Treatment of post-operative surgical pain

Abstract

The present invention relates to a clostridial neurotoxin for use in treating post-operative surgical pain in a patient, said method comprising administering to a patient a clostridial neurotoxin more than

Description

[Technical Field]

[0001] This invention relates to the treatment of surgical pain (e.g., postoperative surgical pain) and / or surgery-induced anxiety (e.g., postoperative anxiety) caused by surgery. More specifically, this invention provides a treatment method comprising the administration of clostridial neurotoxin, and more particularly, a method for treating postoperative surgical pain and anxiety using botulinum neurotoxin. [Previous Technology]

[0002] Postoperative surgical pain is an unpleasant sensation caused by the surgical procedure. Postoperative surgical pain may be caused by surgical interventions, the surgical procedure itself, wound closure, and damage to tissues caused by any forces applied during the surgical procedure. Postoperative surgical pain (e.g., postoperative pain) may also stem from factors accompanying the surgery. For example, a patient may experience back pain due to their position on the operating table, or chest pain due to surgical interventions in the chest area. Sore throat may also occur after general anesthesia because the insertion of a breathing tube can cause irritation. However, the most common cause of postoperative surgical pain is surgical interventions that cut into the skin and muscles.

[0003] For example, a surgical intervention (or more specifically, a surgical incision) may represent a "noxious stimulus" that causes pain. A noxious stimulus is a stimulus that triggers tissue damage, which can activate the release of neurotransmitters from sensory receptive afferent terminals and the release of neuropeptides from sensory terminals such as substance P and calcitonin gene-related peptide (CGRP). The noxious information is then transmitted from the peripheral nervous system to the central nervous system, where the individual perceives pain.

[0004] Postoperative pain can be caused by a combination of inflammation and nerve tissue damage at the surgical intervention site. Any inflammation and / or nerve tissue damage reinforces postoperative pain. For example, degranulation of fat cells activated in response to tissue damage can lead to the release of various substances, including proteases, cytokines, and serotonin. These substances can sensitize primary afferent neurons (activating them at a lower threshold) to produce a pain hypersensitivity response. Due to the extensive nerve innervation of tissues, any part of the body is susceptible to nerve damage caused by surgery.

[0005] Postoperative anxiety may result in physical symptoms and behavioral changes, including, but not limited to, fatigue, difficulty concentrating and sleeping, and muscle tension. In addition, patients may experience emotional symptoms of anxiety, including irritability, irritability, difficulty controlling fear or worry, fear, and panic. Postoperative anxiety may be caused by the effects of anesthesia, the procedure itself, postoperative surgical pain, and / or stress in the hospital environment. For example, surgical patients are usually under a considerable degree of mental, physical, and emotional stress before surgery (e.g., stress caused by anticipation of surgery) and after surgery, which manifests as symptoms of anxiety.

[0006] Existing methods for the treatment of surgical (e.g., postoperative) pain typically target neurotransmitters and peptides and include nonsteroidal anti-inflammatory drugs ((NSAIDS), opioids, local anesthetic blockade, or a combination of them.However, such treatments produce a wide variety of side effects, especially usually inducing dependence (e.g., addiction).Furthermore, these treatments only provide postoperative acute pain relief for a short period of time pain relief, thus requiring continuous / repeated administration (exacerbating the patient's problems with analgesic dependence / addiction) This increases the patient's chances of developing chronic postoperative surgical pain when acute postoperative pain is not effectively controlled This type of pain management approach (requiring continuous administration of agents) also often leads to drug resistance Additional issues associated with previous methods of dealing with postoperative surgical pain include the need for adverse analgesic administration doses.

[0007] Thus, current methods for the treatment of surgical pain are not suitable (e.g., insufficient) for the management / reduction of postoperative surgical pain, especially for moderate to severe postoperative surgical pain, and they do not provide appropriate management of the postoperative anxiety experienced by patients (the latter often requires alternative / additional medications). Therefore, there is an increasing need for alternative / improved methods for the treatment of postoperative surgical pain and / or postoperative anxiety.

[0008] The present invention addresses one or more of these problems by providing a treatment for long-term postoperative surgical pain / anxiety (including a tendency to reduce chronic postoperative surgical pain), e.g., even after a single (e.g., acute) administration. A better sample of the invention allows effective postoperative surgical pain or postoperative anxiety management at the time the patient emerges from surgery (favorably reducing postoperative pain or postoperative anxiety that would otherwise be considered an effect of systemic / local anesthetic regression) based on the astonishing observation that treatment (administration) is initiated prior to surgery on the patient, and provides specific preoperative administration at a specific time point for administration based on treatment based on Clostridium neurotoxin. [Invention Contents]

[0009] Summary of the Invention

[0010] More specifically, the present invention is based on the surprising discovery that postoperative surgical pain and postoperative anxiety can be treated and reduced or suppressed by intradermal or intrathecal administration of Clostridium neurotoxin (such as botulinum neurotoxin) more than 5 days prior to surgery. This is completely unexpected, as prior art methods report optimal analgesic activity when Clostridium neurotoxin is administered close to the time of surgery and via a selective administration route.

[0011] Advantageously, the inventors have demonstrated that by administering Clostridium neurotoxin intradermally or intrathecally more than 5 days prior to surgery, as early as one hour after surgery, Clostridium neurotoxin effectively treats postoperative surgical pain and provides continuous control / treatment of postoperative surgical pain for several days (or even weeks) after surgery without the need for continuous administration, and without the side effects associated with conventional analgesics / anesthetics. Thus, as the effects of any "general" or "local" anesthetic (used during surgery) gradually diminish, Clostridium neurotoxin can provide relief from postoperative surgical pain. In other words, administration before surgery (more than 5 days prior) advantageously allows the analgesic effect of Clostridium neurotoxin to occur (e.g., reach maximum efficacy / effect) at the point when the patient begins to perceive postoperative surgical pain or postoperative anxiety due to the gradual diminishing effects of the primary anesthetic / analgesic used during surgery. Therefore, treatment can be initiated before the onset of postoperative surgical pain, thereby preventing any associated (potentially significant) discomfort and distress in the patient. As described in more detail below, this early management of postoperative surgical pain (e.g., acute moderate to severe postoperative surgical pain) can be beneficial in reducing the onset of chronic postoperative surgical pain.

[0012] This contrasts with treatment observed when Clostridium neurotoxin is administered close to the time of surgery (or throughout the peri-operative period), where administration throughout the peri-operative period resulted in a significant lag phase or "activation period" observed post-operatively until any surgical pain relief effect of Clostridium neurotoxin was achieved.

[0013] Another surprising observation by the inventors is that, when Clostridium neurotoxin is administered intradermally, it is a particularly effective (e.g., rapid) treatment for postoperative pain / postoperative anxiety. Indeed, when compared with alternative routes of administration such as subcutaneous and intramuscular administration, the inventors observed that intradermal administration provides increased relief of postoperative pain and / or postoperative anxiety. This was entirely unexpected, as Clostridium neurotoxin has no apparent disadvantage in efficacy when routinely administered in other indications via such alternative routes of administration (e.g., subcutaneous / intramuscular).

[0014] Another advantageous discovery by the inventors is that Clostridium neurotoxins can act at a site distal to the administration site. For example, after administering Clostridium neurotoxins at or near the surgical intervention site, the inventors observed SNARE protein cleavage (e.g., SNAP-25 protein cleavage) in the spinal cord (with minimal or no SNARE protein cleavage at or near the surgical intervention site), suggesting that the Clostridium neurotoxins are transported retrogradely from their administration site into the spinal cord. This allows the Clostridium neurotoxins to be administered at a site distant from any site of injury (e.g., the surgical intervention site) that may cause discomfort to the patient, thus minimizing any further pain experienced by the patient.

[0015] Specific embodiments of the present invention will now be described by way of example only, with reference to the following figures and examples.

Implementation Method

[0017] Detailed Description

[0018] Similarly, the present invention provides a Clostridium neurotoxin for treating postoperative surgical pain in patients, the method comprising administering the Clostridium neurotoxin to the patient more than 5 days prior to surgery, wherein the Clostridium neurotoxin is administered in the following manner: i) intradermal; or ii) intrathecal.

[0019] In other words, one aspect of the present invention provides a method for treating postoperative surgical pain in patients, the method comprising administering a Clostridium neurotoxin to the patient more than 5 days prior to surgery, wherein the Clostridium neurotoxin is administered in the following manner: i) intradermal; or ii) intrathecal.

[0020] In one embodiment, the present invention provides a Clostridium neurotoxin for use in treating postoperative pain, the method comprising administering (e.g., intradermal) Clostridium neurotoxin to a patient more than 5 days prior to surgery. In other words, one embodiment of the present invention provides a method for treating postoperative pain, the method comprising administering (e.g., intradermal) Clostridium neurotoxin to a patient more than 5 days prior to surgery.

[0021] In a related observation, it has been found that pre-administration of Clostridium neurotoxin (before surgery) reduces or inhibits / prevents (e.g., completely prevents) postoperative anxiety episodes (postoperative anxiety) in patients. Therefore, another surprising technical effect provided by the present invention is the anti-anxiety effect achievable by pre-operative administration of Clostridium neurotoxin.

[0022] Therefore, another aspect of the present invention provides a Clostridium neurotoxin for reducing or suppressing postoperative anxiety, the method comprising administering a Clostridium neurotoxin to a patient prior to surgery, wherein the Clostridium neurotoxin is administered in the following manner: i) intradermal; or ii) intrathecal.

[0023] In other words, one aspect of the present invention provides a method for reducing or suppressing postoperative anxiety, the method comprising administering a Clostridium neurotoxin to a patient prior to surgery, wherein the Clostridium neurotoxin is administered in the following manner: i) intradermal; or ii) intrathecal.

[0024] Preferably, a method for reducing or suppressing postoperative anxiety includes administering Clostridium neurotoxin more than 5 days before surgery; for example, Clostridium neurotoxin may be administered more than 5 days before surgery.

[0025] Another aspect of the present invention provides a Clostridium neurotoxin for reducing or suppressing postoperative anxiety, the method comprising administering (e.g., intradermal) Clostridium neurotoxin to a patient prior to surgery (e.g., more than 5 days prior to surgery). In other words, one aspect of the present invention provides a method for reducing or suppressing postoperative anxiety, the method comprising administering (e.g., intradermal) Clostridium neurotoxin to a patient prior to surgery.

[0026] Preferably, a method for reducing or suppressing postoperative anxiety includes administering Clostridium neurotoxin more than 5 days before surgery; for example, Clostridium neurotoxin may be administered more than 5 days before surgery.

[0027] Preferably, Clostridium neurotoxin can be administered intradermally.

[0028] Alternatively or by means of intrathecal administration (e.g., by intrathecal administration / injection) of Clostridium neurotoxin.

[0029] Various additional (optional) specific embodiments of the present invention will now be described. It should be noted that each of the following specific embodiments is applicable to any method or Clostridium neurotoxin used herein.

[0030] In one specific embodiment, the administration of Clostridium neurotoxin does not include intramuscular administration.

[0031] In one specific embodiment, Clostridium neurotoxin may be administered 5-50 days prior to surgery, for example 6-50 days prior to surgery, or alternatively 5-40 days prior to surgery. For example, Clostridium neurotoxin may be administered 5-30 days prior to surgery, preferably 5-20 days prior to surgery, and more preferably 5-15 days prior to surgery.

[0032] In one specific embodiment, Clostridium neurotoxin (preferably in the method for treating postoperative surgical pain described herein) may be administered >5 days prior to surgery, optionally in a single administration step. For example, Clostridium neurotoxin may be administered 10-20 days prior to surgery; or 14-16 days prior to surgery (preferably in the method for treating postoperative surgical pain described herein), optionally in a single administration step.

[0033] The Clostridium neurotoxin may be administered more than 15 days before the operation, preferably about 15 days before the operation.

[0034] In a preferred embodiment, Clostridium neurotoxin (preferably the method for treating postoperative pain described herein) may be administered >5 to ≤15 days prior to surgery, optionally in a single administration step.

[0035] In one specific embodiment, Clostridium neurotoxin (preferably the method for treating postoperative pain described herein) is administered more than 5 days prior to surgery.

[0036] In one specific embodiment, Clostridium neurotoxin (preferably the method for treating postoperative pain described herein) is administered more than 12 days prior to surgery.

[0037] In one specific embodiment, Clostridium neurotoxin is administered intradermally more than 15 days prior to surgery. In a preferred embodiment, Clostridium neurotoxin is administered intradermally approximately 15 days prior to surgery.

[0038] In one specific embodiment, Clostridium neurotoxin is administered intrathecally at least 15 days prior to surgery. In a preferred embodiment, Clostridium neurotoxin is administered intrathecally approximately 15 days prior to surgery.

[0039] Clostridium neurotoxins treat postoperative surgical pain by providing analgesia. Therefore, the terms "treatment" or "management" as used herein are intended to encompass analgesic treatment. The terms "treatment" or "management" encompass treating postoperative surgical pain so that the patient no longer perceives surgical pain (or perceives less surgical pain compared to control patients who were not treated with Clostridium neurotoxins).

[0040] Similarly, Clostridium neurotoxins suppress postoperative anxiety by providing an anxiolytic effect. Therefore, the terms "suppression" or "repression" encompass the suppression of postoperative anxiety (e.g., its symptoms) in patients by the anxiolytic effect provided by administration of Clostridium neurotoxins. Suppression can be provided concurrently with postoperative pain management, for example, as a result of postoperative pain management. Therefore, without being limited to any theory, Clostridium neurotoxins can provide an anxiolytic effect through their analgesic effects. Clostridium neurotoxins can suppress postoperative anxiety symptoms related to (or caused by) the effects of surgical anesthesia, the surgery itself, postoperative pain, and / or stress (e.g., from the hospital environment).

[0041] Therefore, a therapeutically effective amount or a preventatively effective amount (preferably a preventatively effective amount) of Clostridium neurotoxin can be administered to the subject. "Therapeuticly effective amount" refers to any amount of Clostridium neurotoxin that, when administered alone or in combination to the subject for the treatment of postoperative surgical pain and / or postoperative anxiety, is sufficient to achieve such treatment. "Preventatively effective amount" refers to any amount of Clostridium neurotoxin that, when administered alone or in combination to the subject, inhibits or delays the onset of postoperative surgical pain and / or postoperative anxiety. In some specific embodiments, the preventatively effective amount completely prevents the onset of postoperative anxiety. "Inhibit" onset means reducing the likelihood of postoperative surgical pain and / or postoperative anxiety, or completely preventing the onset.

[0042] Preferably, the effective dose for treatment and / or prevention is a dose that does not cause muscle paralysis. The term "muscle paralysis" preferably refers to long-term muscle paralysis, as transient muscle paralysis may occur shortly after administration.

[0043] In this document, the terms "subject," "individual," and "patient" are used interchangeably to refer to a mammalian subject. In one embodiment, a "subject" is a human, a companion animal (e.g., a pet such as a dog, cat, and / or rabbit), livestock (e.g., a pig, sheep, cattle, and / or goat), and / or a horse. In a preferred embodiment, the subject (patient) is a human.

