Treatment methods for functional constipation in children

Abstract

A method for the treatment of pediatric functional constipation in a subject is disclosed that includes injecting a therapeutically effective amount of a botulinum toxin composition into an anal sphincter of the patient. The botulinum toxin composition may include a botulinum neurotoxin serotype A. The botulinum neurotoxin serotype A may be daxibotulinumtoxinA. The botulinum toxin composition may include daxibotulinumtoxinA-lanm (DAXXIFY®). The daxibotulinumtoxinA-lanm (DAXXIFY®) may be injected at a dosage of about 12 units per kilogram of body mass of the patient. The daxibotulinumtoxinA-lanm (DAXXIFY®) may be injected at a maximum dosage of about 200 units. The daxibotulinumtoxinA-lanm (DAXXIFY®) may include a duration of action of about six months.

Description

[0001] TITLE OF THE INVENTION

[0002] TREATMENT METHODS FOR FUNCTIONAL CONSTIPATION IN CHILDREN

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority from U. S. Provisional Application Serial No. 63 / 748,302 filed on January 22, 2025, which is incorporated herein by reference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] Not applicable.

[0007] MATERIAL INCORPORATED-BY-REFERENCE

[0008] Not applicable.

[0009] FIELD OF THE INVENTION

[0010] The present disclosure generally relates to methods of treatment for functional constipation in children.

[0011] BACKGROUND OF THE INVENTION

[0012] Disordered defecation (DD) is defined as constipation with inability to effectively evacuate stool from the rectum, resulting from a wide array of etiologies including functional disorders, anatomic abnormalities, and pelvic floor dyssynergia. For pediatric functional constipation (FC), diagnostic guidelines are provided in the Rome IV criteria. Worldwide, the prevalence of pediatric constipation is between 3-9.5%, constituting one-fourth of all outpatient gastroenterology visits. The resultant impact on healthcare utilization and associated cost burden is immense, with annual medical costs for children with DD being more than three times that of counterparts without constipation. Despite the availability of diagnostic criteria, delay in treatment is common and is negatively associated with symptom resolution. DD has also been shown to negatively influence psychosocial wellbeing and quality of life (QoL) for patients and caregivers. Data also implicate psychosocial stressors as a predisposition toward FC. Disordered defecation may also play a detrimental role in participation in activities of daily life as well as educational and extra-curricular performance.

[0013] Management of constipation typically relies on a multi-dimensional approach irrespective of the underlying etiology, including non- pharmacologic, pharmacologic, and procedural therapies. Among the procedural therapies, internal anal sphincter (IAS) BOTOX®, which mediates muscle relaxation via inhibition of pre-synaptic acetylcholine release, has been increasingly utilized as an adjunct to standard pharmacologic / non-pharmacologic management in pediatric constipation. For the past four decades, IAS BOTOX® has been used in pediatrics for a variety of indications from anismus to outlet dysfunction, such as those with pelvic floor dyssynergia or Hirschsprung disease. Moreover, regardless of IAS metrics as determined by anorectal manometry, IAS BOTOX® has been shown to generate analogous efficacy in children with constipation. Furthermore, safety of IAS BOTOX® has been established In several studies, with infrequent, minor side effects that were all transient.

[0014] More recently, a prospective cohort study evaluated the effect of IAS BOTOX® (onabotulinumtoxinA [BOTOX®; Allergan, North Chicago, IL, USA]) as an adjunct to standard management for pediatric constipation on patient / caregiver quality of life metrics in conjunction with corresponding clinical outcomes. At three months after IAS BOTOX®, there was a significant reduction in odds of meeting functional constipation criteria and a significant increase in quality of life metrics for both the patient and their caregivers. Furthermore, side effects, which were reported in 11% and included scant bleeding, increase in fecal incontinence, and anal discomfort, were short-lived when present. The global impact on outcomes for pediatric constipation suggests the need to appropriately integrate IAS BOTOX® into the typical management algorithm. Despite these positive findings, most patients noted a gradual loss of efficacy starting at a median of 70 days (IQR 39-83) after IAS BOTOX®, and such, the suggestion in current clinical practice is to repeat injection every three months. Based on clinical experience, the peak efficacy of IAS BOTOX® is thought to be between 6-8 weeks postinjection.SUMMARY OF THE INVENTION

[0015] In one aspect, a method for the treatment of pediatric functional constipation in a subject is disclosed that includes injecting a therapeutically effective amount of a botulinum toxin composition into an anal sphincter of the patient. In some aspects, the botulinum toxin composition includes a botulinum neurotoxin serotype A. In some aspects, the botulinum neurotoxin serotype A includes daxibotulinumtoxinA. In some aspects, the botulinum toxin composition includes daxibotulinumtoxinA-lanm (DAXXIFY®). In some aspects, the daxibotulinumtoxinA-lanm (DAXXIFY®) is injected at a dosage of about 12 units per kilogram of body mass of the patient. In some aspects, the daxibotulinumtoxinA-lanm (DAXXIFY®) is injected at a maximum dosage of about 200 units. In some aspects, the daxibotulinumtoxinA-lanm (DAXXIFY®) includes a duration of action of about six months.

[0016] DESCRIPTION OF THE DRAWINGS

[0017] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes oniy. The drawings are not intended to limit the scope of the present teachings in any way.

[0018] FIG. 1 is a flow chart summarizing a study population and survey administration time points used in a study described in the examples below. All surveys were completed by the same caregiver on behalf of their child FIG. 2 is a flow chart summarizing Rome IV outcomes from the study summarized in FIG. 1. Cl, confidence interval; FC, functional constipation; RR, relative risk; OR, odds ratio.

[0019] FIG. 3 is a graph summarizing PedsQL survey outcomes from the study summarized in FIG. 1. * Denotes statistically significant improvement in comparison to baseline.

