Management of postoperative surgical pain

Abstract

Treatment of postoperative surgical pain The present invention relates to a Clostridial neurotoxin for use in treating post-operative surgical pain in a patient, said method comprising administering a Clostridial neurotoxin to the patient more than 5 days prior to surgery, and wherein the Clostridial neurotoxin is administered: i) intradermally; or ii) intrathecally. Corresponding methods of treatment and dosages of Clostridial neurotoxins are also provided.

Description

[Technical Field]

[0001] The present invention relates to the treatment of surgical pain resulting from surgery (e.g., postoperative surgical pain) and / or surgery-induced anxiety (e.g., postoperative anxiety). More specifically, the present invention provides a method of treatment comprising the administration of Clostridium neurotoxin, and in particular a method of treating postoperative surgical pain and anxiety using botulinum neurotoxin. [Background technology]

[0002] Postoperative surgical pain is an unpleasant sensation resulting from a surgical procedure. It can be caused by tissue damage resulting from the surgical intervention, the surgical procedure itself, wound closure, or any force applied during the procedure. Postoperative surgical pain (e.g., postoperative surgical pain) can also stem from factors associated with the surgery. For example, a patient may experience back pain due to how they are positioned on the surgical table, or chest pain due to surgical intervention in the thoracic region. Furthermore, sore throat may occur after general anesthesia because the insertion of a breathing tube can cause irritation. However, the most common cause of postoperative surgical pain is incisions in the skin and muscles resulting from the surgical intervention.

[0003] For example, surgical intervention (or more specifically, surgical incision) can represent a “noxious stimulus” that causes pain. A noxious stimulus, i.e., a stimulus that can cause tissue damage, can activate the release of neurotransmitters from nociceptive afferent terminals and the release of neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) from sensory terminals. This noxious information is then transmitted from the peripheral nervous system to the central nervous system, where pain is perceived by the individual.

[0004] Post-surgical pain can be caused by a combination of inflammation and nerve tissue damage at the site of surgical intervention. Some inflammation and / or nerve tissue damage occurs in addition to post-surgical pain. For example, as a result of degranulation of mast cells activated in response to tissue trauma, the release of various substances including proteases, cytokines, and serotonin can occur. These substances can sensitize primary afferent neurons (activate them at lower thresholds), producing hyperalgesia. Since tissues are innervated extensively, any region of the body is sensitive to nerve injury from surgery.

[0005] Post-surgical anxiety can be associated with patients experiencing physical symptoms and behavioral changes, including but not limited to fatigue, difficulty concentrating and sleeping, and muscle tension. Additionally, patients may experience emotional symptoms of anxiety, including restlessness, irritability, difficulty controlling fear or worry, dread, and panic. Post-surgical anxiety may be caused by the effects of anesthesia, the surgery itself, post-surgical pain, and / or stress from the hospital environment. For example, surgical patients are often under a significant amount of mental, physical, and emotional stress (e.g., stress due to anticipation of surgery before the operation) both before and after surgery, and such stress manifests as symptoms of anxiety.

[0006] Existing methods for the treatment of surgical (e.g., postoperative) pain typically target neurotransmitters and peptides and involve the use of non-steroidal anti-inflammatory drugs (NSAIDs), opioids, local anesthetic blocks, or combinations thereof. However, these treatment methods produce various side effects and, notably, often induce dependence (e.g., addiction). In addition, these treatment methods only provide relief from acute postoperative surgical pain, e.g., they provide relief from surgical pain only for a short period after administration and thus require continuous / repeated administration (worsening the problem of patient dependence / addiction to analgesics). Failure to effectively manage acute postoperative surgical pain may increase the patient's chance of developing chronic postoperative surgical pain. Such pain management methods (which require continuous administration of drugs) also often result in drug tolerance. A further problem associated with prior methods for managing postoperative surgical pain is the need for high doses of analgesic drugs to provide an analgesic effect, often accompanied by side effects that increase in proportion to that dose.

[0007] Thus, current methods of treating surgical pain are not adequate (e.g., sufficient) to manage / relieve postoperative surgical pain, particularly moderate to severe postoperative surgical pain, nor do they provide adequate management of postoperative anxiety experienced by the patient (the latter generally requires alternative / additional drugs). Therefore, there is an increasing need for alternative / improved methods for treating postoperative surgical pain and / or postoperative anxiety.

[0008] The present invention addresses one or more of these problems by providing a method for the long-term, sustained treatment (including a tendency to reduce chronic postoperative surgical pain) of postoperative surgical pain / anxiety, even after, for example, a single (e.g., acute) administration. A preferred aspect of the present invention is based on the surprising observation that initiating treatment (administration) before subjecting the patient to surgery enables effective management of postoperative surgical pain or postoperative anxiety when the patient comes out of surgery (advantageously reducing postoperative surgical pain or postoperative anxiety that might otherwise be felt as a waning of the effect of general / local anesthesia), and provides a detailed preoperative administration time point that is uniquely suitable for treatment with the Clostridium neurotoxin system. [Overview of the Initiative]

[0009] More specifically, the present invention is based on the surprising finding that preoperative administration of Clostridium neurotoxin (such as botulinum neurotoxin) by intradermal or intrathecal administration more than five days prior to surgery treats postoperative surgical pain and reduces or suppresses postoperative anxiety. This was entirely unexpected, as prior art methods had reported optimal analgesic effects when Clostridium neurotoxin was administered closer to the time of surgery and by an alternative route of administration.

[0010] Advantageously, the inventors have demonstrated that by administering Clostridium neurotoxin intradermally or intrathecally more than five days prior to surgery, Clostridium neurotoxin is effective in treating postoperative surgical pain as early as one hour postoperatively, and continues to manage / treat postoperative surgical pain for several days (and even weeks) postoperatively without the need for continuous administration and without the side effects associated with traditional analgesics / anesthetics. Thus, Clostridium neurotoxin can provide relief of postoperative surgical pain as the effects of any "systemic" or "local" anesthetic (used during surgery) diminish. In other words, preoperative administration (more than five days prior to surgery) advantageously allows the analgesic effect of Clostridium neurotoxin to emerge (e.g., reach its maximum efficacy / effect) at the point when the patient might otherwise begin to perceive postoperative surgical pain or anxiety due to the tapering effects of the primary anesthetic / analgesic used during surgery. Therefore, by treating postoperative surgical pain before it develops, any accompanying (potentially significant) discomfort and pain in the patient can be prevented. As will be described in more detail below, such early management of postoperative surgical pain (e.g., acute moderate to severe postoperative surgical pain) can advantageously reduce the onset of chronic postoperative surgical pain.

[0011] This contrasts with the treatment observed when Clostridium neurotoxins are administered near the time of surgery (or perioperatively), in which case a clear delay or "activation period" is observed until any surgical pain-relieving effect of Clostridium neurotoxins is provided postoperatively.

[0012] A particularly surprising observation by the inventors is that when Clostridium neurotoxin is administered intradermally, it provides a particularly effective (e.g., rapid) treatment for postoperative surgical pain / postoperative anxiety. In fact, the inventors observed that intradermal administration can provide enhanced relief of postoperative surgical pain and / or postoperative anxiety compared to alternative administration routes such as subcutaneous and intramuscular administration. This was entirely unexpected, as Clostridium neurotoxin is usually administered via these alternative routes (e.g., subcutaneous / intramuscular) without any apparent disadvantage in terms of efficacy for other conditions.

[0013] Another advantageous finding by the inventors is that Clostridium neurotoxin can exert its effects distal to the site of administration. For example, after administration of Clostridium neurotoxin at or proximal to the site of surgical intervention, the inventors observed SNARE protein cleavage (e.g., SNAP-25 protein cleavage) in the spinal cord (and observed minimal or no SNARE protein cleavage at or proximal to the site of surgical intervention), which suggests that Clostridium neurotoxin travels from the site of administration to the spinal cord by retrograde transport. This makes it possible to administer Clostridium neurotoxin at a site away from the site of trauma (e.g., the site of surgical intervention) that could cause discomfort to the patient, and thus minimize any further pain perceived by the patient. [Modes for carrying out the invention]

[0014] In one embodiment, the present invention provides a Clostridium neurotoxin for use in treating postoperative surgical pain in a patient, the method comprising administering the Clostridium neurotoxin to the patient more than 5 days prior to surgery, wherein the Clostridium neurotoxin is administered as follows: i) intradermally; or ii) Intrathecal.

[0015] In other words, one aspect of the present invention provides a method for treating postoperative surgical pain in a patient, the method comprising administering Clostridium neurotoxin to the patient more than 5 days prior to surgery, wherein the Clostridium neurotoxin is administered as follows: i) intradermally; or ii) Intrathecal.

[0016] In one embodiment, the present invention provides a Clostridium neurotoxin for use in treating postoperative pain, the method comprising administering the Clostridium neurotoxin to a patient (e.g., intradermally) more than five days before surgery. In other words, one aspect of the present invention provides a method for treating postoperative pain, the method comprising administering the Clostridium neurotoxin to a patient (e.g., intradermally) more than five days before surgery.

[0017] In related observations, pre-operative administration of Clostridium neurotoxin has been found to reduce or suppress / prevent (e.g., completely prevent) the onset of anxiety (postoperative anxiety) in patients after surgery. Therefore, the even more remarkable technical effect provided by the present invention is the anxiolytic effect achievable as a result of pre-operative administration of Clostridium neurotoxin.

[0018] Thus, another aspect of the present invention provides a clostridium neurotoxin for use in reducing or suppressing postoperative anxiety, the method comprising administering the clostridium neurotoxin to a patient before surgery, wherein the clostridium neurotoxin is administered as follows: i) intradermally; or ii) Intrathecal.

[0019] In other words, one aspect of the present invention provides a method for reducing or suppressing postoperative anxiety, the method comprising administering Clostridium neurotoxin to a patient before surgery, wherein Clostridium neurotoxin is administered as follows: i) intradermally; or ii) Intrathecal.

[0020] Preferably, methods for reducing or suppressing postoperative anxiety include administering Clostridium neurotoxin at least five days before surgery; for example, Clostridium neurotoxin may be administered more than five days before surgery.

[0021] Another aspect of the present invention provides a Clostridium neurotoxin for use in reducing or suppressing postoperative anxiety, the method comprising administering the Clostridium neurotoxin to the patient (e.g., intradermally) before surgery (e.g., five days or more before surgery). In other words, one aspect of the present invention provides a method for reducing or suppressing postoperative anxiety, the method comprising administering the Clostridium neurotoxin to the patient (e.g., intradermally) before surgery.

[0022] Preferably, methods for reducing or suppressing postoperative anxiety include administering Clostridium neurotoxin at least five days before surgery; for example, Clostridium neurotoxin may be administered more than five days before surgery.

[0023] Preferably, Clostridium neurotoxin can be administered intradermally.

[0024] In addition, Clostridium neurotoxin may be administered intrathecally (e.g., intrathecal administration / injection).

[0025] Herein, various additional (optional) embodiments of the present invention are described. Each of the embodiments described below may be applied to any of the methods or uses of Clostridium neurotoxin described herein.

[0026] In one embodiment, the administration of Clostridium neurotoxin does not include intramuscular administration.

[0027] In one embodiment, Clostridium neurotoxin may be administered 5 to 50 days before surgery, for example 6 to 50 days before surgery, or optionally 5 to 40 days before surgery. For example, Clostridium neurotoxin may be administered 5 to 30 days before surgery; preferably 5 to 20 days before surgery; more preferably 5 to 15 days before surgery.

[0028] In one embodiment, Clostridium neurotoxin may be administered in an optional single dose step > 5 days prior to surgery (preferably in the method for treating postoperative surgical pain described herein). For example, Clostridium neurotoxin may be administered in an optional single dose step 10 to 20 days prior to surgery (preferably in the method for treating postoperative surgical pain described herein); or 14 to 16 days prior to surgery.

[0029] Clostridium neurotoxin may be administered at least 15 days before surgery, preferably about 15 days before surgery.

[0030] In a preferred embodiment, Clostridium neurotoxin may be administered (preferably in a method for treating postoperative surgical pain as described herein) in an optional single-dose step, >5 to ≤15 days prior to surgery.

[0031] In one embodiment, Clostridium neurotoxin is administered at least five days before surgery (preferably in the method for treating postoperative surgical pain described herein).

[0032] In one embodiment, Clostridium neurotoxin is administered at least 12 days before surgery (preferably in the method for treating postoperative surgical pain described herein).

[0033] In one embodiment, Clostridium neurotoxin is administered intradermally at least 15 days before surgery. In a preferred embodiment, Clostridium neurotoxin is administered intradermally about 15 days before surgery.

[0034] In one embodiment, Clostridium neurotoxin is administered intrathecally at least 15 days before surgery. In a preferred embodiment, Clostridium neurotoxin is administered intrathecally about 15 days before surgery.

[0035] Clostridium neurotoxin treats postoperative surgical pain by providing an analgesic effect. Therefore, the terms “treat” or “treating” as used herein are intended to encompass analgesic treatment. The terms “treat” or “treating” encompass treating postoperative surgical pain so that the patient no longer perceives surgical pain (or perceives less surgical pain compared to a control patient not treated with Clostridium neurotoxin).

[0036] Similarly, Clostridium neurotoxins suppress postoperative anxiety by providing an anxiolytic effect. Therefore, the term “suppress” or “suppressing” encompasses the suppression of postoperative anxiety (e.g., its symptoms) in patients through the anxiolytic effect provided by the administration of Clostridium neurotoxins. This suppression may be provided in conjunction with and, for example, as a result of postoperative surgical pain management. Therefore, without being bound by any theory, Clostridium neurotoxins may provide anxiolytic effects through their analgesic effect. Clostridium neurotoxins may suppress the symptoms of postoperative anxiety associated with (or arising from) the effects of anesthesia employed for surgery, the surgery itself, postoperative surgical pain, and / or stress (e.g., from the hospital environment).

