Clostridial neurotoxins for treatment of bladder pain syndrome

Abstract

The present invention provides a method of treating a patient with bladder pain syndrome, the method comprising injecting a solution comprising a clostridial neurotoxin into the bladder of the patient; increasing the volume of the solution containing clostridial neurotoxin in the bladder, thereby applying a mechanical force to the inner surface of the urinary tract epithelium; a solution containing the clostridial neurotoxin within the bladder is maintained at a volume that does not cause urination of the patient and maintained for at least 30 minutes such that the clostridial neurotoxin diffuses through the urinary tract epithelium and into the intrinsic layer in which the clostridial neurotoxin binds to the primary sensory afferent nerve fibers, inhibits secretion of neurotransmitters therefrom, and inhibits secretion of neurotransmitters from the primary sensory afferent nerve fibers. Therefore, bladder pain is relieved.

Description

Technical Field

[0001] The present invention relates to the treatment of painful bladder syndrome (BPS), in particular to the treatment of interstitial cystitis (IC). Background Art

[0002] Painful bladder syndrome is a chronic bladder health problem that affects an estimated 6-14 million people in the United States (i.e., 5-11% of the U.S. adult population). The International Continence Society (ICS) defines painful bladder syndrome as a condition characterized by chronic (>6 months) pelvic pain, pressure, or discomfort that is believed to be bladder-related and is accompanied by at least one other urinary symptom, such as persistent urinary urgency or frequency.

[0003] Frequent urination means urinating more often than normal. The average person urinates no more than seven times a day and gets up no more than once at night to urinate. People with painful bladder syndrome, on the other hand, need to urinate frequently throughout the day and night, and as the urinary frequency increases, it can lead to an urgency to urinate. For some people, this urgency to urinate does not go away even after they have just urinated.

[0004] Interstitial cystitis is a more severe or advanced form of painful bladder syndrome and is also characterized by "classic cystoscopic and histologic features." For example, patients with interstitial cystitis have a higher incidence and degree of epithelial desquamation, ulceration, pyuria, and / or submucosal inflammation than patients with painful bladder syndrome without interstitial cystitis. Interstitial cystitis is more appropriately described as a chronic submucosal inflammatory disease.

[0005] Although the exact cause of painful bladder syndrome is unknown, the following factors are thought to be involved:

[0006] Defects in bladder tissue that allow potential irritants in urine to penetrate the bladder wall;

[0007] Mast cell hyperactivity and excessive secretion of inflammatory signals (such as histamine);

[0008] Contaminants in the urine that damage the bladder wall;

[0009] Hypersensitivity of local afferent nerves, resulting in pain from events that would not normally cause pain (such as bladder filling);

[0010] Autoimmunity.

[0011] Urine is produced by the nephrons of the kidneys and is transported to the bladder for storage before being excreted through the urethra. This cyclical filling and emptying process is called urination. As the bladder fills, the bladder wall stretches, stimulating afferent signals. Conversely, efferent signals cause the bladder muscles to contract and the urethral sphincter to relax, respectively. In addition to mechanoreceptors, various psychological factors (such as stress, acceptability of the surrounding environment, and emotional state) also play a crucial role in the timing and environment of urination. Obviously, the bladder has excellent elastic properties. These properties are derived from the structure of the bladder wall, which is divided into the following layers from the inside to the outside:

[0012] Epithelial layer;

[0013] · Lamina propria;

[0014] ·Muscularis propria;

[0015] Serosa / adventitia.

[0016] The bladder epithelium forms a critical barrier that prevents irritants in the urine from passing through the bladder lining and reaching the underlying cells and connective tissue surrounding the bladder lumen. In fact, the bladder epithelium forms a highly specialized stratified epithelium, the urothelium, to achieve this specific purpose. In more detail, the urothelium is composed of three layers:

[0017] The superficial layer, which is the innermost layer, is the main barrier between the bladder lumen and the underlying tissues. This highly specialized layer consists of a single layer of cells ("umbrella cells") that work together via tight intercellular junctions to form an impermeable barrier. Another important role of umbrella cells is to adapt to the stretching of the bladder. This is achieved by releasing uroplaque proteins (via fusiform vesicles containing uroplaque proteins) that form a surface plaque layer covering the umbrella cells. When the bladder wall relaxes, these uroplaque proteins are returned to the umbrella cells via SNARE-mediated endocytosis;

[0018] The middle layer, consisting of two to three layers of polygonal cells;

[0019] The basal layer consists of two to three layers of small cuboidal cells.

[0020] In a flaccid (i.e., unexpanded) bladder, the urothelium is five to seven cell layers thick. In this unexpanded state, the typical bladder capacity of a healthy adult is about 500 ml. When the bladder fills with urine, the bladder wall stretches to accommodate the increased volume, and in this expanded state, the urothelium reorganizes into two to three layers without any structural damage.

[0021] The lamina propria forms an extracellular matrix that separates the urothelium from the underlying muscularis propria (detrusor muscle). This matrix contains many specialized cell types (e.g., elastic fibers, capillaries, afferent nerve endings, interstitial cells of Cajal, a thin layer of smooth muscle, and muscularis mucosa) and serves as the "functional center" of the bladder. In this regard, the lamina propria regulates the afferent limbs of the micturition reflex, and the interstitial cells of Cajal are thought to be transducers of nerve signals to the smooth muscle cells of the bladder. Thus, the lamina propria is a capacity-regulating layer of the bladder.

[0022] The muscularis propria is also called the detrusor muscle. It is innervated by the efferent (motor) nerves and consists of three layers: the inner longitudinal muscle, the middle circular muscle, and the outer longitudinal muscle.

[0023] The serosa and adventitia are thin connective tissue layers that make up the outermost layer of the bladder.

[0024] Together, these layers are responsible for maintaining the homeostasis of the bladder (e.g., urination), while the bladder wall lining functions to isolate and protect adjacent tissues and organs from exposure to urine stored in the bladder, including toxic solutes and metabolites in the urine. In this way, the bladder wall lining provides a highly efficient barrier to urine penetration. However, an unfavorable consequence of this highly specialized function is that the bladder wall also impedes the penetration of potential therapeutic molecules. This presents a significant challenge for any treatment of bladder pain syndrome and / or interstitial cystitis, as therapeutic molecules must first cross the bladder wall barrier before they can have a clinically relevant effect on target cells located deeper in the bladder wall.

[0025] While non-invasive approaches have attracted some attention, a common problem with these approaches is that they provide limited beneficial effects at best and are very slow to take effect (may take up to 6 months). In addition, frequent repeat dosing is required. Pentosan polysulfate sodium remains the only FDA-approved oral medication for the treatment of painful bladder syndrome / interstitial cystitis. Pentosan polysulfate sodium is structurally similar to the natural glycosaminoglycan coating of the bladder lining and is thought to temporarily repair the bladder lining. Although its exact mechanism of action is unknown, it is known that It adheres to the luminal side of the bladder wall and forms a layer, so it acts as a chemical filler or sealant, effectively covering any damaged areas of the urothelium.

[0026] Invasive approaches address these shortcomings and are the preferred intervention for the treatment of painful bladder syndrome and / or interstitial cystitis. Although invasive approaches often involve some form of physical intervention, this brings with it additional patient management issues ranging from anesthesia to intravenous sedation. One of the most common physical intervention procedures is intradetrusor injection (see Figure 1 ), a procedure that requires careful insertion of an instrument through the urethral opening and down the urethra into the patient's bladder. The needle is then manipulated sequentially to multiple predetermined injection sites on the lining of the bladder wall under cystoscopic guidance. Each injection requires the needle to penetrate the lining of the bladder wall (into the detrusor muscle) and deliver a dose of the therapeutic agent at each injection site. Adverse consequences of this procedure include local bleeding and pain, and may result in significant patient management issues. In particular, side effects of this procedure, such as local bleeding and pain, may result in lower overall patient satisfaction, which in turn may result in reduced patient compliance with this treatment regimen.

[0027] Another method is intravesical instillation (see Figure 2 ), which involves inserting a catheter into the patient's urethra and draining any urine present in the bladder. A small amount (e.g., 50 ml) of medication is then slowly injected into the bladder through the catheter. The catheter is removed and the patient is instructed to resume normal daily activities but not to empty the bladder for at least 15 minutes, and preferably at least 90 minutes. In this way, the medication can contact and potentially treat the entire lining of the bladder wall. Unfortunately, however, this procedure fails to address the impermeable barrier posed by the bladder wall lining and is therefore limited in that it is only suitable for treatments that act on the luminal side of the bladder wall lining.

[0028] Attempts have been made to modify this procedure to expand the range of treatment options, including targeting cells located deeper in the bladder wall. One such approach relies on the use of chemical peeling agents (e.g., dimethyl sulfoxide (DMSO), protamine sulfate, hyaluronic acid-phosphatidylethanolamine) that are dissolved in a liquid instillation mixture and administered to the patient as part of a standard instillation regimen. For example, DMSO may be instilled at a 50% concentration as a single agent or, more commonly, as part of a “cocktail” with methylprednisolone or hydrocortisone, alkalinized lidocaine, and heparin sulfate. Once administered, the peeling agents attack any cells they come into contact with, gradually stripping cells from the urothelial layer and permeabilizing the bladder wall.

[0029] Unfortunately, instillations with a stripping agent have not proven to be an ideal approach for the treatment of painful bladder syndrome and / or interstitial cystitis. There are several reasons for this, the most important of which is that chemical strippers are toxic (and often non-selective) irritants, and therefore prolonged exposure by the patient or physician should be avoided. As a result, instillations with a stripping agent are not suitable for repeated use. Furthermore, once initiated, it is difficult to adequately control the stripping process, which in turn makes the procedure unpredictable and therefore unreliable. Relevant factors include chemical agent variables (e.g., choice and concentration of agent), instillation variables (e.g., contact time with the agent), and patient-specific variables (e.g., extent of existing bladder wall damage and responsiveness to the chosen therapeutic molecule or regimen).

[0030] The lining of the bladder wall (urothelium) is not the only structural challenge that needs to be addressed when considering the delivery of therapeutic molecules to relevant target cells for the treatment of bladder pain syndrome and / or interstitial cystitis. For example, when the target cells are located in the deep layers of the bladder wall, the therapeutic molecules must be able to penetrate the bottom layers of the bladder wall to reach these target cells and exert their therapeutic effects. Therefore, it is not surprising that the physical constraints imposed by the structure of the bladder wall are inherently more favorable to small molecule therapeutic drugs, making this type of molecule the preferred molecular class for the treatment of bladder pain syndrome and / or interstitial cystitis.

[0031] Therapeutic interventions are further complicated by the need to ensure that the therapeutic molecule is selectively delivered to the relevant target cells, thereby avoiding or minimizing any undesirable off-target effects. In the context of treating painful bladder syndrome and / or interstitial cystitis, this requires preferential selective targeting of sensory afferent nerve fibers in the underlying tissue of the bladder wall over efferent nerve fibers.

[0032] The use of clostridial neurotoxins, particularly botulinum neurotoxin A (BoNT / A), for the treatment of painful bladder syndrome by intradetrusor injection is known. Intradetrusor injection of BoNT / A is highly invasive and simultaneously targets all three levels of innervation of the bladder wall, namely: the urothelium; the afferent nerve endings of the lamina propria; and the efferent nerve endings of the detrusor muscle (see Figure 3 ). This approach does not achieve selective targeting of BoNT / A to sensory afferent fibers (vs. efferent fibers). In addition, binding of BoNT / A to detrusor efferent nerve endings may result in undesirable side effects such as bleeding.

[0033] On the other hand, intravesical instillation of BoNT / A (without injection) does not deliver BoNT / A to the bladder wall due to the impermeability of the bladder urothelium to large molecules and the degradation of BoNT / A by proteases in urine - see Khera, M. et al. (2005) Urology, 66, 208-212; and Shimizu, S. (2012) J. Urol., 187, e370. Attempts have been made to use liposome-encapsulated BoNT / A to address the degradation issue.

[0034] Although some degree of delivery of BoNT / A has been reported (when co-administered via intravesical instillation with DMSO, protamine sulfate, or hyaluronan-phosphatidylethanolamine), this approach using a stripping agent has not provided a recognized, significant improvement over intradetrusor injection of BoNT / A, which remains the current therapeutic intervention of choice for the treatment of bladder painful syndrome.

[0035] Some degree of delivery of BoNT / A has also been reported when administered in hydrogel or liposome-encapsulated formulations (via intravesical instillation). However, similar to approaches using exfoliants, the use of hydrogels or liposomes has not provided a well-established, significant improvement over intradetrusor injection of BoNT / A, which remains the current therapeutic intervention of choice for the treatment of bladder pain syndrome.

[0036] Therefore, there is a need in the art for a method of treating painful bladder syndrome and / or interstitial cystitis that selectively and preferentially targets sensory afferent fibers in the bladder wall over efferent fibers while being minimally invasive (e.g., less invasive than intradetrusor injection). There is also a need to avoid the use of dissecting agents.

[0037] The present invention solves one or more of the problems set forth above. SUMMARY OF THE INVENTION

[0038] The present invention relates to the use of Clostridial neurotoxins, such as botulinum neurotoxins (BoNTs), as therapeutic agents for treating painful bladder syndrome (BPS), particularly interstitial cystitis (IC), in a subject in need thereof.

[0039] In more detail, the present inventors surprisingly discovered for the first time that administration of a clostridial neurotoxin (e.g., BoNT / A) by a unique method, i.e., gentle pressure instillation of a solution containing the neurotoxin into the bladder, can reduce or eliminate pain associated with bladder pain syndrome, particularly interstitial cystitis, significantly improve the quality of life of patients, and has virtually no side effects, unlike conventional methods.

[0040] A surprising discovery of the inventors is that this approach generates a mild mechanical force on the urothelial layer of the bladder sufficient to diffuse the clostridial neurotoxin across the urothelial layer and into the lamina propria, while limiting further diffusion of the neurotoxin into deeper layers of the bladder wall, particularly the detrusor muscle. This mild pressure-driven diffusion of the clostridial neurotoxin enables the toxin to target primary sensory afferent nerves located within the lamina propria, inhibiting the release of neurotransmitters therein, thereby relieving pain perception.

[0041] This is possible because the urothelial layer of the bladder of subjects suffering from painful bladder syndrome, particularly interstitial cystitis, is damaged and porous, so the solution is able to pass through this layer with the aid of the light pressure infusion technique of the present invention. A further advantage is that no needles or irritating excipients are required, so only a simple saline-based solution is required. This is in contrast to existing methods that use painful needle injections or highly irritating formulations to deliver clostridial neurotoxins (such as BoNT) to the lamina propria and deeper layers. Therefore, unlike traditional methods that intentionally damage the urothelial layer and deeper tissues, the drug delivery technology of the present invention (light pressure infusion) has no or little effect on the integrity of the urothelial cell layer, for example, it will not cause more severe damage than existing damage.

[0042] In practice, the patient's bladder is typically flushed / cleaned prior to gentle instillation. Afterwards, the bladder is instilled with the clostridial neurotoxin solution, for example, via a simple catheter, which applies gentle pressure to the urothelial lining. This instillation is continued up to, but not beyond, the point at which the patient experiences the urge to urinate. In healthy adults, this occurs (usually) when the bladder holds approximately 300-400 ml of fluid, although in patients with painful bladder syndrome, this typically occurs when the bladder holds 200-300 ml of fluid, and sometimes even less. The urge to urinate occurs when stretch receptors in the bladder signal the parasympathetic nervous system to stimulate the muscarinic receptors of the detrusor muscle to contract, thereby initiating urination. Therefore, it may take some trial and error for each patient to determine the threshold volume of fluid that will trigger bladder emptying. Then, simply work backwards from this threshold volume to calculate a reduced volume (e.g., 5% or 10% reduction) to establish a safety factor to ensure that the patient will not urinate while receiving the clostridial neurotoxin solution. Once the desired volume of the clostridial neurotoxin solution has been infused, the catheter is closed and the solution is allowed to diffuse across the urothelium.