[0020] FIG. 4 is a graph summarizing PedsQL-FIM survey outcomes from the study summarized in FIG. 1. * Denotes statistically significant improvement in comparison to baseline.

[0021] FIG. 5 is a bar graph summarizing PedsQL-FIM survey outcomes from the study summarized in FIG. 1

[0022] FIG. 6 is a flow chart summarizing Rome IV outcomes from the study summarized in FIG. 1, including separate scores for subjects experiencing <2stools per week.

[0023] FIG. 7 is a flow chart comparing the first-time BOTOX® recipient and the prior BOTOX® recipient sub-populations of the cohort summarized in FIG.

[0024] 1.

[0025] FIG. 8 is a flow chart comparing the Rome IV survey outcomes for first-time BOTOX® recipient and the prior BOTOX® recipient sub-populations of the cohort summarized in FIG. 1.

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] The present disclosure is based, at least in part, on the discovery that IAS DAXXIFY® treatments have a positive effects on patient and caregiver QoL metrics in association with corresponding clinical outcomes for pediatric patients with DD. The administration of IAS DAXXIFY®, as an adjunct to standard management, would improve QoL and caregiver well-being / family functioning in conjunction with a reduction in stooling difficulties for pediatric patients with DD.

[0028] In various aspects, the IAS DAXXIFY® treatment comprises DAXXIFY® injections completed under general anesthesia with guidance of either a pediatric gastroenterologist (AT, SJ, ECU) or pediatric surgeon (BAS). Regardless of provider, 12 units per kilogram dosing (maximum dose: 200 units) of DAXXIFY® is used and equally divided into four separate syringes by the hospital pharmacy. Prior to injection a speculum is carefully introduced into the anal canal to improve visualization of the dentate line.

[0029] Each syringe was injected using a 25-gauge needle into one of four IAS quadrants, identified within the proximal aspect ofthe dentate line and confirmed by palpation of the IAS muscle complex.

[0030] In various aspects, IAS DAXXIFY® is not utilized as a monotherapy for DD but rather as part of a multimodal approach in conjunction with non-pharmacologic (i.e., counseling with behavioral modifications, pelvic floor physical therapy) and pharmacologic therapies.

[0031] In some aspects, the maximum dose may range from 12 units to 200 units. In various other aspects, the maximum dose may range from 12 units to 20 units, from 15 units to 25 units, from 20 units to 30 units, from 25 units to 35 units, from 30 units to 40 units, from 35 units to 45 units, from 40 units to 50 units, from 45 units to 55 units, from 50 units to 60 units, from 55 unitsto 65 units, from 60 units to 70 units, from 65 units to 75 units, from 70 units to 80 units, from 75 units to 85 units, from 80 units to 90 units, from 85 units to 95 units, from 90 units to 100 units, from 95 units to 105 units, from 100 units to 110 units, from 105 units to 115 units, from 110 units to 120 units, from 115 units to 125 units, from 120 units to 130 units, from 125 units to 135 units, from 130 units to 140 units, from 135 units to 145 units, from 140 units to 150 units, from 145 units to 155 units, from 150 units to 160 units, from 155 units to 165 units, from 160 units to 170 units, from 165 units to 175 units, from 170 units to 180 units, from 175 units to 185 units, from 180 units to 190 units, from 185 units to 195 units, and from 190 units to 200 units.

[0032] in various aspects, the syringe may be injected using any suitable needle gauge including, but not limited to, a 25 gauge needle.

[0033] FORMULATION

[0034] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0035] The term ’’formulation” refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation” can inciude pharmaceutically acceptable excipients, including diluents or carriers.

[0036] The term "pharmaceutically acceptable” as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeia! Convention, Inc, Rockville, Maryland, 2005 (" USP / NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of theFDA. Other useful components that are not described in the USP / NF, etc. may also be used.

[0037] The term “pharmaceutically acceptable excipient," as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in therapeutic compositions is contemplated.

[0038] Supplementary active ingredients can also be incorporated into the compositions.

[0039] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 °C and about 60 °C, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0040] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0041] Controlled-reiease (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce the dosage frequency.Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s)that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled release of an agent may be stimulated by various inducers, e g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0042] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for the treatment of the disease, disorder, or condition.

[0043] Botulinum Toxins

[0044] The term “botulinum toxin" as used herein is meant to refer to any of the known types of botulinum toxin without limitation, whether produced by the bacterium or by recombinant techniques, as well as any such types that may be subsequently discovered including engineered variants or fusion proteins. Seven immunologically distinct botulinum neurotoxins have been characterized, namely botulinum neurotoxin serotypes A, B, C, D, E, F and G, each of which is distinguished by neutralization with type-specific antibodies. The botulinum toxin serotypes are available from Sigma-Aldrich and from Metabiologics, Inc.

[0045] (Madison, Wis.), as well as from other sources. The different serotypes of botulinum toxin vary in the anima! species that they affect and in the severity and duration of the paralysis they evoke.

[0046] BOTOX® (onabotulinumtoxinA) has been used to treat functional constipation (FC) and other defecation disorders. Typically, IAS BOTOX® treatments have been reserved for the perioperative management of Hirschsprung disease, IAS achalasia, or anal fissure. Recently, this paradigmhas shifted to include patients with severe FC, given the comparable efficacy of BOTOX® regardless of IAS dynamics. Safety of such practice for pediatric FC was demonstrated in a large, retrospective cohort study in which the overall complication rate was less than 1%, all being transient effects needing no further intervention.

