after injection tests 1 and 2. They will be invited to record at home after discharge their current NRS scores hourly up to 8 hours and between 20 and 24 hours post-injection test in this diary.

[j] Subjects will be asked what were their average and worst NRS scores within 24 hours before the visit.

[k] Subjects will be asked what is their current NRS score pre-injection test. They will report their pain relief within 30 minutes after the injection test if any. They will be asked for their current NRS score at 30 minutes and 1 hour post-injection test. They will be discharged home and collect their current NRS scores hourly up to 8 hours and between 20 and 24 hours post-injection test.

[L] The Actiwatch^® will alarm and alert the subjects twice a day to record their worst and average NRS scores over the preceding 12 hours. The Actiwatch^® will allow electronic NRS score collection and storage from the Actiwatch^® training to EoS. Data will be extracted from the Actiwatch^® at each visit. A paper diary will also be provided to subjects in the unlikely event of an Actiwatch^® malfunction during the collection period. One week before post-dosing visit to the CRU, the subjects might be requested to complete the paper diary in case of Actiwatch^® malfunction or data quality check.

[m] QST (light touch, pressure, thermal) to be performed before D-3, at week 6 and week 12. Predose NRS score is the current pain.

Statistical Analyses

Analyses Populations

The following populations will be used for statistical analyses:

• Screened population: All subjects screened (i.e. who signed the informed consent),

• Run-in population: All subjects who received at least one injection in the pre randomisation run-in period (Part A),

• Randomised population: All subjects randomised in the double-blind period (Part B),

• Safety population: All subjects who received at least one dose of the study drug during the randomised double-blind period (Part B),

• Per protocol (PP) population: All subjects from the randomised population for whom no major protocol deviation occurred.

Sample Size Calculation

No prospective calculations of statistical power is made.

An appropriate sample size cannot be determined statistically as no previous human data from previous clinical trials are available. A sample size of six subjects per dose of Dysport has been selected. Approximately 24 subjects will be randomised in the double-blind period. Subjects who withdraw within the six first weeks from dosing will be replaced.

Statistical Methods

A statistical analysis pain (SAP) describing the planned statistical analysis in detail with tables, figures, and listings (TFLs) templates will be developed as a stand-alone document.

TFLs will be presented by treatment group (placebo, Dysport 2.5, 10 and 20 U). Some tables will also be described by total dose received in the painful injection area and the total dose received in the painful area divided by the painful area. This will be fully detailed in the SAP.

Pharmacodynamic/ Efficacy Evaluation

Pre-Randomisation Run-in Period

Only listing on NRS data will be provided.

Randomised Double-blind Period Analysis of the primary endpoints

Four endpoints have been defined to meet the study primary objective. They are presented in Table 4 with their associated estimate and estimand.

Only descriptive statistics will be provided. No statistical test will be performed.

As described in Table 4, the estimand will be based on "treatment policy" strategy, which is the estimate of the treatment effect regardless of whether the subject has an intercurrent event during the study.

The following potential intercurrent events will be considered:

· The short-term use of rescue medication,

• The change in use of rescue medication.

Supplementary and sensitivity analysis will be performed to take into account these intercurrent events and to handle missing data. These analyses will be detailed in the SAP. Table 4: Primary Objective - Analysis of Primary Endpoints

Analysis of the secondary endpoints

Endpoints have been defined to meet the study secondary objectives. They are presented in Table 5 with their associated estimate and estimand.

Table 5: Secondary Objectives - Analysis of Secondary Endpoints

Only descriptive statistics will be provided. No statistical test will be performed. Supplementary and sensitive analysis will be performed to take into account the intercurrent event and to handle missing data. Exploratory efficacy endpoints

Endpoints have been defined to meet the study exploratory objectives. They are presented in Table 6 with their associated estimate and estimand.

Table 6: Exploratory Endpoints - Analysis of Exploratory Endpoints

Example 5 Pre-Screening Protocol to Determine Suitability for Participation in a Clinical Trial

Investigating a Novel Opioid Peptide Analog

To determine whether a subject is deemed suitable for participation in a clinical trial investigating a novel opioid peptide analog for the treatment of pain associated with peripheral neuropathy, a cross-over screening protocol of the invention is performed.

Upon consent and after successful assessment of medical eligibility and within 21 days maximum of the first dose of the clinical trial, subjects are injected in a double-blind fashion with a first injection test of either lidocaine (local anaesthetic) or saline (placebo). Spontaneous current pain perception is assessed using a NRS both prior to the first injection test and within one hour following the first injection test.

Approximately one week later and within 14 days maximum before the first dose of the clinical trial, subjects are crossed over and injected with the other agent, i.e. those subjects who received an injection of lidocaine at the first injection test are injected with placebo, and those subjects who received placebo at the first injection test are injected with lidocaine. Spontaneous current pain perception is assessed using a NRS both prior to the second injection test and within 1 hour following the second injection test.

After completion of this second injection test, the blind is broken by the trial investigator to determine whether a subject is deemed a responder and thus suitable for participation in the clinical trial. Responders are identified using the formula below:

[NRS predose - NRS 1 hour post-injection test] lidocaine [NRS predose NRS 1 hour post-injection test] placebo— 2

The pain perception score of one of the responders is shown below:

• Pre-lidocaine NRS: 8

• 1 hour post-injection lidocaine NRS: 2

• Pre-placebo NRS: 8

• 1 hours post-placebo NRS: 6

Thus, the subject's score is 4 and is therefore considered a responder.

