Belmosudil liquid formulation

Abstract

The present disclosure provides liquid formulations comprising belmosudil, processes for preparing liquid formulations of belmosudil, and liquid pharmaceutical compositions comprising belmosudil that can be used to treat 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 belmosudil. The present disclosure further relates to methods for preparing liquid formulations comprising belmosudil, and methods for treating subjects, including adult and pediatric populations, with the liquid formulations.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to PCT U.S. Patent Application Publication No. PCT / US2022 / 044256, filed September 21, 2021, which is incorporated by reference herein in its entirety. [Background technology]

[0003] Belmosudil is an oral, selective inhibitor of rho-associated coiled-coil-containing protein kinase 2 (ROCK2). ROCK2 inhibition acts on fibrosis resulting from a dysregulated adaptive immune system and abnormal tissue repair. Belmosudil inhibits ROCK2 and ROCK1 with IC values ​​of approximately 100 nM and 3 μM, respectively. 50 Inhibit by value.

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

[0005] Belmosudil is also known as KD025.

[0006] Belmosudil mesylate is currently marketed in the United States and other countries under the trade name REZUROCK® (Kadmon Corp. / Sanofi) for the treatment of patients with chronic graft-versus-host disease (cGVHD), in some cases after the failure of at least two prior lines of systemic therapy. The active pharmaceutical ingredient in REZUROCK® is belmosudil mesylate, with the molecular formula C 27 H 28 N6O5S, molecular weight 548.62 g / mol, 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 belmosudil mesylate is as follows: [ka]

[0008] Belmosudil binds to and inhibits the activity of ROCK1 and ROCK2 serine / threonine kinases. Belmosudil downregulated proinflammatory responses by modulating STAT3 / STAT5 phosphorylation and shifting the Th17 / Treg balance in ex vivo and in vitro human T cell assays. Belmosudil also inhibited aberrant profibrotic signaling in vitro. In vivo, belmosudil demonstrated activity in an animal model of cGVHD. Therefore, belmosudil is useful for treating diseases, disorders, and conditions regulated by ROCK, including autoimmune and fibrotic disorders, acute and chronic GVHD, idiopathic pulmonary fibrosis, and moderate to severe psoriasis, among other indications.

[0009] A process for preparing berumosudil is disclosed in U.S. Pat. No. 8,357,693 (the '693 patent), particularly in Example 82 thereof. The process disclosed in the '693 patent provides berumosudil as a crude solid product that is purified by high performance liquid chromatography (HPLC). Belmosudil and processes for producing this compound are also described in U.S. Pat. Nos. 9,815,820, 10,183,931, and 10,696,660.

[0010] This method of administering belmosudil involves formulating belmosudil mesylate into pharmaceutically acceptable capsules and tablets for oral administration.However, certain patients who may benefit from treatment with belmosudil are unable to swallow, have difficulty swallowing, and / or are reluctant to do so, such as certain adult patients, patients with dysphagia, and / or pediatric patients.Therefore, a liquid formulation of belmosudil would be useful for administering the compound to a wider range of patient populations.In addition, a liquid formulation may provide the advantage of improving the flexibility of drug administration methods.

[0011] Liquid formulations also offer the advantage of being able to flexibly administer clinically recommended doses to patients according to their weight.For example, in the case of liquid formulations, the amount of drug administered can be adjusted by adjusting the scale according to the patient's weight.In contrast, currently available solid oral dosage forms contain 200 mg of belmosudil in each tablet.

[0012] However, belmosudil is a weakly basic compound that is substantially insoluble in water. It is also insoluble or substantially insoluble in many other solvents or carriers typically used in pharmaceutical development. Due to the low solubility of belmosudil, developing a liquid formulation presents challenges. Furthermore, liquid formulations must be homogeneous upon suspension and resuspension (e.g., when a single vial is used multiple times). Obtaining a liquid formulation that can be homogeneously resuspended requires the use of specific equipment that may generate foaming. This is particularly true for hydrophobic compounds such as belmosudil. Therefore, there remains a need for a liquid formulation containing belmosudil that can address these challenges, as well as a method for producing a homogeneous, resuspendable preparation. Summary of the Invention [Means for solving the problem]

[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 (bermosudil). In one embodiment, the liquid formulation comprises jet-milled or pin-milled berumosudil suspended in a diluent in combination with a suspending agent and a thickening agent. The liquid formulation containing berumosudil may also contain a preservative, a pH adjuster to achieve a pH in the range of about 2.5 to 4.0, if necessary, and a sweetener and / or flavoring agent.

[0014] In a further 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 (bermosudil), in combination with a suspending agent, a thickening agent, and an antifoaming agent. Liquid formulations containing bermosudil may also contain one or more of a preservative, a pH adjuster (if necessary) to achieve a pH in the range of about 2.5 to 4.0, and a sweetener and / or flavoring agent. In one embodiment, the liquid formulation comprises jet-milled bermosudil. In one embodiment, the antifoaming agent comprises simethicone. In one embodiment, the antifoaming agent comprises about 30% simethicone emulsion.

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

[0016] Another aspect of the present disclosure provides a liquid formulation of belmosudil produced by mixing jet-milled or pin-milled belmosudil in a vehicle, the vehicle comprising at least an antifoaming agent in a diluent.

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

[0018] In another embodiment, the present disclosure provides a pharmaceutical composition comprising an effective amount of a liquid formulation of belmosudil useful for treating diseases, disorders and conditions modulated by ROCK, as further described herein. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is a flow chart illustrating a process for preparing a liquid formulation of Belmosudil. [Figure 2A] 1 shows particle size distribution (PSD) data for pin-milled Belmosudil in sterile water for irrigation, as described in Example 3. [Figure 2B] 1 shows particle size distribution (PSD) data for pin-milled Belmosudil in sterile water for irrigation, as described in Example 3. [Figure 3A] 1 shows PSD data for pin-milled Belmosudil liquid formulation containing Natrosol (Sample 3A of Example 4). [Figure 3B] 1 shows PSD data for a jet-milled Belmosudil liquid formulation containing Natrosol (Sample 3B of Example 4). [Figure 3C] 1 shows PSD data for pin-milled Belmosudil liquid formulation containing hypromellose K4M (Sample 4A of Example 4). [Figure 3D] 1 shows PSD data for pin-milled Belmosudil liquid formulation containing povidone (Sample 6A from Example 4). [Figure 4] 1 shows the results of precipitation assays of samples obtained from six liquid formulations composed of belmosudil, including three formulations containing pin-milled belmosudil (Samples 13A-15A) and three formulations containing jet-milled belmosudil (Samples 13B-15B), as described in Example 6. [Figure 5] 1 is a flow chart illustrating an additional process for preparing a liquid formulation of belmosudil. [Figure 6] 1 is a flowchart illustrating yet a further process for preparing a liquid formulation of belmosudil. DETAILED DESCRIPTION OF THE INVENTION

[0020] definition As used herein, "about" includes not only the exact amount modified by the term "about," but also an amount that is expected to be within experimental error, such as within 15%, 10%, or 5%, depending on the context. For example, "about 200 mg" means "200 mg," and also means a range of mg within experimental error, such as ±15%, 10%, or 5% of 200 mg. As used herein, the term "about" can be used to modify a range and a specific value.

[0021] As used herein, "administer" or "administer to" (e.g., with respect to administering bermosudil or a liquid formulation containing bermosudil) refers to the act of prescribing a medication containing bermosudil to a subject for administration during treatment, the act of prescribing a medication protocol to be taken by a subject, the act of dispensing the medication to a subject, the act of reconstituting powdered bermosudil in a liquid formulation, and / or the act of physically receiving or ingesting the medication. Thus, bermosudil can be "administered" by a physician or other medical professional who writes a prescription for bermosudil; and / or by a pharmacist who fills the prescription; and / or by a person who prepares a reconstituted liquid formulation using powdered bermosudil; and / or by a patient or subject who ingests bermosudil; and / or by their partner or caregiver who delivers bermosudil to a subject.

[0022] "API" means "active pharmaceutical ingredient" and is synonymous with the definition of belmosudil (or KD025) in this specification.

[0023] "Allogeneic hematopoietic stem cell transplantation (allo-HSCT)" or "allo-HCT," also known as bone marrow transplantation or stem cell transplantation, 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 (PBSCs) or bone marrow (BM). In some situations, umbilical cord blood can be used. The donor and recipient can be matched by human leukocyte antigen (HLA) genes, such as siblings. The donor and recipient can be haploidentical (haploidentical) parent and child.

[0024] As used herein, "belmosudil" may encompass any form of the compound belmosudil, as well as pharmaceutically acceptable salts thereof, unless the context clearly indicates otherwise. The term "belmosudil" refers to both the compound belmosudil (e.g., in free base form, amorphous form, or crystalline form), pharmaceutically acceptable salts of belmosudil, such as the mesylate form used as REZUROCK®, and any form of belmosudil that can be used to prepare a liquid formulation or pharmaceutical composition for administering the compound to a patient.

[0025] "Clinical endpoint" or "study endpoint" refers to an event or outcome in a clinical trial that can be objectively measured to determine the outcome and potential beneficial effects of a drug or administration protocol designed in the clinical trial. Examples of clinical endpoints include the following: Overall response rate (ORR) is the percentage of people in the study or treatment group who have a partial response (PR) or complete response (CR) to treatment within a certain period of time. Failed treatment survival (FFS) means the time from the first dose of belmosudil to a failure event, or the interval from the initiation of belmosudil to the addition of a new cGVHD therapy, recurrence of the underlying disease, or non-relapse mortality (NRM). Overall survival (OS) means the length of time from the date of disease diagnosis or the start of treatment. Duration of response (DOR) means the time from the time of initial response (e.g., PR or CR) to documented progression from the best cGVHD response, the time from the initial response to the initiation of additional systemic cGVHD therapy, or death. Time to next therapy (TTNT) refers to the time to initiation of subsequent systemic cGVHD therapy.

[0026] "Clinically recommended amount" or "clinically recommended dosage" refers to the amount or dosage of belmosudil that is recommended and / or approved for administration to a subject by one skilled in the art of medicinal chemistry to treat the disease state in question following clinical trials, as described, for example, in publications, clinical trial results, and approved drug labeling. In one embodiment, the clinically recommended dosage of belmosudil without administration of a CYP3A inducer or PPI, as indicated in the drug labeling of belmosudil, is 200 mg once daily.

[0027] As used herein, "co-administration," "in combination with," and / or "co-administered" with respect to administration of belmosudil with other substances (e.g., CYP3A inducers and / or PPIs) means that during the patient's course of treatment with belmosudil, the patient is also receiving one or more doses of one or more other therapeutic agents. A compound need not be administered at the same time or on the same day as belmosudil to be considered "co-administered" under this definition.

[0028] A "high-fat, high-calorie meal" refers to a meal containing about 800 to 1,000 calories, with about 50% of the total calorie content coming 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 full-fat milk.

[0029] "Homogenizer" refers to a piece of laboratory or industrial equipment used to homogenize 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 devices, such as cell lysers, dispersers, high-shear mixers, homogenizers, Polytrons, rotor-stator homogenizers, ultrasonicators, tissue tearers, etc. Homogenizers can achieve mixing at various shear and rotational speeds using different types of paddles, rotor-stator assemblies, and screens. One advantageous homogenizer is a SILVERSON® homogenizer. One advantageous homogenizer assembly includes a high-shear screen.

[0030] "Immunosuppressive therapy" (IST) refers to therapy typically administered for at least 6 months after allo-HSCT to attempt to prevent GVHD. Examples of IST include sirolimus, prednisone, and calcineurin inhibitors such as tacrolimus and cyclosporine.

[0031] "Label claim" refers to the amount or dose of API equivalent to that indicated on the approved drug label. For example, in the United States, the currently approved drug label for REZUROCK® states that the recommended dosage is 200 mg, taken orally once daily with a meal, or 200 mg twice daily if coadministered with a strong CYP3A inducer and a PPI. The equivalent dose of belmosudil in the liquid formulation (corresponding to the 200 mg labeled dose) is calculated as 40 mg / mL of free base, which, when adjusted for a salt correction factor of 1.2124, corresponds to 48.496 mg / mL of belmosudil mesylate.

[0032] The Lee Symptom Scale (LSS) total score measures the impact on patient function and health. The Lee Symptom Scale is a 30-item scale developed to measure the 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.

[0033] "Line of treatment" or "line of therapy" describes the order or sequence in which different therapies are given to a patient as their disease progresses. The first treatment (first-line treatment) may not work or stop working after a period of time. A second, different treatment (second-line treatment) may be given after the first-line treatment is discontinued. A subsequent line of treatment may be given if the second-line treatment does not work or stops working. Some patients may receive multiple lines of treatment over the course of their disease.

[0034] First-line treatment for moderate to severe chronic graft-versus-host disease (cGVHD), as defined by the National Institutes of Health (NIH), can be corticosteroids alone or in combination with sirolimus or a calcineurin inhibitor (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: BMT CTN 0801. Haematologica 103:1915-1924, 2018).

[0035] Examples of corticosteroid therapy for treating cGVHD include, but are not limited to, prednisone, prednisolone, methylprednisolone, and budesonide. Examples of prior systemic therapy for treating cGVHD include, but are not limited to, prednisone, tacrolimus, extracorporeal photopheresis (ECP), sirolimus, ibrutinib, ruxolitinib, mycophenolate mofetil (MMF), rituximab, methotrexate (MTX), cyclosporine, imatinib, ixazomib, and ofatumumab.

[0036] As used herein, "liquid formulation" or "liquid" means that the formulation is in a substantially liquid state rather than a solid (or gaseous) state, and includes suspensions in which solid particles (e.g., particles of bermosudil) are dispersed throughout the liquid without being dissolved in the liquid.

[0037] "Myeloablative transplant" refers to a transplant process that uses very high doses of chemotherapy or radiation before transplantation with autologous or allogeneic hematopoietic stem cells. Non-myeloablative transplantation, or reduced-intensity transplantation, involves patients receiving less intense chemotherapy before transplantation with allogeneic hematopoietic stem cells.

[0038] The "NIH Pulmonary Symptom Score" or "NIH cGVHD Pulmonary Score" is a clinical symptom-based score 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 when climbing 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 shortness of breath requiring oxygen.

[0039] "Or" is used in its inclusive sense (equivalent to "and / or") unless the context requires otherwise.

[0040] As used herein, a "patient" or "subject" includes an animal or a human. In one embodiment, the term "patient" refers to a human subject.

[0041] As used herein, "pediatric patient" refers to a non-adult patient (i.e., under the age of 18). In one embodiment, it refers to a patient under the age of 12. In another embodiment, it refers to a patient between the ages of 3 months and 12 years.

[0042] "Pharmaceutically acceptable salts" refers to non-toxic inorganic and organic acid addition salts of belmosudil. In one embodiment, the pharmaceutically acceptable salt of belmosudil herein is the mesylate salt.

[0043] "Povidone" (also referred to as "povidon") refers to a water-soluble polymer made from the monomer N-vinylpyrrolidone, also commonly known as polyvinylpyrrolidone or polyvidone. The molecular formula of povidone is (C6H9NO) n and has the appearance of a white to slightly off-white powder. The number k in the name of povidone refers to the average molecular weight of povidone. Thus, povidone 90 has a higher molecular weight than povidone K30.

[0044] As used herein, a "protocol" refers to a method or plan used to administer belmosudil to a patient in need of treatment. The term "protocol" is intended to encompass not only the overall detailed plan of patient care, but also individual or partial steps that are part of the overall plan. For example, a protocol may include the dosage of belmosudil that the patient will receive (or is receiving), the drug combination that the patient will receive, the timing and method of administration of belmosudil (e.g., taking into account DDIs, food effects, and the effects that different formulations or delivery modes may have on absorption and bioavailability), and side effect management, as well as an overall plan that takes into account the dosage, combination, timing and method of administration, and side effects taken together.

[0045] "Proton pump inhibitors" or "PPIs" are drugs that inhibit the H + / K + PPIs are drugs that inhibit the ATPase proton pump, causing a decrease in gastric acid production. Because PPIs reduce gastric acid production, they can increase gastric pH, which affects the solubility and potential bioavailability of orally delivered medications. Examples of PPIs include omeprazole, lansoprazole, dexlansoprazole, esomeprazole, pantoprazole, rabeprazole, and ilaprazole.

[0046] As used herein, "silica" or "silica agent" refers to any silica product that may 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, including silica products sold under the SYLOID® and AEROSIL® trade names.

[0047] AEROSIL® 200 is a hydrophilic fumed silica product containing more than 99.8% SiO2 and having a specific surface area of ​​200 m2 / g.

[0048] SYLOID® 244 FP is an alternative silica (SiO2) product available from W.R. Grace & Co. (Conn., USA) sold in the form of an amorphous, white, free-flowing powder. SYLOID® 244 FP silica is a high-pore-volume silica gel with a high internal surface area. This silica gel has a strong affinity for moisture and organic liquids. SYLOID® 244 FP silica can adsorb up to 1.6 ml of liquid per gram. SYLOID® 244 FP silica is advantageous as a glidant, tableting aid, and carrier for pharmaceutical and personal care products.

[0049] "Simethicone" (also known as "simethicone emulsion") refers to an anti-foaming agent. The molecular formula of simethicone is CH 18 O4Si3, and is available as a 30% aqueous emulsion that appears as an off-white homogeneous liquid. An advantageous 30% simethicone emulsion is Medical Antifoam C Emulsion, made with 30% simethicone USP, methylcellulose, sorbic acid, and water.