[0043] Importantly, the inventors observed that this light pressure-driven diffusion of the clostridial neurotoxin is generally confined to the lamina propria. In particular, this diffusion does not extend to the bladder muscle layer, so that the clostridial neurotoxin does not target the efferent nerve endings of the detrusor muscle as traditional intra-bladder detrusor injections do, thereby avoiding adverse side effects such as bleeding and muscle paralysis. Instead, the new method enables precise delivery of the clostridial neurotoxin, allowing the neurotoxin to diffuse systemically in the urothelial layer and lamina propria of the bladder wall to target afferent nerve endings (sensory endings capable of transmitting pain signals) located in the lamina propria (see Figure 4 The advantage of the novel administration method (light pressure infusion) of the present invention is that it provides a simple, painless and non-invasive way to administer the drug (clostridial neurotoxin without a needle), so the operation can be performed not only by nurses but also by patients themselves. The non-invasive nature of this method combined with the desired treatment effect improves the overall patient satisfaction and thus enhances patient compliance with this treatment regimen.

[0044] Detailed Description

[0045] In one aspect, the present invention provides a method of treating a patient suffering from painful bladder syndrome, the method comprising:

[0046] Injecting a solution containing a clostridial neurotoxin into the patient’s bladder;

[0047] Increasing the volume of a solution containing clostridial neurotoxins in the bladder, thereby exerting mechanical forces on the inner surface of the urothelium;

[0048] Maintaining the intravesical volume of the solution containing the clostridial neurotoxin at a volume that does not induce urination for at least 30 minutes allows the clostridial neurotoxin to diffuse across the urothelium and into the lamina propria where it binds to primary sensory afferent nerve fibers and inhibits the secretion of neurotransmitters therefrom, thereby relieving bladder pain.

[0049] In one aspect, the present invention provides a solution comprising a clostridial neurotoxin for use in a method of treating a patient suffering from bladder pain syndrome, the method comprising:

[0050] Injecting a solution containing a clostridial neurotoxin into the patient’s bladder;

[0051] Increasing the volume of a solution containing clostridial neurotoxins in the bladder, thereby exerting mechanical forces on the inner surface of the urothelium;

[0052] Maintaining the intravesical volume of the solution containing the clostridial neurotoxin at a volume that does not induce urination for at least 30 minutes allows the clostridial neurotoxin to diffuse across the urothelium and into the lamina propria where it binds to primary sensory afferent nerve fibers and inhibits the secretion of neurotransmitters therefrom, thereby relieving bladder pain.

[0053] The bladder wall consists of three cell layers: the urothelium, the lamina propria, and the detrusor muscle. The urothelium is the innermost layer of the bladder wall and is a unique, highly specialized epithelial lining that acts as a barrier separating the contents of the bladder lumen from the tissues beneath the urothelium. The lamina propria is a layer of loose connective tissue that separates the urothelium from the detrusor muscle, which is composed of longitudinal and circular smooth muscle fibers.

[0054] Advantageously, the inventors have observed that filling the bladder of a patient with painful bladder syndrome / interstitial cystitis to, but not beyond, the point at which the patient experiences the urge to urinate with a solution containing a clostridial neurotoxin exerts mild pressure on the urothelial layer, allowing the clostridial neurotoxin to diffuse through that layer and into the lamina propria, driven by the mild pressure.

[0055] A variety of methods can be used to determine a patient's threshold volume of fluid that triggers bladder emptying. For example, urination diaries, uroflowmetry, ultrasound scanning, and cystometry can be used.

[0056] A voiding diary can be a record of a patient's daily fluid intake and urination (including accidental urine leakage) over a 24-hour period.

[0057] Uroflowmetry may involve having the patient urinate, with the uroflowmeter recording the total volume, rate, and length of time that urine passes from the bladder. A bladder scan may then be performed to assess whether the patient has residual urine volume in the bladder. The threshold volume of fluid that triggers bladder emptying can then be calculated based on the results of the uroflowmeter and bladder scan.

[0058] An ultrasound scan is a scan of the pelvic area of ​​a patient with a full bladder (before urinating) to determine the amount of fluid in the patient's bladder. An ultrasound scan can provide information about the size of a patient's bladder, how full it is, and / or the bladder wall.

[0059] Cystometry is performed by inserting a 5-F catheter into the patient's bladder while the patient is in a sitting position and instilling saline solution at a rate of 50 ml per minute. The amount of saline solution instilled into the bladder at which the patient can no longer hold urine is recorded (in milliliters) to determine the threshold volume of fluid that triggers bladder emptying.

[0060] It will be appreciated by those skilled in the art that for patients with painful bladder syndrome / interstitial cystitis (BPS / IC), the threshold volume of liquid that triggers urination may vary from individual to individual. In addition, it will be appreciated by those skilled in the art that the threshold volume may also differ between BPS / IC patients and healthy patients.

[0061] In order to prevent the patient from urinating during treatment while applying gentle pressure to the urothelium, a solution containing a clostridial neurotoxin can be administered at a volume below the threshold volume of fluid that triggers urination. The volume of the solution containing a clostridial neurotoxin administered to the patient can be reduced by at least 5%, 10%, 15%, 20%, 25%, 30% compared to the threshold volume of fluid that triggers urination in the same patient. The volume of the solution containing a clostridial neurotoxin administered to the patient can be reduced by less than or equal to 70%, 60%, 50%, or 40% compared to the threshold volume of fluid that triggers urination in the same patient. The volume of the solution containing a clostridial neurotoxin administered to the patient can be reduced by 5-70%, 10-60%, 15-50%, 20-40%, or 25-30% compared to the threshold volume of fluid that triggers urination in the same patient.

[0062] The reduced volume of the solution containing the clostridial neurotoxin administered to the patient may be at least 150, 175, 200, 225, 250, 275, or 300 milliliters. The reduced volume of the solution containing the clostridial neurotoxin administered to the patient may be less than or equal to 400, 375, 350, 325, 300, 275, or 250 milliliters. The reduced volume of the solution containing the clostridial neurotoxin administered to the patient may be 150-400, 175-375, 200-350, 225-325, or 250-300 milliliters.

[0063] In one embodiment, the duration of a single therapeutic infusion with a solution containing a clostridial neurotoxin is a prescribed duration, for example, a duration of at least 15 minutes, 20 minutes, 25 minutes, 30 minutes, or 40 minutes, preferably at least 45 minutes, and more preferably at least 50 minutes (e.g., at least 1 hour). In one embodiment, the duration of a single therapeutic infusion with a solution containing a clostridial neurotoxin is less than or equal to 2 hours, 1 hour 45 minutes, 1 hour 30 minutes, or 1 hour 25 minutes, preferably less than or equal to 1 hour 15 minutes, more preferably less than or equal to 1 hour 10 minutes (e.g., at least 1 hour). In one embodiment, the duration of a single therapeutic infusion with a solution containing a clostridial neurotoxin is 15 minutes to 2 hours, 30 minutes to 1 hour 45 minutes, 45 minutes to 1 hour 30 minutes, preferably 45 minutes to 1 hour 15 minutes, and more preferably 50 minutes to 1 hour 10 minutes (e.g., 1 hour). Thereafter, the physician may ask the patient to urinate to expel the solution from the patient's bladder.

[0064] The patient may receive a course of treatment that includes multiple therapeutic perfusions over a specified time period, such as multiple therapeutic perfusions over a 2-week period. In one embodiment, as part of a course of treatment, a solution containing a clostridial neurotoxin is administered by gentle pressure perfusion, wherein the course of treatment includes multiple discrete perfusions over a specified time period of at least 2 weeks, 3 weeks, or 4 weeks, preferably at least 5 weeks, and more preferably at least 6 weeks (e.g., at least 7 weeks). In one embodiment, as part of a course of treatment, a solution containing a clostridial neurotoxin is administered by gentle pressure perfusion, wherein the course of treatment includes multiple discrete perfusions over a specified time period of less than or equal to 12 weeks, 11 weeks, or 10 weeks, preferably less than or equal to 9 weeks, and more preferably less than or equal to 8 weeks (e.g., less than or equal to 7 weeks). In one embodiment, as part of a course of treatment, a solution containing a clostridial neurotoxin is administered by gentle pressure perfusion, wherein the course of treatment includes multiple discrete perfusions over a specified time period of 2-12 weeks, 3-11 weeks, or 4-10 weeks, preferably 5-9 weeks, and more preferably 6-8 weeks (e.g., 7 weeks).

[0065] Multiple discrete perfusions can be administered at a frequency of at least 1 or 2 times per week. Multiple discrete perfusions can be administered at a frequency of less than or equal to 4 times, 3 times, 2 times or 1 times per week. Multiple discrete perfusions can be administered at a frequency of 1-4 times, 1-3 times or 2-3 times per week, preferably 2-3 times per week.

[0066] The term "hydrodistention" refers to a method of using a cystoscope to infuse sterile liquid into the bladder until the bladder is overdistended. Hydrodistention may involve filling the bladder with a solution at a pressure of 60-80 cmH0 (Inoue et al., "Hydrodistention of the bladder in patients with interstitial cystitis--clinical efficacy and its association with immunohistochemical findings for bladder tissues." Hinyokika Kiyo 52(10)(2006):765-8). Hydrodistention relies on the use of high pressure to overdistend the bladder.

[0067] The method of administration of the present invention is different from and not equivalent to hydrodilation.

[0068] Thus, in one embodiment, administering a solution containing a clostridial neurotoxin to a patient does not involve hydrodistention of the patient's bladder. In one embodiment, administering a solution containing a clostridial neurotoxin to a patient does not involve distention of the bladder to a pressure of at least 50, 60, 70, or 80 cmH0. In one embodiment, administering a solution containing a clostridial neurotoxin to a patient does not involve distention of the bladder to a pressure of less than or equal to 50-100, 60-90, or 70-80 cmH0.

[0069] In one embodiment, the clostridial neurotoxin selectively binds to primary sensory afferent nerve fibers. In one embodiment, the clostridial neurotoxin is substantially retained within the lamina propria (preferably, the clostridial neurotoxin does not diffuse into the detrusor muscle of the bladder wall). Thus, advantageously, unlike conventional methods, the methods of the present invention avoid targeting efferent nerve endings located in the detrusor muscle (which can result in bleeding and muscle paralysis).

[0070] In one embodiment, a solution containing a clostridial neurotoxin diffuses systemically in the lamina propria of the bladder wall, and wherein the clostridial neurotoxin inhibits neurotransmitter release from substantially all parasympathetic afferent neurons therein. Thus, unlike conventional methods (e.g., intra-detrusor injection into the bladder), the methods of the present invention can achieve local administration of clostridial neurotoxins with little or no diffusion to surrounding areas and little systemic circulation (better targeting of sensory fibers).

[0071] The term "systemically" refers to the distribution of the solution throughout the tissue, preferably the lamina propria of the bladder wall. The solution can be evenly dispersed in up to two layers of the bladder wall. The solution can be dispersed in the urothelium and / or the lamina propria. The solution can diffuse systemically in the lamina propria without diffusing into deeper layers of the bladder wall, such as the detrusor muscle.

[0072] In the context of inhibiting the release of neurotransmitters from parasympathetic afferent neurons, the term "substantially" can mean inhibiting the release of neurotransmitters from 50-100%, 60-90%, or 70-80% of the parasympathetic afferent neurons in the lamina propria. Preferably, the Clostridial neurotoxin can inhibit the release of neurotransmitters from 100% of the parasympathetic afferent neurons in the lamina propria.

[0073] In one embodiment, the solution is injected into the bladder via a catheter. In one embodiment, the method is substantially non-invasive, and preferably, the method does not cause substantial physical damage to the urothelium and / or lamina propria. Thus, an advantage of the present invention is that the clostridial neurotoxin is administered by a non-invasive method that avoids the use of needles and is therefore painless.

[0074] In one embodiment, the solution can avoid further damage to urothelial cells and lamina propria cells. In one embodiment, the solution comprises (preferably consists of) a clostridial neurotoxin and a physiologically inert buffer. Preferably, the physiologically inert buffer can be phosphate buffered saline. In one embodiment, the method of the present invention avoids the use of a stripping agent, thereby preventing further damage to the bladder wall. The stripping agent can be DMSO, protamine sulfate and / or hyaluronic acid-phosphatidylethanolamine. In other words, unlike the traditional method of using chemical irritants (such as DMSO) to permeabilize bladder wall cells, the present invention can administer a harmless (does not contain irritating excipients such as DMSO) solution containing a clostridial neurotoxin.

[0075] The term "inert" means that a solution may be chemically inactive, eg, not causing irritation or tissue damage.

[0076] In one embodiment, the method does not cause the patient to urinate (preferably, the Clostridial neurotoxin does not bind to parasympathetic efferent neurons to stimulate muscarinic receptors in the detrusor muscle to contract the detrusor muscle). Thus, advantageously, the method provides sufficient time for the solution containing the Clostridial neurotoxin to diffuse across the urothelium and before the patient urinates, allowing the patient to benefit from the therapeutic effects of the solution.

[0077] In one embodiment, a single therapeutic perfusion with a solution comprising a clostridial neurotoxin is performed for a period of at least 1 hour.

[0078] In summary, the present invention effectively treats bladder pain syndrome by reducing or eliminating the pain level perceived by the patient and improving the patient's quality of life.

[0079] Thus, in one embodiment, bladder pain is reduced following treatment with a solution containing a clostridial neurotoxin using the methods of administration of the invention. For example, pain levels in patients with BPS / IC treated with a solution containing a clostridial neurotoxin using the methods of administration of the invention can be reduced or eliminated compared to pain levels in (control) patients not treated with a solution containing a clostridial neurotoxin.

[0080] In one embodiment, bladder pain is reduced for at least 2 days following gentle instillation of the bladder with a solution comprising a clostridial neurotoxin.

[0081] In one embodiment, after the bladder is gently perfused with a solution containing a clostridial neurotoxin, bladder pain is relieved for at least 1 day, preferably at least 2 days, more preferably at least 3 days (e.g., at least 4 days). In one embodiment, after the bladder is gently perfused with a solution containing a clostridial neurotoxin, bladder pain is relieved for less than or equal to 9 days, 8 days, or 7 days, preferably less than or equal to 6 days, more preferably less than or equal to 5 days (e.g., less than or equal to 4 days). In one embodiment, after the bladder is gently perfused with a solution containing a clostridial neurotoxin, bladder pain is relieved for 1-7 days or 2-6 days, preferably 2-5 days, more preferably 3-5 days (e.g., 4 days).

[0082] In one embodiment, after gentle pressure instillation of a solution containing a clostridial neurotoxin into the bladder, bladder pain is relieved for at least 3 months, 4 months, 5 months, or 6 months, preferably at least 7 months, and more preferably at least 8 months (e.g., at least 9 months). In one embodiment, after gentle pressure instillation of a solution containing a clostridial neurotoxin into the bladder, bladder pain is relieved for less than or equal to 14 months, 13 months, or 12 months, preferably less than or equal to 11 months, and more preferably less than or equal to 10 months (e.g., less than or equal to 9 months). In one embodiment, after gentle pressure instillation of a solution containing a clostridial neurotoxin into the bladder, bladder pain is relieved in 6-14 months or 7-13 months, preferably in 8-12 months, and more preferably in 9-11 months (e.g., less than or equal to 9 months).

[0083] One type of pain that may represent painful bladder syndrome is allodynia. Allodynia means "other pain." It is pain caused by a stimulus that does not normally cause pain. Patients with "tactile" allodynia (also called static tactile allodynia or mechanical allodynia) may experience pain while at rest, such as pain, pressure, and tenderness in the abdomen, or pain during urination. Therefore, allodynia is considered "pain caused by a stimulus that does not normally cause pain," which is different from hyperalgesia.

[0084] As used herein, the term "hyperalgesia" refers to an increased pain response to stimuli that would normally cause pain. Tissue or nerve damage can induce hyperalgesia, resulting in an increased perception of pain in the patient. Injury-induced hyperalgesia can be divided into two subtypes: primary hyperalgesia (resulting in increased pain perception at the local site of injury) and secondary hyperalgesia (pain is perceived in other parts of the body away from the site of injury).

[0085] Thus, in one embodiment, bladder pain comprises allodynia or hyperalgesia.

[0086] In one embodiment, after administration of a clostridial neurotoxin using the methods of the invention, the patient's pain threshold may be increased. In one embodiment, the patient's pain threshold may be increased by at least 30%, 40%, 50%, 60%, 70%, or 80% compared to a patient who did not receive a solution containing a clostridial neurotoxin. In one embodiment, the patient's pain threshold may be increased by 30-100%, 40-90%, 50-80%, or 60-70% compared to a patient who did not receive a solution containing a clostridial neurotoxin.

