Liquid formulations of besudil

Abstract

The present disclosure provides a liquid formulation comprising besudil, a method for preparing a liquid formulation of besudil, and a liquid pharmaceutical composition comprising besudil useful in the treatment of a subject.

Description

Technical Field

[0001] The present disclosure relates to liquid formulations comprising 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, also known as belumosudil. The present disclosure further relates to methods of preparing liquid formulations comprising belumosudil and methods of treating subjects including adults and pediatric populations with the liquid formulations. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to PCT application number PCT / US2022 / 044256, filed on September 21, 2021, which is incorporated herein by reference in its entirety. Background Art

[0003] Besudild is an oral, selective inhibitor of rho-associated coiled-coil-containing protein kinase-2 (ROCK2). ROCK2 inhibition plays a role in the dysregulated adaptive immune system and fibrosis due to abnormal tissue repair. Besudild inhibits ROCK2 and ROCK1, IC 50 The values ​​are approximately 100 nM and 3 μM, respectively.

[0004] The compound besudil has a chemical name: 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide and is represented by the following formula I:

[0005] Besudil is also known as KD025.

[0006] The mesylate salt of besudil is currently marketed in the United States and other countries under the trade name (Kadmon Corp. / Sanofi) for the treatment of patients with chronic graft-versus-host disease (cGVHD), in some cases after failure of at least two prior lines of systemic therapy. The active ingredient is besudeli methanesulfonate, with a molecular formula of C 27 H 28 N6O5S, molecular weight 548.62 g / mol, and chemical name 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide methanesulfonate (1:1).

[0007] The chemical structure of Besudiol Mesylate is as follows:

[0008] Besudiol binds to ROCK1 and ROCK2 and inhibits their serine / threonine kinase activity. In ex vivo or in vitro human T cell assays, besudiol downregulates proinflammatory responses by regulating STAT3 / STAT5 phosphorylation and changing the Th17 / Treg balance. In vitro, besudiol also inhibits abnormal profibrotic signaling. In vivo, besudiol exhibits activity in animal models of cGVHD. Therefore, besudiol can be used to treat diseases, disorders and conditions regulated by ROCK, including autoimmune and fibrotic disorders, acute and chronic GVHD, idiopathic pulmonary fibrosis and moderate to severe psoriasis and other indications.

[0009] A method for preparing besudil is disclosed in U.S. Pat. No. 8,357,693 (the '693 patent), specifically in Example 82 thereof. The method disclosed in the '693 patent provides besudil as a crude solid product, which is purified by high performance liquid chromatography (HPLC). Besudil and methods for preparing the compound are also described in U.S. Pat. No. 9,815,820, U.S. Pat. No. 10,183,931, and U.S. Pat. No. 10,696,660.

[0010] The method of administering besudil of the present invention involves formulating the mesylate salt of besudil into pharmaceutically acceptable capsules and tablets for oral administration. However, some patients who may benefit from treatment with besudil cannot, or have difficulty and / or do not like to swallow solid dosage forms, such as some adult patients, patients with dysphagia and / or children. Therefore, liquid formulations of besudil will be useful for administering the compound to a wider range of patient populations. Liquid formulations can also provide advantages in terms of enhancing the flexibility of drug administration methods.

[0011] Liquid formulations may also provide advantages in enabling flexible administration of clinically recommended doses to patients based on body weight. For example, when using a liquid formulation, the amount of drug to be administered can be adjusted on a sliding scale based on the patient's body weight. In contrast, when using currently marketed solid oral dosage forms, each tablet contains 200 mg of besudil.

[0012] However, besudil is a weakly alkaline compound that is almost insoluble in water. Besudil is also insoluble or almost insoluble in many other solvents or carriers that are commonly used in drug development. The low solubility of besudil has brought challenges in developing liquid formulations. In addition, liquid formulations should be homogeneous when suspended and resuspended (for example, when a single vial is used multiple times). In order to obtain a liquid formulation that can be homogeneously resuspended, it is necessary to use a specific device that may cause foaming. For hydrophobic compounds such as besudil, this is especially true. Therefore, there is still a need for a liquid formulation containing besudil that can solve these challenges and a method for producing a homogeneous preparation that can be resuspended. Summary of the invention

[0013] In one aspect, the present disclosure provides a liquid formulation comprising 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or a pharmaceutically acceptable salt thereof (besudil). In one embodiment, the pharmaceutical formulation comprises jet-milled or pin-milled besudil suspended in a diluent in combination with a suspending agent and a thickening agent. The liquid formulation comprising besudil may also include a preservative, a pH adjuster to achieve a pH in the range of about 2.5 to 4.0 as desired, and a sweetener and / or flavoring agent.

[0014] In other aspects, the present disclosure provides a liquid formulation comprising 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or a pharmaceutically acceptable salt thereof (besudil) in combination with a suspending agent, a thickener and a defoamer. The liquid formulation comprising besudil may also include a preservative, a pH adjuster to achieve a pH in the range of about 2.5 to 4.0 as needed, and one or more of a sweetener and / or flavoring agent. In one embodiment, the liquid formulation comprises jet-milled besudil. In one embodiment, the defoamer comprises simethicone. In one embodiment, the defoamer comprises about 30% simethicone emulsion.

[0015] Another aspect of the present disclosure provides a liquid formulation of besudil produced by mixing jet-milled or pin-milled besudil in a vehicle, wherein the vehicle comprises at least a suspending agent and a thickening agent in a diluent.

[0016] Another aspect of the present disclosure provides a liquid formulation of besudil produced by mixing jet-milled or pin-milled besudil in a vehicle, wherein the vehicle comprises at least a defoaming agent in a diluent.

[0017] In another embodiment, the present disclosure provides a kit comprising jet-milled or pin-milled besudil prepared as a dry powder and provided with a reconstitution vehicle comprising at least a suspending agent and a thickening agent in a diluent, wherein the besudil can be reconstituted with the reconstitution vehicle prior to use. The present disclosure also provides a method for preparing a liquid formulation of besudil, as further described herein.

[0018] In another embodiment, the present disclosure provides pharmaceutical compositions comprising an effective amount of a liquid formulation of besudil, which are useful for treating diseases, disorders and conditions modulated by ROCK, as further described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a flow chart showing a process for preparing a liquid formulation of besudil.

[0020] Figure 2A-2B Particle size distribution (PSD) data for needle-milled besudil in sterile irrigation water, as described in Example 3, are shown.

[0021] Figure 3A PSD data for the needle-milled besudil liquid formulation with Natrosol (Sample 3A of Example 4) are shown.

[0022] Figure 3B PSD data for a jet-milled besudil liquid formulation with Natrosol (Sample 3B of Example 4) are shown.

[0023] Figure 3C PSD data of the pin-milled besudil liquid formulation with hypromellose K4M (Sample 4A of Example 4) are shown.

[0024] Figure 3D PSD data for the needle-milled besudil liquid formulation with povidone (Sample 6A of Example 4) are shown.

[0025] Figure 4 Shown are the results of deposition assays of samples obtained from six liquid formulations containing besudil, including three formulations containing pin-milled besudil (Samples 13A-15A) and three formulations containing jet-milled besudil (Samples 13B-15B), as described in Example 6.

[0026] Figure 5 is a flow chart showing an additional process for preparing a liquid formulation of besudil.

[0027] Figure 6 is a flow chart illustrating yet another method for preparing a liquid formulation of besudil. DETAILED DESCRIPTION definition

[0028] As used herein, "about" includes an exact amount modified by the term about, as well as an amount expected to be within experimental error (such as, for example, within 15%, 10%, or 5%), depending on the context. For example, "about 200 mg" means "200 mg" and a range of mg within experimental error (e.g., plus or minus 15%, 10%, or 5% of 200 mg). As used herein, the term "about" can be used to modify ranges as well as specific values.

[0029] As used herein, "administering" or "administering to" (e.g., with respect to besudil or a liquid formulation containing besudil) refers to the act of prescribing one or more drugs containing besudil for a subject to take during treatment, the act of prescribing a drug regimen to be taken by a subject, the act of dispensing one or more drugs to a subject, the act of reconstituting besudil powder in a liquid formulation, and / or the act of physically receiving or ingesting one or more drugs. Thus, besudil can be "administered" by a physician or other medical professional who writes a prescription for besudil; and / or "administered" by a pharmacist who fills the prescription, and / or "administered" by a person who prepares a reconstituted liquid formulation using besudil powder; and / or "administered" by a patient or subject who ingests besudil and / or his or her partner or caregiver who delivers besudil to the subject.

[0030] "API" means "active pharmaceutical ingredient", which is synonymous with the definition of besudil (or KD025) herein.

[0031] "Allogeneic hematopoietic stem cell transplantation (allo-HSCT)" (also called bone marrow transplantation or stem cell transplantation) or "allogeneic hematopoietic cell transplantation (allo-HCT)" refers to the procedure of transplanting hematopoietic cells from a donor into a recipient who is not an identical twin. The source of hematopoietic stem cells for allogeneic transplantation can be peripheral blood stem cells (PBSC) or bone marrow (BM). In some cases, umbilical cord blood can be used. The donor and recipient can be matched at the human leukocyte antigen (HLA) gene, such as siblings. The donor and recipient can be parents and children who are only half-matched (haploid identical).

[0032] Unless the context clearly indicates otherwise, as used herein, "besudil" may encompass the compound besudil in any form and its pharmaceutically acceptable salts. The term "besudil" refers to both the compound besudil (e.g., in free base form, amorphous form or crystalline form), and to pharmaceutically acceptable salts of besudil (e.g., as The term "besudil" refers to any form of besudil that can be used to prepare a liquid formulation or pharmaceutical composition for administration of the compound to a patient.

[0033] "Clinical endpoint" or "study endpoint" refers to the event or outcome of the outcome and potential beneficial effect of the drug or administration scheme designed in the clinical trial that can be objectively measured in the clinical trial. The example of clinical endpoint includes the following. The overall response rate (ORR) is the percentage of people with partial response (PR) or complete response (CR) to treatment in a certain period of time in the study group or treatment group. Failure-free survival (FFS) means the time from the first dose of besudiil to the failure event, or the interval between starting besudiil and adding new cGVHD therapy, recurrence of basic disease or non-relapse death (NRM). Overall survival (OS) means the length of time from the diagnosis date of the disease or the start of treatment. Duration of response (DOR) means from the initial reaction (for example, PR or CR) until the time recorded from the best response progress of cGVHD, from the initial reaction to the start of other systemic cGVHD therapy or death. The time to the next treatment (TTNT) means the time to start subsequent systemic cGVHD therapy.

[0034] "Clinically recommended amount" or "clinically recommended dose" refers to the amount or dose of besudild recommended and / or approved by a person skilled in the art of medicinal chemistry for administration to a subject to treat the disease state in question following a clinical trial, for example, as indicated in a publication, in the results of a clinical trial, and on an approved drug label. In one embodiment, the clinically recommended dose of besudild is 200 mg once daily, as indicated on the drug label of besudild, in the absence of a CYP3A inducer or PPI.

[0035] As used herein with respect to the administration of besudil with other substances (e.g., CYP3A inducers and / or PPIs), "co-administration", "combination with", and / or "co-administered" means that during the course of treatment of the patient with besudil, the patient is also receiving one or more doses of one or more other treatments. These compounds do not need to be administered concomitantly with besudil or on the same day to be considered "co-administered" under this definition.

[0036] A "high-fat, high-calorie meal" is a meal containing about 800 to 1,000 calories, wherein about 50% of the total calorie content of the meal comes from fat foods. For example, in one embodiment, a high-fat breakfast may consist of hash browns, bacon, fried eggs, white bread, and 240 mL of whole milk.

[0037] "Homogenizer" refers to a piece of laboratory or industrial equipment used for homogenization of various types of materials, such as excipients, diluents, and APIs. Those skilled in the art will appreciate that there are many different types of homogenization equipment, including Cell Lysor, disperser, high shear mixer, homogenizer, Polytron, rotor-stator homogenizer, ultrasonic generator, TissueTearor, etc. Homogenizers can use different types of blades, rotor-stator assemblies, and screens and mix at different shear rates and speeds. One advantageous homogenizer is Homogenizer. One advantageous homogenizer assembly includes a high shear screen.

[0038] "Immunosuppressive therapy" (IST) refers to therapy that is usually administered for at least six months after allo-HSCT in an attempt to prevent GVHD. Examples of IST include sirolimus, prednisone, and calcineurin inhibitors such as tacrolimus and cyclosporine.

[0039] The “label claim” is the amount or dose of the API that corresponds to the amount or dose indicated in the approved drug label. The drug label of 200 mg / mL states that the recommended dose is 200 mg taken orally once daily with meals, and 200 mg twice daily when co-administered with strong CYP3A inducers and PPIs. The equivalent dose of besudil in liquid formulation (corresponding to the 200 mg label dose) is calculated to be 40 mg / mL free base, which is equivalent to 48.496 mg / mL besudil mesylate when adjusted with a salt correction factor of 1.2124.

[0040] The Lee Symptom Scale (LSS) summary score measures the impact on the patient's function and well-being. The Lee Symptom Scale is a 30-item scale developed to measure symptoms of cGVHD and is described in Lee SJ et al., Development and validation of a scale to measure symptoms of chronic graft-versus host disease. Biol Blood Marrow Transplant 2002; 8: 444-452.

[0041] "Treatment line" or "therapy line" describes the order or sequence in which different therapies are given to a patient as his or her disease progresses. The initial treatment (first-line therapy) may not work, or may stop working after a period of time. After the first-line therapy is discontinued, a second different treatment (second-line therapy) may be given. When the second-line therapy does not work or stops working, a subsequent therapy line may be given. Some patients may be administered multiple therapy lines during the course of the disease.

[0042] First-line therapy for moderate to severe chronic graft-versus-host disease (cGVHD) defined by the National Institutes of Health (NIH) can be corticosteroids alone or in combination with sirolimus or calcineurin inhibitors. (Carpenter PA et al.: A phase II / III randomized, multicenter trial of prednisone / sirolimus versus prednisone / sirolimus / calcineurin inhibitor for the treatment of chronic graft-versus-host disease [prednisone / sirolimus for the treatment of chronic graft-versus-host disease compared to prednisone / sirolimus / calcineurin inhibitors II / III phase randomization, multicenter trial]: BMT CTN 0801. Haematologica [Hematology] 103: 1915-1924, 2018).

[0043] The example of corticosteroid therapy for the treatment of cGVHD includes but is not limited to prednisone, prednisolone, methylprednisolone and budesonide. The example of previous systemic therapy for the treatment of cGVHD includes but is not limited to prednisone, tacrolimus, extracorporeal phototherapy (ECP), sirolimus, ibrutinib, ruxolitinib, mycophenolate mofetil (MMF), rituximab, methotrexate (MTX), cyclosporin, imatinib, ixazomib and ofatumumab.

[0044] As used herein, "liquid formulation" or "liquid" means that the formulation is substantially liquid rather than solid (or gaseous), and includes suspensions in which solid particles (eg, besudil particles) are dispersed throughout the liquid rather than dissolved therein.

[0045] "Myeloablative transplant" refers to a transplant procedure that uses very high doses of chemotherapy or radiation before transplanting with autologous or allogeneic hematopoietic stem cells. Nonmyeloablative transplants or reduced-intensity transplants involve patients undergoing less-intensive chemotherapy before transplanting with allogeneic hematopoietic stem cells.

[0046] The "NIH pulmonary symptom score" or "NIH cGVHD pulmonary score" is a score based on clinical symptoms, ranging from 0 to 3. A score of 0 is used for no symptoms, a score of 1 is used for symptoms of shortness of breath going up stairs, a score of 2 is used for symptoms of shortness of breath on level ground, and a score of 3 is used for shortness of breath at rest or requiring oxygen.

[0047] Unless the context requires otherwise, "or" is used in an inclusive sense (equivalent to "and / or").

[0048] As used herein, "patient" or "subject" includes animals or humans; in one embodiment, the term "patient" refers to a human subject.

[0049] As used herein, "pediatric patient" means a non-adult patient (ie, less than 18 years of age); in one embodiment, it means a patient less than 12 years of age; in another embodiment, it means a patient between the ages of 3 months and 12 years of age.

[0050] "Pharmaceutically acceptable salt" refers to non-toxic inorganic acid addition salts and organic acid addition salts of besudil. In one embodiment, the pharmaceutically acceptable salt of besudil herein is a methanesulfonate salt.

[0051] "Povidone" (also known as povidon) refers to a water-soluble polymer made from the monomer N-vinylpyrrolidone, also commonly known as polyvinylpyrrolidone or polyvinylpyrrolidone. The molecular formula of povidone is (C6H9NO) n , and is a white to slightly off-white powder. The k number associated with the name of povidone refers to the average molecular weight of povidone. Therefore, povidone 90 has a higher molecular weight than povidone K30.

[0052] As used herein, "protocol" refers to a method or plan for administering besudil to a subject in need of treatment. The term "protocol" is intended to encompass an overall detailed care plan for a patient, as well as individual or partial steps that are part of the overall plan. For example, a regimen may include the dose of besudil that the patient will (or is) receiving, the drug combination that the patient receives, the timing and method of administering besudil (e.g., considering the effects of DDI, food effects, and different formulations or delivery methods that may have on absorption and bioavailability), and the management of side effects, as well as a total plan that covers the doses, combinations, timing and methods of administration, and side effects considered together.

[0053] "Proton pump inhibitors" or "PPIs" are drugs that inhibit the production of H + / K +Drugs that inhibit the ATPase proton pump and cause a decrease in gastric acid production. Because PPIs reduce gastric acid production, they can increase the pH of the stomach, thereby affecting the solubility and potential bioavailability of orally delivered drugs. Examples of PPIs include omeprazole, lansoprazole, dexlansoprazole, esomeprazole, pantoprazole, rabeprazole, and ilaprazole.

[0054] As used herein, "silicon dioxide" or "silicon dioxide agent" refers to any silicon dioxide product that can be used as a glidant and / or anti-caking agent, such as silicon dioxide, fumed silica, colloidal hydrated silica, precipitated silica, and magnesium aluminum silicate, and includes those sold under the trade names and Silica products for sale.

[0055] 200 is a hydrophilic fumed silica product containing more than 99.8% SiO2 and having a specific surface area of ​​200 m2 / g.

[0056] 244FP is an alternative silica (SiO2) product sold as an amorphous white free-flowing powder available from WR Grace & Co. (Connecticut, USA). 244FP silica is a high pore volume silica gel with a large internal surface area. It has a strong affinity for moisture and organic based liquids. 244FP silica can absorb up to 1.6 ml of liquid per gram. 244FP silica is advantageous as a glidant, tableting aid and carrier for pharmaceutical and personal care products.

[0057] "Simethicone" (also called "Simethicone emulsion") refers to a defoaming agent. The molecular formula of Simethicone is C6H 18 O4Si3, and is available as a 30% water emulsion that is an off-white homogeneous liquid. An advantageous 30% simethicone emulsion is a medical defoamer C emulsion made with 30% simethicone USP, methylcellulose, sorbic acid, and water.

[0058] "Steroid-refractory" (SR) cGVHD is defined as cGVHD progression while on steroids or corticosteroids; in one embodiment, cGVHD progression while on prednisone.

