Treatment of bladder pain syndrome

Abstract

The present invention provides a method of treating a patient suffering from bladder pain syndrome, comprising administering a solution containing a Clostridial neurotoxin to the patient's bladder; increasing the volume of the solution containing the Clostridial neurotoxin present in the bladder, thereby applying a mechanical force to the inner surface of the urothelial layer; and maintaining the volume of the solution containing the Clostridial neurotoxin present in the bladder at a volume that does not cause the patient to urinate for a period of at least 30 minutes, thereby allowing the Clostridial neurotoxin to diffuse across the urothelial layer and into the lamina propria, where the Clostridial neurotoxin binds to primary sensory afferent nerve fibers and inhibits neurotransmitter secretion therefrom, thereby alleviating bladder pain.

Description

[Technical Field]

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

[0002] BPS is a chronic bladder health problem that affects approximately 6 to 14 million patients in the United States (i.e., 5% to 11% of the U.S. adult population). The International Continence Society (ICS) defines BPS as a condition characterized by chronic (greater than 6 months) pelvic pain, pressure, or discomfort recognized as bladder-related, and accompanied by at least one other urinary symptom, such as a persistent urge or frequency to urinate.

[0003] Urinary frequency is the need to urinate more frequently than usual. The average person does not urinate more than seven times per day and does not need to get up more than twice a night to go to the bathroom. People with BPS need to urinate frequently, day and night, and as the frequency becomes more intense, this leads to urinary urgency. In some patients, this need does not go away, even immediately after urination.

[0004] IC is a more severe or advanced form of BPS and is further characterized by the presence of "typical cytoscopic and histological features." For example, compared with BPS (non-IC) patients, IC patients have a higher incidence and severity of epithelial desquamation, ulcers, pyuria, and / or submucosal inflammation. IC may also be referred to as a chronic submucosal inflammatory disease.

[0005] The specific cause of BPS is unknown, but the following are thought to be contributing factors: - Defects in the bladder tissue that allow potential irritants present in urine to penetrate the bladder wall; - hyperactivity of mast cells and excessive secretion of inflammatory signals (e.g., histamine), - the presence of contaminants in the urine that damage the bladder wall, Hypersensitivity of local afferent nerves, such that pain is evoked by events that are not normally painful (e.g., bladder filling), and - Autoimmunity.

[0006] Urine is formed by the nephrons in the kidneys, transported to the bladder for storage, and then expelled through the urethra. This cyclical filling and emptying process is known as micturition. As the bladder fills, it stretches, mimicking an afferent signal. In contrast, efferent signals cause contraction of the bladder musculature and relaxation of the urethral sphincter, respectively. In addition to mechanoreceptors, various psychological factors (e.g., stress, perception of an acceptable environment, and emotional state) play an important role in the timing and circumstances of urination. Needless to say, the bladder has excellent elastic properties. These properties are due to the structure of the bladder wall, which is composed of the following layers (from the inside to the outside): - epithelial lining, - lamina propria, - muscularis propria, and - Serosa / adventitia.

[0007] The epithelial cells lining the bladder provide an important barrier that prevents irritants present in urine from passing through the bladder lining and contacting the underlying cells and connective tissue surrounding the bladder lumen. In fact, the epithelial cells of the bladder form a highly specialized stratified epithelium, the urothelium, that serves this specific purpose. More specifically, the urothelium is composed of three layers: - The apical layer is the innermost layer and serves as the main barrier between the bladder lumen and the underlying tissue. This highly specialized layer is made up of a single layer of cells ("thecal cells"), which are linked together through tight intercellular junctions to form an impermeable barrier. Another important role played by thecal cells is that they absorb bladder stretch. This is achieved by the release of uroplakin (via uroplakin-containing spindle-shaped vesicles), which forms a superficial plaque layer that covers the thecal cells. Then, when the bladder wall relaxes, this reservoir of uroplakin is returned to the thecal cells by SNARE-mediated endocytosis. - an intermediate layer formed by two to three layers of polygonal cells; and - Basal layer formed by 2-3 layers of small cuboidal cells.

[0008] In a relaxed (i.e., undistended) bladder, the urothelium is five to seven layers thick. In this undistended configuration, the typical capacity of a healthy (human) bladder is approximately 500 ml. When the bladder fills with urine, the bladder wall stretches to accommodate the increased volume, and in this distended form, the urothelium reorganizes into two or three layers without any structural damage.

[0009] 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, the ill-defined smooth muscle layer, and the muscularis mucosae) and serves as the "functional center" of the bladder. In this regard, it is believed that the lamina propria regulates the afferent limb of the micturition reflex, and the interstitial cells of Cajal function as neural signaling agents for the smooth muscle cells of the bladder. Thus, the lamina propria functions as the capacitive layer of the bladder.

[0010] The muscularis propria, also known as the detrusor muscle, is innervated by efferent (motor) nerves and consists of three layers: the inner longitudinal layer, the middle circular layer, and the outer longitudinal layer.

[0011] The serosa and adventitia are thin connective tissue layers that form the outermost layer of the bladder.

[0012] These layers collectively play a role in maintaining bladder homeostatic control (e.g., urination), while the bladder wall lining insulates and protects adjacent tissues and organs from contact with stored urine (including toxic solutes and metabolites present in urine). In doing so, the bladder wall lining provides a highly efficient barrier that is impermeable to urine. However, a negative consequence of this highly specialized function is that the bladder wall is also impermeable to potential therapeutic molecules. This poses a significant challenge for any therapeutic treatment of BPS and / or IC, as therapeutic molecules must first penetrate the bladder wall barrier before they can exert clinically relevant effects on targeted target cells located in the deeper underlying layers of the bladder wall.

[0013] Although noninvasive approaches have garnered some interest, a common problem with these methods is that, at best, they offer limited beneficial effects and their effects act very slowly (taking up to six months). Frequent repeated administration is also required. To date, Elmiron® (i.e., pentosan polysulfate sodium) remains the only drug approved by the FDA for the treatment of BPS / IC via oral administration. Pentosan polysulfate is structurally similar to the natural glycosaminoglycan coating of the bladder lining and is thought to provide temporary repair of the lining. While its exact mechanism of action is unknown, Elmiron® is understood to adhere to the luminal side of the bladder wall, forming a layer. Thus, Elmiron® functions as a type of chemical filler or sealant, effectively masking any damaged areas of the urothelium.

[0014] Invasive techniques address these shortcomings and are the preferred interventional method for treating BPS and / or IC. Despite this, invasive methods typically involve some degree of physical intervention, resulting in additional patient management considerations, from anesthesia to intravenous sedation. The most common physical intervention procedure is intradetrusor injection (see FIG. 1 ), which involves carefully maneuvering a device through an opening, along the length of the urethra, into the patient's bladder. Cytoscopic guidance then allows for sequential navigation of a needle to multiple pre-determined intramuscular injection sites located throughout the bladder wall lining. Each injection requires the needle to penetrate the bladder wall lining (to the detrusor muscle), resulting in the delivery of a metered dose of therapeutic agent at each injection site. Adverse effects of this procedure include localized bleeding and pain, which can pose significant patient management challenges. In particular, side effects of this procedure, such as localized bleeding and pain, can lead to a decrease in overall patient satisfaction and, in turn, to decreased patient compliance with the treatment plan.

[0015] Another technique is intravesical instillation (see FIG. 2 ), which involves inserting a catheter into the patient's urethra and draining any urine present in the bladder. A small volume (e.g., 50 ml) of medication is then slowly instilled into the bladder through the catheter. The catheter is removed, and the patient resumes normal daily activities, but is instructed not to empty the bladder for at least 15 minutes, preferably at least 90 minutes. This allows the medication to contact and potentially treat the entire bladder wall lining. Unfortunately, however, this procedure is limited in that it cannot address the problem of inability to penetrate the barrier posed by the bladder wall lining, and is therefore only suitable for therapeutic agents that act on the luminal side of the bladder wall lining.

[0016] Modifications to this procedure have been made to broaden available therapeutic options to include targeting cells located in deeper layers of the bladder wall. One such approach relies on the use of chemical exfoliants (e.g., dimethyl sulfoxide (DMSO), protamine sulfate, hyaluronic acid-phosphatidylethanolamine), which are dissolved in a liquid infusion mixture and administered to the patient as part of a standard infusion protocol. For example, DMSO may be administered as a single infusion at a 50% concentration, or more commonly as part of a "cocktail" with methylprednisolone or hydrocortisone, alkalized lidocaine, and heparin sulfate. Once administered, the exfoliants attack any cells they come into contact with, gradually stripping them from the urothelial layer and rendering the bladder wall permeable.

[0017] Unfortunately, instillation of stripping agents has not proven successful as the method of choice for treating BPS and / or IC. This is for several reasons, not least because chemical stripping agents are toxic (typically indiscriminate) irritants, and therefore any long-term exposure to the patient or physician should be avoided. Therefore, instillation of stripping agents is unsuitable for repeated use. Furthermore, once initiated, maintaining adequate control of the stripping process is difficult, which in turn makes the process unpredictable and therefore unreliable. Contributing factors include chemical variables (e.g., agent selection and concentration), instillation variables (e.g., duration of exposure to agent), and patient-specific variables (e.g., the degree of pre-existing bladder wall damage and response to the selected therapeutic molecule or treatment regimen).

[0018] When considering delivering therapeutic molecules to relevant target cells to treat BPS and / or IC, the bladder wall lining (urothelium) is not the only structural challenge that must be addressed. For example, if the target cells are located in deeper layers of the bladder wall, the therapeutic molecule must be able to penetrate the lower layers of the bladder wall to reach the target cells, where it can exert its therapeutic effect. As expected, the physical constraints imposed by the bladder wall structure inherently favor small molecule therapeutics, making such molecules a preferred class of molecules for treating BPS and / or IC.

[0019] Therapeutic intervention is further complicated by the need to ensure selective delivery of therapeutic molecules to the relevant target cells, thereby avoiding or minimizing any undesired off-site targeting effects. In the context of treating BPS and / or IC, this requires selective targeting of sensory afferent nerve fibers over efferent nerve fibers present in the underlying tissue of the bladder wall.

[0020] Clostridial neurotoxins, particularly BoNT / A, are known to be used to treat BPS via intradetrusor injection. Intradetrusor injection of BoNT / A is highly invasive and simultaneously targets all three innervation levels of the bladder wall: the urothelium, the afferent terminals of the lamina propria, and the efferent terminals of the detrusor muscle (see Figure 3). This procedure does not allow for selective targeting of BoNT / A to sensory afferent fibers (preferentially over efferent fibers). Furthermore, binding of BoNT / A to the efferent terminals of the detrusor muscle can result in undesirable side effects, such as bleeding.

[0021] On the other hand, intraluminal instillation (not injection) of BoNT / A has not been able to deliver BoNT / A to the bladder wall because the bladder urothelium is impermeable to large macromolecules and BoNT / A is degraded by urinary proteases (see Khera, M. et al. (2005) Urology, 66, 208-212 and Shimizu, S. (2012) J. Urol., 187, e370). Attempts have been made to address the degradation effect by using liposomally encapsulated BoNT / A.

[0022] Although limited delivery of BoNT / A has been reported (when co-administered via intraluminal instillation of DMSO, protamine sulfate, or hyaluronic acid-phosphatidylethanolamine), this abrasive approach has failed to provide any approved method that offers a significant improvement over intradetrusor injection of BoNT / A, which remains the current method of choice for therapeutic intervention in BPS.

[0023] Additionally, limited delivery of BoNT / A has been reported when administered (via intraluminal instillation) in the form of hydrogel or liposome-encapsulated formulations. However, similar to the abrasive approach, the use of hydrogels or liposomes has failed to provide any approved method that offers a significant improvement over intradetrusor injection of BoNT / A, which remains the current method of choice for therapeutic intervention of BPS.

[0024] Thus, there is a need in the art for a method of treating BPS and / or IC that selectively targets sensory afferent fibers in preference to efferent fibers present in the bladder wall, yet is minimally invasive (e.g., less invasive than intradetrusor injection). There is also a need to avoid the use of abrasive agents.

[0025] The present invention addresses one or more of the problems identified above. Summary of the Invention

[0026] The present invention relates to the use of clostridial neurotoxins, such as botulinum neurotoxins (BoNTs), as therapeutic agents for the treatment of bladder pain syndrome (BPS), particularly interstitial cystitis (IC), in a subject in need thereof.

[0027] More specifically, the present inventors have surprisingly found for the first time that administration of a Clostridial neurotoxin (e.g., BoNT / A) by a unique method involving gently filling the bladder with a solution containing the neurotoxin can dramatically improve the quality of life of patients by reducing or eliminating the pain associated with BPS, particularly IC, with little to no side effects, unlike conventional methods.

[0028] The inventors' surprising finding is that this approach generates a mild mechanical force against the urothelial layer of the bladder that is sufficient to diffuse the clostridial neurotoxin across the urothelial layer into the lamina propria, while simultaneously limiting further diffusion of the neurotoxin into deeper layers of the bladder wall, particularly the detrusor muscle. This pressure-driven diffusion of the clostridial neurotoxin allows the toxin to target primary sensory afferent nerves located within the lamina propria, inhibiting neurotransmitter release therefrom and reducing pain sensation.

[0029] This is possible because the urothelial layer of the bladder in subjects with BPS, particularly IC, is damaged and porous, allowing the solution to pass through the layer with the aid of the gentle packing technique of the present invention. A further advantage is that no needle or abrasive excipient is required, requiring only a simple saline-based solution. This contrasts with existing methods that use painful needle injections or highly irritating formulations to deliver clostridial neurotoxins (e.g., BoNT) into the lamina propria and beyond. Therefore, unlike conventional methods that intentionally damage the urothelial cell layer and beyond, the administration technique of the present invention (gentle packing) has no or very little impact on the integrity of the urothelial cell layer, e.g., does not cause any additional damage beyond what is already present.

[0030] In practice, the patient's bladder is typically flushed / washed before gentle filling. A Clostridial neurotoxin solution is then infused into the bladder, e.g., via a simple catheter, exerting gentle pressure on the urothelial layer. This infusion can be continued until, but not beyond, the patient experiences a sense of urgency. In healthy adults, this typically occurs when the bladder contains approximately 300-400 ml of fluid; in patients with BPS, this typically occurs when the bladder holds 200-300 ml, or sometimes even less. The urge to urinate occurs when stretch receptors present in the bladder signal the parasympathetic nervous system, stimulating muscarinic receptors in the detrusor muscle to contract, thereby initiating urination. Therefore, establishing the relevant threshold volume of fluid that triggers bladder emptying may require some trial and error for each patient. This threshold volume of fluid is then simply calculated backward to calculate the reduced volume (e.g., by 5% or 10%) and a comfort factor is incorporated to ensure that the patient does not empty their bladder during treatment with the Clostridial neurotoxin solution. Once the desired volume of Clostridial neurotoxin has been infused, the catheter is closed and the solution is allowed to diffuse across the urothelial layer.