This subject and other subjects deemed to be responders based on the above calculation are enrolled in the clinical trial investigating a novel opioid peptide analog for the treatment of pain associated with peripheral neuropathy.

Example 6 - Pre-Screening Protocol to Determine Suitability for Participation in a Clinical Trial

Investigating Dysport^®

In order to identify subjects who respond strongly to placebo, a cross-over screening protocol in accordance with the present invention is performed. Identification of these subjects allows investigators to eliminate these subjects from the pool of subjects for a clinical trial investigating the efficacy of Dysport^® for the treatment of chronic back pain.

Upon consent and after successful assessment of medical eligibility and within 21 days maximum of the first dose of Dysport^®, subjects are injected in a double blind fashion with a first injection test of either benzocaine (local anaesthetic) or saline (placebo). Spontaneous current pain perception is assessed using a NRS both prior to the first injection test and within one hour following the first injection test.

Approximately one week later and within 14 days maximum before the first dose of Dysport^®, subjects are crossed over and injected with the other agent, i.e. those subjects who received an injection of benzocaine at the first injection test are injected with placebo, and those subjects who received placebo at the first injection test are injected with benzocaine. Spontaneous current pain perception is assessed using a NRS both prior to the second injection test and within 1 hour following the second injection test.

After completion of this second injection test, the blind is broken by the trial investigator to determine whether a subject is deemed to a have strong placebo effect and therefore should not proceed to the clinical trial.

Subjects who did not elicit a strong placebo effect during the cross-over screening protocol are enrolled in the clinical trial investigating the efficacy of Dysport^® for the treatment of chronic back pain.

Example 7 - Clinical Trial Investigating Dysport ⁹ in which Subjects were Pre-Screened to Determine their Suitability for Participation in the Clinical Trial

In order to identify subjects who respond strongly to placebo, a cross-over screening protocol in accordance with the present invention was performed.

60 subjects were assessed for eligibility for Part A (as per Example 3) of the study, 46 of these subjects were eligible for inclusion in the Part A study. In Part A all subjects received all of the planned volume of study treatment. Of the 46 subjects who were included in Part A, 17 were responders, 27 were non-responders and two subjects did not complete Part A due to adverse event, lost-to follow up and other (one subject each). Of the 17 responders, one withdrew and 16 subjects were randomised into Part B (as per Example 4) of the study (Table 7).

Table 7: Subject Disposition of Part A responders taken forward for Part B

N=number of subjects in group; U=unit(s). Demographics and baseline characteristics, including age, race, ethnicity and body mass index (BMI) were generally well balanced between treatment groups (Table 8). The majority of subjects in most treatment groups were White, with the exception of the 10 U Dysport^® group, where three subjects (60.0%) were Asian. The mean and median BMI were also lower in this treatment group (23.60 kg/m² and 25.50 kg/m², respectively) compared to other treatment groups (mean values between 28.60 kg/m² and 35.13 kg/m²; median values between 26.45 kg/m² and 38.60 kg/m²).

Urine drug of abuse tests and serology were conducted for each subject for the randomised population. Overall, positive results only occurred in three subjects (positive for opiate use in two [66.7%] subjects treated with 2.5 U Dysport^®/injection site and one [20.0%] treated with 10 U Dysport^®/injection site).

Disease specific and regional characteristics were generally well balanced between Part B treatment groups (Table 9). There was some variation in total painful area between treatment groups, with values between 4.5 cm² and 27.5 cm² reported for individual subjects. The mean and median painful areas in the 20 U Dysport^®/injection site (19.63 cm² and 21.00 cm², respectively) group were higher than other treatment groups. Consequently, the total Dysport^® dose received, when expressed in U/cm², was similar in the 10 U Dysport^®/injection site and 20 U Dysport^®/injection site treatment groups (Table 10).

There were no notable differences between the medical and surgical history of the randomised population in the treatment groups.

Prior and concomitant medications, non-drug therapies, and concomitant surgical procedures were recorded.

Prior medications were only reported for five (31.3%) subjects in the safety population. The most commonly reported prior medications were in the therapeutic classes of analgesics and anti inflammatory and antirheumatic products (two [12.5%] subjects each). There were no notable trends in prior medications between treatment groups.

During the study, 15 (93.8%) subjects received concomitant medications, with the most frequently used concomitant medications including paracetamol (nine [56.3%] subjects), omeprazole (6 [37.5%]), ibuprofen, levothyroxine, paracetamol-codeine and sertraline (three [18.8%] subjects each). There were no notable differences in concomitant medications between treatment groups. Concomitant medications related to medical history were reported in 13 (81.3%) subjects, with the most frequent being omeprazole (four [25.0%] subjects), levothyroxine, paracetamol and sertraline (three [18.8%] subjects each). There were no notable differences in concomitant medications related to medical history between treatment groups. Table 8: Demographic of Part B subjects

BMI=body mass index; max=maximum; min=minimum; SD=standard deviation; U=unit(s). Percentages are based on the number of subjects in the randomised population.