[0087] The term "pain threshold" refers to the level of noxious stimulation required for a patient to perceive pain.

[0088] Further details of the Clostridial neurotoxins encompassed by the present invention, as well as technical background information, are provided below.

[0089] Bacteria in the genus Clostridium produce highly potent and specific protein toxins that are toxic to neurons and other cells on which they act. Examples of such clostridial neurotoxins include the neurotoxin produced by C. tetani (TeNT), the neurotoxin produced by C. botulinum serotypes AG and X (see WO 2018 / 009903 A2), and the neurotoxins produced by C. baratii and C. butyricum.

[0090] Clostridial neurotoxins are some of the most potent toxins known. For example, the median lethal dose (LD50) values ​​of botulinum neurotoxin in mice vary depending on the serotype, ranging from 0.5 to 5 ng / kg. Both tetanus toxin and botulinum neurotoxin act by inhibiting the function of the affected neurons, specifically the release of neurotransmitters. Botulinum toxin acts at the neuromuscular junction, inhibiting cholinergic transmission in the peripheral nervous system, while tetanus toxin acts in the central nervous system.

[0091] In nature, clostridial neurotoxins are initially synthesized as single-chain polypeptides that are modified post-translationally by proteolytic cleavage events to form two polypeptide chains linked together by disulfide bonds. Cleavage occurs at a specific cleavage site, often referred to as the activation site, which is located between the cysteine ​​residues that form the interchain disulfide bonds. It is this two-chain form that is the active form of the toxin. The two chains are referred to as the heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and the light chain (L chain), which has a molecular weight of approximately 50 kDa. The H chain contains an N-terminal transport component (HN domain) and a C-terminal targeting component (HC domain). The cleavage site is located between the L chain and the translocation domain components. After the HC domain binds to the target neuron and the bound toxin is internalized into the cell via the endosome, the HN domain transports the L chain across the endosomal membrane into the cytoplasm, and the L chain provides the protease function (also known as the non-cytotoxic protease).

[0092] Non-cytotoxic proteases act by proteolytically cleaving an intracellular transporter called a SNARE protein (e.g. SNAP-25, VAMP or Syntaxin) - see Gerald K (2002) Cell and Molecular Biology (4th edition, John Wiley & Sons, Inc.). The acronym SNARE is derived from "soluble N-ethylmaleimide-sensitive factor attachment receptor", where NSF stands for ethylmaleimide-sensitive factor. SNARE proteins are essential for intracellular vesicle fusion and therefore for the secretion of molecules from the cell via vesicular transport. The protease function is a zinc-dependent endopeptidase activity with a high substrate specificity for SNARE proteins. Therefore, once delivered to the desired target cell, the non-cytotoxic protease is able to inhibit cellular secretion of the target cell. The L chain protease of Clostridial neurotoxin is an example of a non-cytotoxic protease that is able to cleave SNARE proteins.

[0093] Given the ubiquity of SNARE proteins, clostridial neurotoxins such as botulinum neurotoxin have been successfully applied in a wide range of therapeutic areas.

[0094] For example, William J. Lipham in Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004) describes the use of clostridial neurotoxins, such as botulinum neurotoxins (BoNTs), BoNT / A, BoNT / B, BoNT / C1, BoNT / D, BoNT / E, BoNT / F, and BoNT / G, and tetanus neurotoxin (TeNT), to inhibit neuronal transmission in a variety of therapeutic and cosmetic applications - for example, BOTOX™ is currently approved for the following therapeutic indications: treatment of pain (see U.S. Patient Guidelines for Use in Patients with Pain and Other Disorders). Patents 6,869,610; 6,641,820; 6,464,986 and 6,113,915); treating muscle injuries (see U.S. Patent 6,423,319); treating sinus headaches (see U.S. Patent 6,838,434); treating neurological diseases such as Parkinson's disease (see U.S. Patents 6,620,415; 6,306,403) and treating neuropsychiatric diseases (see U.S. Patent Applications 2004 / 0180061; 2003 / 0211121). All of the above publications are incorporated herein by reference in their entirety.

[0095] Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum as large protein complexes consisting of the BoNT itself in complex with a number of accessory proteins. Nine different classes of botulinum neurotoxins exist, namely: botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, and X, all of which have similar structures and modes of action. The different BoNT serotypes can be distinguished by inactivation by specific neutralizing antisera, and this classification by serotype is correlated with the percentage sequence identity at the amino acid level. BoNT proteins of a given serotype are further subdivided into different subtypes based on the percentage amino acid sequence identity.

[0096] BoNTs can be absorbed in the gastrointestinal tract and enter the blood circulation, where they bind to the presynaptic membrane of cholinergic nerve endings and prevent the release of the neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave synaptobrevin / VAMP; BoNT / C1, BoNT / A, and BoNT / E cleave 25kDa synaptosome-associated protein (SNAP-25); and BoNT / C1 also cleaves Syntaxin. BoNT / X has been found to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and Syntaxin 1.

[0097] Tetanus toxin is produced by Clostridium tetani and has only one serotype. Clostridium butyricum produces BoNT / E, while Clostridium pasteurianum produces BoNT / F.

[0098] Despite these differences, BoNT / A remains the serotype of choice for treatment, with three common commercial preparations available on the market ( and ), and there is only one BoNT / B product on the market To date, these purified BoNT / A and BoNT / B products from Clostridium strains are the only two BoNT serotypes currently approved by regulatory agencies for use in humans, including, but not limited to, spasticity, bladder dysfunction, or hyperhidrosis (for BoNT / A) (e.g., see https: / / www.medicines.org.uk / emc / medicine / 112, https: / / www.medicines.org.uk / emc / medicine / 870, https: / / www.medicines.org.uk / emc / medicine / 2162, the contents of which are incorporated herein by reference in their entireties) and cervical dystonia (for BoNT / B) (e.g., see https: / / www.medicines.org.uk / emc / medicine / 20568, the contents of which are incorporated herein by reference in their entireties).

[0099] Unlike cytotoxic proteases (e.g., ricin, diphtheria toxin, Pseudomonas exotoxin), which act by killing their natural target cells, clostridial neurotoxins are non-cytotoxic proteases that act by temporarily inactivating the cellular function of their natural target cells. Importantly, non-cytotoxic proteases do not kill the natural target cells they act on. With the exception of clostridial neurotoxins (e.g., Dysport TM 、Neurobloc TM and Botox TM In addition to botulinum neurotoxins sold under the names of ANTA®, some of the best-known examples of non-cytotoxic proteases include IgA proteases (see, for example, WO 99 / 032272 ) and antarease proteases (see, for example, WO 2011 / 022357 ).

[0100] The term "clostridial neurotoxin" encompasses any polypeptide produced by Clostridium bacteria that is capable of entering neurons and inhibiting the release of neurotransmitters, as well as such polypeptides produced by recombinant or chemical techniques. For the purposes of the present invention, the term includes the above-mentioned functionally equivalent non-cytotoxic proteases. Preferably, the clostridial neurotoxin is a botulinum neurotoxin (BoNT).

[0101] An example of a BoNT / A neurotoxin amino acid sequence is encoded by SEQ ID NO:2, whose nucleotide sequence is set forth in SEQ ID NO:1. An example of a BoNT / B neurotoxin amino acid sequence is set forth in SEQ ID NO:3 (UniProt Accession No. B1INP5). An example of a BoNT / C neurotoxin amino acid sequence is set forth in SEQ ID NO:4 (UniProt Accession No. P18640). An example of a BoNT / D neurotoxin amino acid sequence is set forth in SEQ ID NO:5 (UniProt Accession No. P19321). An example of a BoNT / E neurotoxin amino acid sequence is set forth in SEQ ID NO:6 (Accession No. WP_003372387). An example of a BoNT / F neurotoxin amino acid sequence is set forth in SEQ ID NO:7 (UniProt Accession No. Q57236) or in SEQ ID NO:8 (UniProt / UniParc Accession No. UPI0001DE3DAC). An example of a BoNT / G neurotoxin amino acid sequence is shown in SEQ ID NO:9 (Accession No. WP_039635782). An example of a BoNT / DC neurotoxin amino acid sequence is shown in SEQ ID NO:10 (Accession No. BAM65681). An example of a BoNT / X neurotoxin amino acid sequence is shown in SEQ ID NO:12 (Accession No. BAQ12790.1). In one embodiment, the BoNT selected is BoNT / A, such as a wild-type BoNT / A.

[0102] The term "HC domain" as used herein refers to a functionally distinct region in the heavy chain of a neurotoxin, with a molecular weight of approximately 50 kDa, which enables the neurotoxin to bind to receptors on the surface of target cells. The HC domain consists of two structurally distinct subdomains, the "HCN subdomain" (the N-terminal portion of the HC domain) and the "HCC subdomain" (the C-terminal portion of the HC domain), each with a molecular weight of approximately 25 kDa.

[0103] As used herein, the term "LHN domain" refers to a neurotoxin that does not contain a HC domain and consists of an endopeptidase domain (the "L" or "light chain") and a domain responsible for transporting the endopeptidase into the cytoplasm (the HN domain of the heavy chain).

[0104] As mentioned above, clostridial neurotoxins are formed by two polypeptide chains, the heavy chain (H chain) has a molecular weight of about 100 kDa and the light chain (L chain) has a molecular weight of about 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or HC domain) and an N-terminal transport component (HN domain).

[0105] Examples of light chain reference sequences include:

[0106] Botulinum neurotoxin type A: amino acid residues 1-448

[0107] Botulinum neurotoxin type B: amino acid residues 1-440

[0108] Botulinum neurotoxin type C1: amino acid residues 1-441

[0109] Botulinum neurotoxin type D: amino acid residues 1-445

[0110] Botulinum neurotoxin type E: amino acid residues 1-422

[0111] Botulinum neurotoxin type F: amino acid residues 1-439

[0112] Botulinum neurotoxin type G: amino acid residues 1-441

[0113] Tetanus neurotoxin: amino acid residues 1-457

[0114] For the recently discovered BoNT / X, whose L chain is reported to correspond to amino acids 1-439, the L chain boundary may differ by approximately 25 amino acids (eg, 1-414 or 1-464).

[0115] The reference sequences identified above should be considered as a guide, as there may be slight variations depending on the subtype. For example, U.S. Patent Application 2007 / 0166332 (incorporated herein by reference in its entirety) cites a slightly different Clostridium sequence:

[0116] Botulinum neurotoxin type A: amino acid residues M1-K448

[0117] Botulinum neurotoxin type B: amino acid residues M1-K441

[0118] Botulinum neurotoxin type C1: amino acid residues M1-K449

[0119] Botulinum neurotoxin type D: amino acid residues M1-R445

[0120] Botulinum neurotoxin type E: amino acid residues M1-R422

[0121] Botulinum neurotoxin type F: amino acid residues M1-K439

[0122] Botulinum neurotoxin type G: amino acid residues M1-K446

[0123] Tetanus neurotoxin: amino acid residues M1-A457

[0124] The term "activation loop" refers to a polypeptide domain comprising a proteolytic cleavage site. Activation loops of neurotoxins have been described in the art, for example in WO2016156113 (incorporated herein by reference in its entirety).

[0125] In one embodiment, the Clostridial 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 one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:12.

[0126] In one embodiment, the Clostridial 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 SEQ ID NO:2.

[0127] In one embodiment, the Clostridial neurotoxin consists of or comprises the amino acid sequence of SEQ ID NO: 2 (eg, BoNT / A).

[0128] The term "clostridial neurotoxin" is also intended to encompass modified clostridial neurotoxins and derivatives thereof, including, but not limited to, those described below. A modified clostridial neurotoxin or derivative may contain one or more modified amino acids compared to the native (unmodified) form of the clostridial neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form. For example, a modified clostridial neurotoxin may have a modified amino acid sequence in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence. These modifications may alter the functional properties of the toxin, such as biological activity or persistence.

[0129] A preferred modified BoNT / A comprises modifications at one or more amino acid residues selected from the group consisting of ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277. Such modified BoNT / As exhibit reduced or no side effects compared to known BoNT / As. The increased tissue retention properties of the modified BoNT / As of the present invention also provide greater efficacy and / or a longer duration of action, and may allow for the use of lower doses (or higher doses without any additional adverse effects) compared to known Clostridial toxin therapeutics, thereby providing further advantages.

[0130] The modifications are modifications compared to the unmodified BoNT / A as set forth in SEQ ID NO:2, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO:2. Since the methionine residue at position 1 in SEQ ID NO:2 (and the corresponding SEQ ID NOs of the modified BoNT / A polypeptides described herein) is optional, one skilled in the art will take into account the presence or absence of the methionine residue when determining the amino acid residue numbering. For example, if SEQ ID NO:2 includes a methionine, the position numbering will be as defined above (e.g., ASN 886 will be ASN 886 in SEQ ID NO:2). Alternatively, if the methionine is not present in SEQ ID NO:2, the amino acid residue numbering should be adjusted to minus 1 (e.g., ASN 886 will be ASN 885 in SEQ ID NO:2). Similar considerations apply when the methionine at position 1 in other polypeptide sequences described herein is present / absent, and one skilled in the art will readily determine the correct amino acid residue numbering using routine techniques in the art.

[0131] The amino acid residues indicated for modification are surface exposed amino acid residues.

[0132] A modified BoNT / A can comprise modifications at one or more amino acid residues selected from the group consisting of ASN886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277. The modified BoNT / A can be encoded by a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, and 19. For example, a nucleic acid sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, 17, and 19. Preferably, the modified BoNT / A for use in the present invention may consist of (or consist of) SEQ ID NOs: 13, 15, 17, and 19.

[0133] Preferably, the modified BoNT / A for use in the present invention may be encoded by a nucleic acid sequence comprising (or consisting of) SEQ ID NOs: 13, 15, 17, and 19. The modified BoNT / A may comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 14, 16, 18, and 20. 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: 14, 16, 18, and 20. More preferably, the modified BoNT / A for use in the present invention may comprise (more preferably consist of) a polypeptide sequence selected from SEQ ID NOs: 14, 16, 18, and 20.

[0134] When used in the context of a modified BoNT / A, the term "one or more amino acid residues" preferably refers to at least 2, 3, 4, 5, 6, or 7 of the indicated amino acid residues. Thus, a modified BoNT / A can comprise at least 2, 3, 4, 5, 6, or 7 (preferably 7) modifications at the indicated amino acid residues. A modified BoNT / A can comprise 1-30, 3-20, or 5-10 amino acid modifications. More preferably, when used in the context of a modified BoNT / A, the term "one or more amino acid residues" refers to all of the indicated amino acid residues.

[0135] Preferably, apart from the one or more amino acid modifications at the indicated amino acid residues, the modified BoNT / A does not comprise any further amino acid modifications compared to SEQ ID NO:2.

[0136] Most preferably, the modified BoNT / A comprises (more preferably consists of) a modification at one or more amino acid residues selected from the group consisting of ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, and GLN 1229. The modified BoNT / A can be encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 13. For example, a nucleic acid sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 13. Preferably, the modified BoNT / A for use in the present invention can be encoded by a nucleic acid sequence comprising (or consisting of) SEQ ID NO: 13. The modified BoNT / A can comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 14. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 14. More preferably, the modified BoNT / A for use in the present invention may comprise (more preferably consist of) SEQ ID NO:14.

[0137] The modification may be selected from:

[0138] i. replacing acidic surface exposed amino acid residues with basic amino acid residues;

[0139] ii. replacing acidic surface exposed amino acid residues with uncharged amino acid residues;

[0140] iii. replacing uncharged surface exposed amino acid residues with basic amino acid residues;

[0141] iv. inserting a basic amino acid residue; and

[0142] v. Deletion of acidic surface exposed amino acid residues.

[0143] The above modifications result in a modified BoNT / A having an increased positive surface charge and an increased isoelectric point compared to the corresponding unmodified BoNT / A.

[0144] The isoelectric point (pI) is a specific property of a given protein. As is well known in the art, proteins are made up of specific sequences of amino acids (also called amino acid residues in proteins). Each of the standard twenty amino acids has a different side chain (or R group), which means that each amino acid residue in a protein exhibits different chemical properties, such as charge and hydrophobicity. These properties can be affected by the surrounding chemical environment, such as temperature and pH. The overall chemical properties of a protein will depend on the sum of these various factors.