[0031] Importantly, the inventors confirmed that the light pressure-induced diffusion of clostridial neurotoxins is typically limited to diffusion within the lamina propria. Notably, because the diffusion does not extend to the bladder muscle layer, unlike conventional intradetrusor injection, the clostridial neurotoxins do not target the efferent terminals of the detrusor muscle, thus avoiding undesirable side effects such as bleeding and muscle paralysis. Instead, this new method enables precise delivery of clostridial neurotoxins, where the neurotoxins are distributed systemically throughout the urothelial layer and lamina propria of the bladder wall and target afferent nerve endings (sensory endings capable of transmitting pain signals) localized within the lamina propria (see Figure 4). The new administration method (light pressure injection) of the present invention is advantageous in that it allows for a simplified, painless, and non-invasive administration mode (needle-free administration of clostridial neurotoxins), potentially enabling this procedure to be performed not only by nurses but also by patients themselves. The non-invasive nature of this procedure, combined with the desirable treatment outcomes, leads to improved overall patient satisfaction, which in turn improves patient compliance with this treatment regimen. DETAILED DESCRIPTION OF THE INVENTION

[0032] In one aspect, the present invention provides a method of treating a patient suffering from bladder pain syndrome, said method comprising: administering a solution containing a Clostridial neurotoxin into the patient's bladder; increasing the volume of a solution containing a Clostridial neurotoxin present in the bladder, thereby exerting a mechanical force against the inner surface of the urothelial layer; and Maintaining the volume of solution containing the Clostridial neurotoxin present in the bladder at a volume that does not cause the patient to urinate for a period of at least 30 minutes, thereby allowing the Clostridial neurotoxin to diffuse across the urothelial layer into the lamina propria, where it binds to primary sensory afferent nerve fibers, inhibiting neurotransmitter secretion therefrom and relieving bladder pain.

[0033] In one aspect, the present invention provides a solution comprising a Clostridial neurotoxin for use in a method of treating a patient suffering from BPS, said method comprising: administering a solution containing a Clostridial neurotoxin into the patient's bladder; increasing the volume of a solution containing a Clostridial neurotoxin present in the bladder, thereby exerting a mechanical force against the inner surface of the urothelial layer; and Maintaining the volume of solution containing the Clostridial neurotoxin present in the bladder at a volume that does not cause the patient to urinate for a period of at least 30 minutes, thereby allowing the Clostridial neurotoxin to diffuse across the urothelial layer into the lamina propria, where it binds to primary sensory afferent nerve fibers, inhibiting neurotransmitter secretion therefrom and relieving bladder pain.

[0034] The bladder wall contains three cell layers: the urothelium, the lamina propria, and the detrusor muscle. The urothelium, the innermost layer of the bladder wall, is a unique and highly specialized epithelial lining that acts as a barrier separating the contents of the bladder cavity from the tissues underlying 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.

[0035] Advantageously, the inventors have determined that by filling the bladder of a patient suffering from BPS / IC with a solution containing a Clostridial neurotoxin up to, but not beyond, the time when the patient feels the need to urinate, this exerts a gentle pressure on the urothelial layer, allowing the Clostridial neurotoxin to diffuse across said layer into the lamina propria upon gentle pressure.

[0036] Various methods can be used to ascertain a patient's threshold volume of fluid that will trigger bladder emptying, for example, voiding diaries, uroflowmetry, ultrasound scanning, and cystometry.

[0037] A voiding diary can be a record of a patient's daily fluid intake and timing of urination (including accidental leakage) over a 24-hour period.

[0038] Uroflowmetry may involve having the patient urinate and recording the total volume, rate, and length of time that urine passes through the bladder with a uroflowmeter. A bladder scan may then be performed to assess whether the patient's bladder has residual volume. Based on the results from the uroflowmeter and bladder scan, a threshold volume of fluid that will trigger bladder emptying can then be calculated.

[0039] Ultrasound scanning may involve scanning the pelvic region of a patient with a full bladder (before urination) to measure the amount of fluid in the patient's bladder. Ultrasound scanning can provide information about the size, fullness, and / or bladder lining of the patient's bladder.

[0040] Cystometry can be performed by placing a 5-F catheter into the patient's bladder (with the patient in a sitting position) and infusing saline into the bladder at a rate of 50 ml per minute. The threshold volume of fluid that triggers bladder emptying can be determined by noting the amount of saline (in ml) infused into the patient's bladder until the patient is unable to postpone urination.

[0041] Those skilled in the art will appreciate that the threshold volume of fluid that triggers urination may vary from patient to patient when the patient has BPS / IC, and furthermore, those skilled in the art will appreciate that the difference in threshold volume may vary between BPS / IC patients and healthy patients.

[0042] To prevent urination during treatment while simultaneously applying gentle pressure to the urothelial layer, a reduced volume of a solution containing a Clostridial neurotoxin (e.g., below the threshold volume of fluid that triggers urination) can be administered. The volume of the solution containing a Clostridial neurotoxin administered to a patient can be reduced by at least 5%, 10%, 15%, 20%, 25%, or 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 a patient can be reduced by 70% or less, 60% or less, 50% or less, or 40% or less 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 a patient can be reduced by 5% to 70%, 10% to 60%, 15% to 50%, 20% to 40%, or 25% to 30% compared to the threshold volume of fluid that triggers urination in the same patient.

[0043] The reduced volume of the solution containing a Clostridial neurotoxin administered to a patient can be at least 150 ml, at least 175 ml, at least 200 ml, at least 225 ml, at least 250 ml, at least 275 ml, or at least 300 ml. The reduced volume of the solution containing a Clostridial neurotoxin administered to a patient can be 400 ml or less, 375 ml or less, 350 ml or less, 325 ml or less, 300 ml or less, 275 ml or less, or 250 ml or less. The reduced volume of the solution containing a Clostridial neurotoxin administered to a patient can be 150 ml to 400 ml, 175 ml to 375 ml, 200 ml to 350 ml, 225 ml to 325 ml, or 250 ml to 300 ml.

[0044] In one embodiment, a single therapeutic infusion of a solution containing a Clostridial neurotoxin is administered over a defined period of time, for example, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, or at least 40 minutes, preferably at least 45 minutes, more preferably at least 50 minutes (e.g., at least 1 hour). In one embodiment, a single therapeutic infusion of a solution containing a Clostridial neurotoxin is administered over a period of 2 hours or less, 1 hour 45 minutes or less, 1 hour 30 minutes or less, or 1 hour 25 minutes or less, preferably 1 hour 15 minutes or less, more preferably 1 hour 10 minutes or less (e.g., at least 1 hour). In one embodiment, a single therapeutic infusion of a solution containing a Clostridial neurotoxin is administered over a period of 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, more preferably 50 minutes to 1 hour 10 minutes (e.g., 1 hour). Following this, the patient may then be asked by a physician to urinate to clear the solution from the patient's bladder.

[0045] A patient may receive a course of treatment that includes multiple treatment infusions over a defined period, such as multiple treatment infusions over a two-week period. In one embodiment, the solution containing a Clostridial neurotoxin is administered by light pressure infusion as part of the course of treatment, which includes multiple individual infusions over a defined period of at least two weeks, at least three weeks, at least four weeks, preferably at least five weeks, and more preferably at least six weeks (e.g., at least seven weeks). In one embodiment, the solution containing a Clostridial neurotoxin is administered by light pressure infusion as part of the course of treatment, which includes multiple individual infusions over a defined period of 12 weeks or less, 11 weeks or less, or 10 weeks or less, preferably 9 weeks or less, and more preferably 8 weeks or less (e.g., 7 weeks or less). In one embodiment, the solution containing a Clostridial neurotoxin is administered by light pressure infusion as part of the course of treatment, which includes multiple individual infusions over a defined period of 2 to 12 weeks, 3 to 11 weeks, or 4 to 10 weeks, preferably 5 to 9 weeks, and more preferably 6 to 8 weeks (e.g., 7 weeks).

[0046] The multiple separate infusions may be administered at least once or twice per week. The multiple separate infusions may be administered no more than four times, no more than three times, no more than two times, or no more than one time per week. The multiple separate infusions may be administered 1 to 4 times, 1 to 3 times, or 2 to 3 times per week, preferably 2 to 3 times per week.

[0047] The term "hydrodistention" can refer to a method in which a cystoscope is used to fill the bladder with sterile fluid until it becomes overdistended. Hydrodistention can involve filling the bladder with solution at a pressure of 60 to 80 cmH2O (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.

[0048] The administration method of the present invention is different from and not equivalent to hydrodistention cystoscopy.

[0049] Thus, in one embodiment, administering a solution containing a Clostridial neurotoxin to a patient does not include hydrocystodistention of the patient's bladder. In one embodiment, administering a solution containing a Clostridial neurotoxin to a patient does not include distending the bladder to a pressure of at least 50 cmH2O, at least 60 cmH2O, at least 70 cmH2O, or at least 80 cmH2O. In one embodiment, administering a solution containing a Clostridial neurotoxin to a patient does not include distending the bladder to a pressure of 50 cmH2O to 100 cmH2O or less, 60 cmH2O to 90 cmH2O or less, or 70 cmH2O to 80 cmH2O or less.

[0050] In one embodiment, the clostridial neurotoxin selectively binds to primary sensory afferent nerve fibers. In one embodiment, the clostridial neurotoxin remains substantially within the lamina propria (preferably, the clostridial neurotoxin does not diffuse into the detrusor muscle of the bladder wall). Thus, unlike conventional methods, the method of the present invention advantageously avoids targeting efferent nerve endings localized in the detrusor muscle (which can cause bleeding and muscle paralysis).

[0051] In one embodiment, a solution containing a Clostridial neurotoxin is dispersed throughout the lamina propria of the bladder wall, where the Clostridial neurotoxin inhibits neurotransmitter release from substantially all parasympathetic afferent neurons present therein. Thus, unlike conventional methods (such as intradetrusor injection), the method of the present invention may allow for local administration of the Clostridial neurotoxin with little or no spread to surrounding areas and with little systemic circulation (better targeting sensory fibers).

[0052] The term "systemically" can refer to the distribution of the solution throughout the tissue, preferably the lamina propria of the bladder wall. The solution can be distributed evenly through up to two layers of the bladder wall. The solution can be distributed throughout the urothelial layer and / or the lamina propria. The solution can be distributed systematically throughout the lamina propria without dispersing into deeper layers of the bladder wall, such as the detrusor muscle.

[0053] The term "substantially" in the context of inhibiting neurotransmitter release from parasympathetic afferent neurons can mean inhibiting neurotransmitter release from 50% to 100%, 60% to 90%, or 70% to 80% of the parasympathetic afferent neurons present in the lamina propria. Preferably, the clostridial neurotoxin can inhibit neurotransmitter release from 100% of the parasympathetic afferent neurons present in the lamina propria.

[0054] In one embodiment, the solution is administered into the bladder via a catheter. In one embodiment, the method is substantially non-invasive, wherein preferably, the method does not substantially physically damage the urothelial layer and / or the lamina propria. Thus, the present invention is advantageous in that it eliminates the use of needles, thereby providing a painless, non-invasive method for administering the Clostridial neurotoxin.

[0055] 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 an exfoliant, thereby preventing further damage to the bladder wall. The exfoliant can include DMSO, protamine sulfate, and / or hyaluronic acid-phosphatidylethanolamine. In other words, unlike conventional methods that administer chemical irritants such as DMSO to permeabilize bladder wall cells, the present invention allows the administration of a harmless solution containing a clostridial neurotoxin (without exfoliative excipients such as DMSO).

[0056] The term "inert" can refer to a solution that may be chemically inert, for example, that does not cause irritation or tissue damage.

[0057] In one embodiment, the method may not cause the patient to urinate (preferably, the Clostridial neurotoxin does not bind to parasympathetic efferent neurons and does not stimulate muscarinic receptors in the detrusor muscle to contract), and thus advantageously allows the patient to benefit from the therapeutic effects of the solution containing the Clostridial neurotoxin for sufficient time for the solution to diffuse across the urothelial layer before the patient empties their bladder.

[0058] In one embodiment, a single treatment infusion of a solution containing a Clostridial neurotoxin is administered for at least 1 hour.

[0059] In summary, the present invention advantageously treats bladder pain syndrome by reducing or eliminating the level of perceived pain and improving the patient's quality of life.

[0060] Thus, in one embodiment, bladder pain is alleviated after treatment with a solution containing a Clostridial neurotoxin using the administration method of the present invention. For example, pain levels in a BPS / IC patient after treatment with a solution containing a Clostridial neurotoxin using the administration method of the present invention can be reduced or eliminated compared to pain levels in a patient not treated with a solution containing a Clostridial neurotoxin (control).

[0061] In one embodiment, bladder pain is relieved for at least two days after gentle filling of the bladder with a solution comprising a Clostridial neurotoxin.

[0062] In one embodiment, after gently filling the bladder with a solution containing a Clostridial neurotoxin, bladder pain is alleviated for at least 1 day, preferably at least 2 days, and more preferably at least 3 days (e.g., at least 4 days). In one embodiment, after gently filling the bladder with a solution containing a Clostridial neurotoxin, bladder pain is alleviated for 9 days or less, 8 days or less, or 7 days or less, preferably 6 days or less, and more preferably 5 days or less (e.g., 4 days or less). In one embodiment, after gently filling the bladder with a solution containing a Clostridial neurotoxin, bladder pain is alleviated for 1 to 7 days, or 2 to 6 days, preferably 2 to 5 days, and more preferably 3 to 5 days (e.g., 4 days).

[0063] In one embodiment, after gently filling the bladder with a solution containing a Clostridial neurotoxin, bladder pain is relieved for at least 3 months, at least 4 months, at least 5 months, or at least 6 months, preferably at least 7 months, and more preferably at least 8 months (e.g., at least 9 months). In one embodiment, after gently filling the bladder with a solution containing a Clostridial neurotoxin, bladder pain is relieved for 14 months or less, 13 months or less, or 12 months or less, preferably 11 months or less, and more preferably 10 months or less (e.g., 9 months or less). In one embodiment, after gently filling the bladder with a solution containing a Clostridial neurotoxin, bladder pain is relieved for 6 to 14 months, or 7 to 13 months, preferably 8 to 12 months, and more preferably 9 to 11 months (e.g., 9 months or less).

[0064] One type of pain that may be a symptom of bladder pain syndrome is allodynia. Allodynia means "other pain." Allodynia is pain caused by stimuli that are not normally painful. People with "tactile" allodynia (also known as static tactile or mechanical allodynia) may experience pain such as pain, pressure, and tenderness in the abdomen at rest, or pain during urination. Thus, allodynia is considered to be "pain from stimuli that do not normally cause pain," in contrast to hyperalgesia.

[0065] As used herein, the term "hyperalgesia" refers to increased pain from a stimulus that normally causes pain. Hyperalgesia can be induced by injury to tissue or nerves, resulting in an increased perception of pain by the patient. Injury-induced hyperalgesia can be divided into two subtypes: primary hyperalgesia (resulting in increased pain sensation at the local site of injury) and secondary hyperalgesia (pain is perceived in other body areas distant from the site of injury).

[0066] Thus, in one embodiment, the bladder pain comprises allodynia or hyperalgesia.

[0067] In one embodiment, after administering a Clostridial neurotoxin using the methods of the present invention, the patient's nociceptive threshold is increased. In one embodiment, the patient's nociceptive threshold may be increased by at least 30%, 40%, 50%, 60%, 70%, or 80% compared to a patient not receiving a solution containing a Clostridial neurotoxin. In one embodiment, the patient's nociceptive threshold may be increased by 30% to 100%, 40% to 90%, 50% to 80%, or 60% to 70% compared to a patient not receiving a solution containing a Clostridial neurotoxin.

[0068] The term "nociceptive threshold" may refer to the level of nociceptive stimulus required for a patient to perceive pain.

[0069] Further details of the clostridial neurotoxins encompassed by the present invention are provided below, along with technical background information.