[0059] A "suspension" refers to a mixture of a solid finely distributed in a liquid, wherein the solid is not dissolved in the liquid. As used herein, the term "liquid formulation" or "formulation" includes a suspension. For example, the term "liquid formulation comprising besudil," "liquid formulation of besudil," and / or "liquid formulation of besudil" is intended to cover a suspension of besudil, or a heterogeneous or homogeneous mixture in which solid besudil particles are distributed in a liquid but not dissolved in the liquid.

[0060] A "therapeutically effective amount" of an API means an amount sufficient to achieve treatment of the disease state being treated when administered to a human to treat a disease (e.g., cGVHD). When applied to human cGVHD, "treating" or "treatment" includes (1) reducing the risk of developing cGVHD and / or inhibiting cGVHD, i.e., preventing or reducing the development of cGVHD or its clinical symptoms; and (2) alleviating cGVHD, i.e., causing regression, reversal or improvement of cGVHD, or reducing the number, frequency, duration or severity of its clinical symptoms.

[0061] The therapeutically effective amount of the API may vary depending on the health and physical condition of the subject to be treated, the extent of disease progression, the assessment of the medical condition, and other relevant factors. It is expected that the therapeutically effective amount may fall within a range that can be determined by experimentation and by reference to clinical trial data and results, for example, as described in the Examples herein and in the scientific literature.

[0062] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a solid filler, diluent, excipient or manufacturing aid. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Overview

[0063] The present disclosure provides a useful liquid formulation containing besudil that meets an acceptable Quality Target Product Profile (QTPP) for administering besudil to a patient in a manner that does not require swallowing of a solid dosage form.

[0064] In order to provide a liquid formulation containing besudil as disclosed herein, various excipients were studied, including viscosity modifiers, preservatives, suspending agents, pH modifiers, sweeteners, thickeners and defoamers. The physical stability problems encountered when using these agents were studied over time and at different temperature and pH systems. The applicant found that satisfactory dispersion and resuspendibility of besudil can be achieved at a suitable inclusion body level to meet the concentration corresponding to the adult labeled amount and the concentration for the pediatric population using the colloidal silica system.

[0065] Formulations were evaluated with combinations of suspending / thickening agents, preservatives, sweeteners, flavoring agents, and antifoaming agents to identify the most favorable system for liquid formulations containing besudil.

[0066] An evaluation of jet-milled and pin-milled besudil was also performed to select the most suitable input material for use in a liquid formulation containing besudil. Comparison of formulations made with alternative milling techniques for besudil initially indicated that the pin-milled API would provide the most suitable material for use in the formulation and that the jet-milled API was problematic and presented various obstacles. However, upon further investigation, the applicant discovered that the jet-milled API provided a preferred form for achieving uniform homogeneity and meeting the QTPP.

[0067] Knowledge of the behavior of besudil in liquids combined with an understanding of the importance of ingredients plays an important role in the development of such a stable formulation.Jet-milled API can be used in this formulation instead of pin-milled API to reduce the amount of sedimentation and foaming.

[0068] In addition, suspending agents such as silicon dioxide or colloidal silicon dioxide may be used to minimize sedimentation and combined with povidone (or polyvinyl pyrrolidone [PVP]) to improve dispersion. Povidone is a polymer and thickening agent that can help maintain the viscosity of the medium.

[0069] In addition, preservatives may be used. In fact, as disclosed herein, it was found that the use of a preservative method can provide advantages in protecting liquid formulations containing besudil. After experiments, sodium benzoate was selected as an effective preservative for use in formulations. Sodium benzoate has both antibacterial and antifungal properties due to undissociated benzoic acid. Therefore, a liquid formulation of besudil without the use of a preservative method would be less advantageous for preparing a ready-to-use liquid pharmaceutical formulation.

[0070] Liquid formulations as suspensoids should be robust so that they are homogeneous after suspension and resuspension, and ideally do not foam when mixed (including during manufacture). Such formulations may be difficult to prepare. During formulation manufacture, high-speed mixing with a dedicated mixing device (such as a paddle shape and a screen) can be used to reduce particle agglomeration and provide a homogeneous and stable dispersion. The side effect of high-speed mixing (especially for hydrophobic drugs) is increased foaming. Compositions and manufacturing methods designed to overcome foaming problems can obtain suspensoids with desired characteristics.

[0071] In addition, defoamers can be used. In fact, as disclosed herein, it was found that the use of defoamers provides advantages in homogeneous and stable liquid formulations containing besudild. After experiments, about 30% simethicone emulsion was selected as an effective defoamer for use in the formulation. Therefore, for the preparation of a ready-to-use liquid pharmaceutical formulation that can be resuspended multiple times in a homogeneous manner, a liquid formulation of besudild without the use of a defoaming method would be less advantageous.

[0072] An evaluation of mixing methods was also performed to select the most appropriate manufacturing conditions for use in preparing a liquid formulation containing besudil. Comparison of formulations made using alternative processing steps initially indicated that using a two-vessel (side phase) process, incorporating a hold time step, and using relatively low speed mixing (i.e., using a rotation speed of less than about 1000 rpm) would provide the most suitable manufacturing method for preparing a liquid besudil formulation. However, upon further investigation, the applicant discovered that adding a defoamer to the formulation and using a higher speed mixing via a homogenizer with a high shear screen to manufacture the formulation provided a preferred form for achieving uniform homogeneity and meeting the QTPP.

[0073] In one embodiment, a homogenization step can be used to improve the uniformity of the formulation. Homogenization can be performed using a standard or high shear rotor-stator assembly, and can include the use of a shear screen. In an embodiment, homogenization is performed at a speed of about 900 to 10000 rpm, such as at least about 1000 rpm, about 1000 to 8000 rpm, about 4000 to 6000 rpm, or about 5000 rpm. In an embodiment, homogenization is performed using a high shear screen. In an embodiment, homogenization is performed using a paddle. In an embodiment, homogenization is performed using a rotor and a stator.

[0074] Flavoring and sweetener combinations that showed improved palatability when compared to unflavored and unsweetened control systems were identified from human volunteer taste studies, and development activities were conducted to optimize thickener / suspending systems for besudil.

[0075] Reagents and excipients for use in liquid formulations containing besudil are commercially available. For example, Silica suspending agents are commercially available from Grace Company, Connecticut, USA; Silica products sold are commercially available from Evonik Degussa (Essen, Germany) and / or Azelis Ltd (Hertford, UK); thickeners povidone K30 and povidone K90F are commercially available from BASF Corp. (Florham Park, NJ, USA); sucralose and sodium benzoate are commercially available from Merck KGaA (Darmstadt, Germany); flavoring agents such as lemon and tropical fruit blends are commercially available from Givaudan International SA (Switzerland) and / or IMCD Group; and defoamers such as simethicone are commercially available from DuPont (Wilmington, DE, USA) and Dow Corning (Midland, MI, USA).

[0076] Identify alternative formulations, and select the main formulation after stability testing. The stability of the formulation is an important attribute of its feasibility in practical applications (especially when the goal is to deliver the formulation containing besudil in a pre-prepared liquid form). To this end, the applicant evaluated various alternative besudil formulations to evaluate their chemical stability (absence of degradation); solution stability (limited pH changes over time); physical stability (absence of irreversible deposition or ability to reconstitute the formulation via agitation); and microbial stability (preservatives are used to ensure that there is no microbial / bacterial growth).

[0077] The besudil formulation disclosed herein enables flexible dosing by making it easy to change the dosage volume according to the patient's age and / or weight. In one embodiment, besudil is administered according to a weight-based dosing regimen, wherein the subjects receive approximately the same dose based on mg / kg. This weight-based dosing regimen is not achievable using fixed-dose tablets.

[0078] This liquid formulation comprising besudil according to the present disclosure herein is suitable for patients of all ages, including adults and children, including adults and children who cannot, are difficult to or do not like to swallow solid oral dosage forms. For example, liquid formulations comprising besudil suitable for children aged 3 months to 12 years or 3 months to 18 years are contemplated. The liquid formulation can be manufactured on a large scale for use in commercial and relative bioavailability assessments, and compared with solid oral dosage forms (such as 200 mg tablets approved for commercial sale).

[0079] In one embodiment, the present disclosure provides a liquid suspension form of besudil that can be administered by oral route, for example, to adult patients who are unable, difficult and / or do not like to swallow oral tablets or capsules and / or to pediatric patients.

[0080] In one embodiment, the liquid formulations comprising besudil disclosed herein can be provided in a ready-to-use liquid form, wherein besudil has been distributed throughout the liquid vehicle. In other embodiments, besudil can be prepared and manufactured as a dry powder, and provided as a kit with a reconstitution vehicle, wherein besudil is reconstituted with the vehicle before use. Exemplary Embodiments

[0081] In one embodiment, a liquid formulation having the components shown in Table 1 is provided. Table 1: Preparation ingredients Components %w / w Besudil (API) 2-8 preservative 0.02-0.08 Sweeteners 0.1-0.4 Suspending Agents 0.206-0.824 Thickener 2.061-8.247 pH Adjusters Adjust to pH 2.5 to 4.0 Thinner Appropriate amount to volume

[0082] In one embodiment, besudil mesylate is used in the formulations described in Table 1. In one embodiment, besudil mesylate has been jet milled and / or pin milled. In some embodiments, besudil (or besudil mesylate) has been jet milled.

[0083] In another embodiment, the preservative used in the formulation of Table 1 is sodium benzoate.

[0084] In another embodiment, the sweetener used in the formulation of Table 1 is sucralose.

[0085] In another embodiment, the suspending agent used in the formulation of Table 1 is a silicon dioxide product selected from the group consisting of colloidal silicon dioxide, silicon dioxide, and fumed silicon dioxide. In another embodiment, the suspending agent is colloidal silicon dioxide; in one embodiment, the suspending agent is SYLOID In another embodiment, the suspending agent is 200.

[0086] In another embodiment, the thickener used in the formulation of Table 1 is a water-soluble polymer made from the monomer N-vinyl pyrrolidone. In another embodiment, the thickener is povidone. In another embodiment, the thickener is selected from povidone K30 and povidone 90F. In another embodiment, the thickener is povidone 90F.

[0087] In another embodiment, the pH adjuster used in the formulation of Table 1 is tartaric acid. In another embodiment, the pH adjuster is added to achieve a pH in the range of 3.0 ± 0.5.

[0088] In another embodiment, the diluent used in the formulation of Table 1 is sterile irrigation water.

[0089] In one embodiment, the flavoring agent is added to the formulation described in Table 1. In one embodiment, the flavoring agent is added to the liquid formulation along with other excipients and / or ingredients when the liquid formulation is prepared. In another embodiment, the flavoring agent is stored and / or provided in a separate container and is optionally added to the liquid formulation as needed and / or desired approximately at the time of administration.

[0090] In one embodiment, the flavoring agents are selected from those flavoring agents that are commercially available and suitable for use in medicine.

[0091] In another embodiment, the flavoring is selected from tropical fruit blend flavoring, lemon flavoring, and citrus flavoring; in another embodiment, the flavoring is tropical fruit blend flavoring.

[0092] In one embodiment, the flavoring agent is lemon flavoring agent PHS-135460 available from Immigration Group; in another embodiment, the flavoring agent is tropical fruit blend flavoring agent PHS-145298 available from Immigration Group.

[0093] In another embodiment, a liquid formulation having the components shown in Table 2 below is provided. Table 2: Besudil formulation components a The amount of drug substance (mg) is given as besudelidol mesylate, with the free base equivalent in parentheses. The label dose is specified based on the free base. Potency adjustments may be made if necessary.

[0094] In one embodiment, an antifoaming agent is added to the formulation described in Table 1 or Table 2. In one embodiment, the antifoaming agent comprises simethicone. In another embodiment, the antifoaming agent is about 30% simethicone emulsion.

[0095] In another embodiment, a liquid formulation having the following components is provided: 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or a pharmaceutically acceptable salt thereof (besudil), a suspending agent, a thickening agent, a preservative, a pH adjuster to achieve a pH in the range of about 2.5 to 4.0 as needed, a sweetener, an antimicrobial agent, and an antifoaming agent.

[0096] In another embodiment, a liquid formulation having the components shown in Table 3 below is provided. Table 3: Besudil formulation components Components Concentration (%w / w) Besudil (API) 2-8 Preservatives / antimicrobial agents 0.02-0.08 Sweeteners 0.1-0.4 Suspending Agents 0.2-0.8 Thickener 2.1-8.2 Defoaming agent 0.25-4.0 pH Adjusters Adjust to pH 2.5 to 4.0 vehicle Appropriate amount up to 100

[0097] In one embodiment, besudil mesylate is used in the formulations described in Table 3. In one embodiment, besudil mesylate has been jet milled and / or pin milled. In some embodiments, besudil (or besudil mesylate) has been jet milled.

[0098] In another embodiment, the preservative used in the formulation of Table 3 is sodium benzoate.

[0099] In another embodiment, the sweetener used in the formulation of Table 3 is sucralose.

[0100] In another embodiment, the suspending agent used in the formulation of Table 3 is a silicon dioxide product selected from the group consisting of colloidal silicon dioxide, silicon dioxide, and fumed silicon dioxide. In another embodiment, the suspending agent is colloidal silicon dioxide; in one embodiment, the suspending agent is SYLOID In another embodiment, the suspending agent is 200.

[0101] In another embodiment, the thickener used in the formulation of Table 3 is a water-soluble polymer made from the monomer N-vinyl pyrrolidone. In another embodiment, the thickener is povidone. In another embodiment, the thickener is selected from povidone K30 and povidone 90F. In another embodiment, the thickener is povidone 90F.

[0102] In another embodiment, the defoaming agent used in the formulation of Table 3 is simethicone. In another embodiment, the defoaming agent is about 30% simethicone emulsion. In one embodiment, the defoaming agent is 30% simethicone emulsion medical defoamer C emulsion.

[0103] In another embodiment, the pH adjusting agent used in the formulation of Table 3 is tartaric acid. In another embodiment, the pH adjusting agent is added to achieve a pH in the range of 3.0 ± 0.5. In some embodiments, the pH adjusting agent is added to achieve a pH in the range of 2.5-4.0. In some embodiments, the pH adjusting agent is added to achieve a pH in the range of 2.7-3.3.

[0104] In another embodiment, the diluent used in the formulation of Table 3 is sterile irrigation water. In another embodiment, the diluent used in the formulation of Table 3 is purified water.

[0105] In another embodiment, a liquid formulation having the components shown in Table 4 below is provided. Table 4: Besudil formulation components

[0106] In another embodiment, a liquid formulation is provided having the components of Table 1, Table 2, Table 3 or Table 4 and meeting a target product quality profile (QTPP) having the following requirements: 200 mg besudil dose (free base equivalent), a single oral 5 mL dose (dose expressed as free base equivalent and based on a salt correction factor of 1.2124, where 200 mg free base is equivalent to 242.48 mg mesylate); no taste / mouthfeel issues, uniform color; and a shelf life long enough for packaging and distribution, optionally using refrigeration (in some embodiments, at 2°C-8°C).

[0107] In one embodiment, the present disclosure provides a liquid formulation comprising besudil, the besudil concentration of the liquid formulation being equivalent to about 40 mg / mL free base. In another embodiment, a more dilute formulation is provided to facilitate the administration of lower doses (e.g., a low dose administered via a high concentration formulation will require a low volume that may be more difficult to measure). In another embodiment, a more concentrated formulation is provided to facilitate the administration of higher doses (e.g., a high dose administered via a low concentration formulation will require a higher volume that may be more difficult for the patient to accept).

[0108] In one embodiment, the preparation comprises about 242mg besudiol mesylate (equivalent to 200mg free alkali) of every 5mL liquid preparation. In another embodiment, the preparation is diluted to provide about 242mg besudiol mesylate (equivalent to 200mg free alkali) of every 10mL liquid preparation. In one embodiment, the 5mL dosage includes the equivalent dose of about 100mg free alkali besudiol. In another embodiment, the preparation is further diluted to provide about 242mg besudiol mesylate (equivalent to 200mg free alkali) of every 20mL liquid preparation. In one embodiment, the 5mL dosage includes the equivalent dose of about 50mg free alkali besudiol.

[0109] In another embodiment, a formulation as described in Table 1, Table 2, Table 3 or Table 4 is processed with a homogenization step. In one embodiment, a mixing vessel is used to prepare the formulation, and the vessel is placed in a homogenizer, which is operated to homogenize the contents during at least one step of the formulation preparation. In one embodiment, the homogenization is performed at a speed between about 4000 rpm and 6000 rpm. In an embodiment, the homogenization is performed using a high shear screen. In an embodiment, the homogenization is performed using a rotor and a stator.

[0110] In another embodiment, a formulation is provided, the formulation being prepared by a process of mixing jet-milled or pin-milled besudil (in one embodiment, besudil mesylate) in a vehicle comprising a suspending agent and a thickening agent in a diluent.

[0111] In another embodiment, a liquid formulation comprising besudil is provided, the liquid formulation having components that meet a QTPP with the following requirements: a 200 mg besudil dose (as free base equivalent), a single oral 5 mL dose (the dose is expressed as free base equivalent and based on a salt correction factor of 1.2124, where 200 mg free base is equivalent to 242.48 mg mesylate); no taste / mouthfeel issues, uniform color; and a shelf life long enough for packaging and distribution, optionally using refrigeration (in some embodiments, at 2°C-8°C).

[0112] In another embodiment, the flavoring agent is added to the formulation. In one embodiment, the flavoring agent is added to the liquid formulation along with other excipients and / or ingredients when the liquid formulation is prepared. In another embodiment, the flavoring agent is stored and / or provided in a separate container and is optionally added to the liquid formulation as needed and / or desired approximately at the time of administration.

[0113] In another embodiment, the flavoring agent is selected from commercially available flavoring agents suitable for use in medicine. In certain embodiments, the flavoring agent is selected from tropical fruit blend flavoring and lemon flavoring. In another embodiment, it is tropical fruit blend flavoring.

[0114] In one embodiment, a method is provided for preparing a liquid formulation containing besudiidil according to the following steps: (a) dispensing a first portion of a diluent into a mixing container of appropriate size; (b) dispensing and mixing a preservative, a suspending agent, a thickening agent, and optionally a sweetener and / or a flavoring agent into the mixing container; (c) optionally, performing a visual inspection after each excipient addition; (d) transferring the mixing container to a homogenizer and homogenizing the formulation; (e) optionally, performing a visual inspection; (f) adding an amount of besudiidil mesylate to the homogenized solution and mixing; (g) optionally, performing a visual inspection; (h) measuring the pH of the formulation; (i) if necessary, adding a pH adjuster to the formulation in aliquots and mixing until the desired pH is reached; and (j) adding a final portion of the diluent to produce a besudiidil formulation. Thereafter, the liquid formulation containing besudil may be homogenized and / or allowed to stand until any foam or froth has dissipated, and thereafter, may optionally be visually inspected.The formulation may be dispensed by weight into final container closures and labeled.

[0115] In another embodiment, a liquid formulation comprising besudil is provided, wherein the liquid formulation is prepared according to the steps set forth in the previous paragraph.