[0145] Certain amino acid residues (described below) have ionizable side chains that may exhibit a charge depending on the surrounding pH. At a given pH, whether such a side chain is charged depends on the pKa of the associated ionizable group, where pKa is the negative logarithm of the acid dissociation constant (Ka) for a particular proton in the conjugate base.

[0146] For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with a pKa value of approximately 4.1 (the exact pKa value may depend on temperature, ionic strength, and the microenvironment of the ionizable group). Therefore, at a pH of 7.4 (often referred to as "physiological pH"), these side chains have a negative charge. At low pH values, these side chains will be protonated and lose their charge.

[0147] In contrast, basic residues such as lysine and arginine have nitrogen-containing side chain groups with pKa values ​​of approximately 10-12. Therefore, at pH 7.4, these side chains carry a positive charge. At high pH values, these side chains will deprotonate and lose their charge.

[0148] Therefore, the overall (net) charge of a protein molecule depends on the number of acidic and basic residues present in the protein (and their surface exposure) and the surrounding pH. Changing the surrounding pH changes the overall charge of the protein. Therefore, for each protein there is a specific pH at which the number of positive and negative charges is equal and the protein exhibits no overall net charge. This point is called the isoelectric point (pI). The isoelectric point is a standard concept in protein biochemistry and should be familiar to those skilled in the art.

[0149] The isoelectric point (pI) is thus defined as the pH value at which a protein exhibits a net charge of zero. An increase in the pI value means that a higher pH value is required for the protein to exhibit a net charge of zero. Thus, an increase in the pI value represents an increase in the net positive charge of the protein at a given pH value. Conversely, a decrease in the pI value means that a lower pH value is required for the protein to exhibit a net charge of zero. Thus, a decrease in the pI value represents a decrease in the net positive charge of the protein at a given pH value.

[0150] Methods for determining protein pI values ​​are known in the art and should be familiar to those skilled in the art. For example, the pI value of a protein can be calculated based on 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 ExPASy's Compute pI / MW Tool (https: / / web.expasy.org / compute_pi / ), which is a preferred method for calculating pI values ​​according to the present invention. Comparisons of pI values ​​between different molecules should be performed using the same computational technique / program.

[0151] Where appropriate, the calculated pI value of a protein may be experimentally confirmed using an isoelectric focusing technique ("observed pI value"). This technique uses electrophoresis to separate proteins according to their pI values. Isoelectric focusing is typically performed using a gel with a fixed pH gradient. When an electric field is applied, the protein migrates in the pH gradient until it reaches a pH value where its net charge is zero, and this point is the pI value of the protein. The results provided by isoelectric focusing are generally relatively low resolution in nature, and therefore the inventors believe that the results provided using the above-described calculated pI values ​​(as described) are more suitable for use.

[0152] In this specification, "pI" means "calculated pI value" unless otherwise specified.

[0153] The pI value of a protein can be increased or decreased by changing the number of basic and / or acidic groups displayed on its surface. This can be achieved by modifying one or more amino acids of the protein. For example, the pI value can be increased by reducing the number of acidic residues or increasing the number of basic residues.

[0154] The modified BoNT / A of the 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 the unmodified BoNT / A (e.g., SEQ ID NO: 2). Preferably, the modified BoNT / A has a pi value of at least 6.6, such as at least 6.8.

[0155] The characteristics of the 20 standard amino acids are shown in the following table:

[0156] Amino Acids Sidechain Aspartic acid Asp D Charged (acidic) Glutamate Glu E Charged (acidic) Arginine Arg R Charged (alkaline) Lysine Lys K Charged (alkaline) Histidine His H No charge (polarity) Asparagine Asn N No charge (polarity) Glutamine Gln Q No charge (polarity) Serine Ser S No charge (polarity) Threonine Thr T No charge (polarity) Tyrosine Tyr Y No charge (polarity) Methionine Met M No charge (polarity) Tryptophan Trp W No charge (polarity) Cysteine Cys C No charge (polarity) Alanine Ala A Non-charged (hydrophobic) Glycine Gly G Non-charged (hydrophobic) Valine Val V Non-charged (hydrophobic) Leucine Leu L Non-charged (hydrophobic) Isoleucine Ile I Non-charged (hydrophobic) Proline Pro P Non-charged (hydrophobic) Phenylalanine Phe F Non-charged (hydrophobic)

[0157] The following amino acids are considered charged amino acids: aspartic acid (negative charge), glutamic acid (negative charge), arginine (positive charge), and lysine (positive charge).

[0158] 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.

[0159] The following amino acids are considered to be uncharged, polar (meaning they can participate in hydrogen bonding) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0160] The following amino acids are considered to be uncharged, hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0161] In an amino acid insertion, an additional amino acid residue (one that is not normally present) is incorporated into a BoNT / A polypeptide sequence, thereby increasing the total number of amino acid residues in the sequence. In an amino acid deletion, an amino acid residue is removed from a Clostridial toxin amino acid sequence, thereby decreasing the total number of amino acid residues in the sequence.

[0162] Preferably, the modification is a substitution, which advantageously maintains the number of amino acid residues in the modified BoNT / A unchanged. In an amino acid substitution, an amino acid residue in the BoNT / A polypeptide sequence is replaced with a different amino acid residue. The substituted amino acid residue can be one of the 20 standard amino acids described above. Alternatively, in an amino acid substitution, the substituted amino acid can be a non-standard amino acid (an amino acid that does not belong to the set of the standard 20 amino acids described above). For example, the substituted amino acid can be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid, or the D-isomer of lysine, arginine, and ornithine). Methods for introducing non-standard amino acids into proteins are known in the art, including the use of an E. coli auxotrophic expression host for recombinant protein synthesis.

[0163] In one embodiment, the substitution is selected from the group consisting of: 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, when the substitution is substitution of an acidic amino acid residue with an uncharged amino acid residue, the acidic amino acid residue is substituted by its corresponding uncharged amide amino acid residue (i.e., aspartic acid is substituted by asparagine, and glutamic acid is substituted by glutamine).

[0164] Preferably, the basic amino acid residue is a lysine residue or an arginine residue. In other words, the substitution is with lysine or arginine. Most preferably, the modification is with lysine.

[0165] A translocation domain is a molecule that enables the transport of a protease into a target cell, thereby allowing the protease activity to function in the cytoplasm of the target cell. Whether any molecule (eg, a protein or peptide) has the desired transport function of the present invention can be confirmed by any of a number of routine assays.

[0166] For example, Shone C. (1987) described an in vitro assay using liposomes that were challenged with a test molecule. +The presence of the desired transport function can be confirmed by the release of 5-nitropropene and / or labeled NAD, which can be readily monitored [see Shone C. (1987) Eur. J. Biochem; Vol. 167(1): pp. 175-180].

[0167] Another example is provided by Blaustein R. (1987), who described a simple in vitro assay using a planar phospholipid bilayer membrane. The membrane is challenged with a test molecule and the desired transport function is confirmed by an increase in the conductivity of the membrane [see Blaustein (1987) FEBS Letts; Vol. 226, No. 1: pp. 115-120].

[0168] Other methods for assessing membrane fusion and thereby identifying translocation domains suitable for use in the present invention are provided in Methods in Enzymology, Vol. 220 and 221, Parts A and B (Academic Press 1993).

[0169] The present invention also encompasses variant translocation domains, as long as the variant domain still shows the desired transport activity. For example, the variant may have at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% or at least 98% amino acid sequence homology with the reference translocation domain. When used for a translocation domain, the term "fragment" refers to a peptide with at least 20, preferably at least 40, more preferably at least 80, and most preferably at least 100 amino acid residues of the reference translocation domain. For a clostridial translocation domain, the fragment preferably has at least 100, preferably at least 150, more preferably at least 200, and most preferably at least 250 amino acid residues of the reference translocation domain (e.g., HN domain). The transport "fragment" of the present invention encompasses fragments of variant translocation domains based on reference sequences.

[0170] The translocation domain is preferably capable of forming ion permeable pores in lipid membranes under low pH conditions. Preferably, it has been found to use only those parts of the protein molecule that are capable of forming pores in endosomal membranes.

[0171] The translocation domain can be obtained from a microbial protein source, in particular from a bacterial or viral protein source. Thus, in one embodiment, the translocation domain is a translocation domain of an enzyme, such as a bacterial toxin or a viral protein.

[0172] It is well documented that domains of certain bacterial toxin molecules are capable of forming such pores. It is also known that translocation domains of membrane fusion proteins expressed by certain viruses are capable of forming such pores. These domains can be used in the present invention.

[0173] The translocation domain may be derived from Clostridium, such as the HN domain (or a functional component thereof). HN refers to a portion or fragment of the heavy chain of a Clostridium neurotoxin, approximately corresponding to the N-terminal half of the heavy chain, or a domain in the intact heavy chain corresponding to the fragment.

[0174] Examples of suitable (reference) translocation domains include:

[0175] Botulinum neurotoxin type A - amino acid residues (449-871)

[0176] Botulinum neurotoxin type B - amino acid residues (441-858)

[0177] Botulinum neurotoxin type C - amino acid residues (442-866)

[0178] Botulinum neurotoxin type D - amino acid residues (446-862)

[0179] Botulinum neurotoxin type E - amino acid residues (423-845)

[0180] Botulinum neurotoxin type F - amino acid residues (440-864)

[0181] Botulinum neurotoxin type G - amino acid residues (442-863)

[0182] Tetanus neurotoxin - amino acid residues (458-879)

[0183] The reference sequences identified above should be considered as a guide, as there may be slight variations depending on the subtype. For example, U.S. Patent Application 2007 / 0166332 (incorporated herein by reference in its entirety) cites a slightly different Clostridium sequence:

[0184] Botulinum neurotoxin type A - amino acid residues (A449-K871)

[0185] Botulinum neurotoxin type B - amino acid residues (A442-S858)

[0186] Botulinum neurotoxin type C - amino acid residues (T450-N866)

[0187] Botulinum neurotoxin type D - amino acid residues (D446-N862)

[0188] Botulinum neurotoxin type E - amino acid residues (K423-K845)

[0189] Botulinum neurotoxin type F - amino acid residues (A440-K864)

[0190] Botulinum neurotoxin type G - amino acid residues (S447-S863)

[0191] Tetanus neurotoxin - amino acid residues (S458-V879)

[0192] In the context of the present invention, a variety of clostridial neurotoxin HN regions comprising translocation domains can be used in various aspects of the present invention, provided that these active fragments can promote the release of non-cytotoxic proteases (e.g., clostridial L chains) from intracellular vesicles into the cytoplasm of target cells, thereby participating in the overall cellular mechanism of proteolytic cleavage of substrates by clostridial neurotoxins. The HN region of the clostridial neurotoxin heavy chain is about 410-430 amino acids in length and comprises a translocation domain. Studies have shown that the entire HN region length of the clostridial neurotoxin heavy chain is not necessary for the transport activity of the translocation domain. Therefore, various aspects of this embodiment may include a clostridial neurotoxin HN region comprising a translocation domain, for example, a length of at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, and at least 425 amino acids. Other aspects of this embodiment may include a clostridial neurotoxin HN region comprising a translocation domain, for example, a length of at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, and at most 425 amino acids.

[0193] For more detailed information on the genetic basis of toxin production by Clostridium botulinum and Clostridium tetani, see Henderson et al. (1997), The Clostridia: Molecular Biology and Pathogenesis, Academic press.

[0194] The term HN encompasses naturally occurring neurotoxin HN portions, as well as modified HN portions having amino acid sequences not occurring in nature and / or synthetic amino acid residues, as long as the modified HN portion still exhibits the above-mentioned transport function.

[0195] Alternatively, the translocation domain may be derived from a non-Clostridium. Examples of non-Clostridium (reference) translocation domain sources include, but are not limited to, the translocation domain of diphtheria toxin [O'Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp. 25-51], the translocation domain of Pseudomonas exotoxin A [Prior et al., Biochemistry (1 992) 31, 3555-3559], the translocation domain of anthrax toxin [Blanke et al., Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442], a variety of fusion or hydrophobic peptides with transport function [Plank et al., J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al. (1992) PNAS, 89, pp. 7934-7938], and amphipathic peptides [Murata et al. (1992) Biochem., 31, pp. 1986-1992]. The translocation domain may be similar to the translocation domain present in naturally occurring proteins, or may include amino acid changes, as long as these changes do not destroy the transport ability of the translocation domain.

[0196] Examples of Clostridial neurotoxin HC domain reference sequences include:

[0197] ·BoNT / A-N872-L1296

[0198] ·BoNT / B-E859-E1291

[0199] ·BoNT / C1-N867-E1291

[0200] BoNT / D-S863-E1276

[0201] ·BoNT / E-R846-K1252

[0202] ·BoNT / F-K865-E1274

[0203] ·BoNT / G-N864-E1297

[0204] ·TeNT-I880-D1315

[0205] For the recently discovered BoNT / X, the HC domain is reported to correspond to amino acids 893-1306, with domain boundaries likely to differ by approximately 25 amino acids (eg, 868-1306 or 918-1306).

[0206] The clostridial neurotoxins described herein may also comprise a transport facilitating domain. Such domains facilitate delivery of non-cytotoxic proteases to the cytoplasm of target cells and are described, for example, in WO 08 / 008803 and WO 08 / 008805, both of which are incorporated herein by reference in their entirety.

[0207] For example, suitable transport facilitating domains include enveloped virus fusion peptide domains, for example, suitable fusion peptide domains include influenza virus fusion peptide domains (e.g., 23 amino acids of influenza A virus fusion peptide domain), alphavirus fusion peptide domains (e.g., 26 amino acids of Sindbis Forest virus fusion peptide domain), vesicular stomatitis virus fusion peptide domains (e.g., 21 amino acids of vesicular stomatitis virus fusion peptide domain), respiratory virus fusion peptide domains (e.g., 25 amino acids of Sendai virus fusion peptide domain), measles virus fusion peptide domains (e.g., 25 amino acids of canine distemper virus fusion peptide domain), avian paramyxovirus fusion peptide domains (e.g., 25 amino acids of Newcastle disease virus fusion peptide domain), henipavirus fusion peptide domains (e.g., 25 amino acids of Hendra virus fusion peptide domain), metapneumovirus fusion peptide domains (e.g., 25 amino acids of human metapneumovirus fusion peptide domain), or foamy virus fusion peptide domains, such as simian foamy virus fusion peptide domains; or fragments or variants thereof.