[0044] This invention relates systematically to postoperative surgical pain, which differs from other types of pain, such as inflammatory pain and neuropathic pain. In this respect, inflammatory pain is usually caused by infection, irritants, or an overactivated immune response, while neuropathic pain is usually caused by neurological disorders / syndromes. In contrast, this invention is unrelated to pain caused by such stimuli.

[0045] In one specific embodiment, administration of Clostridium neurotoxin to treat postoperative surgical pain preferentially over inflammatory pain. In one specific embodiment, administration of Clostridium neurotoxin to treat minimal to no inflammatory pain. In one specific embodiment, administration of Clostridium neurotoxin to treat postoperative surgical pain and minimal to no inflammatory pain.

[0046] The term "surgical intervention" refers to a medical procedure involving the treatment of an injury or disease in a subject, including an incision made into a part of the body (optionally to remove or repair the injured part). Although the level of invasiveness (e.g., the level of the required surgical incision) may vary depending on the type of surgery, it is intended to encompass procedures with a level of invasiveness that causes postoperative surgical pain and / or postoperative anxiety in the subject after the procedure. Postoperative surgical pain is typically caused by surgical incisions that cut through the patient's skin and / or fascia and / or muscles and / or bones and / or organs. Therefore, surgical pain is usually experienced at or near the site of the surgical intervention.

[0047] Surgical intervention may involve incisions in the skin and / or fascia and / or muscles. Preferably, the surgical intervention involves incisions in the skin.

[0048] Surgical interventions are not limited to procedures that can be performed by a physician, but also include, for example, dental interventions. Non-limiting examples of surgical interventions include appendectomy, breast examination, breast augmentation or reduction, cosmetic surgery, cholecystectomy, coronary artery bypass surgery, debridement (e.g., wounds, burns or infections), skin grafts, organ transplants and tonsillectomy.

[0049] Preferably, "postoperative" may refer to a period of time beginning at most one day after the surgery (e.g., postoperative). In other words, the term "postoperative" may refer to a period of time beginning no more than one day after the surgery. For example, the term "postoperative" may refer to a time point beginning 1-20 hours after surgery; alternatively, 2-15 hours after surgery; alternatively, 5-10 hours after surgery. Such a time may represent a period of time beginning at a temporal interface, during which the analgesic effect from the surgical anesthetic administered to the patient diminishes (e.g., gradually decreases) and the patient begins to perceive surgical pain.

[0050] Furthermore, the term “postoperative” can be used interchangeably with the term “postoperative”, since “surgery” is used in this text in the sense of “surgical procedure”.

[0051] Similarly, the term "postoperative surgical pain" can refer to surgical pain perceived (or more specifically, the onset of perception) within a period of up to one day after surgery. In other words, the term "postoperative surgical pain" can refer to surgical pain perceived by the patient within a period of no more than one day after surgery. For example, the term "postoperative surgical pain" can refer to surgical pain perceived within a period of 1–20 hours after surgery; alternatively, 2–15 hours after surgery; alternatively, 5–10 hours after surgery.

[0052] This time period can be 1-50 weeks after surgery; for example, 5-45 weeks, 10-40 weeks or 10-35 weeks.

[0053] This contrasts with the term "peri-operative," which may refer to, for example, the time period before or after a patient undergoes surgery (e.g., the time a patient spends in the operating room), appropriately the period that begins at least one hour before surgery and / or ends less than one hour after surgery.

[0054] The postoperative treatment of the present invention can be combined with the whole surgical and / or postoperative treatment strategies to enhance efficacy, preferably increase the duration or suppress postoperative surgical pain (e.g., in patients at high risk of surgical pain).

[0055] In one specific embodiment, the method of the present invention may include administering additional analgesics to the patient throughout the surgical procedure and / or postoperatively (preferably postoperatively). In other words, additional analgesics may be administered during the surgical procedure (e.g., at least 1 hour before surgery and / or less than 1 hour after surgery). In one specific embodiment, additional analgesics may be administered postoperatively (e.g., 10, 20, 40, or 50 weeks after surgery).

[0056] Postoperative surgical pain can be caused by the release of neurotransmitters from sensory receptors and / or by the release of neuropeptides such as substance P and calcitonin gene line peptide (CGRP) from sensory receptors, for example, pain induced by harmful stimuli of the surgery (preferably the surgical incision). For example, harmful messages (generated by harmful stimuli) can then be transduced from the peripheral nervous system to the central nervous system, where the patient will perceive surgical pain.

[0057] In one specific embodiment, Clostridium neurotoxin treats surgical pain by inhibiting the exocytosis of pain neuromodulators (such as substance P and CGRP).

[0058] Postoperative pain can be caused by inflammation or nerve damage at the surgical site, or a combination thereof. Any inflammation and / or nerve damage reinforces postoperative pain. For example, degranulation of activated fat cells in response to tissue damage can release various substances, including proteases, cytokines, and serotonin. These substances can sensitize primary afferent neurons (activating them at a lower threshold) to produce a pain hypersensitivity response. Because tissues are extensively innervated, any area of ​​the body can be susceptible to surgically induced nerve damage.

[0059] In other words, pain can be sensory receptive pain, for example, postoperative surgical pain caused by tissue damage and perceived by the response of nociceptors to harmful stimuli.

[0060] In one specific embodiment, postoperative surgical pain may be neuropathic pain (e.g., pain caused by damage or disease affecting the somatosensory nervous system). For example, postoperative surgical pain may be peripheral neuropathy (also known as peripheral pain), such as pain caused by damage to nerves (peripheral nerves) outside the brain and spinal cord.

[0061] Postoperative surgical pain can manifest as other types of pain, such as allodynia. Allodynia means "other types of pain." Allodynia is pain caused by stimuli that are not normally painful. Patients with "tactile" allodynia (also known as static tactile pain or mechanical tactile pain) may experience tenderness when the surgical incision is placed on a bed or when wearing clothing that comes into contact with the area. Therefore, allodynia is considered to be "pain caused by stimuli that are not normally painful," rather than hyperalgesia (increased pain caused by stimuli that are normally painful).

[0062] Postoperative surgical pain can preferably be acute postoperative surgical pain; for example, surgical pain that lasts for less than 3 months (postoperatively).

[0063] In one specific embodiment, the postoperative surgical pain is chronic postoperative surgical pain; for example, a type of surgical pain that can last for more than 3 months (after surgery) and can continue to be perceived after tissue damage (e.g. due to surgical incision) has healed.

[0064] More specifically, as used herein, the term "chronic postoperative surgical pain" preferably refers to pain that persists for more than 3 months after the resolution of intrinsic injury (e.g., damage to muscles caused by a surgical incision), such as postoperative pain lasting for more than 3 months. For example, "chronic postoperative surgical pain" may result from inadequate (or lack thereof) treatment of acute postoperative surgical pain (e.g., a type of surgical pain that typically lasts less than 3 months). Poor management of "acute postoperative surgical pain" may increase the chance that such acute surgical pain will become chronic postoperative surgical pain. Therefore, advantageously, by controlling acute postoperative surgical pain at an early stage (advantageously due to the administration of analgesia preoperatively, which provides analgesia shortly after surgery), the present invention reduces the occurrence of chronic postoperative surgical pain.

[0065] Chronic postoperative surgical pain may be perceived at or around the scar (the scar formed at the surgical incision site). In a preferred embodiment, chronic postoperative surgical pain is chronic scar pain. The term "chronic scar pain" refers to pain caused by the formation of tissue scars. "Chronic scar pain" may develop due to damage to the skin and / or muscle tissue and / or nerve tissue, as well as nerve regeneration.

[0066] In a preferred embodiment, postoperative surgical pain is surgical pain perceived at the site of surgical intervention and / or at a site close to the site of surgical intervention, preferably wherein surgical pain is perceived within tissue (e.g., skin, muscle) that has been damaged by the surgical intervention. Postoperative surgical pain may also be surgical pain perceived in tissues / organs within the body that have already undergone biopsy.

[0067] Preferably, administration of Clostridium neurotoxin reduces the level of perceived surgical pain in patients after surgery. For example, the level of perceived surgical pain in patients after surgery may be reduced compared to the level of perceived surgical pain in patients (controls) who did not receive Clostridium neurotoxin before surgery (e.g., more than 5 days before surgery).

[0068] In one specific embodiment, the patient's perception of postoperative surgical pain is reduced within 24 hours after surgery. In other words, administration of Clostridium neurotoxin within 24 hours after surgery can reduce the patient's perception of postoperative surgical pain. For example, administration of Clostridium neurotoxin within 6 hours after surgery, preferably within 1 hour after surgery, can reduce the patient's perception of postoperative pain.

[0069] Postoperative surgical pain may decrease at least 3 days, 6 days, or 9 days after surgery; for example, at least 15 days after surgery; in another example, at least 30 days after surgery. In a preferred embodiment, postoperative surgical pain may decrease up to 3 months (inclusive) after surgery.

[0070] Reduced postoperative pain perception in patients can be maintained for at least 5 days, at least 7 days, or at least 9 days postoperatively, preferably at least 9 days postoperatively.

[0071] In one embodiment, administration of Clostridium neurotoxin substantially reduces the patient's perceived postoperative surgical pain, and the reduced perceived postoperative surgical pain is maintained for 24 hours immediately following the surgery. In another embodiment, the patient's perceived postoperative surgical pain is substantially reduced and maintained for 2 days immediately following the surgery. In one embodiment, substantially all of the reduced perceived postoperative surgical pain is maintained for 3 days immediately following the surgery. In one embodiment, substantially all of the reduced postoperative pain is maintained for 4 days immediately following the surgery. In one embodiment, substantially all of the reduced postoperative pain is maintained for 5 days immediately following the surgery. In one embodiment, substantially all of the reduced postoperative pain is maintained for 6 days immediately following the surgery. In one embodiment, substantially all of the reduced postoperative pain is maintained for 7 days immediately following the surgery. Preferably, substantially all of the reduced postoperative pain is maintained for 8 days immediately following the surgery.

[0072] In one specific embodiment, the level of reduction in pain perception observed immediately after surgery at a defined time point (as described in the preceding paragraph) is at least 50% of the maximum level of reduction in pain perception observed at any time after administration of the Clostridium neurotoxin. For example, immediately after surgery, the level of reduction in pain perception observed at a defined time point (as described in the preceding paragraph) is at least 55%, at least 60%, at least 65%, at least 70%, preferably at least 75% of the maximum level of reduced pain perception observed at any time after administration of the Clostridium neurotoxin.

[0073] More specifically, the reference to "reduction" (in terms of postoperative surgical pain) preferably means that, compared with the level of surgical pain experienced by subjects (e.g., patients) who have undergone surgery and have not received clostridium neurotoxin (or placebo), the level of perceived surgical pain is lower. For example, when compared with subjects (who have undergone surgery) who have not received clostridium neurotoxin (or placebo), the administration of clostridium neurotoxin may reduce the level of perceived surgical pain by at least 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, or 95%. For example, when compared with subjects (who have undergone surgery) who have not received clostridium neurotoxin (or placebo), the administration of clostridium neurotoxin may reduce the level of perceived surgical pain by at least 75%, preferably at least 85%, and more preferably at least 95%.

[0074] Various methods for assessing pain perception are known to those of ordinary skill in the art. For example, the assessment of mechanical tactile pain (either static or dynamic) is routinely used in human pain research, as described by Pogatzki-Zahn et al. (Pain Rep. 2017 Mar; 2(2): e588), which is incorporated herein by reference.

[0075] Suitable (even if not limited) methods for assessing pain perception in subjects include the following: Numerical Rating Scale (NRS) scoring; although other methods that may be used additionally or alternatively, as known to those skilled in the art, such as sensory thresholds, pain perception thresholds, static mechanical tactile pain, dynamic mechanical tactile pain, temporal summation, pressure pain thresholds, conditioned pain modulation, and temperature thresholds.

[0076] Other non-limiting examples of pain perception measurements include: changes in SF-36 scores from baseline at each predetermined time point; the amount of emergency medication taken during the study period and the time of first administration of emergency medication. These may be considered "exploratory" endpoints or pain perception assessment measurements.

[0077] Therefore, in a preferred embodiment, after administration of Clostridium neurotoxin, postoperative surgical pain perception can be assessed by one or more of the following: (a) Numerical Rating Scale (NRS); (b) Stimulus-induced NRS; (c) Temperature of the pain area; (d) Size of the pain area; (e) Time of onset of analgesia; (f) Peak analgesia; (g) Time to reach peak analgesia; (h) Duration of analgesia; and (i) SF-36 quality of life.

[0078] Such methods for assessing pain perception are known to those skilled in the art. For convenience, a further description of the Numerical Rating Scale and the Short Form of the Quality of Life Questionnaire-36 is provided below.

[0079] Numerical Rating Scale (NRS): Generally, the surgical pain perception according to the present invention uses the Numerical Rating Scale (NRS). The NRS is an 11-point scale used to assess the subject's surgical pain perception. Subjects are asked to give a number between 0 and 10 that best represents their surgical pain intensity. Zero represents "no surgical pain at all," while the upper limit of 10 represents "the most severe possible surgical pain."

[0080] The NRS can be used to assess many aspects of surgical pain, including spontaneous average surgical pain, spontaneous maximum surgical pain, and spontaneous current surgical pain. Spontaneous average surgical pain is assessed by asking the subject to select a number that best describes the subject's average surgical pain (e.g., perceived surgical pain) over a period of time (e.g., at least 6 hours, 12 hours, 24 hours, or at least 48 hours). Spontaneous maximum surgical pain is assessed by asking the subject to select a number that best describes the subject's most severe surgical pain during a specified period, such as at least the previous 6 hours, 12 hours, 24 hours, or the previous 48 hours. Spontaneous current surgical pain is assessed by asking the subject to select a number that best describes the level of surgical pain experienced by the subject at the time of assessment.

[0081] The NRS can also be used to assess a subject's perception of surgical pain in response to various stimuli. To assess the degree of perceived surgical pain in response to stimuli, subjects will receive stimuli of various properties applicable to the pain region. Subjects will be asked about their current NRS scores before and after administration.

[0082] Examples of stimuli used include: (i) light touch (which can be assessed by measuring pain on the surface of the pain area by applying the von Frey filament as described herein); (ii) pressure (pressure pain threshold), which can be assessed by asking the subject to give an NRS score as the pressure is increased using a pressure sensor, as described herein; and (iii) temperature (which can be assessed by asking the subject to give an NRS score for warm, cold, and hot stimulation by attaching a thermometer to the pain area), as described herein.

[0083] Preferably, when compared with the NRS scores of control patients who were not given Clostridium neurotoxin, the administration of Clostridium neurotoxin described herein reduced the postoperative NRS scores of patients (e.g., from a grade of ≥7 to a grade of ≤6).

[0084] Short Version of Quality of Life Questionnaire-36 (SF-36): The SF-36 Quality of Life Questionnaire can be used to assess a subject's perceived surgical pain. The SF-36 is a 36-item subject-reported health survey. The SF-36 consists of eight rating items (vitality, physical functioning, physical pain, general health, bodily role functioning, emotional role functioning, social role functioning, and mental health). Assuming equal weight for each item, each scale is directly converted to a 0-100 scale. Higher scores in the SF-36 indicate a lower degree of disability.