[0047] In various aspects, the botulinum toxin used to treat disordered defecation (DD) as disclosed herein is DAXXIFY®, defined herein as daxibotulinumtoxinA-lanm (Revance Therapeutics Inc, Nashville, TN, USA). DAXXIFY® injection is currently approved in the United States for temporary improvement in the appearance of glabellar lines (40 units total) and treatment for cervical dystonia (125-250 units total) in adults. In comparison to BOTOX® which is thought to have duration of action of approximately 3 months, DAXXIFY® has demonstrated duration of action out to 6 months, which may be related to less diffusion from the injection site related to differences in stabilizer differences between BOTOX® and DAXXIFY®. While efficacy and safety have been established in adults, including a boxed warning for the potential effect of distant spread of toxin, data are currently unavailable in pediatrics (<18 years of age). A comparable safety profile has been demonstrated for daxibotulinumtoxinA-lanm and onabotulinumtoxinA in adults.

[0048] Without being limited to any particular theory, the dosing of DAXXIFY® is not equivalent to BOTOX® injections in corresponding treatments, including the treatment of DD as disclosed herein. Current dosing forms available for DAXXIFY® include 50 or 100 unit single-dose vials. Given the longer-acting formulation of DAXXIFY® relative to BOTOX®, the long-acting formulation of DAXXIFY® would reduce anesthesia time for children and also be cost-effective for patients and payers as compared to existing DD treatments using BOTOX®.

[0049] Without being limited to any particular theory, BOTOX® includes human serum albumin as a stabilizer, whereas DAXXIFY® utilizes a proprietary synthetic peptide excipient configured to enhance tissue interaction. This synthetic peptide excipient is functional rather than only stabilizing, as is the case with the human serum albumin stabilizer of BOTOX®. Consequently, the mechanism of action beyond the toxin in DAXXIFY® includes the effect of the peptide excipient that enhances local retention and binding, thus altering localpharmacokinetics as well as potency. These alterations may modulate the risk profile and predictability when moving DAXXIFY® into smooth muscle because smooth muscle can be highly sensitive to diffusion and autonomic regulation. While DAXXIFY® is able to achieve x2 duration without increasing toxin dose, prolonged duration could theoretically impact incontinence, obstruction, or dysmotility in unexpected ways. The unpredictability of dosing is further heightened in children, where smooth muscle tone and compensatory mechanisms differ substantially from adults, and where the effect prolonged neuromodulation is relatively poorly characterized.

[0050] Without being limited to any particular theory, prior study has characterized DAXXIFY® as a treatment for disorders of excessive skeletal muscle contraction. Formulation matters more in smooth muscle, including anal sphincter tissue, than skeletal muscle, because smooth muscle lacks voluntary override, continuously functions, and is integrated into reflex arcs. Small pharmacokinetic changes may result in large physiologic consequences. This introduces a degree of unpredictability to the clinical response of distinct botulinum toxin formulations in the treatment of DD as described herein.

[0051] The claimed dosing range of DAXXIFY® in the disclosed treatment method represents a solution to an unpredictable pharmacologic problem unique to DAXXIFY® in pediatric smooth muscle. DAXXIFY® units are not interchangeable with BOTOX® units - prior BOTOX® dosing cannot be extrapolated to define DAXXIFY® dosing given differences in the unit definitions between formulations, tissue dispersement, and duration of effect.

[0052] In some embodiments, the botulinum toxin formulations are prepared in a powder form for ease of handling, transport, or storage. The powder form may be prepared by any method known in the art. Non-limiting examples of such methods include lyophilization, vacuum-drying, drum-drying and spray drying, with lyophilization and vacuum-drying being particularly preferred. In other embodiments, the botulinum toxin formulations are stored in liquid form, rather than solid form.

[0053] THERAPEUTIC METHODS

[0054] Also provided is a process of treating, preventing, or reversing a diseasein a subject in need of administration of a therapeutically effective amount of an IAS DAXXIFY® treatment, so as to treat a functional constipation in a pediatric patient.

[0055] Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a disease. A determination of the need for treatment will typically be assessed by a history, physical exam, or diagnostic tests consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans or chickens. For example, the subject can be a human subject.

[0056] Generally, a safe and effective amount of an IAS DAXXIFY® treatment is, for example, an amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of an IAS DAXXIFY® treatment described herein can substantially inhibit a disease, slow the progress of a disease, or limit the development of a disease.

[0057] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intratumoral, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0058] When used in the treatments described herein, a therapeutically effective amount of an IAS DAXXIFY® treatment can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit / risk ratio applicable to any medical treatment, in a sufficient amount to treat a disease.

[0059] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject or host treated and the particular mode ofadministration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0060] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50 / ED5O, where larger therapeutic indices are generally understood in the art to be optimal.

[0061] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincot Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4thed., Lippincot Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing, reversing, or delaying the appearance of clinical symptoms in a mamma! that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.

[0062] Administration of an IAS DAXXIFY® treatment can occur as a single event or over a time course of treatment. For example, an IAS DAXXIFY® treatment can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0063] Treatment in accordance with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for a disease.

[0064] An IAS DAXXIFY® treatment can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, an IAS DAXXIFY® treatment can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory. Simultaneous administration can occur through the administration of separate compositions, each containing one or more of an IAS DAXXIFY® treatment, an antibiotic, an anti-inflammatory, or another agent. Simultaneous administration can occur through the administration of one composition containing two or more of an IAS DAXXIFY® treatment, an antibiotic, an anti-inflammatory, or anotheragent. An IAS DAXXIFY® treatment can be administered sequentially with an antibiotic, an anti-inflammatory, or another agent. For example, an IAS DAXXIFY® treatment can be administered before or after the administration of an antibiotic, an anti-inflammatory, or another agent.

[0065] ADMINISTR TION

[0066] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

[0067] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricuiar, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.

[0068] Agents and compositions described herein can be administered in a variety of methods well-known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., upto 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e g., 1-100 pm), reservoir devices, a combination of any ofthe above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

[0069] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar tothat used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site.

[0070] Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof, in addition, a controiled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

[0071] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule / agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in viva prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific del ivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste at the product; or improve shelf life of the product.