[0116] In another embodiment, a method is provided for preparing a liquid formulation comprising besudil according to the following steps: (a) dispensing a first portion of sterile rinse water into a mixing container of appropriate size; (b) dispensing and mixing sodium benzoate, sucralose, colloidal hydrated silicon dioxide, and povidone into the mixing container; (c) optionally, performing a visual inspection after each excipient addition; (d) transferring the mixing container to a homogenizer and homogenizing the formulation; (e) optionally, performing a visual inspection; (f) adding an amount of besudil mesylate to the homogenized solution and mixing; (g) optionally, performing a visual inspection; (h) measuring the pH of the formulation; (i) adding tartaric acid to the formulation in aliquots and mixing until the desired pH is reached; and (j) adding a final portion of sterile rinse water to produce a besudil formulation. Thereafter, the liquid formulation containing besudil may be homogenized and / or allowed to stand until any foam or froth has dissipated, and thereafter, may optionally be visually inspected.The formulation may be dispensed by weight into final container closures and labeled.

[0117] In another embodiment, a liquid formulation comprising besudil is provided, the liquid formulation being prepared using the steps set forth in the previous paragraph.

[0118] In one embodiment, for each excipient and ingredient as shown in Table 1, Table 2, Table 3, or Table 4, the foregoing steps are performed using weight percentages.

[0119] In another embodiment, a method for preparing a liquid formulation containing besudil is provided according to the following steps: (a) dispensing a first portion of purified water into a first appropriately sized mixing container; (b) adding an amount of povidone to the solution and mixing; (c) optionally, performing a visual inspection; (d) adding an amount of besudil mesylate to the solution and mixing; (e) optionally, performing a visual inspection; (f) dispensing a second portion of purified water into a second appropriately sized mixing container; (g) mixing sodium benzoate, sucralose, and colloidal hydrate; Silicon dioxide is dispensed and mixed into a second mixing container; (h) optionally, a visual inspection is performed after each excipient addition; (i) the material from the second mixing container is transferred to the first mixing container and the formulation is mixed; (j) tartaric acid is added to the formulation in aliquots and mixed until the desired pH is reached; (k) a final portion of purified water is added; (l) the formulation is mixed; (m) optionally, a visual inspection is performed; and (n) the liquid formulation containing besudil is allowed to stand until any foam or froth has dissipated to produce a besudil formulation. Thereafter, a visual inspection may optionally be performed. The formulation may be dispensed by weight into the final container closure and labeled.

[0120] In another embodiment, a liquid formulation comprising besudil is provided, the liquid formulation being prepared using the steps set forth in the previous paragraph.

[0121] In another embodiment, the mixing step of the aforementioned steps is performed using a paddle mixer. In another embodiment, the mixing step of the aforementioned steps is performed at a rotation speed between about 300 rpm and 600 rpm.

[0122] In one embodiment, for each excipient and ingredient as shown in Table 1 or Table 2, the foregoing steps are performed using weight percentages.

[0123] In another embodiment, a method for preparing a liquid formulation containing besudil is provided according to the following steps: (a) dispensing a portion of purified water into a mixing container of appropriate size; (b) dispensing and mixing sodium benzoate, sucralose, colloidal hydrated silicon dioxide, and povidone into a mixing container; (c) adding an amount of about 30% simethicone emulsion to the formulation and mixing; (d) adding an amount of besudil mesylate to the solution and mixing; (e) optionally, performing a visual inspection; (f) adding tartaric acid to the formulation in aliquots and mixing until the desired pH is reached; (g) adding a final portion of purified water; (h) mixing the formulation; and (i) optionally, performing a visual inspection. The formulation can be dispensed by weight into a final container closure and labeled.

[0124] In another embodiment, a liquid formulation comprising besudil is provided, the liquid formulation being prepared using the steps set forth in the previous paragraph.

[0125] In another embodiment, one or more of the mixing steps of the aforementioned steps are performed using a homogenizer. In another embodiment, step (h) of the aforementioned steps is performed using a homogenizer. In another embodiment, one or more of the mixing steps of the aforementioned steps are performed using a homogenizer. In another embodiment, one or more of the mixing steps of the preceding steps are performed using a high shear screen. In another embodiment, one or more of the mixing steps of the preceding steps are performed at a speed of at least about 1000 rpm. In another embodiment, one or more of the mixing steps of the preceding steps are performed at a speed between about 4000 rpm and 6000 rpm.

[0126] In another embodiment, a method for preparing a liquid formulation containing besudil is provided according to the following steps: (a) dispensing a portion of purified water into a mixing container of appropriate size; (b) adding sodium benzoate to the formulation and mixing; (c) adding sucralose to the formulation and mixing; (d) adding povidone to the formulation and mixing; (e) adding colloidal hydrated silicon dioxide to the formulation and mixing; (f) adding about 30% simethicone emulsion to the formulation; (g) homogenizing the formulation at 5000 rpm; (h) adding an amount of besudil mesylate to the formulation and mixing; (i) homogenizing the formulation using a high shear screen at 5000 rpm; (j) optionally, performing a visual inspection; (k) adding tartaric acid to the formulation and mixing until a pH of 3.0 is reached; (l) adding a final portion of purified water; (m) mixing the formulation; and (n) optionally, performing a visual inspection. The formulation may be dispensed by weight into final container closures and labeled.

[0127] In another embodiment, the mixing of the aforementioned step is carried out at a speed between about 500 rpm and 600 rpm. In another embodiment, the homogenization of the aforementioned step is carried out using In another embodiment, the homogenization of the preceding step is performed using a high shear screen. In another embodiment, the homogenization of the preceding step is performed at a speed of at least about 1000 rpm. In another embodiment, the homogenization of the preceding step is performed at a speed between about 4000 rpm and 6000 rpm. In another embodiment, the homogenization of the preceding step is performed at a speed of about 5000 rpm.

[0128] In one embodiment, for each excipient and ingredient as shown in Table 3 or Table 4, the foregoing steps are performed using weight percentages.

[0129] In another embodiment, the excipients and ingredients are added to the formulation using the following weight percentages: about 2 to 8 weight percent besudil, about 0.02 to 0.08 weight percent preservative; about 0.1 to 0.4 weight percent sweetener; about 0.2 to 0.8 weight percent suspending agent; and about 1 to 8 weight percent thickening agent.

[0130] In another embodiment, the excipients and ingredients are added to the formulation using the following weight percentages: about 2 to 8 weight percent besudil, about 0.02 to 0.08 weight percent preservative; about 0.1 to 0.4 weight percent sweetener; about 0.2 to 0.8 weight percent suspending agent; about 2.1 to 8.2 weight percent thickening agent; and about 0.25 to 4.0 weight percent antifoaming agent.

[0131] In another embodiment, the excipients and ingredients are added to the formulation using the following weight percentages: (a) for besudil, about 2 to 6 weight percent besudil, in another embodiment, about 3 to 5 weight percent besudil (optionally, about 4.5 to 5 weight percent besudil, or optionally, about 4.75 weight percent besudil); (b) for preservatives, about 0.035 to 0.1 weight percent preservative (or optionally, about 0.05 weight percent preservative); (c) For sweeteners, about 0.1 to 0.2 weight percent sweetener (or optionally, about 0.15 weight percent sweetener); (d) for suspending agents, about 0.3 to 1 weight percent suspending agents, optionally, about 0.4 to 0.6 weight percent suspending agents (or optionally, about 0.5 suspending agents); and (e) for thickeners, about 4 to 6 weight percent thickeners (or optionally, about 4.5 to 5.5 weight percent thickeners; or in another embodiment, about 4.9 weight percent thickeners).

[0132] In another embodiment, the excipients and ingredients are added to the formulation using the following weight percentages: (a) for besudil, about 2 to 6 weight percent besudil, in another embodiment, about 3 to 5 weight percent besudil (optionally, about 4.5 to 5 weight percent besudil, or optionally, about 4.75 weight percent besudil); (b) for preservatives, about 0.035 to 0.1 weight percent preservative (or optionally, about 0.05 weight percent preservative); (c) for sweeteners, about 0.1 to 0.2 weight percent sweetener (or optionally, about 0.15 weight percent (d) for suspending agents, about 0.3 to 1 weight percent suspending agents, optionally, about 0.4 to 0.6 weight percent suspending agents (or optionally, about 0.5 weight percent suspending agents); (e) for thickening agents, about 4 to 6 weight percent thickening agents (or optionally, about 4.5 to 5.5 weight percent thickening agents; or in another embodiment, about 4.9 weight percent thickening agents); and (f) for defoaming agents, about 0.25 to 4 weight percent defoaming agents (or optionally, about 0.5 to 3.5 weight percent defoaming agents; or in another embodiment, about 2.0 weight percent defoaming agents).

[0133] In another embodiment, a liquid formulation comprising besudil is provided, the liquid formulation comprising about 2 to 8 weight percent besudil; in another embodiment, about 2 to 6 weight percent besudil.

[0134] In another embodiment, a liquid formulation comprising besudil is provided, the liquid formulation comprising about 0.02 to 0.08 weight percent sodium benzoate as a preservative; in another embodiment, about 0.035 to 0.1 weight percent sodium benzoate.

[0135] In another embodiment, a liquid formulation comprising besudil is provided, the liquid formulation comprising about 0.1 to 0.4 weight percent sucralose as a sweetener; in another embodiment, about 0.1 to 0.2 weight percent sucralose.

[0136] In another embodiment, a liquid formulation comprising besudil is provided, the liquid formulation comprising about 0.2 to 0.8 weight percent colloidal silicon dioxide as a suspending agent; in another embodiment, about 0.4 to 0.6 weight percent colloidal silicon dioxide.

[0137] In another embodiment, a liquid formulation comprising besudil is provided, the liquid formulation comprising about 1 to 8 weight percent povidone as a thickener. In another embodiment, the liquid formulation comprises about 4 to 6 percent povidone. In certain embodiments, the povidone is povidone 90F.

[0138] In another embodiment, a liquid formulation comprising besudil is provided, the liquid formulation comprising about 0.25 to 4 weight percent of a defoaming agent. In another embodiment, a liquid formulation comprising besudil is provided, the liquid formulation comprising about 0.25 to 4 weight percent of about 30% simethicone emulsion as a defoaming agent. In another embodiment, the liquid formulation comprises about 0.5 to 3.5 percent of about 30% simethicone emulsion as a defoaming agent. In another embodiment, the liquid formulation comprises about 2.0 percent of about 30% simethicone emulsion as a defoaming agent. In certain embodiments, the about 30% simethicone emulsion is a medical defoamer C emulsion.

[0139] In another embodiment, besudil is pin-milled or jet-milled prior to being added to the formulation (eg, according to step [f] in the above method). In another embodiment, jet-milled besudil mesylate is added to the formulation.

[0140] In another embodiment, flavoring agents are added to the formulation.In another embodiment, flavoring agents are added during step (b) along with preservatives, sweeteners, suspending agents, and thickening agents.

[0141] In one embodiment, a liquid formulation of Besudil is analyzed using a reversed phase gradient HPLC method. In one embodiment, the liquid formulation is assayed during manufacture of the liquid formulation (e.g., in one embodiment, via HPLC). In one embodiment, the first, intermediate, and last bottles filled during manufacture are assayed.

[0142] In one embodiment, the liquid formulation comprising besudil is formulated to deliver a single dose of 200 mg besudil (as free base equivalent) in a volume of about 5 mL. In another embodiment, liquid besudil is formulated to deliver a single dose in the range of 12 to 200 mg (as free base equivalent) in a single volume dose of about 1 to 10 mL (in another embodiment, in a volume of about 5 mL).

[0143] In another embodiment, the liquid formulation comprising besudil is formulated to deliver a single dose ranging from 10 to 400 mg (as free base equivalent) in a single dose volume of about 1 to 10 mL (in another embodiment, in a single dose volume of about 5 mL).

[0144] In another embodiment, the liquid formulation comprising besudil is formulated to deliver a single dose of API in the range of about 50 to 200 mg (free base equivalent) in a volume of about 1 mL to 10 mL (in another embodiment, in a volume of about 5 mL). In another embodiment, besudil is formulated to deliver an API dose of about 50, 100 or 200 mg (free base equivalent) in a volume of about 1 to 10 mL.

[0145] In another embodiment, a liquid formulation comprising besudil is prepared to deliver a dose of about 50, 100, 200 or 400 mg (as free base equivalent) of the API in a single volume dose of about 5 mL.

[0146] In one embodiment, the liquid formulation containing besudil is provided in a bottle containing 10 to 100 mL of the formulation; in another embodiment, the liquid formulation containing besudil is provided in a bottle containing 20 to 60 mL of the liquid formulation. In another embodiment, the liquid formulation containing besudil is provided in a bottle containing about 30 mL ± 2 mL of the formulation; in some embodiments, the bottle containing the liquid formulation of besudil includes a total of about 5-6 unit doses.

[0147] In one embodiment, the liquid formulation containing besudil is provided with an appropriately sized oral syringe, and a single unit dose is measured into the syringe prior to administration. In one embodiment, the syringe is sized to withdraw 1 to 10 mL of the liquid formulation containing besudil for administration. In another embodiment, the syringe is sized to withdraw up to 5 mL of the liquid formulation for unit administration.

[0148] In another embodiment, the liquid formulation comprising besudil is provided with an appropriately sized dosing cup, and a single unit dose is measured into the dosing cup prior to administration. In one embodiment, the dosing cup is sized to accommodate 1 to 10 mL of the liquid formulation comprising besudil for administration. In another embodiment, the dosing cup contains a fill line that reflects the level required to fill the dosing cup to approximately 5 mL of the liquid formulation for unit administration.

[0149] In one embodiment, the liquid preparation comprising besudil is packaged in a medicine bottle. In another embodiment, the bottle for packaging the liquid preparation comprising besudil contains a child-resistant cover or cap. In another embodiment, the liquid preparation comprising besudil is packaged in a colored bottle (e.g., an amber bottle or a blue bottle), and / or an opaque high-density polyethylene plastic bottle. In one embodiment, the liquid preparation comprising besudil is packaged in a Type III amber glass container of appropriate size with a tamper-proof cover. In one embodiment, the bottle has a polyethylene cover. In one embodiment, the liquid preparation comprising besudil is packaged in a medicine bottle containing a polyethylene liner.

[0150] In one embodiment, the liquid formulation is refrigerated at a temperature in the range of 2° C. to 8° C. In one embodiment, the liquid formulation stored at refrigerated temperature is accompanied by instructions stating that the formulation should be administered within a specified time period when the product is in its final dosage form.

[0151] In another embodiment, the liquid formulation is stored at ambient temperature (e.g., about 15° C.-25° C.). In one embodiment, the liquid formulation stored at ambient temperature is accompanied by instructions stating that the formulation should be administered within a specified period of time after reconstitution and / or placement in its final form.

[0152] In another embodiment, the liquid formulation is accompanied by instructions stating that once the liquid formulation is aspirated or drawn into a syringe, the formulation should be used within a specified time period (in one embodiment, within four hours) or should be discarded. Pharmaceutical compositions, uses and administration

[0153] In one aspect, the present invention provides pharmaceutically acceptable liquid compositions comprising a therapeutically effective amount of besudil formulated in a liquid diluent together with one or more pharmaceutically acceptable excipients.

[0154] In one aspect, the present invention provides pharmaceutically acceptable liquid compositions comprising a therapeutically effective amount of besudil formulated in a liquid diluent at a concentration equivalent to about 40 mg / mL besudil free base together with one or more pharmaceutically acceptable excipients.

[0155] In one aspect, the invention provides pharmaceutically acceptable liquid compositions comprising a therapeutically effective amount of besudelin formulated in a liquid diluent with a concentration equivalent to about 40 mg / mL besudelin free base together with one or more pharmaceutically acceptable excipients, wherein the dosage is between about 2.5 and 5.0 mg / kg, particularly about 3.0, 3.5, 4.0, 4.5 and 5.0 mg / kg, and more particularly about 4.0 mg / kg. In an embodiment, the dosage is about 3.0 mg / kg. In an embodiment, the dosage is about 3.5 mg / kg. In an embodiment, the dosage is about 4.0 mg / kg. In an embodiment, the dosage is about 4.5 mg / kg. In an embodiment, the dosage is about 5.0 mg / kg.

[0156] In one aspect, the present invention provides pharmaceutically acceptable liquid compositions comprising a therapeutically effective amount of besudild formulated in a liquid diluent at a concentration equivalent to about 40 mg / mL besudild free base together with one or more pharmaceutically acceptable excipients, wherein the administered dose is between about 2.5 and 5.0 mg / kg, particularly about 3.0, 3.5, 4.0, 4.5 and 5.0 mg / kg, and more particularly about 4.0 mg / kg, and wherein the maximum administered dose corresponds to about 200 mg besudild free base once or twice a day.

[0157] The patient can be an adult or pediatric patient. In embodiments, the patient is an adult patient. In embodiments, the patient is a pediatric patient.

[0158] In one embodiment, the present disclosure provides a method of administering a liquid formulation comprising besudil to a patient (including an adult patient) having a body weight of approximately equal to or greater than 40 kg at an equivalent dose of 200 mg once daily. In another embodiment, the present disclosure provides a method of administering a liquid formulation comprising besudil to a patient (including an adult patient) having a body weight of approximately equal to or greater than 40 kg at an equivalent dose of 200 mg twice daily or 400 mg once daily.

[0159] In one embodiment, the present disclosure provides a method of administering a liquid formulation comprising besudil at an equivalent dose of about 10 mg to 50 mg once daily to a pediatric patient having a body weight in the range of about 6 kg to less than 20 kg; in another embodiment, the present disclosure provides a method of administering a liquid formulation comprising besudil at an equivalent dose of about 50 mg once daily to a pediatric patient having a body weight in the range of about 10 kg to less than 20 kg; in another embodiment, in another embodiment, the present disclosure provides a method of administering a liquid formulation comprising besudil at an equivalent dose of about 100 mg once daily to a pediatric patient having a body weight in the range of about 20 kg to less than 40 kg; and in another embodiment, the present disclosure provides a method of administering a liquid formulation comprising besudil at an equivalent dose of 200 mg once daily to a pediatric patient having a body weight of approximately equal to or greater than 40 kg.

[0160] In one embodiment, the present disclosure provides a method of administering a liquid formulation comprising besudild to a patient at a dose between about 2.5 mg / kg and 5.0 mg / kg (e.g., about 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg / kg). In an embodiment, the present disclosure provides a method of administering a liquid formulation comprising besudild to a patient at a dose based on the patient's weight. In an embodiment, the present disclosure provides a method of administering a liquid formulation containing besudil to a patient at a dose of about 2.5 mg / kg; in another embodiment, the present disclosure provides a method of administering a liquid formulation containing besudil to a patient at a dose of about 3.0 mg / kg; in another embodiment, the present disclosure provides a method of administering a liquid formulation containing besudil to a patient at a dose of about 3.5 mg / kg; in another embodiment, the present disclosure provides a method of administering a liquid formulation containing besudil to a patient at a dose of about 4.0 mg / kg; in another embodiment, the present disclosure provides a method of administering a liquid formulation containing besudil to a patient at a dose of about 4.5 mg / kg; and in another embodiment, the present disclosure provides a method of administering a liquid formulation containing besudil to a patient at a dose of about 5.0 mg / kg. In an embodiment, the dose is administered once daily. In an embodiment, the dose is administered twice daily. In an embodiment, the patient is an adult patient. In an embodiment, the patient is a pediatric patient. In an embodiment, the maximum administered dose corresponds to about 200 mg of besudil free base once or twice daily.