[0085] Relevant parameters for treating surgical pain that are commonly tested in clinical trials are known in the art and can be readily selected by those skilled in the art. Examples of such parameters include, but are not limited to, NRS; stimulus-induced NRS; temperature of the pain area; size of the pain area; time of onset of analgesia; peak analgesia; time to reach peak analgesia; duration of analgesia; and / or SF-36 quality of life as described herein. Methods for assessing these parameters are also known in the art and can be performed by those skilled in the art using conventional methods and procedures.

[0086] Preferably, when compared with the SF-36 scores of control patients who were not given Clostridium neurotoxin, the administration of Clostridium neurotoxin described herein increases the postoperative SF-36 score of patients (e.g., from a score ≤50 to a score ≥50).

[0087] Turning now to “postoperative anxiety,” references to the same content (i.e., postoperative anxiety) may refer to situations in which patients experience physical symptoms and behavioral changes, including, but not limited to, fatigue, difficulty concentrating and sleeping, and muscle tension. In addition, patients may experience emotional symptoms of anxiety, including irritability, irritability, difficulty controlling fear or worry, fear, and panic. Postoperative anxiety may be caused by the effects of anesthesia, the procedure itself, postoperative surgical pain and stress in the hospital environment.

[0088] Thus, in a specific embodiment, postoperative anxiety is caused by postoperative surgical pain.

[0089] Postoperative anxiety can be defined as panic disorder, phobia, post-traumatic stress disorder, social anxiety disorder (social phobia) or generalized anxiety disorder (GAD) (e.g., causing you to feel anxious about a wide range of situations and problems rather than anxiety about a specific event).

[0090] In a specific embodiment, the method of administering Clostridium neurotoxin to reduce or suppress postoperative anxiety or include administering Clostridium neurotoxin to a patient prior to surgery is administered more than 5 days prior to surgery, preferably wherein Clostridium neurotoxin is administered more than 5 days prior to surgery.

[0091] In a specific embodiment, administration of Clostridium neurotoxin substantially reduces the patient's postoperative anxiety perception, and wherein the reduced postoperative anxiety perception is maintained for 24 hours immediately after surgery. In one specific embodiment, substantially all reduced postoperative anxiety perception is maintained for 2 days immediately after surgery. In one specific embodiment, substantially all reduced postoperative anxiety perception is maintained for 3 days immediately after surgery. In one specific embodiment, substantially all reduced postoperative anxiety perception is maintained for 4 days immediately after surgery. In one specific embodiment, substantially all reduced postoperative anxiety perception is maintained for 5 days immediately after surgery. In one specific embodiment, substantially all reduced postoperative anxiety perception is maintained for 6 days immediately after surgery. In one specific embodiment, substantially all reduced postoperative anxiety perception is maintained for 7 days immediately after surgery. In one specific embodiment, substantially all reduced postoperative anxiety perception is maintained for 8 days immediately after surgery. Preferably, substantially all reduced postoperative anxiety perceptions were maintained for 9 days immediately after surgery.

[0092] Preferably, the administration of Clostridium neurotoxin described herein reduces symptoms of postsurgical anxiety (e.g., a 30%, 50%, 75%, or 95% reduction) when compared to control patients who were not administered Clostridium neurotoxin. Examples of symptoms of postoperative anxiety include irritability, irritability, difficulty controlling fear or worry, fear, and panic.

[0093] More specifically, the term "reduction" (in terms of postoperative anxiety) preferably means that subjects given clostridium neurotoxin (e.g., patients) perceive a lower level of postoperative anxiety compared to subjects who did not receive clostridium neurotoxin (or received a placebo) (who have also undergone surgery). For example, when compared to subjects who did not receive clostridium neurotoxin (or received a placebo) (who have also undergone surgery), administration of clostridium neurotoxin may reduce the perceived level of anxiety by at least 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, or 95%. For example, when compared to subjects who did not receive clostridium neurotoxin (or received a placebo) (who have also undergone surgery), administration of clostridium neurotoxin may reduce the perceived level of anxiety by at least 75%, preferably at least 85%, and more preferably at least 95%.

[0094] In one specific embodiment, postoperative anxiety experienced by the patient is suppressed within 24 hours after surgery. In other words, administration of Clostridium neurotoxin within 24 hours after surgery can reduce postoperative anxiety in patients. For example, postoperative anxiety can be reduced or suppressed within 2 hours, 4 hours, or 24 hours after surgery, preferably within 4 hours after surgery.

[0095] The inventors have demonstrated that Clostridium neurotoxin can be administered to treat postoperative pain and suppress postoperative anxiety. Thus, in one specific embodiment, Clostridium neurotoxin treats postoperative pain and reduces or suppresses postoperative anxiety.

[0096] In summary, by reducing the level of surgical pain and anxiety and other aspects perceived after surgery, the present invention improves the patient's quality of life and advantageously increases the patient's overall "postoperative health".

[0097] Thus, in one specific embodiment, administration of Clostridium neurotoxin promotes postoperative health.

[0098] Further details and technical background information regarding the Clostridium neurotoxins covered by this invention are provided below.

[0099] Bacteria in the genus *Clostridia* produce potent and specific protein toxins that can poison neurons and other cells to which they are delivered. Examples of such clostridia neurotoxins include neurotoxins produced by *C. tetani* (TeNT) and *C. botulinum* (BoNT) serotype AG, as well as those produced by *C. baratii* and *C. butyricum*.

[0100] Clostridium neurotoxins (e.g., in nature) cause muscle paralysis by inhibiting cholinergic transmission in the peripheral nervous system, particularly at the neuromuscular junction, and can therefore be fatal. In nature, Clostridium neurotoxins are synthesized as single-chain polypeptides, which are post-translational modified by protease cleavage events to form two polypeptide chains linked together by disulfide bonds. Cleavage occurs at a specific cleavage site, commonly referred to as the activation site, located between cysteine ​​residues that provide the inter-chain disulfide bond. This is the double-chain form, the most active form of the toxin. These two chains are called the heavy chain (H-chain), which has a molecular weight of approximately 100 kDa; and the light chain (L-chain), which has a molecular weight of approximately 50 kDa. This H-chain contains an N-translocation component (HN domain) and a C-targeting component (HC domain). The cleavage site is located between the L-chain and the HN domain.

[0101] The mechanism of action of Clostridium neurotoxin depends on five different steps: (1) binding of the HC domain to its target neuron, followed by (2) internalization of the bound toxin into the cell via the endosome, (3) translocation of the L-chain through the HN domain across the endosome membrane and into the cytosol, (4) proteolytic cleavage of intracellular transport proteins known as SNARE proteins by the L-chain, which provides non-cytotoxic protease function, and (5) inhibition of cellular secretion by the target cell.

[0102] Non-cytotoxic proteases act by 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 is derived from the term Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-Sensitive Factor. SNARE proteins are essential for intracellular vesicle fusion and therefore essential for the secretion of molecules transported from the cell via vesicles. This protease functions as a zinc-dependent endopeptidase and exhibits high receptor specificity for SNARE proteins. Therefore, once delivered to the target cell, this non-cytotoxic protease can inhibit the cellular secretion of the target cell. The L-chain protease of Clostridium neurotoxin is a non-cytotoxic protease that cleaves SNARE protein.

[0103] Due to their unique properties, Clostridium neurotoxins such as botulinum toxin have been successfully used in a wide range of therapeutic applications, particularly for motor and autonomic nervous system disorders, to restore, for example, the activity of overactive nerve endings to normal levels. At least seven antigenically distinct BoNT serotypes have been described, namely, BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, and BoNT / G (Rossetto, O. et al., "Botulinum neurotoxins: genetic, structural and mechanistic insights." Nature Reviews Microbiology 12.8 (2014): 535-549).

[0104] Despite this diversity, BoNT / A remains the serotype of choice for treatment, with three commonly used commercial formulations (Botox®, Dysport®, and Xeomin®), while only one BoNT / B product (Neurobloc® / Myobloc®) is available on the market. To date, these BoNT / A and BoNT / B products, which are toxins purified from Clostridium difficile strains, are the only two BoNT serotypes currently approved by regulatory agencies for human use, ranging from spasms, bladder dysfunction, or hyperhidrosis (in BoNT / A) (see, for example: https: / / www.medicines.org.uk / emc / medicine / 112, https: / / www.medicines.org.uk / emc / medicine / 870, https: / / www.medicines.org.uk / emc / medicine / 2162, all of which are incorporated herein by reference in their entirety) to cervical dystonia (in BoNT / B) (see, for example, https: / / www.medicines.org.uk / emc / medicine / 20568, all of which are incorporated herein by reference in their entirety).

[0105] In contrast to cytotoxic proteases that act by killing their natural target cells (e.g., ricin, diphtheria toxin, Pseudomonas exotoxin), Clostridium neurotoxins are non-cytotoxic proteases that act by temporarily incapacitating the cellular function of their natural target cells. Importantly, non-cytotoxic proteases do not kill the natural target cells during their action. Besides Clostridium neurotoxins (e.g., botulinum neurotoxin, marketed under trademarks such as Dysport™, Neurobloc™, and Botox™), some of the most well-known examples of non-cytotoxic proteases include IgA proteases (see, for example, WO99 / 032272) and antarease proteases (see, for example, WO2011 / 022357).

[0106] As used herein, the term "clostridium neurotoxin" refers to any polypeptide that enters neurons and inhibits the release of neurotransmitters. This process includes the binding of the neurotoxin to low- or high-affinity receptors, the internalization of the neurotoxin, the translocation of the endopeptidase portion of the neurotoxin into the cytoplasm, and the enzymatic modification of the neurotoxin receptor. More specifically, the term "neurotoxin" includes any polypeptide (clostridium neurotoxin) produced by Clostridium that enters neurons and inhibits the release of neurotransmitters, and such polypeptide is derived from recombinant or chemical techniques. Preferably, the clostridium neurotoxin is botulinum neurotoxin (BoNT).

[0107] BoNT serotypes A to G can be distinguished based on inactivation by specific neutralizing antisera, with this serotype classification being correlated with the percentage of sequence identity at the amino acid level. BoNT proteins of a given serotype are further subdivided into different subtypes based on the percentage of sequence identity at the amino acid level.

[0108] An example of the BoNT / A neurotoxin amino acid sequence is provided as SEQ ID NO: 1 (UniProt accession number A5HZZ9) and SEQ ID NO: 13, which is encoded by the nucleotide sequence provided as SEQ ID NO: 12. An example of the BoNT / B neurotoxin amino acid sequence is provided as SEQ ID NO: 2 (UniProt accession number B1INP5). The BoNT / C neurotoxin amino acid sequence is provided as SEQ ID NO: 3 (UniProt accession number P18640). An example of the BoNT / D neurotoxin amino acid sequence is provided as SEQ ID NO: 4 (UniProt accession number P19321). An example of the BoNT / E neurotoxin amino acid sequence is provided as SEQ ID NO: 5 (accession number WP_003372387). An example of the BoNT / F neurotoxin amino acid sequence is provided as SEQ ID NO: 6 (UniProt accession number Q57236) or SEQ ID NO: 9 (UniProt / UniParc accession number UPI0001DE3DAC). An example of the BoNT / G toxin amino acid sequence is provided as SEQ ID NO: 7 (accession number WP_039635782). An example of the BoNT / DC neurotoxin amino acid sequence is provided as SEQ ID NO: 8 (accession number BAM65681). An example of the BoNT / X neurotoxin amino acid sequence is provided as SEQ ID NO: 11 (accession number BAQ12790.1). Preferably, BoNT is BoNT / A, more preferably wild-type BoNT / A.

[0109] As used herein, the term "HC domain" refers to a functionally unique region of a neurotoxin heavy chain having a molecular weight of approximately 50 kDa, which enables the neurotoxin to bind to a receptor located on the surface of a target cell. The HC domain consists of two structurally unique subdomains, the "HCN subdomain" (the N-terminal portion of the HC domain) and the "HCC subdomain" (the C-terminal portion of the HC domain), each having a molecular weight of approximately 25 kDa. The HCC domain is capable of binding to Clostridium neurotoxin protein receptors.

[0110] As used herein, the term "LHN domain" refers to a neurotoxin region distinct from the HC domain, which consists of an endopeptidase domain ("L" or "light chain") and a domain responsible for transferring endopeptidase into the cytoplasm (the HN domain of the heavy chain). The endopeptidase domain ("L" or "light chain") is capable of cleaving SNARE proteins.

[0111] The L, HN, HCN and HCC domains are shown in Table 1.

[0112] Table 1 – Examples of L, HN, HCN and HCC domains BoNT Login ID SEQ ID NO: L H N H CN H CC BoNT / A1 A5HZZ9 1 1-448 449-872 873-1094 1095-1296 BoNT / B1 B1INP5 2 1-441 442-859 860-1081 1082-1​​291 BoNT / C1 P18640 3 1-449 450-867 868-1095 1096-1291 BoNT / D P19321 4 1-442 443-863 864-1082 1083-1276 BoNT / E1 WP_003372387 5 1-423 424-846 847-1069 1070 -1252 BoNT / F1 Q57236 6 1-439 440-865 866-1087 1088-1278 BoNT / F7 UPI0001DE3DAC 9 1-508 509-862 863-1076 1077-1268 BoNT / G WP_039635782 7 1-446 447-864 865-1089 1090-1297 BoNT / DC BAM65681 8 1-442 443-863 864-1091 1092-1285 BoNT / X BAQ12790.1 11 1-439 440-892 893-1306

[0113] The reference sequences identified above should be considered as guidelines, as slight variations may occur depending on the subserotype. For example, US 2007 / 0166332 (incorporated here in its entirety by reference) cites slightly different Clostridium sequences.

[0114] The term "activation zone" refers to a polypeptide domain containing a protease cleavage site. Activation zones of neurotoxins have been described in the art, such as WO2016156113 (which is incorporated herein by reference in its entirety).

[0115] In one specific embodiment, the Clostridium neurotoxin consists of or contains the following amino acid sequences, which have at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with any one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 11.

[0116] In one specific embodiment, the Clostridium neurotoxin consists of or contains the following amino acid sequences, which have at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity with SEQ ID NO: 1.

[0117] In one specific embodiment, the Clostridium neurotoxin consists of or contains the amino acid sequence of SEQ ID NO: 1 (e.g., BoNT / A).

[0118] In one specific embodiment, the Clostridium neurotoxin is a chimeric neurotoxin.

[0119] As used herein, the term "chimeric neurotoxin" means a neurotoxin comprising one or more domains derived from a first neurotoxin and one or more domains derived from a second neurotoxin. For example, a chimeric neurotoxin may comprise an LHN domain derived from a serotype or subtype of a first neurotoxin and an HC domain derived from a serotype or subtype of a second neurotoxin. Another example of a chimeric neurotoxin is a neurotoxin comprising an LHNHCN domain derived from a serotype or subtype of a first neurotoxin and an HCC domain derived from a serotype or subtype of a second neurotoxin. Yet another example of a chimeric neurotoxin is a neurotoxin comprising an LHN domain derived from a serotype or subtype of a first neurotoxin and an activation zone derived from a serotype or subtype of a second neurotoxin. Examples of chimeric neurotoxins are provided in WO2017191315 and WO2016156113, both of which are incorporated herein by reference in their entirety.