[0050] "Steroid-refractory" (SR) cGVHD is defined as cGVHD that has progressed while on steroid or corticosteroid treatment, in one embodiment, while on prednisone treatment.

[0051] A "suspension" refers to a mixture of solids finely distributed in a liquid, in which the solids are not dissolved in the liquid. As used herein, the term "liquid formulation" or "formulation" includes a suspension. For example, the terms "liquid formulation containing bermosudil," "liquid formulation of bermosudil," and / or "liquid formulation of bermosudil" are intended to encompass a suspension of bermosudil or a heterogeneous or homogeneous mixture in which solid bermosudil particles are distributed in a liquid but not dissolved therein.

[0052] A "therapeutically effective amount" of an API means an amount that, when administered to a human for treating a disease (e.g., cGVHD), is sufficient to treat the disease state being treated. When applied to human cGVHD, "treating" or "treatment" includes (1) reducing the risk of developing cGVHD and / or inhibiting cGVHD, i.e., suppressing or reducing the onset of cGVHD or its clinical symptoms, and (2) alleviating cGVHD, i.e., causing regression, remission, or improvement of cGVHD, or reducing the number, frequency, duration, or severity of its clinical symptoms.

[0053] A therapeutically effective amount of an API can vary depending on the health and condition of the subject being treated, the extent of disease progression, an evaluation of the medical condition, and other relevant factors. It is expected that a therapeutically effective amount can be in a range that can be determined through testing and by reference to clinical trial data and results, for example, as described in the Examples herein and in the scientific literature.

[0054] 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.

[0055] overview The present disclosure provides useful liquid formulations comprising belmosudil that meet acceptable quality target product profiles (QTPPs) for use in administering belmosudil to patients in a manner that does not require swallowing a solid dosage form.

[0056] In order to provide a liquid formulation containing belmosudil as disclosed herein, various excipients, including viscosity adjusters, preservatives, suspending agents, pH adjusters, sweeteners, thickeners, and antifoaming agents, were investigated. The physical stability problems encountered with the drug were investigated over time and in various temperature and pH systems. The applicant discovered that satisfactory dispersibility and resuspension of belmosudil can be achieved at a suitable content level to meet the label claims for adults and concentrations corresponding to the pediatric population, including colloidal silica systems.

[0057] Formulations were evaluated combining suspending / thickening agents, preservatives, sweeteners, flavors, and antifoaming agents to identify the most advantageous system for a liquid formulation containing belmosudil.

[0058] To select the most suitable input material for use in a liquid formulation containing belmosudil, jet-milled and pin-milled belmosudil were also evaluated. Initially, a comparison of formulations produced using alternative milling techniques for belmosudil suggested that pin-milled API provided the most suitable material for use in the formulation, while jet-milled API presented problems and various barriers. However, upon further investigation, the applicant discovered that jet-milled API provided a morphology that was favorable for achieving uniform homogeneity and meeting QTPP.

[0059] Knowledge of the behavior of vermosudil in liquids, coupled with an understanding of the importance of the ingredients, played a key role in developing this stable formulation. Jet-milled API may be used in this formulation rather than pin-milled API to reduce the amount of precipitation and foaming.

[0060] Additionally, suspending agents such as silica or colloidal silica may be used to minimize settling and may be used in combination with povidone (or polyvinylpyrrolidone [PVP]) to improve dispersibility. Povidone is a polymer and a thickening agent that can help maintain the viscosity of the vehicle.

[0061] In addition, a preservative may be used. In fact, as disclosed herein, it has been found that the use of a preservation method provides an advantage in protecting a liquid formulation containing belmosudil. After experimentation, sodium benzoate was selected as an effective preservative for use in the formulation. Sodium benzoate is a non-dissociated form of benzoic acid, and therefore has both bacteriostatic and antifungal properties. Therefore, a liquid formulation of belmosudil without a preservation method is disadvantageous for preparing a ready-to-use liquid pharmaceutical formulation.

[0062] Liquid formulations that are suspensions must be robust, homogeneous when suspended and resuspended, and ideally not foam when mixed, including during manufacturing. Such formulations can be difficult to prepare. During formulation manufacturing, high-speed mixing with specialized mixing equipment (such as paddle shapes and screens) can be used to reduce particle agglomeration and create a homogeneous, stable dispersion. A side effect of high-speed mixing, especially with hydrophobic drug products, is increased foaming. Compositions and manufacturing processes designed to overcome the foaming problem can result in suspensions with desired characteristics.

[0063] In addition, antifoaming agent may be used.In fact, as disclosed herein, it has been found that the use of antifoaming agent brings advantages to the liquid formulation containing homogeneous and stable bermosudil.After experiment, about 30% simethicone emulsion is selected as an effective antifoaming agent for use in the formulation.Therefore, the liquid formulation of bermosudil that does not include antifoaming method is disadvantageous for preparing ready-to-use liquid pharmaceutical formulation that can be resuspended multiple times in a homogeneous manner.

[0064] To select the most appropriate manufacturing conditions for use in the preparation of a liquid formulation containing veremoszil, an evaluation of the mixing process was also conducted. First, by comparing formulations produced using alternative process steps, it was suggested that using a two-vessel (side phase) process incorporating a hold time step and relatively slow mixing (i.e., using a rotational speed of less than about 1000 rpm) provides the most appropriate manufacturing process for preparing a liquid veremoszil formulation. However, upon further investigation, the Applicant has discovered that by adding an anti-foaming agent to the formulation and manufacturing the formulation using high-speed mixing with a homogenizer equipped with a high-shear screen, a preferred form is provided to achieve uniform homogeneity and meet the QTPP.

[0065] In one embodiment, a homogenization step may be used to improve the uniformity of the formulation. Homogenization may be performed using a standard or high-shear rotor-stator assembly and may include using a shear screen. In one embodiment, homogenization is performed at a rotational 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 one embodiment, homogenization is performed using a high-shear screen. In one embodiment, homogenization is performed using a paddle. In one embodiment, homogenization is performed using a rotor and a stator.

[0066] From human volunteer taste tests, combinations of flavors and sweeteners that showed improved palatability when compared to a flavorless and sweetener-free control system were identified, and development activities were conducted to optimize the thickener / suspension system of veremoszil.

[0067] Drugs and excipients for use in liquid formulations containing belmosudil are commercially available. For example, silica suspending agents sold under the trade name SYLOID® are available from W.R. Grace & Co. (Conn. USA); silica products sold under the trade name AEROSIL® 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 blend flavors are commercially available from Givaudan International SA (Switzerland) and / or IMCD Group; and antifoaming agents such as simethicone are commercially available from DuPont (Wilmington, DE, USA) and Dow Corning (Midland, MI, USA).

[0068] Alternative formulations were identified, and a lead formulation was selected after stability testing. Formulation stability is an important attribute for feasibility, particularly in practical applications where the goal is to deliver a formulation containing belmosudil in a pre-prepared liquid form. Therefore, the applicant evaluated various alternative belmosudil formulations to assess their chemical stability (no decomposition); solution stability (limited pH change over time); physical stability (no irreversible precipitation or the formulation can be reconstituted by stirring); and microbiological stability (use of preservatives to prevent microbial / bacterial growth).

[0069] The belmosudil formulation disclosed herein allows for flexible administration by easily varying the dose volume according to the patient's age and / or weight. In one embodiment, belmosudil is administered in a weight-based administration regime, and subjects receive approximately the same dose on a mg / kg basis. Such a weight-based administration regime is not possible with fixed-dose tablets.

[0070] The liquid formulations containing belmosudil according to the present disclosure are suitable for patients of all ages, including adults and children who are unable, have difficulty, or avoid swallowing solid oral dosage forms. For example, liquid formulations containing belmosudil suitable for children aged 3 months to 12 years, or 3 months to 18 years, are contemplated. The liquid formulations may be manufactured on a large scale for use in commercial applications and for comparative bioavailability evaluations, and compared to solid oral dosage forms, such as commercially approved 200 mg tablets.

[0071] In one embodiment, the present disclosure provides belmosudil in a liquid, suspension form that can be administered by the oral route, for example to adult and / or pediatric patients who are unable, have difficulty, and / or prefer not to swallow oral tablets or capsules.

[0072] In one embodiment, the liquid formulations comprising belmosudil disclosed herein may be provided in a ready-to-use liquid form, with belmosudil already distributed throughout the liquid vehicle. In another embodiment, belmosudil may be prepared and manufactured as a dry powder and provided as a kit with a reconstitution vehicle, with belmosudil being reconstituted with the vehicle prior to use.

[0073] Illustrative Embodiments In one embodiment, a liquid formulation is provided having the components set forth in Table 1.

[0074] [Table 1]

[0075] In one embodiment, bermosudil mesylate is used in a formulation described in Table 1. In one embodiment, the bermosudil mesylate is jet-milled and / or pin-milled. In some embodiments, the bermosudil (or bermosudil mesylate) is jet-milled.

[0076] In another embodiment, the preservative used in the formulations of Table 1 is sodium benzoate.

[0077] In another embodiment, the sweetener used in the formulations of Table 1 is sucralose.

[0078] In another embodiment, the suspending agent used in the formulations of Table 1 is a silica product selected from colloidal silica, silicon dioxide, and fumed silica. In another embodiment, the suspending agent is colloidal silica. In one embodiment, the suspending agent is SYLOID® 244FP. In another embodiment, the suspending agent is AEROSIL® 200.

[0079] In another embodiment, the thickener used in the formulations of Table 1 is a water-soluble polymer made from the monomer N-vinylpyrrolidone. 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.

[0080] In another embodiment, the pH adjuster used in the formulations of Table 1 is tartaric acid. In another embodiment, a pH adjuster is added to achieve a pH in the range of 3.0±0.5.

[0081] In another embodiment, the diluent used for the formulations of Table 1 is sterile water for irrigation.

[0082] In one embodiment, a flavoring agent is added to a 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, optionally, added to the liquid formulation at the approximate time of administration, if needed and / or desired.

[0083] In one embodiment, the flavoring agents are selected from those commercially available and suitable for use in pharmaceutical products.

[0084] In another embodiment, the flavor is selected from tropical fruit blend flavor, lemon flavor, and orange flavor, hi another embodiment, the flavor is a tropical fruit blend flavor.

[0085] In one embodiment, the fragrance is Lemon fragrance PHS-135460 available from IMCD Group, hi another embodiment, the fragrance is Tropical Fruit Blend fragrance PHS-145298 available from IMCD Group.

[0086] In another embodiment, a liquid formulation is provided having the components set forth in Table 2 below.

[0087] [Table 2]

[0088] a The active ingredient (mg) is listed as bermosudil mesylate, with the free base equivalent in parentheses. The labeled dose is specified on the free base basis. Potency adjustments may be made as needed.

[0089] In one embodiment, an anti-foaming agent is added to a formulation described in Table 1 or Table 2. In one embodiment, the anti-foaming agent comprises simethicone. In another embodiment, the anti-foaming agent is about 30% simethicone emulsion.

[0090] In another embodiment, a liquid formulation is provided having the components 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide or a pharmaceutically acceptable salt thereof (bermosudil), 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 if necessary, a sweetener, an antibacterial agent, and an antifoaming agent.

[0091] In another embodiment, a liquid formulation is provided having the components set forth in Table 3 below.

[0092] [Table 3]

[0093] In one embodiment, belamosudil mesylate is used in the formulations described in Table 3. In one embodiment, the belamosudil mesylate is jet milled and / or pin milled. In some embodiments, the belamosudil (or belamosudil mesylate) is jet milled.

[0094] In another embodiment, the preservative used in the formulations of Table 3 is sodium benzoate.

[0095] In another embodiment, the sweetener used in the formulations of Table 3 is sucralose.

[0096] In another embodiment, the suspending agent used in the formulations of Table 3 is a silica product selected from colloidal silica, silicon dioxide, and fumed silica. In another embodiment, the suspending agent is colloidal silica. In one embodiment, the suspending agent is SYLOID® 244FP. In another embodiment, the suspending agent is AEROSIL® 200.

[0097] In another embodiment, the thickening agent used in the formulations of Table 3 is a water-soluble polymer made from the monomer N-vinylpyrrolidone. In another embodiment, the thickening agent is povidone. In another embodiment, the thickening agent is selected from povidone K30 and povidone 90F. In another embodiment, the thickening agent is povidone 90F.

[0098] In another embodiment, the antifoaming agent used in the formulations of Table 3 is simethicone. In another embodiment, the antifoaming agent is about a 30% simethicone emulsion. In one embodiment, the antifoaming agent is Medical Antifoam C Emulsion, which is a 30% simethicone emulsion.

[0099] In another embodiment, the pH adjuster used in the formulations of Table 3 is tartaric acid. In another embodiment, a pH adjuster is added to achieve a pH in the range of 3.0±0.5. In some embodiments, a pH adjuster is added to achieve a pH in the range of 2.5 to 4.0. In some embodiments, a pH adjuster is added to achieve a pH in the range of 2.7 to 3.3.

[0100] In another embodiment, the diluent used for the formulations in Table 3 is sterile water for irrigation. In another embodiment, the diluent used for the formulations in Table 3 is purified water.

[0101] In another embodiment, a liquid formulation is provided having the components set forth in Table 4 below.

[0102] [Table 4]

[0103] In another embodiment, a liquid formulation is provided having the components of Table 1, Table 2, Table 3, or Table 4 that meets a Quality Target Product Profile (QTPP) with the following requirements: a 200 mg dose of bermosudil (free base equivalent), a single oral 5 mL dose (dose expressed as free base equivalent based on a salt correction factor of 1.2124, where 200 mg of free base is equivalent to 242.48 mg of mesylate); uniform color with no taste / feel issues; and sufficient shelf life for packaging and distribution, optionally refrigerated (in some embodiments, 2-8°C).

[0104] In one embodiment, the present disclosure provides a liquid formulation comprising berumosudil, having a concentration of berumosudil equivalent to about 40 mg / mL of the free base. In another embodiment, a more dilute formulation is provided to facilitate administration of lower doses (e.g., a lower dose administered by a highly concentrated formulation requires a smaller volume, which may be more difficult to measure). In another embodiment, a more concentrated formulation is provided to facilitate administration of higher doses (e.g., a higher dose administered by a less concentrated formulation requires a larger volume, which may be more difficult to administer to a patient).

[0105] Thus, in one embodiment, the formulation contains about 242 mg of bermosudil mesylate (equivalent to 200 mg of free base) per 5 mL of liquid formulation. In another embodiment, the formulation is diluted to provide about 242 mg of bermosudil mesylate (equivalent to 200 mg of free base) per 10 mL of liquid formulation. In one embodiment, a 5 mL dose contains an equivalent dose of about 100 mg of bermosudil free base. In another embodiment, the formulation is further diluted to provide about 242 mg of bermosudil mesylate (equivalent to 200 mg of free base) per 20 mL of liquid formulation. In one embodiment, a 5 mL dose contains an equivalent dose of about 50 mg of bermosudil free base.

[0106] In another embodiment, a formulation such as those set forth in Table 1, Table 2, Table 3, or Table 4 is subjected to a homogenization step. In one embodiment, a mixing vessel is used to prepare the formulation, and the vessel is placed in a homogenizer that operates to homogenize the contents during at least one step of preparing the formulation. In one embodiment, homogenization is performed at a rotational speed of about 4000-6000 rpm. In one embodiment, homogenization is performed using a high shear screen. In one embodiment, homogenization is performed using a rotor and stator.

[0107] In another embodiment, there is provided a formulation prepared by a process of mixing jet-milled or pin-milled bermosudil (in one embodiment, bermosudil mesylate) in a vehicle comprising a suspending agent and a thickening agent in a diluent.

[0108] In another embodiment, a liquid formulation containing belmosudil is provided having components that meet the QTPP requirements with the following requirements: a 200 mg belmosudil dose (as free base equivalent), a single oral 5 mL dose (dosage expressed as free base equivalent based on a salt correction factor of 1.2124, where 200 mg of free base is equivalent to 242.48 mg of mesylate); uniform color with no taste / feel issues; and sufficient shelf life for packaging and distribution, optionally refrigerated (in some embodiments, 2-8°C).

[0109] In another embodiment, a 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, optionally, added to the liquid formulation at the approximate time of administration, if needed and / or desired.

[0110] In another embodiment, the flavor is selected from commercially available flavors suitable for use in pharmaceutical products. In some embodiments, the flavor is selected from tropical fruit blend flavor and lemon flavor. In another embodiment, the flavor is a tropical fruit blend flavor.

[0111] In one embodiment, a method for preparing a liquid formulation containing berumosudil is provided, comprising the steps of: (a) dispensing a first portion of a diluent into a suitable mixing vessel; (b) dispensing and mixing a preservative, a suspending agent, a thickening agent, and optionally a sweetener and / or a flavoring into the mixing vessel; (c) optionally visually inspecting after each excipient addition; (d) transferring the mixing vessel to a homogenizer and homogenizing the formulation; (e) optionally visually inspecting; (f) adding and mixing an amount of berumosudil mesylate to the homogenized solution; (g) optionally visually inspecting; (h) measuring the pH of the formulation; (i) optionally adding and mixing a pH adjuster to the formulation in aliquots until the desired pH is achieved; and (j) adding a final portion of the diluent to produce the berumosudil formulation. The liquid formulation containing berumosudil may then be homogenized and / or allowed to stand until any foam or effervescence has disappeared, after which it may be optionally visually inspected. The formulation may be dispensed by weight into final containers, which are sealed and labeled.