[0161] The liquid formulations and pharmaceutical compositions disclosed herein are useful for inhibiting ROCK1 and / or ROCK2 enzymes, preferably ROCK2, and are therefore useful for treating diseases regulated by ROCK enzymes, such as autoimmune disorders and / or fibrotic disorders, including GVHD (chronic and acute), pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, radiation-induced fibrosis, or arterial fibrosis, cardiac fibrosis, endomyocardial fibrosis, renal fibrosis or liver fibrosis; moderate to severe psoriasis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), Crohn's disease, dermatitis (e.g., atopic dermatitis) and eczema, as well as other indications.

[0162] The liquid formulation may further be used to treat bronchiolitis obliterans syndrome (BOS), a potentially serious complication following lung or allogeneic hematopoietic stem cell transplantation (allo-HSCT).

[0163] The liquid formulation may further be used to treat chronic lung allograft dysfunction (CLAD) following lung transplantation.

[0164] The following abbreviations may be useful in considering the disclosure herein. abbreviation Examples Example 1: Excipient and preservative selection

[0165] Initially, the excipients listed in Table 5 below were identified for possible inclusion in besudil formulations. In exploring the use of these excipients, their suitability for use in a pediatric patient population was considered, for example, by reviewing regulations related to age-appropriate excipients for children in the target age group of 3 months to 12 years. Table 5: Excipients studied

[0166] All excipients listed in Table 5 were initially identified as suitable for evaluation. Banana 501013AP0551 flavor was the only excipient identified as unacceptable for use in children's formulations because it included residual amounts of benzyl alcohol, which was considered unacceptable for use in children's populations without sufficient justification.

[0167] Certain excipients are identified as having specified acceptable daily intake (ADI) levels. When considering the ADI levels, the maximum level included in the children's formulation of besudil was calculated taking into account the expected maximum daily dose and average weight (5.5 kg) for female children aged 3 months based on the 2000 CDC growth charts for the United States (representing the minimum weight for the target age group of 3 months to 12 years).

[0168] Compatibility testing of the excipients listed in Table 5 in combination with besudil was also performed. Binary mixtures of besudil and selected excipients were prepared at a 1:1 ratio of besudil to excipient, except for binary mixtures containing flavorings or preservatives, which were prepared at a ratio of 10:1. The appearance and related substances of the dried binary mixtures were analyzed after storage for 14 and 28 days at 2°C-8°C (control storage conditions) and 40°C / 75% RH. The visual appearance of the binary mixtures at the time of initial mixing was also tested.

[0169] Results from this compatibility study indicated that besudil was incompatible with hydrochloric acid and maleic acid over a 14 day period at 2°C-8°C and 40°C / 75% RH. Therefore, hydrochloric acid and maleic acid were not evaluated further for inclusion in liquid formulations.

[0170] The T=14 natural form compatibility data also showed poor chromatographic resolution between known impurities and besudil when combined with the following preservatives: sodium methylparaben, sodium ethylparaben, sodium propylparaben, and ethylparaben. Therefore, these preservatives were not further evaluated for use in preparing liquid formulations.

[0171] The T=28 day formulation compatibility data showed a small interaction of the potassium sorbate sample with an unknown related substance eluting at RRT 0.96 (which had an area % of 0.09% at 40°C / 75% RH). A pH preservative stability screening study was then conducted to identify suitable target pH, pH adjusters, and preservative systems suitable for inclusion in future formulations to be developed. For example, samples were prepared for evaluation that included the following components: 1.1 Besudil and water (control); 1.2 Potassium sorbate (without pH adjuster); 1.3 Sodium benzoate (without pH adjuster); 1.4 Sodium benzoate (adjusted to pH 3 with citric acid); 1.5 sodium benzoate (adjusted to pH 3 with tartaric acid); 1.6 sodium benzoate (adjusted to pH 3.0 with 0.1 M citrate buffer); 1.7 potassium sorbate (adjusted to pH 5.4 with 0.1 M citrate buffer); and 1.8 Sodium benzoate (adjusted to pH 4.0 with 0.1 M citrate buffer).

[0172] The above samples (1.1-1.8) were stored at 2-8°C, 25°C / 60% RH and 40°C / 75% RH for testing at T=0 (visual appearance and pH only), 14 days and 28 days. After storage, all samples were evaluated for visual appearance, pH and / or related substances and assays.

[0173] The results showed that after 14 days of storage at 40°C / 75% RH, chemical interactions were observed in sample 1.2 containing 2.0 mg / mL potassium sorbate and no pH adjuster, and the chemical interactions showed a significant increase by day 28. Therefore, it is not recommended to include this pH preservative system in future formulation prototypes.

[0174] In addition, sample 1.7 (containing potassium sorbate, adjusted to pH 5.4 with 0.1M citrate buffer) and sample 1.8 (containing sodium benzoate, adjusted to pH 4.0 with 0.1M citrate buffer) were not easily dispersed with manual agitation and showed significant physical changes upon visual observation after storage for up to 28 days at 25°C / 60% RH and 40°C / 75% RH. These samples (1.7 and 1.8) could not be dispersed by shaking, so these samples were not considered during further studies in the development of besudil formulations. All other samples were easily dispersed with manual agitation to form yellow opaque dispersions with yellow foam and were considered suitable for inclusion in future formulation prototypes. Example 2: Stability study using pH adjusters

[0175] A pH preservative stability screening study was conducted to evaluate the stability of besudil in selected aqueous liquid formulations using selected pH adjusters and preservatives to achieve a pH range of 3.0-5.0. Samples containing sodium benzoate as a preservative and pH adjusted with citric acid or tartaric acid were prepared and compared to samples without pH adjusters. Table 6 describes the samples used in this study. Table 6: Samples used in pH stability study

[0176] The above samples were prepared for XRPD analysis in three steps: solids were isolated by centrifugation at 3000 rpm for 20 minutes; the supernatant was decanted; and the samples were vacuum dried at ambient temperature for 18-24 hours overnight. XRPD data were obtained for solid samples. The XRPD data showed that the API (besudil) was stable in the formulation, which is a positive indicator for providing a stable formulation. The XRPD data showed that there were consistent additional peaks for the solids isolated from the samples containing sodium benzoate and no pH adjuster and the samples containing sodium benzoate and adjusting the pH with citric acid or tartaric acid, which is promising.

[0177] However, during the study, a downward shift in pH and significant physical changes were observed for samples containing potassium sorbate and citrate buffers. XRPD data for solid samples obtained from formulations containing citrate buffer (samples 2.5 and 2.6 in Table 6) showed changes as shown in the XRPD. Based on these observed changes, citrate buffer was not selected for use in the formulation. Example 3: Needle milling and jet milling using povidone

[0178] For suspension formulations, achieving uniform dispersion of the API particles throughout the liquid medium presents a challenge.The use of jet-milled and pin-milled forms of besudil in liquid media was investigated by evaluating the particle size distribution and homogeneity of the formulation both upon initial preparation and over time. (a) Comparison of needle grinding and jet grinding in water

[0179] To study the pin- and jet-milled dispersions of besudil in sterile irrigation water, samples were prepared by weighing a portion of the API into a clear glass bottle and adding sterile irrigation water to 40 mg / mL.

[0180] The samples were evaluated using a Sympatec Helos particle size analyzer using the general method for wet dispersions as outlined below: -R5 lens and R3 lens -Add sample until 10%-15% C is reached opt - Run the method to generate 3 replicates (samples were stirred and sonicated throughout the measurement run)

[0181] Figure 2A and Figure 2B Particle size distribution (PSD) data of needle-milled API in sterile irrigation water using R3 and R5 lenses are presented in Figure 1. Results of needle-milled API show reproducibility between replicates using both R5 and R3 lenses. When repeated testing, PSD data is not comparable to the original evaluation. It is difficult to obtain a starting C of 10%-15%.opt (likely due to poor dispersion of the API); therefore, additional sample was added to achieve the desired C opt .

[0182] The evaluation of wet dispersions using jet-milled Besudil gave poor results, with readings inconsistent between measurement runs. This is believed to be due to poor dispersion of the API. These results reflect that the use of jet-milled materials can be problematic and limit formulation development. For further use of jet-milled materials, additional obstacles in stabilizing formulations and improving the dispersion of jet-milled materials must be addressed. (b) Comparison of needle milling and jet milling using various suspending agents

[0183] To further evaluate and compare the pin and jet milling methods in developing liquid formulations, eleven different formulations were prepared, the composition details of which are shown in Table 5, Table 6, Table 7 below, and Table 8 below. In each of Tables 7, 8, 9, and 10, besudil mesylate was added at 48.496 mg / mL, which is equivalent to 40 mg / mL of free base when adjusted with a salt correction factor of 1.2124. Table 7: Details of formulations of needle-milled API (Samples 1A-3A) Table 8: Details of formulations of needle-milled API (Samples 4A-6A) Table 9: Details of the formulations of jet-milled API (Samples 1B-3B) Table 10: Details of the formulations of jet-milled API (Samples 4B-5B)

[0184] In preparing the formulations described in Table 7, Table 8, Table 9 and Table 10, the following steps were used: 1. Add approximately 40 mL of sterile water to a pre-calibrated beaker for rinsing. 2. While stirring, add sodium benzoate and mix until dissolved; 3. Add the suspending agent and mix until a homogeneous mixture is observed; 4. While mixing, add Besudil and mix until a homogeneous mixture is observed; 5. Measure the pH and, if necessary, add tartaric acid in aliquots until pH 3.5 ± 0.2 is reached; 6. Add sterile water to make up the volume for flushing and record the pH; and 7. Transfer the liquid formulation containing Besudil into a 60 mL clear glass bottle.

[0185] The samples were evaluated for ease of dispersion of the API, level of sedimentation, and ease of redispersion of any sediment.

[0186] In the absence of a suspending agent, the API dispersed easily for samples 1A (pin milled) (Table 7) and 1B (jet milled) (Table 9). After about 24 hours, a small amount of flocculation was observed, but the flocculation was easily resuspended by shaking. Both the pin milled and jet milled APIs foamed when shaken, but the foaming of the jet milled API (Sample G) was less obvious, with only a small upper layer of foam after about 30 seconds.

[0187] In contrast, in the batches (i.e., sample 2A and 2B / table 7 and table 9, respectively) containing xanthan gum (Xantural 75), API is not easy to disperse. These samples need homogenization to help API to disperse, and even after homogenization, two batches do not present homogeneous preparations. Draw a conclusion based on this observation, xanthan gum is not an ideal suspending agent. Sample 5A (table 8) containing sodium carboxymethylcellulose demonstrates serious flocculation, and is also difficult to disperse API even in the case of homogenization. After the content of the glass beaker is transferred to a glass bottle, a large amount of residues are also observed at the bottom of the glass beaker.

[0188] For other samples using pin grinding and jet grinding and hydroxyethylcellulose (Natrosol250HX) (samples 3A and 3B); Hydroxypropyl methylcellulose K4M (samples 4A and 4B) and povidone K30 (samples 6A and 5B), API was easily dispersed to form a yellow opaque suspension using a magnetic stirrer, with no observed differences between batches. When transferred from a beaker to a bottle, small aggregates of undispersed material were observed as residues, which suggests that homogenization may still be required. In general, it is observed that the use of povidone provides improved results.

[0189] For both samples 4B and 5B containing jet-milled API (Table 10), the API was easily dispersed to form a yellow opaque suspension using only a magnetic stirrer. No differences were observed between the samples. These observations are consistent with those previously recorded for equivalent batches incorporating needle-milled API (i.e., samples 4A and 6A [Table 8], which contain hypromellose K4M and povidone K30, respectively).

[0190] All samples in Table 7, Table 8, Table 9 and Table 10 were monitored at ambient temperature over 4 days and 2 weeks and evaluated for visual appearance. Samples 4B and 5B (Table 10) were made approximately 1 week after all other samples and were therefore evaluated only at T = 4 days. The results are summarized in Table 11 (needle milled API) and Table 12 (jet milled API). Table 11: Visual observations of needle milled formulations after 4 days and 2 weeks *After 20 days at ambient temperature, the sample converted to a solid gel-like formulation. Table 12: Visual observations of jet milled formulations after 4 days and 2 weeks

[0191] The observations showed that samples containing no suspending agent, Natrosol 250HX, Xantural 75 or sodium carboxymethylcellulose (made with both jet-milled API and pin-milled API) all showed various signs of separation, flocculation or solidification. Although samples (samples 4A and 4B) incorporating hypromellose K4M were observed to be yellow opaque suspensions and the API was visually dispersed after 4 days and / or 2 weeks of storage at ambient temperature, sample 4A (3 mg / mL hypromellose K4M with pin-milled API) had become a solid gel-like material when observed after 20 days at ambient temperature. When shaken to liquefy and redisperse, the API began to aggregate in the formulation. Therefore, it was determined that hypromellose K4M was not the best choice for further development.

[0192] After 4 days of storage at ambient temperature and after approximately 2 weeks, samples using Povidone K30 (made with both jet-milled and pin-milled API) remained yellow opaque suspensions and the API was visually dispersed. Based on this study, Povidone K30 was considered the primary suspending agent for further evaluation. Example 4: Particle size distribution analysis

[0193] Based on the results of the visual observation analysis in Example 3, four liquid formulations containing besudil were selected for further evaluation with respect to their particle size distribution. The following four formulations were measured: Sample 3A (pin-milled formulation with hydroxyethylcellulose); Sample 3B (jet-milled formulation with hydroxyethylcellulose); Sample 4A (pin-milled formulation with hypromellose K4M); and Sample 6A (pin-milled formulation with povidone).

[0194] Samples were evaluated using a Sympatec Helos particle size analyzer and sample was added until 10%-15% C was achieved.opt Two different lens assemblies were used, giving the following range for particle size assessment: R3 lens (0.5-175 μm) and R5 lens (4.8-875 μm). Three replicates were performed as part of the measurement run, with the samples being stirred and sonicated as part of the method.

[0195] For samples 3A and 3B, the R5 lens was used for evaluation. The results are shown in Figure 3A (Sample 3A) and Figure 3B (Sample 3B). The results showed that consistent replication was achieved for both batches, but the particle size recorded was larger than expected (when compared to the dry PSD of the milled API). It was noted during the study that the formulation containing Natrosol 250HX formed solid aggregates upon dilution with water, rather than dispersing.

[0196] This is probably a result of severe flocculation, which is consistent with the PSD measurements giving unexpectedly high values, with this effect being significantly more severe for the jet-milled sample (Sample 3B) comparing the two API samples.

[0197] For Sample 4A (needle-milled hypromellose K4M formulation), PSD was evaluated using both R5 and R3 lenses. The most consistent replication was obtained when using the R3 lens, and the results are shown in Figure 3C Multiple peaks of larger size were observed and believed to be due to bubbles. The stirrer speed was reduced from 80% to 50% to minimize aeration and bubble generation, resulting in consistent replication.

[0198] For Sample 6A (needle mill formulation with povidone), the sample was evaluated using both R5 and R3 lenses. As with Sample 4A, the most consistent replication was obtained when using the R3 lens; the results are shown in Figure 3D For this formulation, no flocculation / aggregation was observed as was present in samples 3A and 3B with Natrosol 250HX.

[0199] The D10, D50, and D90 values ​​for Sample 6A were consistent with those for Sample 4A, both of which were made using pin milling of Besudil. The results provide confidence that the method was able to produce accurate and consistent results for both formulation prototypes.

[0200] In summary, when testing samples 3A and 3B (containing hydroxyethylcellulose / Natrosol 250HX), aggregates were observed when diluted in water in the mix tank, which resulted in inconsistent data between replicates. Therefore, the results do not represent the expected particle size distribution of the API. Differences were observed between the pin-milled and jet-milled APIs, with larger aggregates and flocculation observed for sample 3B made using the jet-milled API compared to sample 3A (using pin-milling). This observation confirms the conclusion from Example 3 that jet-milled materials can be problematic and present an obstacle in formulation development.

[0201] When analyzing samples 4A and 6A (pin milled formulations with hypromellose K4M and povidone K30), consistent repetitions were obtained when using an R3 lens and after reducing the stirring speed from 80% to 50%. Occasional peaks at larger values ​​were observed, which were attributed to bubbles. The results for both samples 4A and 6A were similar. This suggests that these formulation prototypes allow for more consistent PSD results compared to particle size evaluation of wet dispersions of API in water alone, as shown in Table 1. Figure 2A and Figure 2B As shown in and discussed above in Example 3 (part (a)). Based on this evaluation, the needle milling process was considered suitable for developing liquid formulations containing besudil. Example 5: Study on xanthan gum and sodium CMC thickener system

[0202] In Example 3, samples containing 3 mg / mL xanthan gum (Xantural 75) (Sample 2A) and 3 mg / mL sodium CMC (Blanose CMC 7H3SXF) (Sample 5A) were shown to exhibit severe flocculation and difficulty dispersing the API. In this experiment, four additional formulations were studied to determine whether flocculation could be resolved via: -Add suspending agent after API is fully dispersed; - Reduced levels of xanthan gum and sodium CMC; - including silica products (i.e. 200 and 244FP) to prevent clumping and aid in resuspension.

[0203] According to the composition details presented in Table 13, Samples 7A, 8A, 9A and 10A were manufactured in 50 mL batch sizes.

[0204] The formulation preparation was planned according to the following steps: (1) to a suitable beaker, add approximately 40 mL of sterile water; (2) while mixing using a magnetic stirrer, add sodium benzoate and mix until dissolved; (3) while mixing, add besudil (pin milled) and mix for 15 minutes until a uniform dispersion is obtained; (4) while mixing, add Xantural 75 or Blanose CMC 7H3SXF and mix for 30 minutes until uniform; (5) measure the pH and, if necessary, add tartaric acid to adjust to pH 3.5 ± 0.2; and (6) add sterile water to make up the volume and mix the formulation for 15 minutes until uniform. Table 13: Formulations used for xanthan gum and sodium CMC experiments* *48.496 mg / mL is equivalent to 40 mg / mL adjusted by a correction factor of 1.2124.

[0205] However, all samples (7A, 8A, 9A and 10A) showed signs of flocculation after adding API (step 4), as previously observed in Example 3. Samples 7A and 8A containing xanthan gum were homogenized for about 1 minute using a Silverson homogenizer equipped with a universal disintegration head. The severe flocculation observed previously was still observed. Due to severe flocculation, manufacturing was stopped after step 4.