Table 9 : Disease Specific and Regional Characteristics (Randomised Population)

Dl=Day 1; min=minimum; max=maximum; NRS=Numerical Rating Scale; SD=standard deviation; U=unit(s).

For spontaneous NRS score, baseline is defined as the mean of all pre-dose data (from day -7 and including pre-dose on Day 1).

Concomitant non-drug therapies were received by four (25.0%) subjects, all of which were in the SOC of surgical and medical procedures, however, no notable differences between treatment groups were observed. No subjects received any prior non-drug therapies.

Three (18.8%) subjects in the safety population received concomitant surgical procedures (one subject each in the placebo group and the 2.5 U and 20 U Dysport^® injection site groups). None of these procedures was related to study treatment and no trends were observed.

In Part B all subjects received all of the planned volume of study treatment. A summary of exposure data by treatment group in Part B is presented in Table 10. There were no notable differences between treatment groups aside from the higher dose received by subjects in the higher dose groups. When total Dysport^® dose is presented in U/cm², mean values there were no notable differences between the 10 U Dysport^®/injection site and 20 U Dysport^®/injection site treatment groups. This is a consequence of the relatively larger painful area at baseline in the 20 U Dysport^® /injection site group. There was no clear difference in study duration between the treatment groups. Table 10: Study Treatment Administration and Extent of Exposure (Randomised Population)

N=number of subjects in group; SD=standard deviation; U=unit(s).

Total Dose (in U) = Number of injection site * Number of units of Dysport received.

Total dose (in U/cm²) = Total dose / (Total painful area at day 1 pre-dose).

n represents the number of subjects who performed the assessment. N represents the number of subjects in each treatment group.

Primary Efficacy Endpoints

The time to onset of effect (defined as the time to a decrease from baseline of two points or greater in the spontaneous NRS score) was measured in terms of worst and average pain over a 12 hour period. Current NRS scores were collected pre-injection and at 30 minutes, 1 hour and 8 hours post dose.

Reductions in NRS score corresponding to treatment effect were reported in all groups, including the placebo group. Time to onset of effect data are therefore available for 12 subjects for worst NRS score (10 subjects in Dysport^® groups and two subjects in the placebo group) and 12 subjects for average NRS score (9 subjects in Dysport^® groups and three subjects in the placebo group). There was considerable variability in time to onset of effect, both between and within treatment groups (not shown). There was no evidence of any effect of Dysport^® dose on time to onset of pain relief.

Peak effects (defined as the maximal decrease from baseline NRS score) were measured in terms of worst and average pain over a 12 hour period (Table 11). Peak effect data are summarised in box and whisker plot in Figure 5.

Mean and median peak effects in worst NRS scores were larger in Dysport^® treatment groups compared to placebo, suggesting some pain relief with Dysport^® treatment. There was no evidence of a dose effect and the range of peak effects was similar for all treatment groups. Differences in mean and median peak effects between Dysport^® and placebo treatment groups were less marked when considering the average NRS score. Peak effects in subjects treated with Dysport^® varied between -7.3 and 0.4 points for worst NRS score, and -6.1 and 0.6 points for average NRS score (where greater reductions correspond to greater pain relief). Table 11: Peak Effect (Randomised Population)

Max=maximum; min=minimum; NRS=numerical rating scale; SD=standard deviation.

The time to peak effect was measured in terms of worst and average pain over a 12 hour period. There was considerable variability in time to peak effect, both between and within each treatment group and between Dysport^® groups. There was no evidence of any effect of Dysport^® dose on time to peak effect (data not shown).

The duration of effect (defined as the duration between time to onset and last time-point for which change from baseline in the spontaneous NRS score was >2 points) was measured in terms of worst and average pain over a 12 hour period in Table 12. Table 12: Duration of Effect in Days (Randomised Population)

Max=maximum; min=minimum; NRS=numerical rating scale; SD=standard deviation.

Duration of effect in terms of worst NRS score was similar in all treatment groups. While The mean and median durations of effect in average NRS score were greater for the higher Dysport^® doses groups compared to the placebo and 2placebo.5 U/injection site treatment groups, the maximum duration of effect is similar in all treatment groups.

Secondary Efficacy Endpoints

Consistent with data for the primary efficacy endpoints, mean changes from baseline in worst and average NRS score (spontaneous and stimulus evoked) were similar between treatment arms (data not shown). There was no evidence of a dose effect in subjects treated with Dysport^® over time. Morning and evening scores in mean change from baseline in spontaneous worst and average NRS scores were consistent within treatment groups.

Spontaneous NRS scores were analysed using time weighted sum pain intensity differences

(SPID) and area under the effect (AUE) at week 6, week 12 and at the end of the study. These were based on the worst pain and the average within the last 12 hours. End of study SPID and AUE data are summarised in Table 13.

Mean SPID and AUE data suggested greater improvements in NRS scores (and therefore pain relief) in Dysport^® treatment arms. In particular, a number of subjects (Subject 82600100015 in the 2.5 U treatment group, Subject 82600100054 in the 10 U treatment group and Subjects 82600100027 and 82600100051 in the 20 U treatment group) demonstrated observably greater pain relief compared with the effect recorded in the placebo subjects.