[0072] KITS

[0073] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate the performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include but are not limited to DAXXIFY®. Such packaging of the components separately can. if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. Thepack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing the activity of the components.

[0074] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0075] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and / or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet website specified by the manufacturer or distributor of the kit.

[0076] A control sample or a reference sample as described herein can be a sample from a healthy subject. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.

[0077] Compositions and methods described herein utilizing molecular biologyprotocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et a!. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988.

[0078] Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10:

[0079] 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10:0954523253).

[0080] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0081] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about." In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may containcertain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0082] In some embodiments, the terms “a” and ton” and “the” and similar references used in the context of describing a particular embodiment (especially In the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise, in some embodiments, the term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0083] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises," “has" or “includes" one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0084] Al! methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0085] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination withother members of the group or other elements found herein. One or more members of a group can be inducted in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0086] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0087] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

[0088] EXAMPLES

[0089] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0090] Example 1: Botulinum Toxin {BOTOX® Treatment of Pediatric Disordered Defecation

[0091] To evaluate the effect of IAS BOTOX® on patient and caregiver QoL metrics in association with corresponding clinical outcomes for pediatricpatients with DD, the foliowing experiments were conducted. Internal anal sphincter (IAS) BOTOX® injections were used as an adjunct to standard treatment for disordered defecation (DD), defined by the inability to effectively evacuate stool from the rectum and resulting in constipation.

[0092] Patient / caregiver quality of life (QoL) metrics in association with corresponding clinical outcomes were obtained for a cohort of IAS patients. Completed surveys at baseline, two weeks, and three months post-procedure were completed for consecutive children undergoing IAS BOTOX® for DD. Time points included assessment of clinical symptoms (Rome IV Questionnaire), QoL (Pediatric Quality of Life Inventory [PedsQL]), and caregiver well-being / family functioning (PedsQL-Family Impact Module [PedsQL-FIM]).

[0093] Methods

[0094] All pediatric patients who underwent IAS BOTOX® for DD were eligible for inclusion in this single-institution, prospective cohort study. Children with DD, defined by severe constipation with the inability to effectively evacuate stool from the rectum, were identified through participation in a multidisciplinary colorectal clinic. Exclusion criteria consisted of patients undergoing BOTOX® for management of anal fissure and age > 18 years. Using baseline data from children with constipation and other chronic health conditions, we anticipated a 10% improvement in baseline PedsQL and PedsQL-FI M scores following IAS BOTOX®, and with a power of 0.9, our study required a cohort size of at least 37 patients.

[0095] Sixty of 72 approached consecutive patients and families agreed to participate. As summarized in Table 1 herein and in Figure 1, the clinical study population consisted of 32 (53.3%) first-time BOTOX® recipients and 28 (46.7%) prior BOTOX® recipients. Interventions trialed prior to IAS BOTOX® for DD, including pharmacologic (laxatives, colonic stimulants, suppositories, enemas) and non-pharmacologic (psychology, physical therapy) therapies, were similar between the groups (p ≥ 0.149; Figure 5).

[0096] Clinical data was extracted from the electronic medical record for each participant, including demographics, medical history, therapies trialed prior to IAS BOTOX®, number of bowel cleanouts in the past year, number ofantibiotic courses for HD-associated enterocolitis if applicable, and number of prior IAS BOTOX® injections.

[0097] BOTOX® injections were completed under general anesthesia with the guidance of either a pediatric gastroenterologist or pediatric surgeon.

[0098] Regardless of provider, 6 units per kilogram dosing (maximum dose: 100 units) of onabotulinumtoxin A (BOTOX; Allergan, North Chicago, IL, USA) was used and equally divided into four separate syringes by the hospital pharmacy. Prior to injection, a speculum was carefully introduced into the anal canal to improve visualization of the dentate line. Each syringe was injected using a 25-gauge needle into one of four I AS quadrants, identified within the proximal aspect of the dentate line and confirmed by palpation of the IAS muscle complex.

[0099] Surveys were collected at three separate time points (Figure 1 ), and the same caregiver was asked to complete ail surveys on behalf of their child. The Rome IV criteria, published in 2016, leverage a symptom-based diagnostic approach in the detection of pediatric functional gastrointestinal disorders. Questions pertaining to the diagnostic criteria for FC and fecal incontinence (Fl) were utilized. Children not toilet-trained at the time of the study were excluded from the Fl analysis. The PedsQL version 4.0 is a 23-question inventory that measures QoL in relation to health. The PedsQL-FIM is a 36-question caregiver-report inventory aimed at evaluating the influence of a child's chronic medical condition on the caregiver’s health and family functioning. Caregivers were asked to frame responses in the context of their child's DD.

[0100] Within the follow-up surveys, caregivers provided answers regarding the first effect of IAS BOTOX®, any noted side effects, changes in bowel regimen, the first loss of effect, and current effectiveness and overall satisfaction with IAS BOTOX® as treatment for their child’s DD. Current effectiveness was evaluated using a 100-point visual analog scale from “not effective at all” to “extremely effective,” while overall satisfaction was similarly evaluated using a 100-point visual analog scale from “completely unsatisfied” to “completely satisfied.”

[0101] Clinical characteristics and survey responses were compared amongst first-time IAS BOTOX® recipients and prior recipients within and betweensurvey time points. Categorical variables were reported as frequency with percentage. Continuous variables were reported as medians with associated interquartile range (IQR). Categorical data within each time point were compared using Chi-square or Fisher exact tests, depending on whether the expected frequencies were less than five. Continuous data within each time point were compared using the Wilcoxon rank sum test. PedsQL total score and PedsQL-FIM total score were compared between visits using a linear mixed model, accounting for random subject effect with a compound

[0102] symmetry covariance structure. Odds ratio for stool frequency and Fl between visits was estimated using a generalized estimation equations model with compound symmetry covariance structures to account for correlated repeated measures within subjects. Relative risk for meeting diagnostic criteria for FC was calculated using modified Poisson regression with robust standard errors. All statistical tests were two-sided. Differences were considered statistically significant when p < 0.05. Statistical analysis was performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).