[0120] For example, the chimeric neurotoxin may include an LHN domain from a first neurotoxin covalently linked to an HC domain from a second neurotoxin, preferably wherein the first and second neurotoxins are different, wherein the C-terminal amino acid residue of the LHN domain corresponds to a first amino acid residue of the 310 helix separating the LHN and HC domains in the first neurotoxin, and wherein the N-terminal amino acid residue of the HC domain corresponds to a second amino acid residue of the 310 helix separating the LHN and HC domains in the second neurotoxin.

[0121] In one specific embodiment, the Clostridium neurotoxin is a chimeric neurotoxin comprising an HC domain derived from BoNT / B and an LHN domain derived from BoNT / A, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G.

[0122] For example, in one specific embodiment, the HC domain consists of or contains the following amino acid sequences, which correspond to amino acid residues 860 to 1291 of SEQ ID NO: 2 (e.g., BoNT / B), or have at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with them; and the LHN domain consists of or contains the following amino acid sequences, which are selected from the group consisting of: - amino acid residues 1 to 872 of SEQ ID NO: 1 (e.g., BoNT / A), or sequences having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with them, - amino acid residues 1 to 867 of SEQ ID NO: 3, or sequences having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with them, - SEQ ID NO: 2 - SEQ ID NO: 4 amino acid residues 1 to 863, or sequences having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith; - SEQ ID NO: 5 amino acid residues 1 to 846, or sequences having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith; - SEQ ID NO: 6 amino acid residues 1 to 865, or sequences having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith; - SEQ ID NO: 7 amino acid residues 1 to 864, or sequences having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith; - SEQ ID - Amino acid residues 1 to 863 of NO: 8, or sequences having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith, and - Amino acid residues 1 to 862 of SEQ ID NO: 9, or sequences having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity therewith.

[0123] In a preferred embodiment, the Clostridium neurotoxin is a chimeric neurotoxin comprising an HC domain derived from BoNT / B and an LHN domain derived from BoNT / A.

[0124] In a preferred embodiment, the HC domain is composed of or contains the following amino acid sequences, which correspond to amino acid residues 860 to 1291 of SEQ ID NO: 2, or have at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with them; and the LHN domain contains the amino acid sequences corresponding to amino acid residues 1 to 872 of SEQ ID NO: 1, or have at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with them.

[0125] In a specific embodiment, the Clostridium neurotoxin comprises an HC domain derived from BoNT / B (e.g., in the case of a Clostridium neurotoxin being BoNT / B, or a chimeric neurotoxin comprising an HC domain derived from BoNT / B). The Clostridium neurotoxin may have one or more modifications to the amino acid sequence of the heavy chain providing the "modified heavy chain" (e.g., in the HC domain). Preferably, the modified heavy chain binds to the target nerve cell with a higher (or lower) affinity than the native neurotoxin. Such modifications in the HC domain may include modifications to amino acid residues at the ganglioside binding site of the HCC domain, which may alter the binding of gangliosides to the target nerve cell, and / or modifications to amino acid residues at the protein receptor binding site of the HCC domain, which may alter the binding to the protein receptor of the target nerve cell. Examples of such modified neurotoxins are described in WO2006027207 and WO2006114308, both of which are incorporated herein by reference in their entirety.

[0126] In this article, Clostridium neurotoxins with one or more modifications to the amino acid sequence of the heavy chain are referred to as "modified Clostridium neurotoxins".

[0127] When compared with the natural HCC domain of the BoNT serotype, in a specific embodiment of the modified Clostridium neurotoxin according to the present invention, the HCC domain derived from BoNT / B is modified.

[0128] In a preferred embodiment, the HCC domain derived from the BoNT / B neurotoxin contains a mutation in at least one amino acid residue that, compared to the native BoNT / B HCC domain, increases the binding affinity of the HCC domain to human Syt II. Furthermore, preferably, compared to the native BoNT / B HCC domain, this mutation in at least one amino acid residue increases the binding affinity of the HCC domain to human Syt II by at least 50%.

[0129] Such suitable amino acid residue mutations in the BoNT / B HCC domain have been described in WO2013180799 and WO2016154534 in this field of art, both of which are incorporated herein by reference in their entirety.

[0130] In particular, when compared with the natural BoNT / B HCC domain, the mutation of at least one amino acid residue suitable for increasing the binding affinity of the BoNT / B HCC domain to human Syt II by at least 50% is a substitution, addition or deletion of an amino acid residue selected from the group consisting of: 1118M, 1183M, 1191M, 1191I, 1191Q, 1191T, 1199Y, 1199F, 1199L, 1201V, 1191C, 1191V, 1191L, 1191Y, 1199W, 1199E, 1199H, 1178Y, 1178Q, 1178A, 1178S, 1183C, 1183P and any combination thereof. Preferably, the mutation of at least one amino acid residue in the BoNT / B HCC domain is composed of substitution, addition or deletion of two amino acid residues selected from the group consisting of: 1191M and 1199L, 1191M and 1199Y, 1191M and 1199F, 1191Q and 1199L, 1191Q and 1199Y, 1191Q and 1199F, 1191M and 1199W, 1191M and 1178Q, 1191C and 1199W, 1191C and 1199Y, 1191C and 1178Q, 1191Q and 1199W, 1191V and 1199W, 1191V and 1199Y, or 1191V and 1178Q. More preferably, the mutation of at least one amino acid residue in the BoNT / B HCC domain is composed of the substitution, addition, or deletion of three amino acid residues: 1191M, 1199W, and 1178Q. More preferably, the mutation of at least one amino acid residue in the BoNT / B HCC domain is composed of the substitution, addition, or deletion of two amino acid residues: 1191M and 1199Y.

[0131] In a preferred embodiment, when compared with the natural BoNT / B HCC domain, the mutation of at least one amino acid residue suitable for increasing the binding affinity of the BoNT / B HCC domain to human Syt II by at least 50% is a substitution, addition, or deletion of an amino acid residue selected from the group consisting of: V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, and any combination thereof. Preferably, at BoNT / B The mutation of at least one amino acid residue in the HCC domain consists of the substitution of two amino acid residues selected from the group consisting of: E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q. Furthermore, preferably, the mutation of at least one amino acid residue in the BoNT / B HCC domain is composed of substitutions of three amino acid residues: E1191M, S1199W, and W1178Q. More preferably, the mutation of at least one amino acid residue in the BoNT / B HCC domain is composed of substitutions of two amino acid residues: E1191M and S1199Y.

[0132] In a preferred embodiment, the BoNT / B HCC domain to be modified corresponds to amino acid residues 1082 to 1291 of SEQ ID NO: 2 (the natural BoNT / B HCC domain), or corresponds to an amino acid sequence having at least 70%, preferably at least 80%, 85%, 90%, 95% or 99% sequence identity with it.

[0133] In one specific embodiment, as described above, the Clostridium neurotoxin of the present invention may be both chimeric and modified. For example, in a preferred embodiment, the Clostridium neurotoxin comprises (or consists of) the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with it.

[0134] In one specific embodiment, as described above, the Clostridium neurotoxin of the present invention may be both chimeric and modified. For example, in a preferred embodiment, the Clostridium neurotoxin comprises (or consists of) the amino acid sequence of SEQ ID NO: 10 (e.g., BoNT / ABMY), or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with it.

[0135] The Clostridium neurotoxin of the present invention can be produced using recombinant technology. Thus, in one specific embodiment, the Clostridium neurotoxin of the present invention is a recombinant Clostridium neurotoxin.

[0136] The use of such recombinant neurotoxins can advantageously expand the selection of Clostridium neurotoxins for use in the methods described herein, for example, based on properties such as potency and duration of action deemed suitable for any given procedure. Suitable (known) recombinant Clostridium neurotoxins include modified botulinum neurotoxin A (BoNT / A) that preferably has a longer duration of action when compared with unmodified BoNT / A (e.g., Dysport®). This duration of action can be at least greater than 1.25x, 1.5x, 1.75x, 2.0x, or 2.25x. The duration of action of modified BoNT / A can be between 6 and 9 months. For example, the duration of action can be at least: 4.5 months (from start), 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months, or 9.0 months.

[0137] Suitable modified BoNT / A peptides (and the nucleotide sequences encoding them, in the presence) are described in WO 2015 / 004461 A1 and WO 2017 / 191315, both of which are incorporated herein by reference in their entirety.

[0138] More specifically, in one particular embodiment, the Clostridium neurotoxin is a modified recombinant BoNT / A neurotoxin. In one specific embodiment, the modified BoNT / A comprises modification with one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1215, GLN 1216, GLN 1229, GLN 1216, GLN 1218 ... 1242, ASN 1243, SER 1274, and THR 1277, preferably wherein the modification is selected from: (i) replacing an amino acid residue exposed on an acidic surface with a basic amino acid residue; (ii) replacing an amino acid residue exposed on an acidic surface with an uncharged amino acid residue; (iii) replacing an amino acid residue exposed on an uncharged surface with a basic amino acid residue; (iv) inserting a basic amino acid residue; and (v) deleting an amino acid residue exposed on an acidic surface.

[0139] When compared with the unmodified BoNT / A shown in SEQ ID NO: 1, the modification may be a modification in which the amino acid residue number is determined by comparison with SEQ ID NO: 1. Since the methionine residue at position 1 of SEQ ID NO: 1 (and the SEQ ID NO corresponding to the modified BoNT / A polypeptide described herein) is optional, when determining the amino acid residue number, those skilled in the art will consider the presence / absence of the methionine residue. For example, if SEQ ID NO: 1 includes methionine, the position number will be as defined above (e.g., ASN 886 will be ASN 886 of SEQ ID NO: 1). Alternatively, if methionine is not present in SEQ ID NO: 1, the amino acid residue number should be modified by -1 (e.g., ASN 886 will be ASN 885 of SEQ ID NO: 1). When methionine is present or absent at position 1 of other polypeptide sequences described herein, similar considerations apply, and those skilled in the art will readily determine the correct amino acid residue number using conventional techniques in the art. This also applies to any other BoNTs described herein (e.g., the chimeric BoNTs described above).

[0140] The amino acid residue referred to in the modification is a surface-exposed amino acid residue.

[0141] The modified BoNT / A may be encoded by a nucleic acid sequence having at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 14, 16, 18, and 20. For example, a nucleic acid sequence having at least 80%, 90%, 95%, or 99.9% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 14, 16, 18, and 20. Preferably, the modified BoNT / A used in this invention may be encoded by a nucleic acid sequence comprising (or consisting of) SEQ ID NOs: 14, 16, 18, or 20. The modified BoNT / A may comprise a polypeptide sequence having at least 70% sequence identity with a polypeptide sequence selected from SEQ ID NOs: 15, 17, 19, and 21. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity with a polypeptide sequence selected from SEQ ID NOs: 15, 17, 19, and 21. Preferably, the modified BoNT / A used in this invention may contain (more preferably consist of) a polypeptide sequence selected from SEQ ID NO: 15, 17, 19, and 21.

[0142] When used in the context of modified BoNT / A, the term "one or more amino acid residues" preferably means at least 2, 3, 4, 5, 6, or 7 of the referred amino acid residues. Thus, the modified BoNT / A may contain at least 2, 3, 4, 5, 6, or 7 (preferably 7) modifications to the referred amino acid residues. The modified BoNT / A may contain 1-30, 3-20, or 5-10 amino acid modifications. More preferably, when used in the context of modified BoNT / A, the term "one or more amino acid residues" means all of the referred amino acid residues.

[0143] Preferably, apart from one or more amino acid modifications at the indicated amino acid residues, the modified BoNT / A does not contain any further amino acid modifications when compared with SEQ ID NO: 1.

[0144] Preferably, the modified BoNT / A comprises (more preferably consists of) a modification located at one or more amino acid residues selected from the following: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, and GLN 1229. The modified BoNT / A may be encoded by a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO:14. For example, a nucleic acid sequence having at least 80%, 90%, 95%, or 99.9% sequence identity with SEQ ID NO:14. More preferably, the modified BoNT / A used in this invention may be encoded by a nucleic acid that comprises (or consists of) SEQ ID NO:14. The modified BoNT / A may comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:15. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity with SEQ ID NO: 15. Preferably, the modified BoNT / A used in this invention may comprise (more preferably consist of) SEQ ID NO: 15.

[0145] This modification may be selected from: (i) replacing an amino acid residue exposed on an acidic surface with a basic amino acid residue; (ii) replacing an amino acid residue exposed on an acidic surface with an uncharged amino acid residue; (iii) replacing an amino acid residue exposed on an uncharged surface with a basic amino acid residue; (iv) inserting a basic amino acid residue; and (v) deleting an amino acid residue exposed on an acidic surface.

[0146] The modifications mentioned above result in the modified BoNT / A having an increased positive surface charge and an increased isoelectric point when compared with the corresponding unmodified BoNT / A.

[0147] The isoelectric point (pI) is a characteristic of a specified protein. More specifically, the isoelectric point (pI) is defined as the pH value at which a protein exhibits a net charge of zero. An increase in pI means that a higher pH value is required for a protein to exhibit a net charge of zero. Thus, an increase in pI represents an increase in the net positive charge of the protein at a given pH. Conversely, a decrease in pI means that a lower pH value is required for a protein to exhibit a net charge of zero. Thus, a decrease in pI represents a decrease in the net positive charge of the protein at a given pH.

[0148] Methods for determining the pI of proteins are known in the art and are familiar to those skilled in the art. For example, the pI of a protein can be calculated from the average pKa value of the amino acids present in the protein ("calculated pI"). This calculation can be performed using computer programs known in the art, such as the Compute pI / MW Tool from ExPASy (https: / / web.expasy.org / compute_pi / ), which is a preferred method for calculating pI according to the present invention. Comparisons of pI values ​​between different molecules should be performed using the same calculation technique / program. Where appropriate, the calculated pI of a protein can be confirmed using isoelectric focusing ("observed pI"). This technique uses electrophoresis to separate proteins based on their pI. Isoelectric focusing is generally performed using a colloid with a fixed pH gradient. When an electric field is applied, the protein moves along the pH gradient until it reaches its pH with zero net charge, which is the pI of the protein. The results provided by the isoelectric focal aggregation method are generally of relatively low resolution, so the inventors believe that the results provided by the calculated pI (as described above) are more applicable.

[0149] Unless otherwise stated, throughout this specification, "pI" means "calculated pI". The pI of a protein can be increased or decreased by changing the number of basic and / or acidic groups displayed on the protein surface. This can be achieved by modifying one or more amino acids of the protein. For example, an increase in pI can be provided by reducing the number of acidic residues or by increasing the number of basic residues.

[0150] The modified BoNT / A of the present invention may have a pI value that is at least 0.2, 0.4, 0.5 or 1 pI unit higher than that of the unmodified BoNT / A (e.g., SEQ ID NO: 1). Preferably, the modified BoNT / A may have a pI of at least 6.6, for example, at least 6.8 pI.