Baseline QST assessment was carried out. Stimulus evoked NRS scores for static and dynamic mechanical allodynia were measured as described above (Example 1). Marked reductions in NRS scores evoked by static and dynamic allodynia at the painful area were reported for the Dysport^® treatment groups at week 6, with less marked reductions at Week 12. Improvements were relatively modest compared to placebo. There was no evidence of a dose effect.

Table 13: Summary Statistics on Time-Weighted SPID and AUE Parameters (Randomised Population)

AUE=area under the effect; max=maximum; min=minimum; NRS=numerical rating scale; SD=standard deviation; SPID=sum pain intensity differences.

Tactile detection threshold (light touch) was measured as described above (Example 1). There was no evidence of improvements in Von Frey sensory threshold or pain perception threshold in any of the treatment groups. Stimulus evoked NRS scores for temporal summation was measured as described above (Example 1). Changes in NRS scores evoked by temporal summation were similar between treatment arms. There was no evidence of greater pain relief with Dysport^® A treatment compared to placebo, and no evidence of a dose effect. Similarly, pressure pain threshold and thermal detection and pain threshold were measured as described above (Example 1). Using these measures, there was no evidence of greater pain relief, or a change in temperature perception/painful temperature threshold with Dysport^® treatment compared to placebo, and no evidence of a dose effect.

Painful area data (longitudinal axis and total painful area) by time-point for the randomised population was measured as described above (Example 1). There was no evidence of a reduction in painful area in any treatment arms. There was no difference between placebo and Dysport^® treatment groups and no evidence of a dose effect.

Exploratory Efficacy Endpoints

Individual response to the SF 36 questionnaire were recorded (as per Example 1 above). Mean SF 36 scores were generally higher after study treatment compared to baseline, suggesting slightly improved QoL. There were, however, no clear differences between Dysport^® treatment and placebo, and no evidence of greater improvements with higher doses.

Two subjects (one subject each in the Dysport^® 10 U and Dysport^® 20 U treatment groups) received rescue medication (paracetamol in two subjects and ibuprofen in one subject). Time to first rescue medication use was not calculated due to the low number of subjects receiving rescue medication.

Individual Response Data

Overall, response to treatment (defined as a reduction in NRS score by two points from baseline) was reported for eleven subjects (two subjects in the placebo treatment group and nine subjects in Dysport^® treatment groups). No response was reported for five subjects (two subjects in the placebo treatment group and three patients in Dysport ^® treatment groups). For three of the subjects who reported a response the data were confounded by missing NRS data (either more than 30% of time-points missing, or data missing for key study visits: subjects 82600100001 and 82600100006), or by variability in data which made interpretation of response data difficult (subject 826100036).

Profiles for the confirmed treatment responders are set out below, with results summarised for all subjects in Table 14. Table 14: Pharmacodynamic endpoints on spontaneous NRS scores (randomised population)

ID=identifier; ND=not determined; NRS=numerical rating scale; U=units.

Error! Bookmark not defined. Subjects 82600100001 and 82600100006 had a significant number of missing NRS data (>30% or significant NRS data missing).

b Subject 82600100036 had high variability of pain perception recorded during the baseline collection period. Variability in NRS scores continued after dosing, with most NRS scores during the treatment period being within the range reported at baseline

c Subject 82600100047 is considered a responder for worst NRS, but not for average NRS.

Subject 82600100026 (Placebo)

This subject was a White female aged 66 years at screening, The subject had a normal BMI (21.0 kg/m²) and reported scar pain following cosmetic breast reduction surgery less than two years prior to study entry.

The subject had significant ongoing medical history at randomisation, including joint pain, back pain, Raynaud's syndrome, peripheral neuropathy, cervical spondylosis and depression. As a consequence, the subject was heavily medicated during the study, with significant stable medication including pregbalin, escitalopram and paracetamol. At screening, spontaneous average and worst NRS scores were 7 and 8, respectively.

The subject's response profile during Part A indicated good response to lidocaine, but not placebo. Baseline pain scores for this subject were variable, with average NRS between 0 and 5, and worst scores between 2 and 6. The subject's reported pain scores reduced between Weeks 3 and Week 8 post dose, after which NRS scores returned to baseline levels. Increase in NRS scores after treatment was coincident with reduction in pregbalin dose on Day 78. Improvements in NRS were not reflected in QST analysis, while SF-36 QoL scores improved at both Week 6 (increase of SF-36 total score by 430 points from baseline) and Week 12 (increase by 745 points).

The improvements in the subject's NRS scores from Week 3 and Week 8 are consistent with a response to study treatment (placebo). Analysis of QST and QoL scores did not follow the profile of NRS scores.

Subject 82600100015 (Dysport¹¹¹ 2.5 U/injection site)

This subject was a White female aged 44 years at screening. The subject had a high BM I (38.6 kg/m²) and reported scar pain following cholecystectomy approximately one year prior to study entry.

Ongoing medical conditions at baseline included post traumatic stress disorder and degenerative disc disease. Concomitant medication included sertraline for stress disorder and gabapentin for degenerative disc disease. Ongoing treatment for scar pain included stable paracetamol and codeine as well as tramadol as needed. No tramadol intake was recorded for the subject during the study. At screening, spontaneous average and worst NRS scores were 4 and 6, respectively.