[0070] Bacteria of the genus Clostridium produce highly potent and specific protein toxins that can be delivered to poison neurons and other cells. Examples of such clostridial neurotoxins include those produced by C. baratii and C. butyricum, as well as the neurotoxins (TeNT) produced by C. tetani and the neurotoxins (BoNT) produced by C. botulinum serotypes A-G and X (see WO 2018 / 009903 A2).

[0071] Clostridial neurotoxins are among the most potent known toxins. For example, botulinum neurotoxins have median lethal doses (LD) for mice ranging from 0.5 ng / kg to 5 ng / kg, depending on the serotype. 50Both tetanus and botulinum neurotoxins act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. Botulinum toxin acts at the neuromuscular junction, inhibiting cholinergic transmission in the peripheral nervous system, whereas tetanus toxin acts in the central nervous system.

[0072] In nature, clostridial neurotoxins are synthesized as single-chain polypeptides, which are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains held together by a disulfide bond. Cleavage occurs at a specific cleavage site, often called the activation site, located between the cysteine ​​residues that provide the interchain disulfide bond. This two-chain form is the active form of the toxin. The two chains are called heavy chains (H chains), with a molecular weight of approximately 100 kDa, and light chains (L chains), with a molecular weight of approximately 50 kDa. The H chains are connected to an N-terminal translocation component (H N domain) and a C-terminal targeting component (H C The cleavage site is located between the L chain and the translocation domain component. C The domain binds to its target neuron, and the bound toxin is internalized via endosomes, followed by H N The domain translocates the L chain across the endosomal membrane into the cytosol, where the L chain provides the protease function (also known as a non-cytotoxic protease).

[0073] Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g., SNAP-25, VAMP, or syntaxin) (see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc.). The acronym SNARE is derived from the term Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-Sensitive Factor. SNARE proteins are essential for intracellular vesicle fusion and, ultimately, secretion of molecules from cells via vesicle transport. Protease function is zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins. Therefore, when delivered to desired target cells, non-cytotoxic proteases can inhibit cellular secretion from the target cells. The light chain proteases of clostridial neurotoxins are non-cytotoxic proteases that cleave SNARE proteins.

[0074] In light of the ubiquitous nature of SNARE proteins, clostridial neurotoxins, such as botulinum neurotoxin, have been used successfully in a wide range of therapeutics.

[0075] For example, William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004) describes the use of clostridial neurotoxins, such as botulinum neurotoxins (BoNTs), i.e., BoNT / A, BoNT / B, BoNT / C1, BoNT / D, BoNT / E, BoNT / F, and BoNT / G, and tetanus neurotoxin (TeNT), to inhibit nerve transmission in a wide variety of therapeutic and cosmetic applications; as an example, BOTOX™ is currently approved as a therapeutic agent for the following indications: for the treatment of pain (see US 6,869,610, US 6,641,820, US 6,464,986, and US 6,113,915), for the treatment of muscle injuries (see US 6,423,319), for the treatment of headaches associated with sinusitis (see US 6,423,319), and for the treatment of steroids (see US 6,423,319). Nos. 6,838,434), for the treatment of neurological disorders such as Parkinson's disease (see U.S. Pat. No. 6,620,415, U.S. Pat. No. 6,306,403), and for the treatment of neuropsychiatric disorders (see U.S. Pat. No. 2004 / 0180061, U.S. Pat. No. 2003 / 0211121). All of the above publications are incorporated herein by reference in their entireties.

[0076] Botulinum neurotoxins (BoNTs) are produced by C. botulinum in the form of large protein complexes consisting of BoNT itself complexed with numerous accessory proteins. Currently, there are nine distinct classes of botulinum neurotoxins: botulinum neurotoxin serotypes A, B, C1, D, E, F, G, H, and X, all of which share a similar structure and mode of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralizing antisera, and this serotype classification correlates with the percentage of sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes based on the percentage of amino acid sequence identity.

[0077] BoNTs are absorbed in the gastrointestinal tract and enter the systemic circulation. After binding to the presynaptic membranes of cholinergic nerve terminals, they prevent the release of the neurotransmitter acetylcholine. BoNT / B, BoNT / D, BoNT / F, and BoNT / G cleave synaptobrevin / vesicle-associated membrane protein (VAMP). BoNT / C1, BoNT / A, and BoNT / E cleave 25 kDa synaptosomal-associated protein (SNAP-25). BoNT / C1 cleaves syntaxin. BoNT / X has been shown to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.

[0078] Tetanus toxin is produced in a single serotype by C. tetani: C. butyricum produces BoNT / E, whereas C. baratii produces BoNT / F.

[0079] Despite this diversity, BoNT / A remains the serotype of choice in therapy, with three commonly available commercial preparations (Botox®, Dysport®, and Xeomin®), while only one BoNT / B product is available on the market (Neurobloc® / Myobloc®). To date, these BoNT / A and BoNT / B products, which are toxins purified from the genus Clostridial, are the only two BoNT serotypes approved by regulatory agencies for use in humans for applications ranging from spasticity, bladder dysfunction, or hyperhidrosis (in the case of BoNT / A) (see, e.g., https: / / www.medicines.org.uk / emc / medicine / 112, https: / / www.medicines.org.uk / emc / medicine / 870, https: / / www.medicines.org.uk / emc / medicine / 2162, which are incorporated herein by reference in their entireties) to cervical dystonia (in the case of BoNT / B) (see, e.g., https: / / www.medicines.org.uk / emc / medicine / 20568, which is incorporated herein by reference in its entirety), among others.

[0080] In contrast to 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 transiently disabling the cellular function of their natural target cells. Importantly, non-cytotoxic proteases do not kill the natural target cells they act upon. In addition to clostridial neurotoxins (e.g., botulinum neurotoxins, sold under names such as Dysport™, Neurobloc™, and Botox™), some of the best-known examples of non-cytotoxic proteases include IgA protease (see, e.g., WO 99 / 032272) and antarase protease (see, e.g., WO 2011 / 022357).

[0081] The term "clostridial neurotoxin" includes any polypeptide produced by Clostridium bacteria that enters neurons and inhibits neurotransmitter release, as well as such polypeptides produced by recombinant or chemical techniques. For purposes of the present invention, the term includes functionally equivalent non-cytotoxic proteases, as described above. Preferably, the clostridial neurotoxin is a botulinum neurotoxin (BoNT).

[0082] An exemplary amino acid sequence of a BoNT / A neurotoxin is shown as SEQ ID NO: 1, which is encoded by the nucleotide sequence shown as SEQ ID NO: 2. An exemplary amino acid sequence of a BoNT / B neurotoxin is shown as SEQ ID NO: 3 (UniProt Accession No. B1INP5). An exemplary amino acid sequence of a BoNT / C neurotoxin is shown as SEQ ID NO: 4 (UniProt Accession No. P18640). An exemplary amino acid sequence of a BoNT / D neurotoxin is shown as SEQ ID NO: 5 (UniProt Accession No. P19321). An exemplary amino acid sequence of a BoNT / E neurotoxin is shown as SEQ ID NO: 6 (Accession No. WP_003372387). An exemplary amino acid sequence of a BoNT / F neurotoxin is shown as SEQ ID NO: 7 (UniProt Accession No. Q57236) or SEQ ID NO: 8 (UniProt / UniParc Accession No. UPI0001DE3DAC). An exemplary amino acid sequence of a BoNT / G neurotoxin is shown as SEQ ID NO: 9 (Accession No. WP_039635782). An example of the amino acid sequence of a BoNT / DC neurotoxin is set forth as SEQ ID NO: 10 (Accession No. BAM65681). An example of the amino acid sequence of a BoNT / X neurotoxin is set forth as SEQ ID NO: 12 (Accession No. BAQ12790.1). In one embodiment, the BoNT selected is BoNT / A, e.g., wild-type BoNT / A.

[0083] As used herein, "H C The term "domain" refers to a functionally distinct region of a neurotoxin heavy chain having a molecular weight of approximately 50 kDa that enables the neurotoxin to bind to a receptor located on the surface of a target cell. C The domain is divided into two structurally distinct subdomains, namely "H" and "H" with molecular weights of approximately 25 kDa each. CN Subdomains (H C N-terminal part of the domain) and "H CC Subdomains (H C It consists of the C-terminal part of the domain.

[0084] As used herein, "LH"N The term "domain" is used in C domain and contains an endopeptidase domain ("L" or "light chain") and a domain responsible for translocation of the endopeptidase to the cytoplasm (the H domain of the heavy chain). N It refers to a neurotoxin consisting of the ATP domain.

[0085] As discussed above, clostridial neurotoxins are formed from two polypeptide chains: a heavy chain (H chain) having a molecular weight of approximately 100 kDa and a light chain (L chain) having a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or H chain). C domain) and the N-terminal translocation component (H N domain).

[0086] Examples of light chain reference sequences include: Botulinum neurotoxin type A: amino acid residues 1-448 Botulinum neurotoxin type B: amino acid residues 1-440, Botulinum neurotoxin type C1: amino acid residues 1-441, Botulinum neurotoxin type D: amino acid residues 1-445, Botulinum neurotoxin type E: amino acid residues 1-422 Botulinum neurotoxin type F: amino acid residues 1-439, Botulinum neurotoxin type G: amino acid residues 1-441, Tetanus neurotoxin: amino acid residues 1-457.

[0087] In the case of the recently identified BoNT / X, the L chain is reported to correspond to amino acids 1-439, where the boundaries of the L chain can vary by approximately 25 amino acids (e.g., 1-414 or 1-464).

[0088] The reference sequences identified above should be considered as a guideline, as slight variations may exist depending on the serovar. By way of example, US 2007 / 0166332 (hereby incorporated by reference in its entirety) cites the following slightly different Clostridial sequences: Botulinum type A neurotoxin: amino acid residues M1 to K448, Botulinum type B neurotoxin: amino acid residues M1 to K441; Botulinum type C1 neurotoxin: amino acid residues M1 to K449, Botulinum type D neurotoxin: amino acid residues M1 to R445, Botulinum type E neurotoxin: amino acid residues M1 to R422, Botulinum type F neurotoxin: amino acid residues M1 to K439, Botulinum type G neurotoxin: amino acid residues M1 to K446, Tetanus neurotoxin: amino acid residues M1 to A457.

[0089] The term "activation loop" refers to the polypeptide domain that contains the proteolytic cleavage site. Activation loops of neurotoxins are described in the art, such as in WO2016156113, which is incorporated herein by reference in its entirety.

[0090] 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 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.

[0091] 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.

[0092] In one embodiment, the Clostridial neurotoxin consists of or comprises the amino acid sequence of SEQ ID NO: 2 (eg, BoNT / A).

[0093] 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 amino acids that are modified compared to the native (unmodified) form of the clostridial neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial neurotoxin. By way of example, a modified clostridial neurotoxin may have an amino acid sequence that is modified in one or more domains relative to the sequence of a native (unmodified) clostridial neurotoxin. Such modifications can alter functional aspects of the toxin, such as biological activity or persistence.

[0094] Preferred modified BoNT / As are ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243 ... The modified BoNT / A of the present invention comprises a modification in one or more amino acid residues selected from THR 1274, and THR 1277. Such modified BoNT / As exhibit reduced or no side effects compared to the use of known BoNT / As. The increased tissue retention properties of the modified BoNT / As of the present invention also result in increased potency and / or duration of action, which may allow for reduced dosages to be used (or increased dosages without any additional adverse effects) compared to known clostridial toxin therapeutics, providing further advantages.

[0095] The alterations may be alterations relative to the unmodified BoNT / A shown as SEQ ID NO:2, where the numbering of the amino acid residues is determined by alignment with SEQ ID NO:2. Because the presence of a methionine residue at position 1 of SEQ ID NO:2 (and the SEQ ID NOs corresponding to the modified BoNT / A polypeptides described herein) is optional, one of skill in the art would take the presence / absence of the methionine residue into account when determining the numbering of the amino acid residues. For example, if SEQ ID NO:2 contains a methionine, the numbering of the position would be as defined above (e.g., ASN 886 would become ASN 886 in SEQ ID NO:2). Alternatively, if a methionine is absent in SEQ ID NO:2, the numbering of the amino acid residues should be corrected by -1 (e.g., ASN 886 would become ASN 885 in SEQ ID NO:2). Similar considerations apply to the presence / absence of a methionine at position 1 of the other polypeptide sequences described herein, and one of skill in the art would readily determine the correct numbering of the amino acid residues using routine techniques in the art.

[0096] The amino acid residues indicated for modification are surface-exposed amino acid residues.

[0097] The modified BoNT / A can include a modification at one or more amino acid residues selected from ASN 886, 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 SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 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 SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19. Preferably, a modified BoNT / A for use in the present invention can be encoded by a nucleic acid comprising (or consisting of) SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO: 19. A modified BoNT / A can comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 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 NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20. Preferably, a modified BoNT / A for use in the present invention can comprise (more preferably consist of) a polypeptide sequence selected from SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20.

[0098] The term "one or more amino acid residues," when used in the context of a modified BoNT / A, preferably means at least two, at least three, at least four, at least five, at least six, or at least seven of the indicated amino acid residues. Thus, a modified BoNT / A can contain at least two, at least three, at least four, at least five, at least six, or at least seven (preferably seven) modifications to the indicated amino acid residues. A modified BoNT / A can contain 1 to 30, 3 to 20, or 5 to 10 amino acid modifications. More preferably, the term "one or more amino acid residues," when used in the context of a modified BoNT / A, means all of the indicated amino acid residues.

[0099] Preferably, the modified BoNT / A does not contain any additional amino acid modifications when compared to SEQ ID NO:2 beyond one or more amino acid modifications at the indicated amino acid residues.

[0100] Most preferably, the modified BoNT / A comprises (and more preferably consists of) a modification in one or more amino acid residues selected from 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 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. Preferably, a modified BoNT / A for use in the present invention may comprise (and more preferably consist of) SEQ ID NO:14.

[0101] The modification may be selected from the following: i. Substitution of surface-exposed acidic amino acid residues with basic amino acid residues; ii. Substitution of surface-exposed acidic amino acid residues with uncharged amino acid residues; iii. Substitution of surface-exposed uncharged amino acid residues with basic amino acid residues; iv. insertion of a basic amino acid residue, and v. Deletion of surface-exposed acidic amino acid residues.

[0102] The modifications set forth above result in a modified BoNT / A with an increased positive surface charge and an increased isoelectric point compared to the corresponding unmodified BoNT / A.

[0103] 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 amino acid sequences (also called amino acid residues when within a protein). Each amino acid in the standard set of 20 has a different side chain (i.e., R group). This means that each amino acid residue within 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 be determined by the sum of these various factors.

[0104] Certain amino acid residues (discussed in more detail below) have ionizable side chains that may exhibit a charge depending on the pH of their surroundings. Whether such side chains are charged at a given pH depends on the pKa of the associated ionizable moiety, where pKa is the negative logarithm of the acid dissociation constant (Ka) for a particular proton from the conjugate base.

[0105] For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with pKa values ​​of approximately 4.1 (the exact pKa value may depend on temperature, ionic strength, and the microenvironment of the ionizable group). Thus, these side chains exhibit a negative charge at a pH of 7.4 (often referred to as "physiological pH"). At lower pH values, these side chains become protonated and lose their charge.

[0106] In contrast, basic residues such as lysine and arginine have nitrogen-containing side chains with pKa values ​​of approximately 10-12. Therefore, these side chains exhibit a positive charge at pH 7.4. At higher pH values, these side chains become deprotonated and lose their charge.