[0112] In another embodiment, a liquid formulation comprising bermosudil is provided, prepared according to the steps described in the immediately preceding paragraph.

[0113] In another embodiment, a method for preparing a liquid formulation containing belmosudil is provided, comprising the steps of: (a) dispensing a first portion of sterile water for irrigation into a suitably sized mixing vessel; (b) dispensing and mixing sodium benzoate, sucralose, silica colloid hydrate, and povidone into the mixing vessel; (c) optionally visually inspecting after each excipient addition; (d) transferring the mixing vessel to a homogenizer and homogenizing the formulation; (e) optionally visually inspecting; (f) adding and mixing an amount of belmosudil mesylate to the homogenized solution; (g) optionally visually inspecting; (h) measuring the pH of the formulation; (i) adding and mixing tartaric acid in aliquots to the formulation until the desired pH is achieved; and (j) adding a final portion of sterile water for irrigation to produce the belmosudil formulation. The liquid formulation containing belmosudil may then be homogenized and / or allowed to stand until any bubbles or foam have disappeared, after which it may be optionally visually inspected. The formulation may be dispensed by weight into final containers, which are sealed and labeled.

[0114] In another embodiment, a liquid formulation comprising bermosudil is provided, prepared using the process described in the immediately preceding paragraph.

[0115] In one embodiment, the foregoing steps are carried out using the weight percentages of each excipient and ingredient as set forth in Table 1, Table 2, Table 3, or Table 4.

[0116] In another embodiment, a method is provided for preparing a liquid formulation comprising bermosudil according to the following steps: (a) dispensing a first portion of purified water into a first suitably 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 bermosudil mesylate to the solution and mixing; (e) optionally, performing a visual inspection; (f) dispensing a second portion of purified water into a second suitably sized container; (g) adding sodium benzoate, sucralose, and silica. (h) optionally, visual inspection is performed after each addition of each excipient; (i) the materials are transferred from the second mixing vessel to the first mixing vessel and the formulation is mixed; (i) tartaric acid is added to the formulation in aliquots and mixed until the desired pH is achieved; (k) the final portion of purified water is added; (l) the formulation is mixed; (m) optionally, visual inspection is performed; and (n) the liquid formulation containing bermosudil is allowed to stand until any bubbles or foam disappears to produce the bermosudil formulation, which may then be visually inspected. The formulation may be dispensed by weight into final containers, sealed, and labeled.

[0117] In another embodiment, a liquid formulation comprising bermosudil is provided, prepared using the process described in the immediately preceding paragraph.

[0118] In another embodiment, the mixing step of the aforementioned process is performed using a paddle mixer. In another embodiment, the mixing step of the aforementioned process is performed at a rotation speed of about 300 to 600 rpm.

[0119] In one embodiment, the foregoing steps are carried out using the weight percentages of each excipient and ingredient as set forth in Table 1 or Table 2.

[0120] In another embodiment, a method for preparing a liquid formulation containing bermosudil is provided, comprising the steps of: (a) dispensing a portion of purified water into a suitable sized mixing vessel; (b) dispensing and mixing sodium benzoate, sucralose, silica colloid hydrate, and povidone into the mixing vessel; (c) adding and mixing an amount of about 30% simethicone emulsion to the formulation; (d) adding and mixing an amount of bermosudil mesylate to the solution; (e) optionally, visually inspecting; (f) adding and mixing tartaric acid to the formulation in aliquots until the desired pH is achieved; (g) adding the final portion of purified water; (h) mixing the formulation; and (i) optionally, visually inspecting. The formulation may be dispensed by weight into final containers, which are sealed and labeled.

[0121] In another embodiment, a liquid formulation comprising bermosudil is provided, prepared using the process described in the immediately preceding paragraph.

[0122] In another embodiment, one or more of the mixing steps of the foregoing processes are performed using a homogenizer. In another embodiment, step (h) of the foregoing processes is performed using a homogenizer. In another embodiment, one or more of the mixing steps of the foregoing processes are performed using a Silverson® homogenizer. In another embodiment, one or more of the mixing steps of the foregoing processes are performed using a high shear screen. In another embodiment, one or more of the mixing steps of the foregoing processes are performed at a rotational speed of at least about 1000 rpm. In another embodiment, one or more of the mixing steps of the foregoing processes are performed at a rotational speed of about 4000-6000 rpm.

[0123] In another embodiment, a method is provided for preparing a liquid formulation comprising belmosudil, according to the following steps: (a) dispensing a portion of purified water into a suitably sized mixing vessel; (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 silica colloid hydrate to the formulation and mixing; (f) adding approximately 30% of simethicone emulsion to the formulation; (g) homogenizing the formulation at 5000 rpm; (h) adding an amount of belmosudil mesylate to the formulation and mixing; (i) homogenizing the formulation at 5000 rpm using a high shear screen; (j) optionally, visually inspecting; (k) adding tartaric acid to the formulation and mixing until a pH of 0.3 is achieved; (l) adding a final portion of purified water; (m) mixing the formulation; and (n) optionally, visually inspecting. The formulation may be dispensed by weight into final containers, which are sealed and labeled.

[0124] In another embodiment, the mixing step is performed at a rotational speed of about 500-600 rpm. In another embodiment, the homogenizing step is performed using a Silverson® homogenizer. In another embodiment, the homogenizing step is performed using a high shear screen. In another embodiment, the homogenizing step is performed at a rotational speed of at least about 1000 rpm. In another embodiment, the homogenizing step is performed at a rotational speed of about 4000-6000 rpm. In another embodiment, the homogenizing step is performed at a rotational speed of about 5000 rpm.

[0125] In one embodiment, the foregoing steps are carried out using the weight percentages of each excipient and ingredient as set forth in Table 3 or Table 4.

[0126] In another embodiment, the following weight percentages are used to add excipients and ingredients to the formulation: about 2-8 weight percent vermosudil, about 0.02-0.08 weight percent preservative; about 0.1-0.4 weight percent sweetener; about 0.2-0.8 weight percent suspending agent; and about 1-8 weight percent thickener.

[0127] In another embodiment, the following weight percentages are used to add excipients and ingredients to the formulation: about 2-8 weight percent vermosudil, about 0.02-0.08 weight percent preservative; about 0.1-0.4 weight percent sweetener; about 0.2-0.8 weight percent suspending agent; about 2.1-8.2 weight percent thickener; and about 0.25-4.0 weight percent antifoaming agent.

[0128] In another embodiment, the following weight percentages are used to add excipients and ingredients to the formulation: (a) in the case of berumosdil, about 2-6 weight percent berumosdil, in another embodiment, about 3-5 weight percent berumosdil (or optionally, about 4.5-5 weight percent berumosdil, or optionally, about 4.75 weight percent berumosdil); (b) in the case of preservatives, about 0.035-0.1 weight percent preservatives (or optionally, about 0.05 weight percent preservatives). ); (c) in the case of a sweetening agent, about 0.1 to 0.2 weight percent sweetening agent (or optionally about 0.15 weight percent sweetening agent; (d) in the case of a suspending agent, about 0.3 to 1 weight percent suspending agent, optionally about 0.4 to 0.6 weight percent suspending agent (or optionally about 0.5 suspending agent); and (e) in the case of a thickening agent, about 4 to 6 weight percent thickening agent (or optionally about 4.5 to 5.5 weight percent thickening agent; or in another embodiment, about 4.9 weight percent thickening agent).

[0129] In another embodiment, the following weight percentages are used to add excipients and ingredients to the formulation: (a) in the case of bermosdil, about 2-6 weight percent bermosdil, in another embodiment, about 3-5 weight percent bermosdil (or optionally about 4.5-5 weight percent bermosdil, or optionally about 4.75 weight percent bermosdil); (b) in the case of a preservative, about 0.035-0.1 weight percent preservative (or optionally about 0.05 weight percent preservative); (c) in the case of a sweetener, about 0.1-0.2 weight percent sweetener (or optionally about 0.15 weight percent sweetening agent; (d) if a suspending agent, about 0.3 to 1 weight percent suspending agent, optionally about 0.4 to 0.6 weight percent suspending agent (or optionally about 0.5 suspending agent); (e) if a thickening agent, about 4 to 6 weight percent thickening agent (or optionally about 4.5 to 5.5 weight percent thickening agent; or in another embodiment, about 4.9 weight percent thickening agent); and (f) if an antifoaming agent, about 0.25 to 4 weight percent antifoaming agent (or optionally about 0.5 to 3.5 weight percent antifoaming agent; or in another embodiment, about 2.0 weight percent antifoaming agent).

[0130] In another embodiment, a liquid formulation comprising bermosudil is provided, comprising about 2 to 8 weight percent bermosudil, and in another embodiment, about 2 to 6 weight percent bermosudil.

[0131] In another embodiment, a liquid formulation containing bermosudil is provided, which contains about 0.02 to 0.08 weight percent sodium benzoate as a preservative, and in another embodiment, about 0.035 to 0.1 weight percent sodium benzoate.

[0132] In another embodiment, a liquid formulation containing bermosudil is provided, which contains about 0.1 to 0.4 weight percent sucralose as a sweetener, and in another embodiment, about 0.1 to 0.2 weight percent sucralose.

[0133] In another embodiment, a liquid formulation comprising bermosudil is provided, which comprises about 0.2 to 0.8 weight percent colloidal silica as a suspending agent, and in another embodiment, about 0.4 to 0.6 weight percent colloidal silica.

[0134] In another embodiment, a liquid formulation containing bermosudil is provided, comprising about 1 to 8 weight percent povidone as a thickening agent. In another embodiment, the liquid formulation comprises about 4 to 6 weight percent povidone. In a specific embodiment, the povidone is povidone 90F.

[0135] In another embodiment, a liquid formulation comprising bermosudil is provided, comprising about 0.25 to 4 weight percent of an antifoaming agent. In another embodiment, a liquid formulation comprising bermosudil is provided, comprising about 0.25 to 4 weight percent of about 30% simethicone emulsion as an antifoaming agent. In another embodiment, the liquid formulation comprises about 0.5 to 3.5 percent of about 30% simethicone emulsion as an antifoaming agent. In another embodiment, the liquid formulation comprises about 2.0 percent of about 30% simethicone emulsion as an antifoaming agent. In a specific embodiment, the about 30% simethicone emulsion is Medical Antifoam C Emulsion.

[0136] In another embodiment, beromosudil is pin-milled or jet-milled prior to addition to the formulation (e.g., for step [f] of the process described above). In another embodiment, jet-milled beromosudil mesylate is added to the formulation.

[0137] 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.

[0138] In one embodiment, a liquid formulation of belmosudil is analyzed using a reversed-phase gradient HPLC method. In one embodiment, the liquid formulation is assayed (e.g., in one embodiment, by HPLC) during its manufacture. In one embodiment, the first, middle, and last bottles filled during manufacturing are assayed.

[0139] In one embodiment, the liquid formulation containing bermosudil is formulated to deliver a single dose of 200 mg of bermosudil (as free base) in a volume of about 5 mL. In another embodiment, the liquid bermosudil is formulated to deliver a single dose in the range of 12 to 200 mg (as free base) in a single volume dose of about 1 to 10 mL, and in another embodiment, in a volume of about 5 mL.

[0140] In another embodiment, a liquid formulation containing belmosudil is formulated to deliver a single dose in the range of 10 to 400 mg (as free base) in a single volumetric dose of about 1 to 10 mL, and in another embodiment, in a single volumetric dose of about 5 mL.

[0141] In another embodiment, the liquid formulation comprising belmosudil is formulated to deliver a single dose of API in the range of about 50-200 mg (free base equivalent) in a volume of about 1-10 mL, and in another embodiment, about 5 mL. In another embodiment, belmosudil is formulated to deliver a dose of about 50, 100, or 200 mg (free base equivalent) of API in a volume of about 1-10 mL.

[0142] In another embodiment, a liquid formulation comprising bermosudil is prepared to deliver a dose of about 50, 100, 200, or 400 mg of API (as free base equivalent) in a single volume dose of about 5 mL.

[0143] In one embodiment, the liquid formulation containing bermosudil is provided in a bottle containing 10 to 100 mL of the formulation, in another embodiment, the liquid formulation containing bermosudil is provided in a bottle containing 20 to 60 mL of the liquid formulation, in another embodiment, the liquid formulation containing bermosudil is provided in a bottle containing about 30 mL ± 2 mL of the formulation, and in some embodiments, the bottle of liquid formulation containing bermosudil contains a total of about 5 to 6 unit doses.

[0144] In one embodiment, the liquid formulation containing bermosudil is provided in a suitably sized oral syringe, and a single unit dose is metered into the syringe prior to administration. In one embodiment, the syringe is sized to dispense 1 to 10 mL of the liquid formulation containing bermosudil for administration. In another embodiment, the syringe is sized to dispense up to 5 mL of the liquid formulation for unit administration.

[0145] In another embodiment, the liquid formulation comprising bermosudil is provided in a suitably 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 hold 1 to 10 mL of the liquid formulation comprising bermosudil for administration. In another embodiment, the dosing cup includes a fill line indicating the level required to fill the dosing cup to approximately 5 mL of the liquid formulation for unit administration.

[0146] In one embodiment, the liquid formulation comprising belmosudil is packaged in a medical bottle. In another embodiment, the bottle for packaging the liquid formulation comprising belmosudil contains a child-resistant lid or cap. In another embodiment, the liquid formulation comprising belmosudil 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 formulation comprising belmosudil is packaged in a suitably sized Type III amber glass container with a tamper-evident cap. In one embodiment, the bottle is equipped with a polyethylene cap. In one embodiment, the liquid formulation comprising belmosudil is packaged in a medical bottle comprising a polyethylene liner.

[0147] In one embodiment, the liquid formulation is refrigerated at a temperature ranging from 2-8° C. In one embodiment, a liquid formulation stored at refrigerated temperatures is accompanied by instructions that the formulation should be administered within a specific time period while the product is in its final dosage form.

[0148] In another embodiment, the liquid formulation is stored at ambient temperature, for example, about 15-25° C. In one embodiment, a liquid formulation stored at ambient temperature is accompanied by instructions that the formulation should be administered within a specified time period after reconstitution and / or placement in its final dosage form.

[0149] In another embodiment, the liquid formulation is accompanied by instructions that once the liquid formulation is pipetted or withdrawn into a syringe, the formulation must be used or discarded within a specific period of time (in one embodiment, within 4 hours).

[0150] Pharmaceutical Compositions, Uses and Administration In one aspect, the present invention provides a pharmaceutically acceptable liquid composition comprising a therapeutically effective amount of bermosudil formulated with one or more pharmaceutically acceptable excipients in a liquid diluent.

[0151] In one aspect, the present invention provides a pharmaceutically acceptable liquid composition comprising a therapeutically effective amount of belmosudil formulated in a liquid diluent with one or more pharmaceutically acceptable excipients at a concentration equivalent to about 40 mg / mL of belmosudil free base.

[0152] In one aspect, the present invention provides a pharmaceutically acceptable liquid composition comprising a therapeutically effective amount of berumosudil formulated in a liquid diluent with one or more pharmaceutically acceptable excipients at a concentration equivalent to about 40 mg / mL of berumosudil free base, wherein the administered dose is about 2.5 to 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 one embodiment, the administered dose is about 3.0 mg / kg. In one embodiment, the administered dose is about 3.5 mg / kg. In one embodiment, the administered dose is about 4.0 mg / kg. In one embodiment, the administered dose is about 4.5 mg / kg. In one embodiment, the administered dose is about 5.0 mg / kg.

[0153] In one aspect, the present invention provides a pharmaceutically acceptable liquid composition comprising a therapeutically effective amount of berumosudil formulated in a liquid diluent with one or more pharmaceutically acceptable excipients at a concentration equivalent to about 40 mg / mL of berumosudil free base, wherein the administration dose is about 2.5 to 5.0 mg / kg, particularly about 3.0, 3.5, 4.0, 4.5, and 5.0 mg / kg, more particularly about 4.0 mg / kg, with a maximum administration dose equivalent to 200 mg of berumosudil free base administered once daily or twice daily.

[0154] The patient may be an adult or a pediatric patient. In one embodiment, the patient is an adult patient. In one embodiment, the patient is a pediatric patient.

[0155] In one embodiment, the present disclosure provides a method of administering a liquid formulation comprising belmosudil to a patient, e.g., an adult patient weighing about 40 kg or more, at an equivalent dose of 200 mg once daily. In another embodiment, the present disclosure provides a method of administering a liquid formulation comprising belmosudil to a patient, e.g., an adult patient weighing about 40 kg or more, at an equivalent dose of 200 mg twice daily or 400 mg once daily.

[0156] In one embodiment, the present disclosure provides a method for administering a liquid formulation containing belmosudil to a pediatric patient having a body weight in the range of about 6 kg to less than 20 kg at an equivalent dose of about 10 to 50 mg administered once daily. In another embodiment, the present disclosure provides a method for administering a liquid formulation containing belmosudil to a pediatric patient having a body weight in the range of about 10 kg to less than 20 kg at an equivalent dose of about 50 mg administered once daily. In another embodiment, the present disclosure provides a method for administering a liquid formulation containing belmosudil to a pediatric patient having a body weight in the range of about 20 kg to less than 40 kg at an equivalent dose of about 100 mg administered once daily. In another embodiment, the present disclosure provides a method for administering a liquid formulation containing belmosudil to a pediatric patient having a body weight of about 40 kg or more at an equivalent dose of 200 mg administered once daily.