[0037] Therefore, Clostridium neurotoxin may be administered to a subject in a therapeutically effective amount or a prophylactically effective amount (preferably a prophylactically effective amount). “Therapeutically effective amount” means any amount of Clostridium neurotoxin sufficient to perform such treatment of postoperative surgical pain and / or postoperative anxiety when administered to a subject alone or in combination. “Prophylactically effective amount” means any amount of Clostridium neurotoxin that inhibits or delays the onset of postoperative surgical pain and / or postoperative anxiety when administered to a subject alone or in combination. In some embodiments, a prophylactically effective amount completely prevents the onset of postoperative anxiety. “Inhibiting” the onset means either reducing the likelihood of postoperative surgical pain and / or postoperative anxiety, or completely preventing its onset.

[0038] Preferably, the therapeutically and / or prophylactically effective dose is one that does not lead to muscle paralysis. The term "muscle paralysis" preferably refers to long-term muscle paralysis, as transient muscle paralysis may occur for a short period after administration.

[0039] The terms “subject,” “individual,” and “patient” may be used interchangeably herein to refer to mammalian subjects. In one embodiment, “subject” means human, companion animal (e.g., dog, cat, and / or pet such as rabbit), livestock (e.g., pig, sheep, cattle, and / or goat), and / or horse. In a preferred embodiment, the subject (patient) is human.

[0040] This invention specifically relates to postoperative surgical pain, which is distinctly different from other types of pain such as inflammatory pain and neuropathic pain. Inflammatory pain typically arises from infection, irritants, or an overactive immune response, while neuropathic pain typically arises from nervous system disorders / syndromes. In contrast, this invention does not relate to pain resulting from these irritants.

[0041] In one embodiment, administration of Clostridium neurotoxin treats postoperative surgical pain in preference to inflammatory pain. In one embodiment, administration of Clostridium neurotoxin treats minimal inflammatory pain or does not treat inflammatory pain at all. In one embodiment, administration of Clostridium neurotoxin treats postoperative surgical pain and treats minimal inflammatory pain or does not treat inflammatory pain at all.

[0042] The term "surgical intervention" refers to a medical procedure involving the treatment of trauma or disease in a subject, including the incision of a part of the body (optionally, removal or repair of a damaged part of the body). The level of invasiveness (e.g., the level of surgical incision required) may vary between types of surgery, but it is intended to encompass any surgery that, upon completion, has a level of invasiveness that causes postoperative surgical pain and / or postoperative anxiety in the subject. Postoperative surgical pain is typically caused by the surgical incision that cuts open the skin and / or fascia and / or muscles and / or bones and / or organs in the patient. Therefore, surgical pain is typically experienced at or proximal to the site of the surgical intervention.

[0043] Surgical intervention may include an incision to the skin and / or fascia and / or muscle. Preferably, the surgical intervention includes an incision to the skin.

[0044] Surgical interventions are not limited to those performed by a physician, but include, for example, oral surgical interventions. Non-limiting examples of surgical interventions include appendectomy, breast biopsy, breast augmentation or reduction, cheek blepharoplasty, cholecystectomy, coronary artery bypass surgery, debridement (e.g., for wounds, burns, or infections), skin grafts, organ transplants, and tonsillectomy.

[0045] Preferably, “postoperative” may refer to the period beginning up to one day after the surgery (e.g., after the surgery). In other words, the term “postoperative” may refer to the period beginning one day or less after the surgery. For example, the term “postoperative” may refer to a point in time beginning 1 to 20 hours after surgery; optionally 2 to 15 hours after surgery; optionally 5 to 10 hours after surgery. Such times may represent the period beginning at the temporal boundary where the analgesic effect from the surgical anesthetic administered to the patient begins to diminish (e.g., tapering) and therefore the patient begins to perceive surgical pain.

[0046] Furthermore, since "operative" is used herein to mean "surgery," the term "post-operative" may be used interchangeably with the term "post-surgical."

[0047] Similarly, the term “postoperative surgical pain” can refer to surgical pain perceived (or more precisely, beginning to be perceived) during the period following surgery (e.g., post-surgery) that begins up to one day. In other words, the term “postoperative surgical pain” can refer to surgical pain perceived by a patient during the period that begins less than one day after surgery. For example, the term “postoperative surgical pain” can refer to pain perceived during the period that begins between 1 and 20 hours post-surgery; optionally between 2 and 15 hours post-surgery; or optionally between 5 and 10 hours post-surgery.

[0048] The aforementioned period may be 1 to 50 weeks after surgery; for example, 5 to 45 weeks, 10 to 40 weeks, or 10 to 35 weeks.

[0049] This is in contrast to the term "perioperative period," which may refer, for example, to the period when a patient is undergoing surgery (e.g., when the patient is in the operating room) or the period immediately before or after, preferably the period beginning at least one hour before surgery and / or ending less than one hour after surgery.

[0050] The postoperative treatment of the present invention may be combined with perioperative and / or postoperative treatment strategies to improve effectiveness and preferably increase the duration or suppression of postoperative surgical pain (for example, in patients at high risk of developing surgical pain).

[0051] In one embodiment, the method of the present invention may include administering additional analgesics to the patient perioperatively and / or postoperatively (preferably postoperatively). In other words, additional analgesics may be administered during surgery (for example, during a period beginning at least one hour before surgery and / or ending less than one hour after surgery). In one embodiment, additional analgesics may be administered after surgery (for example, at 10, 20, 40, or 50 weeks postoperatively).

[0052] Postoperative surgical pain may be pain caused by the release of nociceptive afferent terminals of neurotransmitters and / or the release of neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) from sensory terminals, which are induced, for example, by noxious stimuli during surgery (preferably surgical incisions). For example, noxious information (resulting from a noxious stimulus) may then be transmitted from the peripheral nervous system to the central nervous system, where surgical pain is perceived by the patient.

[0053] In one embodiment, Clostridium neurotoxin treats surgical pain by inhibiting the exocytosis of pain neuromodulators such as substance P and CGRP.

[0054] Postoperative surgical pain can be caused by inflammation or nerve tissue damage at the site of surgical intervention, or a combination thereof. Some inflammation and / or nerve tissue damage may occur in addition to postoperative surgical pain. For example, the release of various substances, including proteases, cytokines, and serotonin, may occur as a result of degranulation of mast cells activated in response to tissue trauma. These substances can sensitize primary afferent neurons (activate them at the lower threshold), leading to pain hypersensitivity. Because tissues are innervated over a wide area, any area of ​​the body can be sensitive to nerve damage from surgery.

[0055] In other words, pain can be nociceptive pain, for example, in which case postoperative surgical pain arises from tissue damage and is perceived through the activation of nociceptors (pain receptors) in response to noxious stimuli.

[0056] In one embodiment, postoperative surgical pain may be neuropathic pain (e.g., pain caused by injury or disease affecting the somatosensory nervous system). For example, postoperative surgical pain may be peripheral neuropathy (also known as peripheral pain), in which case the pain may be due to injury to nerves outside the brain and spinal cord (peripheral nerves).

[0057] Postoperative surgical pain can also manifest as other types of pain, such as allodynia. Allodynia means "other pain." It is pain caused by stimuli that are not normally painful. Victims of "tactile" allodynia (also known as static tactile allodynia or mechanical allodynia) may experience pain in response to touch, such as when resting the surgically incised (body) site on a bed or when wearing clothing that comes into contact with the site. Thus, allodynia is considered "pain caused by stimuli that are not normally painful," in contrast to hypersensitivity (increased pain from stimuli that are not normally painful).

[0058] Postoperative surgical pain may preferably be acute postoperative surgical pain, for example, a type of surgical pain that may last for less than three months (post-surgery).

[0059] In one embodiment, postoperative surgical pain is a type of surgical pain that is chronic; for example, it may last for more than three months (post-surgery) and may continue to be perceived after tissue damage (e.g., resulting from a surgical incision) has healed.

[0060] More specifically, the term “chronic postoperative surgical pain,” as used herein, preferably refers to pain that persists for more than three months beyond the resolution of the underlying trauma (e.g., muscle injury from a surgical incision), for example, pain that persists for more than three months post-surgery. “Chronic postoperative surgical pain” can arise, for example, from inadequate treatment (or lack thereof) of acute postoperative surgical pain (e.g., a type of surgical pain that typically lasts less than three months). Poor management of postoperative “acute surgical pain” can increase the chances that such acute surgical pain will develop into chronic postoperative surgical pain. Therefore, by managing acute postoperative surgical pain at an early stage (advantageously, due to preoperative administration that provides analgesic effectiveness shortly after surgery), the present invention reduces the development of chronic postoperative surgical pain.

[0061] Chronic postoperative surgical pain may be perceived at or around the scar (the scar formed at the site of a surgical incision). In a preferred embodiment, chronic postoperative surgical pain is chronic scar pain. The term “chronic scar pain” refers to pain that arises as a result of tissue scarring. “Chronic scar pain” may arise from damage to the skin and / or muscle tissue and / or nerve tissue, as well as nerve regeneration.

[0062] In a preferred embodiment, postoperative surgical pain is surgical pain perceived at the site of the surgical intervention and / or surgical pain perceived at a site proximal to the site of the surgical intervention, preferably herein, the surgical pain is perceived within the tissue (e.g., skin, muscle) damaged through the surgical intervention. Postoperative surgical pain may also be surgical pain perceived at a site of internal tissue / organ relative to the body subjected to biopsy.

[0063] Preferably, administration of Clostridium neurotoxin reduces the level of surgical pain perceived by patients after surgery. For example, the level of surgical pain perceived by patients may be reduced after surgery compared to the level of surgical pain perceived in patients who were not administered Clostridium neurotoxin before surgery (e.g., more than 5 days before surgery) (control group).

[0064] In one embodiment, the patient's postoperative surgical pain perception is reduced within 24 hours post-surgery. In other words, administration of Clostridium neurotoxin can reduce the patient's postoperative surgical pain perception within 24 hours post-surgery. For example, administration of Clostridium neurotoxin can reduce the patient's postoperative pain perception within 6 hours post-surgery, preferably within 1 hour post-surgery.

[0065] The patient's postoperative surgical pain perception may be reduced for at least 3 days, at least 6 days, or at least 9 days postoperatively; for example, at least 15 days postoperatively; in another example, at least 30 days postoperatively. In a preferred embodiment, the patient's postoperative surgical pain may be reduced for up to 3 months (including 3 months) postoperatively.

[0066] The reduced postoperative surgical pain perception in patients may be maintained for at least 5 days, at least 7 days, or at least 9 days postoperatively, preferably at least 9 days postoperatively.

[0067] In one embodiment, administration of Clostridium neurotoxin substantially reduces the patient's postoperative surgical pain perception, and this reduction is maintained for 24 hours immediately following surgery. In another embodiment, the patient's postoperative surgical pain perception is substantially reduced and maintained for 2 days immediately following surgery. In one embodiment, substantially all of the reduced postoperative surgical pain perception is maintained for 3 days immediately following surgery. In one embodiment, substantially all of the reduced postoperative surgical pain perception is maintained for 4 days immediately following surgery. In one embodiment, substantially all of the reduced postoperative surgical pain perception is maintained for 5 days immediately following surgery. In one embodiment, substantially all of the reduced postoperative surgical pain perception is maintained for 6 days immediately following surgery. In one embodiment, substantially all of the reduced postoperative surgical pain perception is maintained for 7 days immediately following surgery. Preferably, substantially all of the reduced postoperative surgical pain perception is maintained for 8 days immediately following surgery.

[0068] In one embodiment, the reduced level of pain perception observed at a specified time immediately after surgery (as described in the preceding paragraph) is at least 50% of the maximum level of reduced pain perception observed at any time after the administration of Clostridium neurotoxin. For example, the reduced level of pain perception observed at a specified time immediately after surgery (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 the administration of Clostridium neurotoxin.

[0069] More specifically, the reference to “reduced” (with respect to postoperative surgical pain) preferably means that subjects (e.g., patients) who have been administered Clostridium neurotoxin perceive a lower level of surgical pain compared to subjects who have not been administered Clostridium neurotoxin (or who have been administered a placebo) (similarly subjected to surgery). For example, the level of perceived surgical pain may be reduced by at least 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, or 95% after administration of Clostridium neurotoxin compared to subjects who have not been administered Clostridium neurotoxin (or who have been administered a placebo) (similarly subjected to surgery). For example, the level of perceived surgical pain may be reduced by at least 75%; preferably at least 85%; more preferably at least 95% after administration of Clostridium neurotoxin compared to subjects who have not been administered Clostridium neurotoxin (or who have been administered a placebo) (similarly subjected to surgery).

[0070] Various methods for evaluating pain perception are known to those skilled in the art. For example, the evaluation of mechanical allodynia (either static or dynamic) is routinely used in human pain studies, such as that described by Pogatzki-Zahn et al. (Pain Rep. 2017 Mar;2(2): e588), which is incorporated herein by reference.

[0071] Suitable (but not limited to) methods for evaluating pain perception in subjects include: Numerical Rating Scale (NRS) scores; however, those skilled in the art are aware of other methods that may be used additionally or alternatively, such as sensory thresholds, pain perception thresholds, static mechanical allodynia, dynamic mechanical allodynia, temporal weighting, pressure pain thresholds, conditioned pain modulation, and temperature thresholds.

[0072] Other non-limiting examples of pain perception scales include: the change from baseline in the SF-36 score at each scheduled time point; the amount of emergency medication taken during the study; and the time to the first dose of emergency medication. These may be considered “exploratory” assessment items or pain perception assessment scales.

[0073] Therefore, in a preferred embodiment, postoperative surgical pain perception after administration of Clostridium neurotoxin can be evaluated by one or more of the following: (a) Numerical Rating Scale (NRS); (b) Stimulus-induced NRS; (c) Temperature of the painful area; (d) Size of the painful area; (e) Time to onset of analgesia; (f) Peak analgesia; (g) Time to peak analgesia; (h) Duration of analgesia; and (i) SF-36 quality of life.

[0074] Those skilled in the art are familiar with these methods for evaluating pain perception. For convenience, the Numerical Rating Score and the Quality of Life Questionnaire Short Form-36 are described further below.

[0075] Numerical Rating Scale (NRS): Typically, surgical pain perception according to the present invention uses a Numerical Rating Scale (NRS). The NRS is an 11-point scale for evaluating a subject's surgical pain perception. Subjects are asked to give a number between 0 and 10 that best fits their surgical pain intensity. Zero represents "no surgical pain," and the upper limit of 10 represents "the worst possible surgical pain."