[0107] Therefore, the overall (net) charge of a protein molecule depends on the number of acidic and basic residues present in the protein (and their degree of surface exposure) as well as the surrounding pH. Changing the surrounding pH will change the overall charge of the protein. Therefore, for each protein, there is a given pH at which the number of positive and negative charges is equal and the protein exhibits no net charge overall. This point is known as the isoelectric point (pI). The isoelectric point is a standard concept in protein biochemistry that is well known to those skilled in the art.

[0108] Therefore, the isoelectric point (pI) is defined as the pH value at which a protein exhibits a net charge of zero. An increase in pI means that a protein requires a higher pH value to exhibit a net charge of zero. Therefore, an increase in pI represents an increase in the net positive charge of a protein at a given pH. In contrast, a decrease in pI means that a protein requires a lower pH value to exhibit a net charge of zero. Therefore, a decrease in pI represents a decrease in the net positive charge of a protein at a given pH.

[0109] Methods for determining the pI of a protein are known in the art and will be familiar to those skilled in the art. For example, the pI of a protein can be calculated from the average pKa value of each amino acid present in the protein ("calculated pI"). Such calculations can be performed using computer programs known in the art, such as the Compute pI / MW Tool from ExPASy (https: / / web.expasy.org / compute_pi / ), which is the preferred method for calculating pI according to the present invention. Comparison of pI values ​​between different molecules should be performed using the same calculation technique / program.

[0110] Optionally, the calculated pI of a protein can be experimentally confirmed using the technique of isoelectric focusing ("observed pI"). This technique uses electrophoresis to separate proteins according to their pI. Isoelectric focusing is typically performed using a gel with an immobilized pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches a pH at which it has zero net charge (this point is the protein's pI). Because the results obtained by isoelectric focusing typically have a relatively low sensitivity limit by nature, the inventors believe that the results obtained by calculated pI (as described above) are more suitable for use.

[0111] Throughout this specification, unless otherwise specified, "pI" means "calculated pI."

[0112] The pI of a protein can be increased or decreased by altering 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, an increase in pI can be obtained by decreasing the number of acidic residues or by increasing the number of basic residues.

[0113] The modified BoNT / A of the present invention can have a pI value that is at least 0.2 pI units higher, at least 0.4 pI units higher, at least 0.5 pI units higher, or at least 1 pI unit higher than the pI value of an unmodified BoNT / A (e.g., SEQ ID NO: 2). Preferably, the modified BoNT / A can have a pI of at least 6.6, e.g., at least 6.8.

[0114] The properties of the 20 common amino acids are shown in the table below: [Table A]

[0115] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

[0116] At a pH of 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) are negatively charged, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) are positively charged. Aspartic acid and glutamic acid are called acidic amino acid residues. Arginine and lysine are called basic amino acid residues.

[0117] The following amino acids are considered uncharged polar (meaning they can participate in hydrogen bonds) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0118] The following amino acids are considered uncharged hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0119] In an amino acid insertion, an additional amino acid residue (one not normally present) is incorporated into the 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 the amino acid sequence of a clostridial toxin, thereby decreasing the total number of amino acid residues in the sequence.

[0120] Preferably, the modification is a substitution, which advantageously maintains the same number of amino acid residues in the modified BoNT / A. In an amino acid substitution, an amino acid residue that forms part of the BoNT / A polypeptide sequence is replaced with a different amino acid residue. The replacing amino acid residue can be one of the 20 standard amino acids, as described above. Alternatively, the replacing amino acid in an amino acid substitution can be a non-standard amino acid (an amino acid that is not part of the above-mentioned 20 standard set). By way of example, the replacing amino acid can be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid, or the D-isomers of lysine, arginine, and ornithine. Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using an auxotrophic expression host, E. coli.

[0121] In one embodiment, the substitution is selected from substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue. In one embodiment, when the substitution is substitution of an acidic amino acid residue with an uncharged amino acid residue, the acidic amino acid residue is replaced with its corresponding uncharged amide amino acid residue (i.e., aspartic acid is replaced with asparagine, glutamic acid is replaced with glutamine).

[0122] Preferably, the basic amino acid residue is a lysine or arginine residue, i.e., the substitution is with lysine or arginine. Most preferably, the modification is with lysine.

[0123] A translocation domain is a molecule that allows the translocation of a protease to a target cell, such that functional expression of protease activity occurs in the cytosol of the target cell. Whether any molecule (e.g., a protein or peptide) has the translocation function required for the present invention can be confirmed by any one of several conventional assays.

[0124] For example, Shone C. (1987) describes an in vitro assay using liposomes challenged with a test molecule. The presence of the required translocation function is determined by the release of K from the liposomes. + and / or by the release of labeled NAD, which can be easily monitored [see Shone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180].

[0125] A further example is provided by Blaustein R. (1987), who describes a simple in vitro assay using planar phospholipid bilayer membranes: the membrane is challenged with a test molecule, and an increase in conductance across the membrane confirms the required translocation function [see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp. 115-120].

[0126] Additional methods that allow for the assessment of membrane fusion and therefore the identification of translocation domains suitable for use in the present invention are described in Methods in Enzymology Vol 220 and 221, Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

[0127] The present invention also encompasses variant translocation domains, so long as the variant translocation domain still exhibits the requisite translocation activity. By way of example, variants 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. The term fragment, when used in connection with a translocation domain, refers to a peptide having 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. In the case of a clostridial translocation domain, the fragment preferably has at least 20 amino acid residues of the reference translocation domain (e.g., H N A translocation "fragment" of the present invention includes fragments of variant translocation domains based on a reference sequence.

[0128] The translocation domain is preferably capable of forming ion-permeable pores in lipid membranes under conditions of low pH. It has been found to be preferable to use only those parts of the protein molecule that are capable of pore formation in the endosomal membrane.

[0129] The translocation domain may be obtained from a microbial protein source, in particular a bacterial or viral protein source. Thus, in one embodiment, the translocation domain is the translocation domain of an enzyme, such as a bacterial toxin or a viral protein.

[0130] It is well documented that certain domains of bacterial toxin molecules are capable of forming such pores. It is also known that certain translocation domains of virally expressed membrane fusion proteins are capable of forming such pores. Such domains can be used in the present invention.

[0131] The translocation domain is H NThe domain (or functional components thereof) may be of clostridial origin. N means a portion or fragment of the H chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H chain, or the domain corresponding to that fragment within the intact H chain.

[0132] Examples of suitable (reference) translocation domains include: Botulinum neurotoxin type A - amino acid residues (449-871), Botulinum neurotoxin type B - amino acid residues (441-858), Botulinum neurotoxin type C - amino acid residues (442-866), Botulinum neurotoxin type D - amino acid residues (446-862), Botulinum neurotoxin type E - amino acid residues (423-845), Botulinum neurotoxin type F - amino acid residues (440-864), Botulinum neurotoxin type G - amino acid residues (442-863), Tetanus neurotoxin - amino acid residues (458-879).

[0133] The reference sequences identified above should be considered as a guideline, as slight variations may exist depending on the serovar. By way of example, US 2007 / 0166332 (incorporated herein by reference) cites the following slightly different Clostridial sequences: Botulinum neurotoxin type A - amino acid residues (A449 to K871), Botulinum neurotoxin type B - amino acid residues (A442 to S858), Botulinum neurotoxin type C - amino acid residues (T450 to N866), Botulinum neurotoxin type D - amino acid residues (D446 to N862), Botulinum neurotoxin type E - amino acid residues (K423 to K845), Botulinum neurotoxin type F - amino acid residues (A440 to K864), Botulinum neurotoxin type G - amino acid residues (S447 to S863), Tetanus neurotoxin - amino acid residues (S458 to V879).

[0134] In the context of the present invention, the H of various clostridial neurotoxins containing a translocation domain N Regions may be useful in embodiments of the present invention, provided that these active fragments promote the release of non-cytotoxic proteases (e.g., clostridial light chains) from intracellular vesicles into the cytoplasm of target cells, thus participating in the execution of the overall cellular machinery by which clostridial neurotoxins proteolytically cleave substrates. N The region is approximately 410-430 amino acids long and contains the translocation domain. Studies have shown that the H N It has been shown that the entire length of the region is not required for the translocation activity of the translocation domain. Thus, aspects of this embodiment include, for example, H of a clostridial neurotoxin comprising a translocation domain having a length of at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, and at least 425 amino acids. N Other aspects of this embodiment include, for example, H domains of clostridial neurotoxins that include translocation domains having lengths of up to 350 amino acids, up to 375 amino acids, up to 400 amino acids, and up to 425 amino acids. N It may include a region.

[0135] For further details regarding the genetic basis of toxin production in Clostridium botulinum and C. tetani, see Henderson et al (1997) The Clostridia: Molecular Biology and Pathogenesis, Academic Press.

[0136] H N The term is a modified H NThe H of naturally occurring neurotoxins can be used as long as the moiety still exhibits the translocation function described above. N and modified H having non-naturally occurring amino acid sequences and / or synthetic amino acid residues. N It includes parts and

[0137] Alternatively, the translocation domain may be of non-clostridial origin. Examples of sources of non-clostridial (reference) translocation domains include, but are not limited to, the diphtheria toxin translocation domain [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 Pseudomonas exotoxin type A translocation domain [Prior et al. Biochemistry (1992) 31, 3555-3559], the anthrax toxin translocation domain [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442], various fusogenic or hydrophobic peptides with translocation 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]. Translocation domains can reflect translocation domains present in naturally occurring proteins or can contain amino acid mutations so long as the mutations do not destroy the translocation ability of the translocation domain.

[0138] Clostridial neurotoxin H C Examples of domain reference sequences include: BoNT / A - N872-L1296 BoNT / B - E859-E1291 BoNT / C1 - N867-E1291 BoNT / D - S863-E1276 BoNT / E - R846-K1252 BoNT / F - K865-E1274 BoNT / G - N864-E1297 TeNT - I880-D1315

[0139] In the case of the recently identified BoNT / X, H C The domain is reported to correspond to amino acids 893-1306, where the domain boundaries can vary by approximately 25 amino acids (eg, 868-1306 or 918-1306).

[0140] The clostridial neurotoxins described herein may further comprise a translocation-facilitating domain, which facilitates delivery of a non-cytotoxic protease into the cytosol of a target cell, and is described, for example, in WO 08 / 008803 and WO 08 / 008805, each of which is incorporated herein by reference.

[0141] By way of example, suitable translocation-facilitating domains include fusogenic peptide domains of enveloped viruses, such as the fusogenic peptide domain of influenza viruses (e.g., the 23 amino acid fusogenic peptide domain of influenza A virus), the fusogenic peptide domain of alphaviruses (e.g., the 26 amino acid fusogenic peptide domain of Semliki Forest virus), the fusogenic peptide domain of vesiculoviruses (e.g., the 21 amino acid fusogenic peptide domain of vesicular stomatitis virus), and the fusogenic peptide domain of respiroviruses (e.g., the 25 amino acid fusogenic peptide domain of Sendai virus). Examples of such a fusogenic peptide domain include a fusogenic peptide domain of a spumavirus, such as a fusogenic peptide domain of a morbillivirus (e.g., a 25-amino acid fusogenic peptide domain of a canine distemper virus), a fusogenic peptide domain of an avulavirus (e.g., a 25-amino acid fusogenic peptide domain of a Newcastle disease virus), a fusogenic peptide domain of a henipavirus (e.g., a 25-amino acid fusogenic peptide domain of a Hendra virus), a fusogenic peptide domain of a metapneumovirus (e.g., a 25-amino acid fusogenic peptide domain of a human metapneumovirus), or a fusogenic peptide domain of a simian foam virus, or a fragment or variant thereof.

[0142] As a further example, the translocation facilitating domain may be H of a clostridial neurotoxin. CN More particularly, the H domain of a clostridial neurotoxin may be a clostridial neurotoxin. CN The translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, or at least 275 amino acids. CN The translocation facilitating domain preferably has a length of 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: Botulinum neurotoxin type A - amino acid residues (872-1110), Botulinum neurotoxin type B - amino acid residues (859-1097), Botulinum neurotoxin type C - amino acid residues (867-1111), Botulinum neurotoxin type D - amino acid residues (863-1098), Botulinum neurotoxin type E - amino acid residues (846-1085), Botulinum neurotoxin type F - amino acid residues (865-1105), Botulinum neurotoxin type G - amino acid residues (864-1105), Tetanus neurotoxin - amino acid residues (880-1127).

[0143] The above sequence positions may vary slightly depending on the serotype / subtype and should be used in conjunction with the appropriate (reference) clostridial neurotoxin H CN Further examples of domains include: Botulinum neurotoxin type A - amino acid residues (874-1110), Botulinum neurotoxin type B - amino acid residues (861-1097), Botulinum neurotoxin type C - amino acid residues (869-1111), Botulinum neurotoxin type D - amino acid residues (865-1098), Botulinum neurotoxin type E - amino acid residues (848-1085), Botulinum neurotoxin type F - amino acid residues (867-1105), Botulinum neurotoxin type G - amino acid residues (866-1105), Tetanus neurotoxin - amino acid residues (882-1127).

[0144] Any of the above facilitating domains can be combined with any of the above translocation domain peptides suitable for use in the present invention. Thus, by way of example, a non-clostridial facilitating domain can be combined with a non-clostridial translocation domain peptide or a clostridial translocation domain peptide. Alternatively, the H of a clostridial neurotoxin can be combined with a non-clostridial translocation domain peptide or a clostridial translocation domain peptide. CNThe translocation facilitating domain can be combined with a non-clostridial translocation domain peptide. Alternatively, the H of a clostridial neurotoxin can be combined with a non-clostridial translocation domain peptide. CN The facilitating domain can be combined with a clostridial translocation domain peptide, examples of which include: Botulinum neurotoxin type A - amino acid residues (449-1110), Botulinum neurotoxin type B - amino acid residues (442-1097), Botulinum neurotoxin type C - amino acid residues (450-1111), Botulinum neurotoxin type D - amino acid residues (446-1098), Botulinum neurotoxin type E - amino acid residues (423-1085), Botulinum neurotoxin type F - amino acid residues (440-1105), Botulinum neurotoxin type G - amino acid residues (447-1105), Tetanus neurotoxin - amino acid residues (458-1127).

[0145] Naturally occurring clostridial neurotoxins H C The peptide contains approximately 400-440 amino acid residues and is divided into two functionally distinct domains of approximately 25 kDa each: the N-terminal domain (generally H CN peptide or domain) and the C-terminal region (generally H CCIt consists of a peptide or domain. This fact is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. 4: 788-792, Herreros J (2000) Biochem. J. 347: 199-204, Halpern J (1993) J. Biol. Chem. 268: 15, pp. 11188-11192, Rummel A (2007) PNAS 104: 359-364, Lacey DB (1998) Nat. Struct. Biol. 5: 898-902, Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25: 90, Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7: 1751-1759, and Rummel A (2004) Mol. Microbiol. 51(3), 631-643. Furthermore, the C-terminal region (H CC ) is well documented to be responsible for the binding of clostridial neurotoxins to their natural cellular receptors, i.e., nerve endings at the neuromuscular junction, a fact confirmed by the publications mentioned above.

[0146] Clostridium H CC Examples of reference sequences are shown below: Botulinum neurotoxin type A - amino acid residues (Y1111 to L1296), Botulinum neurotoxin type B - amino acid residues (Y1098 to E1291), Botulinum neurotoxin type C - amino acid residues (Y1112 to E1291), Botulinum neurotoxin type D - amino acid residues (Y1099 to E1276), Botulinum neurotoxin type E - amino acid residues (Y1086 to K1252), Botulinum neurotoxin type F - amino acid residues (Y1106 to E1274), Botulinum neurotoxin type G - amino acid residues (Y1106 to E1297), Tetanus neurotoxin - amino acid residues (Y1128 to D1315).