[0157] In one embodiment, the present disclosure provides a method for administering a liquid formulation comprising belmosudil to a patient at a dose of about 2.5 to 5.0 mg / kg, e.g., about 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mg / kg. In one embodiment, the present disclosure provides a method for administering a liquid formulation comprising belmosudil to a patient at a dose based on the patient's body weight. In one embodiment, the present disclosure provides a method for administering a liquid formulation comprising belmosudil to a patient at a dose of about 2.5 mg / kg. In another embodiment, the present disclosure provides a method for administering a liquid formulation comprising belmosudil to a patient at a dose of about 3.0 mg / kg. In another embodiment, the present disclosure provides a method for administering a liquid formulation comprising belmosudil to a patient at a dose of about 3.5 mg / kg. In another embodiment, the present disclosure provides a method for administering a liquid formulation comprising belmosudil 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 comprising bermosudil to a patient at a dose of about 4.5 mg / kg. In another embodiment, the present disclosure provides a method of administering a liquid formulation comprising bermosudil to a patient at a dose of about 5.0 mg / kg. In one embodiment, the dose is administered once daily. In one embodiment, the dose is administered twice daily. In one embodiment, the patient is an adult patient. In one embodiment, the patient is a pediatric patient. In one embodiment, the maximum administered dose corresponds to about 200 mg of bermosudil free base once or twice daily.

[0158] The liquid formulations and pharmaceutical compositions disclosed herein may be useful for inhibiting ROCK1 and / or ROCK2 enzymes, preferentially ROCK2, and thus may be useful for treating diseases regulated by ROCK enzymes, such as autoimmune and / or fibrotic disorders, e.g., GVHD (chronic and acute), pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, radiation-induced fibrosis, or arterial, cardiac, endomyocardial, renal, or hepatic fibrosis; moderate to severe psoriasis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), Crohn's disease, dermatitis (e.g., atopic dermatitis), and eczema, among other indications.

[0159] The liquid formulation may also be useful in treating bronchiolitis obliterans syndrome (BOS), a potentially serious complication following lung or allogeneic hematopoietic stem cell transplantation (allo-HSCT).

[0160] The liquid formulation may also be useful in treating chronic pulmonary allograft dysfunction (CLAD) after lung transplantation.

[0161] The following abbreviations may be useful in consideration of this disclosure:

[0162] [Table 5]

[0163] [Table 6] [Example]

[0164] Example 1: Selection of excipients and preservatives First, the excipients listed in Table 5 below were identified as being suitable for inclusion in Belmosudil formulations. In investigating the use of these excipients, we considered whether they were suitable for use in pediatric patient populations by reviewing regulations related to age-appropriate excipients for children in the target age group of 3 months to 12 years.

[0165] [Table 7]

[0166] Initially, all excipients listed in Table 5 were identified as suitable for evaluation. The only excipient identified as not useful for use in pediatric formulations was banana 501013 AP0551 flavoring, in view of containing residual amounts of benzyl alcohol, which is deemed unacceptable in the pediatric population without sufficient justification.

[0167] Certain excipients were identified as having specific Acceptable Daily Intake (ADI) levels. When considering the ADI levels, the maximum levels to be included in pediatric Belmosudil formulations were calculated taking into account the intended maximum daily dose and the average weight (5.5 kg) of a 3-month-old girl according to the 2000 CDC growth charts in the United States (representing the minimum weight for the target age group of 3 months to 12 years).

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

[0169] The results of this compatibility study demonstrated that Belmosudil was incompatible with hydrochloric acid and maleic acid over a 14-day period at 2-8°C and 40°C / 75% relative humidity. Therefore, hydrochloric acid and maleic acid were not further evaluated for inclusion in the liquid formulation.

[0170] Additionally, excipient compatibility data at T=14 days showed poor chromatographic resolution between known impurities and bermosudil when combined with the following preservatives: sodium methyl hydroxybenzoate, sodium ethyl hydroxybenzoate, sodium propylparaben, and ethylparaben. Therefore, these preservatives were not further evaluated for use in preparing liquid formulations.

[0171] Excipient compatibility data at T=28 days showed minor interactions with the potassium sorbate sample, with an unknown related substance eluting at a relative retention time of 0.96, which had an area % of 0.09% at 40°C / 75% relative humidity. pH storage stability screening studies were then performed to identify target pH, pH adjusters, and storage systems suitable for inclusion in future formulations to be developed. For example, samples containing the following components were prepared for evaluation: 1.1 Vermosudil 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% relative humidity, and 40°C / 75% relative humidity for testing at T=0 (appearance and pH only), 14 days, and 28 days. After storage, all samples were evaluated and assayed for appearance, pH, and / or related substances.

[0173] Results demonstrated that chemical interactions were observed in Sample 1.2, containing 2.0 mg / mL potassium sorbate and no pH adjuster, after 14 days of storage at 40°C / 75% relative humidity, showing a significant increase by Day 28. Therefore, this pH preservative system was not recommended for inclusion in future formulation prototypes.

[0174] Additionally, Sample 1.7 (containing potassium sorbate adjusted to pH 5.4 with 0.1 M citrate buffer) and Sample 1.8 (containing sodium benzoate adjusted to pH 4.0 with 0.1 M citrate buffer) did not disperse easily with manual agitation and showed significant physical changes upon visual observation after storage for up to 28 days at 25°C / 60% relative humidity and 40°C / 75% relative humidity. These samples (1.7 and 1.8) could not be dispersed by shaking and were therefore not considered for further development of Belmosudil formulations. All other samples dispersed easily with manual agitation to form yellow, opaque dispersions containing a yellow foam, which were considered suitable for inclusion in future formulation prototypes.

[0175] Example 2: Stability test using pH adjuster To evaluate the stability of belmosudil in selected aqueous liquid formulations, a pH storage stability screening test was conducted using pH adjusters and preservatives selected to achieve a pH range of 3.0 to 5.0. Samples containing sodium benzoate as a preservative and with the pH adjusted with either citric acid or tartaric acid were prepared and compared with samples without a pH adjuster. Table 6 lists the samples used in this test.

[0176] [Table 8]

[0177] The above samples were prepared for XRPD analysis in three steps: the solid was isolated by centrifugation at 3000 rpm for 20 minutes; the supernatant was decanted; and the sample was dried overnight under vacuum at room temperature for 18-24 hours. XRPD data was acquired for the solid samples. The XRPD data demonstrated that the API (bermosudil) was stable in the formulation, which was a positive indicator of providing a stable formulation. The XRPD data demonstrated the expected presence of consistent additional peaks in the solids isolated from the samples containing sodium benzoate but without a pH adjuster, and the samples containing sodium benzoate and where the pH was adjusted with either citric acid or tartaric acid.

[0178] However, during testing, samples containing potassium sorbate and citrate buffer were observed to drift downward in pH, along with significant physical changes. XRPD data from solid samples obtained from formulations containing citrate buffer (samples 2.5 and 2.6 in Table 6) demonstrated the changes as shown by XRPD. Based on these observed changes, citrate buffer was not selected for use in the formulations.

[0179] Example 3: Comparison of pin milling and jet milling with povidone Suspension formulations present challenges in achieving uniform dispersion of API particles throughout the liquid medium. The use of jet-milled and pin-milled forms of Belmosudil in a liquid medium was investigated by evaluating the particle size distribution and homogenization of the formulation initially and over time.

[0180] (a) Comparison of pin mill grinding and jet mill grinding in water To study pin-milled and jet-milled dispersions of Belmosudil in sterile water for irrigation, samples were prepared by weighing a portion of the API into a clear glass bottle and adding sterile water for irrigation to 40 mg / mL.

[0181] Samples were evaluated on a Sympatec Helos particle size analyzer using the general method for wet dispersions as outlined below: -R5 and R3 lenses -10~15% C opt Add sample until - Method runs resulting in three replicates (samples were agitated and sonicated between measurement runs)

[0182] Particle size distribution (PSD) data for pin-milled API in sterile water for irrigation using R3 and R5 lenses are shown in Figures 2A and 2B, respectively. Results for pin-milled API demonstrate reproducibility between replicates using both R5 and R3 lenses. When the study was repeated, the PSD data was not comparable to the original evaluation. Starting C of 10-15% opt It was difficult to obtain the required C (probably due to insufficient distribution of APIs). opt Additional sample was added to achieve

[0183] Evaluation of wet dispersions using jet-milled vermosudil produced less than satisfactory results, with inconsistent measurements between runs. This was attributed to insufficient dispersion of the API. These results reflected the potential challenges of using jet-milled materials and their potential limitations in formulation development. Further use of jet-milled materials requires addressing additional obstacles in terms of improving formulation stabilization and dispersibility of jet-milled materials.

[0184] (b) Comparison of pin milling and jet milling with various suspending agents To further evaluate and compare the pin milling and jet milling processes in the development of liquid formulations, 11 different formulations were prepared with composition details as shown in Tables 5, 6, 7, and 8 below. In each of Tables 7, 8, 9, and 10, bermosudil mesylate was added at 48.496 mg / mL, which corresponds to 40 mg / mL of free base when adjusted for a salt correction factor of 1.2124.

[0185] [Table 9]

[0186] [Table 10]

[0187] [Table 11]

[0188] [Table 12]

[0189] The following steps were used in preparing the formulations described in Tables 7, 8, 9 and 10: 1. Add approximately 40 mL of sterile water for irrigation to a pre-calibrated beaker. 2. Add sodium benzoate while stirring and mix until dissolved. 3. Add the suspending agent and mix until a uniform mixture is observed. 4. Add the vermosudil while mixing and mix until a uniform mixture is obtained. 5. Measure the pH and, if necessary, add tartaric acid in aliquots until a pH of 3.5±0.2 is achieved; 6. Add sterile water to adjust volume for irrigation and record pH, and 7. The liquid formulation containing Belmosudil was transferred into a 60 mL clear glass bottle.

[0190] The samples were evaluated for ease of dispersion of the API, sedimentation level, and ease of redispersion of any sediment.

[0191] The API dispersed readily without a suspending agent in Samples 1A (pin-milled) (Table 7) and 1B (jet-milled) (Table 9). After approximately 24 hours, a small amount of flocculation was observed, which was easily resuspended by shaking. Both the pin-milled and jet-milled API foamed upon shaking, but the jet-milled API (Sample G) foamed less clearly and only contained a small foamy top layer after approximately 30 seconds.

[0192] Conversely, in the batches containing xanthan gum (Xantural 75), the API did not readily disperse (i.e., Samples 2A and 2B / Tables 7 and 9, respectively). These samples required homogenization to aid in dispersing the API, and neither batch appeared to be a homogenous formulation, even after homogenization. From this observation, it was concluded that xanthan gum is not an ideal suspending agent. Sample 5A (Table 8), which contained sodium carboxymethylcellulose, exhibited severe flocculation, making it difficult to disperse the API, even with homogenization. Additionally, a large amount of residue was observed at the bottom of the glass beaker after the contents of the glass beaker were transferred to a glass bottle.

[0193] In other samples using pin-milled and jet-milled hydroxyethylcellulose (Natrosol 250HX) (Samples 3A and 3B); hypromellose K4M (Samples 4A and 4B), and povidone K30 (Samples 6A and 5B), the API was easily dispersed using a magnetic stirrer to form a yellow, opaque suspension, with no observed differences between batches. Upon transfer from the beaker to the bottle, small aggregates of undispersed material were observed as residue, suggesting that further homogenization may be required. Overall, improved results were observed with the use of povidone.

[0194] In both Samples 4B and 5B (Table 10), which contained jet-milled API, the API was easily dispersed using only a magnetic stirrer, forming a yellow, opaque suspension. There were no observed differences between the samples. These observations were consistent with those previously noted for equivalent batches incorporating pin-milled API (i.e., Samples 4A and 6A [Table 8], which contained hypromellose K4M and povidone K30, respectively).

[0195] All samples in Tables 7, 8, 9, and 10 were monitored and evaluated for appearance over 4 days and 2 weeks at ambient temperature. Samples 4B and 5B (Table 10) were produced approximately 1 week after all other samples and were therefore evaluated only at T=4 days. Results are summarized in Table 11 (pin milled API) and Table 12 (jet milled API).

[0196] [Table 13]

[0197] [Table 14]

[0198] Observations demonstrated that samples (manufactured with both jet-milled and pin-milled API) that did not contain any of the suspending agents, Natrosol 250HX, Xantural 75, or sodium carboxymethylcellulose, all showed various signs of either separation, flocculation, or caking. Samples incorporating hypromellose K4M (Samples 4A and 4B) were observed as yellow, opaque suspensions with visually dispersed API after 4 days and / or 2 weeks of storage at ambient temperature, whereas 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. Upon shaking to liquefy and redisperse, the API began to aggregate in the formulation. Therefore, it was determined that hypromellose K4M was not the optimal choice for further development.

[0199] Samples using povidone K30 (manufactured with both jet-milled and pin-milled API) remained yellow, opaque suspensions, and the API was visibly dispersed after 4 days at ambient temperature and after approximately 2 weeks of storage. From this study, povidone K30 was considered the primary suspending agent for further evaluation.

[0200] Example 4: Particle size distribution analysis Based on the results from the visual observation analysis in Example 3, four of the liquid formulations containing Belmosudil were selected and further evaluated for their particle size distribution. Measurements were performed on the following four formulations: Sample 3A (a pin-milled formulation containing hydroxyethylcellulose); Sample 3B (a jet-milled formulation containing hydroxyethylcellulose); Sample 4A (a pin-milled formulation containing hypromellose K4M); and Sample 6A (a pin-milled formulation containing povidone).

[0201] 10-15% C optSamples were evaluated using a Sympatec Helos particle size analyzer, with sample being added until a particle size of 0.5-175 μm was achieved. Two different lens assemblies representing the following ranges were used to evaluate particle size: R3 lens (0.5-175 μm) and R5 lens (4.8-875 μm). Three replicates were performed as part of the measurement run, and samples were agitated and sonicated as part of the method.

[0202] Samples 3A and 3B were evaluated using an R5 lens. The results are shown in Figures 3A (Sample 3A) and 3B (Sample 3B). The results demonstrate that although the recorded particle sizes were larger than expected (when compared to the dry PSD of the milled API), consistent replicates were obtained for both batches. During investigation, it was noted that the formulation containing Natrosol 250HX formed solid aggregates when diluted with water instead of dispersing.

[0203] This may be the result of severe flocculation, which is consistent with the PSD measurements, which show unexpectedly high values ​​comparing both API samples. This effect is particularly exacerbated in the jet-milled sample (Sample 3B).

[0204] For Sample 4A (pin-milled hypromellose K4M formulation), PSD was evaluated using both R5 and R3 lenses. The most consistent replicates were obtained using the R3 lens, and the results are shown in Figure 3C. Multiple peaks were observed at larger dimensions, which were believed to be due to air bubbles. The agitation speed was reduced from 80% to 50% to minimize aeration and bubble generation and achieve consistent replicates.

[0205] For Sample 6A (a pin-milled formulation containing povidone), the sample was evaluated using both R5 and R3 lenses. As with Sample 4A, the most consistent replicates were obtained using the R3 lens, and the results are shown in Figure 3D. The flocculation / aggregates present in Samples 3A and 3B containing Natrosol 250HX were not observed with this formulation.

[0206] The D10, D50, and D90 values ​​of Sample 6A were consistent with those of Sample 4A, both of which were manufactured using pin-milled Vermosudil, providing confidence that the method was accurate and capable of producing consistent results for these two formulation prototypes.

[0207] In summary, when Samples 3A and 3B (containing hydroxyethyl cellulose / Natrosol 250HX) were tested, aggregates were observed upon dilution with water in the mixing tank, resulting in inconsistent data between replicates. Therefore, the results did not represent the expected particle size distribution of the API. Differences were observed between the pin-milled and jet-milled API, with greater aggregates and flocculation observed in Sample 3B, which was produced using jet-milled API, compared to Sample 3A (which used pin milling). This observation confirmed the conclusion from Example 3 that jet-milled material is problematic and can be an obstacle in formulation development.

[0208] Analysis of Samples 4A and 6A (pin-milled formulations containing hypromellose K4M and povidone K30) showed consistent replicates after using the R3 lens and reducing the stirring speed from 80% to 50%. Occasionally, large peaks were observed, which were due to air bubbles. Results were similar for both Samples 4A and 6A. This suggests that these formulation prototypes can achieve more consistent PSD results compared to the particle size evaluation of the API-only wet dispersion in water shown in Figures 2A and 2B and described above in Example 3 (section (a)). From this evaluation, the pin-milling method was deemed suitable for developing liquid formulations containing bermosudil.

[0209] Example 5: Investigation of xanthan gum and sodium CMC thickener systems In Example 3, it was found that samples containing 3 mg / mL xanthan gum (Xantural 75) (Sample 2A) and 3 mg / mL sodium CMC (Blanose CMC 7H3SXF) (Sample 5A) exhibited severe flocculation and difficulty dispersing the API. In this experiment, four additional formulations were investigated to determine whether flocculation could be addressed by doing the following: - Add the suspending agent after the API is completely dispersed; - Reducing the levels of xanthan gum and sodium CMC; - Contains silica products (i.e., AEROSIL® 200 and SYLOID® 244 FP) to prevent caking and aid resuspension.

[0210] Samples 7A, 8A, 9A and 10A were prepared in 50 mL batch sizes according to the composition details provided in Table 13.