The subject's response profile during Part A indicated good response to lidocaine, but not placebo. Post dose NRS scores were mostly below baseline for the entire 16 week treatment period, with scores of 0 (indicating total pain relief) occurring within one week of dosing with study treatment (Dysport^® 2.5 U/injection site). There were no notable changes in QST scores, but QoL data indicated a progressive improvement in QoL (255- and 945 point increase at Week 6 and Week 12, respectively). Improvements in QoL were mostly related to improvements in role limitations due to emotional problems and pain.

Improvements in NRS, and concomitant improvements in QoL scores, for this subject are consistent with a response to study treatment (Dysport^® 2.5 U/injection site) for this subject.

This subject was a White female aged 49 years at screening. The subject had a high BMI (44.3 kg/m²) and reported scar pain following hysterectomy approximately five years prior to study entry.

The subject's medical history at randomisation included fibromyalgia and depression, and concomitant medications during the study included stable doses of pregablin, codeine and citalopram). At screening, spontaneous average and worst pain scores were 5 and 10 respectively.

The subject's response profile during Part A indicated good response to lidocaine, but not placebo. Average NRS scores varied between 3 and 6 at baseline. Worst NRS scores varied between 7 and 5. The subject's NRS scores decreased from baseline immediately post-dose, but returned to baseline levels after approximately 1 month. There were no notable changes in QST or QoL assessments during the study.

The rapid pain relief after dosing, followed by return to baseline, is consistent with a transient pain relief response to study treatment (Dysport^® 2.5 U/injection site).

This subject was an Asian female aged 35 years at screening, The subject had a BMI of 19.1 kg/m² and reported scar pain following caesarean section approximately two and a half years prior to study entry.

The subject had no significant ongoing medical conditions at baseline, and no concomitant medications were recorded. At screening, spontaneous average and worst NRS scores were 7 and 8, respectively.

The subject could not provide overall NRS score following lidocaine administration in Part A. Separate NRS scores were recorded for the left and right parts of the painful area. The worst score (corresponding to the right side of the scar) was recorded in the CRF. The subject's response profile during Part A indicated good response to lidocaine, but not placebo.

After dosing with study treatment (Dysport^® 10 U) most of the NRS data were below baseline values, indicating pain relief. While the subject was not receiving concomitant medication for scar pain, and did not use rescue medication, the subject received analgesics (paracetamol) for other conditions (headache, runny nose and head cold) during the treatment period. There was no evidence of improvements in QST or QoL assessments.

Sustained improvements in the subject's NRS scores during the treatment period are consistent with a response to study treatment (Dysport^® 10 U/injection site).

This subject was an Asian male aged 57 years at screening. The subject had a BMI of 26.6 kg/m² and reported scar pain following inguinal hernia repair approximately 5 years prior to study entry.

Ongoing medical conditions at baseline included migraine, epicondylitis, seasonal allergy and asthma. Concomitant medications during the study included salbutamol for asthma, plus sumatriptan and paracetamol and codeine for migraines, ibuprofen for headaches and paracetamol for headache, migraines and head cold. At screening, spontaneous average and worst NRS scores were 6 and 8, respectively.

The subject's response profile during Part A was atypical: the subject responded to lidocaine within two hours, as expected; however, there was a later response to placebo. Both lidocaine and placebo responses were ongoing at 8 hours post-dose. The atypical response was considered clinically plausible, and approval for randomisation into the study was agreed between the investigator and medical monitor.] During the treatment period, the subject reported pain relief from 2 weeks post-dose, with decreases in both average and worst NRS scores, and a maximum change of 2.9 points from baseline. Scores were consistently below baseline up to the end of the study. There were no notable changes in QST parameters or QoL scores.

The subject's prolonged post-dose pain relief, as measured by improvement in NRS score, was consistent with a response to study treatment (Dysport^® 10 U/injection site) for this subject. Occasional (single day) administrations of ibuprofen, paracetamol or sumatriptan for other indications did not appear to impact scar pain perception, as measured by NRS score.

This subject was an Asian female aged 57 years at screening. The subject had a BMI of 20.9 kg/m² and reported scar pain following hysterosalpingo-oophorectomy approximately five and a half years prior to study entry.

No ongoing medical conditions other than scar pain were reported at study entry. The subject received ongoing concomitant medications for scar pain during the study, including stable ibuprofen, as well as paracetamol, paracetamol and codeine, and aspirin, paracetamol and codeine as needed. At screening, spontaneous average and worst NRS scores were 7 and 10, respectively.

The subject's response profile during Part A was atypical, with a delayed response to lidocaine injection. The case was discussed by the investigator and medical monitor prior to randomisation in the study. Despite high baseline NRS scores (average scores between 5 and 7; worst scores between 6 and 9) the subject reported rapid onset of pain relief from study treatment (Dysport^® 10 U/injection site) with response reported from Day 2, and peak effect by Day 7. Pain relief response was maintained throughout the study. Average and worst NRS scores of 0 (indicating no pain at all) were reported following dosing. Concomitant improvements were reported in QST (sensory threshold, pain perception threshold, static and dynamic mechanical allodynia) and QoL assessments (improvement by 1165 and 1120 points at Week 6 and Week 12, respectively). The subject received paracetamol as rescue medication from Day 75 until the end of the study. Pain perception scores remained below baseline during this period.