[0147] The reference sequences identified above should be viewed as a guideline, as slight variations may exist depending on the serosubtype.

[0148] Modified Clostridial neurotoxins contain one or more modifications in the amino acid sequence of the heavy chain (modified H C domain), where the modified heavy chain binds to target neurons with greater or less affinity than the native (unmodified) Clostridial neurotoxin. C Such modifications in the domain include H domains that alter binding to ganglioside receptors and / or protein receptors on target neurons. C These include modifications of residues in the ganglioside binding site or protein (SV2 or synaptotagmin) binding site of the domain. Examples of such modified Clostridial neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated herein by reference in their entireties.

[0149] The modified Clostridial neurotoxin may have one or more modifications in the amino acid sequence of the light chain, for example, modifications in the substrate binding domain or catalytic domain, which may alter or change the SNARE protein specificity of the modified L chain. Examples of such modified Clostridial neurotoxins are described in WO 2010 / 120766 and US 2011 / 0318385 (both of which are incorporated by reference in their entireties).

[0150] Thus, the term "clostridial neurotoxin" is intended to encompass hybrid clostridial neurotoxins and chimeric clostridial neurotoxins. In one embodiment, the modified clostridial neurotoxin may be a hybrid clostridial neurotoxin or a chimeric clostridial neurotoxin, provided that the clostridial neurotoxin is a modified BoNT / AH neurotoxin of the present invention. CC The term "hybrid clostridial neurotoxin" refers to a clostridial neurotoxin that comprises a domain. A hybrid clostridial neurotoxin comprises at least a portion of a light chain from one clostridial neurotoxin or subtype thereof and at least a portion of a heavy chain from another clostridial neurotoxin or clostridial neurotoxin subtype. In one embodiment, a hybrid clostridial neurotoxin may comprise a light chain from one clostridial neurotoxin subtype and the entire heavy chain from another clostridial neurotoxin subtype. In one embodiment, a chimeric clostridial neurotoxin may comprise a portion of the heavy chain (e.g., a binding domain) of one clostridial neurotoxin subtype together with another portion of the heavy chain derived from another clostridial neurotoxin subtype. Similarly or alternatively, a therapeutic element may comprise light chain portions of different clostridial neurotoxins. Such hybrid or chimeric clostridial neurotoxins are useful as a means of exerting the therapeutic benefits of such clostridial neurotoxins, for example, in patients who are immunologically tolerant to a given clostridial neurotoxin subtype, who may have lower than average concentrations of receptors for the heavy chain binding domain of a given clostridial neurotoxin, or who may have protease-resistant mutants of membrane toxin 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 publication is incorporated herein by reference in its entirety.

[0151] For example, a chimeric neurotoxin may be prepared by combining the LH from a first neurotoxin with the LH from a second neurotoxin. N domain and H from the second neurotoxin Cand a domain covalently linked thereto, preferably the first neurotoxin and the second neurotoxin are different, wherein the LH N The C-terminal amino acid residue of the domain is the LH domain of the first neurotoxin. N Domain and H C Separating the domain 3 10 corresponding to the first amino acid residue of the helix, and C The N-terminal amino acid residue of the domain is the LH domain of the second neurotoxin. N Domain and H C Separating the domain 3 10 It corresponds to the second amino acid residue of the helix.

[0152] In one embodiment, the clostridial neurotoxin is a hydroxylase inhibitor of BoNT / B. C domain and LH from BoNT / A, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, or BoNT / X N It is a chimeric neurotoxin containing the domain.

[0153] For example, in one embodiment, H C The domain consists of or comprises an amino acid sequence corresponding to amino acid residues 860 to 1291 of SEQ ID NO: 3 (e.g., BoNT / B), or an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto, and is an LH N The domain consists of or comprises an amino acid sequence selected from the group consisting of: - amino acid residues 1 to 872 of SEQ ID NO: 2 (e.g., BoNT / A), or a sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto; - 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, - 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; - 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, - 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, - 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, and - 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, - 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.

[0154] In one embodiment, the clostridial neurotoxin is a BoNT / X (e.g., a hybrid or chimeric form of BoNT / X) that includes at least one domain from a non-BoNT / X clostridial neurotoxin. For example, in one embodiment, the clostridial neurotoxin can include: i. BoNT / XL chain and non-BoNT / XH N Domain and H C domain, ii. BoNT / XH N Domains and non-BoNT / XL chains and H C domain, iii. BoNT / XH C Domains and non-BoNT / XL chains and H N domain, iv. BoNT / XL chain and H N Domain and non-BoNT / XH C domain, v. BoNT / XL chain and H C Domain and non-BoNT / XH N Domain, or vi. BoNT / XH N Domain and H C domains and non-BoNT / XL chains.

[0155] In a preferred embodiment, the clostridial neurotoxin is H from BoNT / B. C domain and LH from BoNT / A N It is a chimeric neurotoxin containing the domain.

[0156] In a more preferred embodiment, H C The 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 is an LH N The 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.

[0157] Clostridial neurotoxins are H from BoNT / B C domain (e.g., the clostridial neurotoxin is BoNT / B or a H domain from BoNT / B) C In embodiments, the clostridial neurotoxin is a chimeric neurotoxin comprising a heavy chain (H C The neurotoxin may have one or more modifications in the amino acid sequence of the H domain, resulting in an "modified heavy chain," which preferably binds to target neurons with higher (or lower) affinity than the native neurotoxin. CSuch modifications in the domain include H, which can alter the binding of target neurons to gangliosides. CC Modification of amino acid residues in the ganglioside-binding site of the H domain and / or H domains that can alter binding to protein receptors on target neurons. CC This may involve modifications of amino acid residues in the protein receptor binding site of the domain. Examples of such modified neurotoxins are described in WO2006027207 and WO2006114308 (both of which are incorporated herein by reference in their entirety).

[0158] In one embodiment, the clostridial neurotoxin of the present 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 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.

[0159] In one embodiment, the clostridial neurotoxin of the present invention can be both chimeric and modified as described above. For example, in a preferred embodiment, the clostridial neurotoxin has the amino acid sequence 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.

[0160] The term "clostridial neurotoxin" also encompasses 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 oryzae-encoded toxin called BoNT / Wo (NCBI Reference Sequence: WP_027699549.1) that cleaves VAMP2 at W89-W90, the Enterococcus faecium-encoded toxin (GenBank: OTO22244.1) that cleaves VAMP2 and SNAP25, and the Chryseobacterium pipero-encoded toxin (NCBI Reference Sequence: WP_034687872.1).

[0161] The term "clostridial neurotoxin" is intended to encompass retargeted clostridial neurotoxins. In a retargeted clostridial neurotoxin, the clostridial neurotoxin has been modified to include an exogenous ligand known as a targeting moiety (TM). The TM is selected to exhibit binding specificity for a desired target cell, and as part of the retargeting process, the clostridial neurotoxin's native binding moiety (e.g., H C Domain or H CC domain) may be removed.

[0162] The term "clostridial neurotoxin" may encompass catalytically inactive clostridial neurotoxins. As used herein, the term "catalytically inactive" with respect to a clostridial neurotoxin L chain means that the L chain exhibits substantially no non-cytotoxic protease activity. Preferably, the term "catalytically inactive" with respect to a clostridial neurotoxin L chain means that the L chain exhibits substantially no non-cytotoxic protease activity. In one embodiment, a catalytically inactive clostridial neurotoxin L chain does not cleave proteins of the exocytic fusion apparatus in target cells. The term "substantially free of non-cytotoxic protease activity" means that a clostridial neurotoxin L chain has less than 5% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L chain, for example, less than 2%, less than 1%, or preferably less than 0.1% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L chain. Non-cytotoxic protease activity can be determined in vitro by incubating a test Clostridial neurotoxin L chain with a SNARE protein and comparing the amount of SNARE protein cleaved by the test Clostridial neurotoxin L chain with the amount of SNARE protein cleaved by a catalytically active Clostridial neurotoxin L chain under the same conditions. Routine techniques such as SDS-PAGE and Western blotting can be used to quantitate the amount of cleaved SNARE protein. A suitable in vitro assay is described in WO 2019 / 145577 A1 (which is incorporated herein by reference).

[0163] The present invention also encompasses clostridial neurotoxins having non-naturally occurring protease cleavage sites. In such clostridial neurotoxins, the natural protease cleavage site (also known as the activation site, as described above) has been modified or replaced with a protease cleavage site that is not naturally present in the clostridial neurotoxin (i.e., an exogenous cleavage site). Such sites require an exogenous protease for cleavage, allowing for improved control over the timing and location of the cleavage event. Non-naturally occurring protease cleavage sites that can be used in clostridial neurotoxins include the following: TEV (Tobacco Etch Virus) (ENLYFQ↓G) (SEQ ID NO: 26) Thrombin (LVPR↓GS) (SEQ ID NO: 27) PreScission (LEVLFQ↓GP) (SEQ ID NO: 28) Enterokinase (DDDDK↓, SEQ ID NO: 29) Factor Xa (IEGR↓ / IDGR↓, SEQ ID NO: 30 and SEQ ID NO: 31).

[0164] Additional protease cleavage sites include recognition sequences cleaved by non-cytotoxic proteases, such as the light chain of a clostridial neurotoxin. These recognition sequences include SNARE (e.g., SNAP-25, syntaxin, VAMP) protein recognition sequences cleaved by non-cytotoxic proteases, such as the light chain of a clostridial neurotoxin. 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 encompasses inteins, which are self-cleaving sequences. Self-splicing reactions can be controlled, for example, by changing the concentration of reducing agents present.

[0165] The present invention also encompasses clostridial neurotoxins comprising a "destructive cleavage site." In said clostridial neurotoxins, a non-native protease cleavage site is incorporated into the clostridial neurotoxin at a position selected such that cleavage at said site reduces or inactivates the activity of the clostridial neurotoxin. In the event that the clostridial neurotoxin migrates to a non-target location after administration, the destructive protease cleavage site may become susceptible to cleavage by a local protease. Suitable non-native protease cleavage sites include those described above. Clostridial neurotoxins comprising destructive cleavage sites are described in WO 2010 / 094905 and WO 2002 / 044199 (both of which are incorporated herein by reference in their entireties).

[0166] The modified clostridial neurotoxins of the present invention, particularly their light chain components, may be PEGylated, which may help improve the stability of the light chain component, e.g., extend its duration of action. PEGylation is particularly preferred when the light chain comprises BoNT / A protease, BoNT / B protease, or BoNT / C1 protease. PEGylation preferably involves the addition of PEG to the N-terminus of the light chain component. For example, the N-terminus of the light chain may be extended with one or more amino acid (e.g., cysteine) residues, which may be the same or different. One or more of the amino acid residues may have their own PEG molecule attached thereto (e.g., covalently attached). An example of this technology is described in WO2007 / 104567, which is incorporated herein by reference in its entirety.

[0167] The dosage (eg, dose) of a Clostridial neurotoxin can be measured in nanograms.

[0168] The dose of clostridial neurotoxin according to the present invention should be understood as the dose of the active dichain clostridial neurotoxin, i.e., does not include the amount of the complexed protein with which the neurotoxin may be associated. In other words, this refers to the dose of the active dichain clostridial neurotoxin, regardless of whether the neurotoxin is administered in association with the complexed protein or without the complexed protein. As is well known to skilled practitioners, the active dichain clostridial neurotoxin can bind to a membrane (e.g., cell membrane) receptor, translocate the light chain to the cytoplasm, and cleave SNARE proteins, whereas the complexed protein does not exhibit such biological activity (i.e., is not "active").

[0169] Additionally or alternatively, the dose of the clostridial neurotoxin may be measured in "units" (U) of the clostridial neurotoxin. For example, when administering BoNT / A (or more particularly, for example, Dysport®), measuring the dose in units may be particularly appropriate.

[0170] Indeed, as is well known to skilled practitioners, the potency of clostridial neurotoxins is related to the amount (e.g., nanograms) of neurotoxin required to achieve an LD50 (lethal dose 50) unit, where 1 LD50 unit is defined as the median lethal intraperitoneal dose (measured in mice). However, currently commercially available BoNT pharmaceutical preparations contain various amounts of the 150 kD neurotoxin, which is also an LD50 unit. Furthermore, in these preparations, the neurotoxin may or may not be associated (i.e., combined) with a non-toxic neurotoxin-associated protein (NAP), also known as a complexing protein. For ease of conversion (as reported in Field et. al, "AbobotulinumtoxinA (Dysport®), OnabotulinumtoxinA (Botox®), and IncobotulinumtoxinA (Xeomin®) Neurotoxin Content and Potential Implications for Duration of Response in Patients". Toxins 2018, 10(12), 535): - 100 units of Botox® (also known as onabotulinumtoxinA) contains approximately 0.9 ng of the 150 kD BoNT / A as well as complexed proteins. 500 units of Dysport® (also known as abobotulinumtoxinA) contain approximately 2.69 ng of the 150 kD BoNT / A as well as complexed proteins, and 1 unit of Dysport® contains approximately 5.38 pg of BoNT / A. - 100 units of Xeomin® (also known as incobotulinumtoxinA) contains approximately 0.40 ng of the 150 kD BoNT / A, but does not contain complexing proteins.

[0171] Note that conversion values ​​may vary slightly. For example, the conversion values ​​reported in Frevert, 2012 ("Content of botulinum neurotoxin in Botox® / Vistabel®, Dysport® / Azzalure®, and Xeomin® / Bocouture®"; Drugs R D. 2010;10(2):67-73) are as follows: - 100 units of Botox® (also known as onabotulinumtoxinA) contains approximately 0.73 ng of the 150 kD BoNT / A as well as complexed proteins. - 100 units of Dysport® (also known as abobotulinum toxin A) contains approximately 0.65 ng of the 150 kD BoNT / A as well as complexed proteins. - 100 units of Xeomin® (also known as incobotulinumtoxinA) contains approximately 0.44 ng of the 150 kD BoNT / A, but does not contain complexed proteins. - 100 units of Neurobloc / Myobloc® (also known as rimabotulinumtoxinB) contains about 0.2 ng to about 1 ng of the 150 kD BoNT / B as well as complexed proteins.

[0172] The amount of clostridial neurotoxin can be measured by a skilled practitioner according to methods conventionally used in the art for quantifying proteins, preferably at the nanogram level, including, inter alia, mass spectrometry such as isotope dilution mass spectrometry (Munoz et al., Quantification of protein calibrants by amino acid analysis using isotope dilution mass spectrometry, Anal. Biochem. 2011, 408, 124-131), or fluorimetry (Poras et al., Detection and Quantification of Botulinum Neurotoxin Type A by a Novel Rapid In Vitro Fluorimetric Assay, Appl Environ Microbiol. 2009 Jul; 75(13): 4382-4390).

[0173] In one embodiment, the patient is administered at least 5000 units, at least 6000 units, at least 7000 units, at least 8000 units, or at least 9000 units, preferably at least 9500 units, more preferably at least 9900 units (e.g., at least 10,000 units) of Clostridial neurotoxin per 500 ml of solution, hi one embodiment, the patient is administered no more than 55,000 units, no more than 50,000 units, no more than 45,000 units, or no more than 40,000 units, preferably no more than 35,000 units, more preferably no more than 31,000 units (e.g., no more than 30,000 units) of Clostridial neurotoxin per 500 ml of solution. In one embodiment, the patient is administered 5000 Units to 35,000 Units, 6000 Units to 34,000 Units, 7000 Units to 33,000 Units, preferably 8000 Units to 32,000 Units, more preferably 9000 Units to 31,000 Units (e.g., preferably 10,000 Units to 30,000 Units) of Clostridial neurotoxin per 500 ml of solution.