[0103] TABLE 1: Clinical Characteristics of the Study Population

[0104] Total (n = 60) First-time Prior recipients

[0105] recipients (n = 28)

[0106]

[0107] Median age in years (IQR) 7 (4–10) 5 (2.5–8) 8.5 (5.5–12.5) 0.006

[0108] Male sex (%) 34 (56.7%) 16 (50%) 18 (64.3%) 0.265

[0109] Race (%) 0.331 Caucasian 56 (93.3%) 31 (96.9%) 25 (89.3%)

[0110] African American 4 (6.7%) 1 (3.1%) 3 (10.7%)

[0111] Hispanic ethnicity (%) 1 (1.7%) 0 (0%) 1 (3.6%) 0.467

[0112] Median BMI (kg / m²) (IQR) 16.7 (15.0– 15.8 (14.8– 18.8 (15.5–22.3) 0.011

[0113] 19.4) 17.7)

[0114] Past medical history (%) 39 (65%) 17 (53.1%) 22 (78.6%) 0.039 Neurologic disorder 23 (59.0%) 12(70.6%) 11 (50.0%) 0.387 Neuromuscular disorder 4 (10.3%) 3 (17.6%) 1 (4.5%) 0.616 Endocrine disorder 4 (10.3%) 0 (0%) 4 (18.2%) 0.042

[0115] Celiac disease 1 (2.6%) 0 (0%) 1 (4.5%) 0.467 Hirschsprung disease 7 (17.9%) 0 (0%) 7 (31.8%) 0.003

[0116] Genetic diagnosis 8 (20.5%) 2 (11.8%) 6 (27.3%) 0.130 Rheumatology disorder 1 (1.7%) 1 (3.1%) 0 (0%) > 0.999Dermatologic disorder 5 (8.3%) 3 (9.4%) 2 (7.1%) > 0.999 Pulmonary / allergy disorder 11 (18.3%) 3 (9.4%) 8 (28.6%) 0.055 Cardiovascular disorder 6 (10.0%) 4 (12.5%) 2(7.1%) 0.675 Urologic / renal disorder 2 (3.3%) 0 (0%) 2 (7.1%) 0.214 Past surgical history (%) 35 (58.3%) 16 (50%) 19 (57.9%) 0.162 Abdominal 16 (45.7%) 6 (37.5%) 10 (52.6%) 0.453 Median age of constipation onset in 11.5 (3–36) 10 (3.5– 12 (1.1–46.5) 0.734 months (IQR) 29)

[0117] Median age of bowel regimen initiation 36 (12–57) 24 (12–45) 36 (9–60) 0.871 in months (IQR)

[0118] Median number of home cleanouts in 3 (2–6) 3 (2–9.5) 3 (1–6) 0.518 the past 12 months (IQR)

[0119] Median number of inpatient cleanouts 1 (0–2) 1 (0–2) 1 (0–2) 0.669 in the past 12 months (IQR

[0120] Prior doses of iAS BOTOX® received

[0121] (%)

[0122] 1 13 (21.6%) N / A 13 (46.4%)

[0123] 2 5 (8.3%) N / A 5 (17.9%)

[0124] 3 3 (5.0%) N / A 3 (10.7%)

[0125] 4 or more 7 (11.7%) N / A 7 (25.0%)

[0126] Note: Results are displayed as n (% or IQR). Bold values Indicate the Statistically significant differences. Abbreviations: BMI, body mass index; IAS, internal anal sphincter; IQR, interquartile range.

[0127] Results

[0128] Sixty of 72 approached consecutive patients and families agreed to participate (Table 1), consisting of 32 (53.3%) first-time BOTOX® recipients and 28 (46.7%) prior BOTOX® recipients. Interventions trialed prior to IAS BOTOX® for DD, including pharmacologic (laxatives, coionic stimulants, suppositories, enemas) and non-pharmacologic (psychology, physical therapy) therapies, were similar between the groups (p ≥ 0.149; Figure 5). Two-Week Caregiver Response

[0129] Of the 53 responses (30 first-time and 23 prior recipients), 36 (67.9%) reported a positive effect of IAS BOTOX®; a larger proportion of prior recipients (19 [82.6%]) had noted an effect of BOTOX® on DD in comparison to first-time recipients (17 [56.7%]; p = 0.045). In those patients who observed an effect, the first effect was noted at a median of 6 days (IQR 3-14) after injection, with caregivers rating the current effect on their child’s DD at a median of 70.5 (IQR 62.5-77.5) on a 100-point scale. Side effects were reported in 6 (11%) and were mild and transient, including anal discomfort, scant bleeding, and an increase in Fl.Three-Month Caregiver Response

[0130] Of the 55 responses (29 first-time and 26 prior recipients), less than one-half (43.6%) had a change in their bowel regimen since BOTOX® injection. Thirty-nine (70.9%) reported a positive effect on their child’s DD; a larger proportion of prior recipients had an observed effect of BOTOX® on their DD compared to first-time recipients (22 [84.6%] versus 17 [58.6%]; p = 0.034). in those patients who observed an effect, the first effect was noted at a median of 6 days (IQR 3-14). There were no inpatient bowel cleanouts within the cohort during the 3-month period, and only 10 patients (18.2%; 6 first-time and 4 prior recipients) reported the need for a home bowel cleanout. Two-thirds (26) had also endorsed loss of effect, which was first noticeable at a median of 70 days (IQR 39-83) after BOTOX® injection. The overall satisfaction with BOTOX® as a treatment for DD was a median of 76 (IQR 50–95) on a 100-point scale.