[0076] The Number Rating Scale (NRS) can be used to assess multiple aspects of surgical pain, including spontaneous mean surgical pain, spontaneous worst surgical pain, and spontaneous current surgical pain. Spontaneous mean surgical pain is assessed by asking subjects to select a number that best describes their average surgical pain (e.g., perceived surgical pain) over a period of time, e.g., at least 6, 12, 24, or 48 hours. Spontaneous worst surgical pain is assessed by asking subjects to select a number that best describes their worst surgical pain during a specific period, e.g., at least the preceding 6, 12, 24, or 48 hours. Spontaneous current surgical pain is assessed by asking subjects to select a number that best describes their level of surgical pain at the time of assessment.

[0077] The NRS can also be used to assess surgical pain perception in subjects in response to a variety of different stimuli. To assess surgical pain perception in response to stimuli, subjects are subjected to stimuli of various natures applied to the painful area. Subjects are asked what their current NRS score is before administration and after stimulation.

[0078] Examples of stimuli used include: (i) light touch (which can be assessed by measuring pain on the surface of the painful area on the radial spokes after application of von Frey filaments as described herein); (ii) pressure (pressure pain threshold), which can be assessed by asking subjects to provide NRS scores while increasing pressure is applied using a pressure analgesic as described herein; and (iii) temperature (which can be assessed by asking subjects to provide NRS scores for warm, cold, and hot stimuli using a thermal pole applied to the painful area as described herein).

[0079] Preferably, administration of the Clostridium neurotoxin described herein reduces the NRS score of a postoperative patient compared to the NRS score of a control patient who has not been administered Clostridium neurotoxin (for example, from a score of ≥7 to a score of ≤6).

[0080] Quality of Life Questionnaire Short Form 36 (SF-36): The SF-36 Quality of Life Questionnaire can be used to assess a subject's perception of surgical pain. The SF-36 is a 36-item subject-reported survey of a subject's health. The SF-36 consists of eight scaled scores: vitality, physical functioning, bodily pain, overall health perception, physical role functioning, mental role functioning, social role functioning, and mental health. Each scale is directly converted to a 0-100 scale, assuming that each question has equal weight. A higher score recorded on the SF-36 indicates less physical impairment.

[0081] The relevant parameters commonly tested in clinical trials for the treatment of surgical pain 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, the NRS (Natural Rating Scale); stimulus-induced NRS; temperature of the painful area; size of the painful area; time to onset of analgesia; peak analgesia; duration of analgesia; and / or SF-36 quality of life, as described herein. Methods for evaluating these parameters are also known in the art and can be performed by those skilled in the art using routine methods and procedures.

[0082] Preferably, administration of the Clostridium neurotoxin described herein increases the SF-36 score of a postoperative patient compared to the SF-36 score of a control patient who has not received Clostridium neurotoxin (for example, from a score of ≤50 to a score of ≥50).

[0083] In this context, "postoperative anxiety" refers to a state in which a patient experiences physical symptoms and behavioral changes, including but not limited to fatigue, difficulty concentrating and sleeping, and muscle tension. Furthermore, patients may experience emotional symptoms of anxiety, including restlessness, irritability, difficulty controlling fear or worry, phobias, and panic. Postoperative anxiety can be caused by the effects of anesthesia, the surgery itself, postoperative surgical pain, and stress from the hospital environment.

[0084] Therefore, in one embodiment, postoperative anxiety is caused by postoperative surgical pain.

[0085] Postoperative anxiety can be defined as panic disorder, phobia, post-traumatic stress disorder, social anxiety disorder (social phobia), or generalized anxiety disorder (GAD) (for example, a long-term condition in which you feel anxious about a broad range of situations and issues rather than one specific event).

[0086] In one embodiment, a method comprising administering Clostridium neurotoxin to a patient before surgery for use in reducing or suppressing postoperative anxiety is administered at least five days before surgery, preferably the Clostridium neurotoxin is administered more than five days before surgery.

[0087] In one embodiment, administration of Clostridium neurotoxin substantially reduces the patient's perception of postoperative anxiety, and this reduced perception of postoperative anxiety is maintained for 24 hours immediately following surgery. In one embodiment, substantially all of the reduced perception of postoperative anxiety is maintained for 2 days immediately following surgery. In one embodiment, substantially all of the reduced perception of postoperative anxiety is maintained for 3 days immediately following surgery. In one embodiment, substantially all of the reduced perception of postoperative anxiety is maintained for 4 days immediately following surgery. In one embodiment, substantially all of the reduced perception of postoperative anxiety is maintained for 5 days immediately following surgery. In one embodiment, substantially all of the reduced perception of postoperative anxiety is maintained for 6 days immediately following surgery. In one embodiment, substantially all of the reduced perception of postoperative anxiety is maintained for 7 days immediately following surgery. In one embodiment, substantially all of the reduced perception of postoperative anxiety is maintained for 8 days immediately following surgery. Preferably, substantially all of the reduced perception of postoperative anxiety is maintained for 9 days immediately following surgery.

[0088] Preferably, administration of the Clostridium neurotoxin described herein reduces postoperative anxiety symptoms by (e.g., 30%, 50%, 75%, or 95%) compared to the symptoms of control patients who have not received the Clostridium neurotoxin. Examples of postoperative anxiety symptoms include restlessness, irritability, difficulty controlling fear or worry, phobias, and panic.

[0089] More specifically, the reference to “reduced” (with respect to postoperative anxiety) preferably means that subjects (e.g., patients) who received Clostridium neurotoxin perceived a lower level of anxiety compared to subjects who did not receive Clostridium neurotoxin (or received a placebo) (similarly subjected to surgery). For example, the level of perceived anxiety may be reduced by at least 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, or 95% after administration of Clostridium neurotoxin compared to subjects who did not receive Clostridium neurotoxin (or received a placebo) (similarly subjected to surgery). For example, the level of perceived anxiety may be reduced by at least 75%; preferably at least 85%; more preferably at least 95% after administration of Clostridium neurotoxin compared to subjects who did not receive Clostridium neurotoxin (or received a placebo) (similarly subjected to surgery).

[0090] In one embodiment, postoperative anxiety experienced by the patient is suppressed within 24 hours post-surgery. In other words, administration of Clostridium neurotoxin may reduce or suppress the patient's postoperative anxiety within 24 hours post-surgery. For example, the patient's postoperative anxiety may be reduced or suppressed within 2 hours post-surgery, within 4 hours post-surgery, or within 24 hours post-surgery; preferably within 4 hours post-surgery.

[0091] The inventors have demonstrated that Clostridium neurotoxin can be administered for both the treatment of postoperative pain and the suppression of postoperative anxiety. Therefore, in one embodiment, Clostridium neurotoxin treats postoperative pain and reduces or suppresses postoperative anxiety.

[0092] In summary, the present invention advantageously improves the patient's overall "postoperative health condition" and thus improves the patient's quality of life by reducing the levels of both surgical pain and anxiety that would otherwise be perceived after surgery / operation.

[0093] Therefore, in one embodiment, administration of Clostridium neurotoxin promotes a healthy postoperative state.

[0094] Further details of the Clostridium neurotoxin included in the present invention, along with technical background information, are provided below.

[0095] Bacteria of the genus Clostridium produce extremely potent and specific protein toxins that can poison neurons and other cells to which they are delivered. Examples of such Clostridium toxins include neurotoxins produced by Clostridium tetani (TeNT) and Clostridium botulinum (BoNT) serotypes A-G, as well as those produced by Clostridium barati and Clostridium butyricum.

[0096] Clostridium neurotoxin (in nature, for example) can cause muscle paralysis and thus death by inhibiting cholinergic transmission in the peripheral nervous system, particularly at the neuromuscular junction. In nature, Clostridium neurotoxin is synthesized as a single-chain polypeptide, which is post-translationally modified by a protein cleavage event to form two polypeptide chains linked together by disulfide bonds. The cleavage often occurs at a specific cleavage site, often called the activation site, located between cysteine ​​residues that provide the interchain disulfide bond. This two-chain form is the active form of the toxin. These two chains are called the heavy chain (H chain) and the light chain (L chain), with the heavy chain having a molecular weight of approximately 100 kDa and the light chain having a molecular weight of approximately 50 kDa. The H chain contains an N-terminal transporter (HN domain) and a C-terminal targeting component (HC domain). The cleavage site is located between the L chain and the HN domain.

[0097] The mechanism of action of Clostridium neurotoxin relies on the following five distinct steps: (1) binding of the HC domain to the cell membrane of its target neuron, followed by (2) internalization of the bound toxin into the cell via endosomes, (3) translocation of the L chain across the endosomal membrane by the HN domain 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 cytosecretion from target cells.

[0098] Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g., SNAP-25, VAMP, or syntaxin) (see Gerald K (2002) "Cell and Molecular Biology" (4th edition), John Wiley & Sons, Inc.). The acronym SNARE stands for Soluble NSF Attachment Receptor, where NSF stands for N-ethyl maleimide-Sensitive Factor. SNARE proteins are essential for intracellular vesicle fusion and, therefore, for the secretion of molecules via vesicle transport from cells. This protease function is zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins. Thus, once delivered to the desired target cells, non-cytotoxic proteases can inhibit cellular secretion from the target cells. Clostridium neurotoxin's light chain protease is a cytotoxic protease that cleaves SNARE proteins.

[0099] Thanks 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 disorders, for example, to restore the activity of hyperactive nerve endings to normal levels. To date, at least seven antigenically distinct BoNT serotypes have been described: 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).

[0100] Despite this diversity, BoNT / A remains the serotype of choice in therapy, with three commonly available commercial formulations (Botox®, Dysport®, and Xeomin®), while only one BoNT / B product is available on the market (Neurobloc® / Myobloc®). To date, these BoNT / A and BoNT / B products, toxins purified from Clostridium strains, are the only two BoNT serotypes currently approved by regulatory authorities for use in humans, ranging from (for BoNT / A) spasticity, bladder dysfunction, or hyperhidrosis (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) to (for BoNT / B) spasmodic torticollis (see, for example, https: / / www.medicines.org.uk / emc / medicine / 20568, all of which are incorporated herein by reference).

[0101] Unlike cytotoxic proteases (e.g., lysine, diphtheria toxin, Pseudomonas exotoxin) that act by killing their natural target cells, Clostridium neurotoxins are acytotoxic proteases that act by transiently incapacitating the cellular functions of their natural target cells. Importantly, acytotoxic proteases do not kill the natural target cells on which they act. In addition to Clostridium neurotoxins (e.g., botulinum neurotoxin sold under names such as Dysport®, Neurobloc®, and Botox®), some of the best-known examples of acytotoxic proteases include IgA proteases (e.g., see WO99 / 032272) and Antarease proteases (e.g., see WO2011 / 022357).

[0102] As used herein, the term “Clostridium neurotoxin” means any polypeptide that enters neurons and inhibits the release of neurotransmitters. This process includes the binding of the neurotoxin to low-affinity or high-affinity receptors, internalization of the neurotoxin, translocation of the endopeptidase portion of the neurotoxin into the cytoplasm, and enzymatic modification of the neurotoxin substrate. More specifically, the term “neurotoxin” includes any polypeptide produced by Clostridium bacteria that enters neurons and inhibits the release of neurotransmitters (Clostridium neurotoxin), and such polypeptides produced by recombinant or chemical techniques. Preferably, the Clostridium neurotoxin is botulinum neurotoxin (BoNT).

[0103] BoNT serotypes A–G can be distinguished based on inactivation with specific neutralizing antisera, and such serotyping is correlated with percentage sequence identity at the amino acid level. A given serotype of BoNT protein can be further divided into different subtypes based on percentage amino acid sequence identity.

[0104] Examples of BoNT / A neurotoxin amino acid sequences are provided as SEQ ID NO: 1 (UniProt accession number A5HZZ9) and SEQ ID NO: 13, which are encoded by the nucleotide sequence provided as SEQ ID NO: 12. An example of BoNT / B neurotoxin amino acid sequence is provided as SEQ ID NO: 2 (UniProt accession number B1INP5). An example of BoNT / C neurotoxin amino acid sequence is provided as SEQ ID NO: 3 (UniProt accession number P18640). An example of BoNT / D neurotoxin amino acid sequence is provided as SEQ ID NO: 4 (UniProt accession number P19321). An example of BoNT / E neurotoxin amino acid sequence is provided as SEQ ID NO: 5 (accession number WP_003372387). An example of 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 neurotoxin 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, and more preferably wild-type BoNT / A.

[0105] The term “H C As used herein, "domain" refers to a functionally distinct region of a neurotoxin heavy chain having a molecular weight of approximately 50 kDa that enables the binding of the neurotoxin to receptors located on the surface of target cells. C The domain consists of two structurally different subdomains, "H CN subdomain (H C The N-terminal portion of the domain) and "H CC subdomain (H C It consists of a C-terminal domain (also called the Hcc domain), each of which has a molecular weight of approximately 25 kDa. CCThe domain can bind to a Clostridium neurotoxin protein receptor.

[0106] The term "LH N domain", as used herein, refers to a neurotoxin region different from the H C domain, and consists of an endopeptidase domain ("L" or "light chain") and a domain (the H N domain of the heavy chain) responsible for transferring the endopeptidase to the cytoplasm. The endopeptidase domain ("L" or "light chain") can cleave SNARE proteins.

[0107] Representative L, H N 、H CN 、and H CC domains are shown in Table 1.

Table 1

[0108] Since slight variations may occur depending on the serum subtype, the reference sequences identified above should be regarded as a guide. As an example, US 2007 / 0166332 (incorporated herein by reference in its entirety) cites slightly different Clostridium sequences.

[0109] The term "activation loop" refers to a polypeptide domain containing a proteolytic cleavage site. The activation loop of the neurotoxin has been described in the art, such as in WO2016156113 (incorporated herein by reference in its entirety).

[0110] In one embodiment, the Clostridium neurotoxin consists of or comprises an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any 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: eight, SEQ ID NO: 9, or SEQ ID NO: 11.

[0111] In one embodiment, the Clostridium neurotoxin consists of or comprises an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with respect to SEQ ID NO: 1.

[0112] In one embodiment, the Clostridium neurotoxin consists of or includes the amino acid sequence of SEQ ID NO: 1 (e.g., BoNT / A).

[0113] In one embodiment, Clostridium neurotoxin is a chimeric neurotoxin.