The subject reported rapid and sustained improvements in pain perception post-dose, which is consistent with response to study treatment (Dysport^® 10 U/injection site) for this subject. The subject reported total pain relief for some time-points, despite high pain perception at baseline.

This subject was a White female aged 44 years at screening. The subject had a BM I of 25.2 kg/m² and reported scar pain following cardioverter defibrillator approximately four years prior to study entry.

The subject had painful ongoing medical conditions at randomisation, including osteoarthritis and torticollis; however, no ongoing analgesic treatment was reported. At screening, spontaneous average and worst NRS scores were 5 and 7, respectively.

The subject's response profile during Part A was atypical, with a delayed response to lidocaine injection. The case was discussed by the investigator and medical monitor prior to randomisation in the study. There was considerable variability in NRS scores at baseline, with average scores between 2 and 9, and worst scores between 3 and 10. A rapid decrease in NRS scores was reported immediately after dosing with study treatment (Dysport^® 20 U/injection site) with NRS scores of 0 reported, indicating complete pain relief. Reported pain relief persisted up to the end of the study. There were no notable changes in QST assessments. Improvements in QoL scores were reported at both Week 6 and Week 12 (improvements of 600 and 580 points, respectively. The patient received rescue medication (ibuprofen) to treat scar pain during Part A. No rescue medication use was reported post-dose. The rapid and persistent reduction in NRS scores, with concomitant improvements in QoL assessments, are consistent with pain relief response to study treatment (Dysport^® A 20 U/injection site).

This subject was a White female aged 43 years at screening. The subject had a BMI of 27.6 kg/m² and reported scar pain following caesarean section approximately 8 years prior to study entry.

Ongoing medical conditions at randomisation included arthralgia, and concomitant medications included stable doses of paracetamol and ibuprofen (both for scar pain4). At screening, spontaneous average and worst NRS scores were 7 and 9, respectively.

The subject's response profile during Part A indicated good response to lidocaine, but not placebo, despite some missing NRS scores following placebo administration. Post-dose NRS scores were consistently below baseline levels, with the exception of a few excursions. Decreases in NRS score to 0, indicating complete pain relief, were reported. There were no notable changes in QST or QoL scores.

The rapid and persistent reduction in NRS are consistent with pain relief response to study treatment (Dysport^® 20 U/injection site) for this subject.

Efficacy Results Summary

Analysis of the population level data for the primary efficacy endpoints (time to effect, peak effect, time to peak effect and duration of effect) as assessed by change in NRS score, showed similar pain relief with Dysport^® treatment and placebo. Greater peak effects were reported with Dysport^® treatment compared to placebo; however, there was no evidence of a dose effect.

Analysis of spontaneous NRS scores suggest a trend toward higher pain relief in Dysport^® treatment groups compared to placebo, notably for worst NRS scores.

Painful area mapping, QST and QoL assessments were consistent with primary efficacy endpoint analyses, and did not indicate improvements with Dysport^® treatment groups compared to placebo. Rescue medication use in the study was very low.

Treatment response profiles for individual subjects indicated treatment response for two subjects in the placebo group (50%). In the Dysport^® treatment groups nine subjects (75.0%) reported a treatment response. Response data for three treatment responders were confounded by missing or inconclusive data. If these subjects are not considered, treatment response was reported for one subject (33.3%) in the placebo group and seven subjects (70.0%) in the Dysport^® treatment groups. Total pain relief, as reflected by NRS scores of 0, were reported by four subjects who received Dysport^® (Subjects 82600100015, 82600100054, 82600100027 and 82600100051). These subjects reported concomitant improvements in QoL scores.

Safety Results

Eleven of the 12 subjects (91.7%) in the Dysport^® dose groups experienced a total of 40 treatment-emergent adverse effects (TEAEs) (Table 15). None of the TEAEs was of severe intensity. With the exception of one TEAE reported in one subject (25.0%) in the placebo group, the TEAEs were not considered related to study treatment. No TEAEs leading to discontinuation were reported. One subject (33.3%) in the 2.5 U Dysport^® dose group experienced a serious TEAE (PT: pancreatogenous diabetes). One subject (25.0%) in the placebo group experienced a TEAE with a PT corresponding to a term predefined as an AESI (injection site rash). There was no evidence of an increase in TEAEs with a higher total Dysport^® dose in this study.

Table 15: Overall Summary of Adverse Effects (Safety Population)

AE=adverse event; AESI=adverse event of special interest; SAE=serious adverse event; TEAE=treatment-emergent adverse event.

Error! Bookmark not defined, data are presented as: number of subjects (percentage of subjects) [number of events]. The most frequently reported TEAEs in the Dysport^® dose groups were in the SOC of nervous system disorders. Within this SOC, TEAEs of headache and dizziness were most common. There were no TEAEs of headache or dizziness reported in the placebo group. The only TEAE reported by more than one subject in the placebo group was nasopharyngitis. In the placebo group, the most frequently reported TEAEs were in the SOC of infections and infestations, with nasopharyngitis the most common.