[0206] This experiment shows that changing the order of addition will not prevent flocculated material as previously observed. Reducing the suspending agent does show some improvement, but flocculation and thickening still occur, and therefore the manufacture of all four formulations is stopped. Based on these observations, xanthan gum and sodium CMC are excluded as suitable suspending agents for use in Besudil pharmaceutical formulations. Example 6: Experiment on povidone suspension system

[0207] Additional formulations as outlined below were made incorporating different levels of Povidone K30 as the primary suspending agent. 200 and 244FP was incorporated into these formulations to evaluate their effect on preventing agglomeration. Each formulation was made with a 100 mL batch size using both the pin-milled API and the jet-milled API to allow for comparative evaluation of the two. Composition details are shown in Tables 14A and 14B (pin-milled API) and 15A and 15B (jet-milled API). In each of Tables 14A, 14B, 15A, and 15B, besudiol mesylate was added at 48.496 mg / mL, which is equivalent to 40 mg / mL free base when adjusted with a 1.2124 salt correction factor. Table 14A: Needle Mill Formulations for Povidone Studies (Samples 11A-13A) Table 14B: Needle Mill Formulations for Povidone Studies (Samples 14A-16A) Table 15A: Jet Milled Formulations for Povidone Studies (Samples 11B-13B) Table 15B: Jet Milled Formulations for Povidone Studies (Samples 14B-16B)

[0208] The formulations in Tables 14A-14B and 15A-15B were prepared using the following steps: 1. Add approximately 80% of the total volume of sterile water to a suitable beaker for rinsing. 2. While mixing using a magnetic stirrer, add the sodium benzoate and mix until dissolved; 3. While mixing, add povidone K30 and mix for 30 minutes until uniform; 4. While mixing, add Besudil (pin milled or jet milled) and mix for 15 minutes until a uniform dispersion is obtained; 5. Measure pH. If necessary, adjust to pH 3.5 ± 0.2 with tartaric acid 6. Make up to volume with sterile irrigation water and mix for 15 minutes until homogeneous.

[0209] A 12 mL portion of each formulation was transferred to a 15 mL plastic centrifuge tube to test the sedimentation rate. A separate 30 mL portion of each formulation was also transferred to a 60 mL glass bottle for storage at ambient temperature and 50° C. for 7 days. After storage, all samples were evaluated for visual appearance, sedimentation level, ease of resuspension, and pH. (a) Initial deposition and appearance test

[0210] The visual appearance and sediment levels of samples in 15 mL centrifuge tubes stored at ambient temperature were recorded at the following time points; 1 hour, 2 hours and 24 hours, 4 days and 7 days. The results are presented in Table 16 (pin milled formulations) and Table 17 (jet milled formulations). In Table 16, "loosely packed" material was only observable by shining light through the sample. Table 16: Deposition levels over time at different povidone levels (needle grinding) *Note 1: Sample begins to thicken; noticeable when gently tilted, **Note 2: Sample thickened significantly; noticed upon gentle tilting. Table 17: Sedimentation levels over time at different povidone levels (jet milling) *Note 1: Evidence of solid material adhering to the centrifuge walls. **Note 2: A large amount of solid material adhered to the centrifuge walls; it is difficult to determine the amount of sediment **Note 3: Sample thickened and became solid-like. Can be held upside down and retain gel-like material.

[0211] Differences in sedimentation behavior were observed between samples containing pin-milled API and jet-milled API as reflected in Tables 16 and 17. For the pin-milled API, as the level of povidone K30 increased, the amount of sedimented material also increased. 200 and The inclusion of 244FP appears to reduce the sedimentation rate. For samples containing jet-milled API, as the level of povidone K30 increased and 200 or With the addition of 244FP, the second layer of sediments was reduced.

[0212] From this experiment, an important observation was made that the inclusion of colloidal silicon dioxide was beneficial in preventing agglomeration and allowing for ease of redispersion. The 244FP sample required the least number of inversions to completely redisperse the API.

[0213] The visual appearance of samples stored at ambient temperature in 30 mL glass bottles was also recorded after 24 hours and after 7 days at ambient temperature, as well as the visual appearance of these samples at 50° C. for 7 days. Elevated (50° C.) temperature was used to accelerate the conditions and stress the formulations. The "% Sediment" was calculated by measuring the height (mm) and total volume (mm) of the sediment in the glass bottle using a caliper. The results are presented in Table 18 (pin milling) and Table 19 (jet milling). Table 18: Visual appearance over time at ambient temperature (pin milling and jet milling) Table 19: Visual appearance after 7 days at 50°C (pin milling and jet milling) (b) Repeated deposition tests and determination results over time

[0214] To confirm the trends and rates of visually identified deposition, repeated deposition tests were performed using the following six samples previously described in Tables 14A-14B (pin milled) and Tables 15A-15B (jet milled):

[0215] Samples 13A and 13B (9.0 mg / mL Povidone K30 - needle milled and jet milled); Samples 14A and 14B (6.0 mg / mL Povidone K30 and 5.0 mg / mL Needle milling and jet milling); and Samples 15A and 15B (6.0 mg / mL Povidone K30 and 5.0 mg / mL 244FP, needle milling and jet milling).

[0216] The test was repeated on the above samples (samples were approximately 14 days old) that had been previously filled into 60 mL clear glass bottles. The samples were shaken vigorously 24 hours prior to the start of the study to resuspend any sediment.

[0217] On the analysis day, every kind of preparation was violently shaken for 20 seconds (vertical oscillation twice per second in 30cm distance), and it was settled for 5 minutes to eliminate some foams and inflation of preparation. After sample sedimentation, 5.0mL was taken and weighed in a 200mL flask with a tip below the suspension surface 5-10mm by using an automatic pipette. Then it was filled to about 180mL with diluent (50:50MeCN:H o), and supersound process 10 minutes. Allow sample equilibrium to room temperature, then make up volume.

[0218] Samples were tested at T=0, 4 hours and 24 hours. The samples were not shaken at the T=4h or T=24h time points to see how much besudil had settled out of the formulation. After sampling at T=24h, the remaining sample was shaken again and a sample was taken to see if the besudil was indeed resuspended. The assay results, which were not corrected for sample weight, are shown in Table 20 and plotted on Figure 4 middle. Table 20: Deposition measurement results of samples 13-15A / B over time

[0219] Initial Sampling: Results from the initial sampling phase (T=0) showed results within the proposed specification limits of 90%-110% of the labeled amount (or target concentration) of 40 mg / mL for all formulations. These results indicate that most, if not all, of the sediment in the samples was disrupted and resuspended.

[0220] Samples 15A and 14B had unexpectedly low measurements at the initial time point. These results suggest that the besudil may have adhered to the bottle walls where the foam had dried and may not have been fully redispersed. Even though the initial values ​​are low, they provide a baseline to indicate the deposition rate during the 24 hour test.

[0221] After the initial shaking, the amount of foam generated by all samples was similar. However, after the initial 5 minutes, the jet-milled API sample had almost no foam, while the pin-milled sample had a large amount of foam adhering to the pipette tip that had to be removed before transferring the sample. Although the earlier results suggest that jet-milled formulations can be problematic, the reduced foaming with the jet-milled samples is advantageous.

[0222] T = 4 hours: According to the results, the jet milled API remained better suspended with a value greater than 88%, compared to the values ​​of samples containing the pin milled API ranging from 58% to 84%. Only the pin milled sample (13A) containing povidone K30 had the lowest value, confirming that the addition of colloidal silica aided in the suspension of besudil. As in all samples, the samples containing The highest values ​​were obtained for the jet-milled samples.

[0223] After standing for 4 hours, the foam in the pin-milled samples was still visible but was easier to sample with most of the foam adhering to the vial walls rather than the pipette tip. At 4 hours, the foam in the jet-milled samples was minimal, reflecting the consistent reduction in foam using the jet-milled formulations at both T=0 and T=4h.

[0224] T = 24 hours: At the 24 hour time point, all formulations assayed below 85%, but the sample made with jet-milled API still assayed higher than the sample with pin-milled API. As with the T = 4 hour sample, the sample containing povidone K30 alone was significantly higher than the sample containing povidone K30 alone. and The formulation with had a higher assay value, which confirms that at 24 hours, the addition of colloidal silicon dioxide helps to suspend besudil. The samples containing jet-milled API in the povidone-only formulation provided higher assay results compared to the formulation containing The deposition rate was higher in the samples with 20% MgO. However, previous deposition tests have shown that this type of sediment is more loosely packed.

[0225] After 24 hours of standing, the foam from the pin-milled sample was completely adhered to the bottle wall and the formulation had a clear separation between the API sediment and the vehicle; for the jet-milled sample, the layers were not so clear.

[0226] T = 24 hours (after redispersion by shaking): The results at the 24 hour time point after shaking showed high assay values ​​that exceeded the proposed specification limits of 90%-110% of the target concentration. This is attributed to the low assay values ​​at the T = 4 hour and T = 24 hour time points, which means that there is more API to be redispersed in a smaller volume. Although these results are outside the specification limits, they do show that besudil is easily resuspended in the vehicle by shaking.

[0227] The pH of samples 11A to 16A and 11B to 16B was also recorded at ambient temperature and after 7 days at 50°C. After 7 days at ambient temperature, no significant changes were observed in any of the samples (pH values ​​were in the range of 3.2 to 3.6 both in the initial test and after 7 days). After 7 days at 50°C, a decrease in pH was observed, i.e., the pH of all samples was in the range of 2.7 to 2.9.

[0228] Based on this study, it was concluded that samples containing silica performed best in terms of ease of resuspension after 7 days of storage. Provides advantages over Some benefits of 244FP, but shown to contain The 244FP sample clumps less and is easier to resuspend. Since all samples have visually low viscosities over time and high sedimentation rates, ease of redispersion is considered an important characteristic, especially when considering products with longer shelf lives. In terms of ease of handling during manufacturing, 244FP also provides advantages over 200. For these reasons, it is generally believed that 244FP is a better choice.

[0229] Determining the level of povidone requires a delicate balance. Higher levels of povidone K30 are needed to reduce deposition; however, the maximum inclusion level of povidone K30 is affected by the acceptable daily limit (ADI) of 50 mg / kg body weight for this compound; therefore, the level needs to be consistent with the dosing regimen used for administration to the subject.

[0230] The choice of pin-milled API versus jet-milled API also involves consideration of many complex factors. As discussed in Examples 3, 4, and 5, various experiments have shown that pin-milling is preferred. Pin-milled APIs are easier to handle than jet-milled APIs, and better flow is observed. Pin-milled APIs are also easier to wet. When flocculation occurs, it is more severe in samples containing jet-milled APIs, and the jet-milled API is observed to adhere to the centrifuge walls.

[0231] Both the pin and jet milled samples were easily redispersed with no significant differences between the APIs. After 7 days, a smaller amount of sediment was observed in the batch containing the jet milled API.

[0232] Upon agitation, foaming occurred in the samples containing both the pin-milled and jet-milled APIs. The sample containing the jet-milled API exhibited slightly less foaming.

[0233] Thus, there are many reasons why one would initially conclude that needle milling of the API would be the preferred option for drug development. Nevertheless, the applicants herein have found that jet milling is the preferred method for drug development, particularly in light of long term stability considerations and lower sedimentation using jet milled products (which may only be more evident over longer storage periods). (c) Further Povidone Comparison Tests

[0234] As shown in Example 6 (a) and (b) above, formulations containing povidone K30 have relatively high sedimentation rates. In this study, a longer chain PVP povidone 90F was studied to determine whether it could provide a higher viscosity suspension to reduce sedimentation rates. To this end, formulations having the compositions shown in Table 21 were prepared. Table 21: Composition of Povidone 90 Samples with Added Tropical Fruit Mix Flavoring

[0235] The formulations of Table 21 were prepared using the following steps: 1. Add approximately 80 mL of sterile rinse water to a pre-calibrated beaker. 2. While stirring, add sodium benzoate, sucralose, and tropical fruit blend flavoring and mix until dissolved; 3. While mixing, add Povidone 90F and mix until a homogeneous mixture is observed; 4. If necessary, add while mixing 200 or 244FP and mix until homogeneous; 5. While mixing, add Besudil and mix until a homogeneous mixture is observed; 6. Measure the pH and, if necessary, add tartaric acid in aliquots until pH 3.5 ± 0.2 is reached; and 7. Add sterile rinse water to make up volume and record the final pH.

[0236] Samples 16-01 to 16-04 were tested for deposition. 2 mL of each sample was added to a 15 mL plastic centrifuge tube and the deposition was monitored over 7 days at room temperature. The deposition results were recorded initially, after 2 hours, and after 24 hours.

[0237] The results from this deposition testing are summarized in Table 22. Previous results including samples 14A and 15A (Table 16 above) were used for comparison. Table 22: Sedimentation results of formulations containing povidone 90 • The results previously obtained and reported in Table 16 are included here for comparison.

[0238] Sedimentation testing showed that the use of Povidone 90F reduced sedimentation levels when compared to the Povidone K30 samples (Samples 14A and 15A), with the majority remaining suspended after up to 7 days at ambient temperature.

[0239] When containing When compared with sample 16-04, it contains Sample 16-03 of 244FP required less inversion to redisperse the sediment after 7 days at ambient temperature, which may be more beneficial for longer term storage. 244FP is the preferred silica for inclusion in besudil formulations.

[0240] Samples 16-01 to 16-04 were also evaluated over time under accelerated storage conditions. 30 mL of each sample was transferred to 2 x 60 mL glass bottles and stored at ambient temperature and 40 ° C / 75% RH to test visual appearance, sedimentation level, redispersion and pH of any sediment after 7 days. Through this study, it was found that when stored at ambient temperature for T = 7 days, all 4 samples containing povidone 90F stored in 60 mL transparent glass bottles were easily reconstituted by gently shaking for about 10 seconds. After 7 days of storage at ambient temperature, foaming was observed after redispersion, even if the sample contained flavoring.

[0241] For samples with T=7 at 40°C / 75%RH, all four samples containing Povidone 90F appeared more viscous after storage at 40°C / 75%RH, but redispersed by gentle shaking for about 10 seconds. No foaming was observed.

[0242] Finally, four samples were selected for study by XRPD analysis, specifically, samples 15A and 16A (Table 14B), sample 17 (Table 23), and sample 16-03 (Table 21). 30 mL of each sample was transferred to a 1 x 60 mL glass bottle for analytical testing initially and after seven days at ambient temperature and accelerated storage conditions (i.e., 50° C.) (where the sample was agitated to redisperse any sediment). Separate samples were used for XRPD analysis. The XRPD data showed that all samples were consistent with the original form of the API, and the pH drift was unrelated to changes in the API form. There was no problem with the level of sodium benzoate included at 0.5 mg / mL. Example 7: Preservatives and Sodium Benzoate Levels

[0243] Based on the low natural pH of the API in water of approximately pH 3 and the instability observed in the samples containing potassium sorbate described in Example 1, sodium benzoate was selected as the preferred choice for the preservative.

[0244] Four formulations were prepared to evaluate the effect of adjusting the sodium benzoate level and to select a preferred level for use in a besudil formulation. Specifically, four samples 17 to 20 described in Table 23 were prepared, which included high, medium, and low levels of sodium benzoate with 9 mg / mL of povidone K30 and 5 mg / mL of 244FP, which was based on previous examples 3 (povidone) and 6 ( 244FP) was identified as a preferred suspending agent. Sample 20 without preservative was prepared as a reference. Table 23: Formulations used to assess sodium benzoate levels *48.496 mg / mL is equivalent to 40 mg / mL free base adjusted with a salt correction factor of 1.2124

[0245] The above formulations (Samples 17-20) were prepared to a volume of 150 mL using the following manufacturing steps: 1. Add 130 mL of sterile flushing water to a 250 mL glass beaker; 2. While stirring using a magnetic stirrer, add sodium benzoate and mix until completely dissolved; 3. While stirring, add povidone K30 and mix until completely dissolved; 4. While stirring, add FP and mix until dispersed and a homogeneous mixture is obtained; 5. While stirring, add Besudil (pin milled) and mix until dispersed and a homogeneous mixture is obtained; 6. Record the pH and, if necessary, add tartaric acid to adjust to a target pH of 3.5 ± 0.2; 7. Sterile irrigation water was added to make up the volume and the formulation was mixed until homogeneous.

[0246] Samples 17 to 20 were PET tested. The results showed that all batches containing sodium benzoate at all levels (0.25 mg / mL, 0.50 mg / mL and 1.00 mg / mL) provided suitable preservative efficacy. The results confirmed that all levels of sodium benzoate can be considered alternative choices, and the intermediate level of 0.50 mg / mL would be a suitable level for including sodium benzoate (to allow for any potential degradation of sodium benzoate to longer term stability). Sample 20, which did not contain sodium benzoate, failed to provide adequate protection against microbial growth. Therefore, it is advantageous to include a preservative in a ready-to-use liquid formulation containing besudil. Example 8: Effects of Sweeteners and Flavorings

[0247] A range of vehicles including sweeteners and flavorings were developed to identify those vehicles suitable for reconstituting besudil in a bottle and potentially administering to a subject. Initial formulation development studies were conducted in small-scale batches of up to 100 mL. First, vehicles were manufactured according to the composition details shown in Tables 24A-24B and Tables 25A-25B.

[0248] The sweetener level was selected based on the finding that the API has a bitter taste profile, necessitating an improvement in the palatability of the formulation for effective subject acceptance. The vehicle was prepared by: (a) adding the desired amount of sterile rinse water to a 125 mL clear glass bottle; (2) adding the desired amount of sweetener and mixing using a vortex mixer until visually dissolved; and (3) adding the desired amount of flavoring (where applicable) and mixing using a vortex mixer until visually dissolved. Table 24A: Composition details of vehicles containing sucralose (Vehicles 1-3) Table 24B: Composition details of vehicles containing sucralose (Vehicles 4-6) Table 25A: Composition details of vehicles containing acesulfame potassium (Vehicles 7-9) Table 25B: Composition details of vehicles containing acesulfame potassium (vehicles 10-12)

[0249] The visual appearance and pH of each vehicle 01-12 were characterized. The visual observation of vehicles 01-02 (containing sucralose) and vehicles 07-08 (containing acesulfame potassium) was clear colorless solution. The visual appearance of the vehicles containing the combination of sweeteners and flavoring agents was different, where all batches were observed to be turbid liquids, but the vehicles containing citrus flavoring (vehicles 06 and 12) were off-white opaque liquids. The pH range of these vehicles was a low of 4.3 (vehicles 03 and 09) to a high of 7.0 (vehicles 01 and 02), which fell within the proposed acceptance criteria.

[0250] Samples of reconstituted besudil were then prepared to evaluate the performance of vehicles 01-12. For the preparation of reconstituted besudil in a bottle, 727.44 mg of besudil mesylate (600 mg besudil free base) was transferred to a 60 mL Type III clear glass bottle. 15 mL of each vehicle listed in Tables 24A-24B and 25A-25B was added to the bottle and the sample was agitated to reconstitute.

[0251] All samples were reconstructed by gently stirring for 10 seconds. All samples containing acesulfame potassium (using vehicle 07-12) showed signs of flocculation and significant thickening after about 5 minutes. Therefore, it is believed that all samples containing acesulfame potassium are not suitable for use in besudild formulations. The visual appearance of the samples containing sucralose (using vehicle 01-06) is an opaque yellow dispersion and shows no signs of flocculation. Therefore, it is believed that these vehicles containing sucralose are suitable for use in formulations. In addition, the pH of the reconstructed samples using vehicle 01-06 falls within the range of 3.1 to 3.2, which is a pH suitable for the final formulation and does not require pH adjustment.