[0208] For another example, the translocation promoting domain may comprise a clostridial neurotoxin HCN domain or a fragment or variant thereof. In more detail, the length of the clostridial neurotoxin HCN translocation promoting domain may be at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids. In this regard, the length of the clostridial neurotoxin HCN translocation promoting domain is preferably at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids. Specific (reference) examples include:

[0209] Botulinum neurotoxin type A - amino acid residues (872-1110)

[0210] Botulinum neurotoxin type B - amino acid residues (859-1097)

[0211] Botulinum neurotoxin type C - amino acid residues (867-1111)

[0212] Botulinum neurotoxin type D - amino acid residues (863-1098)

[0213] Botulinum neurotoxin type E - amino acid residues (846-1085)

[0214] Botulinum neurotoxin type F - amino acid residues (865-1105)

[0215] Botulinum neurotoxin type G - amino acid residues (864-1105)

[0216] Tetanus neurotoxin - amino acid residues (880-1127)

[0217] The above sequence positions may vary slightly depending on the serotype / subtype. Other examples of suitable (reference) Clostridial neurotoxin HCN domains include:

[0218] Botulinum neurotoxin type A - amino acid residues (874-1110)

[0219] Botulinum neurotoxin type B - amino acid residues (861-1097)

[0220] Botulinum neurotoxin type C - amino acid residues (869-1111)

[0221] Botulinum neurotoxin type D - amino acid residues (865-1098)

[0222] Botulinum neurotoxin type E - amino acid residues (848-1085)

[0223] Botulinum neurotoxin type F - amino acid residues (867-1105)

[0224] Botulinum neurotoxin type G - amino acid residues (866-1105)

[0225] Tetanus neurotoxin - amino acid residues (882-1127)

[0226] Any of the above-mentioned facilitating domains can be combined with any of the previously mentioned translocation domain peptides applicable to the present invention. Thus, for example, a non-Clostridium facilitating domain can be combined with a non-Clostridium translocation domain peptide or a Clostridium translocation domain peptide. Alternatively, a Clostridium neurotoxin HCN translocation facilitating domain can be combined with a non-Clostridium translocation domain peptide. Alternatively, a Clostridium neurotoxin HCN facilitating domain can be combined with a Clostridium translocation domain peptide, as exemplified below:

[0227] Botulinum neurotoxin type A - amino acid residues (449-1110)

[0228] Botulinum neurotoxin type B - amino acid residues (442-1097)

[0229] Botulinum neurotoxin type C - amino acid residues (450-1111)

[0230] Botulinum neurotoxin type D - amino acid residues (446-1098)

[0231] Botulinum neurotoxin type E - amino acid residues (423-1085)

[0232] Botulinum neurotoxin type F - amino acid residues (440-1105)

[0233] Botulinum neurotoxin type G - amino acid residues (447-1105)

[0234] Tetanus neurotoxin - amino acid residues (458-1127)

[0235] The HC peptide of the native clostridial neurotoxin contains approximately 400-440 amino acid residues and is composed of two functionally distinct domains, each approximately 25 kDa, namely the N-terminal region (commonly referred to as the HCN peptide or domain) and the C-terminal region (commonly referred to as the HCC peptide or domain). This fact is confirmed by the following references, each of which is incorporated by reference in its entirety: Umland TC (1997) Nature Structural Biology 4:788-792; Herreros J (2000) Journal of Biol. Chem. 347:199-204; Halpern J (1993) Journal of Biol. Chem. 268:15, pp. 11188-11192; Rummel A (2007) Proc. Natl. Acad. Sci. USA 104:359-364; Lacey DB (1998) Nature Structural Biology 5:898-902; Knapp (1998) Abstracts of the American Crystallographic Society 25:90; Swaminathan and Eswaramoorthy (2000) Nature Structural Biology 7:1751-1759; and Rummel J (2003) Proc. Nat. Acad. Sci. USA 104:359-364. A (2004) Molecular Microbiology 51(3), 631-643. Furthermore, it has been well documented that the C-terminal region (HCC), which constitutes the C-terminal 160-200 amino acid residues, is responsible for the binding of clostridial neurotoxins to their natural cell receptors, i.e. to nerve endings at the neuromuscular junction - a fact also confirmed by the above-mentioned literature.

[0236] An example of a Clostridium HCC reference sequence is as follows:

[0237] Botulinum neurotoxin type A - amino acid residues (Y1111-L1296)

[0238] Botulinum neurotoxin type B - amino acid residues (Y1098-E1291)

[0239] Botulinum neurotoxin type C - amino acid residues (Y1112-E1291)

[0240] Botulinum neurotoxin type D - amino acid residues (Y1099-E1276)

[0241] Botulinum neurotoxin type E - amino acid residues (Y1086-K1252)

[0242] Botulinum neurotoxin type F - amino acid residues (Y1106-E1274)

[0243] Botulinum neurotoxin type G - amino acid residues (Y1106-E1297)

[0244] Tetanus neurotoxin - amino acid residues (Y1128-D1315)

[0245] Given that slight differences may occur depending on the subserotype, the reference sequences determined above should be considered as a guide.

[0246] A modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (e.g., a modified HC domain), wherein the modified heavy chain has a higher or lower binding affinity to target neural cells than a native (unmodified) clostridial neurotoxin. Such modifications in the HC domain may include modifying residues in a ganglioside binding site or a protein (SV2 or synaptotagmin) binding site in the HC domain that alters binding to ganglioside receptors and / or protein receptors of target neural cells. Examples of such modified clostridial neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated by reference in their entirety.

[0247] Modified clostridial neurotoxins may have one or more modifications in the amino acid sequence of the light chain, such as modifications in the substrate binding or catalytic domains, which may alter or modify the specificity of the modified light chain for SNARE proteins. Examples of such modified clostridial neurotoxins are described in WO 2010 / 120766 and US 2011 / 0318385, both of which are incorporated by reference in their entirety.

[0248] Thus, the term "clostridial neurotoxin" is intended to encompass hybrid and chimeric clostridial neurotoxins. In one embodiment, a modified clostridial neurotoxin can be a hybrid or chimeric clostridial neurotoxin, provided that the clostridial neurotoxin comprises a modified BoNT / AHCC domain of the invention. A hybrid clostridial neurotoxin comprises at least a portion of a light chain from one clostridial neurotoxin or a subtype thereof, and at least a portion of a heavy chain from another clostridial neurotoxin or clostridial neurotoxin subtype. In one embodiment, a hybrid clostridial neurotoxin may comprise a complete light chain from one clostridial neurotoxin subtype and a heavy chain from another clostridial neurotoxin subtype. In one embodiment, a chimeric clostridial neurotoxin may comprise a portion of a heavy chain (e.g., a binding domain) of one clostridial neurotoxin subtype, while another portion of the heavy chain is from another clostridial neurotoxin subtype. Similarly or alternatively, a therapeutic element may comprise a portion of a light chain from a different clostridial neurotoxin. Such hybrid or chimeric clostridial neurotoxins are useful, for example, as a means of providing the therapeutic benefit of such clostridial neurotoxins to patients who are immunoresistant to a given clostridial neurotoxin subtype, patients who have below-average receptor concentrations for a given clostridial neurotoxin heavy chain binding domain, or patients who have protease-resistant variants for membrane or vesicular toxin substrates (e.g., SNAP-25, VAMP, and syntaxin). Hybrid and chimeric clostridial neurotoxins are described in U.S. Pat. No. 8,071,110, which is incorporated by reference in its entirety.

[0249] For example, a chimeric neurotoxin may comprise a LHN domain from a first neurotoxin covalently linked to a HC domain from a second neurotoxin, preferably, wherein the first and second neurotoxins are different, wherein the C-terminal amino acid residue of the LHN domain corresponds to the first amino acid residue of the 310 helix separating the LHN and HC domains in the first neurotoxin, and wherein the N-terminal amino acid residue of the HC domain corresponds to the second amino acid residue of the 310 helix separating the LHN and HC domains in the second neurotoxin.

[0250] In one embodiment, the Clostridial neurotoxin is a chimeric neurotoxin comprising a HC domain from BoNT / B and a LHN domain from BoNT / A, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / X.

[0251] For example, in one embodiment, the HC domain consists of or comprises an amino acid sequence corresponding to amino acid residues 860 to 1291 of SEQ ID NO: 3, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, and the LHN domain consists of or comprises an amino acid sequence selected from the group consisting of:

[0252] amino acid residues 1 to 872 of SEQ ID NO: 2, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto,

[0253] amino acid residues 1 to 867 of SEQ ID NO: 4, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto,

[0254] amino acid residues 1 to 863 of SEQ ID NO: 5, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto,

[0255] amino acid residues 1 to 846 of SEQ ID NO: 6, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto,

[0256] amino acid residues 1 to 865 of SEQ ID NO: 7, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto,

[0257] amino acid residues 1 to 862 of SEQ ID NO: 8, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto,

[0258] amino acid residues 1 to 864 of SEQ ID NO: 9, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto,

[0259] - Amino acid residues 1 to 863 of SEQ ID NO: 10, or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0260] In one embodiment, the Clostridial neurotoxin is a BoNT / X that comprises at least one domain from a non-BoNT / X Clostridial neurotoxin (e.g., a BoNT / X hybrid or chimera). For example, in one embodiment, a Clostridial neurotoxin may comprise:

[0261] i. BoNT / X light chain and non-BoNT / X HN and HC domains;

[0262] ii. BoNT / X HN domain and non-BoNT / X light chain and HC domain;

[0263] iii. BoNT / X HC domain and non-BoNT / X light chain and HN domain;

[0264] iv. BoNT / X light chain and HN domain and non-BoNT / X HC domain;

[0265] v. a BoNT / X light chain and HC domain and a non-BoNT / X HN domain; or

[0266] vi. BoNT / X HN domain and HC domain and non-BoNT / X light chain.

[0267] In a preferred embodiment, the Clostridial neurotoxin is a chimeric neurotoxin comprising the HC domain from BoNT / B and the LHN domain from BoNT / A.

[0268] In a more preferred embodiment, the HC domain consists of or comprises an amino acid sequence corresponding to amino acid residues 860 to 1291 of SEQ ID NO: 3, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto, and the LHN domain comprises an amino acid sequence corresponding to amino acid residues 1 to 872 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.

[0269] In embodiments where the clostridial neurotoxin comprises an HC domain from BoNT / B (e.g., the clostridial neurotoxin is BoNT / B, or a chimeric neurotoxin comprising an HC domain from BoNT / B), the clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (e.g., in the HC domain) to provide a "modified heavy chain," preferably, wherein the modified heavy chain has a higher (or lower) binding affinity to target neural cells compared to the native neurotoxin. Such modifications in the HC domain may include modification of amino acid residues in the ganglioside binding site of the HCC domain, which may alter binding to target neural cell gangliosides, and / or modification of amino acid residues in the protein receptor binding site of the HCC domain, which may alter binding to target neural cell protein receptors. Examples of such modified neurotoxins are described in WO2006027207 and WO2006114308, both of which are incorporated by reference in their entirety.

[0270] In one embodiment, the clostridial neurotoxin of the invention can be both chimeric and modified, as described above. For example, in a preferred embodiment, the clostridial neurotoxin comprises (or consists of) the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0271] In one embodiment, the clostridial neurotoxins of the invention may be both chimeric and modified, as described above. For example, in a preferred embodiment, the clostridial neurotoxin comprises (or consists of) SEQ ID NO: 11 (e.g., BoNT / AB MY ), or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0272] The term "Clostridial neurotoxin" may also encompass newly discovered members of the botulinum neurotoxin protein family expressed by non-Clostridial microorganisms, such as the Enterococcus-encoded toxin with the closest sequence identity to BoNT / X, the Weissella millei-encoded toxin named BoNT / Wo (NCBI Reference Sequence: WP_027699549.1), which cleaves VAMP2 at W89-W90, the Enterococcus faecium-encoded toxin (GenBank: OTO22244.1), which cleaves VAMP2 and SNAP25, and the Flavobacterium piperi-encoded toxin (NCBI Reference Sequence: WP_034687872.1).

[0273] The term "clostridial neurotoxin" is intended to encompass retargeted clostridial neurotoxins. In a retargeted clostridial neurotoxin, the clostridial neurotoxin is modified to include an exogenous ligand called a targeting moiety (TM). The TM is selected to provide binding specificity to a desired target cell, and as part of the retargeting process, the native binding portion of the clostridial neurotoxin (e.g., HC domain or HCC domain) may be removed.

[0274] The term "clostridial neurotoxin" may encompass catalytically inactive clostridial neurotoxins. The term "catalytically inactive" as used herein with respect to a clostridial neurotoxin light chain means that the light chain does not substantially exhibit non-cytotoxic protease activity, preferably, the term "catalytically inactive" as used herein with respect to a clostridial neurotoxin light chain means that the light chain does not exhibit non-cytotoxic protease activity. In one embodiment, a catalytically inactive clostridial neurotoxin light chain refers to a light chain that does not cleave exocytic fusion apparatus proteins in a target cell. The term "substantially no non-cytotoxic protease activity" means that the non-cytotoxic protease activity of a clostridial neurotoxin light chain is less than 5% of that of a catalytically active clostridial neurotoxin light chain, for example, less than 2%, 1% or preferably less than 0.1% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin light chain. The non-cytotoxic protease activity can be determined by incubating a test clostridial neurotoxin light chain with a SNARE protein in vitro and comparing the amount of SNARE protein cleaved by the test clostridial neurotoxin light chain with the amount of SNARE protein cleaved by the catalytically active clostridial neurotoxin light chain under the same conditions. Conventional techniques such as SDS-PAGE and protein immunoblotting can be used to quantify the amount of cleaved SNARE proteins. Suitable in vitro assays are described in WO 2019 / 145577 A1, which is incorporated herein by reference.

[0275] The present invention also includes clostridial neurotoxins with non-native protease cleavage sites. In such clostridial neurotoxins, the native protease cleavage site (also referred to as the activation site, as described above) is modified or replaced with a non-native protease cleavage site (i.e., an exogenous cleavage site) of the clostridial neurotoxin. Such sites require exogenous proteases to cleave, thereby providing better control over the timing and location of the cleavage event. Non-native protease cleavage sites that can be used for clostridial neurotoxins include:

[0276] TEV (Tobacco Etch Virus) (ENLYFQ↓G) (SEQ ID NO: 26)

[0277] Thrombin (LVPR↓GS) (SEQ ID NO: 27)

[0278] ·PreScission (LEVLFQ↓GP)(SEQ ID NO:28)

[0279] Enterokinase (DDDDK↓, SEQ ID NO: 29)

[0280] · Coagulation factor Xa (IEGR↓ / IDGR↓, SEQ ID NOs: 30 and 31)

[0281] Other protease cleavage sites include recognition sequences that can be cleaved by non-cytotoxic proteases, such as cleaved by the light chain of clostridial neurotoxins. These include SNARE (such as SNAP-25, syntaxin, VAMP) protein recognition sequences that can be cleaved by non-cytotoxic proteases (such as the light chain of clostridial neurotoxins). Clostridial neurotoxins containing non-natural protease cleavage sites are described in US 7,132,259, EP 1206554-B2 and US 2007 / 0166332, all of which are incorporated herein by reference in their entirety. The term protease cleavage site also includes inteins, which are self-cleaving sequences. The self-splicing reaction is controllable, for example, by changing the concentration of the reducing agent present.

[0282] The present invention also includes clostridial neurotoxins comprising a "destructive cleavage site". In the clostridial neurotoxin, a non-native protease cleavage site is introduced into the clostridial neurotoxin, and the position selected is such that cleavage at this site reduces the activity of the clostridial neurotoxin or inactivates it. If the clostridial neurotoxin migrates to a non-target location after administration, the destructive protease cleavage site can be cleaved by a local protease. Suitable non-native protease cleavage sites include those described above. Clostridial neurotoxins comprising a destructive cleavage site are described in WO 2010 / 094905 and WO 2002 / 044199, both of which are incorporated herein by reference in their entirety.

[0283] The modified Clostridial neurotoxins of the invention, particularly the light chain components thereof, can be pegylated (PEGylated) - this may help to improve stability, such as the duration of action of the light chain component. PEGylation is particularly preferred when the light chain comprises a BoNT / A, B or C1 protease. PEGylation preferably comprises adding polyethylene glycol to the N-terminus of the light chain component. For example, the N-terminus of the light chain can be extended with one or more amino acid (e.g., cysteine) residues, which can be the same or different. Each of the one or more amino acid residues can be linked (e.g., covalently linked) to its own polyethylene glycol molecule. An example of this technique is described in WO2007 / 104567, which is incorporated herein by reference in its entirety.

[0284] The amount of clostridial neurotoxin administered (eg, dose) may be measured in nanograms.

[0285] The dose of the clostridial neurotoxin according to the present invention is to be understood as the dose of the active two-stranded clostridial neurotoxin, i.e. excluding the amount of complex protein to which the neurotoxin may be bound. In other words, it refers to the dose of the active two-stranded clostridial neurotoxin, regardless of whether the neurotoxin is administered to the patient in conjunction with or without complex protein. As is well known to those skilled in the art, the active two-stranded clostridial neurotoxin is able to bind to membrane (e.g. cell membrane) receptors, transport the light chain into the cytoplasm and cleave SNARE proteins, while the complex protein does not show this biological activity (i.e. "inactive").

[0286] Additionally or alternatively, the dosage of a clostridial neurotoxin can be measured in clostridial neurotoxin "units" (U). For example, when administering a BoNT / A (or more specifically, e.g. ), it may be particularly appropriate to measure the dosage in units.

[0287] In practice, as is well known to those skilled in the art, the potency of Clostridial neurotoxins is related to the amount of neurotoxin (e.g., nanograms) required to achieve a median lethal dose (LD50) unit; one LD50 unit is defined as the median lethal intraperitoneal dose (measured in mice). However, BoNT pharmaceutical preparations currently on the market contain varying amounts of 150 kD neurotoxin and varying amounts of LD50 units. In addition, in these preparations, the neurotoxin may or may not be bound (i.e., combined) to a non-toxic neurotoxin-associated protein (NAP, also called a complexing protein). For ease of conversion (as described in Field et al., “AbobotulinumtoxinA OnabotulinumtoxinA andIncobotulinumtoxinA Neurotoxin Content and Potential Implicationsfor Duration of Response in Patients”, Toxins, 2018, 10(12), 535):

[0288] 100 units (also known as OnabotulinumtoxinA) contains about 0.9 ng of 150kDBoNT / A, as well as complex proteins;

[0289] 500 units (also known as AbobotulinumtoxinA) contains approximately 2.69 ng of 150kDBoNT / A, as well as complex proteins; 1 unit Contains about 5.38 pg of BoNT / A;

[0290] 100 units (also known as IncobotulinumtoxinA) contains approximately 0.40 ng of 150 kD BoNT / A and is free of complexing proteins.