[0114] The term "chimeric neurotoxin," as used herein, 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 is LH derived from the serotype or subtype of the first neurotoxin. N Domain and H derived from the second neurotoxin serotype or subtype C It may contain domains. Another example of a chimeric neurotoxin is LH derived from the first neurotoxin serotype or subtype. N H CN Domain and H derived from the second neurotoxin serotype or subtype CC It is a neurotoxin containing a domain. Further examples of chimeric neurotoxins include LH from the first neurotoxin serotype or subtype. N The neurotoxin comprises a domain and an activation loop from a second neurotoxin serotype or subtype. Examples of chimeric neurotoxins are provided in WO2017191315 and WO2016156113, both of which are incorporated herein by reference in their entirety.

[0115] For example, chimeric neurotoxins are produced by H from a second neurotoxin. C LH from the first neurotoxin covalently bound to the domain N It may include a domain, but preferably the first neurotoxin and the second neurotoxin are different, and here the LH N The C-terminal amino acid residue of the domain is LH in the first neurotoxin. N Domain and H C3 Separating the domain 10 This corresponds to the first amino acid residue of the helix, and here, the H C The N-terminal amino acid residue of the domain is LH in the second neurotoxin. N Domain and H C 3 Separating the domain 10 This corresponds to the second amino acid residue of a helix.

[0116] In one embodiment, Clostridium neurotoxin is H from BoNT / B C LH from the domain and BoNT / A, BoNT / C, BoNT / D, BoNT / E, BoNT / F, or BoNT / G N It is a chimeric neurotoxin containing a domain.

[0117] For example, in one embodiment, H C The domain consists of or includes an amino acid sequence corresponding to amino acid residues 860-1291 of SEQ ID NO: 2 (e.g., BoNT / B), or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and LH N A domain consists of or contains an amino acid sequence selected from the following group: • A sequence having sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% of amino acid residues 1-872 of sequence number 1 (e.g., BoNT / A), or thereto. • A sequence having sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% of amino acid residues 1-867 of SEQ ID NO: 3, or a sequence having sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% thereto. • A sequence having sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% of amino acid residues 1-863 of SEQ ID NO: 4, or a sequence having sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% thereto. • A sequence having sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% of amino acid residues 1-846 of SEQ ID NO: 5, or a sequence having sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% thereto. • A sequence having sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% of amino acid residues 1-865 of SEQ ID NO: 6, • A sequence having sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% of amino acid residues 1-864 of SEQ ID NO: 7, or a sequence having sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% thereto. • A sequence having sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% of amino acid residues 1-863 of SEQ ID NO: 8, and • A sequence having sequence identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% of amino acid residues 1 to 862 of SEQ ID NO: 9.

[0118] In a preferred embodiment, Clostridium neurotoxin is H from BoNT / B C LH from domain and BoNT / A N It is a chimeric neurotoxin containing a domain.

[0119] In a more preferred embodiment, H C The domain consists of or includes an amino acid sequence corresponding to amino acid residues 860-1291 of SEQ ID NO: 2, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and LH N The domain contains an amino acid sequence corresponding to amino acid residues 1 to 872 of SEQ ID NO: 1, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0120] Clostridium neurotoxin from BoNT / B C Embodiments including a domain (for example, here, Clostridium neurotoxin is BoNT / B, or H from BoNT / B) CClostridium neurotoxins, being chimeric neurotoxins containing domains, provide a "modified heavy chain" (e.g., H C The heavy chain (in the domain) may have one or more modifications in its amino acid sequence, preferably the modified heavy chain binds to target nerve cells with higher (or lower) affinity than the unmodified (native) neurotoxin. C Such modifications in the domain include H2, which can alter the binding of gangliosides to target neurons. CC Modification of amino acid residues in the ganglioside binding site of the domain, and / or alteration of binding to protein receptors on target neurons. CC Modifications of amino acid residues in the protein receptor binding site of the domain can be cited. Examples of such modified neurotoxins are described in their entirety in WO2006027207 and WO2006114308, which are incorporated herein by reference.

[0121] Clostridium neurotoxins having one or more modifications to the heavy chain amino acid sequence are referred to as "modified Clostridium neurotoxins" in this specification.

[0122] In one embodiment of the modified Clostridium neurotoxin according to the present invention, H from BoNT / B CC The domain is the native H of the BoNT serotype. CC It is modified in comparison to the domain.

[0123] In a preferred embodiment, H from BoNT / B neurotoxin CC The domain is the aforementioned H CC The binding affinity of the domain to human SytII is compared to that of natural BoNT / B H CC This includes a mutation of at least one amino acid residue that increases the domain compared to the domain. Preferably, the at least one amino acid residue mutation is H CC The binding affinity of the domain to human SytII is compared to that of natural BoNT / BH CC Increase the domain size by at least 50%.

[0124] BoNT / BH CC Such preferred amino acid residue mutations in the domain are described in the art in WO2013180799 and WO2016154534, both of which are incorporated herein by reference in their entirety.

[0125] In particular, BoNT / BH CC The binding affinity of the domain to human SytII is compared to natural BoNT / BH CC The at least one amino acid residue mutation suitable for increasing the domain 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 BoNT / BH CC The mutation of at least one amino acid residue in the domain consists of the 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. Preferably BoNT / BH CC The mutation of at least one amino acid residue in the domain consists of the substitution, addition, or deletion of the following three amino acid residues: 1191M, 1199W, and 1178Q. More preferably, BoNT / BH CC The mutation of at least one amino acid residue in the domain consists of the substitution, addition, or deletion of the following two amino acid residues: 1191M and 1199Y.

[0126] In a more preferred embodiment, BoNT / BH CC The binding affinity of the domain to human SytII is compared to natural BoNT / BH CC A mutation of at least one amino acid residue suitable for increasing the domain by at least 50% is a substitution 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 BoNT / BH CC The mutation of at least one amino acid residue in the 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. Preferably BoNT / BH CC The mutation of at least one amino acid residue in the domain consists of the following three amino acid residue substitutions: E1191M, S1199W, and W1178Q. More preferably, BoNT / BH CC The mutation of at least one amino acid residue in the domain consists of the following two amino acid residue substitutions: E1191M and S1199Y.

[0127] In a preferred embodiment, the modified BoNT / BH CC The domain is amino acid residues 1082-1291 of Sequence ID No. 2 (natural BoNT / BH CCThis corresponds to a domain, or an amino acid sequence having at least 70%, preferably at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0128] In one embodiment, the Clostridium neurotoxin of the present invention may be both chimeric and modified, as described above. For example, in a preferred embodiment, the Clostridium neurotoxin comprises (or consists of) an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, or amino acid sequence SEQ ID NO: 10.

[0129] In one embodiment, the Clostridium neurotoxin of the present invention may be both chimeric and modified as described above. For example, in a preferred embodiment, the Clostridium neurotoxin may be an amino acid sequence of SEQ ID NO: 10 (e.g., BoNT / AB). MY ), or comprising (or consisting of) an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

[0130] The Clostridium neurotoxin of the present invention can be produced using recombinant technology. Therefore, in one embodiment, the Clostridium neurotoxin of the present invention is a recombinant Clostridium neurotoxin.

[0131] The use of such recombinant neurotoxins is advantageous in that it broadens the options for the use of Clostridium neurotoxins in the methods described in this invention, which are selected based on properties such as resistance to action and duration of action that are deemed appropriate for any given surgery. Suitable (known) recombinant Clostridium neurotoxins include modified botulinum neurotoxin A (BoNT / A) which has a longer duration of action compared to unmodified BoNT / A (e.g., Dysport®). The duration of action can be at least 1.25 times, 1.5 times, 1.75 times, 2.0 times, or 2.25 times longer. The duration of action of modified BoNT / A may be between 6 and 9 months. For example, the duration of action may be at least: 4.5 months (from the 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.

[0132] Preferred modified BoNT / A polypeptides (and, if present, nucleotide sequences encoding them) are described in WO2015 / 004461A1 and WO2017 / 191315, both of which are incorporated herein by reference as a whole.

[0133] More specifically, in one embodiment, Clostridium neurotoxin is a modified recombinant BoNT / A neurotoxin. In one embodiment, the modified BoNT / A includes modifications at one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP 1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, preferably wherein the modification is selected from: (i) substitution of an amino acid residue exposed to an acidic surface with a basic amino acid residue; (ii) substitution of an amino acid residue exposed to an acidic surface with an uncharged amino acid residue; (iii) substitution of an amino acid residue exposed to an uncharged surface with a basic amino acid residue; (iv) insertion of a basic amino acid residue; and (v) deletion of an amino acid residue exposed to an acidic surface.

[0134] The modifications described herein may be modifications compared to an unmodified BoNT / A sequence shown as SEQ ID NO: 1, where the numbering of amino acid residues is determined by alignment with SEQ ID NO: 1. Since the presence of a methionine residue at position 1 of SEQ ID NO: 1 (and the SEQ ID NOs corresponding to the modified BoNT / A polypeptide described herein) is optional, those skilled in the art will consider the presence or absence of the methionine residue when determining the numbering of amino acid residues. For example, if SEQ ID NO: 1 contains methionine, the position numbering will be as described above (e.g., ASN886 is ASN886 of SEQ ID NO: 1). Alternatively, if methionine is not present in SEQ ID NO: 1, the numbering of amino acid residues should be corrected by -1 (e.g., ASN886 is ASN885 of SEQ ID NO: 1). Similar considerations apply to the presence or absence of methionine at position 1 of other polypeptide sequences described herein, and those skilled in the art will readily determine the correct numbering of amino acid residues using the common art in that field. The same applies to any other BoNTs described herein (e.g., the chimeric BoNTs described above).

[0135] The amino acid residues specified for modification are those exposed on the surface.

[0136] Modified BoNT / A can be encoded by a nucleic acid sequence having at least 70% sequence identity to 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 to a nucleic acid sequence selected from SEQ ID NOs: 14, 16, 18, and 20. Preferably, Modified BoNT / A for use in the present invention can be encoded by a nucleic acid sequence including (or consisting of) SEQ ID NOs: 14, 16, 18, or 20. Modified BoNT / A may include a polypeptide sequence having at least 70% sequence identity to 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 to a polypeptide sequence selected from SEQ ID NOs: 15, 17, 19, and 21. Preferably, the modified BoNT / A for use in the present invention may include (more preferably consist of) polypeptide sequences selected from SEQ ID NOs: 15, 17, 19, and 21.

[0137] The term "one or more amino acid residues," when used in the context of modified BoNT / A, preferably means at least 2, 3, 4, 5, 6, or 7 of the specified amino acid residues. Therefore, modified BoNT / A may include at least 2, 3, 4, 5, 6, or 7 (preferably 7) modifications on the specified amino acid residues. Modified BoNT / A may include 1 to 30, 3 to 20, or 5 to 10 amino acid modifications. More preferably, the term "one or more amino acid residues," when used in the context of modified BoNT / A, means all of the specified amino acid residues.

[0138] Preferably, modified BoNT / A contains no further amino acid modifications other than one or more amino acid modifications at the specified amino acid residue, compared to SEQ ID NO: 1.

[0139] Most preferably, modified BoNT / A comprises (more preferably consists of) modifications at one or more amino acid residues selected from the following: ASN886, ASN930, SER955, GLN991, ASN1026, ASN1052, and GLN1229. Modified BoNT / A can be encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 14. For example, a nucleic acid sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 14. Preferably, modified BoNT / A for use in the present invention can be encoded by a nucleic acid comprising (or comprising) SEQ ID NO: 14. Modified BoNT / A may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 15. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 15. Preferably, modified BoNT / A for use in the present invention may comprise (more preferably consist of) SEQ ID NO: 15.

[0140] The modifications may be selected from the following: (i) substitution of an amino acid residue exposed to an acidic surface with a basic amino acid residue; (ii) substitution of an amino acid residue exposed to an acidic surface with an uncharged amino acid residue; (iii) substitution of an amino acid residue exposed to an uncharged surface with a basic amino acid residue; (iv) insertion of a basic amino acid residue; and (v) deletion of an amino acid residue exposed to an acidic surface.

[0141] The modifications specified above result in modified BoNT / A having an increased positive surface charge and an increased isoelectric point compared to the corresponding unmodified BoNT / A.

[0142] The isoelectric point (pI) is a specific property of a given 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 the protein to exhibit a net charge of zero. Therefore, 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 the protein to exhibit a net charge of zero. Therefore, a decrease in pI represents a decrease in the net positive charge of the protein at a given pH.

[0143] Methods for determining the pI of a protein are known in the art and are well known to those skilled in the art. For example, the pI of a protein can be calculated from the average pKa value of each amino acid present in the protein ("calculated pI"). Such calculations 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. Comparison 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 experimentally verified using the technique of isoelectric focusing ("observed pI"). This technique separates proteins by their pI using electrophoresis. Isoelectric focusing is typically performed using a gel with a fixed pH gradient. When an electric field is applied, the protein moves through the pH gradient until it reaches a pH at which it has a net charge of zero, and this point is the pI of the protein. The results obtained by isoelectric focusing are typically of relatively low resolution, and therefore, the inventors believe that the results obtained by computational pI (as described above) are more suitable for use.

[0144] Throughout this specification, "pI" means "calculated pI" unless otherwise specified. The pI of a protein can be increased or decreased by altering the number of basic and / or acidic groups present on its surface. This can be achieved by modifying one or more amino acids in the protein. For example, an increase in pI may be provided by decreasing the number of acidic residues or increasing the number of basic residues.

[0145] 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 the pI value of 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.

[0146] The properties of 20 standard amino acids are shown in the table below. [Table A]

[0147] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0148] 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 called acidic amino acid residues. Arginine and lysine are called basic amino acid residues.

[0149] The following amino acids are considered uncharged polar (meaning they can participate in hydrogen bonding): asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan. The following amino acids are considered uncharged hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0150] Amino acid insertion involves incorporating an additional amino acid residue (which is normally absent) into the BoNT / A polypeptide sequence, thereby increasing the total number of amino acid residues in the sequence. Amino acid deletion involves removing an amino acid residue from the Clostridium toxin amino acid sequence, thereby decreasing the total number of amino acid residues in the sequence.