[0131] Rome IV Outcomes

[0132] At baseline, 54 (90.0%) patients within the total cohort met Rome IV criteria for FC, including 100.0% (32 / 32) of first-time and 78.6% (22 / 28) of prior recipients (p = 0.008; Table 2 and Figure 8). By 2 weeks, 44 (83.0%) met Rome IV criteria for FC. Although there was no significant difference in the proportion of the cohort meeting FC criteria as a whole (26 [86.7%] firsttime versus 18 [78.3%] prior), first-time recipients trended toward lower risk of fulfilling criteria (RR 0.87, 95% Cl: 0.76-1.00; p = 0.049) and demonstrated lower odds of experiencing < 2 stools a week in comparison to baseline (OR 0.35, 95% Cl: 0.13-0.91; p = 0.032). At 3 months, 40 (72.7%) met Rome IV criteria for FC, including 21 (72.4%) first-time and 19 (73.1%) prior recipients. There was significant improvement in stool frequency, using a threshold of < 2 times a week at 3 months, in comparison to baseline (p = 0.001). Furthermore, in comparison to baseline, there was a lower risk of meeting Rome IV criteria for FC in first-time recipients (RR 0.73, 95% Cl: 0.58-0.91; p = 0.005); this remained significant even after adjusting for any change in bowel regimen since receiving BOTOX® (OR 0.33, 95% Cl: 0.13-0.74; p = 0.012; Figures 2 and 6). In comparison to baseline and 2-week follow-up, there was no difference in the proportion of patients reporting Fl nor was there a difference in the frequency of incontinence episodes.PedsQL Outcomes

[0133] The median baseline total score for the cohort was 60.9 (IQR 50.0- 80.4), with no difference between groups (p = 0.438). By 2 weeks and 3 months, the median total score was 76.2 (IQR 60.9-94.0) and 80.4 (IQR 57.1-91.3), respectively. For first-time recipients, there was a significant improvement in total score at both 2 weeks (11.79 point improvement, 95% Cl: 6.36-17.22; p < 0.0001) and 3 months (13.97 point improvement, 95% Cl: 8.47-19.47; p < 0.0001) in comparison to baseline (Figure 3). There was no significant difference in total score at 2 weeks compared to 3 months (p = 0.433). Prior recipients had significant improvement in total score in comparison to baseline at 2 weeks (6.67 point improvement, 95% Cl: 0.65- 12.69; p = 0.030); however, this did not persist out to 3 months (p = 0.235).

[0134] TABLE 2. Rome IV questionnaire responses by time point.

[0135] Baseline Response Total (n = 60) First-time Prior recipients recipients (n = 28)

[0136]

[0137] (n = 32) Frequency of stools (%) 0.0021 More than 2 times a week 28 (46.7%) 9 (28.1%) 19 (67.9%)

[0138] 2 times a week or less often 32 (53.3%) 23 (71.9%) 9 (32.1%)

[0139] Fecal incontinence (%) 46 (76.7%) 24 (77.4%) 22 (78.6%) 0.9151 Frequency of fecal incontinence (n = 46) 0.4061

[0140] 1 time a week or more (%) 39 (84.8%) 22 (91.7%) 17 (77.3%)

[0141] Meet criteria for functional 54 (90%) 32 (100%) 22 (78.6%) 0.0075 constipation (%)

[0142] 2-week response Total (n = 53) First-time Prior p recipients (n recipients = 30) (n = 23) Frequency of stools (%) 0.0607 More than 2 times a week 34 (64.2%) 16 (53.3%) 18 (78.3%) 2 times a week or less often 19 (35.8%) 14 (46.7%) 5 (21.7%) Fecal incontinence (%) 35 (66.0%) 19 (63.3%) 16 (69.6%) 0.9506 Frequency of fecal incontinence (n = 35) 0.0218 1 time a week or more (%) 24 (68.6%) 17 (89.5%) 7 (43.8%)ᵃ Meet criteria for functional 44 (83.0%) 26 (86.7%) 18 (78.3%) 0.4780 constipation (%) 3-month response Total (n = 55) First-time Prior p recipients recipients

[0143]

[0144] (n = 29) (n = 26) Frequency of stools (%) 0.3525 More than 2 times a week 39 (70.9%) 19 (65.5%) 20 (78.9%)2 times a week or less often 16 (29.1%) 10 (34.5%)ᵃ 6 (23.1%)

[0145] Fecal incontinence (%) 33 (60.0%) 15 (51.7%) 18 (69.2%) 0.9377 Frequency of fecal incontinence 0.7151

[0146] (n= 33)

[0147] 1 time a week or more (%) 27 (81.8%) 13 (86.7%) 14 (77.8%)

[0148] Meets criteria for functional 40 (72.7%) 21 (72.4%)ᵃ 19 (73.1%) 0.9560 constipation (%)

[0149] Note: Results are displayed as n (%). Bold values indicate the Statistically significant differences. ᵃ Denotes statistically significant reduction in odds / risk in comparison to baseline.

[0150] PedsQL-FIM Outcomes

[0151] At baseline, the median total score for the cohort was 58.3 (IQR 45.8-74.3), with no difference between the groups (p = 0.197). By 2 weeks and 3 months, the median total score was 71.5 (IQR 47.2-87.5) and 72.2 (50.7-86.1), respectively. First-time recipients demonstrated significant improvement in total score at both 2 weeks (9.33 point improvement, 95% Cl: 3.77-14.89; p = 0.001) and 3 months (11.57 point improvement, 95% Cl: 5.94-17.20; p < 0.0001) in comparison to baseline; however, there was no difference between the 2-week and 3-month total scores (p = 0.432; Figure 4). Conversely, there was no significant score improvement at either time point for prior recipients. Breakdown by Hirschsprung Disease Status