[0211] The formulation preparation was planned according to the following steps: (1) to a suitable beaker, approximately 40 mL of sterile water was added; (2) while mixing using a magnetic stirrer, sodium benzoate was added and mixed until dissolved; (3) while mixing, vermosudil (pin milled) was added and mixed for 15 minutes until a uniform dispersion was obtained; (4) while mixing, Xantural 75 or Blanose CMC 7H3SXF was added and mixed for 30 minutes until uniform; (5) the pH was measured and adjusted to pH 3.5±0.2 by adding tartaric acid, if necessary; (6) sterile water was added to adjust the volume and the formulation was mixed for 15 minutes until uniform.

[0212] [Table 15]

[0213] However, all samples (7A, 8A, 9A, and 10A) showed signs of flocculation after the API was added (step 4), as previously observed in Example 3. Samples 7A and 8A, which contained xanthan gum, were homogenized for approximately 1 minute using a Silverson homogenizer fitted with a universal disintegrating head. The severe flocculation previously observed was still observed. Production was stopped after step 4 due to severe flocculation.

[0214] This experiment demonstrated that changing the order of addition did not prevent the flocculation of the material as previously observed. While reducing the amount of suspending agent showed some improvement, flocculation and thickening still occurred, resulting in the discontinuation of production of all four formulations. Based on these observations, xanthan gum and sodium CMC were eliminated as suitable suspending agents for use in pharmaceutical formulations of Belmosudil.

[0215] Example 6: Povidone Suspension System Experiments Additional formulations, summarized below, were manufactured incorporating various levels of povidone K30 as the lead suspending agent. AEROSIL® 200 and SYLOID® 244 FP were incorporated into these formulations to evaluate their effect on caking prevention. Each formulation was manufactured in 100 mL batch sizes using both pin-milled and jet-milled API to allow for a comparative evaluation of the two. Composition details are provided in Tables 14A and 14B (pin-milled API) and Tables 15A and 15B (jet-milled API). In each of Tables 14A, 14B, 15A, and 15B, bermosudil mesylate was added at 48.496 mg / mL, which corresponds to 40 mg / mL of free base when adjusted for a salt correction factor of 1.2124.

[0216] [Table 16]

[0217] [Table 17]

[0218] [Table 18]

[0219] [Table 19]

[0220] The formulations in Tables 14A-14B and 15A-15B were prepared by the following steps: 1. Add approximately 80% of the total volume of sterile water for irrigation to a suitable beaker; 2. While mixing using a magnetic stirrer, add sodium benzoate and mix until dissolved. 3. Add povidone K30 while mixing and mix for 30 minutes until uniform. 4. Add the Vermosudil (pin or jet milled) while mixing and mix for 15 minutes until a uniform dispersion is obtained. 5. Measure the pH and adjust to 3.5±0.2 using tartaric acid if necessary. 6. Adjust volume with sterile water for irrigation and mix for 15 minutes until uniform.

[0221] A 12 mL portion of each formulation was transferred to a 15 mL plastic centrifuge tube and tested for sedimentation rate. Separate 30 mL portions of each formulation were also transferred to 60 mL glass bottles and stored at ambient temperature and 50° C. for 7 days. After storage, all samples were evaluated for appearance, sedimentation level, ease of resuspension, and pH.

[0222] (a) Initial sedimentation and appearance test The appearance and sediment levels of the samples in 15 mL centrifuge tubes stored at room temperature were recorded at the following time points: 1 hour, 2 hours, and 24 hours, and 4 and 7 days. The results are presented in Table 16 (pin-milled formulations) and Table 17 (jet-milled formulations). In Table 16, the "loosely packed" material could only be seen by shining a light through the sample.

[0223] [Table 20]

[0224] [Table 21]

[0225] [Table 22]

[0226] As reflected in Tables 16 and 17, differences in precipitation behavior were observed between samples containing pin-milled and jet-milled API. For pin-milled API, as the level of povidone K30 increased, the amount of precipitated material also increased. The inclusion of AEROSIL® 200 and SYLOID® 244 FP appeared to reduce the rate of precipitation and appeared to be superior to AEROSIL®. For samples containing jet-milled API, the second layer of precipitate was reduced with increasing levels of povidone K30 and the addition of AEROSIL® 200 or SYLOID® 244 FP.

[0227] An important observation from this experiment is that the inclusion of colloidal silica is beneficial in preventing caking and facilitating redispersion, as the sample containing SYLOID® 244FP required the fewest number of inversions to fully redisperse the API.

[0228] Additionally, the appearance of samples stored in 30 mL glass bottles at ambient temperature was recorded after 24 hours and 7 days at ambient temperature and at 50°C for 7 days. High temperature (50°C) was used to accelerate conditions and stress the formulation. The "% sediment" was calculated by measuring the height (mm) and total volume (mm) of sediment in the glass bottles using calipers. The results are presented in Table 18 (pin mill grinding) and Table 19 (jet mill grinding).

[0229] [Table 23]

[0230] [Table 24]

[0231] (b) Repeated precipitation tests and time-dependent analysis results To confirm the visually discernible settling tendency and rate, replicate settling tests were conducted using the following six samples previously described in Tables 14A-14B (pin milled) and Tables 15A-15B (jet milled): Samples 13A and 13B (pin-milled and jet-milled 9.0 mg / mL povidone K30); Samples 14A and 14B (pin-milled and jet-milled 6.0 mg / mL Povidone K30 and 5.0 mg / mL AEROSIL®); and Samples 15A and 15B (6.0 mg / mL Povidone K30 and 5.0 mg / mL SYLOID® 244 FP pin milled and jet milled).

[0232] Replicate tests were performed on the above samples (which were approximately 14 days old) previously filled into 60 mL clear glass bottles. 24 hours before the start of the study, the samples were shaken vigorously to resuspend any sediment.

[0233] On the day of analysis, each formulation was shaken vigorously for 20 seconds (twice per second, vertically over a distance of 30 cm) and allowed to settle for 5 minutes to remove some of the formulation bubbles and air entrapment. After the sample had settled, 5.0 mL was collected using an automatic pipette with the tip positioned 5-10 mm below the suspension surface and measured into a 200 mL flask. This was then filled to approximately 180 mL with diluent (50:50 MeCN:HO) and sonicated for 10 minutes. The sample was allowed to equilibrate to room temperature before the volume was adjusted.

[0234] Samples were tested at T=0, 4 hours, and 24 hours. Samples were not shaken at T=4 hours or T=24 hours to determine how much belmosudil had precipitated from the formulation. After the T=24 hour sample was taken, the remaining samples were shaken again and samples were taken to determine whether the belmosudil had resuspended. The assay results, uncorrected for sample weight, are shown in Table 20 and plotted in Figure 4.

[0235] [Table 25]

[0236] Initial Sampling: Results from the initial sampling phase (T=0) showed results within the proposed specification limits of 90-110% of the label claim (or target concentration) of 40 mg / mL for all formulations. These results suggest that most, if not all, of the sediment in the samples was dissolved and resuspended.

[0237] Samples 15A and 14B had unexpectedly low assay values ​​at the early time points. These results suggest that the Vermosudil may have adhered to the bottle walls, where the foam may have dried and not been fully redispersed. Although the initial values ​​were low, they provided a baseline to indicate the settling rate over the 24-hour test period.

[0238] After the initial shaking, all samples produced a similar amount of foam. However, after the first 5 minutes, the jet-milled API sample had little to no foam, while the pin-milled sample had significant foam adhering to the pipette tip that had to be removed before the sample could be transferred. Despite previous results indicating potential problems with jet-milled formulations, the reduced foaming with the jet-milled samples was encouraging.

[0239] T = 4 hours: Assay results showed that the jet-milled API maintained better suspension, with assay values ​​exceeding 88% compared to assay values ​​ranging from 58 to 84% for pin-milled samples containing the API. The pin-milled sample containing only povidone K30 (13A) had the lowest assay value, confirming the benefit of adding colloidal silica as an adjuvant to the suspension of vermosudil. Of all samples, the jet-milled sample containing SYLOID® had the highest assay value.

[0240] After 4 hours of standing, bubbles were still visible in the pin-milled samples, but most of the bubbles were attached to the bottle wall rather than the pipette tip, making sampling easier. At 4 hours, minimal bubbles were present in the jet-milled samples, reflecting a consistent reduction in bubbles with the jet-milled formulations at both T=0 and T-4 hours.

[0241] T=24 Hour: At 24 hours, all formulations had assays below 85%, but the samples produced using jet-milled API still had higher assay results than the samples produced using pin-milled API. As with the samples at T=4 hours, the formulations containing SYLOID® and AREOSIL® had higher assay values ​​compared to the samples containing povidone K30 by itself, confirming that the addition of colloidal silica at 24 hours aids in the suspension of bermosudil. At 24 hours, the samples containing jet-milled API in the povidone-only formulations had higher assay results compared to the formulations containing SYLOID®, suggesting a faster settling rate in the samples containing SYLOID®. However, previous settling tests indicated that this precipitate was more loosely packed.

[0242] After 24 hours of standing, the foam from the pin-milled sample had completely adhered to the bottle wall, and the formulation had a clear separation of API precipitate and vehicle, whereas the jet-milled sample had less distinct layers.

[0243] T=24 hours (after shaking to redisperse): Results after shaking at 24 hours showed a high assay value outside the proposed specification limits of 90-110% of the target concentration. This was due to the low assay values ​​at T=4 hours and T=24 hours, which meant there was more API in a smaller volume to redisperse. Although these results were outside the specification limits, they suggest that belmosudil was easily resuspended in the vehicle by shaking.

[0244] The pH of samples 11A-16A and 11B-16B was also recorded after 7 days at ambient temperature and at 50°C. No significant changes were observed in any of the samples after 7 days at ambient temperature (pH values ​​ranged from 3.2 to 3.6 both initially and after 7 days). A decrease in pH was observed after 7 days at 50°C, i.e., the pH ranged from 2.7 to 2.9 for all samples.

[0245] From this study, it was concluded that the sample containing silicon dioxide performed best in terms of ease of resuspension after 7 days of storage. While the assay results suggest that AEROSIL® offered some advantages over SYLOID® 244 FP in terms of reduced settling rate, the sample containing SYLOID® 244FP exhibited less caking and was easier to resuspend. Because all samples had visually lower viscosities and faster settling rates over time, ease of redispersion was considered an important feature, especially when considering products with longer shelf lives. SYLOID® 244FP also offered the advantage of easier handling during manufacturing compared to AEROSIL® 200. For these reasons, SYLOID® 244FP was considered the better option overall.

[0246] Determining the level of povidone requires a careful balance. Higher levels of povidone K30 are required to reduce precipitation. However, the maximum level of povidone K30 inclusion is affected by the acceptable daily intake (ADI) of this compound, 50 mg / kg body weight, so the level needed to be consistent with the dosing regime for administering it to the subject.

[0247] The selection of jet-milled API compared to pin-milled API involved considering many complex factors. As discussed in Examples 3, 4, and 5, various experiments demonstrated that pin-milled API was preferable. Pin-milled API was easier to handle than jet-milled API, and better flow was observed. Pin-milled API was also more easily wetted. When flocculation occurred, it was more severe in samples containing jet-milled API, and jet-milled API was observed to adhere to the walls of the centrifuge.

[0248] Both the pin-milled and jet-milled samples redispersed easily, with no significant differences between the APIs. Less sediment was observed after 7 days in the batch containing the jet-milled API.

[0249] Once stirred, foaming occurred in the pin-milled and jet-milled samples containing the API, with the jet-milled sample appearing to have slightly less foam.

[0250] Thus, there are many reasons to initially conclude that pin-milled APIs are the preferred option for pharmaceutical development. Nevertheless, applicants herein have discovered that jet milling is the preferred method for pharmaceutical development, especially in light of concerns regarding long-term stability and the reduced amount of sedimentation with jet-milled products, which may only become more pronounced upon longer storage periods.

[0251] (c) Further povidone-controlled trials As shown above in Examples 6(a) and (b), formulations containing povidone K30 had relatively fast settling rates. In this study, povidone 90F, a longer-chain PVP, was investigated to determine whether it could provide a thicker suspension that would reduce the settling rate. To achieve this goal, formulations having the compositions set forth in Table 21 were prepared.

[0252] [Table 26]

[0253] The following steps were used to prepare the formulations in Table 21: 1. Add approximately 80 mL of sterile water for irrigation to a pre-calibrated beaker. 2. While stirring, add the sodium benzoate, sucralose, and tropical fruit blend flavor and mix until dissolved. 3. While mixing, add Povidone 90F and mix until a uniform mixture is observed. 4. If necessary, add AEROSIL® 200 or SYLOID® 244FP while mixing and mix until uniform; 5. Add the vermosudil while mixing and continue mixing until a uniform mixture is observed. 6. Measure the pH and, if necessary, add tartaric acid in aliquots until a pH of 3.5±0.2 is achieved; 7. Sterile irrigation water was added to adjust the volume and the final pH was recorded.

[0254] Samples 16-01 through 16-04 were tested for precipitation. 2 mL of each sample was added to a 15 mL plastic centrifuge tube and monitored for precipitation over a 7-day period at room temperature. Initial, 2-hour, and 24-hour precipitation results were recorded.

[0255] The results of this precipitation test are summarized in Table 22. For comparison, previous results for Samples 14A and 15A (Table 16 above) are included.

[0256] [Table 27]

[0257] Sedimentation studies demonstrated that the use of Povidone 90F reduced the level of sedimentation when compared to the Povidone K30 samples (Samples 14A and 15A), with the majority remaining suspended even after up to 7 days at ambient temperature.

[0258] Sample 16-03, containing SYLOID® 244 FP, required fewer inversions to redisperse the precipitate after 7 days at ambient temperature when compared to Sample 16-04, containing AEROSIL® 200, which may be beneficial for longer storage. Therefore, SYLOID® 244FP was considered the preferred silica for inclusion in Belmosudil formulations.

[0259] Samples 16-01 through 16-04 were also evaluated over time under accelerated storage conditions. 30 mL of each sample was transferred to two 60 mL glass bottles and stored at ambient temperature and 40°C / 75% relative humidity. After 7 days, they were tested for appearance, sediment level, sediment redispersion, and pH. This study revealed that when stored at ambient temperature for T = 7 days, all four samples stored in 60 mL clear glass bottles containing povidone 90F were easily reconstituted by gentle shaking for approximately 10 seconds. Additionally, samples containing flavoring were observed to foam after redispersion after 7 days of storage at ambient temperature.

[0260] For the T=7 samples at 40°C / 75% RH, all four samples containing Povidone 90F appeared to become thicker after storage at 40°C / 75% RH but were redispersed by gentle shaking for approximately 10 seconds. No foaming was observed.

[0261] Finally, four samples were selected for testing by XRPD analysis, specifically Samples 15A and 16A (Table 14B), Sample 17 (Table 23), and Sample 16-03 (Table 21). Thirty mL of each sample was transferred to 1 x 60 mL glass bottle and analytical testing was performed initially and after 7 days (with the samples agitated to redisperse any precipitate) at ambient temperature and accelerated storage conditions (i.e., 50°C). Samples were isolated for XRPD analysis. The XRPD data demonstrated that all samples were consistent with the original form of the API and that the pH drift was not related to a change in the form of the API. The sodium benzoate level contained at 0.5 mg / mL did not pose any concerns.

[0262] Example 7: Preservatives and Sodium Benzoate Levels Based on the API's low natural pH in water of approximately pH 3 and the instability observed in samples containing potassium sorbate as described in Example 1, sodium benzoate was selected as the preferred preservative choice.

[0263] Four formulations were prepared to evaluate the effect of adjusting the sodium benzoate level and select a preferred level for use in Belmosudil formulations. Specifically, four formulations, Samples 17-20, listed in Table 23, were prepared containing high, medium, and low levels of sodium benzoate in combination with 9 mg / mL povidone K30 and 5 mg / mL SYLOID® 244FP (which were identified as preferred suspending agents based on the findings in Example 3 (povidone) and Example 6 (SYLOID® 244FP)). For reference, Sample 20 was prepared without preservatives.

[0264] [Table 28]

[0265] The above formulations (Samples 17-20) were prepared to a volume of 150 mL using the following manufacturing process: 1. Add 130 mL of sterile water for irrigation to a 250 mL glass beaker. 2. Add the sodium benzoate while mixing using a magnetic stirrer until completely dissolved. 3. Add povidone K30 while stirring and mix until completely dissolved. 4. Add SYLOID® 244 FP while stirring and mix until a dispersed and uniform mixture is obtained; 5. Add the pin-milled Vermosudil while stirring and mix until a uniform mixture is obtained. 6. Record the pH and adjust, if necessary, by adding tartaric acid to reach a target pH of 3.5±0.2; 7. Sterile water for irrigation was added to adjust the volume and the formulation was mixed until uniform.

[0266] PET testing was performed on Samples 17-20. The results demonstrated 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 effectiveness. The results confirmed that all levels of sodium benzoate may be considered alternative options, with the intermediate level of 0.50 mg / mL being an appropriate level for containing sodium benzoate (taking into account any potential degradation of sodium benzoate during longer-term stability). Sample 20, which did not contain sodium benzoate, was unable to provide sufficient protection from microbial growth. Therefore, it is advantageous to include a preservative in a ready-to-use liquid formulation containing vermosudil.

[0267] Example 8: Effect of sweeteners and flavors A series of vehicles containing sweeteners and flavorings were developed to identify those suitable for reconstituting belmosudil in a bottle and administering it to a subject. Initial formulation development tests were conducted on small batches of up to 100 mL. Initially, vehicles were manufactured according to the composition details presented in Tables 24A-24B and 25A-25B.