[0291] It should be noted that the converted values ​​may differ slightly. For example, Frevert et al. 2012 (“Content of botulinum neurotoxin in and ”; Drug Development 2010; 10(2): 67-73) reported the following conversion values:

[0292] 100 units (also known as OnabotulinumtoxinA) contains approximately 0.73 ng of 150kDBoNT / A, as well as complex proteins;

[0293] 100 units (also known as AbobotulinumtoxinA) contains approximately 0.65 ng of 150kDBoNT / A, as well as complex proteins;

[0294] 100 units (also known as IncobotulinumtoxinA) contains approximately 0.44 ng of 150kDBoNT / A, without complexing proteins;

[0295] 100 units (also known as RimabotulinumtoxinB) contains about 0.2 ng to about 1 ng of 150 kD BoNT / B, as well as complexing proteins.

[0296] A person skilled in the art can measure the amount of clostridial neurotoxin according to methods routinely used in the art for quantifying proteins (preferably at nanogram levels), including mass spectrometry, such as isotope dilution mass spectrometry ( et al., "Quantification of protein calibrants by amino acid analysis using isotope dilution mass spectrometry," Analytical Biochemistry 2011, 408, 124-131), or fluorometry (Poras et al., "Detection and quantification of botulinum neurotoxin type A by a novel rapid in vitro fluorometric assay," Applied and Environmental Microbiology 2009 Jul;75(13):4382-4390).

[0297] In one embodiment, at least 5000, 6000, 7000, 8000 or 9000 units, preferably at least 9500, more preferably at least 9900 (e.g. at least 10,000) units of clostridial neurotoxin are administered to the patient per 500 ml of solution. In one embodiment, less than or equal to 55,000, 50,000, 45,000 or 40,000 units, preferably less than or equal to 35,000, more preferably less than or equal to 31,000 (e.g. less than or equal to 30,000) units of clostridial neurotoxin are administered to the patient per 500 ml of solution. In one embodiment, 5000-35,000 units, 6000-34,000 units, 7000-33,000 units, preferably 8000-32,000 units, more preferably 9000-31,000 units (such as preferably 10,000-30,000 units) of the clostridial neurotoxin are administered to the patient per 500 ml of solution.

[0298] In one embodiment, at least 16,500 units, 17,000 units, 17,500 units, 18,000 units or 18,500 units, preferably at least 19,000 units, more preferably at least 19,500 units (such as at least 20,000 units) of clostridial neurotoxin are administered to the patient per liter of solution. In one embodiment, less than or equal to 63,000 units, 62,500 units, 62,000 units or 61,500 units, preferably less than or equal to 61,000 units, more preferably less than or equal to 60,500 units (such as less than or equal to 60,000 units) of clostridial neurotoxin are administered to the patient per liter of solution. In one embodiment, 16,000-35,000 units, 17,000-34,000 units, 18,000-33,000 units, preferably 19,000-32,000 units, more preferably 19,500-60,500 units (such as preferably 20,000-60,000 units) of clostridial neurotoxin are administered to the patient per 1 liter of solution.

[0299] In one embodiment, at least 51,000 picograms, 51,500 picograms, 52,000 picograms, or 52,500 picograms, preferably at least 53,000 picograms, more preferably at least 53,500 picograms (e.g., at least 53,800 picograms) of clostridial neurotoxin are administered to the patient per 500 ml of solution. In one embodiment, less than or equal to 164,000 picograms, 163,500 picograms, 163,000 picograms, or 162,500 picograms, preferably less than or equal to 162,000 picograms, more preferably less than or equal to 161,700 picograms (e.g., less than or equal to 161,400 picograms) of clostridial neurotoxin are administered to the patient per 500 ml of solution. In one embodiment, 51,500-163,500 picograms, 52,000-163,000 picograms, or 52,500-162,500 picograms, preferably 53,000-162,000 picograms, more preferably 53,500-161,700 picograms (eg, 53,800-161,400 picograms) of clostridial neurotoxin are administered to the patient per 500 mL of solution.

[0300] In one embodiment, at least 105,000 picograms, 105,500 picograms, 106,000 picograms, or 106,500 picograms, preferably at least 107,000 picograms, more preferably at least 107,400 picograms (e.g., at least 107,600 picograms) of clostridial neurotoxin are administered to the patient per liter of solution. In one embodiment, less than or equal to 325,500 picograms, 325,000 picograms, 324,500 picograms, or 324,000 picograms, preferably less than or equal to 323,500 picograms, more preferably less than or equal to 323,000 picograms (e.g., less than or equal to 322,800 picograms) of clostridial neurotoxin are administered to the patient per liter of solution. In one embodiment, 105,000-325,500 picograms, 105,500-325,000 picograms, 106,000-324,500 picograms, or 106,500-324,000 picograms, preferably 107,000-323,500 picograms, more preferably 107,400-323,000 picograms (such as 107,600-322,800 picograms) of clostridial neurotoxin per liter of solution is administered to the patient.

[0301] In one embodiment, the solution comprises a clostridial neurotoxin and the solution is physiologically inert and / or free of peeling agents. In one embodiment, the solution comprises a clostridial neurotoxin and the solution is saline-based and / or free of peeling agents.

[0302] In one aspect, the present invention provides a pharmaceutical composition comprising a clostridial neurotoxin and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and / or salt.

[0303] In one aspect, the present invention provides a kit for use in the method of the present invention, comprising:

[0304] a) a solution consisting essentially of a clostridial neurotoxin; and

[0305] b) A catheter inserted into the patient's bladder.

[0306] The terms "subject", "individual" and "patient" are used interchangeably herein to refer to a mammalian subject. In one embodiment, the "subject" is a human, a companion animal (e.g., a pet, such as a dog, cat and / or rabbit), livestock (e.g., a pig, sheep, cattle and / or goat) and / or a horse. In a preferred embodiment, the subject (patient) is a human.

[0307] As used herein, the term "disorder" also encompasses "disease." In one embodiment, the disorder is a disease.

[0308] As used herein, the term "treatment" encompasses preventive treatment (such as preventing the onset of a disorder) as well as corrective treatment (treating a subject already suffering from a disorder). Preferably, "treatment" as used herein refers to corrective treatment. As used herein, the term "treatment" refers to a disorder and / or its symptoms.

[0309] Thus, the polypeptides of the invention may be administered to a subject in a therapeutically effective amount or a prophylactically effective amount. Preferably, the clostridial neurotoxins of the invention are administered to a subject in a therapeutically effective amount.

[0310] A "therapeutically effective amount" refers to any amount of a Clostridial neurotoxin that, when administered to a subject for treating a disorder (or a symptom thereof), alone or in combination, is sufficient to effect such treatment for the disorder or a symptom thereof.

[0311] A "prophylactically effective amount" refers to any amount of a clostridial neurotoxin that, when administered alone or in combination to a subject, inhibits or delays the onset or recurrence of a disorder (or a symptom thereof). In some embodiments, a prophylactically effective amount completely prevents the onset or recurrence of a disorder. "Inhibiting" the onset means either reducing the likelihood of an onset of a disorder (or a symptom thereof) or preventing an onset completely.

[0312] Sequence homology

[0313] The percent sequence identity may be determined using any of a variety of sequence alignment methods, including but not limited to global methods, local methods, and hybrid methods, such as fragment methods. Schemes for determining percent sequence identity are routine procedures within the skill of the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by summing the scores for each residue pair and applying a gap penalty. Non-limiting methods include, for example, CLUSTAL W, see, for example, Julie D. Thompson et al., "CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignments by sequence weighting, position-specific gap penalties, and weight matrix selection," Nucleic Acids Research 22 (22) 4673-4680 (1994); and iterative optimization, see, for example, Osamu Gotoh, "Significantly improving the accuracy of multiple protein sequence alignments by iterative optimization: evaluated by reference structure alignment," Journal of Molecular Biology 264 (4) 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs common to all input sequences. Non-limiting methods include, for example, Match-box, see, for example, Eric Depiereux and Ernest Feytmans, "Match-Box: a novel algorithm for the simultaneous alignment of multiple protein sequences," Computers in Applied Biosciences 8(5):501-509 (1992); Gibbs sampling, see, for example, CE Lawrence et al., "Detecting subtle sequence signals: a Gibbs sampling strategy for multiple sequence alignment," Science 262(5131):208-214 (1993); Align-M, see, for example, Ivo Van WaIIe et al., "Align-M - a new algorithm for the multiple alignment of highly divergent sequences," Bioinformatics 20(9):1428-1435 (2004).

[0314] Therefore, the percentage of sequence identity is determined by conventional methods. For example, see Altschul et al., Bulletin of Mathematical Biology 48:603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. In brief, 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 "blosum 62" scoring matrix of Henikoff and Henikoff (supra) (shown below, with amino acids represented by standard single letter codes).

[0315] The "percentage of sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, the percent identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. When calculating the percent identity of a sequence, the number of spaces introduced to achieve optimal alignment of the two or more sequences and the length of each space may also be considered. Sequence comparisons and determination of the percent identity between two or more sequences can be performed using a specific mathematical algorithm, such as BLAST, with which those skilled in the art should be familiar.

[0316] Alignment score used to determine sequence identity

[0317]

[0318]

[0319] The percent identity is then calculated as follows:

[0320] Total number of identical matches

[0321] ________________________________________x 100

[0322] The length of the longer sequence plus the number of gaps introduced into the longer sequence to allow the two sequences to be aligned

[0323] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably minor, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect polypeptide folding or activity; small deletions, usually 1 to about 30 amino acids; and small amino or carboxyl terminal extensions, such as an amino-terminal methionine residue, a small connecting peptide of up to about 20-25 residues in length, or an affinity tag.

[0324] Conservative amino acid substitution

[0325] Basic amino acids: arginine, lysine, histidine

[0326] Acidic amino acids: glutamic acid, aspartic acid

[0327] Polar amino acids: glutamine, asparagine

[0328] Hydrophobic amino acids: leucine, isoleucine, valine

[0329] Aromatic amino acids: phenylalanine, tryptophan, tyrosine

[0330] Small amino acids: glycine, alanine, serine, threonine, methionine

[0331] 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) can replace the amino acid residues in the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can also replace the amino acid residues in the polypeptides. The polypeptides of the present invention may also contain non-naturally occurring amino acid residues.

[0332] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methylene-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be employed in which a chemically aminoacylated suppressor tRNA is used to suppress nonsense mutations. Methods for synthesizing amino acids and aminoacylated tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzym. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Nat. Acad. Sci. USA 90:10145-9, 1993). In a second approach, translation is performed in Xenopus laevis oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In a third approach, E. coli cells are cultured in the absence of the natural amino acid to be substituted (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See Koide et al., Biochemistry 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Science 2:395-403, 1993).

[0333] A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids can replace the amino acid residues of the polypeptides of the present invention.

[0334] Essential amino acids in the polypeptides of the invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science, 244: 1081-5, 1989). Biological interaction sites 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, combined with mutations of putative contact site amino acids. For example, see de Vos et al., Science, 255: 306-12, 1992; Smith et al., J. Molecular Biology, 224: 899-904, 1992; Wlodaver et al., FEBS Letters, 309: 59-64, 1992. The identity of essential amino acids can also be inferred by homology analysis with related components of the polypeptides of the invention, such as translocase or protease components.

[0335] Multiple amino acid substitutions can be made 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 (Proceedings of the National Academy of Sciences of the United States of America 86:2152-6, 1989). In short, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the range of substitutions allowed at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochemistry 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, World Intellectual Property Organization Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0336] Multiple amino acid substitutions can be made 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 (Proceedings of the National Academy of Sciences of the United States of America 86:2152-6, 1989). In short, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the range of substitutions allowed at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochemistry 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, World Intellectual Property Organization Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0337] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 20th Edition, John Wiley and Sons, New York (1994), and Hale and Marham, HarperCollins Dictionary of Biology, Harper Perennial, New York (1991), provide a general dictionary for many of the terms used in the present disclosure to those of skill in the art.

[0338] The present disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the embodiments of the present disclosure. Numerical ranges include the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written from left to right in the direction of 5' to 3'; amino acid sequences are written from left to right in the direction of amino to carboxyl, respectively.

[0339] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0340] The names, three-letter abbreviations or single-letter abbreviations of amino acids are used herein to refer to amino acids. The term "protein" as used herein includes proteins, polypeptides and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or "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. In the present disclosure and claims, conventional single-letter and three-letter codes for amino acid residues may be used. The three-letter code for amino acids is defined according to the provisions of the Joint Committee of the International Union of Pure and Applied Chemistry-International Union of Biochemistry and Molecular Biology (IUPAC-IUB JCBN). It should also be understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

[0341] Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and therefore may vary. It should also be understood that the terms used herein are only used for the purpose of describing specific embodiments and are not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims.

[0342] When a numerical range is provided, it is understood that each intermediate value between the upper and lower limits of the range, to one-tenth of the unit of the lower limit (unless the context clearly dictates otherwise), is also specifically disclosed. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in the stated range is included in the disclosure. The upper and lower limits of these smaller ranges may be independently included in the range, or excluded from the range, and each range in which any, both, or both of the limits are included in the smaller range is also included in the disclosure, subject to any specifically excluded limits in the stated range. When the stated range includes one or both limits, ranges excluding either or both of the stated included limits are also included in the disclosure.

[0343] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a clostridial neurotoxin" includes a plurality of such candidates and reference to "the clostridial neurotoxin" includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0344] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art for the appended claims.

[0345] BRIEF DESCRIPTION OF THE DRAWINGS

[0346] Embodiments of the invention will now be described, by way of example only, with reference to the following figures and examples.

[0347] Figure 1 A schematic diagram of a procedure involving intradetrusor injection is shown. The procedure requires a device to be carefully maneuvered through the urethral opening and into the patient's bladder along the length of the urethra. The needle is then manipulated sequentially to multiple predetermined injection sites on the lining of the bladder wall under cystoscopic guidance. With each injection, the needle penetrates the lining of the bladder wall, delivering a dose of the therapeutic agent at each injection site.

[0348] Figure 2 A diagram showing the gentle instillation method. A catheter is inserted into the patient's urethra to empty the bladder of existing urine. A small amount (e.g., 50 mL) of medication is then slowly infused into the bladder through the catheter. After the catheter is removed, the patient is instructed to resume normal daily activities but not to empty the bladder for at least 15 minutes, and preferably at least 90 minutes. This allows the medication to contact and potentially treat the entire lining of the bladder wall.

[0349] Figure 3 Schematic diagram showing administration of botulinum neurotoxin type A (BoNT / A) by intradetrusor injection. Intradetrusor injection of BoNT / A is highly invasive and simultaneously targets three innervated layers of the bladder wall: the urothelium; afferent nerve endings in the lamina propria; and efferent nerve endings in the detrusor muscle.

[0350] Figure 4 A schematic diagram of the method of the present invention is shown. The method of the present invention is less invasive and targets afferent nerve endings in the urothelium and lamina propria.

[0351] Figure 5 The study design evaluating the effect of Dysport in a chronic rat model of cyclophosphamide-induced interstitial cystitis / bladder pain syndrome is presented.

[0352] Figure 6 The effects of Dysport (10, 20 and 30 units / rat, intravesically injected) on cyclophosphamide-induced chronic visceral pain (based on nociception scores) are shown. Nociception scores (expressed in %) of rats in the Dysport group and vehicle-treated group on day 10 (A) and day 12 (B). The results are expressed as mean ± standard error. Two-way repeated measures ANOVA was used for comparison between groups, and Sidak post hoc test was performed with cyclophosphamide / vehicle group (###p<0.001).