[0174] In one embodiment, the patient is administered at least 16,500 units, at least 17,000 units, at least 17,500 units, at least 18,000 units, or at least 18,500 units, preferably at least 19,000 units, more preferably at least 19,500 units (e.g., at least 20,000 units) of Clostridial neurotoxin per liter of solution, hi one embodiment, the patient is administered no more than 63,000 units, no more than 62,500 units, no more than 62,000 units, or no more than 61,500 units, preferably no more than 61,000 units, more preferably no more than 60,500 units (e.g., no more than 60,000 units) of Clostridial neurotoxin per liter of solution. In one embodiment, the patient is administered 16,000 Units to 35,000 Units, 17,000 Units to 34,000 Units, 18,000 Units to 33,000 Units, preferably 19,000 Units to 32,000 Units, more preferably 19,500 Units to 60,500 Units (e.g., preferably 20,000 Units to 60,000 Units) of Clostridial neurotoxin per liter of solution.

[0175] In one embodiment, the patient is administered at least 51,000 pg, at least 51,500 pg, at least 52,000 pg, or at least 52,500 pg, preferably at least 53,000 pg, more preferably at least 53,500 pg (e.g., at least 53,800 pg) of Clostridial neurotoxin per 500 ml of solution. In one embodiment, the patient is administered no more than 164,000 pg, no more than 163,500 pg, no more than 163,000 pg, or no more than 162,500 pg, preferably no more than 162,000 pg, more preferably no more than 161,700 pg (e.g., no more than 161,400 pg) of Clostridial neurotoxin per 500 ml of solution. In one embodiment, a patient is administered 51,500 pg to 163,500 pg, 52,000 pg to 163,000 pg, or 52,500 pg to 162,500 pg, preferably 53,000 pg to 162,000 pg, more preferably 53,500 pg to 161,700 pg (e.g., 53,800 pg to 161,400 pg) of Clostridial neurotoxin per 500 ml of solution.

[0176] In one embodiment, the patient is administered at least 105,000 pg, at least 105,500 pg, at least 106,000 pg, or at least 106,500 pg, preferably at least 107,000 pg, more preferably at least 107,400 pg (e.g., at least 107,600 pg) of Clostridial neurotoxin per liter of solution. In one embodiment, the patient is administered no more than 325,500 pg, no more than 325,000 pg, no more than 324,500 pg, or no more than 324,000 pg, preferably no more than 323,500 pg, more preferably no more than 323,000 pg (e.g., no more than 322,800 pg) of Clostridial neurotoxin per liter of solution. In one embodiment, the patient is administered 105,000 pg to 325,500 pg, 105,500 pg to 325,000 pg, 106,000 pg to 324,500 pg, or 106,500 pg to 324,000 pg, preferably 107,000 pg to 323,500 pg, more preferably 107,400 pg to 323,000 pg (e.g., 107,600 pg to 322,800 pg) of Clostridial neurotoxin per liter of solution.

[0177] In one embodiment, the solution comprises a Clostridial neurotoxin, wherein the solution is physiologically inactive and / or does not contain a release agent, hi one embodiment, the solution comprises a Clostridial neurotoxin, wherein the solution is saline-based and / or does not contain a release agent.

[0178] 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.

[0179] In one aspect, the present invention provides a kit for use in a method of the present invention comprising: a) a solution consisting essentially of a clostridial neurotoxin, and b) A catheter that is inserted into the patient's bladder.

[0180] As used herein, the terms "subject," "individual," and "patient" may be used interchangeably to refer to a mammalian subject. In one embodiment, a "subject" is a human, a companion animal (e.g., a pet such as a dog, cat, and / or rabbit), a livestock animal (e.g., a pig, sheep, cow, and / or goat), and / or a horse. In a preferred embodiment, the subject (patient) is a human.

[0181] As used herein, the term "disorder" also encompasses "disease." In one embodiment, the disorder is a disease.

[0182] As used herein, the term "treat" or "treating" encompasses corrective treatment (treatment of a subject already suffering from a disorder) as well as preventative treatment (e.g., preventing the onset of a disorder). Preferably, "treat" or "treating" as used herein refers to corrective treatment. As used herein, the term "treat" or "treating" relates to a disorder and / or its symptoms.

[0183] Thus, the polypeptides of the present invention can be administered to a subject in a therapeutically effective amount or a prophylactically effective amount. Preferably, the Clostridial neurotoxin of the present invention is administered to a subject in a therapeutically effective amount.

[0184] A "therapeutically effective amount" is any amount of a Clostridial neurotoxin that, when administered alone or in combination to a subject for treating said disorder (or a symptom thereof), is sufficient to effect such treatment of the disorder or symptom thereof.

[0185] A "prophylactically effective amount" is any amount of a Clostridial neurotoxin that, when administered alone or in combination to a subject, prevents or delays the onset or recurrence of a disorder (or its symptoms). In some embodiments, a prophylactically effective amount completely prevents the onset or recurrence of a disorder. "Preventing" onset means reducing the likelihood of onset of a disorder (or its symptoms) or preventing onset altogether.

[0186] sequence homology

[0187] Any of a variety of sequence alignment methods can be used to determine percent identity, including, but not limited to, global methods, local methods, and hybrid methods such as, for example, segmental approaches. Protocols for determining percent identity are routine procedures within the skill of those in the art. Global methods align sequences from the beginning to the end of the molecule, sum up the scores of individual residue pairs, and apply gap penalties to determine the best alignment. Non-limiting methods include, for example, CLUSTAL W (see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994)) and iterative refinement (see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996)). Local methods align sequences by identifying one or more conserved motifs shared by all input sequences.Non-limiting methods include, for example, Match-box (see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992)), Gibbs sampling (see, e.g., C.E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993)), and Align-M (see, e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004)).

[0188] Thus, percent sequence identity is determined by conventional methods. See, e.g., Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.), as shown below (amino acids are indicated by their standard single-letter code):

[0189] The "percent 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. The calculation of percent sequence identity may also take into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. The comparison of sequences and determination of percent identity between two or more sequences can be performed using specific mathematical algorithms, such as BLAST, which are well known to those skilled in the art.

[0190] Alignment score to determine sequence identity

number

[0191] The percent identity is then calculated as follows: [total number of identical matches × 100] / [length of the longer sequence + number of gaps introduced in the longer sequence to align the two sequences]

[0192] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor in nature, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide, typically small deletions of from 1 to about 30 amino acids, and small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue, a small linker peptide of up to about 20 to 25 residues, or an affinity tag.

[0193] Conservative amino acid substitutions Basicity: Arginine lysine histidine Acidic: glutamic acid Aspartic acid polarity: glutamine Asparagine Hydrophobicity: Leucine Isoleucine Balin Aromatic: phenylalanine Tryptophan Tyrosine small: glycine Alanine Serine Threonine methionine

[0194] In addition to the 20 standard amino acids, amino acid residues in the polypeptides of the present invention may be substituted with non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine). Amino acid residues in the polypeptides may also be substituted with a limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids. The polypeptides of the present invention may also include non-naturally occurring amino acid residues.

[0195] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, 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 used to suppress nonsense mutations using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system containing Escherichia coli (E. coli) S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography (see, e.g., Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is performed in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996).In a third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired unnatural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The unnatural amino acid is incorporated into the polypeptide in place of its natural counterpart. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to unnatural species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the scope of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0196] Amino acid residues in the polypeptides of the invention may be replaced with a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids.

[0197] Essential amino acids in the polypeptides of the invention can be identified using procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structures determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations of putative contact site amino acids. See, e.g., de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of essential amino acids can also be inferred from analysis of homology with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0198] Large numbers of amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods in which two or more positions within a polypeptide are simultaneously randomized, functional polypeptides are selected, and the mutagenized polypeptides are then sequenced to determine the spectrum of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al. U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0199] Large numbers of amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods in which two or more positions within a polypeptide are simultaneously randomized, functional polypeptides are selected, and the mutagenized polypeptides are then sequenced to determine the spectrum of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al. U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0200] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0201] The present disclosure is not limited by the exemplary methods and materials disclosed herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges include the numbers defining the range. Unless otherwise indicated, nucleic acid sequences are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0202] The headings provided herein do not limit the various aspects or embodiments of the disclosure.

[0203] Amino acids are referred to herein using the amino acid name, three-letter abbreviation, or one-letter abbreviation. As used herein, the term "protein" includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein." In some 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 this disclosure and claims, conventional one-letter and three-letter abbreviations for amino acid residues may be used. The three-letter abbreviations for amino acids are defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

[0204] Other definitions of terms may be found throughout the specification. Before describing exemplary embodiments in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure is defined only by the appended claims.

[0205] Where a range of values ​​is given, it is understood that each intervening value between the upper and lower limits of that range is specifically disclosed, down to the tenth of the unit of the lower limit, unless the context clearly indicates otherwise. Each smaller range between any specified value or value within a specified range and any other specified value or value within that specified range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the disclosure, subject to any specifically excluded upper and lower limits in the specified range. Where a specified range includes one or both of the upper and lower limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0206] It must be noted that as used in this specification and 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 candidate agents, 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.

[0207] 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 are prior art to the claims appended hereto.

[0208] Embodiments of the present invention will now be described, by way of example only, with reference to the following figures and examples. [Brief explanation of the drawings]

[0209] [Figure 1] 1 shows a schematic diagram of a procedure involving intradetrusor injection. The procedure involves carefully maneuvering a device through an opening, along the length of the urethra, into the patient's bladder. Cytoscopic guidance then allows the needle to be sequentially maneuvered to multiple pre-determined injection sites located throughout the bladder wall lining. Each injection requires the needle to penetrate the bladder wall lining, resulting in the delivery of a metered dose of therapeutic agent at each injection site. [Figure 2] A schematic diagram of the gentle pressure filling technique is shown. A catheter is inserted into the patient's urethra and all urine present in the bladder is drained. A small volume (e.g., 50 ml) of medication is then slowly filled 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, preferably at least 90 minutes. This allows the medication to contact the entire bladder wall lining and potentially provide treatment. [Figure 3] Schematic diagram showing the administration of BoNT / A via intradetrusor injection. Intradetrusor injection of BoNT / A is highly invasive and simultaneously targets all three levels of innervation of the bladder wall: the urothelium, the afferent endings of the lamina propria, and the efferent endings of the detrusor muscle. [Figure 4] 1 is a schematic diagram illustrating the method of the present invention, which is less invasive and targets the afferent endings of the urothelium and lamina propria. [Figure 5]Figure 1 shows the study design to evaluate the effects of Dysport in a chronic rat model of interstitial cystitis / bladder pain syndrome (CYP-induced interstitial cystitis / bladder pain syndrome). [Figure 6] Figure 1 shows the effect of Dysport (10 U, 20 U, and 30 U per rat, intravesically) on CYP-induced chronic visceral pain (based on nociceptive scores). Nociceptive scores (%) at D10 (A) and D12 (B) in Dysport-treated and vehicle-treated rats. Results are expressed as mean ± sem. Between-group comparisons: two-way RM ANOVA with Sidac's post-hoc test for CYP / vehicle (###p<0.001). [Figure 7] The effects of test and reference substances on CYP-induced chronic allodynia (based on nociceptive threshold) are shown. Nociceptive threshold (g) in the Dysport, DMSO, Ialuril, and vehicle groups before ("D0") and after injection of saline or CYP, but before the start of pharmacological treatment ("D7") (A), and at D10 (B) and D12 (C). Results are expressed as mean ± sem. No statistical analysis was performed. [Figure 8] The effects of test and reference substances on CYP-induced chronic allodynia (based on AUC 1g-6g) are shown. AUC calculated from 1g-6g before ("D0") and after injection of saline or CYP, but before the start of pharmacological treatment ("D7") (A), and on D10 (B) and D12 (C) in the Dysport, DMSO, Ialuril, and vehicle groups. Results are expressed as mean ± sem. No statistical analysis was performed. [Figure 9] The effects of test and reference substances on CYP-induced chronic hyperalgesia (based on AUC 6g-26g) are shown. AUC calculated from 6g-26g before ("D0") and after injection of saline or CYP, but before the start of pharmacological treatment ("D7") (A), and on D10 (B) and D12 (C) in the Dysport, DMSO, Ialuril, and vehicle groups. Results are expressed as mean ± sem. No statistical analysis was performed. [Figure 10] Schematic diagram showing the setup for ex vivo bladder electrophysiology. The bladder was catheterized apically through the urethra. The pelvic nerve (PN) and hypogastric nerve (HGN) bundles were inserted into glass recording electrodes. The technical setup on the right visualizes how the input from the pressure transducers and electrodes is modified by the hardware and Spike2 software to yield the example augmented image at the top. Diagram created using BioRender. [Figure 11] Intravesical BoNT / A treatment significantly decreased bladder mechanosensitivity and increased the pressure-volume relationship. A) The afferent response to distension was significantly decreased 30, 60, and 90 minutes after treatment (p<0.0001, n=9, two-way ANOVA). B) After intravesical Dysport, the pressure-volume relationship was significantly increased compared to controls (p=0.0003, n=9, two-way ANOVA). [Figure 12] Effects of BoNT / B on bladder mechanosensitivity are shown. A) After BoNT / B treatment, the afferent response to distension was reduced in a time-dependent manner (p<0.0001, n=6, two-way ANOVA). B) After BoNT / B treatment, the pressure-volume relationship was significantly higher (p<0.0001, two-way ANOVA). [Figure 13] Effects of BoNT / E on bladder mechanosensitivity. A) After BoNT / E treatment, the afferent response to distension was significantly decreased in a time-dependent manner (p<0.0001, n=5, two-way ANOVA). B) After BoNT / E treatment, the pressure-volume relationship was significantly higher (p<0.0001, two-way ANOVA).

[0210] Sequence Listing

[0211] Where the first Met amino acid residue or the corresponding first codon is shown in any of the following SEQ ID NOs, said residue / codon is optional.