[0268] The sweetener level was selected based on the finding that the API has bitter characteristics that are needed to improve palatability in order to effectively administer the formulation to a subject. The vehicle was prepared by (a) adding the required amount of sterile water for irrigation to a 125 mL clear glass bottle; (2) adding the required amount of sweetener and mixing using a vortex mixer until visually dissolved; and (3) adding the required amount of flavoring (if applicable) and mixing using a vortex mixer until visually dissolved.

[0269] [Table 29]

[0270] [Table 30]

[0271] [Table 31]

[0272] [Table 32]

[0273] Vehicles 01-12 were each characterized for appearance and pH. Vehicles 01-02 (containing sucralose) and vehicles 07-08 (containing acesulfame k) were visually observed as clear, colorless solutions. The appearance of the vehicles containing the sweetener and flavor combinations varied among all batches and were observed as cloudy liquids, except for the vehicles containing orange flavor (vehicles 06 and 12), which were off-white, opaque liquids. The pH of the vehicles ranged from a low of 4.3 (vehicles 03 and 09) to a high of 7.0 (vehicles 01 and 02), falling within the proposed acceptance criteria.

[0274] Next, reconstituted samples of belmosudil were prepared to evaluate the performance of vehicles 01 to 12. To prepare the reconstituted belmosudil in a bottle, 727.44 mg of belmosudil mesylate (600 mg of belmosudil free base) was transferred to a 60 mL Type III clear glass bottle. To this bottle, 15 mL of each vehicle listed in Tables 24A-24B and 25A-25B was added, and the sample was reconstituted by vortexing.

[0275] All samples were reconstituted by gentle stirring for 10 seconds. All samples containing acesulfame K (using vehicles 07-12) showed signs of flocculation and significantly thickened after approximately 5 minutes. Therefore, all samples containing acesulfame K were deemed unsuitable for use in Belmosudil formulations. Samples containing sucralose (using vehicles 01-06) appeared as opaque yellow dispersions and showed no signs of flocculation. Therefore, these vehicles containing sucralose were deemed suitable for use in formulations. Furthermore, the pH of the samples reconstituted using vehicles 01-06 was reduced to the range of 3.1-3.2, which is suitable for final formulations without the need for pH adjustment.

[0276] Further studies were conducted to investigate the effect of adding flavor to bermosudil formulations on foaming after stirring. For this evaluation, a tropical fruit blend flavor was selected, and experiments were conducted using the previously prepared Sample 11A (Table 14A) and Sample 12B (Table 15A) (Example 6 above). Each sample contained sodium benzoate and povidone K30 as preservatives without added silica / suspending agent, with Sample 11A containing pin-milled bermosudil and Sample 12B containing jet-milled drug.

[0277] For each sample, approximately 30 mL was transferred to a 60 mL glass bottle. To evaluate the effects of effervescence and the addition of flavorings, the following experiment was performed. 1. Agitate each sample by shaking for 30 seconds (approximately 2 inversions per second) and note appearance and foaming; 2. Allow each sample to stand until no bubbles / air entrapment are present; 3. To each sample, 0.12 g of tropical fruit blend flavor (approximately 0.4% w / v) was added, which was then dissolved by mixing using a vortex mixer.

[0278] Table 26 summarizes the composition of the liquid formulations containing bermosudil tested in this study after adding flavoring.

[0279] [Table 33]

[0280] The following observations were noted:

[0281] Without flavoring, a large amount of foaming and air entrapment was observed after 30 seconds of stirring. After standing for 5 minutes, there was a significant difference in foaming between the pin-milled and jet-milled API without flavoring. Sample 11A (pin-milled API) had a longer foaming period compared to Sample 12B (jet-milled API).

[0282] After flavor addition, only a very small amount of air entrainment was observed after 30 seconds of mixing. The addition of the tropical fruit blend flavor did not significantly change the amount of foam or air entrainment in samples containing pin-milled API compared to jet-milled API. Therefore, it was concluded that the tropical fruit blend flavor is suitable for use in vermosudil formulations, and the other alternative flavors listed in Tables 24A and 24B were expected to have a similar effect on effervescence based on previous observations with these flavors.

[0283] Example 9: Comparative analysis of scale-up and homogenization processes A comparative analysis was performed on a scaled-up system containing 9 mg / mL povidone 90F and an alternative system containing 6 mg / mL povidone K30 with pin-milled API, along with a system containing 9 mg / mL povidone 90F using jet-milled API to allow comparison with the pin-milled API. All formulations used 5 mg / mL SYLOID® 244FP as a suspending agent, contained 0.5 mg / mL sodium benzoate to provide preservative effectiveness, and used lemon flavor.

[0284] Further details of the compositions used in this study are provided in Table 27.

[0285] [Table 34]

[0286] Samples 21-23 were analyzed for their bulk homogeneity as follows: Prior to sampling, the formulations were gently agitated by inversion 10 times to ensure any sediment was resuspended before testing.

[0287] Two replicate samples were taken from each bottle for an n=6. 1.00 mL of the suspension was placed in a 200 mL volumetric flask, adjusted to approximately 180 mL with diluent (50:50 MeCN:HO), and sonicated for 10 minutes. The sample was allowed to equilibrate to room temperature before adjusting the volume with diluent. The sample was then filtered through a 0.45 μm PTFE filter and placed in a vial for analysis, discarding the first 2 mL.

[0288] From the initial assessment of bulk homogeneity, assay values ​​for both sodium benzoate and bermosudil were collected and the results are shown in Table 28.

[0289] [Table 35]

[0290] Bulk homogeneity data demonstrated that the sodium benzoate assay content met the specification limit of RSD ≤ 6% for all batches. However, the bermosudil assay content was significantly higher than the 90-100% acceptance criteria in sample 21, and showed significant variation in samples 22 and 23, where levels did not meet the specification limit of RSD ≤ 6%.

[0291] Viscosity testing was also performed on Samples 21-23 using a Brookfield DV1 LV cone and plate viscometer with spindle CP41 at 30 RPM at 20° C. The results are shown in Table 29.

[0292] [Table 36]

[0293] The viscosity values ​​recorded were much lower than expected, especially for Sample 22, which contained Povidone 90F. While thickening over time was observed in previous systems, this was not observed in Samples 21-23.

[0294] Although batches were successfully manufactured on a smaller scale using overhead mixing alone, this study demonstrated that overhead mixing was insufficient to adequately disperse the API to form a homogenous suspension upon scale-up, which contributed to the significant variability in assay results in this example. It was found to be advantageous to include a homogenization step in the scale-up to obtain a homogenous suspension. Further investigation confirmed that the use of a Silverson homogenizer improved the homogeneity of the API.

[0295] Example 10: System improvements and advantageous parameters Following the scale-up study in Example 9, additional comparative samples were prepared and analyzed with the initial hypothesis that increasing the viscosity of the system would in turn decrease the rate of precipitation in the PVP / formulation system. Two approaches were investigated to optimize the system: 1. Increasing the level of povidone 90F: PVP has been shown to be effective in wetting APIs and can be used as a thickening agent. However, according to the Drug Development and Stewardship Strategy Plan, povidone 90F is to be increased to a level below the acceptable ADI based on the average weight of a 3-month-old female infant (5.5 kg). PVP has an ADI of 50 mg / kg body weight and therefore should not exceed 275 mg / dose (based on one dose / day). 2. Addition of Methocel K4M in Combination with Povidone K30: Previously, samples containing alternative suspending agents to PVP were observed to gel significantly over time and aggregate significantly. Because PVP aids in wetting of the API, a study was conducted to investigate the use of alternative suspending agents in combination with Povidone K30 (based on useful information that shorter-chain PVPs are more likely to aid wetting compared to Povidone 90F). Methocel K4M was selected as an alternative thickening agent because it demonstrated the least severe physical incompatibility with Belmosudil compared to the other suspending agents tested.

[0296] To investigate these approaches, four formulations were prepared at a 200 g scale with the compositional details outlined in Table 30A and Table 30B. Samples were subjected to short-term stability testing and evaluated for appearance, assay, pH, and viscosity at T=14 days and T=1 month at 25°C / 60% relative humidity and 40°C / 75% relative humidity.

[0297] [Table 37]

[0298] [Table 38]

[0299] All four systems (24-01 through 24-04) appeared to be more viscous than those observed in previous formulations. Upon initial mixing, all four systems were fluid, and the API was well wetted. After 24 hours, Sample 24-03, containing methocel K4M, became semi-solid but changed to a fluid state after 10 seconds of shaking. After one week, Samples 24-01, 24-02, and 24-04 all had some sedimentation. Sample 24-01 appeared to have the greatest amount of sediment when compared to Sample 24-02 (containing a higher level of povidone 90F) and Sample 24-04 (containing the same level of povidone 90F but prepared using jet-milled API). Sample 24-03 showed no signs of sedimentation but remained semi-solid.

[0300] At T=14 and T=28 days, the samples were re-evaluated for appearance. Sample 24-03 remained a viscous, opaque, yellow gel but readily liquefied upon shaking to form an opaque, yellow liquid. Series 24-01, 24-02, and 24-04 were free-flowing, opaque, yellow liquids with a small amount of sediment that redispersed upon shaking.

[0301] It was noted that "banding" was observed where the API adhered to the wall of the glass bottle. Interestingly and surprisingly, more API was observed to adhere to the wall in the systems containing pin-milled API compared to jet-milled API (i.e., Samples 24-03 and 24-04). Therefore, it was concluded that: 1. System 24-03 containing Methocel K4M formed a solid / gel-like structure. However, this structure was easily shaken and re-fluidized. It remained liquid but reverted to its structure after 24 hours. 2. Among the remaining samples, 24-04, which contained 30 mg / mL povidone 90F and used jet-milled API, had the least amount of sediment. Sample 24-01, which contained 30 mg / mL povidone 90F and used pin-milled API, had the fastest rate of sedimentation and was most likely to adhere to the glass bottle. 3. Sample 24-02 containing 50 mg / mL Povidone 90F manufactured using pin-milled API exhibited a reduced settling rate compared to Sample 24-01.

[0302] This study revealed that the system containing Povidone 90F with jet-milled Belmosudil (Sample 24-04) had the least amount of sedimentation and unexpectedly performed best on visual inspection.

[0303] All four samples were also analyzed by the sodium benzoate and bermosudil assays. For all samples, both the bermosudil and sodium benzoate assay data were comparable to each other. Samples obtained with the push-in bottle adapter (PIBA) yielded results comparable to those obtained using an automatic pipette, demonstrating the suitability of both sample transfer methods.

[0304] Samples 24-02 through 24-04 at both storage conditions and sample 24-01 at 25°C / 60% relative humidity had assay values ​​for both bermosudil and sodium benzoate that were within the specification limits of 90-110% of the label claim. Samples 24-02 through 24-04 did not show significant changes in assay data when compared to the initial and 14-day time points.

[0305] A summary of the pH values ​​of the four samples at 25°C / 60% relative humidity and 40°C / 75% relative humidity at initial, T=2 weeks, and T=1 month is shown in Table 31.

[0306] [Table 39]

[0307] A downward pH drift was observed in all systems, with the greatest change observed at 40°C / 75% relative humidity. Initial pH storage stability studies indicated that samples exhibiting pH drift tended to have a pH decrease down to approximately pH 2.9. Because benzoic acid is most effective at pH 3, even a downward drift in pH is acceptable for storage efficacy. pH storage stability testing suggested that no chemical changes occurred at this pH. XRPD analysis suggested that there was no morphological change in the pH 3 samples without the presence of citrate buffer, as discussed in Example 2.

[0308] Because the pH tends to drift toward approximately pH 3.0 over time and then remain at a pH value around this range, and because Belmosudil is stable at this value, we have set pH 3.0±0.5 as a target pH range suitable for liquid formulations containing Belmosudil.

[0309] The viscosity of the four systems was also tested using a Brookfield cone and plate viscometer, spindle CP52, and was 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% relative humidity and 40°C / 75% relative humidity for initial, T=2 weeks, and T=4 weeks.

[0310] [Table 40]

[0311] As can be seen, slight viscosity fluctuations were observed, but no significant changes from the initial values ​​were observed at 25°C / 60% RH and 40°C / 75% RH after 2 and 4 weeks. The trends between the systems remained the same at all time points, with the highest viscosity being sample 24-03 (containing 10 mg / mL povidone K30 + 5 mg / mL Methocel K4M, including jet-milled API) and the lowest viscosity being sample 24-01, containing 30 mg / mL povidone 90F. Surprisingly, the high viscosity of sample 24-03 did not correlate with a reduced sedimentation rate, as initially proposed. As shown above, of all samples, sample 24-04, containing 30 mg / mL povidone 90F and using jet-milled API, had the least amount of sediment, but also one of the lower viscosity rates.

[0312] Overall, in the comparative analysis, Sample 24-02 (50 mg / mL Povidone 90F) produced the most consistent assay results, especially compared to Sample 24-01 (30 mg / mL Povidone 90F), which also used pin-milled API. Sample 24-03 exhibited the previously observed thickening / gelling, but was more easily liquefied upon shaking and did not exhibit agglomerates after shaking similar to those previously observed in batches containing methocel K4M but no povidone K30.

[0313] However, it was also observed that the use of jet-milled API resulted in improved reduction of "banding" of the API on the glass bottle and reduced settling rate compared to pin-milled API. This was observed when comparing Samples 24-01 (pin-milled) and 24-04 (jet-milled), both of which contained 30 mg / mL of povidone 90F. By preparing upscaled development samples and increasing the PVP level, the agglomeration noted in earlier development studies using jet-milled API was not observed. Therefore, it was concluded from this study that jet-milled belmosudil is the most advantageous for use in liquid formulations containing belmosudil. See Table 33, which summarizes the components of the liquid formulation.

[0314] [Table 41]

[0315] [Table 42]

[0316] Example 11: Taste and Bioavailability Studies After developing the liquid formulation containing belmosudil as described above, studies were conducted to (i) evaluate the taste attributes (smell, sweetness, bitterness, flavor, mouthfeel / texture, roughness, and aftertaste) and overall acceptability of the liquid formulation containing belmosudil; (ii) determine the relative bioavailability of the liquid formulation containing belmosudil compared to belmosudil oral tablets after a meal; and (iii) determine the effect of food on the PK of belmosudil after administration of the liquid formulation containing belmosudil.

[0317] The first part of this study involved a "sip and spit" taste evaluation to profile the taste characteristics of the formulation and identify suitable flavor systems. The results of this taste test were then considered in the next part to evaluate the relative bioavailability of the liquid formulation compared to the oral tablet. A 48.496 mg / mL belmosudil liquid formulation (40 mg / mL free base) was used in this study. This concentration was chosen to minimize the volume administered (5 mL for a 200 mg dose) in subsequent relative bioavailability studies conducted in healthy volunteers and to facilitate comparison with the reference tablet (200 mg).

[0318] (a) Part I - Taste Test The objective of this first study was to identify the optimal flavor and sweetener combination to promote or improve the palatability of a liquid formulation of belmosudil used to administer the drug orally to a target population of patients aged 3 months to 12 years.

[0319] (i).Method Twelve healthy adult male subjects (mean age 35.3 years, youngest 23 years, oldest 52 years) were enrolled in the taste profile portion of this study. Subjects were admitted to the hospital the day before product administration (Day 1) and discharged on Day 2. On Day 1, subjects were randomized to receive a total of six liquid formulations containing Belmosudil according to one of six treatment sequences (ABFCED, BCADFE, CDBEAF, DECFBA, EFDACB, and FAEBDC), with two subjects assigned to each treatment sequence.

[0320] Each individual treatment (e.g., A, B, C, D, etc.) contained a single 40 mg / mL (200 mg in 5 mL) oral dose of belmosudil. The bottle formulations and vehicles for six different regimens were used as follows: A = sterile water (Vehicle 1), B = low sucralose (Vehicle 2), C = high sucralose (Vehicle 3), D = orange low sucralose (Vehicle 4), E = tropical fruit blend low sucralose (Vehicle 5), and F = lemon low sucralose (Vehicle 6).

[0321] Subjects were given a training questionnaire using an exemplary liquid (e.g., orange juice / squash) prior to their first dose of the liquid formulation containing belmosudil (either on Day 1 or before breakfast was completed [pre-dose] on Day 1). Subjects were tested with the belmosudil by bottle regimen according to the randomization schedule.

[0322] Subjects received the first single oral dose of the regimen two hours after consuming a standard breakfast on the morning of Day 1. Each regimen followed the same study design: subjects were given a single dose of the liquid formulation of belmosudil in a bottle, which they held in their mouths for approximately one minute and then spat out.

[0323] Immediately after spitting, subjects individually and privately completed a questionnaire using a 9-point Likert scale to assess overall acceptability, taking into account seven key taste attributes (smell, sweetness, bitterness, flavor, mouthfeel / texture, roughness, and aftertaste). Specifically, ratings on this scale were as follows: 1 = extremely dislike, 2 = very dislike, 3 = somewhat dislike, 4 = slightly dislike, 5 = neither like nor dislike, 6 = somewhat like, 7 = somewhat like, 8 = extremely like, 9 = extremely like.

[0324] This process was completed for each of the six regimens. Belmosudil was not swallowed. Between tastings for each regimen, there was a drug-free interval of approximately 30 minutes (including palate cleansing). During this time, subjects used tap water (administered ad libitum in 50 mL aliquots) and crackers to clean their palate before tasting again. Tastings for all regimens were conducted on the same day.