[0353] Figure 7The effects of the test and reference substances on cyclophosphamide-induced chronic allodynia (based on the nociceptive threshold) are shown. The nociceptive threshold (in grams) of the Dysport, DMSO, Ialuril, and vehicle groups before administration ("D0"), at the time of injection of saline or cyclophosphamide but before the start of drug treatment ("D7") (A), and on days 10 (B) and 12 (C). The results are expressed as mean ± standard error. No statistical analysis was performed.

[0354] Figure 8 The effects of the test and reference substances on cyclophosphamide-induced chronic allodynia (based on the area under the curve (AUC) in the range of 1-6 g-force) are shown. The AUC in the range of 1-6 g-force was calculated in the Dysport, DMSO, Ialuril and vehicle groups before administration ("D0"), when saline or cyclophosphamide was injected but drug treatment had not yet started ("D7") (A), and on days 10 (B) and 12 (C). The results are expressed as mean ± standard error. No statistical analysis was performed.

[0355] Fig. 9 The effects of the test and reference substances on cyclophosphamide-induced chronic hyperalgesia (based on AUC in the range of 6-26 gf) are shown. In the Dysport, DMSO, Ialuril and vehicle groups, the AUC in the range of 6-26 gf was calculated before administration ("D0"), when saline or cyclophosphamide was injected but drug treatment had not yet started ("D7") (A), and on days 10 (B) and 12 (C). The results are expressed as mean ± standard error. No statistical analysis was performed.

[0356] Fig.10 Schematic diagram of the isolated bladder electrophysiology preparation. The bladder is cannulated at the urethra and top. The pelvic nerve (PN) and hypogastric nerve (HGN) bundles are inserted into glass recording electrodes. The technical setup on the right shows how the input from the pressure sensor and electrodes is processed by the hardware and Spike2 software to generate the example inflation image at the top. This figure was created using BioRender.

[0357] Fig.11 Intravesical BoNT / A treatment resulted in a significant decrease in bladder mechanical sensitivity and an increase in pressure-volume relationships. A) Afferent responses to distension were significantly reduced at 30, 60, and 90 minutes after treatment (p<0.0001, n=9, two-way ANOVA). B) Pressure-volume relationships were significantly increased after intravesical Dysport administration compared to the control group (p=0.0003; n=9; two-way ANOVA).

[0358] Fig.12 The effects of BoNT / B on bladder mechanosensitivity are shown. A) After BoNT / B treatment, afferent responses to distension were reduced in a time-dependent manner (p<0.0001; n=6; two-way ANOVA). B) Pressure-volume relationships were significantly increased after BoNT / B treatment (p<0.0001; two-way ANOVA).

[0359] Fig.13 The effects of BoNT / E on bladder mechanosensitivity are shown. A) After BoNT / E treatment, afferent responses to distension were significantly reduced in a time-dependent manner (p<0.0001; n=5; two-way ANOVA). B) Pressure-volume relationships were significantly increased after BoNT / E treatment (p<0.0001; two-way ANOVA).

[0360] Sequence Listing

[0361] In any of the following SEQ ID NOs., if an initial methionine (Met) amino acid residue or a corresponding start codon is indicated, this residue / codon is optional.

[0362] SEQ ID NO: 1 - Nucleotide sequence of unmodified botulinum neurotoxin type A (BoNT / A)

[0363]

[0364] SEQ ID NO:2 - Polypeptide sequence of unmodified BoNT / A

[0365]

[0366] SEQ ID NO:3-BoNT / B1, accession number B1INP5, amino acid sequence

[0367]

[0368] SEQ ID NO:4-BoNT / C1, accession number P18640, amino acid sequence

[0369]

[0370] SEQ ID NO:5-BoNT / D, accession number P19321, amino acid sequence

[0371]

[0372] SEQ ID NO:6-BoNT / E1, Accession No. WP_003372387, amino acid sequence

[0373]

[0374] SEQ ID NO:7-BoNT / F1, Accession No. Q57236, amino acid sequence

[0375]

[0376] SEQ ID NO:8—BoNT / F7, amino acid sequence

[0377]

[0378] SEQ ID NO:9-BoNT / G, accession number WP_039635782, amino acid sequence

[0379]

[0380] SEQ ID NO: 10-BoNT / DC, accession number BAM65681, amino acid sequence

[0381]

[0382] SEQ ID NO:11 - BoNT / ABMY, Amino Acid Sequence

[0383] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERD 49

[0384] TFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIY 99

[0385] STDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEEL 149

[0386] NLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEES199

[0387] LEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNA 249

[0388] YYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNK 299

[0389] AKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIY 349

[0390] TEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAA 399

[0391] NFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKA 449

[0392] LNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLI 499

[0393] QQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYT 549

[0394] MFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATE 599

[0395] AAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYK 649

[0396] DDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNAL 699

[0397] SKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINY 749

[0398] QYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNS 799

[0399] MIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDI 849

[0400] PFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVE 899

[0401] VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPK 949

[0402] YKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVF 999FEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIA 1049

[0403] NGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKD 1099

[0404] FWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINY 1149

[0405] RDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF 1199

[0406] KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIG 1249

[0407] LIGIHRFIESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKD 1299

[0408] EGWTE 1304

[0409] SEQ ID NO:12-BoNT / X, amino acid sequence (GenBank:BAQ12790.1)

[0410]

[0411] SEQ ID NO: 13 (Nucleotide sequence of modified BoNT / A "Cat-A")

[0412]

[0413] SEQ ID NO: 14 (Polypeptide sequence of modified BoNT / A "Cat-A")

[0414]

[0415] SEQ ID NO: 15 (Nucleotide sequence of modified BoNT / A "Cat-B")

[0416]

[0417] SEQ ID NO: 16 (Polypeptide sequence of modified BoNT / A "Cat-B")

[0418]

[0419] SEQ ID NO: 17 (Nucleotide sequence of modified BoNT / A "Cat-C")

[0420]

[0421] SEQ ID NO: 18 (Polypeptide sequence of modified BoNT / A "Cat-C")

[0422]

[0423] SEQ ID NO: 19 (Nucleotide sequence of modified BoNT / A "Cat-D")

[0424]

[0425] SEQ ID NO: 20 (Polypeptide sequence of modified BoNT / A "Cat-D")

[0426]

[0427] SEQ ID NO: 21 (Polypeptide sequence of modified BoNT / A “Chimera 1”)

[0428] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

[0429] PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

[0430] STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

[0431] GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0432] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

[0433] KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

[0434] LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

[0435] GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0436] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

[0437] KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

[0438] AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

[0439] AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0440] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

[0441] MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

[0442] VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKSEILNNIILNLRYKDNNLIDLSGYGAKVE

[0443] VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYI

[0444] HNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVT

[0445] ITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTEL

[0446] SQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSK

[0447] YNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF

[0448] KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYES

[0449] GIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0450] SEQ ID NO:22 (Polypeptide sequence of modified BoNT / A “Chimera 2”)

[0451] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

[0452] PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

[0453] STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

[0454] GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0455] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

[0456] KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

[0457] LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

[0458] GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0459] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

[0460] KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

[0461] AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

[0462] AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0463] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

[0464] MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

[0465] VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIIELGGGGSELSEILNNIILNLRYKDNN

[0466] LIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRI

[0467] PKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIRED

[0468] ISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFI

[0469] WMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKK

[0470] DSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNL

[0471] NQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDE

[0472] IGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHH

[0473] HHHHHHH

[0474] SEQ ID NO:23 (Polypeptide sequence of modified BoNT / A “Chimera 3A”)

[0475] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

[0476] PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

[0477] STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

[0478] GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0479] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

[0480] KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

[0481] LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

[0482] GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0483] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

[0484] KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

[0485] AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

[0486] AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0487] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

[0488] MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

[0489] VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV

[0490] YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH

[0491] NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI

[0492] TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS

[0493] QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY

[0494] NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK

[0495] KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG

[0496] IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0497] SEQ ID NO: 24 (Polypeptide sequence of modified BoNT / A "Chimera 3B")

[0498]

[0499] SEQ ID NO:25 (Polypeptide sequence of modified BoNT / A “Chimera 3C”) MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

[0500] PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

[0501] STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

[0502] GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0503] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

[0504] KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

[0505] LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

[0506] GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0507] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

[0508] KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

[0509] AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

[0510] AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0511] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

[0512] MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

[0513] VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV

[0514] YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH

[0515] NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI

[0516] TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS

[0517] QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY

[0518] NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK

[0519] KEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG

[0520] IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0521] SEQ ID NO:26 (Tobacco Etch Virus (TEV) cleavage site)

[0522] ENLYFQG

[0523] SEQ ID NO:27 (thrombin cleavage site)

[0524] LVPRGS

[0525] SEQ ID NO:28 (PreScission cleavage site)

[0526] LEVLFQGP

[0527] SEQ ID NO:29 (enterokinase cleavage site)

[0528] DDDDK

[0529] SEQ ID NO:30 (Coagulation factor Xa cleavage site 1)

[0530] IEGR

[0531] SEQ ID NO:31 (Coagulation factor Xa cleavage site 2)

[0532] IDGR

[0533] SEQ ID NO:32 (Influenza virus hemagglutinin)

[0534] GLFGAIAGFIENGWEGMIDGWYG Example

[0535] Materials and Methods

[0536] 1. Animal Model

[0537] Rats treated with cyclophosphamide (CYP) were used in the following studies. A chronic rat model of CYP-induced painful bladder syndrome / interstitial cystitis (BPS / IC) was established by 3 injections (40 mg / kg, ip) every 3 days. For example, the article "Characterization and Validation of a Chronic Model of Cyclophosphamide-Induced Interstitial Cystitis / Bladder Pain Syndrome in Rats" by Augé C et al., published in Front Pharmacol on August 28, 2020, Vol. 11, p. 1305, doi:10.3389 / fphar.2020.01305, PMID:32982733; PMCID:PMC7485435, and the article "The function of P2X3 receptor and NK1 receptor antagonists on The CYP-induced BPS / IC model is described in the study “cyclophosphamide-induced cystitis in rats”. Rats in this model do not suffer from severe weight loss and develop persistent visceral pain characterized by allodynia (painful responses to normally innocuous stimuli) and hyperalgesia (increased responses to noxious stimuli).

[0538] 2. Induction of chronic cystitis

[0539] To induce chronic cystitis, rats were weighed and injected intraperitoneally with CYP at a dose of 40 mg / kg in a final volume of 5 mL / kg on day 0 (D0), D3, and D6. CYP was freshly prepared in normal saline at a final concentration of 8 mg / mL.

[0540] Control rats were injected with saline under the same experimental conditions as the CYP-treated groups.

[0541] 3. Von Frey test

[0542] The Von Frey test is used to assess visceral pain. For example, Garrido-Suarez, B et al., Journal of Pharmacy & Pharmacognosy Research, 2015, vol. 3, pp. 148-161, and Deuis JR et al., Methods Used to Evaluate Pain Behaviors in Rodents, Front Mol Neurosci, 6 Sep 2017, vol. 10, p. 284, doi:10.3389 / fnmol.2017.00284, PMID: 28932184; PMCID: PMC5592204, describe the Von Frey test. Use standardized conditions, including testing all animals by a single experimenter, to minimize variability in behavior-based pain testing. Visceral pain was assessed in a double-blind manner by applying a set of 8 calibrated Von Frey filaments with increasing force values ​​(1, 2, 4, 6, 8, 10, 15 and 26 g) to the lower abdomen near the bladder at a 5-second stimulation interval. Before testing, the abdominal area designed for mechanical stimulation of each animal was shaved. The animals were placed on an elevated metal mesh floor in a separate transparent plexiglass box and adapted for at least 30 minutes before starting the Von Frey test. The filaments were then applied for 1-2 seconds with sufficient force through the metal mesh floor to slightly bend the filaments. Each filament was tested 3 times. Note that different sites in the lower abdominal area near the bladder are stimulated to avoid desensitization.

[0543] Nociceptive behavior was scored for each animal and each filament as shown in Table 1 .

[0544] Table 1 - Nociceptive Behavioral Scores

[0545]

[0546] Presentation and analysis of results

[0547] Visceral pain

[0548] The definitions of nociception parameters are shown in Table 2

[0549] Table 2 - Definition of nociception parameters

[0550]

[0551] *For example, at a given Von Frey force value, an animal with a score of 1 for the first application, 1 for the second, and 2 for the third would have a combined score of 4. The maximum combined score is 9 (3+3+3), and a combined score of 4 is equivalent to 44% of the maximum response (100 x 4 / 9).

[0552] Example 1

[0553] Experimental design

[0554] To evaluate the effects of Dysport on a chronic rat model of cyclophosphamide (CYP)-induced painful bladder syndrome / interstitial cystitis (BPS / IC), the following experimental protocol was performed (eg Figure 5 shown):

[0555] On D-1, rats were placed in individual plexiglass boxes (Von Frey test apparatus) for at least 30 minutes to adapt to the stimulation of Von Frey filaments to reduce the stress level caused by the new environment.

[0556] On D0, a Von Frey test was performed before the first injection of CYP or saline to obtain basal values ​​of nociceptive behavior.

[0557] • On D0, D3, and D6, chronic cystitis was induced to simulate BPS / IC.

[0558] On D7 (before treatment initiation), a Von Frey test was performed to assess the induction of chronic cystitis.

[0559] On D8, intravesical (i.ves.) drug treatment was performed.

[0560] On D10 and D12, Von Frey test was performed to analyze the effect of the test substance (Dysport) and reference substances (DMSO and laluril) on CYP-induced chronic visceral pain.

[0561] Experimental substances

[0562] 1. Reconstitution of Dysport

[0563] On each experimental day, a vial containing 500U Dysport was dissolved in 1mL sterile phosphate buffered saline (PBS) to obtain a final concentration of 500U / mL. The stock solution was diluted 8.33 times, 12.5 times or 25 times with excipients to obtain a concentration of 30U / 500μL, 20U / 500μL or 10U / 500μL, respectively. Dilutions were performed in silica glass tubes (batch number 8072554, Becton-Dickinson, Plymouth, UK).

[0564] 2. Excipients

[0565] The excipient was sterile PBS (batch number 943548) purchased from Eurobio Ingen (Les Sully, France).

[0566] 3. Reference material

[0567] DMSO was freshly prepared on the day of dosing to a final concentration of 50%.

[0568] The appropriate amount of DMSO was dissolved in the excipient at room temperature. DMSO was purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France; batch number RNBH3467). Ready-to-use alaluril (batch number 170501) was purchased from IBSA through Pharmaclic (Gosselies, Belgium).

[0569] Processing

[0570] 1. Experimental Group

[0571] There are 7 experimental groups, and their detailed descriptions are shown in Table 3.

[0572] Table 3 - Experimental groups

[0573]

[0574]

[0575] Abbreviations: intraperitoneal injection (ip), intravesical injection (i.ves.)

[0576] 2. Drug treatment

[0577] Before the experiment, animals were randomly assigned to treatment groups. The randomization design ensured that there was at least one animal per group on each experimental day and that animals from the same group were in different positions in the Von Frey test box. In preparation for gentle pressure perfusion of the bladder with solutions, a polyethylene catheter (inner and outer diameters of 0.76 mm and 1.22 mm, respectively) was inserted into the bladder through the urethral orifice. The test substance (Dysport), reference substances (DMSO and laluril) and vehicle (500 μL / rat) were injected into the bladder via a syringe connected to the catheter.

[0578] The treatment solution was retained in the bladder for 30 minutes. During the entire procedure, the rats were maintained under 2-2.5% isoflurane anesthesia. After bladder instillation, the lower abdomen was gently massaged to empty the bladder.

[0579] Experimental Results

[0580] 1. Nociception Rating

[0581] Treatment with Dysport at all tested doses (10U, 20U, and 30U) resulted in a significant decrease in nociception scores on D10 and D12 compared with rats injected with CYP and treated with vehicle (see Figure 6 ).

[0582] 2. Nociceptive Threshold

[0583] After CYP injection, the nociceptive threshold of CYP-injected rats was lower on D7 compared with D0 (see Figure 7 A), indicating that chronic cystitis had been successfully induced and the rats' threshold for pain perception was reduced. Compared with CYP-injected rats treated with vehicle alone, CYP-injected rats had increased nociceptive thresholds on D10 and D12 after Dysport treatment (see Figure 7 B and Figure 7 C). On D10, the Dysport dose of 20 U / rat reached a statistically significant level (see Figure 7 B), while on D12, all tested doses reached significant levels (see Figure 7 C). The nociceptive threshold was elevated in CYP-injected rats treated with Dysport compared to CYP-injected rats treated with DMSO or laluril, indicating that Dysport is effective in alleviating pain in CYP-treated animals.