[0151] The following table shows the properties of 20 standard amino acids: amino acids side chain amino acids side chain Aspartic acid Asp D Charged (acidic) Methionine Met M Uncharged (polarity) glutamic acid Glu E Charged (acidic) tryptophan Trp W Uncharged (polarity) Arginine Arg R Charged (alkaline) Cysteine Cys C Uncharged (polarity) lysine Lys K Charged (alkaline) alanine Ala A Uncharged (hydrophobic) histidine His H Uncharged (polarity) Glycine Gly G Uncharged (hydrophobic) aspartic acid Asn N Uncharged (polarity) Valine Val V Uncharged (hydrophobic) glutamic acid Gln Q Uncharged (polarity) Leucine Leu L Uncharged (hydrophobic) serine Ser S Uncharged (polarity) Isoleucine Ile I Uncharged (hydrophobic) threonine Thr T Uncharged (polarity) proline Pro P Uncharged (hydrophobic) Tyrosine Tyr Y Uncharged (polarity) Phenylan Phe F Uncharged (hydrophobic)

[0152] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0153] At pH 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) have a negative charge, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) have a positive charge. Aspartic acid and glutamic acid are referred to as acidic amino acid residues. Arginine and lysine are referred to as basic amino acid residues.

[0154] The following amino acids are considered to be non-charged, polar (meaning they can participate in hydrogen bonding) amino acids: aspartic acid, glutamic acid, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan. The following amino acids are considered to be non-charged, hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0155] In the case of amino acid insertion, additional amino acid residues (not normally present) are incorporated into the BoNT / A polypeptide sequence, thus increasing the total number of amino acid residues in the sequence. In the case of amino acid deletion, amino acid residues are deleted from the Clostridium toxin amino acid sequence, thus reducing the total number of amino acid residues in the sequence.

[0156] Preferably, the modification is a substitution that advantageously maintains the same number of amino acid residues in the modified BoNT / A. In the amino acid substitution, the amino acid residues forming part of the BoNT / A polypeptide sequence are replaced with different amino acid residues. The substituted amino acid residues may be one of the 20 standard amino acids mentioned above. Alternatively, in the amino acid substitution, the substituted amino acid may be a non-standard amino acid (an amino acid not part of the 20 standard groups mentioned above). For example, the substituted amino acid may be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid, or lysine, arginine, and the D-isomer of ornithine. Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using an auxotrophic host of *E. coli*.

[0157] In one specific embodiment, the substitution is selected from: replacing an acidic amino acid residue with a basic amino acid residue, replacing an acidic amino acid residue with an uncharged amino acid residue, and replacing an uncharged amino acid residue with a basic amino acid residue. In one specific embodiment, the substitution is replacing an acidic amino acid residue with an uncharged amino acid residue, and the acidic amino acid residue is replaced by its corresponding uncharged amide amino acid residue (i.e., aspartic acid is replaced by aspartic acid, and glutamic acid is replaced by glutamic acid).

[0158] Preferably, the basic amino acid residue is a lysine residue or an arginine residue. In other words, the substitution is a substitution with lysine or arginine. Most preferably, the modification is a substitution with lysine.

[0159] According to the present invention, after modification, the modified BoNT / A can bind to the target cell receptor bound by the unmodified BoNT / A (e.g., SEQ ID NO: 1).

[0160] The previously described suitable modified (recombinant) BoNT / A neurotoxins, for example, having a longer period of action, are followed by a description of suitable modified (recombinant) BoNT / E neurotoxins, which may act relatively quickly and / or have a shorter period of action. This again demonstrates the advantageous flexibility offered by Clostridium neurotoxins based on the therapies of the present invention. For example, for less invasive procedures (e.g., where postoperative pain is not expected to last long), such BoNT / E can be used to provide a shorter period of action.

[0161] BoNT / E (e.g., rBoNT / E) may contain a polypeptide sequence having at least 70% (preferably at least 80%; more preferably at least 90%) sequence identity with SEQ ID NO: 5, wherein the polypeptide sequence comprises one or more (e.g., one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight; preferably all eight) of the following amino acids (wherein the amino acid position numbering begins with the N-terminal methionine amino acid residue of the BoNT / E protein and ends with the C-terminal amino acid residue): glycine at position 177; serine at position 198; alanine at position 340; leucine at position 773; leucine at position 963; glutamine at position 964; alanine at position 967; and aspartic acid at position 1195.

[0162] The amino acid may be a substitution (e.g., a mutation) associated with the wild-type BoNT / E polypeptide sequence (such as the sequence of UniProt Q00496). For example: glycine at position 177 may be replaced by arginine to glycine (R177G); serine at position 198 may be replaced by C198S; alanine at position 340 may be replaced by R340A; leucine at position 773 may be replaced by I173L; leucine at position 963 may be replaced by F963L; glutamic acid at position 964 may be replaced by E964Q; alanine at position 967 may be replaced by R967A; and / or aspartic acid at position 1195 may be an insertion (e.g., between G1194 and N1195 in the polypeptide sequence of the wild-type BoNT / E sequence such as UniProt Q00496).

[0163] In one specific embodiment, as described above, the presence of one or more amino acids provides a BoNT / E protein with improved solubility compared to a BoNT / E protein lacking that amino acid. This improved solubility increases the yield of the protein in a heterologous expression system (such as an E. coli expression system).

[0164] In one specific embodiment, as described above, the presence of the one or more amino acids provides a BoNT / E protein with improved potency compared to a BoNT / E protein lacking the amino acid. This improved potency may preferably be improved in vivo potency (more preferably, improved in vivo potency in human subjects).

[0165] In a specific embodiment, BoNT / E is described in WO 2014 / 068317 A1 (or encoded by the nucleotide sequence described in WO 2014 / 068317), which is incorporated herein by reference.

[0166] Preferably, the Clostridium neurotoxin (e.g., as described herein) is part of a pharmaceutical composition together with at least one pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means any component compatible with other components of the pharmaceutical composition (particularly with the Clostridium neurotoxin) and not harmful to human patients. According to standard pharmaceutical practice, pharmaceutically acceptable carriers may be selected based on the desired route of administration, including, but not limited to, excipients, diluents, adjuvants, propellants, and salts.

[0167] Therefore, the present invention further relates to a pharmaceutical composition for use in treating postoperative surgical pain and / or anxiety in human patients, wherein the composition comprises the Clostridium neurotoxin of the present invention and at least one pharmaceutically acceptable carrier, and the dosage of the Clostridium neurotoxin administered to the patient is as described above. It is also encompassed as a use and method corresponding to the treatment of postoperative surgical pain and / or anxiety, comprising administering the pharmaceutical composition of the present invention to a human patient. In another specific embodiment, the present invention relates to a pharmaceutical composition for use in promoting postoperative health, wherein postoperative health is the reduction of postoperative surgical pain and anxiety.

[0168] The Clostridium neurotoxin of the present invention can be preferably formulated for intradermal administration.

[0169] The preferred route of administration is intradermal administration. Preferably, intradermal administration means intradermal injection.

[0170] Preferably, the BoNT used to treat postoperative surgical pain and / or postoperative anxiety is purified BoNT. As used herein, the term "purified BoNT" means purified from naturally occurring Clostridium strains (naturally occurring Clostridium strains) or botulinum neurotoxin purified using recombinant technology. Purified BoNT / A may or may not contain a complex protein, but preferably does not contain a complex protein. Thus, in one specific embodiment, the Clostridium neurotoxin is associated with a BoNT complex protein, also referred to as non-toxic neurotoxin-associated proteins (NAP). In other words, the Clostridium neurotoxin is associated with or combined with the BoNT complex protein and administered to a human patient. Therefore, in one specific embodiment, the Clostridium neurotoxin is combined with one or more BoNT complex proteins. Commercially available purified and complex protein-associated BoNT / A products include Botox®, Dysport® (associated with BoNT complex protein), and Xeomin® (purified).

[0171] In another specific embodiment, the Clostridium neurotoxin does not contain the BoNT complex protein (or is not associated with or combined with the BoNT complex protein). In other words, the Clostridium neurotoxin is administered to human patients without being associated with or combined with the BoNT complex protein.

[0172] The dosage of Clostridium neurotoxin can be measured by nanogram.

[0173] The dosage of the Clostridium difficile neurotoxin according to the present invention should be understood as the dosage of the active double-stranded Clostridium difficile neurotoxin, i.e., excluding the amount of complex protein that may associate with the neurotoxin. In other words, it refers to the dosage of the active double-stranded Clostridium difficile neurotoxin, regardless of whether the neurotoxin administered to the patient associates with or does not associate with the complex protein. As is well known to those skilled in the art, the active double-stranded Clostridium difficile neurotoxin can bind to membrane (e.g., cell membrane) receptors, transfer light chains into the cytoplasm, and cleave SNARE proteins, while the complex protein does not exhibit this biological activity (i.e., not "active").

[0174] Alternatively or concurrently, the dose of Clostridium neurotoxin may be measured in "units" (U) of Clostridium neurotoxin. For example, unit-based dose measurement may be particularly suitable when administering BoNT / A (or more specifically, for example, Dysport®).

[0175] Indeed, as is well known to those skilled in the art, the potency of Clostridium neurotoxins is related to the amount of neurotoxin (e.g., nac) required to achieve an LD50 (lethal dose of 50) unit; one LD50 unit is defined as the intraperitoneal median lethal dose (as measured in mice). However, currently marketed BoNT pharmaceutical preparations contain varying amounts of 150 kD neurotoxin and also varying amounts of LD50 units. Furthermore, in these preparations, the neurotoxin may or may not associate with (i.e., combine with) a nontoxic neurotoxin-associated protein (NAP), also known as a complex protein. For ease of conversion (as reported in Field et al., "Abobotulinumtoxin A (Dysport®), Onabotulinumtoxin A (Botox®), and Incobotulinumtoxin A (Xeomin®) Neurotoxin Content and Potential Implications for Duration of Response in Patients"). Toxins 2018, 10(12), 535): - 100 units of Botox® (also known as Onabotulinumtoxin A) contain approximately 0.9 ng of 150 kD BoNT / A and a complex protein; - 500 units of Dysport® (also known as Abobotulinumtoxin A) contain approximately 2.69 ng of 150 kD BoNT / A and a complex protein; 1 unit of Dysport® contains approximately 5.38 pg BoNT / A; - 100 units of Xeomin® (also known as Incobotulinumtoxin A) contain approximately 0.40 ng of 150 kD BoNT / A and do not contain complex proteins.

[0176] It should be noted that the conversion value may vary slightly. For example, the conversion values ​​reported in Frevert, 2012 (“Content of botulinum neurotoxin in Botox® / Vistabel®, Dysport® / Azzalure®, and Xeomin® / Bocouture®”; Drugs R D. 2010;10(2):67-73) are as follows: - 100 units of Botox® (also known as Onabotulinumtoxin A) contain approximately 0.73 ng of 150 kD BoNT / A and complex protein; - 100 units of Dysport® (also known as Abobotulinumtoxin A) contain approximately 0.65 ng of 150 kD BoNT / A and complex protein; - 100 units of Xeomin® (also known as Incobotulinumtoxin A) contain approximately 0.44 ng of 150 kD BoNT / A and do not contain complex protein; - 100 units of Neurobloc / Myobloc® (also known as RimabotulinumtoxinB) contain approximately 0.2 ng to approximately 1 ng of 150 kD BoNT / B, as well as complex protein.

[0177] The amount of Clostridium neurotoxin can be quantified by those skilled in the art using methods commonly used in the art, preferably at the nanometer level, including mass spectrometry, such as isotope dilution mass spectrometry (Muñoz et al., Quantification of protein calibrants by amino acid analysis using isotope dilution mass spectrometry, Anal. Biochem. 2011, 408, 124–131), or fluorescence analysis (Poras et al., Detection and Quantification of Botulinum Neurotoxin Type A by a Novel Rapid In Vitro Fluorimetric Assay, Appl Environ Microbiol. 2009 Jul; 75(13): 4382-4390).

[0178] Intradermal administration may involve intradermal injection with a needle such as a 30-gauge needle, preferably with the needle (such as a 30-gauge needle) inserted into the dermis of the skin at an angle of approximately 5°-15° relative to the skin surface at the surgical intervention site (which may be ventral). The injection depth (relative to the skin surface) may be approximately 0.2-0.3 (preferably approximately 0.25) inches.

[0179] Clostridium neurotoxin can be administered to the site of surgical intervention in the body (e.g., a surgical incision, or the proximal part of the surgical incision site).

[0180] In one specific embodiment, the Clostridium neurotoxin can be administered at the patient's surgical intervention site (e.g., at one or more administration sites at the patient's surgical intervention site) (e.g., via intradermal or intrathecal injection).

[0181] Clostridium neurotoxin may be administered at one or more sites, such as at one or more sites near the surgical intervention site. A site "near the surgical intervention site" may be at most 15 cm from the surgical intervention site; for example, at most 10 cm from the surgical intervention site; preferably at most 5 cm from the surgical intervention site; more preferably at most 1 cm from the surgical intervention site.

[0182] In one specific embodiment, after administration, Clostridium neurotoxin is transported to the spinal cord via reverse transport and affects SNARE protein cleavage (SNAP-25 protein cleavage) in the spinal cord.

[0183] In one specific embodiment, when Clostridium neurotoxin is administered intradermally, minimal or no SNARE protein cleavage (SNAP-25 protein cleavage) caused by the Clostridium neurotoxin is observed at or near the intradermal site after administration of the Clostridium neurotoxin. In one specific embodiment, observation is performed 5-7 days after administration of the Clostridium neurotoxin, and minimal or no SNARE protein cleavage (SNAP-25 protein cleavage) caused by the Clostridium neurotoxin is observed at or near the intradermal site after administration of the Clostridium neurotoxin.

[0184] In one specific embodiment, when Clostridium neurotoxin is administered intrasheath, minimal or no SNARE protein cleavage (SNAP-25 cleavage) caused by the Clostridium neurotoxin is observed at or near the intrasheath site after administration of the Clostridium neurotoxin. In one specific embodiment, observation is performed 5-7 days after administration of the Clostridium neurotoxin, and minimal or no SNARE protein cleavage (SNAP-25 cleavage) caused by the Clostridium neurotoxin is observed at or near the intrasheath site after administration of the Clostridium neurotoxin.

[0185] Thus, Clostridium neurotoxin can be administered distal to the surgical intervention site to treat postoperative pain and postoperative anxiety.

[0186] Thus, in a preferred embodiment, when postoperative pain is caused by surgical intervention, Clostridium neurotoxin may be administered distal to the surgical intervention site (e.g., at one or more administration sites distal to the patient's incision site) (e.g., via intradermal or intrathecal injection).

[0187] Clostridium neurotoxin may be administered at one or more sites distal to the surgical intervention site, for example at a site at least 15 cm from the surgical intervention site; at a site at least 50 cm from the surgical intervention site; or at a site at least 100 cm from the surgical intervention site.

[0188] Those skilled in the art will understand that this invention is directed to preoperative administration. The phrase "at or near the surgical intervention site" means administration at or near the site where the surgical intervention will be performed (e.g., subsequently) once the surgery has begun. The phrase "at a site distal to the surgical intervention site" means administration distal to the site where the surgical intervention will be performed (e.g., subsequently) once the surgery has begun.

[0189] Clostridium neurotoxin can be administered at up to 15 sites (preferably up to 10) (e.g., sites near the surgical intervention site). Such sites may extend across the periphery of the surgical intervention site.

[0190] In a preferred embodiment, the dose (i.e., therapeutic dose) of the Clostridium neurotoxin of the present invention administered to human patients for the treatment of surgical pain is in the range of about 0.00025 ng to about 3 ng.