[0252] Further work was conducted to investigate the effect of adding flavorings to the besudil formulation on post-agitation foaming. A tropical fruit blend flavoring was selected for this evaluation, and experiments were conducted using previously prepared Sample 11A (Table 14A) and Sample 12B (Table 15A) (in Example 6 above). Each sample included sodium benzoate and povidone K30 as preservatives without the addition of silicon dioxide / suspending agents, with Sample 11A including pin-milled besudil and Sample 12B including jet-milled drug.

[0253] For each sample, approximately 30 mL was transferred to a 60 mL glass bottle. To evaluate the foaming and the effect on flavor addition, the following experiment was performed. 1. Agitate each sample by shaking for 30 seconds (approximately 2 inversions per second) and record the visual appearance and foaming; 2. Leave each sample undisturbed until no foam / aeration exists; 3. Add 0.12 g tropical fruit blend flavor (about 0.4% w / v) to each sample and mix the samples using a vortex mixer to dissolve.

[0254] Table 26 summarizes the composition of the liquid formulations containing besudil examined in this study after the addition of flavoring. Table 26: Composition of Samples 11A and 12B after adding flavoring

[0255] Note the following observations.

[0256] In the absence of flavoring, a large amount of foam and aeration can be observed after agitation for 30 seconds. After undisturbed standing for 5 minutes, there is a significant difference in foaming using pin-milled and jet-milled APIs without added flavoring. Sample 11A (pin-milled API) has a longer-lasting foam compared to Sample 12B (jet-milled API).

[0257] After the flavoring was added, only minimal aeration was observed after agitation for 30 seconds. Once the tropical fruit blend flavoring was added, there was no significant difference in the amount of foam or aeration in the samples containing the pin milled API compared to the jet milled API. Therefore, it was concluded that the tropical fruit blend flavoring is suitable for use in the besudil formulation, and it is expected that these flavorings will have a similar effect on foaming based on previous observations using other alternative flavorings listed in Table 24A and Table 24B. Example 9: Scale-up of comparative analysis and homogenization procedures

[0258] A comparative analysis was performed of a scaled-up system containing 9 mg / mL povidone 90F and an alternative system containing 6 mg / mL povidone K30 and needle-milled API, as well as a system containing 9 mg / mL povidone 90F (using jet-milled API to allow comparison with needle-milled API). 244FP was used as a suspending agent, sodium benzoate was included at 0.5 mg / mL to provide a preservative effect, and lemon flavoring was used.

[0259] Details of the compositions used in this study are further shown in Table 27. Table 27: Details of compositions used for comparative and stability studies

[0260] The bulk homogeneity of samples 21-23 was analyzed as follows: The formulations were gently agitated by inverting 10 times prior to sampling to ensure any sediment was resuspended prior to testing.

[0261] Two replicates were taken from each bottle to provide n=6. 1.00 mL of the suspension was added to a 200 mL volumetric flask and made up to about 180 mL with diluent (50:50 MeCN:H2O) and sonicated for 10 minutes. The sample was allowed to equilibrate to room temperature and then made up to volume with diluent. The sample was then filtered through a 0.45 μm PTFE filter, the first 2 mL discarded as waste, and then loaded into a vial for analysis.

[0262] The assay values ​​for both sodium benzoate and besudil were collected from the initial assessment of overall homogeneity. The results are shown in Table 28. Table 28: Scaled-up bulk homogeneity data for samples 21-23

[0263] The overall homogeneity data showed that the sodium benzoate assay content of all batches met the specification limit of RSD ≤ 6%. However, the besudil assay content of sample 21 was significantly higher than the acceptance criteria of 90%-100%, and showed significant differences in samples 22 and 23, whose levels did not meet the specification limit of RSD ≤ 6%.

[0264] Samples 21-23 were also tested for viscosity using a Brookfield DV1 LV cone and plate viscometer (using spindle CP41, 30 RPM, at 20° C.). The results are shown in Table 29. Table 29: Viscosity test results of scaled-up comparative samples 21-23

[0265] The viscosity values ​​recorded were unexpectedly very low, especially for Sample 22 containing Povidone 90 F. Previously it had been observed that the system thickened over time, but this was not observed for Samples 21-23.

[0266] Although several batches have been successfully manufactured at a smaller scale using only overhead mixing, it was determined from this study that overhead mixing was not sufficient to adequately disperse the API and form a homogenous suspension when scaled up, which resulted in widely varying assay results in this example. It was determined that it was advantageous to include a homogenization step when scaled up to obtain a homogenous suspension. Further studies confirmed that the use of a Silverson homogenizer improved API homogeneity. Example 10: System Optimization and Favorable Parameters

[0267] Following the scale-up work in Example 9, additional comparative samples were prepared and analyzed with the initial hypothesis that the viscosity of the system would be increased to subsequently reduce the deposition rate in the PVP / formulation system. The following two approaches were investigated to optimize the system: 1. Increase the level of Povidone 90F: PVP has been shown to be effective in wetting the API and can be used as a thickener. However, according to the Pharmaceutical Development and Control Strategy Plan, Povidone 90F should be increased to a level not higher than the acceptable ADI based on the average weight of a 3-month-old female child (5.5 kg). PVP has an ADI of 50 mg / kg body weight, so it should not exceed 275 mg / dose (based on 1 dose / day) 2. Addition of methocel K4M in combination with povidone K30: Previously, samples containing alternative suspending agents with PVP were observed to gel significantly over time and aggregate significantly. Since PVP aids in the wetting of the API, work was conducted to investigate the use of alternative suspending agents in combination with povidone K30 (based on available information that shorter chain PVP is more likely to aid in wetting than povidone 90F). Methocel K4M was selected as an alternative thickening agent because it showed the lowest physical incompatibility with besudil compared to the other suspending agents tested.

[0268] To investigate these methods, four formulations were prepared at 200 g scale with the compositional details outlined in Table 30A and Table 30B. The samples were placed into a short-term stability study and visual appearance, assay, pH and viscosity were evaluated at T=14 days and T=1 month at 25°C / 60% RH and 40°C / 75% RH. Table 30A: Use of formulations in scale-up optimization studies (Samples 24-01 and 24-02) Table 30B: Use of formulations in scale-up optimization studies (Samples 24-03 and 24-04)

[0269] All four systems (24-01 to 24-04) all show more viscous than observed with previous formulations.When initially mixed, all four systems are all flowable and API wets well.After standing for 24 hours, sample 24-03 containing methocel K4M becomes semisolid, but becomes fluid state after 10 seconds of vibration.After one week, sample 24-01, 24-02 and 24-04 all have certain deposition.When compared with sample 24-02 (containing higher level of polyvidone 90F) and sample 24-04 (containing same level of polyvidone 90F but using jet milling API preparation), sample 24-01 shows the sediment with the highest amount.Sample 24-03 shows no signs of deposition, but is semisolid.

[0270] At T=14 days and T=28 days, the visual appearance of the samples was re-evaluated. Sample 24-03 remained highly viscous, opaque yellow, and gel-like, but readily liquefied under shaking to form an opaque yellow liquid. Systems 24-01, 24-02, and 24-04 were flowable opaque yellow liquids with a small amount of sediment that was redispersed by shaking.

[0271] Note that "banding" was observed where the API adhered to the glass vial wall. Interestingly and surprisingly, more API was observed to adhere to the wall when using the system containing the pin-milled API compared to the jet-milled API (i.e., samples 24-03 and 24-04). Therefore, the following conclusions were drawn: 1. System 24-03 containing Methocel K4M formed a solid / gel-like structure. However, the structure was easily shaken to form a liquid again. It remained liquid-like, but recovered its structure after 24 hours. 2. Among the remaining samples, 24-04, which had 30 mg / mL Povidone 90F and used jet milled API, had the lowest amount of sediment. Sample 24-01, which contained 30 mg / mL Povidone 90F and used pin milled API, had the highest sedimentation rate and was most likely to stick to the glass bottle. 3. Sample 24-02 containing 50 mg / mL Povidone 90F manufactured using needle milled API had a reduced deposition rate compared to Sample 24-01.

[0272] It was determined from this study that the system containing Povidone 90F (Sample 24-04) using jet-milled Besudil unexpectedly provided the best results by visual inspection, with the lowest amount of sediment.

[0273] All four samples were also analyzed via the sodium benzoate and besudil assays. For all samples, the assay data for both besudil and sodium benzoate were comparable to each other. Samples obtained with a Push in Bottle Adaptor (PIBA) provided comparable results to samples collected using an automated pipette, indicating that either sample transfer method is appropriate.

[0274] The assay values ​​for both besudil and sodium benzoate for samples 24-02 to 24-04 at both storage conditions and sample 24-01 at 25°C / 60% RH were within the specification limits of 90%-110% of the labeled amount. For samples 24-02 to 24-04, there were no significant changes in the assay data when compared to the initial and 14 day time points.

[0275] The pH values ​​of the four samples at 25°C / 60% RH and 40°C / 75% RH at initial, T=2 weeks and T=1 month are summarized in Table 31. Table 31: pH results of system optimization (samples 24-01 to 24-04)

[0276] Downward pH drift was observed in all systems, with the largest changes observed at 40°C / 75% RH. Initial pH preservative stability work indicated that in samples where pH drift was observed, the pH tended to drop to about pH 2.9. Benzoic acid is most effective at pH 3, so even with a downward drift, this pH would be acceptable for preservative efficacy. pH preservative stability studies indicated that no chemical changes occurred at this pH. XRPD analysis indicated no form changes for samples at pH 3, except in the presence of citrate buffer as described in Example 2.

[0277] Since it was observed that the pH tended to drift towards about pH 3.0 over time and then maintain this approximate pH value, and given the stability of besudil at this value, pH 3.0 ± 0.5 was chosen as a suitable target pH range for liquid formulations containing besudil.

[0278] The viscosity of these four systems was also tested using a Brookfield cone and plate viscometer (spindle CP52) (tested at 20°C). Prior to testing, the samples were shaken vigorously for 10 seconds to disperse any sediment. Table 32 summarizes the viscosity data at 25°C / 60%RH and 40°C / 75%RH at initial, T=2 weeks, and T=4 weeks. Table 32: Viscosity data for samples 24-01 to 24-04 at initial, T=14 days and T=28 days

[0279] As can be observed, a slight change in viscosity is observed, but there is no significant change relative to the initial and after 2 weeks and 4 weeks at 25°C / 60%RH and 40°C / 75%RH. The trend between the systems remains the same at all time points, with the most viscous being sample 24-03 containing 10mg / mL povidone K30+5mg / mL Methocel K4M (with jet-milled API), and the least viscous being sample 24-01 containing 30mg / mL povidone 90F. Surprisingly, the higher viscosity of sample 24-03 is not associated with reduced sedimentation rates, as initially proposed. As indicated above, among all samples, 24-04, which has 30mg / mL povidone 90F and uses jet-milled API, has the lowest amount of sediment, but also has one of the lower viscosity rates.

[0280] Overall, after comparative analysis, sample 24-02 (50 mg / mL povidone 90F) provided the most consistent assay results, especially compared to sample 24-01 (30 mg / mL povidone 90F), which also used needle milled API. Sample 24-03 showed thickening / gelling as observed in the past, but liquefied more easily when shaken, and did not show the same clumps after shaking as previously observed in batches containing methocel K4M but without povidone K30.

[0281] However, it was also observed that the use of jet-milled API showed improvements in the following aspects compared to pin milling: reduction in API "banding" on glass vials, and reduction in sedimentation rate. This was already observed when comparing samples 24-01 (pin milled) to 24-04 (jet milled), both of which contained 30 mg / mL povidone 90F. By preparing scaled-up development samples and increasing levels of PVP, agglomeration was not observed when jet-milling API was used in early development. Therefore, it was concluded from this study that jet-milling besudil would be most advantageous for use in a liquid formulation containing besudil. Reference is made to Table 33 when summarizing the components of the liquid formulation. Table 33: Formulation components and analysis Example 11: Taste and bioavailability study

[0282] Following development of a liquid formulation containing besudil as previously described, studies were conducted to (i) evaluate the taste attributes (aroma, sweetness, bitterness, flavor, mouthfeel / texture, grittiness, and aftertaste) and overall acceptance of the liquid formulation containing besudil; (ii) determine the relative bioavailability of the liquid formulation containing besudil compared to the besudil oral tablet in the fed state; and (iii) determine the effect of food on the PK of besudil after administration of the liquid formulation containing besudil.

[0283] The first part of this study involved a "sip and spit" taste evaluation to profile the flavor profile of the formulation and identify a suitable flavoring system. The outcome of the taste study was then taken into consideration in the next part for the relative bioavailability evaluation of the liquid formulation compared to the oral tablet. For this study, a 48.496 mg / mL besudil liquid formulation (40 mg / mL free base) was used. This concentration was selected to minimize the volume to be administered in the relative bioavailability study conducted in healthy volunteers later (5 mL for the 200 mg dose) and to facilitate comparison with the reference tablet (200 mg). (a) Part I - Taste Studies

[0284] The purpose of this first study was to identify the optimal flavoring and sweetener combination to enhance or improve the palatability of a liquid formulation of besudil for oral administration of the drug to a target patient population of 3 months to 12 years of age. (i) Methods

[0285] Twelve healthy adult male subjects (mean age 35.3 years, minimum 23 years, maximum 52 years) were enrolled in the taste profile portion of the study. Subjects stayed from the day before product administration (Day 1) and left on Day 2. On Day 1, subjects were randomized to receive a total of six liquid formulations containing besudil according to 1 of 6 treatment sequences (ABFCED, BCADFE, CDBEAF, DECFBA, EFDACB, and FAEBDC), with two subjects assigned to each treatment sequence.

[0286] Each individual treatment (e.g., A, B, C, D, etc.) involved a single 40 mg / mL (200 mg in 5 mL) oral dose of besudil. Bottle formulations and 6 different regimen vehicles were used as follows: A = sterile water (Vehicle 1), B = low sucralose (Vehicle 2), C = high sucralose (Vehicle 3), D = citrus low sucralose (Vehicle 4), E = tropical fruit blend low sucralose (Vehicle 5), and F = lemon low sucralose (Vehicle 6).

[0287] Prior to the first administration of the liquid formulation containing besudil (Day 1 or before breakfast is completed on Day 1 [pre-dose]), subjects were given a training questionnaire using an example fluid (e.g., citrus juice / soda). Subjects received test besudil by bottle regimen according to the randomization schedule.

[0288] Subjects received the first single oral dose of their regimen 2 h after taking a standard breakfast in the morning of Day 1. Each regimen followed the same study design: subjects received a single dose of the besudil liquid formulation in a bottle, which they held in their mouth for approximately 1 min before expectorating.

[0289] Immediately after spitting out, the subjects completed the questionnaire individually and privately to rate the overall acceptance using a 9-point Likert scale, which takes into account seven key taste characteristics (smell, sweetness, bitterness, flavor, mouthfeel / texture, grittiness, and aftertaste). Specifically, the ratings according to this scale are as follows: 1 = extremely unpleasant, 2 = very unpleasant, 3 = moderately unpleasant, 4 = slightly unpleasant, 5 = neither like nor dislike, 6 = slightly like, 7 = moderately like, 8 = very like, and 9 = extremely like.

[0290] This method was completed for each of the 6 regimens. Besudil was not swallowed. There was a washout period of approximately 30 min (including palate cleansing) between tasting each regimen. During this time, subjects used tap water (freely administered in 50 mL aliquots) and crackers to clean their palates before another tasting. All regimens were tasted on the same day.

[0291] A single plasma PK sample was collected approximately 1 h after the final dose (before departure from the clinical unit). This sample was retained for analysis only in the event of accidental swallowing of formulation by a subject and / or for the purpose of studying treatment-emergent adverse events (TEAEs) believed to be related to the IMP. No subject reported accidental swallowing of formulation and / or TEAEs believed to be related to the IMP in Part 1 of the study; therefore, this plasma PK sample did not need to be analyzed for any subject and was destroyed.

[0292] Subjects remained on site until 1 h after the final taste / palatability assessment. To ensure the subject's ongoing well-being, a follow-up call was made 3 to 7 days after the final dose. (ii) Results

[0293] The median (Min-Max) scores for each taste / palatability attribute are summarized in Table 34. The median difference data are summarized in Table 35. Table 34: Min-Max Scores for Each Attribute of Besudil Oral Liquid Formulations in Part I Taste Study Table 35: Median Difference Results for Besudil Oral Liquid Formulations in Part I Taste Study

[0294] Vehicle 1 (sterile water - reference) was the lowest scoring vehicle with a median overall acceptability score of 3.0, indicating that the panel had a moderate dislike for the product. The flavoring options (Vehicles 4 [Citrus Low Sucralose], 5 [Tropical Fruit Blend Low Sucralose], and 6 [Lemon Low Sucralose]) received median scores of 6.5, 7.0, and 7.0, respectively, indicating that the addition of flavoring improved the overall taste / palatability profile of the IMP.

[0295] The taste / palatability attributes with the lowest median scores for Vehicle 1 were flavor, mouthfeel / texture, and grittiness (3.5, 3.0, and 3.5, respectively), indicating that these attributes were the cause of the aversion. With the addition of the flavored low sucralose vehicles (Vehicles 4, 5, and 6), the median scores for each of these attributes increased, ranging from 6.5 to 7.0 for flavor, 4.5 to 5.0 for mouthfeel / texture, and 4.0 to 5.5 for grittiness. With the addition of the flavored low sucralose vehicles, the median scores for all other taste / palatability attributes improved, indicating that the subjects preferred the taste profiles of these vehicles.

[0296] For each taste attribute, the Friedman test was statistically significant at the 5% significance level, indicating that at least one formulation had a significantly different taste score than the other treatments (p<0.001 for overall acceptance, taste, sweetness, bitterness, flavor, and mouthfeel / texture; p=0.041 and p=0.001 for grittiness and aftertaste, respectively).

[0297] For all pairwise comparisons and taste attributes, the median pairwise differences were positive, indicating that the use of sweeteners and / or flavorings showed improved acceptance compared to the reference vehicle, sterile water.

[0298] Across all taste attributes, either the tropical fruit blend low sucralose or lemon low sucralose vehicles showed the greatest improvement or joint greatest improvement (based on the median of the pairwise differences) compared to the reference vehicle. No benefit was shown for increasing the sucralose content of the vehicle. (b) Section 2 - Bioavailability (i) Methods

[0299] Part 2 was a single-center, open-label, randomized, three-stage design to evaluate the relative bioavailability of selected besudil liquid formulations compared to besudil oral tablets and the effect of food on liquid formulations containing besudil in 18 healthy male subjects. At the screening visit (Day -28 to Day -2 of Part 2), subjects underwent a preliminary screening procedure to determine their eligibility for Part 2 of the study. Subjects who participated in Part 1 of the study were allowed to participate in Part 2.

[0300] Subjects received a single oral dose of 200 mg of besudil in 3 phases. Three different regimens (G, H, and I) were used in these three phases. In regimen G, besudil was administered in tablet form (reference); and in regimens H and I, besudil was administered in the form of an oral liquid formulation.