[0584] 3. Chronic allodynia (AUC 1-6g)

[0585] After CYP injection, AUC 1-6g was increased in CYP-injected rats on D7 compared with saline-treated rats (see Figure 8 A), and also increased compared with D0. After Dysport treatment, all tested doses increased on D10 compared with CYP-injected rats treated with saline (see Figure 8 B) and D12 days (see Figure 8 C) AUC 1-6g was decreased. AUC 1-6g decreased more in CYP-injected rats treated with Dysport than in CYP-injected rats treated with DMSO or laluril. These results suggest that administration of Dysport can reduce pain perception associated with allodynia in CYP-treated animals.

[0586] 4. Chronic hyperalgesia (AUC 6-26g)

[0587] After CYP injection, an increase in AUC 6-26g was observed on D7 compared with saline-treated rats (see Fig. 9A). After Dysport treatment, all tested doses showed a significant increase in D10 compared to rats injected with CYP and treated with saline (see Fig. 9 B) and D12 days (see Fig. 9 C) AUC 6-26g was decreased in both CYP-injected and DMSO-injected rats. The AUC 6-26g decrease was greater in CYP-injected rats treated with Dysport than in CYP-injected rats treated with DMSO or laluril. These results suggest that Dysport can reduce pain perception associated with hyperalgesia in CYP-treated animals.

[0588] Example 2

[0589] A physician evaluates a patient with painful bladder syndrome (BPS), specifically interstitial cystitis (IC), and performs a series of steps prior to administering a solution containing a clostridial neurotoxin as treatment. Specifically, the physician assesses the state of damage to the urothelium of the patient's bladder to determine the dosage of the clostridial neurotoxin in solution. Upon evaluation by the physician, it is determined that the patient's urothelium is severely damaged.

[0590] The volume of solution containing the clostridial neurotoxin to be administered is then determined by determining the threshold volume of fluid that triggers the patient to urinate by cystometry, uroflowmetry, or a urination diary. The threshold volume determined by cystometry is 250 ml.

[0591] Because the patient's urothelium was severely damaged, doctors prepared a solution containing 53,800 picograms (pg) of clostridial neurotoxin per 500 ml, with a total volume of 225 ml. Doctors inserted a catheter into the patient's bladder and instilled the solution into the bladder. The solution was then retained in the bladder for 1 hour. Afterwards, the patient was asked to urinate, and the bladder was flushed with saline to remove any remaining clostridial neurotoxin.

[0592] Example 3

[0593] A physician evaluates a patient with painful bladder syndrome (BPS), specifically interstitial cystitis (IC), and performs a series of steps prior to administering a solution containing a clostridial neurotoxin as treatment. Specifically, the physician evaluates the state of damage to the patient's bladder urothelium to determine the dosage of the clostridial neurotoxin in solution. Upon evaluation by the physician, it is determined that the patient's urothelium is mildly damaged, with some tissue remaining intact.

[0594] The volume of solution containing the clostridial neurotoxin to be administered is then determined by determining the threshold volume of fluid that triggers the patient to urinate, either by cystometry, uroflowmetry, or a urination diary. The threshold volume determined by the urination diary is 275 ml.

[0595] Because the patient's urothelium was mildly damaged, a solution containing 161,400 picograms of clostridial neurotoxin per 500 ml was prepared for a total volume of 250 ml. The doctor inserted a catheter into the patient's bladder, connected the catheter, and injected the solution into the bladder, where it remained for 1 hour. Afterwards, the patient was asked to urinate, and the bladder was flushed with saline to remove any remaining clostridial neurotoxin.

[0596] All publications mentioned in the above description are incorporated herein by reference. It will be apparent to those skilled in the art that various modifications and variations of the described methods and systems of the present invention will be apparent without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be overly limited to these specific embodiments. In fact, various modifications to the above-described modes of implementing the present invention that are apparent to those skilled in the art of biochemistry, biotechnology, or related fields are intended to fall within the scope of the following claims.

[0597] Example 4

[0598] Inhibitory effects of botulinum neurotoxin type A (BoNT / A) on bladder mechanosensitivity in an in vitro mouse model

[0599] These studies were performed using adult C57BL / 6J mice aged 8 to 12 weeks (Charles River Laboratories, Margate, Kent). On the day of the experiment, mice were sacrificed using increasing concentrations of carbon dioxide in accordance with Schedule 1 of the Animals (Scientific Procedures) Act 1986. The experiments were performed in accordance with ethical approval obtained from the UCLan Animal Welfare and Ethics Review Board (AWERB) (reference number RE / 16 / 11).

[0600] 1. Ex vivo bladder electrophysiology studies

[0601] After killing the mice by carbon dioxide asphyxiation, the hair on the back of the animals, as well as the two hind limbs and the tail were removed. An incision was made in the abdomen, the intestines were removed, and the spinal cord was cut at the L2 level above the kidneys. The entire pelvic region of the mouse was placed in an organ bath continuously perfused with Krebs buffer (components, in mM: NaCl 118.4, NaHCO24.9, CaCl1.9, MgSO 1.2, KHPO1.2, glucose 11.7; all reagents were purchased from Sigma-Aldrich) containing carbonic acid (95% O / 5% CO), and the buffer was maintained at about 35°C to prevent tissue degradation.

[0602] After placement in an organ bath, the tissue was further dissected under a microscope. The ureters were ligated with silk thread (Fisher-Scientific) to prevent possible backflow when the bladder was distended. The pubic symphysis was cut and removed on both sides to expose the urethra below. The urethra was cut, a catheter connected to a syringe pump (New Era Pump Systems, NE-1000) was inserted, and ligated with sutures to prevent leakage. The syringe was filled with phosphate-buffered saline (PBS; Gibco), and the bladder was filled to a point where a syringe needle (BD microlance) could be inserted and passed through the top of the bladder without damaging the sensory nerves in the trigone. A double-lumen catheter was then inserted into the hole at the top and ligated with sutures. One catheter was connected to a pressure transducer (NL108T2 Digitimer) to monitor intravesical pressure, and the other was connected to a tap for bladder filling and emptying. The nerve bundle containing the pelvic nerve and hypogastric nerve was inserted into a glass electrode to facilitate capturing the afferent nerve response caused by bladder stimulation.

[0603] After catheterization, the bladder was distended to ensure that it was a closed system; failure to reach this pressure indicated a leak. The pelvic and hypogastric nerves emerging from the base of the bladder were dissected into longer nerve bundles and inserted into a glass suction electrode (VWR) connected to a Neurolog preamplifier (NL100AK). The preamplifier was connected to an AC preamplifier (NL104) to amplify the signal (10,000 times), filtered through a bandpass filter (NL125), and 50-60 Hz electrical noise was removed by Humbug (QuestScientific). The signal then passed through a 1401 data acquisition interface (Cambridge electronic Design) and was recorded on a computer using Spike2 software (v10.08; Cambridge electronic Design). Multiunit afferent activity was quantified using a Spike processor (D130; Digitimer), which counted the number of field potentials that exceeded a threshold set at the beginning of the experiment (twice the baseline noise level). The experimental setup was set up as shown. Fig.10 shown.

[0604] 2. Experimental Plan

[0605] In ex vivo bladder electrophysiological recordings, the bladder is stimulated by mechanical (distension) means and the neuronal responses are captured to characterize the effects of stimulation parameters on afferent neural activity.

[0606] 3. Statistical methods

[0607] All data related to isolated bladder electrophysiology measurements, including multiunit nerve firing and intravesical pressure, were collected using Spike2 software (v10.08). Responses at 30, 60, and 90 min were normalized to the third reproducible control distension at the beginning of the experiment. The area under the curve was determined based on this normalized data. N refers to the number of animals, and all data are presented as mean ± standard error. Statistical tests performed using GraphPad Prism v8.0.1 included T tests, one-way analysis of variance, and two-way analysis of variance. Assuming that there were no equipment failures, tissue failures, or other issues during the experimental run, all experimentally prepared response data were included in the final analysis. The specific methods for data extraction and analysis are described in detail below.

[0608] 4. Bladder expansion

[0609] The bladder was slowly distended by closing the tap and turning on the syringe pump to fill it at a rate of 150 μL / min. The tap was opened to empty it and the pressure dropped immediately. As the bladder filled, the activation of nerve fibers sensitive to mechanical stretch of the bladder wall was captured and displayed as field potentials on Spike2. Distension was continued until three reproducible responses were achieved simultaneously. Once this condition was achieved, the experiment was started. All ramp distensions were performed 10 min apart and each preparation was allowed to stabilize for at least 30 min before recording began.

[0610] 5. Bladder compliance

[0611] Compliance is a measure of the pressure-volume relationship during bladder filling, that is, the ability of the bladder wall to accommodate increasing volume.

[0612] Volume (μL) = rate (μl / min-1) x time

[0613] This formula is used to calculate the pressure-volume relationship of the bladder based on the filling rate set by the intravesical pump.The change in bladder compliance is also plotted as the percent change relative to the control distension at the start of the experiment.

[0614] 6. Intracavitary application of BoNT / A

[0615] Ex vivo bladder electrophysiology measurements were reproducible over 120 min, as the response curves to distensions performed at 90 min into the experiment were similar to the initial control distension.

[0616] A syringe containing BoNT / A solution was connected to a syringe pump and the bladder was distended three times to ensure absorption of BoNT / A across the urothelium. Afterwards, the syringe was replaced with a syringe containing PBS and distended for an additional 90 minutes to assess the effects of BoNT on bladder physiology. For safety reasons, any BoNT / A present in the intraluminal fluid flowing out of the top catheter was inactivated using Presept (Advanced Sterilization Products).

[0617] Preliminary experiments showed that the concentration of BoNT / A that provided a stable and reproducible response was 100 U / ml. The total toxin amount was calculated to be 3.6 pM, which was the amount of toxin chosen for serotype comparison purposes.

[0618] 7. Experimental Results

[0619] Intravesical application of 100 U / ml of Dysport (a BoNT / A preparation) resulted in a significant reduction in distension-induced afferent nerve firing in the 90-min protocol ( Fig.11 ; n = 9; p < 0.0001). Bladder compliance increased after BoNT / A treatment ( Fig.11 ; n=9; p=0.0003).

[0620] Example 5

[0621] Inhibitory effects of botulinum neurotoxin type B (BoNT / B) on bladder mechanosensitivity in an in vitro mouse model

[0622] Bladder distension was performed until distension-induced neural responses were reproducible, following the same protocol as described in Example 4. After three reproducible distensions with phosphate-buffered saline (PBS), BoNT / B was administered intravesically via a syringe pump, distending the bladder three times at a rate of 150 μl / min, followed by nine more distensions with PBS, each separated by 10 minutes.

[0623] like Fig.12 As shown, over time, BoNT / B reduced afferent nerve firing induced by bladder distension. Analysis showed that this reduction was significant (p<0.0001; n=6). BoNT / B significantly increased the bladder's pressure-volume relationship, which is considered a measure of bladder compliance (p<0.0001). The study found that BoNT / B significantly inhibited the distension-induced response, with only 52.3% (±19.8%) of afferent nerve firing remaining 90 minutes after intravesical BoNT / B treatment.

[0624] BoNT / B appeared to attenuate dilation-induced afferent nerve firing to a greater extent than the inhibition induced by BoNT / A.

[0625] Example 6

[0626] Inhibitory effects of botulinum neurotoxin type E (BoNT / E) on bladder mechanosensitivity in an in vitro mouse model

[0627] The effect of BoNT / E on bladder mechanosensitivity was investigated following the same protocol as described in Example 4. Fig.13 As shown, BoNT / E significantly reduced distension-induced afferent nerve firing (p<0.0001; n=5). Bladder compliance was significantly increased after BoNT / E treatment.

[0628] In contrast to the effects of BoNT / A on bladder mechanosensitivity, BoNT / E exhibited a stronger inhibitory effect than that observed with BoNT / B.

Claims

1. A method for treating a patient suffering from bladder pain syndrome, the method comprising: A solution containing a clostridial neurotoxin is injected into the patient’s bladder; increasing the volume of a solution containing a clostridial neurotoxin within the bladder, thereby applying mechanical forces to the inner surface of the urothelial lining; and The volume of solution containing the clostridial neurotoxin in the bladder is maintained at a level that does not induce urination in the patient for at least 30 minutes to allow the clostridial neurotoxin to diffuse across the urothelium into the lamina propria where it binds to primary sensory afferent nerve fibers and inhibits the secretion of neurotransmitters therefrom, thereby relieving bladder pain.

2. The method of claim 1, wherein the Clostridial neurotoxin selectively binds to primary sensory afferent nerve fibers.

3. The method of claim 1 or 2, wherein the Clostridial neurotoxin is substantially retained within the lamina propria (preferably, the Clostridial neurotoxin does not diffuse into the detrusor muscle of the bladder wall).

4. The method of any of the preceding claims, wherein the solution comprising the clostridial neurotoxin is diffused systemically within the lamina propria of the bladder wall, and wherein the clostridial neurotoxin inhibits neurotransmitter release from substantially all parasympathetic afferent neurons present therein.

5. A method according to any one of the preceding claims, wherein the solution is infused into the bladder via a catheter.

6. A method according to any one of the preceding claims, wherein the method is substantially non-invasive, preferably the method causes substantially no physical damage to the urothelium and / or lamina propria.

7. A method according to any one of the preceding claims, wherein the solution is administered for a duration of at least 1 hour.

8. A method according to any one of the preceding claims, wherein the method does not cause the patient to urinate (preferably the Clostridial neurotoxin does not bind to parasympathetic efferent neurons to stimulate muscarinic receptors in the detrusor muscle to contract the detrusor muscle).

9. The method of any one of the preceding claims, wherein the Clostridial neurotoxin is a botulinum neurotoxin (BoNT).

10. The method of any one of the preceding claims, wherein the Clostridial neurotoxin is botulinum neurotoxin type A (BoNT / A).

11. The method of any one of the preceding claims, wherein the Clostridial neurotoxin comprises a BoNT / A light chain and transport domain, and a BoNT / B receptor binding domain (HC domain).

12. The method of any of the preceding claims, wherein the clostridial neurotoxin comprises a modification at one or more amino acid residues selected from the group consisting of asparagine (ASN) 886, asparagine 905, glutamine (GLN) 915, asparagine 918, glutamic acid (GLU) 920, asparagine 930, asparagine 954, serine (SER) 955, glutamine 991, glutamic acid 992, glutamine 995, asparagine 1006, asparagine 1025, asparagine 1026, asparagine 1030, 032, asparagine 1043, asparagine 1046, asparagine 1052, aspartic acid (ASP) 1058, histidine (HIS) 1064, asparagine 1080, glutamic acid 1081, glutamic acid 1083, aspartic acid 1086, asparagine 1188, aspartic acid 1213, glycine (GLY) 1215, asparagine 1216, glutamine 1229, asparagine 1242, asparagine 1243, serine 1274 and threonine (THR) 1277, wherein the modification is selected from: i. replacing acidic surface exposed amino acid residues with basic amino acid residues; ii. replacing acidic surface exposed amino acid residues with uncharged amino acid residues; iii. replacing uncharged surface exposed amino acid residues with basic amino acid residues; iv. inserting a basic amino acid residue; and v. Deletion of acidic surface exposed amino acid residues.

13. The method of any one of the preceding claims, wherein bladder pain is reduced for at least 2 days following administration of the solution containing the clostridial neurotoxin.

14. The method of any one of the preceding claims, wherein bladder pain is reduced for 4 days following administration of the solution containing the clostridial neurotoxin.

15. The method of any preceding claim, wherein bladder pain comprises allodynia or hyperalgesia.

16. The method of any of the preceding claims, wherein the patient's nociception threshold is increased following administration of the clostridial neurotoxin.

17. A kit for use in the method of any one of the preceding claims, comprising: a) a solution consisting essentially of a clostridial neurotoxin; as well as b) A catheter inserted into the patient's bladder.

18. The method according to any one of claims 1 to 16 or the kit for use therein according to claim 17, wherein the solution: a. Based on normal saline; and / or b. Does not contain exfoliating agents.

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