[0151] Preferably, the modification is a substitution, and the substitution advantageously maintains the same number of amino acid residues in the modified BoNT / A. In an amino acid substitution, an amino acid residue that forms part of the BoNT / A polypeptide sequence is replaced with a different amino acid residue. The substituted amino acid residue may be one of the 20 standard amino acids described above. Alternatively, the substituted amino acid in an amino acid substitution may be a non-standard amino acid (an amino acid that is not part of the 20 standard set described 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-isomers of ornithine). Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using an E. coli nutrient-requiring host.

[0152] In one embodiment, the substitution is selected from: substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue. In one embodiment, the substitution is the substitution of an acidic amino acid residue with an uncharged amino acid residue, where the acidic amino acid residue is replaced by its corresponding uncharged amide amino acid residue (i.e., aspartic acid is replaced by asparagine, and glutamic acid is replaced by glutamine).

[0153] 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.

[0154] Following the modification according to the present invention, the modified BoNT / A is capable of binding to the target cell receptor to which the unmodified BoNT / A (e.g., SEQ ID NO: 1) binds.

[0155] While the preferred modified (recombinant) BoNT / A neurotoxin described above had, for example, a long duration of action, preferred modified (recombinant) BoNT / E neurotoxin, which may act relatively quickly and / or have a short duration of action, is described below. This also demonstrates the advantageous flexibility provided by Clostridium neurotoxin-based therapies of the present invention. For example, BoNT / E, which provides a shorter duration of action, may be employed for minimally invasive surgery (for example, where prolonged postoperative pain is not expected).

[0156] 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 respect to SEQ ID NO: 5, provided that this polypeptide sequence contains one or more of the following amino acids (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8, preferably all 8) (where the amino acid positions are numbered starting at the N-terminal methionine amino acid residue of the BoNT / E protein and ending at 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; asparagine at position 1195.

[0157] The aforementioned amino acids may be substitutions (e.g., mutations) to the wild-type BoNT / E polypeptide sequence (such as the UniProtQ00496 sequence). For example, glycine at position 177 may be a substitution from arginine to glycine (R177G); serine at position 198 may be a C198S substitution; alanine at position 340 may be an R340A substitution; leucine at position 773 may be an I173L substitution; leucine at position 963 may be an F963L substitution; glutamine at position 964 may be an E964Q substitution; alanine at position 967 may be an R967A substitution; and / or asparagine at position 1195 may be an insertion (e.g., an insertion between G1194 and N1195 of the wild-type BoNT / E sequence, such as the UniProtQ00496 polypeptide sequence).

[0158] In one embodiment, the presence of one or more amino acids as described above provides a BoNT / E protein with improved solubility compared to a BoNT / E protein lacking the amino acids. This improved solubility increases the yield of the protein in heterologous expression systems such as E. coli expression systems.

[0159] In one embodiment, the presence of the one or more amino acids described above provides a BoNT / E protein having improved efficacy compared to a BoNT / E protein lacking the amino acids. The improved efficacy is preferably improved in vivo efficacy (more preferably improved in vivo efficacy in human subjects).

[0160] In one embodiment, BoNT / E is described in (or encoded by the nucleotide sequence described herein) in WO 2014 / 068317 A1, which is incorporated herein by reference.

[0161] Preferably, the Clostridium neurotoxin (for example, in the uses described herein) is part of the pharmaceutical composition together with at least one pharmaceutically acceptable carrier. “pharmaceutically acceptable carrier” as used herein means any component that is compatible with the other components of the pharmaceutical composition, particularly the Clostridium neurotoxin, and is not harmful to human patients. pharmaceutically acceptable carriers may be selected, and are not limited to, excipients, diluents, adjuvants, propellants, and salts, based on the desired route of administration and in accordance with standard pharmaceutical practices.

[0162] Accordingly, the present invention further relates to a pharmaceutical composition for use in the treatment of 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 dose of Clostridium neurotoxin to be administered to the patient is as described above. The present invention also encompasses corresponding uses and methods for treating postoperative surgical pain and / or anxiety, which include administering the pharmaceutical composition of the present invention to human patients. In another embodiment, the present invention relates to a pharmaceutical composition for use in promoting a postoperative state of well-being, wherein the postoperative state of well-being is reduced postoperative surgical pain and anxiety.

[0163] The Clostridium neurotoxin of the present invention can preferably be formulated for intradermal administration.

[0164] The preferred route of administration is intradermal administration. Preferably, intradermal administration means intradermal injection.

[0165] Preferably, the BoNT used to treat postoperative surgical pain and / or postoperative anxiety is purified BoNT. As used herein, the term “purified BoNT” means botulinum neurotoxin purified from a Clostridium strain that naturally produces its neurotoxin (a Clostridium strain of natural origin) or purified using recombinant technology. Purified BoNT / A may be associated with or free from a complexing protein, but preferably free from a complexing protein. Thus, in one embodiment, the Clostridium neurotoxin is associated with a BoNT complexing protein, also known as a non-toxic neurotoxin-associated protein (NAP). In other words, the Clostridium neurotoxin is administered to a human patient in association with or in combination with a BoNT complexing protein. Thus, in one embodiment, the Clostridium neurotoxin is complexed with one or more BoNT complexing proteins. Examples of commercially available purified and complexed protein-related BoNT / A include Botox®, Dysport® (associated with BoNT complexed proteins), and Xeomin® (purified).

[0166] In another embodiment, the Clostridium neurotoxin is not associated with (or combined with) the BoNT complex protein. In other words, the Clostridium neurotoxin is administered to a human patient without association with or combination with the BoNT complex protein.

[0167] The dose of Clostridium neurotoxin can be measured in nanograms.

[0168] The dose of Clostridium neurotoxin according to the present invention should be understood as the dose of active double-chain Clostridium neurotoxin, i.e., the amount of the complexing protein to which the neurotoxin may be associated. In other words, it refers to the dose of active double-chain Clostridium neurotoxin whether the neurotoxin is administered to the patient in association with the complexing protein or without the complexing protein. As is well known to experienced experts, active double-chain Clostridium neurotoxin can bind to membrane (e.g., cell membrane) receptors, transfer its light chain into the cytoplasm, and cleave SNARE proteins, while the complexing protein does not exhibit such biological activity (i.e., is not "active").

[0169] In addition, or alternatively, the dose of Clostridium neurotoxin may be measured in units (U) of Clostridium neurotoxin. For example, dose measurement in units may be particularly preferred when BoNT / A (or more particularly, for example, Dysport®) is administered.

[0170] In fact, as is well known to experienced experts, the potency of Clostridium neurotoxin is related to the amount of neurotoxin (e.g., nanograms) required to achieve the LD50 (lethal dose of 50) unit; 1 LD50 unit is defined as the median lethal intraperitoneal dose (as measured in mice). However, BoNT pharmaceutical formulations currently on the market contain different amounts of 150kD neurotoxin, and therefore the LD50 units also differ. Furthermore, in these formulations, the neurotoxin may or may not be associated with (i.e., combined with) non-toxic neurotoxin-associated proteins (NAPs), also known as complex proteins. For ease of conversion (Field et. al, “AbobotulinumtoxinA (Dysport (R) ), OnabotulinumtoxinA (Botox (R) ), and IncobotulinumtoxinA (Xeomin (R)As reported in "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 along with the complexed protein; • 500 units of Dysport® (also known as Abobotulinumtoxin A) contain approximately 2.69 ng of 150 kD BoNT / A along with the complexed protein; 1 unit of Dysport® contains approximately 5.38 pg of 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.

[0171] Note that the conversion values ​​may fluctuate slightly. For example, Frevert, 2012 ("Content of botulinum neurotoxin in Botox (R) / Vistabel (R) Dysport (R) / Azzalure (R) , and Xeomin (R) / Bocouture (R) The conversion values ​​reported in "; 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 along with the complexed protein; Each 100-unit Dysport® (also known as Abobotulinumtoxin A) contains approximately 0.65 ng of 150 kD BoNT / A along with the complexed protein; • 100 units of Xeomin® (also known as Incobotulinumtoxin A) contain approximately 0.44 ng of 150 kD BoNT / A, but do not contain complexed proteins; Each 100-unit pack of Neurobloc / Myobloc® (also known as Rimabotulinumtoxin B) contains approximately 0.2 ng to 1 ng of 150 kD BoNT / B along with the complexed protein.

[0172] The amount of Clostridium neurotoxin can be measured by a skilled expert using methods conventionally used in the art to quantify proteins, preferably at the nanogram level (in particular, mass spectrometry such as isotope dilution mass spectrometry (Munoz et al., Quantification of protein calibrants by amino acid analysis using isotope dilution mass spectrometry, Anal. Biochem. 2011, 408, 124-131) or fluorescence quantitative assay (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)).

[0173] Intradermal administration may include intradermal injection using a needle such as a 30-gauge needle, preferably, the needle (such as a 30-gauge needle) is inserted into the dermis of the skin at an angle of about 5° to 15° to the skin surface at the site of surgical intervention (which may be on the flank). The injection depth (depth to the skin surface) may be about 0.2 to 0.3 inches (preferably about 0.25 inches).

[0174] Clostridium neurotoxin may be administered at a site of the body that will be subjected to surgical intervention (e.g., a surgical incision or a site proximal to a surgical incision).

[0175] In one embodiment, Clostridium neurotoxin may be administered (by intradermal injection or intrathecal injection, etc.) at the site of the patient's surgical intervention (for example, at one or more administration sites at the site of the patient's surgical intervention).

[0176] Clostridium neurotoxin may be administered at one or more sites, for example, one or more sites proximal to the site of surgical intervention. The "site proximal to the site of surgical intervention" may be located up to 15 cm from the site of surgical intervention; for example, up to 10 cm from the site of surgical intervention; preferably up to 5 cm from the site of surgical intervention; more preferably up to 1 cm from the site of surgical intervention.

[0177] In one embodiment, following administration, Clostridium neurotoxin travels to the spinal cord by retrograde transport and performs SNARE protein cleavage (SNAP-25 protein cleavage) in the spinal cord.

[0178] In one embodiment, when Clostridium neurotoxin is administered intradermally, minimal or no SNARE protein cleavage (SNAP-25 protein cleavage) by the Clostridium neurotoxin is observed at or near the intradermally after administration. In one embodiment, observations are made for 5 to 7 days after administration of the Clostridium neurotoxin, and minimal or no SNARE protein cleavage (SNAP-25 protein cleavage) by the Clostridium neurotoxin is observed at or near the intradermally after administration.

[0179] In one embodiment, when Clostridium neurotoxin is administered to an internal site within the cerebrospinal cavity, minimal or no SNARE protein cleavage (SNAP-25 protein cleavage) by the Clostridium neurotoxin is observed in or near the internal site within the cerebrospinal cavity after administration. In one embodiment, observations are made for 5 to 7 days after administration of Clostridium neurotoxin, and minimal or no SNARE protein cleavage (SNAP-25 protein cleavage) by the Clostridium neurotoxin is observed in or near the internal site within the cerebrospinal cavity following administration.

[0180] Therefore, Clostridium neurotoxin may be administered distal to the site of surgical intervention to treat postoperative surgical pain and postoperative anxiety.

[0181] Therefore, in a preferred embodiment, if postoperative surgical pain is caused by surgical intervention, Clostridium neurotoxin may be administered distal to the site of surgical intervention (for example, at one or more administration sites distal to the patient's incision site) (via intradermal injection or intrathecal injection, etc.).

[0182] Clostridium neurotoxin may be administered at one or more sites distal to the site of surgical intervention, for example, at least 15 cm from the site of surgical intervention; at least 50 cm from the site of surgical intervention; or at least 100 cm from the site of surgical intervention.

[0183] Those skilled in the art will understand that the present invention pertains to preoperative administration, meaning that a reference to administration "at or proximal to the site of surgical intervention" refers to administration at or proximal to the site that will be subjected to surgical intervention once (for example, after) the surgery has begun. A reference to administration "distal to the site of surgical intervention" refers to administration distal to the site that will be subjected to surgical intervention once (for example, after) the surgery has begun.

[0184] Clostridium neurotoxin may be administered at up to 15 (preferably up to 10) sites (for example, sites proximal to the site of surgical intervention). These sites may traverse around the site of surgical intervention.

[0185] In a preferred embodiment, the dose (i.e., therapeutic dose) of the Clostridium neurotoxin of the present invention to be administered to treat surgical pain in a human patient is in the range of about 0.00025 ng to about 3 ng.

[0186] In preferred embodiments, the therapeutic dose of Clostridium neurotoxin is within the range of approximately 0.0003 ng to approximately 2 ng, preferably approximately 0.0004 ng to approximately 1.5 ng, approximately 0.0005 ng to approximately 1 ng, and more preferably approximately 0.0006 ng to approximately 0.5 ng.

[0187] For example, the dose (e.g., total dose) of Clostridium neurotoxin containing BoNT / A is preferably in the range of about 1 ng to about 2 ng.

[0188] The patient may be administered 100-500 U of Clostridium neurotoxin. For example, the patient may be administered 150-300 U of Clostridium neurotoxin; preferably 175-250 U; more preferably about 200 U.

[0189] Patients may be administered 80 to 250 picograms (pg) of Clostridium neurotoxin per kilogram (kg) of their body weight (e.g., 850 to 250 pg / kg). For example, patients may be administered 100 to 200 pg / kg, 115 to 175 pg / kg, or 130 to 150 pg / kg.

[0190] As described above, Clostridium neurotoxin may be administered at one or more administration sites, for example, at two or more administration sites. In one embodiment, the patient may be administered 2.5 to 30 U of Clostridium neurotoxin per administration site; preferably, the patient is administered 20 U of Clostridium neurotoxin per administration site. For example, 10 administration sites may receive 20 U each, providing a total dose of 200 U.

[0191] The patient may be administered Clostridium neurotoxin in a total dose of 10 to 170 pg per injection site. In a preferred embodiment, the patient may be administered Clostridium neurotoxin in a dose of 1 to 14 pg / kg (body weight) per injection site.

[0192] In another embodiment, the therapeutic dose of Clostridium neurotoxin is preferably in the range of about 0.001 ng to about 2 ng. Furthermore, for example, the therapeutic dose of Clostridium neurotoxin is preferably in the range of about 0.0003 ng to about 0.05 ng.