Only one treatment-related TEAE was reported during the study: injection site rash (preferred term, PT) in a subject in the placebo group.

There were no TEAEs leading to death or discontinuation during the study. One subject experienced a serious adverse effect (SAE) and one subject experienced an adverse effect of special interest (AESI). The single SAE was of pancreatogenous diabetes and was reported for one subject in the 2.5 U Dysport^® dose group. Pancreatogenous diabetes was noted as a lifelong condition in the subject. In particular, the subject had a history of recurrent pancreatitis which then evolved into pancreatogenous diabetes. The SAE was considered not related to study treatment, and was of moderate intensity. The single AESI was reported in a subject in the placebo group, who experienced a TEAE with a PT corresponding to a term predefined as an AESI (injection site rash). This event was considered related to study treatment and of mild intensity.

There were no clinically significant abnormal values for any laboratory parameters and all urine pregnancy tests were negative. There were no notable changes in mean systolic and diastolic blood pressure or heart rate during the study. There were no clinically significant abnormal ECG findings at screening. There were no new clinically significant abnormal physical examination findings.

Safety Results Summary

Overall, Dysport^® was well tolerated. There were no TEAEs with an outcome of death and no subjects discontinued the study due to TEAEs. None of the TEAEs was of severe intensity, and no TEAEs considered related to treatment were reported in subjects who received Dysport^®. One subject (in the 2.5 U Dysport^® dose group) experienced a serious TEAE of pancreatogenous diabetes. The SAE was considered not related to study treatment, and was of moderate intensity.

One subject (in the placebo group) experienced an AESI of injection site rash, which was considered related to study treatment and of mild intensity.

The most frequently reported TEAEs in the Dysport^® dose groups were headache and dizziness. In the placebo group, the most frequently reported TEAE was nasopharyngitis.

There were no clinically significant abnormal values for any laboratory parameters during the study and no notable changes in vital signs. Discussion

The present study (Study D FR 5210 244) was an exploratory efficacy study investigating the potential analgesic effect of Dysport^® treatment for patients with chronic scar pain. As such no formal statistical analyses were conducted and only descriptive statistics were produced. Recruitment into the study was terminated early due to continued slow enrolment into the trial. 16 subjects were randomised to receive study treatment (Dysport^® or placebo).

The study had a two part design. Part A was a run in period where subjects received both lidocaine and placebo in a randomised sequence. The purpose of this run in period was to identify subjects who responded to placebo treatment and exclude them from the study. Of the 46 subjects who entered Part A of the study, 17 subjects were determined to have a response to lidocaine but not placebo. After inclusion of this run in period, a single placebo confirmed responder was identified following dosing in Part B of the study.

The primary objective of the study was to describe the pharmacodynamic analgesic profile of intradermal doses of Dysport^® in subjects with abdominal or thoracic chronic scar pain.

Looking at pharmacodynamics profiles for individual subjects, treatment response (defined as a decrease in NRS score by two points or greater) was reported for nine subjects (75.0%) in Dysport^® treatment groups and two subjects (50.0%) in the placebo group. Excluding subjects with missing or inconclusive data, treatment response was reported for seven subjects (70.0%) who received Dysport^® and one subject (33.3%) who received placebo. Of the subjects who responded to Dysport^®, some had particularly marked responses, including subjects who reported complete pain relief (as indicated by an NRS score of 0) at some time points. (Subjects 82600100015, 82600100054, 82600100027 and 82600100051).

Analysis of spontaneous NRS scores suggest a trend toward lower pain perception in Dysport^® treatment groups compared to placebo, notably for worst NRS scores. Reductions in NRS score up to 6.1 and 7.3 points for were reported for average and worst NRS scores, respectively, were reported in subjects who received Dysport^® (3.3 and 4.3 points, respectively in those who received placebo). However, population level analysis of other primary efficacy endpoints (time of onset of meaningful analgesic effect, time to peak effect, duration of effect) did not identify a clear benefit of Dysport^® over placebo in terms of pain relief for subjects with chronic scar pain at the population level. Population level analyses of secondary and exploratory efficacy endpoints were consistent with those for the primary endpoints.

There was a degree of heterogeneity in the study population that may have confounded interpretation of study findings at the population level. For example, there were it would not be appropriate to require subjects to stop their existing medication to control chronic pain in a placebo controlled study, so there were relatively few limitations on concomitant pain medications. In addition, while analgesic medications were required to be stable at study entry, several subjects were receiving antidepressants (for example selective serotonin reuptake inhibitors) during the study, which may also have a central pain modulation effect. Study entry criteria were modified to extend the duration from surgery to 10 years. It is not clear what effect, if any, pain chronicity might have on response to treatment.

Safety data collected during the study were consistent with the known safety profile for Dysport^®, and indicated that study treatment was well tolerated. The one SAE reported during the study was consistent with the subject's known medical history and was not considered related to study treatment. Only one treatment related TEAE was reported, and this occurred in the placebo group. There were therefore no new safety concerns identified with intradermal Dysport^® use to treat chronic scar pain.