[0301] In Period 1, Day 1: Subjects were randomized and then administered the first dose of IMP to 1 of 6 treatment sequences (GHI, HIG, IGH, IHG, GIH, and HGI), with 3 subjects assigned to each treatment sequence. On Day 1 of each study period (Phase 1, 2, and 3), subjects received the regimen in Table 36: Table 36: Protocol used in the bioavailability / follow-up taste study

[0302] Regimen H was administered to subjects as a low sucralose liquid formulation without flavoring in the fasting state, and Regimens G and I were administered to subjects in the fed state. Regimen G was administered with a total of 240 mL of water. For Regimens H and I, subjects drank water immediately after IMP administration to a total volume of 240 mL (including the dosing volume).

[0303] Each study phase followed a similar design. Subjects were admitted to the clinical unit the morning before the first IMP administration (Day -1 of Phase 1) for eligibility confirmation and baseline procedures. For Protocol H only, subjects were given a training questionnaire using an example fluid (e.g., citrus juice / soda) before IMP administration to demonstrate how the questionnaire should be completed. This was done on Day -1 (applicable only when Protocol H was administered in Phases 2 or 3) or before dosing on Day 1 (pre-dose). It was originally planned to use the flavoring system determined to be the most favorable according to Part 1 (lemon flavoring and low sucralose solution) in Protocol H in Part 2, however, a low sucralose oral liquid formulation without flavoring was used instead.

[0304] Subjects received a single dose of IMP on the morning of Day 1 after a minimum of 10 h overnight fast (Schedule H, fasting state) or after a standard breakfast (Schedules G and I, postprandial state). Blood samples were collected regularly for PK analysis. Following administration of Schedule H, subjects completed a written taste / palatability questionnaire individually and privately.

[0305] Subjects will reside on the clinical unit for 10 consecutive nights, covering all 3 treatment periods. All subjects will remain on site until 72 h after the final dose for safety and PK assessments. There will be a minimum washout period of 3 days between each IMP administration. To ensure the continued well-being of the subject, a follow-up call will be made 3 to 7 days after the final dose. If a subject reports any AEs after leaving that represent a cause for concern, they will need to come to the clinical unit for a follow-up assessment. This will be an unscheduled visit. (ii) Results a. Taste / palatability

[0306] Overall, most subjects rated the oral liquid formulation (Regimen H) as 5 ("neither liked nor disliked"), 6 ("mildly liked"), or 7 ("moderately liked"). The overall taste profile scores for Regimen H were comparable to those for Regimen B in Part 1 utilizing the same sweetener combination (i.e., low sucralose). b. Bioavailability

[0307] After a single oral administration of the liquid formulation comprising besudil, the absorption of besudil was faster (median Tmax 2h) compared to besudil administered as a tablet reference (median Tmax 3h).

[0308] Besudil was administered as a reference tablet in the fed state. The median Tmax for absorption of besudil occurred at 3.00 h after the dose, after which the concentration showed a rapid decline, resulting in a geometric mean T1 / 2 of 9.393 h, similar to those observed previously. The inter-subject variability associated with exposure (Cmax and AUC) was moderate, ranging from 26.1% to 39.6%. The median Tmax of KD025m1 and KD025m2 after administration of besudil tablets was similar to that of besudil, at 2.00 h and 3.00 h after the dose, respectively. The concentration of KD025m1 was almost impossible to quantify, and the terminal half-life was reliably estimated for only 2 subjects. The geometric mean T1 / 2 of KD025m2 was 2.466 h, with reliable estimates in 8 of the 18 subjects. Compared with the parent, the two metabolites had higher inter-subject variability in relation to exposure, ranging from 37.9%-63.9% and 52.5%-75.3% for KD025m1 and KD025m2, respectively.

[0309] The key geometric mean (geometric coefficient of variation [CV%]) PK parameters of plasma besudil following dosing with besudil are summarized in Table 37 below. Table 37: Comparative PK parameters of plasma besudil administered with tablets versus liquid formulations n: number of subjects with observed results; N: number of subjects in the dataset; NC: not calculated a Median value (range)

[0310] After a single oral administration of besudil as a tablet reference to healthy male volunteers in the fed state (Schedule G), besudil concentrations were evident between 0.5h and 3h in all subjects. After dosing with a liquid formulation containing besudil in the fasting and fed state (Schedules H and I), concentrations were evident in all subjects from 0.5h onwards. Maximum plasma concentrations occurred between 1h and 5h after the dose.

[0311] The concentration then decreases in a biphasic manner and remains quantifiable until between 24h and 72h after the dose. The resulting elimination half-lives range from 4.84h to 33.02h, 5.36h to 23.76h, and 2.38h to 15.73h, respectively. The geometric mean half-life ranges from 6.788h to 9.499h.

[0312] The key geometric mean (geometric coefficient of variation [CV%]) PK parameters for plasma KD025m1 and KD025m2 following dosing with besudil are summarized below in Tables 38 and 39, respectively. Table 38: Comparative PK parameters of plasma KD025m1 administered with tablets versus liquid formulations Table 39: Comparative PK parameters of plasma KD025m2 administered with tablets versus liquid formulations

[0313] Administration of the liquid formulation containing besudil in the fasted state showed an earlier median Tmax (1.5 h) compared to the same dose administered in the fed state.

[0314] Comparison of the GMRs associated with peak (Cmax) and total exposure (AUC(0-last) and AUC(0-inf)) levels for the besudil liquid formulation in the fed state versus the tablets in the fed state showed that the peak and total exposure levels of besudil and KD025m1 for the liquid formulation were largely similar to those for the tablet reference, with the 90% CI for each parameter including 100%. The upper limit of the 90% CI for peak exposure was slightly less than 100% (upper CI 94.39%); although it does exclude unity, this suggests that any true differences are likely to be minimal and unlikely to be clinically significant. In addition, the change in formulation caused little change in AUC, suggesting that the change in formulation had no significant effect on the total exposure of besudil or its two known metabolites.

[0315] Comparison of GMRs associated with peak and total exposure for the fed versus fasted oral liquid formulations indicated a small increase in besudil following the fed regimen, with exposures on average 15% to 19% greater than those observed with the fasted regimen. A modest increase in total exposure was observed for KD025m1, but this increase should be interpreted with caution because the available data for parameter estimates for KD025m1 across all regimens were relatively sparse. The lower limit of the 90% CI for each parameter exceeded 100%. No significant changes based on food effects were observed for KD025m2.

[0316] In conclusion, the absorption of besudil was faster after a single oral administration of the liquid formulation comprising besudil (median Tmax 2h) compared to besudil administered as a tablet reference (median Tmax 3h).

[0317] The median Tmax of KD025m1 and KD025m2 were similarly faster (1.5 h and 2 h, respectively) after administration as liquid formulations compared to the tablet reference (2 h and 3 h, respectively).

[0318] The maximum (Cmax) and overall (AUC) besudil exposure following administration as an oral liquid formulation showed no change following the formulation change compared to the tablet reference, both of which were administered in the fed state.

[0319] The bioavailability of besudil and metabolites (KD025m1 and KD025m2) was similar between the liquid formulation and the tablets as measured by Cmax and AUC. Example 12: Pediatric Dosage Study

[0320] In this study, two models, a population pharmacokinetic (PopPK) model and a physiologically based pharmacokinetic (PBPK) model, were used to estimate recommended pediatric doses of besudil that matched the AUC following administration of a 200 mg once daily (QD) dose of besudil in adults. (a) PopPK Model

[0321] In applying this model, the following steps were taken: (a) determine weight bins for child and adolescent weights between 10 kg (3rd percentile for 2 years) and 88 kg (97th percentile for 18 years); (b) make predictions over a 24-h period at steady state; (c) consider the effect of GVHD on clearance in each subject; (d) assume no co-administration of PPIs for any virtual subject; (e) assign each population to 50% males; and (f) compare final exposure predictions to a virtual adult group in which all adults were administered 200 mg KD025 QD.

[0322] In identifying appropriate weight intervals, the following steps were taken: (a) the child and adolescent weight range (10 kg to 88 kg) was divided into 3 kg intervals, with 1000 virtual subjects within the weight range in each interval; (b) the AUC for subjects was predicted after 50 mg, 100 mg, and 200 mg QD doses; (c) the AUC for the adult population of 5000 virtual subjects was predicted after a 200 mg QD dose; (d) the 25th percentile AUC in adults was compared to the 50th percentile AUC in each weight interval, and intervals with higher median AUCs were combined into a larger weight interval for each dose.

[0323] Adult body weights range from 40 kg to 126 kg. The following weight ranges were used for the final exposure predictions to allow for consistency in dosing with adults and ease of administration: 10 kg to < 20 kg – 50 mg QD 20 kg to < 40 kg – 100 mg QD ≥40kg-200mg QD

[0324] Applying this model, the final predicted weight intervals and doses were determined to be: (1) 50 mg QD for patients weighing 10 kg to <19 kg; (2) 100 mg QD for patients weighing 19 kg to <49 kg; and (3) 200 mg QD for patients weighing 49 kg to <88 kg. The estimated once daily (QD) and twice daily (BID) doses of besudil oral liquid formulation by age group and weight are shown in more detail in Tables 40 to 43 below. Table 40: Estimated QD Dosage of Besudil in Children by Age Group Table 41: Estimated QD Dosage of Besudil in Children by Body Weight Weight group Minimum age (months) Maximum age (months) Default (mg) 6 kg to < 8 kg 3.002 6.998 24 8 kg to < 10 kg 5.012 11.97 33 10 kg to < 13 kg 9.007 24.95 45 13 kg to < 20 kg 21.01 61.9 64 20kg to <40kg 60.02 131 97 >=40kg 127.1 216 167 Table 42: Estimated BID Dosage of Besudil in Children by Age Table 43. Estimated BID Dosage of Besudil in Children by Weight (b) PBPK model

[0325] A physiologically based pharmacokinetic (PBPK) model for belumosudil (KD025) and its major metabolite KD025m2 (chemical name 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetic acid) (also referred to as "M2") was previously developed by incorporating data from in vitro nonclinical and clinical pharmacokinetic studies conducted in healthy volunteers. See, e.g., Schueller, O. et al., "A Phase I Pharmacokinetic Drug Interaction Study of Belumosudil Coadministered With CYP3A4 Inhibitors and Inducers and Proton Pump Inhibitors," Clinical Pharmacology in Drug Development, 2022, 11(7)795-806.

[0326] In this example, a previously developed PBPK model for KD025 and M2 was applied to predict the systemic exposure of KD025 in children (3 months-11 years) and adolescents (12-17 years) with the goal of supporting dose selection.

[0327] The developed PBPK model was applied using the following four steps: 1. Predict the plasma concentrations of KD025 and M2 in healthy adult subjects following multiple oral doses of 2.7 mg / kg QD KD025 (200 mg equivalent adult dose). 2. Predict the plasma concentrations of KD025 and M2 in healthy pediatric subjects (for age groups of 0.25-0.5y, 0.5-1y, 1-2y, 2-5y, 6-11y, and 12-17y) following multiple oral doses of 2.7mg / kg QD KD025. 3. Simulation-based pediatric:adult KD025 AUC 0-t,SS The ratio (calculated from steps 1 and 2) adjusted the initial 2.7 mg / kg dose of KD025. 4. Predict the plasma concentrations of KD025 and M2 in pediatric subjects (for age groups of 0.25-0.5y, 0.5-1y, 1-2y, 2-5y, 6-11y, and 12-17y) following multiple oral doses of adjusted doses of KD025.

[0328] For comparison, allometric scaling was also investigated based on body weight to predict KD025 doses in infants and children (0.25-0.5y, 0.5-1y, 1-2y, 2-5y, 6-11y, and 12-17y) using an adult daily dose of 200mg.

[0329] Step 1. Simulate the plasma concentration-time profiles of KD025 and M2 in healthy adults after multiple oral doses of KD025 (2.7 mg / kg QD, equivalent to 200 mg QD)

[0330] Ten virtual trials of 20 healthy adult subjects (50% female) aged 20 to 50 years were generated using the Sim-healthy volunteer population library. The "Simcyp Child Simulator" allows modeling of pharmacokinetic behavior in neonates, infants and children. The Simcyp simulator includes a complete PBPK model and an extended library of child demographics (age, height, weight, BSA [body surface area]), developmental physiology (liver size, renal function, liver blood flow) and biochemistry (albumin, CYP ontogeny). The algorithms describing these changes are described in the literature. (See, for example, Johnson and Rostami-Hodjegan, Resurgence in the Use of Physiologically Based Pharmacokinetic Models in Pediatric Clinical Pharmacology [Resurgence of pharmacokinetic models based on physiology in children's clinical pharmacology], Pediatr Anesth. [Children's Anesthesia] 21: 291-301

[2011] ).

[0331] Using a simulated average adult body weight of 73.84 kg, the adult dose of interest (200 mg) was converted to a dose of 2.7 mg / kg. Virtual subjects received multiple oral doses of 2.7 mg / kg KD025 QD for 8 days, and AUC 0-t,SS The simulated single value of is integrated.

[0332] Calculation of mean simulated plasma KD025 and M2 concentrations following multiple oral dosing of 200 mg QD KD025 for 8 days. Predicted mean C of KD025 and M2 on day 8 max The AUC values ​​are shown in Table 44. Table 44: Predicted Geometric Mean C of KD025 and M2 in Healthy Adult Subjects max ,t max and AUC values. Step 2. Simulate the plasma concentration-time profiles of KD025 and M2 in pediatric subjects (3 months-17 years) after multiple oral doses of KD025 (2.7 mg / kg QD).

[0333] Pediatric subjects were divided into the following age groups; 0.25-0.5y, 0.5-1y, 1-2y, 2-5y, 6-11y, and 12-17y. Ten virtual trials of 20 pediatric subjects (50% female) were generated for each age group of KD025 receiving 2.7mg / kg QD (200mg adult equivalent) using the default Sim-Child population library. Simulations were performed using the CYP3A4 ontogeny profile from the following article: Upreti and Wahlstrom, Meta-Analysis of Hepatic Cytochrome P450 Ontogenyto Underwrite the Prediction of Pediatric Pharmacokinetics UsingPhysiologically Based Pharmacokinetic Modelling, JClin Pharmacol. [Journal of Clinical Pharmacology] 56:266-283( 2016) .

[0334] Virtual subjects received multiple oral doses of KD025 for 8 days, and AUC 0-t,ssThe simulated single values ​​of are integrated. The equivalent doses in mg for each age group are specified in Table 43 below.

[0335] The pediatric subjects were divided into the following age groups; 0.25-0.5y, 0.5-1y, 1-2y, 2-5y, 6-11y and 12-17y. Ten virtual trials of 20 pediatric subjects (50% female) were generated for each age group receiving adjusted multiple oral doses of KD025 using the default Sim-Child population library. All simulations were performed using the Upreti Wahlstrom 2016 CYP3A4 ontogeny profile.

[0336] Virtual subjects received multiple oral doses of KD025 for 8 days, and AUC 0-t,ss The simulated single values ​​were integrated. The adjusted doses and equivalent doses in mg for each age group are specified in Table 45. Table 45: Adjusted dose (mg / kg) and equivalent dose in Mg for each pediatric age group. a Equivalent to 2.7 mg / kg b For reference only (simulations were performed using doses in mg / kg)

[0337] Predicted plasma concentration-time profiles of KD025 and M2 following multiple oral doses of 2.7 mg / kg in children aged 0.25-0.5 y, 0.5-1 y, 1-2 y, 2-5 y, 6-11 y, and 12-17 y were calculated using the Upreti Wahlstrom (2016) CYP3A4 ontogeny profile, using a concentration-time profile representing the mean of the total virtual population (n=200) for each simulation. Predicted mean KD025 and M2 AUC on day 8 0-t,SS The values ​​are shown in Table 46. Table 46: Predicted mean plasma AUC of KD025 and M2 following multiple oral doses of KD025 (2.7 mg / kg; adult equivalent 200 mg dose QD) in adult and pediatric subjects 0-tSS value.

[0338] Step 3. KD025 AUC in children / adults based on simulations 0-t,SS Ratio-adjusted initial 2.7 mg / kg dose of KD025

[0339] As shown in equation (1), based on the calculated pediatric / adult KD025 AUC 0-t,SS Ratio (simulation described in steps 1 and 2 above), adjusting the pediatric dose from an initial value of 2.7 mg / kg.

[0340]

[0341] The adjusted dose (mg / kg) is calculated within the following age ranges; 0.25-0.5y, 0.5-1y, 1-2y, 2-5y, 6-11y and 12-17y as described in Table 43.

[0342] Sensitivity analyses were also performed for each pediatric age group at several doses around the calculated adjusted dose (in the range of 2.7-3.6 mg / kg) to confirm that the use of the adjusted dose resulted in similar KD025 and M2 exposures in both pediatric and adult populations.

[0343] Using the Upreti-Wahlstrom (2016) CYP3A4 ontogeny profile, calculated pediatric / adult KD025 AUC 0-t,SS Ratio (steps 1 and 2 and simulation described in Table 44), the pediatric dose was adjusted from an initial value of 2.7 mg / kg. The adjusted doses are given in Table 45 above.

[0344] Step 4. Simulate the plasma concentration-time profiles of KD025 and M2 in pediatric subjects (3 months-17 years) after adjusted multiple oral doses of KD025.

[0345] The pediatric subjects were divided into the following age groups; 0.25-0.5y, 0.5-1y, 1-2y, 2-5y, 6-11y and 12-17y. Ten virtual trials of 20 pediatric subjects (50% female) were generated for each age group receiving adjusted multiple oral doses of KD025 using the default Sim-Child population library. All simulations were performed using the Upreti Wahlstrom 2016 CYP3A4 ontogeny profile.

[0346] Virtual subjects received multiple oral doses of KD025 for 8 days, and AUC 0-t,SS The simulated single values ​​were integrated. The adjusted doses and equivalent doses in mg for each age group are specified in Table 45.

[0347] Predicted plasma concentration-time profiles for KD025 and M2 after dose adjustment in children aged 0.25-0.5y, 0.5-1y, 1-2y, 2-5y, 6-11y, and 12-17y were calculated using the Upreti-Wahlstrom (2016) CYP3A4 ontogeny profile, with concentration-time profiles representing the mean of the total virtual population (n=200) included for each simulation. Mean KD025 and M2 AUC at day 8 after dose adjustment and associated predictions 0-t,SS The values ​​are shown in Table 47.

[0348] Simulations were performed using doses in mg / kg. Table 47: Predicted mean plasma AUC of KD025 and M2 after multiple oral doses in pediatric subjects after dose adjustment using Upreti Wahlstrom (2016) ontogeny 0-t,SS value.

[0349] Allometric scaling was performed based on body weight (BW) using a three-quarter power model as shown in equation (2).

[0350] Doses were calculated in the following age ranges: 0.25-0.5y, 0.5-1y, 1-2y, 2-5y, 6-11y, and 12-17y. Child and adult BW were simulated as the mean values ​​for each age range (Table 43).