[0152] All HD patients were stooling more than twice weekly at baseline, while this was true in only 21 (39.6%) of non-HD patients (p = 0.003). This significant difference was maintained at 2 weeks (p = 0.041 ); however, by 3 months all HD patients continued to stool more than twice weekly, while this was also true of 32 (66.7%) non-HD patients (p = 0.093). Furthermore, there was a significant reduction in odds of passing stool two times per week or less often in non-HD patients (OR 0.37, 95% Cl: 0.20-0.70; p = 0.002). From baseline to 3 months, there was a significant reduction in odds of meeting criteria for FC in non-HD patients (OR 0.13, 95% Cl: 0.03-0.58; p = 0.007). There was no significant difference in odds of experiencing fecal incontinence (p = 0.401) or frequency of fecal incontinence (p = 0.176) at any time point between the groups. Regarding PedsQL, non-HD patients had a significantly lower baseline total score in comparison to HD patients (59.8 vs.80.4; p = 0.042). Only patients without HD demonstrated statistically significant improvement in PedsQL total scores at 2 weeks (p < 0.001 ) and 3 months (p <0.001) in comparison to baseline. Likewise, only patients without HD demonstrated statistically significant improvement in PedsQL-FIM total scores at 2 weeks (p < 0.001 ) and 3 months (p < 0.001) in comparison to baseline. Discussion

[0153] First-time recipients trended toward significant reductions in criteria fulfillment by 2 weeks, suggesting gradual improvement in symptoms secondary to Botox with initial onset of effect within 1 week (6 days for our cohort) followed by clinically apparent improvement at 1 month. Moreover, most children who ultimately had a positive response experienced this response by 2 weeks post-injection with a median effect first observed within the first week. Furthermore, for those who noticed a loss of maximal effect, this was first observed at 10 weeks post-injection of repeat injections every 12 weeks if effective.

[0154] IAS Botox improved QoL in children with DD. While first-time recipients appreciated significant QoL improvement at both time points, prior recipients only experienced an improvement at 2 weeks that was not sustained out to 3 months.

[0155] Similar to the PedsQL findings, despite significant improvements in total PedsQL-FIM scores at both time points in comparison to baseline for first-time recipients, the data demonstrated no significant difference in total score between the 2-week and 3-month surveys. This finding was important in understanding that the impact of IAS Botox is far-reaching beyond the patient and has implications for caregiver health and family functioning.

[0156] Conclusion

[0157] The current prospective cohort study of consecutive pediatric patients undergoing IAS BOTOX® as an adjunct to standard management for DD demonstrated significant improvements in QoL and caregiver health / family functioning at both 2 weeks and 3 months after injection, largely driven by effects on first-time BOTOX® recipients. Moreover, irrespective of preceding BOTOX® use, most respondents reported a positive effect and were highly satisfied with BOTOX® as a treatment for DD. Likewise, in conjunction with QoL outcomes, the results of these experiments reveal the positive clinical impact of IAS BOTOX® in first-time recipients as observed by a reduction inthe risk of meeting Rome IV criteria for FC, a relationship that remained significant after adjusting for any change in bowel regimen. Our findings establish far-reaching benefits of IAS BOTOX®, both clinically and psychosocially, and suggest that widespread incorporation into the treatment paradigm may globally enhance outcomes in children with DD.

[0158] Example 2: Botulinum Toxin (Daxxify) Treatment of Pediatric Disordered

[0159]

[0160] Defecation To evaluate the effect of safety and efficacy of daxibotulinumtoxinA- lanm (DAXXIFY®) for internal anal sphincter injection in children with functional constipation, the following experiments will be conducted. These experiments will demonstrate that IAS injection of daxibotulinumtoxinA-lanm as an adjunct to standard management in children with functional constipation will have a comparable safety profile to those experienced in IAS BOTOX® injections in current clinical practice. These experiments will further demonstrate that IAS injection of daxibotulinumtoxinA-lanm is not inferior to onabotulinumtoxinA (Botox) as an adjunct to standard management in reducing the proportion of children meeting Rome IV criteria for functional constipation at p-8 weeks post-injection. These experiments will further demonstrate that IAS injection of daxibotulinumtoxinA-lanm provides a significantly longer duration of effect compared to onabotulinumtoxinA (Botox) in children with functional constipation.

[0161] Pediatric patients presenting for IAS BOTOX® as an adjunct to their treatment of constipation will be eligible. Inclusion criteria include < 18 years of age, weight of > 10kg diagnosis of functional constipation per Rome IV criteria, no prior IAS BOTOX® injection. Exclusions include a history of Hirschsprung disease or anorectal malformation (e., imperforate anus, anteriorly displaced anus, anal stenosis), celiac disease, cystic fibrosis, hypothyroidism that is untreated or treated with thyroid hormone at a dose not stable for 3 months, prior anorectal surgery, prior IAS BOTOX® injection, neurologic (e.g. spinal cord abnormalities, neurofibromatosis, tethered cord, spina bifida, spinal cord trauma) or neuromuscular disorders affecting bowel / bladder control, hypercalcemia, neurodevelopmental disabilities, Down syndrome or any other chromosomal disorder, current use ofaminoglycosides, muscle relaxants, or anticholinergic agents, prior IAS BOTOX® injection, other bodily BOTOX® injections in the past 6 months, and IAS BOTOX® injection for alternative indications beyond constipation such as anal fissure.