[0191] In a preferred embodiment, the therapeutic dose of the Clostridium neurotoxin is in the range of about 0.0003 ng to about 2 ng, more preferably about 0.0004 ng to about 1.5 ng, about 0.0005 ng to about 1 ng, and even more preferably about 0.0006 ng to about 0.5 ng of the Clostridium neurotoxin.

[0192] For example, the dose (e.g., total dose) of the Clostridium neurotoxin containing BoNT / A is preferably in the range of about 1 ng to about 2 ng.

[0193] Patients may be administered 100-500 U of Clostridium neurotoxin. For example, patients may be administered 150-300 U of Clostridium neurotoxin; preferably 175-250 U; more preferably about 200 U.

[0194] Patients may be administered 80-250 picograms (pg) of Clostridium neurotoxin per kilogram of patient body weight (e.g., 850-250 pg / kg). For example, patients may be administered 100-200 pg / kg, 115-175 pg / kg, or 130-150 pg / kg.

[0195] As described above, Clostridium neurotoxin can be administered at one or more sites, for example, at more than one administration site. In one specific embodiment, the patient is administered 2.5-30 U of Clostridium neurotoxin at each administration site; preferably, the patient is administered 20 U of Clostridium neurotoxin at each administration site. For example, 10 administration sites may receive an administration of 20 U at each site, providing a total administration of 200 U.

[0196] Clostridium neurotoxin can be administered to the patient at a total dose of 10-170 pg per administration site. In a preferred embodiment, Clostridium neurotoxin can be administered to the patient at a dose of 1-14 pg / kg (body weight) per administration site.

[0197] In another specific embodiment, the therapeutic dose of Clostridium neurotoxin preferably ranges from about 0.001 ng to about 2 ng. Also, for example, the therapeutic dose of Clostridium neurotoxin preferably ranges from about 0.0003 ng to about 0.05 ng.

[0198] Nevertheless, it should be understood that the required dose range depends on the exact nature of the Clostridium neurotoxin, the maximum tolerated dose for a particular subject (e.g., a human subject), skin condition, route of administration, nature of the formulation, patient age, patient weight, nature, extent or severity of the patient's condition, contraindications (if any), and the judgment of the attending physician. Standard empirical practices used for optimization can be employed to adjust for variations in these dose ranges.

[0199] In one specific embodiment, a patient is given a monotherapy based on a single botulinum neurotoxin serotype (e.g., BoNT / A). Thus, in one specific embodiment, the present invention utilizes the use of a single botulinum neurotoxin serotype (e.g., BoNT / A).

[0200] The specific embodiments relating to the various methods of the present invention are intended to be equally applicable to other methods, Clostridium neurotoxins, such as engineered Clostridium neurotoxins (whether in single-stranded or double-stranded form), uses or pharmaceutical compositions, and vice versa.

[0201] Sequence homology

[0202] A variety of sequence alignment methods can be used to determine the percentage of agreement, including but not limited to global methods, local methods, and hybrid methods, such as, for example, the segment approach method. The experimental procedure for determining the percentage of agreement is a routine procedure within the scope of ordinary knowledge in the art. The global method aligns the sequence from the beginning to the end of the molecule and determines the best alignment by accumulating the scores of each residue pair and by applying gap penalties. Non-limiting methods include, for example, CLUSTAL W, see, for example, Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight MatrixChoice, 22(22) Nucleic Acids Research 4673-4680 (1994); and its iterative refinement, see, for example, Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI.Biol.823-838 (1996). Local methods align sequences by identifying one or more conserved motifs common to all input sequences.Non-restrictive methods include, for example, Match-box, see, for example, 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, for example, CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214(1993); Align-M, see, for example, Ivo Van Waille et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435(2004).

[0203] Thus, the percentage of sequence identity is determined using 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. In short, the two amino acid sequences are aligned to optimize the alignment score 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 represented by standard single-letter codes).

[0204] The "percentage of sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of common positions in the sequences. Thus, the identity % can be calculated as follows: the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids multiplied by 100. The calculation of the sequence identity % can also take into account the number of gaps introduced to optimize the alignment of two or more sequences, and the length of each gap. The sequence comparison and determination of the percentage of identity between two or more sequences can be performed using specific mathematical algorithms, such as BLAST, which are familiar to those with ordinary technical knowledge.

[0205] Alignment score used to determine sequence identity

[0206] Then, the percentage of identity is calculated as follows:

[0207] Essentially homologous peptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor, i.e., retained amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the peptide; small deletions, typically removing 1 to about 30 amino acids; and small amino or carboxyl terminal extensions, such as amino-terminal methionine residues, small linker peptides of up to about 20-25 residues, or affinity tags.

[0208] Retained amino acid substitutions alkaline: Arginine; Lysine; Histamine Acidity: glutamic acid; aspartic acid polarity: Glutamic acid; Aspartic acid Hydrophobicity: Leucine; Isoleucine; Valine Aromatics: Phenylalanine; Tryptophan; Tyrosine Small: Glycine; Alanine; Serine; Threonine; Methionine

[0209] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovalinic acid, and α-methylserine) can be used to replace the amino acid residues of the peptides of the present invention. A limited number of non-reserved amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can replace the amino acid residues of the peptides. The peptides of the present invention may also contain non-naturally occurring amino acid residues.

[0210] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxyethylcysteine, hydroxyethylhomo-cysteine, nitroglutamine, homoglutamine, 2-piperidinecarboxylic acid, tertiary leucine, n-valine, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in this art. For example, an in vitro system can be used in which a chemically aminolated suppressor tRNA is used to suppress nonsense mutations. Methods for synthesizing amino acids and aminolated tRNA are known in the art. Transcription and translation of plastids containing nonsense mutations are carried out in a cell-free system containing E. coli S30 extract and commercially available enzymes and other reagents. Proteins can be 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 the second method, translation occurs in Xenopus oocytes via microinjection of mutated mRNA and chemically aminolated repressed tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, *E. coli* cells are cultured in the absence of the desired natural amino acid (e.g., phenylalanine) and in the presence of the desired non-natural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-natural amino acid is incorporated into the polypeptide to replace its natural counterpart. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted into non-natural types through 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).

[0211] A limited number of non-reserved amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and non-natural amino acids may replace the amino acid residues of the polypeptides of the present invention.

[0212] The essential amino acids in the polypeptides of this 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 structural physics analysis, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations in the presumed 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 identity of essential amino acids can also be inferred from homology analysis of related components (e.g., translocation or protease components) of the polypeptide of the present invention.

[0213] Known mutagenesis and screening methods, 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), can be used to test multiple amino acid substitutions. In short, these authors disclose methods for simultaneously randomizing two or more positions in a peptide, selecting functional peptides, and then sequentially mutagenesizing the peptide to determine the extent of substitution allowed 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., US 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).

[0214] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide a general dictionary of the many terms used in this disclosure for those of ordinary skill in the art.

[0215] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of specific embodiments of this disclosure. Numerical ranges include numbers defining the range. Unless otherwise stated, any nucleic acid sequence is written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an amino to carboxyl direction.

[0216] The headings provided herein are not intended to limit the various forms or specific embodiments disclosed herein.

[0217] Amino acids are referred to herein by their names, three-letter abbreviations, or single-letter abbreviations. The term "protein" as used herein includes proteins, polypeptides, and peptides. The term "amino acid sequence" as used herein is synonymous with the terms "polypeptide" and / or "protein." In some cases, the term "amino acid sequence" is synonymous with the term "peptide." In some cases, the term "amino acid sequence" is synonymous with the term "enzyme." The terms "protein" and "polypeptide" are used interchangeably herein. Common one-letter and three-letter codes for amino acid residues may be used within the scope of this disclosure and the claims. The three-letter codes for amino acids are defined in accordance with the Joint Commission on Biochemical Nomenclature (JCBN) of IUPACIUB. It should also be understood that, due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0218] Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that this disclosure is not limited to the specific embodiments described and is therefore subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the invention, as the scope of this disclosure is limited only by the appended claims.

[0219] When a numerical range is provided herein, it should be understood that, unless the context explicitly indicates otherwise, one-tenth of a unit between each intermediate value of the range and the upper and lower limits of the range is also specifically disclosed. This disclosure includes each smaller range between "any said value or intermediate value within the range" and "any other said or intermediate value within the range". It should be further understood that the numerical range expressed herein as "from a to b" means a numerical range extending from a to b (i.e., including the strict endpoints a and b).

[0220] In addition, it should be understood that the term “about” in this document should be understood as plus or minus (±) 5%, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1% of the value used with it.

[0221] It must be noted that when used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly specifies otherwise. Thus, for example, reference to “a botulinum neurotoxin” includes multiple such candidate agents, and reference to “the botulinum neurotoxin” includes one or more Clostridium neurotoxins and their equivalents known to those skilled in the art.

[0222] The publications discussed herein are provided only for disclosures made by them prior to the filing date of this application. Nothing in this document should be construed as an admission that such publications constitute prior art within the scope of the appended patent application.

[0223] When the sequence listing indicates the initial Met amino acid residue or the corresponding initial codon in any of the following SEQ ID NOs, the residue / codon is optional. Example

[0224] Materials and Methods

[0225] The animal model used in the following studies was a male domestic pig weighing 11-13 kg. Pigs are a suitable model for studying the treatment of postoperative surgical pain because pig skin is similar to human skin in terms of structure, thickness, nerve innervation, pigmentation, collagen and lipid composition, wound healing, and immune response.

[0226] Reconstitution of Dysport: Dysport is provided in vials containing 500U. For dosing, the 500U vials are reconstituted with saline (0.9% NaCl). Subsequent dilutions with saline are performed according to the test dose as follows: Draw 2.5 ml of saline into a 3 ml syringe with a 21 G needle and transfer it to a Dysport 500U vial; the concentration is 200 U / ml = 400 U / 2 ml; gently rotate the vial until the material dissolves. Tilt each vial left and right 2-3 times (to ensure a homogeneous solution); administer 2 ml to the pig using two 1 ml syringes connected to 30 G needles. This solution is used for a 400U dosing.

[0227] Preparation of 200U / 2 ml dose: Reconstitute Dysport as described above; draw 2 ml of the reconstituted Dysport 500U using a 3 ml syringe and a 21 G needle; draw 2 ml of saline using a 3 ml syringe and a 21 G needle; use a vacuum blood collection vial to mix the two solutions; tilt the mixed solution left and right 5-6 times (to ensure the solution is homogeneous); administer 2 ml to the pig using two 1 ml syringes connected to 30 G needles.

[0228] Preparation of 100U / 2 ml dose: Reconstitute Dysport as described above; draw 2 ml of the reconstituted Dysport 500U using a 3 ml syringe and a 21 G needle; draw 2 ml of saline using a 3 ml syringe and a 21 G needle; use a vacuum blood collection tube to mix the two solutions described above; tilt the mixed solution left and right 5-6 times (to ensure uniformity); draw 2 ml of the prepared solution from the vacuum blood collection tube using a 3 ml syringe and a 21 G needle; draw 2 ml of saline using a 3 ml syringe and a 21 G needle; use a new vacuum blood collection tube to mix the two solutions described above; tilt the mixed solution left and right 5-6 times (to ensure uniformity); administer 2 ml to the pig using two 1 ml syringes connected to 30 G needles.

[0229] Postoperative pain was induced by anesthetizing the pigs with an isoflurane / oxygen mixture delivered via a mask. The incision area was cleaned with Septol and Polydine (Iodo-Vit) solution. A 6-7 cm long skin incision was made on the left side, 3 cm (day 1) lateral to the tail and spinal line, or a 7 cm long skin incision was made on the left leg. The fascia was then incised and the muscles retracted (Castel et al., Characterization of a porcine model of post-operative pain, Eur. J. Pain. 2014, 18(4), 496-505; incorporated herein by reference). The subcutaneous tissue was then sutured with 3-0 Vicryl sutures. The skin was sutured with 3-0 silk sutures using a continuous suture technique. After incision closure and material injection, the pigs received antibiotics (Marbocyl 10%). The incision area was covered with a thin layer of Syntomicine 3%. The animal was kept under anesthesia during the surgery and administration of medication (approximately 20 minutes). After the surgery, the animal was returned to its enclosure for recovery and observation.

[0230] Treatment

[0231] Dysport was administered intradermally throughout the surgical period, immediately after suturing the left flank incision. Dysport (experimental drug), saline (negative control), or the reference compound Expallel (positive control) was injected intradermally (or subcutaneously for Expallel) using a 30G needle attached to a 1 ml syringe, into 10 sites around the incision. A fixed volume and level of administration were given at each site. More specifically, four sites (2 cm intervals) were injected along each side of the 7 cm horizontal incision / suture on the left side (e.g., 8 sites), and at each end of the left incision / suture (see Figure 1). The following experimental groups were evaluated:

[0232] Group number treat Animal number Dosage volume Dosage level per animal 1 brine 6 200 μL / injection site brine 2 Exparel 6 20 ml 266 mg 3 Dysport 6 200 μL / injection site 100 U 4 Dysport 6 200 μL / injection site 200 U 5 Dysport 6 200 μL / injection site 400 U

[0233] Dysport was administered subcutaneously throughout the surgical period. Since the animals were injected before the incision (in the left flank or left leg of pigs), a fixed total volume of 2 ml was first used to further tattoo the incision site. Using a 30G needle attached to a 1 ml syringe, Dysport (experimental drug) or saline (negative control) was subcutaneously injected into 10 sites around the incision. A fixed volume and level of administration were given at each site. Administration was performed 15 days, 5 days, or 1 day before surgery. The following evaluations were conducted in the experimental groups (when the incision was made in the left flank of pigs):

[0234] Group number treat Animal number Dosage volume Dosage level per animal Injection day vs. surgery day 1 Dysport 6 200 μL / injection site brine - 15 2 brine 6 200 μL / injection site 200 U / pig, injected at 20 U in 10 sites. 3 Dysport 6 200 μL / injection site brine - 5 4 brine 6 200 μL / injection site 200 U / pig, injected at 20 U in 10 sites. 5 Dysport 6 200 μL / injection site brine - 1 6 brine 6 200 μL / injection site 200 U / pig, injected at 20 U in 10 sites.

[0235] The following evaluations were conducted on the experimental groups (when an incision was made in the left leg of the pig): Group number treat Animal number Dosage volume Dosage level per animal Injection day vs. surgery day 1 brine 6 200 μL / injection site brine - 15 2 Dysport 5 200 μL / injection site 200 U / pig, injected at 20 U in 10 sites. 3 Exparel 6 20 ml 266 mg 1

[0236] Dysport administration via intradermal, intramuscular, or subcutaneous routes: Since the animals were injected 15 days prior to the incision, the incision site was further tattooed. Using a 30G needle attached to a 1 ml syringe, Dysport (experimental item) or saline (negative control) was injected into 10 sites around the incision. A fixed volume and level of administration were given at each site. Administration was performed via intradermal, subcutaneous, or intramuscular routes.