[0351] Allometric dosage projections in pediatric subjects aged 3 months to 17 years using the adult daily dose of 200 mg are provided in Table 48. Table 48: Allometric Dose Predictions in Pediatric Subjects Aged 3 Months to 17 Years

[0352] Results from simulations using the Upreti Wahlstrom (2016) CYP3A4 ontogeny profile in this example indicate that when dosed on a mg / kg basis, there are small differences in exposure of KD025 and M2 in pediatric subjects compared to adults (KD025 and M2 were 2.5 and 3.3 times higher in AUC in adults, respectively). 0-t,SS within 30% and 1.5 times of the

[0353] Simple allometric scaling of the data may lead to an overestimation of the required dose in the youngest group (5 years and younger). For the older age groups (ages 6-17 years), allometric scaling resulted in dose adjustments similar to the Upreti-Wahlstrom ontogeny-derived PBPK predictions.

[0354] Another simulation.

[0355] The model was developed based on the following assumptions: the drug is administered once daily as a tablet; the drug response is assumed to be proportional to the dose; the effect of concomitant administration of CYP inducers or inhibitors is not considered; the dose is administered in the fed state; all pediatric subjects are assumed to have chronic graft-versus-host disease (cGVHD); each virtual population has 1000 subjects, 50% of whom are males; and the effect of concomitant administration of PPIs on drug PK is not considered.

[0356] Predict pediatric exposure using adult equivalent mg / kg dosing.

[0357] Each virtual population spans an age range of 3 months, starting from 6 months and ending at 216 months. A flat dose of 2.5 mg / kg was administered to each subject, equivalent to a 200 mg dose for an 80.65 kg adult. The dose was administered QD. The model was run to steady state for exposure prediction. Cmax at steady state and AUC within a 24 h time period at steady state were used as exposure metrics. Child exposure was compared with adult exposure.

[0358] Predicting pediatric exposures through simulations based on age-based dosing strategies.

[0359] The pediatric population was divided into the following 3 age intervals: 1Y to 6Y, 6Y to 12Y, and 12Y to 18Y. Doses of 3.5, 4, 4.5, and 5 mg / kg QD were administered to the 1Y to 6Y and 6Y to 12Y populations. A dose of 200 mg QD was administered to the 12Y to 18Y dose group. The model was run to steady state for exposure prediction. Cmax at steady state and AUC within a 24h time period at steady state were used as exposure metrics. Child exposure was compared with adult exposure.

[0360] Effects of enzyme ontogeny on drug exposure.

[0361] The 4 mg / kg dose was administered to a population divided into 3-month age intervals starting from 6 months until 60 months. The fractional activity for each subject was calculated using the following three methods: ontogeny only, allometric growth >2 years old, and allometric growth only. The predicted exposure was then divided into 1 kg body weight intervals to evaluate the need for dosing based on different body weights. For pediatric patients >=13 kg, the ontogeny of CYP enzymes had limited effects on drug exposure at the 4 mg / kg dose level. For the virtual population, the maximum weight of patients in the 18m to 21m age group was 13 kg.

[0362] Predicting exposure in children based on weight ranges.

[0363] The 3 mg / kg, 3.5 mg / kg and 4 mg / kg doses were administered to a population divided into 2-month age intervals starting from 6 months to 24 months. The fractional activity for each subject was calculated using enzyme ontogeny. The predicted exposure was then divided into 1 kg body weight intervals to evaluate the effect of dose level on exposure in the body weight intervals. Example 13: Suspension Formulation Study

[0364] The formulation composition and process variables of a liquid besudil formulation at a concentration of 40 mg / mL were studied to provide a homogenous and stable dispersion. The process variables were evaluated to produce a robust suspension formulation.

[0365] Table 49 shows the initial formulation compositions used to manufacture the first five laboratory batches (Batch Nos. 001 to 005) in the study. Table 49: Initial formulation composition of Besudil Suspension 40 mg / mL of Batch No. 001-005.

[0366] After making the first laboratory batch (Batch No. 001), undesirable foaming and agglomeration were observed. Four additional laboratory batches were made using the initial formulation composition, but with a changed order of addition, including slow addition of the API and side phase preparation. The initial formulation compositions shown in Table 49 and Figure 5 The second laboratory batch (batch 002) was prepared using the manufacturing method shown in . However, foaming and agglomeration were also observed in four other batches (batch numbers 002 to 005). It was concluded that the foaming and agglomeration observed during the manufacturing method of the suspension need to be reduced to provide a liquid formulation suitable for commercial use.

[0367] A modified manufacturing process was used to prepare another batch (Batch 006) of besudild suspension at a concentration of 40 mg / mL. The modified process included replacing the A310 paddle wheel used in the mixing step of the process with a Silverson homogenizer L5M-A equipped with a square hole high shear screen, and adding a defoamer to the formulation composition. This Silverson homogenizer was used to manufacture a 2 kg laboratory batch of besudild suspension with a 30% simethicone emulsion at a concentration of 2% w / w as Batch 006.

[0368] The modified process was found to sufficiently reduce the amount of foaming generated during manufacturing and provide a suspension suitable for commercial use. The modified formulation composition used in Batch No. 006 is shown in Table 50, and the modified manufacturing process used in Batch No. 006 is shown in Table 51. Figure 6 middle. Table 50: Modified formulation composition of Besudil Suspension 40 mg / mL.

[0369] The manufacturing method for preparing Besudil suspension 40 mg / mL of batch number 002 is shown in Figure 5 and the initial formulation composition for batch 002 is shown in Table 49. The modified manufacturing method for preparing besudil suspension 40 mg / mL of batch 006 is shown in Figure 6 The modified formulation composition used in batch 006 is shown in Table 50.

[0370] Table 51 compares the manufacturing process details for batches 002 and 006, illustrating a number of specific differences that occurred between the initial manufacturing process and the modified manufacturing process. Table 51 : Details of the manufacturing process followed for Besudil Suspension 40 mg / mL of Batch Nos. 002 and 006.

[0371] Figure 5 The manufacturing method shown for preparing batch 002 includes two phases ("API phase" and "water phase") using two containers; one container is used to mix the povidone PVP and besudil mesylate components, and the other container is used to mix the sodium benzoate, sucralose and Syloid colloidal silicon dioxide components. The two phases are mixed separately, then combined, and tartaric acid is added, the pH and water levels are adjusted, and the formulation is left to stand for 1 hour and 30 minutes. Mixing is performed with an A310 paddle wheel rotating at a speed ranging from 300 rpm to 600 rpm. According to Table 51, a holding time is used to allow the foaming to dissipate, and the mixing time and the holding time together total up to at least 305 minutes.

[0372] Figure 6 The manufacturing method for preparing Batch No. 006 shown in utilises a single manufacturing vessel for mixing all ingredients and does not require a hold time to allow foaming to dissipate as no foaming is generated. Mixing is performed using a Silverson homogeniser equipped with a square hole high shear screen impeller rotating at speeds up to 5000 rpm. According to Table 51, the mixing time totals up to 105 minutes.

[0373] The use of a Silverson homogenizer allows for significantly higher rotational speeds to be used in the mixing step of the process, and the use of a square hole high shear screen allows for high shear rates to be achieved during mixing. The process using the Silverson homogenizer has a shorter total mixing time and does not include a 90 minute hold step, but this hold step is required in the process using the A310 paddle wheel and mixing at a lower speed. The suspension (Batch 006) made using a modified formulation composition including a simethicone emulsion and a Silverson homogenizer did not foam, unlike the suspension (Batch 002) made without simethicone and using an A310 paddle wheel and mixing at a lower speed, which exhibited foaming.

[0374] As described in this article and Figure 6 The liquid besudeil oral suspension shown using a modified formulation composition including a simethicone emulsion and a Silverson homogenizer provides a robust homogeneous and stable dispersion that can be homogeneously resuspended so that it can be oscillated multiple times over time before administration, thereby reproducibly delivering the desired drug dose. During formulation manufacturing, high-speed mixing with a specific paddle shape is often used to reduce particle agglomeration and provide a homogeneous and stable dispersion. However, a side effect of high-speed mixing (especially for hydrophobic drugs) is increased foaming. The modified composition and manufacturing method overcome the foaming problem and obtain a suspension with desired characteristics. Example 14: Additional Suspension Formulation Studies

[0375] Further studies may evaluate additional formulations and process variables of besudil suspension at a concentration of 40 mg / mL. The relationship between input variables (factors) and output variables (responses) will be studied. The studies to be conducted include evaluating the effective concentration of simethicone in besudil suspension 40 mg / mL.

[0376] The formulations and method parameters to be evaluated will be based on the design of experiments (DoE) performed using Minitab 21 software. The formulation compositions to be studied are shown in Table 52. The study ranges of formulation variables are also shown with the composition along with the previously determined independent identical distribution (IID) limits. The effects of effective simethicone concentration, pH, and the effective concentrations of Syloid 244FP and PVP K-90 will also be studied. Table 52: Ranges of formulation variables and IID limits *Salt conversion factor 1.2124, i.e. 242.48 mg of besudinil mesylate API is equivalent to 200 mg of free base. The density of the suspension is 1.02 g / mL

[0377] To evaluate the effective concentration of 30% simethicone emulsion, the design plan was a one-factor-at-a-time (OFAT) approach.

[0378] The same manufacturing process as used for Batch 006 in Example 13 was followed, but the order of addition of PVP K-90 and Syloid 244FP was changed. The different experiments performed to evaluate the effective concentration of 30% simethicone emulsion are shown in Table 53. Table 53: OFAT test used to evaluate the effective concentration of 30% simethicone emulsion. Table 54: Reactions to be studied

[0379] To study the effect of pH: API is soluble at pH 2.0-3.0, so the effect of pH on suspension can be studied. The most effective formulation composition from the above study results will be used in this study. Table 55 shows two additional experiments that will be conducted to study the pH range. Table 55: OFAT test used to study pH range

[0380] For the pH effect study, the same reactions as shown in Table 54 were also studied.

[0381] DoE study for Syloid 244FP, PVP K-90 and pH: The effect of the amount of Syloid 244FP and PVP K-90 and pH will be studied using the DoE plan as shown in Table 56. Design Plan: A fractional factorial design with 3 factors is used with the following design summary: Factor: 3 Basis Design: 3,4 Separation: III Runs: 8 Repeats: 1 Fraction: 1 / 2 Block: 1 Center point (total): 4 Design table (randomization) run Block A B C 1 1 + + + 2 1 0 0 - 3 1 - - - 4 1 + - - 5 1 - - - 6 1 0 0 + 7 1 0 0 + 8 1 0 0 _ Table 56: DoE used to study the effect of the amount of Svloid 244FP, PVP K-90 and pH

[0382] For the DoE studies for Syloid 244FP, PVP K-90 and pH studies, the same reactions as shown in Table 54 will also be studied.

[0383] Experimental design: Figure 6 The method DoE was conducted with the modified manufacturing method shown in . The identified method parameters for the study were mixing time and speed after PVP K-90 addition, and homogenization time and speed after API addition. Table 57 shows the DoE experiments to be conducted to study these method parameters during the manufacture of Besudil suspension 40 mg / mL.

[0384] Design Plan: A fractional factorial design using 4 factors with the following design summary: Factors: 4 Basis Design: 4, 8 Degree of Separation: IV Runs: 11 Repeats: 1 Fraction: 1 / 2 Block: 1 Center point (total): 3 Design table (randomization) run Block A B C D 1 1 + - + - 2 1 0 0 0 0 3 1 0 0 0 0 4 1 - - - - 5 1 0 0 0 0 6 1 - - - - 7 1 + + + + 8 1 - - - - 9 1 - - - - 10 1 - - - + 11 1 - + - -

[0385] Table 57: DoE used to study method parameters

[0386] For the DoE study of the method parameter study, the same reactions as shown in Table 54 will also be studied.

Claims

1. A liquid formulation comprising 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or a pharmaceutically acceptable salt thereof (besudil) in a diluent in combination with a suspending agent, a thickening agent and a defoaming agent.

2. The liquid formulation of claim 1, wherein the antifoaming agent comprises about 30% simethicone emulsion.

3. A liquid formulation comprising 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or a pharmaceutically acceptable salt thereof (besudil) in a diluent in combination with a suspending agent and a thickening agent.

4. The liquid formulation of any one of claims 1 to 3, further comprising one or more of a preservative, a pH adjuster to achieve a pH in the range of about 2.5 to 4.0 as required, and a sweetener.

5. The liquid formulation of any one of claims 1 to 4, wherein the besudil is jet-milled or pin-milled.

6. The liquid formulation of any one of claims 1 to 4, wherein the besudil comprises jet-milled besudil mesylate.

7. The liquid formulation of any one of claims 4 to 6, wherein the preservative is sodium benzoate, the suspending agent is colloidal silicon dioxide, the thickening agent is povidone, the pH adjuster is tartaric acid, and the sweetener is sucralose.

8. The liquid formulation of any one of claims 1 to 7, wherein the thickener is povidone 90F.

9. The liquid formulation according to any one of claims 1 to 8, wherein the diluent is sterile irrigation water.

10. The liquid formulation of any one of claims 1 to 9, further comprising a flavoring agent.

11. The liquid formulation of claim 10, wherein the flavoring agent is a tropical fruit blend flavoring agent or a lemon flavoring agent.

12. The liquid formulation of any one of claims 1 to 11, wherein the liquid formulation is homogenized.

13. The liquid formulation of any one of claims 4 to 6, comprising about 2 to 8 weight percent of besudil, about 0.02 to 0.08 weight percent of a preservative, about 0.1 to 0.4 weight percent of a sweetener, about 0.2 to 0.8 weight percent of a suspending agent, and about 1 to 8 weight percent of a thickening agent.

14. The liquid formulation of any one of claims 4 to 6, comprising about 3 to 5 weight percent besudil, about 0.035 to 0.1 weight percent preservative, about 0.1 to 0.2 weight percent sweetener, about 0.4 to 0.6 weight percent suspending agent, and about 4 to 6 weight percent thickening agent.

15. The liquid formulation of any one of claims 4 to 6, comprising the following weight percentages of besudild, preservatives, suspending agents, thickeners, and sweeteners: about 4.75% besudild mesylate; about 0.035% to 0.1% preservatives; about 0.15% suspending agents; about 0.5% thickeners; and about 0.15% sweeteners.

16. The liquid formulation of any one of claims 13 to 15, wherein the suspending agent is colloidal silicon dioxide, the thickening agent is povidone 90F, the sweetener is sucralose, and the pH adjuster is tartaric acid as required.

17. The liquid formulation of claim 16, further comprising a flavoring agent.

18. The liquid formulation of claim 17, wherein the flavoring agent is tropical fruit blend flavoring agent or lemon flavoring agent.

19. The liquid formulation of any one of claims 1 to 12, formulated to deliver a dose of besudil in the range of about 50 to 400 mg to a patient in a volume of about 1 to 10 mL.

20. The liquid formulation of any one of claims 1 to 12, formulated to deliver a single dose in the range of 50 to 200 mg to a patient in a single volume dose of about 5 mL.

21. A pharmaceutical composition for administration to a subject, comprising a therapeutically effective amount of the liquid formulation of any one of claims 1 to 20.

22. A method for treating graft-versus-host disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the liquid formulation of any one of claims 1 to 20.

23. The method of claim 22, wherein the graft-versus-host disease is chronic graft-versus-host disease.

24. The method of claim 22 or 23, wherein the subject is a pediatric patient ranging in age from 3 months to less than or equal to 18 years.

25. The method of any one of claims 22 to 24, wherein the liquid formulation is administered to the subject at a dose of besudil adjusted based on the patient's weight.

26. The method of claim 25, wherein the liquid formulation is administered to the patient based on the patient's weight as follows: (1) for patients weighing 10 kg to <20 kg, about 50 mg of besudil is administered once daily; (2) for patients weighing 20 kg to <40 kg, about 100 mg of besudil is administered once daily; and (3) for patients weighing 40 kg or more, about 200 mg of besudil is administered once daily.

27. A process for preparing a liquid formulation comprising besudil, the process comprising mixing jet-milled and / or pin-milled besudil in a diluent with a suspending agent and a thickening agent.

28. The method of claim 27, comprising the steps of: (a) dispensing a first portion of the diluent into a container; (b) dispensing a preservative, a sweetener, the suspending agent and the thickening agent into the container; (c) mixing the contents of the container; (d) transferring the container to a homogenizer; (e) operating the homogenizer to homogenize the contents of the container; (f) adding an amount of the besudil to the container and mixing the contents of the container to form a suspension; (g) measuring the pH of the suspension; (h) adding a pH adjuster to the suspension, with mixing, in a sufficient amount to achieve a pH of about 2.5 to about 4.0; as well as (i) adding a second portion of the diluent to the container to reach a predetermined volume to provide the liquid formulation comprising besudil.

29. The method of claim 28, wherein the preservative is sodium benzoate, the sweetener is sucralose, the suspending agent is silicon dioxide, the thickening agent is povidone 90F, the pH adjuster is tartaric acid, and the diluent is sterile water.

30. The method of claim 28, further comprising the steps after step (i): (j) allowing the suspension to stand until any foam or froth has dissipated, and (k) dispensing the liquid formulation comprising besudil into containers.

31. The method of claim 28, further comprising visually inspecting the contents of the container and / or the suspension after one or more of steps (c) to (i).

32. The method of claim 28, further comprising visually inspecting the suspension for foaming and / or sedimentation after step (c), step (e), step (f), and step (j).

33. The method of claim 29, wherein the besudil, the sodium benzoate, the sucralose, the silicon dioxide and the povidone are added to the container according to the following weight percentages calculated in view of the predetermined volume: about 3 to 5 weight percent besudil, about 0.035 to 0.1 weight percent sodium benzoate, about 0.1 to 0.2 weight percent sucralose, about 0.4 to 0.6 weight percent silicon dioxide and about 4 to 6 weight percent povidone 90F.

34. The method of any one of claims 28 to 33, wherein the besudil added to the formulation is jet-milled besudil mesylate.

35. The method of claim 27, comprising the steps of: (a) dispensing a first portion of the diluent into a container; (b) dispensing a preservative, a sweetener, the suspending agent, the thickening agent, an antifoaming agent and a certain amount of the besudil into the container; (c) mixing the contents of the container with a homogenizer containing a high shear screen to form a suspension; (d) measuring the pH of the suspension; (e) adding a pH adjuster to the suspension, with mixing, in a sufficient amount to achieve a pH of about 2.5 to about 4.0; as well as (f) adding a second portion of the diluent to the container to reach a predetermined volume to provide the liquid formulation comprising besudil.

36. The method of claim 35, wherein the mixing with a homogenizer is performed at a rotation speed between about 4000 and 6000 rpm.

37. A liquid formulation comprising besudil produced by the method of any one of claims 27 to 36.

38. A kit for preparing a liquid formulation of besudil, the kit comprising (a) in a first container, an amount of besudil; and (b) in a second container, a reconstitution vehicle comprising a suspending agent and a thickening agent in a diluent.

39. The kit of claim 38, wherein the besudil in the first container is in powder form; and (b) the suspending agent is silicon dioxide and the thickening agent is povidone.

40. The kit of claim 38 or 39, further comprising a sweetener in the reconstituted vehicle.

41. The liquid formulation of any one of claims 1-20 or the pharmaceutical composition of claim 21, wherein the concentration of besudil is 40 mg / ml.

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