[0162] Eligible patients will be called via telephone prior to their upcoming IAS DAXXIFY® procedure for study introduction and potential consent. If interested in participation, informed consent, and if indicated assent, will be obtained electronically. If requiring more time to consider potential enrollment, written informed consent will be obtained on the day of IAS DAXXIFY® procedure. The Rome IV constipation / fecal incontinence questionnaires will be completed on the day of procedure by a caregiver on behalf of their child. In a double-blind fashion, 50 patients will be randomized 1:1 to receive onabotulinumtoxinA or daxibotulinumtoxinA-lanm. The dosing of onabotulinumtoxinA to daxibotulinumtoxinA-lanm will be 1:2 given that this ratio provides equivalent amounts of toxin (i.e., 100 units onabotulinumtoxinA: 200 units daxibotulinumtoxinA-lanm). Injection of either botulinum toxin formulation will occur under general anesthesia. A speculum will be carefully inserted into the anal canal to improve dentate line identification, and palpation will confirm IAS muscle complex location. One-fourth of the total 200 units daxibotulinumtoxinA-lanm dose will be injected into each quadrant of the proximal aspect of the dentate line after confirming the needle tip is not within a blood vessel.

[0163] Participants will be surveyed at day 3. week 1, week 2, week 3, week 4, following IAS DAXXIFY® injection to document the presence of side effects and determine efficacy related to IAS DAXXIFY® injection. Specific side effects reported in the literature for non-daxibotulinumtoxinA-lanm preparations, including anal discomfort, injection site bleeding, and worsened fecai incontinence, among others will be recorded (see Phase 1 below for full details). Regarding non-inferiority related to clinical symptoms, surveys will be administered at week 2, week 8, and week 12 for primary outcome.

[0164] Secondarily, surveys will be administered monthly after 12 weeks until 9 months to understand longer-term outcomes.

[0165] Phase I

[0166] The first 50 participants (25 per group) will be enrolled in a safety lead-in phase. Although onabotulinumtoxinA is not FDA-approved for the indication utilized in this cohort (i.e., FC), its use is supported by several published studies within the pediatric literature, including a previous prospective trial. These participants will be monitored closely for adverse events in the 4 weeks following IAS injection. Safety events will include serious adverse events (SAEs) such as hospitalization, systemic botulism-like effects, or other lifethreatening complications; and moderate / severe local adverse events including persistent fecal incontinence, severe perianal pain, or urinary retention.

[0167] Phase 2: Non-inferiority & Superiority

[0168] Based on preliminary clinical observations and the available literature, it is predicted that approximately 72.4% of children will continue to meet Rome IV criteria at 3 months following botulinum toxin injection. Peak efficacy of IAS botulinum toxin injection is thought to be between 6-8 weeks post-injection. A non-inferiority margin of 15% (A = 0.15) was selected a priori, representing the maximum acceptable difference in Rome IV persistence rates that would still justify the use of an alternative botulinum formulation in clinical practice. This margin reflects the maximum allowable difference in efficacy (in terms of persistence of Rome IV criteria) that would still be considered clinically acceptable, especially in the context of potential benefits of the alternative formulation (e.g., cost, access, or tolerability). Given the non-fatal nature of the condition, the use of two active comparators, and the primary outcome being symptom-based rather than life-threatening, a 15% margin is deemed appropriate and aligns with FDA guidance for non-inferiority designs involving quality-of-life or functional outcomes.

[0169] The primary analysis will assess the difference in the proportion of patients meeting Rome IV criteria between groups at 6-8 weeks, given that peak efficacy is typically noted during this timeframe. A 95% confidence interval for the risk difference abobotulinumtoxinA minus onabotulinumtoxinA) will be calculated. If the upper bound of the confidence interval is less than 15%, non-inferiority will be concluded.

[0170] Secondary outcomes will be analyzed using chi-square or Fisher's exact tests for categorical variables and t-tests or Wilcoxon rank-sum tests for continuous outcomes, depending on data distribution. Change from baselinein 12-week SBM frequency rate will be analyzed using repeat measures models or appropriate longitudinal techniques.

[0171] A secondary, superiority-based analysis will compare the duration of effect between formulations and proportion of patients meeting Rome IV criteria at several time points beyond 3 months, including: Kaplan-Meier survival analysis for time to recurrence of FC and time to repeat injection; Logrank tests for comparing time-to-event distributions between the groups; and Cox proportional hazards regression to estimate hazard ratios for time to FC recurrence.

[0172] Long-Term Outcomes

[0173] To evaluate the durability of treatment response, long-term follow-up will be conducted at 3 through 9 months post-injection. The following outcomes will be assessed: proportion of patients meeting or not meeting Rome IV criteria at follow-up; spontaneous bowel movement (SBM) frequency rate; time to recurrence of symptoms; time to second botulinum toxin injection; quality of life scores; number and type of additional interventions required; spontaneous bowel movement (SBM) frequency rate between groups.

[0174] Patients who receive a second dose of botulinum toxin will be retained in the study and included in exploratory long-term analyses. These participants will be stratified in post hoc analyses based on whether they required a second injection. Kaplan-Meier curves will be used to evaluate time to recurrence or second injection. Logistic regression may be used to evaluate predictors of retreatment. Although the primary trial is powered for noninferiority at 6-8 weeks, exploratory analyses will also compare the duration of effect between formulations as a hypothesis-generating outcome.

Claims

CLAIMSWe claim the following:

1. A method for the treatment of pediatric functional constipation in a subject, the method comprising injecting a therapeutically effective amount of a botulinum toxin composition into an anal sphincter of the patient.

2. The method of claim 1, wherein the botulinum toxin composition comprises a botulinum neurotoxin serotype A.

3. The method of claim 2, wherein the botulinum neurotoxin serotype A comprises daxibotulinumtoxinA.

4. The method of claim 3, wherein the botulinum toxin composition comprises daxibotulinumtoxinA-lanm (DAXXIFY®).

5. The method of claim 4, wherein the daxibotulinumtoxinA-lanm (DAXXIFY®) is injected at a dosage of about 12 units per kilogram of body mass of the patient.

6. The method of claim 5, wherein daxibotulinumtoxinA-lanm (DAXXIFY®) is injected at a maximum dosage of about 200 units.

7. The method of ciaim 6, wherein the daxibotulinumtoxinA-lanm (DAXXIFY®) comprises a duration of action of about six months.

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