[0237] The evaluation of the following experimental groups is as follows: Group number treat Investment channels Dosage volume and dosage level Injection day vs. surgery day 1 Dysport Intramuscular 200 U / 2 ml / pig, divided into 10 200 μL portions. -15 2 brine 3 Dysport Intradermal 200 U / 2 ml / pig, divided into 10 200 μL portions. -15 4 brine 5 Dysport subcutaneous 200 U / 2 ml / pig, divided into 10 200 μL portions. -15 6 brine

[0238] Von Frei measurements were performed on healthy, unoperated animals at 1, 2, 4, and 6 hours post-surgery on day 1 and once daily for 10 days, following Dysport / saline injection. Von Frei fibers (Ugo Basile, Italy) were applied approximately 0.5 cm below the incision line on the skin surface of the flank or leg. The force applied to the skin of the flank or leg increased with the weight of the fiber. The maximum force used was 60 g. The fiber was applied until the animal withdrew from the stimulus. Each fiber was applied 3-5 times. If withdrawal was not achieved, a thicker fiber was applied. If withdrawal was achieved, a thinner fiber was applied (thicker or thinner refers to higher / greater or lower / fineer grams of force). The force required to achieve a withdrawal response was determined and recorded by varying the fiber thickness. The following table lists the dimensions and forces of the Von Frei fibers:

[0239] Inclusion criteria: Animals with a baseline flank retraction force ≥26g (preferably 60g) were included in the study. Postoperatively, a flank retraction force ≤10g was considered to indicate pain (tactile pain). Animals not meeting this criterion were excluded from the study. Due to the relatively low preoperative threshold (≤10g), one animal was excluded from the study.

[0240] Animals were included in the study if their leg retraction force was ≥13 g at baseline. Postoperatively, if the leg retraction force was ≤2 g, pain (tactile pain) was considered to be present. Animals that did not meet this criterion were excluded from the study.

[0241] Approaching Time Test: Before being introduced to the pigs, researchers conducting the approaching time (AT) test first enter the pen. When someone enters the pen, the pigs' normal behavior is to move away from the intruder and then approach the person. The more familiar and comfortable the pigs are with the person, the shorter the time it takes for them to approach. The latency period for the researchers approaching their home-pen is measured in seconds (with a cutoff time of 120 seconds). This test is conducted in the morning, at least one hour after the morning feeding (6:30 AM), before the distress behavior scoring and during the habituation period.

[0242] Distress Behavior Scoring: Animal behavior changes after incision. When approached, animals tend to distance themselves from researchers entering the enclosure, protect the incision side, and sometimes vocalize. This is the most common phenomenon observed after this type of surgery; in rare cases, animals may become agitated or exhibit isolation behavior. Distress behavior is scored from 0 (normal) to 7 (very distressed). The distress behavior scoring test is performed immediately after the approach time test. General animal behavior is monitored in their home enclosure during the morning. The distress behavior score also assesses the animal's overall health. Animal behavior is scored by observers unaware of the treatment, with the total score being the sum of all components shown in the table below.

[0243] Rating section parameter Fraction Part 1 Avoid standing (or lying down). 1 Standing 0 Part 2 Avoid walking 1 walk 0 Part 3 It will protect the incision side when walking. 1 Normal performance 0 Part 4 They would move away when researchers approached. 1 They do not move away when researchers approach. 0 Part 5 Anxiety 1 normal 0 Part 6 Isolate from other animals 1 Together with other animals 0 Part 7 Scream (loudly) 1 Normal vocalization 0

[0244] The assessment of behavioral scores is not conducted in a specific order, but rather based on the animal’s total spontaneous behavior.

[0245] Open Space Test for Movement Activities: The open space was a rectangular area 2.5m wide and 4.7m long. The walls of the area were smooth and 1.6m high. On the morning of the test, all groups of animals were individually guided to the open space, one at a time, for a period of 5 minutes (5). The movement activities of the animals were recorded using a CCTV camera and analyzed using AnyMaze software (Stoelting Co.). The open space test was conducted at the end of the behavioral tests conducted in the enclosure (i.e., approach time, distress behavior, and von Frey). After each open space test, the following parameters were analyzed: total walking distance (m) and percentage of time spent in the center of the area (area E; see Figure 2).

[0246] Distressed and distressed animals tend to walk closer to the walls of the enclosure or the open space installation. Animals that are not distressed will not hesitate to go to the center of the open space installation.

[0247] Example 1: Dysport provides delayed postoperative analgesia and anxiolytic effects throughout the surgical period (left flank incision in pigs).

[0248] Immediately after suturing the left flank incision in pigs (i.e., throughout the entire surgical period), pigs were intradermally injected with saline, Exparol (266 mg fixed dose), or different concentrations of Dysport. Mechanosensitivity in pigs was measured using a Von Frey assay as an assessment of postoperative surgical pain management. Exparol showed analgesic effects for 1 day compared to the saline-treated group, but no effective analgesic activity was observed thereafter. Administration of 400 U of Dysport induced moderate analgesia on day 2 postoperatively. Administration of 200 U or 400 U of Dysport induced greater analgesia on day 4 postoperatively. On day 6 postoperatively, all tested Dysport concentrations completely suppressed postoperative surgical pain. This suggests that Dysport provides an effective and sustained analgesic effect for treating postoperative surgical pain. This data is illustrated in the bar graph in Figure 3A.

[0249] The latency period for pigs to approach their managers was measured. Pigs were injected intradermally with saline, Expareel, or different concentrations of Dysport at the time of incision. By 2 hours post-surgery, all treatment groups showed a delay in approaching their managers. By 6 hours, intradermal injection of 200 U or 400 U of Dysport reduced the time required for pigs to approach their managers; these effects lasted up to 5 days post-surgery, suggesting a potential reduction in postoperative distress and anxiety-like reactions. Pigs treated with saline or Expareel showed no improvement in approaching their managers, suggesting that these treatments did not reduce postoperative distress and anxiety-like reactions. This data is illustrated in Figure 3B.

[0250] Measurement of distress behavior scores in pigs. Unlike the saline and Expallel treatment groups, pigs receiving 100U, 200U, or 400U of Dysport showed a decrease in distress behavior scores 2 days post-surgery. This data is illustrated in Figure 3C.

[0251] The open space test showed no difference in total walking distance between animals before and after saline treatment. Treatment with Expalarel or Dysport for 3 days after administration did not affect total walking distance, suggesting no change in motor function after surgery. This data is illustrated in Figure 4A. Animals treated with 400U Dysport spent more time in the center of the open space apparatus, although this difference was not statistically significant (see Figure 4B).

[0252] Example 2: Preoperative administration of Dysport induces faster analgesia and suppresses postoperative distress and anxiety-like reactions when making a surgical incision in the left flank of pigs.

[0253] Because the analgesic effect of Dysport administered throughout the surgical period is delayed, the analgesic and anxiolytic effects of preoperative Dysport were measured. Saline or 200 U of Dysport was administered intradermally to pigs 15 days (see Figure 5A), 5 days (see Figure 5B), or 1 day (see Figure 5C) before surgery (using a left flank incision). Using the Von Frey assay, the fastest analgesic effect was observed when Dysport was administered 15 days before surgery, reducing postoperative pain up to 1 day postoperatively. In contrast, when Dysport was administered 5 days before surgery, postoperative pain was reduced up to 5 days postoperatively.

[0254] When Dysport was administered intradermally 15 or 5 days prior to surgery, pigs showed a reduction in time spent near their handlers (see Figure 6). Similarly, when Dysport was administered intradermally 15 or 5 days prior to surgery, pigs showed a reduction in distress behavior scores (see Figure 7). Administering Dysport 1 day prior to surgery did not induce a significant anti-anxiety effect. This suggests that preoperative administration of Dysport 15 or 5 days prior to surgery can completely prevent postoperative distress and anxiety-like reactions.

[0255] No difference was observed in total postoperative walking distance in any of the treatment groups (intradermal injection of Dysport 15, 5 or 1 day prior to surgery) (see Figure 8), suggesting that muscle activity was not affected and there was no systemic dissemination of toxins.

[0256] Animals injected with saline spent a similar percentage of time in the open space facility center before and after surgery. Animals treated with Dysport 15 days prior to surgery spent more time in the open space facility center (see Figure 9A). When administered 5 days or 1 day prior to surgery, there was no difference in the percentage of time spent in the central area between animals treated with saline and those treated with Dysport (see Figures 9B and 9C).

[0257] Example 3: Intradermal administration of Dysport provides a beneficial approach to reducing postoperative pain and suppressing postoperative anxiety.

[0258] Different routes of administration (intradermal, subcutaneous, and intramuscular) of 200 U Dysport 15 days prior to surgery were evaluated to assess its ability to induce postoperative analgesia and anxiolytic effects. Surprisingly, intradermal administration provided better results compared to other selected routes (indeed, only intradermal administration of Dysport typically showed rapid analgesic effects (see Figure 10). Subcutaneous and intramuscular administration of Dysport both showed little effect on analgesic activity. When Dysport was administered intradermally 15 days prior to surgery, the time pigs spent approaching their handlers was shortened, and distress behavior scores decreased (see Figures 11 and 12). This suggests that intradermal administration is effective in relieving postoperative surgical pain and preventing the complete onset of postoperative distress and anxiety-like reactions.

[0259] The postoperative walking distance recorded in all saline groups was the same as that recorded before surgery. In addition, the treatment with Dysport and its administration route (intradermal, subcutaneous, or intramuscular) did not affect the total postoperative walking distance (see Figure 13).

[0260] Animals that received saline injections before and after surgery spent a similar percentage of time in the center of the open space device. There was no difference in the percentage of time animals spent in the center of the open space device between different administration routes (see Figure 14).

[0261] Example 4: SNAP-25 incision occurred at the site distal to the Dysport injection, in the ipsilateral dorsal horn of the spinal cord.

[0262] To evaluate the mechanism of action of Dysport (intradermal injection), immunohistochemistry was performed on two tissue samples from the surgical incision site (left flank of the pig) and the spinal cord. No cleaved SNAP-25 was detected in the nerves of the skin sample (see Figure 15). Unexpectedly, cleaved SNAP-25 was observed in the ipsilateral dorsal horn of the spinal cord (see Figure 16), suggesting BoNT / A activity in the spinal cord and indicating that it may provide postoperative pain / anxiety control via central action in the spinal cord. This also highlights that Dysport can be administered directly into the spinal cord via intrathecal delivery.

[0263] Immunohistochemical staining was used to assess the expression levels of two neuropeptides involved in pain regulation in the spinal cord: calcitonin gene line peptide (CGRP) and substance P. No difference in the expression levels of either neuropeptide was observed in the spinal cord of pigs treated with Dysport compared to untreated pigs (see Figure 17).

[0264] Compared with untreated pigs, pigs treated with Dysport showed reduced levels of Iba1, a marker of microglial cell activation in the spinal cord (see Figures 18A and 18B). Similarly, compared with untreated pigs, pigs treated with Dysport showed reduced levels of GFAP, a marker of stellate cell activation, in the spinal cord (Figures 18C and 18D).

[0265] Example 5: When making a surgical incision on the left leg of a pig, Dysport was administered preoperatively to induce rapid analgesia and suppress the occurrence of postoperative distress and anxiety-like reactions.

[0266] The analgesic and anxiolytic effects of preoperative Dysport were measured when different surgical incision sites were made in pigs (for the left leg rather than the left flank). Pigs were injected intradermally with saline or 200U of Dysport 15 days prior to surgery, or injected with Expalarel on the day of surgery (day 1) (see Figure 19A) (for the left leg surgical incision). Rapid analgesia was observed using the Von Frey assay, with a reduction in postoperative surgical pain on day 1 postoperatively, and a persistent reversal of mechanical tactile pain observed on day 4.

[0267] When Dysport was administered intradermally 15 days prior to surgery, compared to administration of saline and Expalarel, pigs (with a sutured incision on the left leg) showed a reduced time spent approaching their handlers (see Figure 19B). Similarly, when Dysport was administered intradermally 15 days prior to surgery, compared to administration of saline and Expalarel, pigs (with a sutured incision on the left leg) exhibited reduced distress behavior scores (see Figure 19C). This suggests that preoperative administration of Dysport 15 days prior to surgery (to the incision on the left leg) completely prevents the occurrence of postoperative distress and anxiety-like responses.

[0268] Overall, this study further supports the rapid analgesic and anxiolytic effects of Dysport administered 15 days prior to surgery.

[0269] Example 6: SNAP-25 incision occurred in the ipsilateral dorsal horn of the spinal cord of a pig with a surgical incision in the left leg.

[0270] To assess whether the same mechanism of action of Dysport occurs when administered intradermally at different sites in pigs, immunohistochemistry was performed on tissue samples from the surgical incision site (left leg of the pig) and different regions of the spinal cord (see Figure 20). In samples collected 5–7 days after incision and Dysport injection, no cleaved SNAP-25 was detected in the nerves of the skin samples (see Figure 21). Cleaved SNAP-25 was observed in the ipsilateral dorsal horn of the spinal cord, particularly in the L5–L6 region of the lumbar region (see Figure 22), similar to findings observed in surgical incisions at the left flank of the pig. These findings suggest BoNT / A activity in the spinal cord and its potential to provide control of postoperative surgical pain / anxiety via central effects within the spinal cord. The localization of cleaved SNAP-25 staining in the ipsilateral dorsal horn differed from that observed in surgical incisions at the left flank of the pig.

[0271] The intensity of cut SNAP-25 staining is graded from 1 to 3, where 0 = uncut SNAP-25 staining, 1 = low-intensity cut SNAP-25 staining, 2 = average-intensity cut SNAP-25 staining, and 3 = high-intensity cut SNAP-25 staining (see Figure 23A). Based on this grading system, pigs with surgical incisions on the left leg have lower intensity cut SNAP-25 staining on the ipsilateral dorsal angle compared to pigs with surgical incisions on the left flank.

[0272] The intensity of cut SNAP-25 staining was quantified (see Figure 23B). The H-score was calculated as a measure of the intensity of cut SNAP-25 staining. The H-score was calculated by multiplying the percentage of positive spinal cord sections by the staining intensity in the dorsal horn. In pigs treated with Dysport (and with a surgical incision in the left leg), the highest level of cut SNAP-25 staining (designated as grade 2 cut SNAP-25 intensity staining) was observed in the spinal cord of the L5-L6 lumbar region, compared to the L3-L4 and L1-L2 lumbar regions and the thoracic and cervical regions of the spinal cord (where traces of cut SNAP-25 staining were observed). No evidence of cut SNAP-25 staining was found in pigs injected with saline or Expallel.

[0273] The above immunohistochemical staining is summarized in Figure 24 and confirms the observation of SNAP-25 cleavage in local areas of the spinal cord. Local areas L5-L6, L3-L4, and L1-L2, as well as the thoracic and cervical vertebrae of the spinal cord, showed positive staining for the cleaved SNAP-25, while other tissues (including the skin at the injection site) showed negative staining for the cleaved SNAP-25.