[0212] SEQ ID NO: 1 - nucleotide sequence, unmodified BoNT / A

[0213] SEQ ID NO:2 - Polypeptide sequence, unmodified BoNT / A

[0214] SEQ ID NO:3 - BoNT / B1, Accession number B1INP5, amino acid sequence

[0215] SEQ ID NO: 4 - BoNT / C1, Accession Number P18640, amino acid sequence

[0216] SEQ ID NO:5 - BoNT / D, Accession Number P19321, amino acid sequence

[0217] SEQ ID NO: 6 - BoNT / E1, Accession number WP_003372387, amino acid sequence

[0218] SEQ ID NO:7 - BoNT / F1, Accession No. Q57236, amino acid sequence

[0219] SEQ ID NO:8 - BoNT / F7, amino acid sequence

[0220] SEQ ID NO: 9 - BoNT / G, Accession number WP_039635782, amino acid sequence

[0221] SEQ ID NO: 10 - BoNT / DC, Accession number BAM65681, amino acid sequence

[0222] SEQ ID NO: 11 - BoNT / AB MY , amino acid sequence [ka]

[0223] SEQ ID NO: 12 - BoNT / X, amino acid sequence (GenBank: BAQ12790.1)

[0224] SEQ ID NO: 13 (nucleotide sequence, modified BoNT / A “Cat-A”)

[0225] SEQ ID NO: 14 (polypeptide sequence, modified BoNT / A "Cat-A")

[0226] SEQ ID NO: 15 (nucleotide sequence, modified BoNT / A “Cat-B”)

[0227] SEQ ID NO: 16 (polypeptide sequence, modified BoNT / A “Cat-B”)

[0228] SEQ ID NO: 17 (nucleotide sequence, modified BoNT / A "Cat-C")

[0229] SEQ ID NO: 18 (polypeptide sequence, modified BoNT / A "Cat-C")

[0230] SEQ ID NO: 19 (nucleotide sequence, modified BoNT / A “Cat-D”)

[0231] SEQ ID NO: 20 (polypeptide sequence, modified BoNT / A "Cat-D")

[0232] SEQ ID NO: 21 (polypeptide sequence, modified BoNT / A "chimera 1") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKSEILNNIILNLRYKDNNLIDLSGYGAKVE VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYI HNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVT ITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTEL SQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSK YNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYES GIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0233] SEQ ID NO: 22 (polypeptide sequence, modified BoNT / A "chimera 2") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIIELGGGGSELSEILNNIILNLRYKDNN LIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRI PKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIRED ISEYINRWFFVTITNNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFI WMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKK DSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNL NQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDE IGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHH HHHHHHH

[0234] SEQ ID NO: 23 (polypeptide sequence, modified BoNT / A "chimera 3A") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0235] SEQ ID NO: 24 (polypeptide sequence, modified BoNT / A "chimera 3B")

[0236] SEQ ID NO: 25 (polypeptide sequence, modified BoNT / A "chimera 3C") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYIH NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNLNNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFTELS QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0237] SEQ ID NO: 26 (TEV cleavage site) ENLYFQG

[0238] SEQ ID NO: 27 (thrombin cleavage site) LVPRGS

[0239] SEQ ID NO: 28 (PreScission cleavage site) LEVLFQGP

[0240] SEQ ID NO: 29 (enterokinase cleavage site) DDDDK

[0241] SEQ ID NO: 30 (Factor Xa cleavage site 1) IEGR

[0242] SEQ ID NO: 31 (Factor Xa cleavage site 2) IDGR

[0243] SEQ ID NO: 32 (influenza virus hemagglutinin) GLFGAIAGFIENGWEGMIDGWYG [Example]

[0244] Materials and Methods

[0245] Animal models

[0246] In the following study, rats treated with cyclophosphamide (CYP) were used to develop a chronic rat model of CYP-induced BPS / IC consisting of three injections (40 mg / kg, i.p.) every three days. For example, Auge C, et al. Characterization and Validation of a Chronic Model of Cyclophosphamide-Induced Interstitial Cystitis / Bladder Pain Syndrome in Rats. Front Pharmacol. 2020 Aug 28;11:1305. doi: 10.3389 / fphar.2020.01305. PMID: 32982733; PMCID: PMC7485435, and Zhang HP, et al. The function of P2X3 receptor and NK1 receptor antagonists on cyclophosphamide-induceditis in rats. World J Urol. 2014 Feb;32(1):91-7. doi: 10.1007 / s00345-013-1098-z. Epub 2013 May 12. PMID: 23666265 describes a CYP-induced BPS / IC model. No significant weight loss occurs. This model exhibits long-lasting visceral pain characterized by both allodynia (painful responses to normally innocuous stimuli) and hyperalgesia (increased responses to noxious stimuli).

[0247] Induction of chronic cystitis

[0248] To induce chronic cystitis, rats were weighed on days 0 (D0), 3, and 6 and given intraperitoneal (ip) injections of CYP at a dose of 40 mg / kg in a final volume of 5 mL / kg. CYP was freshly prepared in saline at a final concentration of 8 mg / mL.

[0249] Control rats were given saline under the same experimental conditions as CYP.

[0250] Von Frey assay

[0251] Visceral pain was assessed using the von Frey assay. For example, Garrido-Suarez, B. et al., (2015). Ovariectomy-induced chronic abdominal hypernociception in rats: Relation with brain oxidative stress. Journal of Pharmacy & Pharmacognosy Research. 3. 148–161, and Deuis JR. et al., Methods Used to Evaluate Pain Behaviors in Rodents. Front Mol Neurosci. 2017 Sep 6;10:284. doi: 10.3389 / fnmol.2017.00284. PMID: 28932184; PMCID: PMC5592204 describe the von Frey assay methodology. Standardized conditions, including single-experimenter testing of all animals, were applied to minimize variability in behavioral pain testing. Visceral pain was assessed blindly by applying a set of eight calibrated von Frey filaments of increasing force (1 g, 2 g, 4 g, 6 g, 8 g, 10 g, 15 g, and 26 g) to the lower abdomen close to the bladder with a 5-second interstimulus interval. Prior to testing, each animal was shaved in the abdominal area where mechanical stimulation was planned. Animals were placed on an elevated wire mesh floor beneath individual clear Plexiglas boxes and allowed to acclimate for at least 30 minutes before the von Frey test began. The filament was then applied for 1–2 seconds through the mesh floor with enough force to produce a slight bend in the filament. Each filament was tested three times. Care was taken to stimulate different areas within the lower abdominal region close to the bladder to avoid desensitization.

[0252] Nociceptive behavior was scored for each animal and each filament as shown in Table 1. [Table 1]

[0253] Presentation and analysis of results

[0254] visceral pain

[0255] The definitions of nociceptive parameters are shown in Table 2. [Table 2]

[0256] Example 1

[0257] Protocol Design

[0258] The following protocol (as shown in Figure 5) was performed to evaluate the effect of Dysport on a chronic rat model of CYP-induced BPS / IC. On D-1, rats were allowed to acclimate to individual Plexiglas boxes (von Frey setup) for a minimum of 30 min to reduce the level of stress posed by the novel environment, and von Frey filaments were applied. On D0, before the first injection of CYP or saline, the von Frey test was performed to obtain a baseline value for nociceptive behavior. Chronic cystitis was induced on D0, D3, and D6 to mimic BPS / IC. On D7 (before treatment began), the von Frey test was performed to assess the induction of chronic cystitis. On D8, pharmacological intravesical treatment was administered. On days D10 and D12, von Frey tests were performed to analyze the effects of the test substance (Dysport) and reference substances (DMSO and Ialuril) on CYP-induced chronic visceral pain.

[0259] material

[0260] Dysport Reconstitution

[0261] On each experimental day, a vial containing 500 U of Dysport was dissolved in 1 mL of sterile phosphate-buffered saline (PBS) to give a final concentration of 500 U / mL. The master solution was diluted 8.33-, 12.5-, or 25-fold in vehicle to give 30 U / 500 μL, 20 U / 500 μL, or 10 U / 500 μL, respectively. Dilutions were performed in siliconized glass tubes (batch number 8072554, Becton-Dickinson, Plymouth, UK).

[0262] Vehicle

[0263] The vehicle, sterile PBS (batch number 943548), was purchased from Eurobio Ingen (Les Ulis, France).

[0264] reference material

[0265] DMSO was prepared fresh on the day of administration at a final concentration of 50%.

[0266] The appropriate volume was dissolved in the vehicle at room temperature. DMSO was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France, batch number RNBH3467). Ready-to-use Ialuril (batch number 170501) was purchased from IBSA via Pharmaclic (Gosselies, Belgium).

[0267] treatment

[0268] Experimental group

[0269] Seven experimental groups were included as described in Table 3. [Table 3]

[0270] Pharmacological treatment

[0271] Before the experiment, animals were randomly assigned to treatment groups. Randomization was planned so that each group had at least one animal on each experimental day, with animals from the same group assigned to different positions in the von Frey chamber. A polyethylene catheter (inner and outer diameters of 0.76 mm and 1.22 mm, respectively) was inserted into the bladder via the urethra to gently fill the bladder with solution. Test substances (Dysport), reference substances (DMSO and Ialuril), and vehicle (500 μL per rat) were administered intravesically via syringes connected to the catheter.

[0272] The procedure was allowed to proceed for 30 minutes. Rats were kept under isoflurane anesthesia (2%-2.5%) throughout the procedure. After filling the bladder, the bladder was emptied by gentle massage of the lower abdomen.

[0273] result

[0274] Nociception score

[0275] Dysport treatment significantly reduced nociceptive scores at D10 and D12 at all doses tested (10 U, 20 U, and 30 U) compared to vehicle-treated CYP-injected rats (see Figure 6).

[0276] Nociceptive threshold

[0277] After CYP injection, the nociceptive threshold of CYP-injected rats was decreased on D7 compared with D0 (see Figure 7A). This indicates that chronic cystitis was induced, lowering the rats' pain perception threshold. On D10 and D12, the nociceptive threshold increased in CYP-injected rats after Dysport treatment compared with CYP-injected rats treated with vehicle alone (see Figures 7B and 7C). On D10, Dysport achieved statistical significance at a dose of 20 U per rat (see Figure 7B), while all tested doses reached significance on D12 (see Figure 7C). CYP-injected rats treated with Dysport showed an increased nociceptive threshold compared with CYP-injected rats treated with DMSO or ialuril. This indicates that Dysport can effectively alleviate pain in CYP-treated animals.

[0278] Chronic allodynia (AUC 1g~6g)

[0279] After CYP injection, an increase in AUC 1g-6g was observed in CYP-injected rats on D7 compared to saline-treated rats (see Figure 8A) and D0. After Dysport treatment, AUC 1g-6g decreased for all doses tested on D10 (see Figure 8B) and D12 (see Figure 8C) compared to saline-treated CYP-injected rats. CYP-injected rats treated with Dysport showed a greater decrease in AUC 1g-6g compared to CYP-injected rats treated with DMSO or ialuril. These results suggest that administration of Dysport can reduce allodynia-related pain perception in CYP-treated animals.

[0280] Chronic hyperalgesia (AUC 6g~26g)

[0281] After CYP injection, an increase in AUC 6g-26g was observed on D7 compared to saline-treated rats (see Figure 9A). After Dysport treatment, AUC 6g-26g decreased on D10 (see Figure 9B) and D12 (see Figure 9C) for all doses tested compared to saline-treated CYP-injected rats. CYP-injected rats treated with Dysport showed a greater decrease in AUC 6g-26g compared to CYP-injected rats treated with DMSO or ialuril. These results suggest that Dysport can reduce pain perception associated with hyperalgesia in CYP-treated animals.

[0282] Example 2

[0283] A physician evaluates the condition of a patient suffering from BPS, particularly IC, and then administers a solution containing a Clostridial neurotoxin as a treatment. Specifically, the physician evaluates the deterioration of the patient's bladder urothelium and determines the dosage of the Clostridial neurotoxin solution. The physician's evaluation confirms that the patient's urothelium has been severely damaged.

[0284] The volume (in ml) of solution containing the Clostridial neurotoxin to administer is then determined by determining the patient's threshold volume of fluid to trigger urination, either by cystometry, uroflowmetry, or a voiding diary, which is determined by cystometry to be 250 ml.

[0285] Because the patient's urothelium is severely damaged, the physician prepares a solution containing Clostridial neurotoxin at a dose of 53,800 pg per 500 ml. The physician prepares this solution in a total volume of 225 ml. The physician places a catheter into the patient's bladder and injects the solution into the bladder. The solution is then retained in the bladder for one hour. The patient is then asked to urinate, after which the bladder is rinsed with saline to remove any remaining Clostridial neurotoxin.

[0286] Example 3

[0287] A physician evaluates the condition of a patient suffering from BPS, particularly IC, and then administers a solution containing a Clostridial neurotoxin as a treatment. Specifically, the physician assesses the deterioration of the patient's bladder urothelium and determines the dosage of the Clostridial neurotoxin solution. The physician's evaluation reveals that the patient's urothelium is mildly damaged, but some areas of the tissue remain intact.

[0288] The volume (in ml) of solution containing the Clostridial neurotoxin to administer is then determined by determining the patient's threshold volume of fluid to trigger urination, either by cystometry, uroflowmetry, or a voiding diary, which is determined to be 275 ml by a voiding diary.

[0289] Because the patient's urothelium is mildly damaged, the physician prepares a solution containing Clostridial neurotoxin at a dose of 161,400 pg per 500 ml. The physician prepares this solution in a total volume of 250 ml. The physician places a catheter into the patient's bladder, connects the catheter, and injects the solution into the bladder, which is then held in the bladder for one hour. The patient is then asked to urinate, and the bladder is rinsed with saline to remove any remaining Clostridial neurotoxin.

[0290] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

[0291] Example 4

[0292] Suppression of bladder mechanosensitivity by BoNT / A in an ex vivo mouse model

[0293] These studies were carried out using adult C57BL / 6J mice (Charles River Laboratories, Margate, Kent) aged 8-12 weeks. On the day of the experiment, mice were handed over and sacrificed using elevated CO2 concentrations in accordance with Schedule 1 of the Animals (Scientific Procedures) Act 1986. Experiments were carried out in accordance with ethical approval (reference RE / 16 / 11) obtained from the UCLan Animal Welfare and Ethics Review Board (AWERB).

[0294] Ex vivo bladder electrophysiology

[0295] After CO2 asphyxiation, the animals' two hind limbs and tails were removed, along with the fur from their backs. An incision was made in the abdomen to remove the intestines, and the spinal cord was severed at the L2 level above the kidneys. The entire pelvic region of the mouse was placed in an organ bath continuously perfused with carbonate-containing (95% O2 / 5% CO2) Krebs buffer (composition, in mM: NaCl 118.4, NaHCO3 24.9, CaCl2 1.9, MgSO4 1.2, KH2PO4 1.2, glucose 11.7, all from Sigma-Aldrich) maintained at approximately 35°C to prevent tissue degradation.

[0296] Once placed in the organ bath, the tissue was further dissected under a microscope. To prevent possible reflux during distention, the ureters were tied with silk sutures (Fisher-Scientific). The pubic symphysis was transected on both the left and right sides and removed to expose the underlying urethra. The urethra was transected, and a catheter attached to a syringe pump (New Era Pump Systems, NE-1000) was inserted and tied with sutures to prevent leakage. The syringe contained phosphate-buffered saline (PBS, Gibco), and the bladder was filled to a specific point where a needle (BD microlance) could be inserted to penetrate the bladder dome without damaging the sensory nerves in the trigone. A double-lumen catheter was then inserted into the dome and tied with sutures. One catheter was attached to a pressure transducer (NL 108T2 Digitimer) to monitor intravesical pressure, and the other catheter was attached to a tap (valve) to allow for bladder filling and emptying. A nerve bundle containing the pelvic and hypogastric nerves was inserted into the glass electrode to facilitate capture of afferent nerve responses to bladder stimulation.

[0297] Once the catheter was inserted, the bladder was inflated to ensure a closed system, as failure to reach this pressure would indicate leakage. The pelvic and hypogastric nerves emerging from the bladder base were cut into long nerve bundles and inserted into a glass suction electrode (VWR) attached to a Neurolog headstage (NL100AK). The headstage was connected to an AC preamplifier (NL104) to amplify the signal (10,000x), filter it with a passband filter (NL125), and remove 50-60 Hz electrical noise using a Humbug (Quest Scientific). The signal was then passed through a 1401 Data Acquisition Interface (Cambridge Electronic Design) and recorded on a computer via Spike2 software (v10.08, Cambridge Electronic Design). Multiunit afferent activity was quantified using a Spike processor (D130, Digitimer) that counted the number of field potentials passing a threshold set at twice the baseline noise level at the beginning of the experiment. The preparation setup is shown in Figure 10.