[0193] That said, it should be understood that the required dose range depends on the exact properties of Clostridium neurotoxin, the maximum tolerable dose in a particular subject (e.g., a human subject), skin condition, route of administration, nature of the prescription, patient's age, patient's weight, nature, degree, or severity of the patient's condition, contraindications if any, and the judgment of the treating physician. These variations in dosage levels can be adjusted using standard empirical procedures for optimization.

[0194] In one embodiment, the patient is administered monotherapy based on a single botulinum neurotoxin serotype (e.g., BoNT / A). Therefore, in one embodiment, the present invention employs the use of a single botulinum neurotoxin serotype (e.g., BoNT / A).

[0195] Embodiments of the various methods of the present invention are intended to be equally applicable to other methods, Clostridium neurotoxins, such as genetically modified Clostridium neurotoxins (in single-chain or double-chain form), uses, or pharmaceutical compositions, and vice versa.

[0196] sequence homology

[0197] Percent identity can be determined using any of the following sequence alignment methods, including but not limited to global methods, local methods, and hybrid methods such as the segment approach. Protocols for determining percentage identity are routine procedures within the scope of the art. The global method determines the best alignment by aligning the sequence from the beginning to the end of the molecule, summing the scores of individual residue pairs, and applying a gap penalty. Non-restrictive methods include, for example, CLUSTAL W, see Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement methods, see Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). The local method aligns sequences by identifying one or more conserved motifs common to all input sequences.Non-restrictive methods include, for example, the match-box method (see 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 CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993)); and Align-M (see Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004)).

[0198] Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. In short, two amino acid sequences are aligned, and the alignment score is optimized using a gap opening penalty of 10, a gap extension penalty of 1, and the Henikoff and Henikoff(ibid.) "blosum62" scoring matrix as shown below (amino acids are identified by standard single-letter codes).

[0199] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions common to those sequences. Therefore, % identity can be calculated by dividing the number of identical nucleotides / amino acids by the total number of nucleotides / amino acids and multiplying by 100. The calculation of % sequence identity may also take into account the number of gaps and the length of each gap that may need to be introduced to optimize the alignment of the two or more sequences. Sequence comparison and determination of percentage identity between two or more sequences can be performed using specialized mathematical algorithms such as BLAST, which are familiar to those skilled in the art.

number

[0200] Percent identity is then calculated as follows: [Total number of identical matches × 100] / [Length of the longer sequence + Number of gaps introduced into the longer sequence to align the two sequences].

[0201] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor nature changes, such as conservative amino acid substitutions (see below) that do not significantly affect polypeptide folding or activity, and are typically small deletions of 1 to about 30 amino acids, and small amino-terminal or carboxyl-terminal extensions such as amino-terminal methionine residues, small linker peptides of about 20 to 25 residues, or affinity tags.

[0202] Conservative amino acid substitutions Basic: Arginine; Lysine; Histidine Acidic: Glutamic acid; Aspartic acid Polarity: Glutamine; Asparagine Hydrophobic: leucine; isoleucine; valine Aromatics: Phenylalanine; Tryptophan; Tyrosine Small: Glycine; Alanine; Serine; Threonine; Methionine

[0203] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may substitute for amino acid residues in the polypeptide of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids may substitute for polypeptide amino acid residues. The polypeptide of the present invention may also contain amino acid residues of unnatural origin.

[0204] Examples of non-natural amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-natural amino acid residues into proteins are known in the art. For example, an in vitro system can be employed in which nonsense mutations are suppressed using chemically aminoacrylated suppressor tRNA. Methods for synthesizing amino acids and aminoacrylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system containing Escherichia coli S30 extract and reagents such as commercially available enzymes. The protein is 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 is performed by microinjection of mutant mRNA and chemically aminoacrylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996) into African clawed frog oocytes. In the third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of a desired non-natural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).Non-natural amino acids are incorporated into polypeptides in place of their natural counterparts. (See Koide et al., Biochem. 33: 7470-6, 1994.) Natural amino acid residues can be converted to non-natural species by in vitro chemical modification. Chemical modification, combined with site-derived mutagenesis, can further expand the range of substitutions (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

[0205] A limited number of non-conserved amino acids, amino acids not coded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids can substitute for amino acid residues in the polypeptide of the present invention.

[0206] The essential amino acids in the polypeptide of the present invention can be identified according to techniques known in the art, such as site-specific mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). The site of biological interaction can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutations of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992. The identity of the essential amino acids can also be estimated from homology analysis with related components of the polypeptide of the present invention (e.g., transporter or protease components).

[0207] Multiple amino acid substitutions can be performed and tested using 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). In short, these authors disclose a method for simultaneously randomizing two or more positions in a polypeptide to select a functional polypeptide, and then sequencing the mutageneised polypeptide to determine a spectrum of acceptable substitutions at each position. Other available methods 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-specific mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0208] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in 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 those skilled in the art with many general dictionaries of the terms used herein.

[0209] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein may be used in the practical application or testing of embodiments of this disclosure. Numerical ranges include the numbers that define the range. Unless otherwise specified, all nucleic acid sequences are written from left to right in the 5' to 3' orientation; each amino acid sequence is written from left to right in the amino to carboxyl orientation.

[0210] The headings given herein are not limitations on the various aspects or embodiments of this disclosure.

[0211] In this specification, amino acids are referred to using their full names, three-letter abbreviations, or one-letter abbreviations. The term “protein” as used herein encompasses proteins, polypeptides, and peptides. As used herein, the term “amino acid sequence” 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. Conventional one-letter and three-letter codes for amino acid residues may be used in this disclosure and claims. Three-letter codes for amino acids, such as those defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that polypeptides may be encoded by two or more nucleotide sequences due to the degeneracy of the genetic code.

[0212] 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 and is therefore subject to change. Since the scope of this disclosure is defined solely by the appended claims, it should also be understood that the terminology used herein is not intended to describe, but rather to limit, any particular embodiment.

[0213] Where a range of values ​​is given herein, unless the context explicitly specifies otherwise, each intermediate value up to one-tenth of that unit between the upper and lower limits of that range should be understood to be specifically disclosed as well. Each smaller range between any of the described values, or intermediate values ​​within a described range, and any other described or intermediate values ​​within that described range are encompassed within this disclosure. Any range of numerical values ​​indicated herein by the expression “a to b” means a range of numerical values ​​extending from a to b (i.e., encompassing the exact endpoints a and b).

[0214] In addition, the term “about” in this specification should be understood as plus or minus (±) 5%, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1% of the numerical value of the number in which it is used.

[0215] When used herein and in the appended claims, it should be noted that the singular forms "a," "an," and "the" encompass multiple references unless the context otherwise explicitly specifies. For example, a reference to "a botulinum neurotoxin" encompasses several such candidate agents, and a reference to "the botulinum neurotoxin" encompasses one or more Clostridium neurotoxins and their equivalents known to those skilled in the art, and so on.

[0216] The publications discussed herein are provided separately for their disclosures prior to the filing date of this application. Nothing in this specification should be construed as an acknowledgment that such publications constitute prior art with respect to the claims attached herein.

[0217] Hereinafter, embodiments of the present invention will be described merely as examples with reference to the following drawings and examples. [Brief explanation of the drawing]

[0218] [Figure 1] The diagram shows the injection site along the surgical incision, where Dysport, physiological saline, or the reference compound Exparel® (bupivacaine) was injected. [Figure 2] This is an outline of the arena used for open-field testing of walking activities. [Figure 3] This study demonstrates the effects of perioperative intradermal administration of either saline, Exparel (control agent), or Dysport at different concentrations (100, 200, and 400 U) on reducing postoperative surgical pain and anxiety. The Von Frey assay was performed as a method for measuring surgical pain perception. The results are shown in (A). The horizontal line crossing the graph is set at 26 g and represents the baseline for not feeling surgical pain (e.g., a threshold above which the subject's postoperative surgical pain may be considered treated). Pain can be defined as moderate / severe if the mechanical sensitivity is 0-15 g, low / moderate if it is 15-26 g, and little / no pain if it is above 26 g. The time (seconds) it took for pigs to approach their handler after perioperative intradermal administration of Exparel or Dysport is shown in (B). Distress behavior after perioperative administration of Exparel or Dysport was scored. This is shown in (C). [Figure 4]The average group total walking distance (meters) (A) and the average percentage of time spent in the center zone of the open field apparatus (B) are shown after perioperative intradermal injection of either physiological saline, Exparel (control agent), or Dysport (100, 200, and 400 U) at different concentrations. [Figure 5] The Von Frey assay results for intradermal administration of saline (control) versus BoNT / A (Dysport) 15 days (A), 5 days (B), or 1 day (C) prior to surgery are shown. [Figure 6] The bar graphs (A-C) show the latency (in seconds) from intradermal administration of either saline or Dysport (200U / pig) 15 days (A), 5 days (B), or 1 day (C) before surgery until the pig approaches the handler. For each time point (day) in the bar graphs (A-C), the left bar (lighter color) shows the results for Dysport treatment, and the right bar (darker color) shows the results for saline treatment. [Figure 7] The graph shows the distress behavior scores of pigs after intradermal administration of either saline or Dysport 15 days (A), 5 days (B), or 1 day (C) before surgery. For each time point (day) in the bar graph (A-C), the left bar (lighter color) shows the results for Dysport treatment, and the right bar (darker color) shows the results for saline treatment. [Figure 8] This shows the total walking distance (in meters) of pigs after intradermal administration of either saline or Dysport 15 days (A), 5 days (B), or 1 day (C) before surgery. [Figure 9] This shows the time course (individual and median) of the time spent in the central zone of the open-field device within a 5-minute period after intradermal administration of either saline or Dysport 15 days (A), 5 days (B), or 1 day (C) prior to surgery. [Figure 10] The Von Frey assay results for saline versus BoNT / A(Dysport) administered via intradermal (A), subcutaneous (B), or intramuscular (C) injection are shown. A total of 200 U of Dysport was administered per pig for each injection route tested. [Figure 11] This shows the latency (in seconds) until a pig approaches its handler after administration of saline or BoNT / A (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] This shows the distress behavior scores of pigs after administration of saline or BoNT / A (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 13] This shows the time course of the total distance (meters) walked by animals in an open field over a 5-minute period after intradermal, subcutaneous, or intramuscular administration of physiological saline or Dysport (individual values ​​and mean ± SEM). [Figure 14] This shows the time course (percentage) spent in the central zone of an open-field microscope after intradermal, subcutaneous, or intramuscular administration of physiological saline or Dysport (individual values ​​and mean ± SEM). [Figure 15] This shows immunohistochemical staining of SNAP-25 in skin samples containing small nerves around arterioles (A), nerves terminating in the arrector pili muscle (B), and small to medium-sized nerves in the dermis (C). [Figure 16] The images show immunohistochemical staining of SNAP-25 in the spinal cord of untreated pigs (A), and SNAP-25 staining of the ipsilateral horn (B) or contralateral horn (C) of Dysport-treated pigs. [Figure 17] The expression levels of calcitonin gene-related peptide (CGRP) and substance P in the spinal cord of pigs in either the untreated (A, C) or administered via intradermal injection of Dysport (B, D) cases are shown. [Figure 18] The expression levels of Iba1 (A, B) and glial fibrillary acidic protein (GFAP) (C, D) in the spinal cord of pigs, either in the untreated or administered via intradermal injection of Dysport, are shown. [Figure 19] The Von Frey assay is shown (A) for intradermal administration of saline (control) or BoNT / A (Dysport) 15 days prior to surgery or intradermal administration of Extarel on the day of surgery (D1), and for pigs whose left leg is used for surgical incision. *p<0.05;**p<0.01;***p<0.001;****p<0.0001, vs. saline group, using one-way ANOVA followed by Tukey test. #p<0.05;##p<0.01,####p<0.0001, Dysport vs. Extarel group, using one-way ANOVA followed by Tukey test. $$p<0.01: time point after surgery vs. -4 days, using paired t-test). (B) shows the latency (seconds) from when the pig approaches the handler after intradermal administration of saline (control) 15 days prior to surgery or BoNT / A (Dysport), or after intradermal administration of Exparel on the day of surgery (D1), and when the pig's left leg is offered for surgical incision ($$p<0.01: postoperative time vs. -4 days, paired t-test used. £££p<0.001: -16 days vs. -4 days, paired t-test used. *p<0.05;**p<0.01;***p<0.001, treatment vs. saline group, one-way ANOVA followed by Tukey test). (C) shows the pain behavior scores of pigs after intradermal administration of saline (control) or BoNT / A (Dysport) 15 days prior to surgery, or after intradermal administration of Exparel on the day of surgery (D1), and when the pigs were subjected to surgical incision of the left leg (*p<0.05, ***p<0.001, and ****p<0.0001, vs. saline group, using one-way ANOVA followed by Tukey test. $$p<0.01, $p<0.05: time point after surgery vs. -4 days, using paired t-test). [Figure 20] This document outlines the tissue samples (formalin-fixed, paraffin-encased tissue) collected for immunohistochemical staining, and shows the specific region from which the tissue samples were taken from the spinal cord. [Figure 21] This image shows immunohistochemical staining of transection SNAP-25 in the skin (A), muscle (B), and dorsal root ganglion (C) of a pig that underwent a surgical incision in the left leg. [Figure 22] The images show a section of the ipsilateral posterior horn of the lumbar vertebra L5-L6 in the spinal cord of a pig that underwent a surgical incision in the left leg, stained with SNAP-25 (A), and a magnified view (B). [Figure 23] The scoring scale used to determine the intensity of SNAP-25 staining of transections is shown. SNAP-25 staining of transections was scored on a scale of 1 to 3. Grade 0 = no SNAP-25 staining of transections, Grade 1 = low intensity SNAP-25 staining of transections, Grade 2 = average intensity SNAP-25 staining of transections, and Grade 3 = high intensity SNAP-25 staining of transections (A). Figure 23 also shows the quantification of staining intensity for different regions of the spinal cord: lumbar spine L5-L6, L3-L4, L1-L2, and thoracic and cervical regions. Staining intensity was measured by the "H-score" method, calculated as % of the positive portion of the spinal cord × staining intensity in the dorsal horn (B). [Figure 24] This document provides an overview of whether SNAP-25 staining of sectioned tissue samples is "positive" or "negative".