[0274] All publications mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific preferred embodiments, it should be understood that the claims should not be unduly limited to such specific embodiments. Indeed, it will be apparent to those skilled in the art of biochemistry and biotechnology or related fields that various modifications to the modes of carrying out the invention fall within the scope of the following claims. [Simplified Explanation of the Diagram]

[0016] Figure 1 shows a schematic diagram of the injection site along the surgical incision, where Dysport, saline, or the reference compound Expareel is injected. Figure 2 shows a schematic diagram of the activity site for the open space test used for locomotion activity. Figure 3 shows the effect of intradermal injection of saline, Expareel (control), or different concentrations of Dysport (100, 200, and 400 U) throughout the surgical period on reducing postoperative surgical pain and anxiety. The Von Frey measurement was performed as a measure of perceived surgical pain, and the results are shown in (A). The horizontal line across the graph is set at 26 g, representing the baseline of no perceived surgical pain (e.g., a threshold above which postoperative surgical pain in the subject may be considered for treatment). Pain was defined as moderate / severe when the mechanosensitivity was 0-15 g, mild / moderate when it was between 15-26 g, and little / no pain when it was above 26 g. The time (in seconds) required for pigs to approach their handlers after intradermal administration of Expareel or Dysport throughout the surgical period is shown in (B). Discomfort behavior was scored after administration of Expareel or Dysport throughout the surgical period and is shown in (C). Figure 4 shows the average total walking distance (in meters) of the group during a 5-minute period (A) and the average percentage of time spent in the central area of ​​the open space apparatus after intradermal injection of saline, Expareel (control), or different concentrations of Dysport (100, 200, and 400 U) throughout the surgical period (B). Figure 5 shows the Von Frey determination of intradermal saline (control) versus BoNT / A (Dysport) at 15 days (A), 5 days (B), or 1 day (C) before surgery. Figure 6 shows the latency (in seconds) for pigs to approach their handlers after intradermal administration of saline or Dysport (200 U / pig) at 15 days (A), 5 days (B), or 1 day (C) before surgery. In the bar chart (AC), at each time point (day), the left (lighter) bars represent the results of Dysport treatment, and the right (darker) bars represent the results of saline treatment. Figure 7 shows the distress behavior scores of pigs after intradermal administration of saline or Dysport at 15 days (A), 5 days (B), or 1 day (C) before surgery. In the bar chart (AC), at each time point (day), the left (lighter) bars represent the results of Dysport treatment, and the right (darker) bars represent the results of saline treatment. Figure 8 shows the total walking distance (in meters) of pigs after intradermal administration of saline or Dysport at 15 days (A), 5 days (B), or 1 day (C) before surgery.Figure 9 shows the time course (individual values ​​and median) of time spent in the central area of ​​the open space device during a 5-minute period following intradermal administration of saline or Dysport 15 days (A), 5 days (B), or 1 day (C) prior to surgery. Figure 10 shows the Von Frey determination of saline relative to BoNT / A (Dysport) when administered via intradermal (A), subcutaneous (B), or intramuscular (C) injection. A total of 200 U of Dysport was administered per pig for each injection route tested. Figure 11 shows the latency (in seconds) of pigs approaching their manager when administered saline or Dysport via intradermal (A), subcutaneous (B), or intramuscular (C) injection. A total of 200 U of Dysport was administered per pig for each injection route tested. Figure 12 shows the distress behavior scores of pigs when administered saline or Dysport via intradermal (A), subcutaneous (B), or intramuscular (C) injection. For each injection route tested, a total of 200 U of Dysport was administered to each pig. Figure 13 shows the time progression (individual and mean ± SEM) of the total distance (in meters) traveled by animals in an open space for 5 minutes after intradermal, subcutaneous, or intramuscular administration of saline or Dysport. Figure 14 shows the time progression (individual and mean ± SEM) of the time spent by animals in the central region of an open space apparatus after intradermal, subcutaneous, or intramuscular administration of saline or Dysport. Figure 15 shows the immunohistochemical staining of SNAP-25 in skin samples, including small nerves around arterioles (A), nerve endings in arrector pili muscles (B), and small to medium-sized nerves in the dermis (C). Figure 16 shows the immunohistochemical staining of cut SNAP-25 in the spinal cord of untreated pigs (A) and cut SNAP-25 staining in the ipsilateral horn (B) or contralateral horn (C) of pigs treated with Dysport. Figure 17 shows the expression levels of calcitonin gene line peptide (CGRP) and substance P in porcine spinal cord when untreated (A, C) or after intradermal administration of Dysport (B, D). Figure 18 shows the expression levels of Iba1 (A, B) and glial fibrillary acidic protein (GFAP) (C, D) in porcine spinal cord when untreated or after intradermal administration of Dysport. Figure 19 shows the Von Frey assay results of intradermal administration of saline (control) or BoNT / A (Dysport) 15 days prior to surgery or intradermal administration of Expallel on the day of surgery (D1), with the following values ​​when the pig's left leg underwent surgical incision: (A) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. saline group, using a single-factor ANOVA followed by a Tukey test.#p<0.05; ##p<0.01 ####p<0.0001 Dysport vs. Exparel group, using one-way ANOVA followed by Tajik test. $$ p<0.01: postoperative time point vs. day -4, using paired T-test. (B) Showing the latency (in seconds) of pigs approaching their handlers after intradermal administration of saline (control) or BoNT / A (Dysport) 15 days prior to surgery or intradermal administration of Expalarel on the day of surgery (D1), and when the pig's left leg underwent the surgical incision ($$ p < 0.01: postoperative time point vs. day -4, using paired t-test. £££ p < 0.001: day -16 vs. day -4, using paired t-test. *p < 0.05; **p < 0.01; ***p < 0.001 treatment vs. saline group, using single-factor ANOVA followed by Tajik test). (C) Shows distress scores in pigs after intradermal administration of saline (control) or BoNT / A (Dysport) 15 days prior to surgery or intradermal administration of Expareel on the day of surgery (D1), and when the pig's left leg underwent surgical incision (*p<0.05, ***p<0.001 and ****p<0.0001 vs. saline group, using single-factor ANOVA followed by Tucky test. $$p<0.01, $p<0.05: postoperative time point vs. day-4, using paired T test). Figure 20 shows a summary of tissue samples collected for immunohistochemical staining (formaldehyde-fixed paraffin-embedded tissue), and specific areas from which tissue samples were collected from the spinal cord. Figure 21 shows immunohistochemical staining of cut SNAP-25 in the skin (A), muscle (B), and dorsal root ganglion (C) of the pig with the left leg surgical incision. Figure 22 shows the cut SNAP-25 staining in the ipsilateral dorsal horn of the L5-L6 lumbar vertebrae in the porcine spinal cord through a surgical incision in the left leg (A) and a magnified view (B). Figure 23 shows the grading scale used to determine the intensity of cut SNAP-25 staining. Cut SNAP-25 staining was graded from 1 to 3, with grade 0 = no cut SNAP-25 staining, grade 1 = low-intensity cut SNAP-25 staining, grade 2 = average cut SNAP-25 intensity staining, and grade 3 = high-intensity cut SNAP-25 staining (A). Figure 23 also shows the quantification of staining intensity in different regions of the spinal cord: L5-L6, L3-L4, L1-L2 lumbar vertebrae, and thoracic and cervical vertebrae. Staining intensity was measured using the "H score" and calculated as % x staining intensity in the dorsal horn of a positive spinal cord section (B). Figure 24 provides a summary of the presence, "positive" or absence, and "negative" of cut SNAP-25 staining in collected tissue samples. [Sequence List]

Claims

1. A Clostridium neurotoxin for use in treating postoperative surgical pain in patients, the method comprising administering the Clostridium neurotoxin to the patient more than 5 days prior to surgery, wherein the Clostridium neurotoxin is administered by: i) intradermal; or ii) intrathecal.

2. A method for treating postoperative surgical pain in a patient, the method comprising administering a Clostridium neurotoxin to the patient more than 5 days prior to surgery, wherein the Clostridium neurotoxin is administered by: i) intradermal; or ii) intrathecal.

3. The Clostridium neurotoxin used as claimed in claim 1 or the method as claimed in claim 2, wherein the Clostridium neurotoxin is administered 6 to 50 days prior to surgery; preferably 10 to 20 days prior to surgery.

4. The Clostridium neurotoxin used as claimed in claim 1 or 3, or the method as claimed in claim 2 or 3, wherein the Clostridium neurotoxin is administered 14 to 16 days prior to the operation; preferably about 15 days prior to the operation.

5. The Clostridium neurotoxin used as claimed in any of claims 1, 3 or 4, or the method of any of claims 2 to 4, wherein administration of the Clostridium neurotoxin substantially reduces the patient’s postoperative surgical pain perception, and wherein the reduced postoperative surgical pain perception is maintained for 24 hours immediately following the surgery.

6. The Clostridium neurotoxin used as claimed in any of claims 1 or 3 to 5, or the method as claimed in any of claims 2 to 5, wherein substantially all reduced postoperative surgical pain perception is maintained for 3 days immediately following the surgery, 5 days immediately following the surgery, 7 days immediately following the surgery, and preferably 8 days immediately following the surgery.

7. The clostridium neurotoxin used as claimed in claim 5 or 6, or the method as claimed in claim 5 or 6, wherein the level of reduction in pain perception observed at a defined time point immediately following the surgery is at least 50% of the maximum level of reduction in pain perception observed at any time after administration of the clostridium neurotoxin, preferably at least 75% of the maximum level of reduction in pain perception observed at any time after administration of the clostridium neurotoxin.

8. A Clostridium neurotoxin for reducing or suppressing postoperative anxiety, the method comprising administering the Clostridium neurotoxin to a patient prior to surgery, wherein the Clostridium neurotoxin is administered via: i) intradermal; or ii) intrathecal.

9. A method for reducing or suppressing postoperative anxiety, the method comprising administering a Clostridium neurotoxin to a patient prior to surgery, wherein the Clostridium neurotoxin is administered via: i) intradermal; or ii) intrathecal.

10. The Clostridium neurotoxin used as claimed in claim 8 or the method as claimed in claim 9, wherein the Clostridium neurotoxin is administered 5 days or more prior to the operation; preferably wherein the Clostridium neurotoxin is administered more than 5 days prior to the operation.

11. The Clostridium neurotoxin used as claimed in claim 8 or 10, or the method as claimed in claim 9 or 10, wherein administration of the Clostridium neurotoxin substantially reduces the patient’s perceived postoperative anxiety, and wherein the reduced perceived postoperative anxiety is maintained for 24 hours immediately following the surgery.

12. The Clostridium neurotoxin used as claimed in claim 11 or the method as claimed in claim 11, wherein substantially all reduced postoperative anxiety perception is maintained for 2 days immediately following the surgery, 5 days immediately following the surgery, 7 days immediately following the surgery, and preferably 9 days immediately following the surgery.

13. The Clostridium neurotoxin used as claimed in any of claims 1, 3 to 8, or 10 to 12, or the method as claimed in any of claims 2 to 7 or 9 to 12, wherein the Clostridium neurotoxin treats postoperative surgical pain and reduces or inhibits postoperative anxiety.

14. The Clostridium neurotoxin used as claimed in any of claims 1, 3 to 8, or 13, or the method as claimed in any of claims 2 to 7 or 13, wherein the postoperative pain is caused by the surgical intervention, and wherein the Clostridium neurotoxin is administered at a location remote from the site of the surgical intervention.

15. The Clostridium neurotoxin used as claimed in claim 14 or the method as claimed in claim 14, wherein the distance from the surgical intervention site is at least 15 cm, 50 cm or 100 cm from the surgical intervention site.

16. The Clostridium neurotoxin used as claimed in any of claims 1, 3 to 8, or 10 to 15, or the method as claimed in any of claims 2 to 7 or 9 to 15, wherein, After administration, the Clostridium neurotoxin migrates to the spinal cord via retrograde transport and affects the SNARE protein cleavage (SNAP-25 protein cleavage) in the spinal cord.

17. The Clostridium neurotoxin used as claimed in any of claims 1, 3 to 8, or 10 to 16, or the method as claimed in any of claims 2 to 7 or 9 to 16, wherein the Clostridium neurotoxin is administered to an intradermal site, and wherein, after administration of the Clostridium neurotoxin, minimal or no SNARE protein cleavage (SNAP-25 protein cleavage) caused by the Clostridium neurotoxin is observed at or near the intradermal site.

18. The Clostridium neurotoxin used as claimed in any of claims 1, 3 to 8, or 10 to 16, or the method as claimed in any of claims 2 to 7 or 9 to 16, wherein the Clostridium neurotoxin is administered at a site in the intrathecal space of the spinal cord, and wherein, after administration of the Clostridium neurotoxin, minimal or no SNARE protein cleavage (SNAP-25 protein cleavage) caused by the Clostridium neurotoxin is observed at or near that site.

19. The Clostridium neurotoxin used as claimed in any of claims 1, 3 to 8, or 10 to 18, or the method as claimed in any of claims 2 to 7 or 9 to 18, wherein the surgical intervention includes an incision of the skin and / or fascia and / or muscle, preferably wherein the surgical intervention includes an incision of the skin.

20. The Clostridium neurotoxin used as claimed in any of claims 1, 3 to 8, or 10 to 19, or the method as claimed in any of claims 2 to 7 or 9 to 19, wherein the Clostridium neurotoxin is botulinum neurotoxin (BoNT).

21. The Clostridium neurotoxin used as claimed in any of claims 1, 3 to 8, or 10 to 20, or the method as claimed in any of claims 2 to 7 or 9 to 20, wherein the Clostridium neurotoxin is botulinum neurotoxin serotype A (BoNT / A).

22. The Clostridium neurotoxin used as claimed in any of claims 1, 3 to 8, or 10 to 21, or the method as claimed in any of claims 2 to 7 or 9 to 21, wherein the postoperative pain is acute postoperative pain.

23. The Clostridium neurotoxin used as in any of claims 1, 3 to 8, or 10 to 22, or the method as in claims 2 to 7 or 9 to 22, wherein the postoperative pain is chronic postoperative pain.

24. The use of Clostridium neurotoxin as claimed in any of claims 1, 3 to 8, or 10 to 23, or the method as claimed in any of claims 2 to 7 or 9 to 23, wherein the use or method does not include intramuscular administration of Clostridium neurotoxin.

25. The Clostridium neurotoxin used as claimed in any of claims 1, 3 to 8, or 10 to 24, or the method as claimed in any of claims 2 to 7 or 9 to 24, wherein the patient is administered 100-500 U of Clostridium neurotoxin; preferably wherein the patient is administered 200 U of Clostridium neurotoxin.

26. The Clostridium neurotoxin used as in any of claims 1, 3 to 8, or 10 to 25, or the method as in claims 2 to 7 or 9 to 25, wherein the patient is administered a total dose of 1-3 ng of Clostridium neurotoxin.

27. The Clostridium neurotoxin used as in any of claims 1, 3 to 8, or 10 to 26, or the method as in claims 2 to 7 or 9 to 26, wherein the patient is administered 80-250 pg of Clostridium neurotoxin per kg (body weight).

28. The Clostridium neurotoxin used as claimed in any of claims 1, 3 to 8, or 10 to 27, or the method as claimed in any of claims 2 to 7 or 9 to 27, wherein the Clostridium neurotoxin is administered at more than one administration site; preferably wherein the patient is administered 2.5-30 U of Clostridium neurotoxin at each administration site; more preferably wherein the patient is administered 20 U of Clostridium neurotoxin at each administration site.

29. The Clostridium neurotoxin used as claimed in any of claims 1, 3 to 8, or 10 to 28, or the method as claimed in any of claims 2 to 7 or 9 to 28, wherein the Clostridium neurotoxin is administered at more than one administration site; preferably wherein the patient is administered 10-170 pg of Clostridium neurotoxin at each administration site; more preferably 1-14 pg / kg body weight at each administration site.

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