[0325] A single plasma PK sample was collected approximately 1 hour after the final dose (prior to discharge from the clinical unit). This sample was retained for analysis only if the subject accidentally swallowed the formulation and / or for the purpose of investigating treatment-emergent adverse events (TEAEs) thought to be related to IMP. In Part 1 of this study, no subjects accidentally swallowed the formulation and / or reported TEAEs thought to be related to IMP. Therefore, plasma PK samples did not need to be analyzed for any subjects and were discarded.

[0326] Subjects remained on-site until 1 hour after the final taste / palatability assessment. A follow-up phone call was conducted 3-7 days after the final dose to ensure the subjects' continued well-being.

[0327] (ii) Results The median (lowest-highest) scores for each taste / palatability attribute are summarized in Table 34. Median difference data are summarized in Table 35.

[0328] [Table 43]

[0329] [Table 44]

[0330] Vehicle 1 (sterile water - reference) was the lowest scoring vehicle, with an overall mean acceptability score of 3.0, indicating that the subject panel moderately disliked the product. The flavored regimens (Vehicles 4 [Orange Low Sucralose], 5 [Tropical Fruit Blend Low Sucralose], and 6 [Lemon Low Sucralose]) achieved mean scores of 6.5, 7.0, and 7.0, respectively, indicating that the addition of flavor improved the overall taste / palatability profile of the IMP.

[0331] The taste / palatability attributes with the lowest mean scores for Vehicle 1 were flavor, mouthfeel / texture, and roughness (3.5, 3.0, and 3.5, respectively), indicating that these were disliked attributes. The addition of flavored, low-sucralose vehicles (Vehicles 4, 5, and 6) increased the mean scores for each of these attributes, ranging from 6.5 to 7.0 for flavor, 4.5 to 5.0 for mouthfeel / texture, and 4.0 to 5.5 for roughness. The mean scores also improved for all other taste / palatability attributes with the addition of flavored, low-sucralose vehicles, indicating that subjects preferred the taste profiles of these vehicles.

[0332] Each taste attribute was statistically significant at the 5% significance level by Friedman's test, indicating that at least one of the formulations had a taste score that was significantly different from the other treatments (p<0.001 for overall acceptability, odor, sweetness, bitterness, flavor, and mouthfeel / texture; 0.041 and p=0.001 for roughness and aftertaste, respectively).

[0333] For all pairwise comparisons and taste attributes, the mean pairwise differences were positive, indicating that the use of sweeteners and / or flavorings improved acceptability compared to the reference vehicle, sterile water.

[0334] For all taste attributes, the tropical fruit blend low-sucralose vehicle or the lemon low-sucralose vehicle demonstrated either the greatest improvement or the greatest common improvement compared to the reference vehicle (based on the average pairwise differences). No benefit was demonstrated by increasing the sucralose content of the vehicle.

[0335] (b) Part 2 - Bioavailability (i) Method Part 2 was a single-center, open-label, randomized, three-period study evaluating the relative bioavailability of selected liquid formulations of belmosudil compared with oral tablets of belmosudil and the effect of food on liquid formulations containing belmosudil in 18 healthy male subjects. Subjects underwent a prescreening procedure at the screening visit (days -28 to -2 of Part 2) to determine eligibility for Part 2 of the study. Subjects who participated in Part 1 of the study were permitted to participate in Part 2.

[0336] Subjects received a single oral dose of 200 mg of belmosudil over three periods. Three different regimens (G, H, and I) were used over the three periods. In regimen G, belmosudil was administered in tablet form (reference), and in regimens H and I, belmosudil was administered in the form of an oral liquid formulation.

[0337] Period 1, Day 1: Prior to administration of the first dose of IMP, subjects were randomized to one of six treatment sequences (GHI, HIG, IHG, GIH, and HGI), with three subjects assigned to each treatment sequence. On Day 1 of each study period (Periods 1, 2, and 3), subjects received the regimen in Table 36.

[0338] [Table 45]

[0339] Regimen H was administered to subjects in a fasting state as an unflavored, low-sucralose liquid formulation, while Regimen G and I were administered to subjects after a meal. Regimen G was administered with a total of 240 mL of water. For Regimen H and I, subjects drank up to a total of 240 mL of water (including the dose) immediately after receiving the IMP.

[0340] Each study period followed a similar design. Subjects were admitted to the clinical unit the morning before their first IMP administration (Day -1 of Period 1) to verify eligibility and baseline treatment. For Regimen H only, subjects were given a training questionnaire prior to receiving IMP, demonstrating how the questionnaire should be completed using exemplary liquids (e.g., orange juice / squash). This was conducted either on Day -1 (only applicable if Regimen H was administered in Periods 2 or 3) or prior to dosing on Day 1 (pre-dose). The flavor system determined to be most preferred from Part 1 (lemon flavor with low-sucralose solution) was originally planned for use in Regimen H in Part 2, but an unflavored, low-sucralose oral liquid formulation was used instead.

[0341] Subjects received a single dose of IMP on the morning of Day 1 after a minimum 10-hour overnight fast (Regimen H, fasting) or after a standard breakfast (Regimens G and I, postprandial). Blood samples were collected at regular intervals for PK analysis. After administration of Regimen H, subjects individually and privately completed a written taste / palatability questionnaire.

[0342] Subjects stayed on the clinical unit for 10 consecutive nights across all three treatment periods. All subjects remained on site until 72 hours after the final dose for safety and PK evaluations. There was a minimum of 3 days of washout between each IMP administration. A follow-up phone call was conducted 3-7 days after the final dose to ensure the subject's continued well-being. If the subject reported any AEs indicating cause for concern after discharge, they were required to return to the clinical unit for a follow-up evaluation. This was a scheduled outpatient visit.

[0343] (ii) Results a. Taste / Deliciousness Overall, the oral liquid formulation (Regimen H) was rated by the majority of subjects as either Grade 5 ("neither like nor dislike"), Grade 6 ("somewhat like"), or Grade 7 ("fairly like") The overall taste profile scores for Regimen H were comparable to those for Regimen B in Part 1, which used the same sweetener combination (i.e., low sucralose).

[0344] b. Bioavailability After a single oral dose of the liquid formulation containing belmosudil, absorption of belmosudil was rapid (mean Tmax of 2 hours) compared with belmosudil administered as a tablet reference (mean Tmax of 3 hours).

[0345] Belmosudil was administered after a meal as a reference tablet. Belmosudil was absorbed with a mean Tmax of 3.00 hours after administration, followed by a rapid decline in concentrations, similar to those observed previously, resulting in a geometric mean T1 / 2 of 9.393 hours. Exposure-related intersubject variability (Cmax and AUC) was moderate, ranging from 26.1% to 39.6%. The mean Tmax of KD025m1 and KD025m2 after administration of belmosudil tablets was similar to that of belmosudil, at 2.00 and 3.00 hours after administration, respectively. KD025m1 concentrations were poorly quantifiable, and the terminal half-life was reliably assessed in only two subjects. The geometric mean T1 / 2 of KD025m2 was 2.466 hours and was reliably assessed in 8 of 18 subjects. Exposure-related intersubject variability was higher for both metabolites compared with the parent, ranging from 37.9% to 63.9% and 52.5% to 75.3% for KD025m1 and KD025m2, respectively.

[0346] The key geometric mean (geometric coefficient of variation [CV%]) PK parameters of belmosudil in plasma after dosing with belmosudil are summarized in Table 37 below.

[0347] [Table 46]

[0348] After a single oral dose of belmosudil (tablet reference) administered to healthy male volunteers after a meal (regimen G), belmosudil concentrations were evident from 0.5 to 3 hours in all subjects. After administration of a liquid formulation containing belmosudil in the fasting state and after a meal (regimen H and I), concentrations were evident from 0.5 hours in all subjects. Maximum plasma concentrations occurred between 1 and 5 hours after administration.

[0349] Concentrations then declined biphasically and remained quantifiable for up to 24 to 72 hours after administration. The resulting elimination half-lives ranged from 4.84 to 33.02 hours, 5.36 to 23.76 hours, and 2.38 to 15.73 hours, respectively. The geometric mean half-lives ranged from 6.788 to 9.499 hours.

[0350] The key geometric mean (geometric coefficient of variation [CV%]) PK parameters of KD025m1 and KD025m2 in plasma after dosing with belmosudil are summarized in Tables 38 and 39 below, respectively.

[0351] [Table 47]

[0352] [Table 48]

[0353] Administration of a liquid formulation containing belmosudil under fasting conditions demonstrated a faster mean Tmax (1.5 hours) compared with the same dose administered after a meal.

[0354] Comparing the GMRs associated with peak (Cmax) and total exposure (AUC(0-last) and AUC(0-inf)) levels for the postprandial tablet compared with the postprandial belmosudil liquid formulation, the peak and total exposure levels of belmosudil and KD025m1 for the liquid formulation were generally similar to those of the tablet reference, with 90% CIs for each parameter including 100%. The upper limit of the 90% CI for peak exposure was slightly below 100% (upper CI 94.39%) but did not include 1, indicating that any true difference was likely minimal and unlikely to be clinically significant. Additionally, the change in formulation resulted in little or no change in AUC, indicating that the change in formulation had no notable effect on overall exposure to belmosudil or its two known metabolites.

[0355] Comparison of GMRs for peak and total exposures for the oral liquid formulation between fed and fasted states demonstrated a slight increase in belmosudil after the fed regimen, averaging 15% to 19% greater than exposure observed with the fasted regimen. For KD025m1, a modest increase in total exposure was observed; however, this increase should be interpreted with caution because data available to estimate KD025m1 parameters were relatively sparse across all regimens. The lower bound of the 90% CI for each parameter exceeded 100%. For KD025m2, no notable changes due to food effects were observed.

[0356] In summary, after a single oral dose of a liquid formulation containing belmosudil, absorption of belmosudil was rapid (mean Tmax of 2 hours) compared with belmosudil administered as a tablet reference (mean Tmax of 3 hours).

[0357] The mean Tmax of KD025m1 and KD025m2 was similarly faster after administration as the liquid formulation (1.5 hours and 2 hours, respectively) compared to after administration as the tablet reference (2 hours and 3 hours, respectively).

[0358] Both the maximum (Cmax) and overall (AUC) exposure of belmosudil after administration as an oral liquid formulation, post-prandial, showed no change compared to the tablet reference after the formulation change.

[0359] Bioavailability, as measured by Cmax and AUC, was similar for belmosudil and metabolites (KD025m1 and KD025m2) in the liquid and tablet formulations.

[0360] Example 12: Pediatric Dosage Study In this study, the recommended pediatric dose of belmosudil corresponding to the AUC after a once-daily (QD) dose of 200 mg belmosudil in adults was evaluated using two models: a population pharmacokinetic (PopPK) model and a physiologically based pharmacokinetic (PBPK) model.

[0361] (a) PopPK model In applying this model, the following steps were taken: (a) weight bins were determined for children and adolescents between 10 kg (3rd percentile at 2 years of age) and 88 kg (97th percentile at 18 years of age); (b) predictions were made over a 24-hour period at steady state; (c) the effect of GVHD on clearance was accounted for for each subject; (d) no concomitant PPI administration was assumed for any hypothetical subjects; (e) 50% males were assigned to each population; and (f) the final exposure predictions were compared to a hypothetical adult population in which all adults received 200 mg of KD025 QD.

[0362] In identifying appropriate weight bins, the following steps were taken: (a) the pediatric and adolescent weight range (10 kg to 88 kg) was divided into 3 kg bins with 1,000 virtual subjects within the weight range in each bin; (b) the AUC in subjects after doses of 50 mg, 100 mg, and 200 mg QD was predicted; (c) the AUC in the adult population of 5,000 virtual subjects after a dose of 200 mg QD was predicted; (d) the 25th percentile AUC in adults was compared to the 50th percentile AUC in each weight bin, and bins with higher mean AUC for each dose were combined into one larger weight bin.

[0363] Adult weights range from 40 kg to 126 kg. The following weight bins were used to predict final exposure and doses were aligned to adults for ease of administration: 10kg to under 20kg - 50mg per QD 20kg to under 40kg - 100mg per QD 40kg and over - 200mg QD

[0364] Applying this model, the final predicted weight bins and doses were determined to be: (1) for patients weighing 10 kg to less than 19 kg—50 mg QD; (2) for patients weighing 19 kg to less than 49 kg—100 mg QD; and (3) for patients weighing 49 kg to less than 88 kg—200 mg QD. The estimated once-daily (QD) and twice-daily (BID) doses of Belmosudil oral liquid formulation by age group and weight are set forth in more detail below in Tables 40 to 43.

[0365] [Table 49]

[0366] [Table 50]

[0367] [Table 51]

[0368] [Table 52]

[0369] (b) PBPK model Physiologically based pharmacokinetic (PBPK) models for Belumosudil (KD025) and its major metabolite, KD025m2 (having the chemical name 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetic acid) (also referred to as "M2"), have been previously investigated by incorporating data from in vitro, nonclinical, and pharmacokinetic studies 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.

[0370] In this example, previously studied PBPK models of KD025 and M2 were applied to predict the systemic exposure of KD025 in children (3 months to 11 years) and adolescents (12 to 17 years) for the purpose of supporting dose selection.

[0371] The studied PBPK model was applied by the following four steps: 1. Plasma concentrations of KD025 and M2 were predicted in healthy adult subjects after repeated oral doses of 2.7 mg / kg KD025 QD (equivalent to an adult dose of 200 mg). 2. Plasma concentrations of KD025 and M2 in healthy pediatric subjects (in the age groups of 0.25-0.5 years, 0.5-1 years, 1-2 years, 2-5 years, 6-11 years, and 12-17 years) after repeated oral doses of KD025 at 2.7 mg / kg QD were predicted. 3. The initial 2.7 mg / kg dose of KD025 was compared with the simulated pediatric:adult AUC 0-t,SS The adjustment was made based on the ratio of (calculated from steps 1 and 2). 4. Plasma concentrations of KD025 and M2 in pediatric subjects (in the age groups of 0.25-0.5 years, 0.5-1 years, 1-2 years, 2-5 years, 6-11 years, and 12-17 years) were predicted after repeated oral administration of adjusted doses of KD025.

[0372] For comparison, relative growth rates were also investigated based on body weight, and the adult daily dose of 200 mg was used to predict KD025 doses for infants and children (0.25-0.5 years, 0.5-1 year, 1-2 years, 2-5 years, 6-11 years, and 12-17 years).

[0373] Step 1. Simulation of 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) Using the Sim-Healthy Volunteer Population Library, we generated a virtual study of 10 cases involving 20 healthy adult subjects (50% female) aged 20-50 years. The Simcyp Pediatric Simulator allows us to model pharmacokinetic behavior in neonates, infants, and children. The Simcyp simulator includes a complete PBPK model with extensive libraries of pediatric demographics (age, height, weight, BSA [body surface area]), developmental physiology (liver size, renal function, hepatic blood flow), and biochemistry (albumin, CYP ontogeny). The algorithms describing these modifications have been described in the literature (see, e.g., Johnson and Rostami-Hodjegan, "Resurgence in the Use of Physiologically Based Pharmacokinetic Models in Pediatric Clinical Pharmacology," Pediatr Anesth. 21;291-301

[2011] ).

[0374] The intended adult dose (200 mg) was converted to a dose of 2.7 mg / kg using a simulated average adult body weight of 73.84 kg. Virtual subjects received repeated oral doses of 2.7 mg / kg KD025 QD for 8 days, and the AUC 0-t,SS The individual simulated values ​​for were integrated on the 8th day (simulation time 168–192).

[0375] After repeated oral doses of 200 mg KD025 QD for 8 days, the mean simulated plasma KD025 and M2 concentrations were calculated. The predicted mean C for KD025 and M2 on day 8 max and AUC values ​​are shown in Table 44.

[0376] [Table 53]

[0377] Step 2. Simulation of plasma concentration-time profiles of KD025 and M2 in pediatric subjects (3 months to 17 years) after repeated oral doses of KD025 (2.7 mg / kg QD). Pediatric subjects were divided into the following age bands: 0.25-0.5 years, 0.5-1 year, 1-2 years, 2-5 years, 6-11 years, and 12-17 years. Using the default Sim-Pediatric population library, we generated 10 virtual trials of 20 pediatric subjects (50% female) for each age band receiving KD025 at 2.7 mg / kg (200 mg adult equivalent) QD. Simulations were performed using the CYP3A4 ontogenetic profile from Upreti and Wahlstrom, "Meta-Analysis of Hepatic Cytochrome P450 Ontogeny to Underwrite the Prediction of Pediatric Pharmacokinetics Using Physiologically Based Pharmacokinetic Modeling," J Clin Pharmacol. 56;266-283 (2016).

[0378] Virtual subjects received multiple oral daily doses of KD025 for 8 days and the AUC 0-t,SS The individual simulated values ​​for were integrated on day 8 (simulation time 168-192). The equivalent doses in mg are specified below by age group in Table 43.

[0379] Pediatric subjects were divided into the following age bands: 0.25-0.5 years, 0.5-1 year, 1-2 years, 2-5 years, 6-11 years, and 12-17 years. Using the default Sim-Pediatric population library, 10 virtual studies of 20 pediatric subjects (50% female) were generated for each age band receiving tailored repeated oral doses of KD025. All simulations were performed using the CYP3A4 ontogenetic profile from Upreti and Wahlstrom 2016.