[0219] Sequence List

[0220] If the first Met amino acid residue or the corresponding start codon is shown in any of the following sequence numbers, then the residue / codon is optional.

[0221] Sequence ID 1 - BoNT / A1, Acceptance Number A5HZZ9, Amino Acid Sequence

[0222] Sequence ID 2 - BoNT / B1, Acceptance Number B1INP5, Amino Acid Sequence

[0223] Sequence ID 3 - BoNT / C1, Acceptance Number P18640, Amino Acid Sequence

[0224] Sequence ID 4 - BoNT / D, Acceptance Number P19321, Amino Acid Sequence

[0225] Sequence ID 5 - BoNT / E1, Acceptance Number WP_003372387, Amino Acid Sequence

[0226] Sequence ID 6 - BoNT / F1, Acceptance Number Q57236, Amino Acid Sequence

[0227] Sequence ID 7 - BoNT / G, Acceptance Number WP_039635782, Amino Acid Sequence

[0228] Sequence ID 8 - BoNT / DC, Acceptance Number BAM65681, Amino Acid Sequence

[0229] Sequence ID 9 - BoNT / F7, amino acid sequence

[0230] Sequence ID 10 - BoNT / AB MY , amino acid sequence [ka]

[0231] Sequence ID 11 - BoNT / X, amino acid sequence (GenBank: BAQ12790.1)

[0232] Sequence ID 12 (nucleotide sequence, unmodified BoNT / A)

[0233] Sequence ID 13 (polypeptide sequence, unmodified BoNT / A)

[0234] Sequence ID 14 (nucleotide sequence, modified BoNT / A “Cat-A”)

[0235] Accession No. 15 (polypeptide sequence, modified BoNT / A “Cat-A”)

[0236] Sequence ID 16 (nucleotide sequence, modified BoNT / A “Cat-B”)

[0237] Accession No. 17 (polypeptide sequence, modified BoNT / A “Cat-B”)

[0238] Sequence ID 18 (nucleotide sequence, modified BoNT / A “Cat-C”)

[0239] Sequence ID 19 (polypeptide sequence, modified BoNT / A “Cat-C”)

[0240] Sequence ID 20 (nucleotide sequence, modified BoNT / A “Cat-D”)

[0241] Sequence ID 21 (polypeptide sequence, modified BoNT / A “Cat-D”) [Examples]

[0242] Materials and methods

[0243] Animal models

[0244] The following studies used male farmed pigs weighing 11–13 kg. Because pig skin is similar to human skin in terms of structure, thickness, innervation, pigmentation, collagen and lipid composition, wound healing, and immune response, pigs are a suitable model for studying the management of postoperative surgical pain.

[0245] Reorganization of Dysport

[0246] Dysport was provided in a vial containing 500 U. For administration, the 500 U vial was reconstituted with physiological saline (0.9% NaCl). Subsequent dilutions with physiological saline were performed according to the test dose as follows: 2.5 ml of physiological saline was withdrawn using a 3 ml syringe with a 21 G needle and transferred to a Dysport 500 U vial; the concentration was 200 U / ml = 400 U / 2 ml; the vial was gently swirled until the material was dissolved. Each vial was tilted 2-3 times from side to side (to ensure homogeneity of the solution); 2 ml was administered to the pigs using two 1 ml syringes connected to 30 G needles. This solution was used to administer 400 U.

[0247] Preparation for a 200U / 2ml dose: Dysport was reconstituted as described above; 2 ml of reconstituted Dysport 500U was withdrawn using a 3 ml syringe and a 21 G needle; 2 ml of physiological saline was withdrawn using a 3 ml syringe and a 21 G needle; the two solutions were mixed using a Vacutainer vial; the mixed solution was tilted 5-6 times from side to side (to ensure homogeneity of the solution); 2 ml was administered to the pigs using two 1 ml syringes connected to 30 G needles.

[0248] Preparation of a 100U / 2ml dose: Dysport was reconstituted as described above. 2 ml of reconstituted Dysport 500U was withdrawn using a 3 ml syringe and a 21 G needle; 2 ml of saline solution was withdrawn using a 3 ml syringe and a 21 G needle; the two solutions were mixed using a Vacutainer vial; the mixed solution was tilted 5-6 times from side to side (to ensure homogeneity of the solution); 2 ml of the prepared solution was withdrawn from the Vacutainer vial using a 3 ml syringe and a 21 G needle; 2 ml of saline solution was withdrawn using a 3 ml syringe and a 21 G needle; the two solutions were mixed using a new Vacutainer; the mixed solution was tilted 5-6 times from side to side (to ensure homogeneity of the solution); 2 ml was administered to the pigs using two 1 ml syringes connected to 30 G needles.

[0249] Induction of postoperative surgical pain

[0250] The pigs were anesthetized with an isoflurane / oxygen mixture. This mixture was delivered through a face 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 flank of the pig, toward the tail end and 3 cm lateral to the spine (Day 1), or a 7 cm long skin incision was made on the left leg. Subsequently, the fascia was cut and the muscles were 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 suture. The skin was sutured with 3-0 silk suture using a continuous suture method. Following incision closure and material injection, the pigs received an antibiotic (Marbosil 10%). The incision area was covered with a thin layer of 3% syntomycin. The animals were kept under anesthesia for the duration of the surgery and medication administration (approximately 20 minutes). Postoperatively, the animals were returned to their cages for recovery and observation.

[0251] treatment

[0252] Intradermal perioperative administration of Dysport

[0253] For perioperative administration, the animals were injected immediately after suturing the incision made on the left abdomen. Dysport (test article), normal saline (negative control), or the reference compound Exparel (positive control) was injected intradermally (or subcutaneously for Exparel) using a 30G needle attached to a 1 ml syringe at 10 sites around the incision. A constant dosage volume and a constant dosage level were injected at each site. More specifically, injections were made at 4 sites (e.g., 8 sites) along each side of a 7 cm horizontal incision / suture on the left abdomen (at 2 cm intervals), and also at the sites at each end of the incision / suture on the left abdomen (see Figure 1). The following experimental groups were evaluated as follows.

Table B

[0254] Intradermal preoperative administration of Dysport

[0255] For preoperative injection, when injecting a total of 2 ml into the animals before incision (either on the left abdomen or left leg of the pig), ink was first applied at the position of the additional incision. Dysport (test article) or normal saline (negative control) was injected intradermally using a 30G needle attached to a 1 ml syringe at 10 sites around the incision. A constant dosage volume and a constant dosage level were injected at each site. The administration was carried out either 15 days, 5 days, or 1 day before the surgery. The following experimental groups (when the incision was made on the left abdomen of the pig) were evaluated as follows.

Table C

[0256] ​​​​​​​​​​ Administration of Dysport via intradermal, intramuscular, or subcutaneous routes.

[0258] Fifteen days prior to incision, the animals were tattooed at the site of further incisions when receiving injections. Dysport (test product) or physiological saline (negative control) was injected into 10 sites around the incision using a 30G needle attached to a 1 ml syringe. A fixed dose volume and dose level were injected at each site. Administration was performed via intradermal, subcutaneous, or intramuscular routes.

[0259] The following experimental groups were evaluated as follows: [Table E]

[0260] von Frey assay

[0261] The von Frey assay was performed once daily for 10 days in healthy, unsurgered animals after injection of Dysport / saline. Von Frey filaments (Ugo Basile, Italy) were applied to the surface of the skin on the flank or leg approximately 0.5 cm proximal to the incision line. The force applied to the skin on the flank or leg increased with increasing filament weight. The maximum force used was 60 g. The filament was applied until the animal retreated from the stimulus. Each filament was applied 3-5 times. If retreat did not occur, a thicker filament was applied. If retreat occurred, a thinner filament was applied (thicker or thinner refers to higher / thicker or lower / thinner gram forces). By changing the filament thickness, the force required to achieve the retreat response was determined and recorded. The sizes and forces of the von Frey filaments are summarized in the table below. [Table F]

[0262] Inclusion Criteria: Animals were included in the study if their flank escape force was ≥26g (preferably 60g) at baseline. Postoperatively, if the flank escape force was ≤10g, pain (allodynia) was considered present. Animals that did not meet this criterion were excluded from the study. One animal was excluded from the study because it showed a relatively low threshold (≤10g) before surgery.

[0263] Animals were included in the study if their leg escape force was ≥13g at baseline. After surgery, pain (allodynia) was considered present if the leg escape force was ≤2g. Animals that did not meet this criterion were excluded from the study.

[0264] Proximity Time Test

[0265] Prior to administering medication to the pigs, researchers conducting the Approach Time (AT) test entered the enclosure for the first time. Normal pig behavior when someone enters an enclosure is to move away from the intruder and then approach them. The more accustomed and comfortable the pig is with the person, the shorter the time it takes to approach. The latency time to approach the researcher entering the enclosure was measured in seconds (end time 120 seconds). This test was conducted in the morning, at least one hour after the morning feeding (6:30 AM), prior to the distress behavior score and during the acclimatization period.

[0266] Pain behavior score

[0267] Following the incision, the animals' behavior changed. When approached, the animals tended to move away from the researcher entering the cage, guard the incision site, and sometimes vocalize. This is a major phenomenon observed after this type of surgery; in rare cases, the animals became restless or exhibited isolated behavior. Painful behavior was scored from 0 (normal) to 7 (very painful). The painful behavior score test was performed immediately after the approach time test. The animals' overall behavior was monitored in their enclosures throughout the morning. The painful behavior score also allows for an assessment of the animals' overall health. The animals' behavior was scored by observers who were unaware of the procedure, and the total score is the sum of all sections shown in the table below. [Table G]

[0268] The behavioral scores were evaluated according to the animals' overall spontaneous behavior, rather than in a specific order.

[0269] Open field testing of walking activity

[0270] The open field was a rectangular arena measuring 2.5m wide and 4.7m long. The arena walls were smooth and 1.6m high. On the morning of the experiment, animals from all groups were individually led into the open field one at a time for 5 minutes (5). The animals' walking and motor activity was recorded using CCTV cameras and analyzed with AnyMaze software (Stoelting Co.). The open field experiment was conducted at the end of the behavioral tests (i.e., approach time, distress behavior, and von Frey) conducted in cages. After each open field experiment, the following parameters were analyzed: total walking distance (m) and the percentage of time spent in the center of the area (zone E; see Figure 2).

[0271] Animals experiencing pain or suffering will typically walk near the walls of the cage or open-field enclosure. Animals without pain will enter the center of the open-field enclosure without hesitation.

[0272] Example 1 - Perioperative administration of Dysport provides delayed analgesic and anxiolytic effects after surgery (incision in the left flank of a pig).

[0273] Immediately after suturing an incision made in the left flank of pigs (i.e., perioperatively), the pigs were administered intradermal injections of either saline, Extarel (a constant dose of 266 mg), or different concentrations of Dysport. The mechanical sensitivity of the pigs was measured by the Von Frey assay to evaluate the management of postoperative surgical pain. Compared to the saline-treated group, Extarel showed an analgesic effect for a duration of 1 day, but thereafter did not show an effective analgesic effect. Administration of 400 U of Dysport induced a moderate analgesic effect 2 days post-surgery. Greater analgesic effects were induced by 4 days post-surgery when pigs were administered 200 U or 400 U of Dysport. All concentrations of Dysport tested completely suppressed postoperative surgical pain at 6 days post-surgery. This suggests that Dysport provides effective and long-lasting analgesic effects for managing postoperative surgical pain. This data is shown in the bar graph in Figure 3A.

[0274] The latency time for pigs to approach their handlers was measured. At the time of incision, pigs were administered intradermal injections of either saline, Exparel, or different concentrations of Dysport. Up to 2 hours postoperatively, all treatment groups showed a delay in approaching their handlers. Up to 6 hours postoperatively, intradermal administration of either 200U or 400U of Dysport reduced the time it took for pigs to approach their handlers, and these effects lasted up to 5 days postoperatively, suggesting a potential reduction in postoperative distress and anxiety-like reactions. Pigs treated with either saline or Exparel showed no improvement in approaching their handlers, suggesting that these treatments did not reduce postoperative distress and anxiety-like reactions. This data is shown in Figure 3B.

[0275] The distress behavior scores of the pigs were measured. Pigs administered 100U, 200U, or 400U of Dysport showed a reduction in distress behavior scores by 2 days after surgery, unlike the saline and Exparel treatment groups. This data is shown in Figure 3C.

[0276] The open-field test showed no difference between the total distance animals walked before surgery and the total distance they walked after surgery following saline treatment. Treatment with Exparel or Dysport did not affect the total walking distance at 3 days post-treatment, suggesting no change in the animals' motor function after surgery. This data is shown in Figure 4A. Although animals treated with 400U of Dysport spent more time in the center of the open-field apparatus, this difference was not statistically significant (see Figure 4B).

[0277] Example 2 - Preoperative administration of Dysport induces a faster analgesic effect and suppresses the appearance of postoperative pain and anxiety-like reactions when surgical incision is made in the left flank of pigs.

[0278] Perioperative administration of Dysport showed a delay in the induction of analgesic effects; therefore, the analgesic and anxiolytic effects of preoperative administration of Dysport were measured. Pigs were administered intradermal injections of either physiological saline or 200 U of Dysport 15 days (see Figure 5A), 5 days (see Figure 5B), or 1 day (see Figure 5C) before surgery (incision in the left flank of the pig). Using the Von Frey assay, the fastest analgesic effect was observed when Dysport was administered 15 days before surgery; in this case, postoperative surgical pain decreased by 1 day after surgery. In comparison, when Dysport was administered 5 days before surgery, postoperative surgical pain decreased by 5 days after surgery.

[0279] When Dysport was administered intradermally 15 or 5 days prior to surgery, pigs showed a reduction in the time it took to approach 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). Administration of Dysport one day prior to surgery did not induce an effective anxiolytic effect. This suggests that preoperative administration of Dysport 15 or 5 days prior to surgery effectively prevents the development of postoperative distress and anxiety-like responses.

[0280] None of the treatment groups (intradermal injection of Dysport 15 days, 5 days, or 1 day before surgery) showed any difference in total walking distance postoperatively (see Figure 8), which suggests that muscle activity was not affected and that there was no systemic spread of the toxin.