[0298] Experimental protocol

[0299] In ex vivo bladder electrophysiological recordings, the bladder was stimulated by mechanical (distention) means and neural responses were obtained to characterize the effects of stimulation parameters on afferent nerve activity.

[0300] statistics

[0301] All data for ex vivo bladder electrophysiology assays, including multiunit neuronal activity and intravesical pressure, were acquired using Spike2 software (v10.08). Responses at 30, 60, and 90 minutes were normalized to the third reproducible control distention at the beginning of the experiment. Area under the curve was calculated from this normalized data. N refers to the number of animals, and all data are expressed as mean + / - SEM. Statistical tests performed included t-tests, one-way ANOVA, and two-way ANOVA using GraphPad Prism v8.0.1. Responses from all runs were retained in the final analysis, and data were included assuming the experiment was performed without problems such as equipment failure or tissue packing failure. Specific methods for data extraction and analysis are described in detail below.

[0302] Expansion

[0303] The bladder was slowly distended by closing the tap, turning on the syringe pump, and filling at a rate of 150 μL / min. The pressure immediately decreased when the tap was opened to empty the contents. Once the bladder was filled, activation of nerve fibers sensitive to mechanical stretch of the bladder wall was acquired and visualized as field potentials in Spike2. Distension continued until three reproducible responses were achieved simultaneously. Once this was achieved, the experiment began. All ramp distensions were performed 10 min apart, and each set was allowed to stabilize for at least 30 min before recording began.

[0304] Bladder compliance

[0305] Compliance is a measure of the pressure-volume relationship during bladder filling, ie, the ability of the bladder wall to accommodate increasing volume. Volume (μL) = speed (μl / min-1) x time This equation was used to calculate the bladder pressure-volume relationship using the fill rate programmed into the intravesical pump, and the change in bladder compliance was plotted as a percentage change compared to control distention at the start of the experiment.

[0306] Intracavitary application of BoNT / A

[0307] The ex vivo bladder electrophysiology assay showed reproducibility over 120 min, as the response profile of distensions performed 90 min into the experiment was similar to that of control distensions at the start of the experiment.

[0308] A syringe filled with BoNT / A-containing solution was connected to a syringe pump, and the bladder was distended three times to ensure uptake of BoNT / A across the urothelium. The syringe was then replaced with one containing PBS, and distension was continued for 90 minutes to assess the effects of BoNT on bladder physiology. For safety reasons, any BoNT / A present in the intraluminal fluid was inactivated using Presept (Advanced Sterilization Products) as it exited the dome catheter.

[0309] Preliminary experiments revealed that the BoNT / A concentration that produced a robust and reproducible response was 100 U / ml. The total toxin dose was calculated to be 3.6 pM, which is the chosen amount of toxin used for serotype comparison purposes.

[0310] result

[0311] Intravesical application of 100 U / ml Dysport resulted in a significant decrease in distension-evoked afferent firing over the 90-minute protocol (Figure 11, n=9, p<0.0001). After BoNT / A treatment, bladder compliance improved (Figure 11, n=9, p=0.0003).

[0312] Example 5

[0313] Suppression of bladder mechanosensitivity by BoNT / B in an ex vivo mouse model

[0314] Distension was performed until distension-evoked neural responses were reproducible, following the same protocol as described in Example 4. After three reproducible distensions with PBS, BoNT / B was applied intravesically using a syringe pump, and the bladder was distended three times at a rate of 150 μL / min, followed by continued distension with PBS every 10 min for nine distensions.

[0315] As shown in Figure 12, BoNT / B reduced afferent firing in response to distension over time, which was found to be significant in the analysis (p<0.0001, n=6). BoNT / B appeared to significantly increase the bladder pressure-volume relationship, interpreted as a measure of bladder compliance (p<0.0001). BoNT / B was found to significantly inhibit distension-evoked responses, as 52.3% (+ / -19.8%) of afferent firing remained 90 minutes after intravesical BoNT / B treatment.

[0316] Compared with the inhibitory effect induced by BoNT / A, BoNT / B appeared to attenuate distension-evoked afferent firing to a greater extent.

[0317] Example 6

[0318] Suppression of bladder mechanosensitivity by BoNT / E in an ex vivo mouse model

[0319] The effect of BoNT / E on bladder mechanosensitivity was investigated according to the same protocol as described in Example 4. As shown in Figure 13, distension-evoked afferent firing was significantly reduced by BoNT / E (p<0.0001, n=5). After BoNT / E treatment, bladder compliance was significantly improved.

[0320] Compared to the effect of BoNT / A on bladder mechanosensitivity, BoNT / E showed even more potent inhibition than that observed with BoNT / B. [Explanation of symbols]

[0321] [Figure 2] BLADDER: Bladder URETHRA: urethra INTERNAL URETHRAL SPHINCTER: internal urethral sphincter CATHERTER: Catheter MEDICATION: [Figure 3] Intradetrusor injections: Intratrusor injections Urothelium: urothelium UROTHELIAL CELLS: urothelial cells ATP release: ATP release BoNT / A injection: BoNT / A injection Lamina propria: lamina propria ACh and ATP co-release: ACh and ATP co-release Neuropeptide release: AFFERENT TERMINALS: afferent terminals Detrusor: Detrusor muscle EFFERENT TERMINALS: Efferent terminals Conventional intradetrusor injections result in SNAP25 cleavage / activity at the three levels of innervation (urothelium + afferents +efferents) (modified from Tyagi, 2019): Conventional intradetrusor injections result in SNAP25 cleavage / activity at the three levels of innervation (urothelium + afferents + efferents) (modified from Tyagi, 2019) FEAR OF PAIN, BLEEDING: [Figure 4] Bladder filling: Bladder filling BoNT / A 'filling': BoNT / A "filling" Urothelium: urothelium Made permeable by the pathology itself: Lamina propria: lamina propria AFFERENT TERMINALS: afferent terminals Detrusor: Detrusor muscle BoNT / A instillation would focus SNAP25 cleavage / activity at a lamina propria level (ie afferents) by combining: ·Pressure induced by the volume of the solution ·Acessibility of the aff vs eff fibers: BoNT / A infusion will focus SNAP25 cleavage / activity to the lamina propria level (i.e., afferent) by combining: Pressure induced by the volume of the solution Accessibility of afferent fibers compared to efferent fibers + NEEDLE-FREE DOSING: + Needle-free medication [Figure 5] Intravesical Dysport or Reference: Intravesical Dysport or Reference Acclimatization: Acclimatization Von Frey testing (basal): Body weight recording (D0, D3, D6, D8, D10 and D12): Von Frey testing (post-CYP): D7, D10 and D12 Bladder collection (processing for histology): Bladder collection (processing for histology) 70 rats, n=10 / groups: 70 rats, n=10 per group Negative control (saline + saline): Negative control (saline + saline) Positive control (saline + CYP): Positive control (saline + CYP) DMSO (intravesic.): DMSO (intravesic.) Ialuril (intravesic.): Ialuril (intravesic.) Dysport (registered trademark) 10, 20, 30U / rat (intravesical): 10U, 20U, 30U of Dysport (registered trademark) (intravesical) per rat [Figure 6] (Post CYP): (After CYP) CYP / Vehicle: CYP / Vehicle CYP / Dysport 10 U: Dysport of CYP / 10U CYP / Dysport 20 U: Dysport of CYP / 20U CYP / Dysport 30 U: Dysport of CYP / 30U Nociceptive scores (%): Nociceptive scores (%) von Frey forces (g): von Frey forces (g) Vehicle: PBS, 500 μL / rat, i.ves., n=10, once at D8: Vehicle: PBS, 500 μL / rat, i.ves., n=10, once at D8 Dysport: 10, 20 and 30 U / rat, i.ves., n=9-10, once at D8: Dysport: 10U, 20U and 30U per rat, i.ves., n=9-10, once at D8 [Figure 7] Before i.ves. treatment: Before i.ves. treatment Nociceptive threshold (g): Nociceptive threshold (g) D0 (Basal): D0 (Basal) D7 (Post Saline or CYP): D7 (Post Saline or CYP) Saline / Vehicle: Physiological saline / vehicle CYP / Vehicle: CYP / Vehicle CYP / Dysport 10 U: Dysport of CYP / 10U CYP / Dysport 20 U: Dysport of CYP / 20U CYP / Dysport 30 U: Dysport of CYP / 30U CYP / DMSO: CYP / DMSO CYP / Ialuril: CYP / Ialuril Post i.ves, treatment: After intravesical treatment Vehicle: PBS, 500 μL / rat, i.ves., n=10, once at D8: Vehicle: PBS, 500 μL / rat, i.ves., n=10, once at D8 Dysport: 10, 20 and 30 U / rat, i.ves., n=9-10, once at D8: Dysport: 10U, 20U and 30U per rat, i.ves., n=9-10, once at D8 DMSO: 50%, i.ves., n=9, once at D8: DMSO: 50%, intravesical, n=9, once at D8 Ialuril: 100%, i.ves., n=10, once at D8: Ialuril: 100%, intravesical, n=10, once at D8 [Figure 8] Before i.ves. treatment: Before i.ves. treatment AUC 1-6 g (%scores x g): AUC 1 g~6 g (%scores x g) D0 (Basal): D0 (Basal) D7 (Post Saline or CYP): D7 (Post Saline or CYP) Saline / Vehicle: Physiological saline / vehicle CYP / Vehicle: CYP / Vehicle CYP / Dysport 10 U: Dysport of CYP / 10U CYP / Dysport 20 U: Dysport of CYP / 20U CYP / Dysport 30 U: Dysport of CYP / 30U CYP / DMSO: CYP / DMSO CYP / Ialuril: CYP / Ialuril Post i.ves, treatment: After intravesical treatment Vehicle: PBS, 500 μL / rat, i.ves., n=10, once at D8: Vehicle: PBS, 500 μL / rat, i.ves., n=10, once at D8 Dysport: 10, 20 and 30 U / rat, i.ves., n=9-10, once at D8: Dysport: 10U, 20U and 30U per rat, i.ves., n=9-10, once at D8 DMSO: 50%, i.ves., n=9, once at D8: DMSO: 50%, intravesical, n=9, once at D8 Ialuril: 100%, i.ves., n=10, once at D8: Ialuril: 100%, intravesical, n=10, once at D8 [Figure 9] Before i.ves. treatment: Before i.ves. treatment AUC 6-26 g (%scores x g): AUC 6 g~26 g (%scores x g) D0 (Basal): D0 (Basal) D7 (Post Saline or CYP): D7 (Post Saline or CYP) Saline / Vehicle: Physiological saline / vehicle CYP / Vehicle: CYP / Vehicle CYP / Dysport 10 U: Dysport of CYP / 10U CYP / Dysport 20 U: Dysport of CYP / 20U CYP / Dysport 30 U: Dysport of CYP / 30U CYP / DMSO: CYP / DMSO CYP / Ialuril: CYP / Ialuril Post i.ves, treatment: After intravesical treatment Vehicle: PBS, 500 μL / rat, i.ves., n=10, once at D8: Vehicle: PBS, 500 μL / rat, i.ves., n=10, once at D8 Dysport: 10, 20 and 30 U / rat, i.ves., n=9-10, once at D8: Dysport: 10U, 20U and 30U per rat, i.ves., n=9-10, once at D8 DMSO: 50%, i.ves., n=9, once at D8: DMSO: 50%, intravesical, n=9, once at D8 Ialuril: 100%, i.ves., n=10, once at D8: Ialuril: 100%, intravesical, n=10, once at D8 [Figure 10] Pressure transducer: TAP: Tap Outflow: Outflow Dome catheter: Dome catheter Bladder: Bladder PN & HGN bundle: PN and HGN bundle Urethral catheter: urethral catheter Recording electrode: Pump: Filter: Filter Amplifier: Headstage: Headstage [Figure 11] control: Control 30 minutes: 30 minutes 60 minutes: 60 minutes 90 minutes: 90 minutes Normalized afferent discharge (%): Normalized afferent discharge (%) Intravesical pressure (mmHg): Intravesical pressure (mmHg) Normalized Δ volume (%): Normalized volume difference (%) [Figure 12] control: Control 30 minutes: 30 minutes 60 minutes: 60 minutes 90 minutes: 90 minutes Normalized afferent discharge (%): Normalized afferent discharge (%) Intravesical pressure (mmHg): Intravesical pressure (mmHg) Normalized Δ volume (%): Normalized volume difference (%) [Figure 13] control: Control 30 minutes: 30 minutes 60 minutes: 60 minutes 90 minutes: 90 minutes Normalized afferent discharge (%): Normalized afferent discharge (%) Intravesical pressure (mmHg): Intravesical pressure (mmHg) Normalized Δ volume (%): Normalized volume difference (%)

Claims

1. 1. A method of treating a patient suffering from bladder pain syndrome, comprising: administering a solution containing a Clostridial neurotoxin to the bladder of said patient; increasing the volume of the solution containing the Clostridial neurotoxin present in the bladder, thereby exerting a mechanical force against the inner surface of the urothelial layer; and Maintaining the volume of solution containing the Clostridial neurotoxin present in the bladder at a volume that does not cause the patient to urinate for a period of at least 30 minutes, thereby allowing the Clostridial neurotoxin to diffuse across the urothelial layer into the lamina propria, where it binds to primary sensory afferent nerve fibers, inhibiting neurotransmitter secretion therefrom and relieving bladder pain.

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

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

4. 10. The method of claim 1, wherein a solution containing the Clostridial neurotoxin is systemically dispersed throughout the lamina propria of the bladder wall, wherein the Clostridial neurotoxin inhibits neurotransmitter release from substantially all parasympathetic afferent neurons present therein.

5. 10. The method of any one of the preceding claims, wherein the solution is administered into the bladder via a catheter.

6. 10. The method of any one of the preceding claims, wherein the method is substantially non-invasive, wherein preferably the method does not cause substantial physical damage to the urothelial layer and / or the lamina propria.

7. 10. The method of any one of the preceding claims, wherein the solution is administered over a period of at least 1 hour.

8. 10. The method of 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 and does not stimulate muscarinic receptors in the detrusor muscle to contract).

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

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

11. The Clostridial neurotoxin contains the light chain and translocation domain of BoNT / A and the receptor binding domain (H) of BoNT / B. C 4. The method of claim 1, further comprising:

12. The clostridial neurotoxins include ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243 ... 1274, and THR 1277, wherein the modification is selected from the following: i. Substitution of surface-exposed acidic amino acid residues with basic amino acid residues; ii. Substitution of surface-exposed acidic amino acid residues with uncharged amino acid residues; iii. Substitution of surface-exposed uncharged amino acid residues with basic amino acid residues; iv. insertion of a basic amino acid residue, and v. Deletion of surface-exposed acidic amino acid residues.

13. 10. The method of any one of the preceding claims, wherein bladder pain is reduced for at least two days following administration of the solution comprising the Clostridial neurotoxin.

14. 10. The method of any one of the preceding claims, wherein bladder pain is reduced for four days following administration of the solution comprising the Clostridial neurotoxin.

15. 10. The method of any one of the preceding claims, wherein the bladder pain comprises allodynia or hyperalgesia.

16. 10. The method of any one of the preceding claims, wherein the patient's nociceptive 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, and b) a catheter for insertion into the patient's bladder.

18. The method according to any one of claims 1 to 16 or the kit for use according to claim 17, wherein the solution is: a. saline-based; and / or b. Contains no release agent.

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