Claims

1. A local anaesthetic for use in screening a subject for suitability for participation in a clinical trial for the treatment of pain, comprising: a. administration of the local anaesthetic or a placebo to said subject;

b. assessing said subject for pain perception;

c. in the case where the local anaesthetic was administered in step a, administration of the placebo, or in the case where the placebo was administered in step a, administration of the local anaesthetic;

d. assessing said subject for pain perception; and

e. determining whether a subject is suitable for participation in the clinical trial on the basis of a comparison of the pain perception assessment of step b and step d; wherein steps a to e are carried out sequentially.

2. The local anaesthetic for use of claim 1, wherein step c is carried out at least three days, at least five days, or at least seven days after step a, and wherein preferably step c is carried out one week after step a.

3. The local anaesthetic for use of claim 1 or 2, wherein pain perception is assessed prior to step a and/or step c, preferably before both steps a and c, to determine a baseline of pain perception.

4. The local anaesthetic for use of any one of the preceding claims, wherein: i. step b is carried out up to 24 hours after step a; and/or

ii. step d is carried out up to 24 hours after step c; wherein preferably step b is carried out up to 24 hours after step a, and step d is carried out up to 24 hours after step c.

5. The local anaesthetic for use of any one of the preceding claims, wherein step b and/or step d comprises at least two separate assessments for pain perception, optionally wherein: i. in step b pain perception is assessed at least one of: 30 minutes after step a; hourly up to 8 hours after step a; and/or hourly between 20-24 hours after step a; and preferably pain perception is assessed at each of these times; and/or ii. in step d pain perception is assessed at least one of: 30 minutes after step c; hourly up to 8 hours after step c; and/or hourly between 20-24 hours after step c; and preferably pain perception is assessed at each of these times.

6. The local anaesthetic for use of any one of the preceding claims, wherein: i. the local anaesthetic is lidocaine; and/or

ii. the placebo is saline; wherein preferably the local anaesthetic is lidocaine and the placebo is saline.

7. The local anaesthetic for use of any one of the preceding claims, wherein steps a to d are conducted in a double-blind manner.

8. The local anaesthetic for use of any one of the preceding claims, wherein pain perception is assessed using the Numeric Rating Scale (NRS).

9. The local anaesthetic for use of claim 8, wherein determining whether a subject is suitable for participation in a clinical trial on the basis of a comparison of the pain perception assessment of step b and step d comprises determining if the pain perception following administration of the local anaesthetic is at least two points lower on the NRS compared with the pain perception following administration of the placebo.

10. The local anaesthetic for use of any one of the preceding claims, wherein the investigational medicinal product (IMP) for investigation in the clinical trial for the treatment of pain is a clostridial neurotoxin, preferably a botulinum neurotoxin (BoNT) or tetanus neurotoxin (TeNT).

11. The local anaesthetic for use of claim 10, wherein the botulinum neurotoxin is a wild-type BoNT, or a modified BoNT, preferably wherein the BoNT is a wild-type BoNT/A, BoNT/B, BoNT/Cl, BoNT/D, BoNT/E, BoNT/F or BoNT/G, more preferably wherein the BoNT is a wild- type BoNT/A.

12. The local anaesthetic for use of any one of the preceding claims, wherein: i. step a takes place a maximum of 21 days before the first administration of the IMP or the corresponding IMP placebo in a clinical trial for the treatment of pain; and/or ii. step c takes place a maximum of 14 days before the first administration of the IMP or the corresponding IMP placebo in a clinical trial for the treatment of pain.

13. The local anaesthetic for use of any one of the preceding claims, wherein the pain is chronic pain, preferably abdominal or thoracic chronic scar pain.

14. The local anaesthetic for use of any one of the preceding claims, wherein the clinical trial is a double-blind trial for investigating the IMP wild-type BoNT/A compared with a placebo, wherein the wild-type BoNT/A is administered at a dose of 2.5 U/injection site, 10 U/injection site or 20 U/injection site and the subject is monitored for 16 weeks post administration of the IMP.

15. The local anaesthetic for use of claim 14, wherein the clinical trial assesses parameters comprising: i. NRS;

ii. stimulus-evoked NRS;

iii. temperature of the painful area;

iv. size of the painful area;

v. time to onset of analgesic effect;

vi. peak analgesic effect;

vii. time to peak analgesic effect;

viii. duration of analgesic effect; and/or

ix. SF-36 quality of life.

16. A method of determining local anaesthetic sensitivity comprising: a. administering the local anaesthetic or a placebo;

b. assessing for pain perception;

c. in the case where the local anaesthetic was administered in step a, administering the placebo, or in the case where the placebo was administered in step a, administering the local anaesthetic; and

d. assessing for pain perception; wherein steps a to d are carried out sequentially.

17. A method for screening a subject for suitability for participation in a clinical trial for the treatment of pain comprising: i. carrying out the method of claim 16; and

ii. comparing the pain perception assessed in step b with the pain perception assessed in step d; and

iii. determining whether a subject is suitable for participation in the clinical trial on the basis of the comparison of step ii.

18. A method for screening a subject for suitability for a treatment for pain comprising: i. carrying out the method of claim 16; and

iii. determining whether a subject is suitable for a treatment for pain on the basis of the comparison of step ii.