[0281] The percentage of time spent in the center of the open-field apparatus was similar for animals injected with saline before and after surgery. Animals treated with Dysport 15 days prior to surgery spent more time in the center of the open-field apparatus (see Figure 9A). When administered either 5 days or 1 day prior to surgery, there was no difference in the percentage of time spent in the center between animals treated with saline and animals treated with Dysport (see Figures 9B and 9C).

[0282] Example 3 - Intradermal administration of Dysport provides a favorable route for reducing postoperative surgical pain and suppressing the development of postoperative anxiety.

[0283] The ability of different routes (intradermal, subcutaneous, and intramuscular injection) of 200U of Dysport administered 15 days prior to surgery to induce analgesic and anxiolytic effects postoperatively was evaluated. Surprisingly, intradermal administration provided better results than the alternative routes (in fact, only the intradermal route of Dysport administration was generally observed to show a rapid analgesic effect (see Figure 10)). Both the subcutaneous and intramuscular routes of Dysport administration showed little to no effect on analgesia. Pigs administered intradermal injection of Dysport 15 days prior to surgery showed a reduction in time to approach handlers and a decrease in distress behavior scores (see Figures 11 and 12). This suggests that the intradermal route of administration is effective in reducing postoperative surgical pain and preventing the full manifestation of postoperative distress and anxiety-like reactions.

[0284] In all saline groups, the walking distance recorded postoperatively was the same as the walking distance recorded preoperatively. Furthermore, the treatment with Dysport and the route of administration (intradermal, subcutaneous, or intramuscular) did not affect the total walking distance postoperatively (see Figure 13).

[0285] The percentage of time spent in the center of the open-field apparatus by animals injected with physiological saline was similar before and after surgery. There was no difference in the percentage of time spent in the center of the open-field apparatus between different administration routes (see Figure 14).

[0286] Example 4 - SNAP-25 transection occurs distal to the Dysport injection site in the ipsilateral posterior horn of the spinal cord.

[0287] To evaluate the mechanism of action of Dysport (intradermal injection), immunohistochemistry was performed on tissue samples from both the surgical incision site (left flank of a pig) and the spinal cord. Transected SNAP-25 was not detected in nerves from the skin samples (see Figure 15). Surprisingly, transected SNAP-25 was visualized in the ipsilateral posterior horn of the spinal cord (see Figure 16), demonstrating BoNT / A activity in the spinal cord and suggesting that control of postoperative surgical pain / anxiety may be provided via central effects in the spinal cord. This also highlights that Dysport can be administered directly to the spinal cord via intrathecal administration.

[0288] The expression levels of two neuropeptides involved in pain regulation, calcitonin gene-related peptide (CGRP) and substance P, were evaluated in the spinal cord by immunohistochemical staining. Neither neuropeptide showed a significant difference in expression levels in the spinal cords of Dysport-treated pigs compared to untreated pigs (see Figure 17).

[0289] The expression level of Iba1, a marker of small glial cell activation, was reduced in the spinal cord of Dysport-treated pigs compared to untreated pigs (see Figures 18A and B). Similarly, the expression level of GFAP, a marker of astrocyte activation, was reduced in the spinal cord of Dysport-treated pigs compared to untreated pigs (Figures 18C and D).

[0290] Example 5 - Preoperative administration of Dysport induces a rapid analgesic effect and suppresses the development of postoperative pain and anxiety-like reactions in pigs undergoing surgical incision in the left leg.

[0291] The analgesic and anxiolytic effects of preoperative administration of Dysport were measured in pigs subjected to surgical incisions at different sites (surgical incisions in the left leg, rather than the left flank). Pigs were administered intradermal injections of saline or 200 U of Dysport 15 days prior to surgery, or Exparel on the day of surgery (day 1) (see Figure 19A) (surgical incision in the left leg). Rapid analgesic effects were observed using the Von Frey assay, with postoperative surgical pain reduced by day 1 postoperatively and prolonged reversal of mechanical allodynia observed by day 4.

[0292] Pigs (with a sutured incision in the left leg) showed reduced time to approach handler when administered intradermal Dyspor 15 days prior to surgery compared with administration of saline and Exparel (see Figure 19B). Similarly, pigs (with a sutured incision in the left leg) showed reduced distress behavior scores when administered intradermal Dyspor 15 days prior to surgery compared with administration of saline and Exparel (see Figure 19C). This suggests that preoperative administration of Dyspor 15 days prior to surgery (incision in the left leg) effectively prevents the development of postoperative distress and anxiety-like reactions.

[0293] Overall, this experiment provides further support for the rapid analgesic and anxiolytic effects of Dysport when administered 15 days prior to surgery.

[0294] Example 6 - SNAP-25 transection occurs in the ipsilateral posterior horn of the spinal cord in a pig that underwent a surgical incision in the left leg.

[0295] To evaluate whether the same mechanism of action of Dysport occurs when administered intradermally to different sites in pigs, immunohistochemistry was performed on tissue samples from the surgical incision site (left leg of the pig) and various regions of the spinal cord (see Figure 20). Transected SNAP-25 was not detected in nerves of skin samples collected 5-7 days after incision and Dysport injection (see Figure 21). Similar to the findings in pigs with surgical incisions made on the left flank, transected SNAP-25 was visualized in the ipsilateral posterior horn of the spinal cord, specifically in the lumbar region L5-L6 (see Figure 22). These findings indicate BoNT / A activity in the spinal cord and suggest that control of postoperative surgical pain / anxiety may be provided via central effects in the spinal cord. The localization of transected SNAP-25 staining in the ipsilateral posterior horn differed from that in pigs with surgical incisions made on the left flank.

[0296] The intensity of SNAP-25 staining of the tissue sections was graded on a scale of 1 to 3 (grade 0 = no SNAP-25 staining of tissue sections, grade 1 = low intensity SNAP-25 staining of tissue sections, grade 2 = average intensity SNAP-25 staining of tissue sections, and grade 3 = high intensity SNAP-25 staining of tissue sections) (see Figure 23A). Based on the above grading system, pigs that underwent surgical incision in the left leg had lower intensity SNAP-25 staining of the ipsilateral posterior horn compared to pigs that underwent surgical incision in the left flank.

[0297] The intensity of SNAP-25 staining in transections was quantified (see Figure 23B). The H-score was calculated as a measure of SNAP-25 staining intensity in transections. The H-score was calculated by multiplying the percentage (%) of positive spinal cord sections by the staining intensity in the posterior horn. In pigs treated with Dysport (and with surgical incision in the left leg), the highest level of SNAP-25 staining in transections (judged as Grade 2 SNAP-25 intensity staining) was observed in the spinal cord in the lumbar region L5-L6 compared to the lumbar regions L3-L4 and L1-L2 of the spinal cord, as well as the thoracic and cervical regions (the cervical region had trace amounts of SNAP-25 staining). No evidence of SNAP-25 staining in transections was found in pigs treated with saline or Exparel injection.

[0298] The immunohistochemical staining results described above are summarized in Figure 24, which supports the observation of SNAP-25 transection in localized areas of the spinal cord. The localized areas of the spinal cord L5-L6, L3-L4, and L1-L2, as well as the thoracic and cervical regions, were positive for SNAP-25 transection staining, while the remaining tissues (including the skin at the injection site) were negative for SNAP-25 transection staining.

[0299] All publications referenced in the above specification are incorporated herein by reference. Various modifications and changes to the methods and systems described in the present invention 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 relation to specific preferred embodiments, it should be understood that the claimed invention is not limited beyond such specific embodiments. In fact, various modifications to the described forms for carrying out the invention, which are obvious to those skilled in the art in biochemistry and biotechnology or related fields, are intended to be included within the scope of the following claims. [Explanation of Symbols]

[0300] [Figure 2] door entrance: door entrance [Figure 3] Saline: Physiological saline solution Withdrawal Force (g): The power of escape (g) Day -1: 1 day ago D1: Day 1 1h: 1 hour 2h: 2 hours 4h: 4 hours 6h: 6 hours Day 2: Day 3: Day 3 Day 4: Day 4 Day 5: Day 5 Day 6: Day 6 B Time (Seconds): Time (seconds) Baseline: Baseline DBS Score (points): DBS score (points) [Figure 4] Total walking distance: walking distance (m): Pre-operation no treatment: No treatment was performed before the operation. Saline: Physiological saline solution Day 3 post-operation: Percentage of time spent in the central zone: [Figures 5, 6, 7, 8, 9, 10, 11, 12, 13, 19] Experiment 1: 15 days pretreatment: Dysport 200U / pigs: Dysport 200U / pig Withdrawal Force (g): The power of escape (g) Day -16: 16 days ago Day -15: 15 days ago Day -13: 13 days ago Day -10: 10 days ago Day -4: 4 days ago Day -1: 1 day ago Day 1: Day 1 1h: 1 hour 2h: 2 hours 4h: 4 hours 6h: 6 hours Day 2: Day 3: Day 3 Day 4: Day 4 Day 5: Day 5 Day 6: Day 6 Day 7: Day 8: Day 8 Day 9: Day 9 Day 10: Day 10 Experiment 2: 5 days before treatment Day -6: 6 days ago Experiment 3: 1 day pretreatment Day -2: 2 days ago Time (sec): Time (sec) Score: [Figure 8] Cohort 1: Cohort 1 meter: meter Cohort 2: Cohort 3: Cohort 3 [Figures 9, 14] Center (%): Center (%) [Figures 10, 11, 12, 13, 14] Intra-Dermal (ID) injections: Intra-Dermal (ID) injections Subcutis (SC) injections: Subcutaneous (SC) injections Intra-muscular (IM) injections: Intramuscular (IM) injections [Figure 12] DBS (points): DBS (points) [Figures 13, 14] Open Field Distance (meter): Baseline: Baseline [Figure 15] Periarterial: Around the arteries Hair erector muscle: Arrector pili muscle Nerve: nerve [Figures 16, 17, 18] Untreated: Untreated Dysport injected ipsi: Dysport injection ipsilateral Dysport injection contra: Contralateral side of Dysport injection Substance P: Substance P Dysport injected: Dysport injection [Figure 19] Study days: Exam days Operation: Surgery Approaching Time (sec): Approaching time (sec) DBS Score (points): DBS score (points) [Figures 20, 24] Block number: Tissue: tissue Skin, injection site: skin, injection site Gastrocnemius muscle right (contralateral): Gastrocnemius muscle left (ipsilateral): spinal cord: spinal cord Dorsal root ganglia, lumbar vertebrae L4-L5-L6 right (contralateral): Dorsal root ganglia, lumbar vertebrae L1-L2-L3 right (contralateral): Dorsal root ganglia Lumber L4-L5-L6 left (ipsilateral): Dorsal root ganglia Lumber L4-L5-L6 left (ipsilateral) Dorsal root ganglia Lumber L1-L2-L3 left (ipsilateral): Dorsal root ganglia Lumber L1-L2-L3 left (ipsilateral) Thoracic spinal cord: Thoracic spinal cord Cervical spinal cord: Cervical spinal cord Lumber: Lumbar vertebrae Block: Block Thoracic: chest Cervical: Cervical [Figure 23] Grade: Grade 0.5: Grade 0.5 H-Score: H-score C-SNAP25 Level: C-SNAP25 Level traces: traces H-Score = % of positive spinal cord sections x staining intensity in the dorsal horns: [Figure 24] Group: Group Negative: negative Positive: positive

Claims

1. A pharmaceutical preparation comprising botulinum neurotoxin serotype A (BoNT / A) for use in the treatment of postoperative surgical pain in patients. The treatment comprises administering BoNT / A to the patient 5 to 20 days prior to surgery, wherein BoNT / A is administered intradermally, and the postoperative surgical pain in the patient is caused by a surgical intervention including (i) skin and (ii) surgical incision of fascia, muscle, bone, and / or organs.

2. The pharmaceutical product according to claim 1. Here, a) BoNT / A is administered 10 to 20 days before surgery; b) BoNT / A is administered 14 to 16 days before surgery; c) Administration of BoNT / A substantially reduces the patient's perception of postoperative surgical pain, and this reduced perception of postoperative surgical pain is maintained for 24 hours immediately following surgery; and / or d) Substantially all reduced postoperative surgical pain sensation is maintained for 3, 4, 5, 6, 7, or 8 days immediately following surgery.

3. BoNT / A is the pharmacopoeia according to claim 1, administered 5 to 15 days before surgery.

4. The pharmaceutical product according to claim 2 c) or d), wherein the level of reduced pain perception observed at a specified time immediately after surgery is at least 50% of the maximum level of reduced pain perception observed at any time after administration of BoNT / A.

5. A pharmaceutical product according to any one of claims 1 to 4. Here, a) BoNT / A treats postoperative surgical pain and reduces or suppresses postoperative anxiety; and / or b) The postoperative surgical pain is caused by the surgical intervention, and BoNT / A is administered distal to the site of the surgical intervention, with the distal site of the surgical incision being at least 15 cm from the site of the surgical intervention.

6. A pharmaceutical product according to any one of claims 1 to 5. Herein, after administration, BoNT / A travels to the spinal cord by retrograde transport and performs SNARE protein cleavage or SNAP-25 protein cleavage in the spinal cord.

7. A pharmaceutical product according to any one of claims 1 to 6, wherein BoNT / A is administered intradermally, and after administration of BoNT / A, minimal SNARE protein cleavage or SNAP-25 protein cleavage by BoNT / A is observed in or near the intradermally, or no cleavage is observed at all.

8. A pharmaceutical product according to any one of claims 1 to 7. Here, a) Postoperative surgical pain is acute postoperative surgical pain; b) Postoperative surgical pain is chronic postoperative surgical pain; and / or c) The above procedure does not include intramuscular administration of BoNT / A.

9. A pharmaceutical product according to any one of claims 1 to 8. Here, a) The patient is administered 100–500 U of BoNT / A; b) The patient is administered a total dose of 1–3 ng of BoNT / A; c) The patient is administered 80–250 pg of BoNT / A per kg (body weight); d) BoNT / A is administered at two or more injection sites.

10. The pharmaceutical product according to claim 9, wherein the patient is administered 10 to 170 pg of BoNT / A per injection site.

11. The pharmaceutical product according to claim 9, wherein the patient is administered BoNT / A at a dose of 1 to 14 pg / kg body weight per injection site.

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