[0380] Virtual subjects received multiple oral daily doses of KD025 for 8 days and the AUC 0-t,SSThe individual simulated values ​​for were integrated on day 8 (simulation time 168-192). The adjusted doses and equivalent doses in mg are specified in Table 45 by age group.

[0381] [Table 54]

[0382] The CYP3A4 ontogenetic profile from Upreti Wahlstrom (2016) was used to calculate predicted plasma concentration-time profiles of KD025 and M2 after repeated oral doses of 2.7 mg / kg in children aged 0.25-0.5 years, 0.5-1 year, 1-2 years, 2-5 years, 6-11 years, and 12-17 years. For each simulation, concentration-time profiles representing the mean of the entire hypothetical population (n=200) were used. The predicted mean AUCs for KD025 and M2 on day 8 were: 0-t,SS The values ​​are shown in Table 46.

[0383] [Table 55]

[0384] Step 3. Simulated pediatric / adult KD025 AUC 0-t,SS Adjustment of the initial 2.7 mg / kg dose of KD025 based on the ratio The calculated AUC of pediatric / adult KD025 as shown in equation (1) 0-t,SS Based on the ratio (simulations described in steps 1 and 2 above), the pediatric dose was adjusted from the initial value of 2.7 mg / kg.

number

[0385] Adjusted doses (mg / kg) were calculated across the following age ranges: 0.25-0.5 years, 0.5-1 years, 1-2 years, 2-5 years, 6-11 years, and 12-17 years, and are listed in Table 43.

[0386] In addition, sensitivity analyses were performed for each pediatric age group at several doses (within the range of 2.7–3.6 mg / kg) around the calculated adjusted dose, and it was confirmed that applying the adjusted dose resulted in similar KD025 and M2 exposure in both pediatric and adult populations.

[0387] AUC of KD025 in children / adults calculated using the CYP3A4 ontogenetic profile of Upreti Wahlstrom (2016) 0-t,SS Based on the ratios (steps 1 and 2 and the simulations described in Table 44), the pediatric dose was adjusted from the initial value of 2.7 mg / kg. The adjusted dose is shown in Table 45 above.

[0388] Step 4. Simulation of plasma concentration-time profiles of KD025 and M2 in pediatric subjects (3 months to 17 years) after adjusted multiple oral doses of KD025. Pediatric subjects were divided into the following age bands: 0.25-0.5 years, 0.5-1 year, 1-2 years, 2-5 years, 6-11 years, and 12-17 years. Using the default Sim-Pediatric population library, 10 virtual studies of 20 pediatric subjects (50% female) were generated for each age band receiving tailored repeated oral doses of KD025. All simulations were performed using the CYP3A4 ontogenetic profile from Upreti and Wahlstrom 2016.

[0389] Virtual subjects received multiple oral doses of KD025 for 8 days and the AUC 0-t,SS The individual simulated values ​​for were integrated on day 8 (simulation time 168-192). The adjusted doses and equivalent doses in mg are specified in Table 45 by age group.

[0390] The CYP3A4 ontogenetic profile from Upreti Wahlstrom (2016) was used to calculate predicted plasma concentration-time profiles of KD025 and M2 after dose adjustment in children aged 0.25-0.5 years, 0.5-1 year, 1-2 years, 2-5 years, 6-11 years, and 12-17 years. Each simulation included concentration-time profiles representing the mean of the entire hypothetical population (n=200). The adjusted dose on day 8 and the associated predicted AUCs of KD025 and M2 were calculated. 0-t,SS The mean values ​​are shown in Table 47.

[0391] Simulations were performed using doses in mg / kg.

[0392] [Table 56]

[0393] Relative growth rates were calculated based on body weight (BW) using the 3 / 4 power law as shown in equation (2).

number

[0394] Doses were calculated across the following age ranges: 0.25-0.5 years, 0.5-1 years, 1-2 years, 2-5 years, 6-11 years, and 12-17 years. BW for children and adults was taken as the mean simulated value from each age range (Table 43).

[0395] Relative growth rate dose predictions for pediatric subjects 3 months to 17 years of age using the 200 mg adult daily dose are shown in Table 48.

[0396] [Table 57]

[0397] Simulation results performed in this example using the Upreti Wahlstrom (2016) CYP3A4 ontogenetic profile suggest that there are smaller differences in KD025 and M2 exposure in pediatric subjects compared to adults when dosed on a mg / kg basis (AUC 0.01 for KD025 and M2, respectively, in adults). 0-t,SS (30% of the original value, within 1.5 times the original value).

[0398] Simple allometric scaling of the data may lead to an overestimation of the dose required in the youngest age group (≤5 years). In older age groups (6-17 years), allometric scaling yielded dose adjustments similar to PBPK predictions derived from Upreti-Wahlstrom ontogeny.

[0399] Further simulation. The following assumptions were used to develop the model: the drug is administered once daily as a tablet; drug response is assumed to be dose-proportional; the effect of co-administered CYP inducers or inhibitors is not considered; doses are administered after meals; all pediatric subjects are assumed to have chronic graft-versus-host disease (cGVHD); there are 1000 subjects in each hypothetical population, 50% of whom are male; and the effect of co-administration of PPIs on drug PK is not considered.

[0400] Estimated pediatric exposure based on adult-equivalent mg / kg dosing. Each hypothetical population spanned a 3-month age range, from 6 months to 216 months. Each subject received a fixed dose of 2.5 mg / kg (equivalent to a 200 mg dose in an 80.65 kg adult). Doses were administered QD. To predict exposure, the model was run to steady state. Cmax at steady state and AUC over 24 hours at steady state were used as exposure metrics. Pediatric exposure was compared to adult exposure.

[0401] Prediction of pediatric exposure through simulation based on age-based dosing strategies. The pediatric population was divided into three age bins: 1-6 years, 6-12 years, and 12-18 years. Doses of 3.5, 4, 4.5, and 5 mg / kg QD were administered to the 1-6 and 6-12 year old cohorts. A dose of 200 mg QD was administered to the 12-18 year old dose group. Models were run to steady state to predict exposure. Cmax at steady state and AUC over 24 hours at steady state were used as exposure metrics. Pediatric exposure was compared to adult exposure.

[0402] The influence of enzyme ontogeny on drug exposure. A 4 mg / kg dose was administered to a cohort of patients binned in 3-month age bins ranging from 6 months up to 60 months. Fractional activity for each subject was calculated using three methods: ontogeny alone, allometric growth at ages >2 years, and allometric growth alone. The predicted exposure was then binned into 1 kg weight bins to assess the need for dosing based on various weights. For pediatric patients weighing 13 kg or more, the ontogeny of CYP enzymes has limited influence on drug exposure at the 4 mg / kg dose level. In the hypothetical cohort, the maximum weight of patients within the 18- to 21-month age group was 13 kg.

[0403] Pediatric exposure is predicted based on weight bins.

[0404] Cohorts binned in 2-month age bins from 6 to 24 months were administered doses of 3 mg / kg, 3.5 mg / kg, and 4 mg / kg. Fractional activity for each subject was calculated using enzyme ontogeny. Predicted exposures were then binned into 1 kg body weight bins to assess the effect of dose level on exposure across body weight bins.

[0405] Example 13: Suspension formulation test The formulation composition and process variables of a liquid bermosudil formulation with a concentration of 40 mg / mL were tested to provide a homogeneous and stable dispersion. The process variables were evaluated to obtain a robust suspension formulation.

[0406] Table 49 shows the initial formulation composition used to manufacture the first five lab batches in the study (Batch Nos. 001-005).

[0407] [Table 58]

[0408] After manufacturing the first laboratory batch (Batch No. 001), undesirable foaming and agglomeration were observed. Four additional laboratory batches were manufactured using the initial formulation composition with a modified addition order, including slow addition of the API and side phase preparation. A second laboratory batch (Batch No. 002) was prepared using the initial formulation composition shown in Table 49 and the manufacturing process shown in Figure 5. However, foaming and agglomeration were also observed in the four additional batches (Batch Nos. 002-005). It was concluded that there was a need to reduce the foaming and agglomeration observed during the suspension manufacturing process to provide a liquid formulation suitable for commercial use.

[0409] A further batch (Batch No. 006) of Belmosudil suspension with a concentration of 40 mg / mL was prepared using a modified manufacturing process. This modified process included replacing the A310 paddle impeller used in the mixing step with a Silverson homogenizer L5M-A equipped with a square-hole high-shear screen and adding an anti-foaming agent to the formulation. Using this Silverson homogenizer, a 2 kg laboratory batch of Belmosudil suspension containing 30% simethicone emulsion at a concentration of 2% w / w was produced as Batch No. 006.

[0410] The improved process was found to completely reduce the amount of foaming that occurs during manufacturing and provide a suspension suitable for commercial use. The improved formulation composition used for Batch No. 006 is shown in Table 50, and the improved manufacturing process used for Batch No. 006 is shown in Figure 6.

[0411] [Table 59]

[0412] The manufacturing process used to prepare Batch No. 002 Belmosudil Suspension 40 mg / mL is shown in Figure 5, and the initial formulation composition used for Batch No. 002 is shown in Table 49. The improved manufacturing process used to prepare Batch No. 006 Belmosudil Suspension 40 mg / mL is shown in Figure 6, and the improved formulation composition used for Batch No. 006 is shown in Table 50.

[0413] Table 51 is a comparison of the manufacturing process details for batch numbers 002 and 006, showing a number of specific differences that occurred between the initial and improved manufacturing processes.

[0414] [Table 60]

[0415] The manufacturing process used to prepare Batch No. 002, shown in Figure 5, involves two phases ("API Phase" and "Aqueous Phase") and two vessels (one for mixing the povidone PVP and vermosudil mesylate components, and the other for mixing the sodium benzoate, sucralose, and colloidal silica components). These two phases are mixed separately, then combined, tartaric acid is added, pH and moisture levels are adjusted, and the formulation is allowed to stand for 1 hour and 30 minutes. Mixing is accomplished with an A310 paddle impeller rotating at speeds ranging from 300 to 600 rpm. According to Table 51, a hold time is used to allow foaming to dissipate, and both the mixing time and hold time total at least 305 minutes.

[0416] The manufacturing process used to prepare Batch No. 006, shown in Figure 6, utilizes a single manufacturing vessel used to mix all ingredients, and since there is no foaming generated, there is no hold time required to allow foaming to dissipate. Mixing is accomplished with a Silverson homogenizer equipped with a square-hole high-shear screen impeller rotating at speeds up to 5000 rpm. According to Table 51, the total mixing time amounts to 105 minutes.

[0417] The use of a Silverson homogenizer allowed for much higher rotational speeds to be used in the mixing step of the process, and the use of a square-hole high-shear screen allowed for higher shear rates to be achieved during mixing. The process using a Silverson homogenizer had shorter total mixing times and did not include a 90-minute hold step as required by the process using an A310 paddle impeller and slow mixing. The suspension produced using the improved formulation composition containing simethicone emulsion and a Silverson homogenizer (Batch No. 006) was free of foaming, unlike the suspension produced without simethicone using an A310 paddle impeller and slow mixing, which exhibited foaming (Batch No. 002).

[0418] The liquid vermosudil oral suspension produced using the improved formulation composition comprising simethicone emulsion and a Silverson homogenizer as described herein and shown in Figure 6 provides a robust, homogeneous, and stable dispersion that can be resuspended uniformly over time and shaken multiple times before administration while reproducibly delivering the desired drug dose. During formulation manufacturing, high-speed mixing with a specific paddle geometry is commonly used to reduce particle agglomeration and produce a homogeneous, stable dispersion. However, a side effect of high-speed mixing, especially with hydrophobic drug products, is increased foaming. The improved composition and manufacturing process overcome the foaming problem and produce a suspension with the desired characteristics.

[0419] Example 14: Additional Suspension Formulation Studies Further testing can evaluate additional formulation and process variables for the 40 mg / mL Belmosudil suspension. The relationships between input variables (factors) and output variables (responses) will be examined. Tests performed include evaluating the effective concentration of simethicone in the 40 mg / mL Belmosudil suspension.

[0420] The formulation and process parameters evaluated are based on a design of experiments (DoE) performed using Minitab 21 software. The formulation compositions tested are shown in Table 52. Also shown with the compositions are the test ranges for the formulation variables along with the independent identically distributed (IID) limits determined so far. The effective simethicone concentration, the effect of pH, and the effective concentrations of Syloid 244 FP and PVP K-90 are also tested.

[0421] [Table 61]

[0422] The design strategy for evaluating the effective concentration of 30% simethicone emulsion is a one-factor-at-a-time (OFAT) approach.

[0423] The same manufacturing process is followed as used in Batch No. 006 of Example 13, except for changing the order of addition of PVP K-90 and Syloid 244 FP. Various experiments performed to evaluate the effective concentration of 30% simethicone emulsion are shown in Table 53.

[0424] [Table 62]

[0425] [Table 63]

[0426] To test the effect of pH: Because the API is soluble at pH 2.0-3.0, the effect of pH on the suspension can be tested. This test uses the most effective formulation composition from the test results described above. Table 55 shows two additional experiments performed to test the pH range.

[0427] [Table 64]

[0428] The same responses shown in Table 54 are also tested for pH impact testing.

[0429] DoE Study for Syloid 244 FP, PVP K-90 and pH: To study the effect of the amount of Syloid 244 FP and PVP K-90 as the pH changes, a DoE design as shown in Table 56 is used. Design Plan: A fractional factorial design with three factors with the following design overview: Factor: 3 Basic Design: 3, 4 Resolution: III Runs: 8 Iterations: 1 Rate: 1 / 2 Blocks: 1 Center points (total): 4

[0430] [Table 65]

[0431] [Table 66]

[0432] The same responses shown in Table 54 are also tested for DoE studies for Syloid 244 FP, PVP K-90 and pH tests.

[0433] Process Experimental Design: A process DoE is performed on the improved manufacturing process shown in Figure 6. The process parameters identified for testing are the mixing time and rate after adding PVP K-90, and the homogenization time and rate after adding the API. Table 57 shows the DoE studies performed to test these process parameters during the production of Belmosudil Suspension 40 mg / mL.

[0434] Design Plan: A fractional factorial design with four factors, with the following design outline: Factor: 4 Basic Design: 4,8 Resolution: IV Runs: 11 Iterations: 1 Rate: 1 / 2 Blocks: 1 Center points (total): 3

[0435] [Table 67]

[0436] [Table 68]

[0437] The same responses shown in Table 54 are also tested for DoE testing related to process parameter testing.

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 (bermosudil), in a diluent in combination with a suspending agent, a thickening agent, and an antifoaming agent.

2. 10. 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 (bermosudil), in a diluent in combination with a suspending agent and a thickening agent.

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

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

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

7. 7. The liquid formulation of claim 4, wherein the preservative is sodium benzoate, the suspending agent is colloidal silica, the thickening agent is povidone, the pH adjuster is tartaric acid, and the sweetener is sucralose.

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

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

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

11. 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. 7. The liquid formulation of any one of claims 4 to 6, comprising about 2 to 8 weight percent bermosudil, 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 thickener.

14. 7. The liquid formulation of any one of claims 4 to 6, comprising about 3 to 5 weight percent bermosudil, 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 thickener.

15. 7. The liquid formulation of any one of claims 4 to 6, comprising the following weight percentages of bermosudil, preservative, suspending agent, thickener, and sweetener: about 4.75% bermosudil mesylate; about 0.035-0.1% preservative; about 0.15% suspending agent; about 0.5% thickener; and about 0.15% sweetener.

16. 16. The liquid formulation of any one of claims 13 to 15, wherein the suspending agent is colloidal silica, the thickening agent is povidone 90F, the sweetening agent is sucralose, and, if necessary, the pH adjusting agent is tartaric acid.

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

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

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

20. 13. 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 volumetric 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. 21. A method for treating graft-versus-host disease in a subject, comprising administering to the subject a therapeutically effective amount of a liquid formulation according to any one of claims 1 to 20.

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

24. 24. The method of claim 22 or 23, wherein the subject is an adult patient.

25. 24. The method of claim 22 or 23, wherein the subject is a pediatric patient.

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

27. 27. The method of any one of claims 22 to 26, wherein the liquid formulation is administered at a concentration equivalent to about 40 mg / ml of bermosudil free base.

28. 28. The method of claim 26 or 27, wherein the liquid formulation is administered at a dose of about 2.5 to 5.0 mg / kg.

29. 29. The method of claim 28, wherein the liquid formulation is administered at a dose of about 3.5 mg / kg.

30. 29. The method of claim 28, wherein the liquid formulation is administered at a dose of about 4.0 mg / kg.

31. The method of any one of claims 22 to 30, wherein the liquid formulation is administered once daily or twice daily.

32. A process for preparing a liquid formulation containing belmosudil, comprising mixing jet-milled and / or pin-milled belmosudil in a diluent containing a suspending agent and a thickening agent.

33. 33. The process of claim 32, (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 said container; (d) transferring the container to a homogenizer; (e) operating the homogenizer to homogenize the contents of the vessel; (f) adding a quantity of said vermosudil to said container and mixing the contents thereof to form a suspension; (g) measuring the pH of the suspension; (h) adding to the suspension, while mixing, a sufficient amount of a pH adjuster to obtain a pH of from about 2.5 to about 4.0; and (i) adding a second portion of the diluent to the container to achieve a predetermined volume to provide the liquid formulation comprising bermosudil.

34. 34. The process of claim 33, wherein the preservative is sodium benzoate, the sweetening agent 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.

35. 34. The process of claim 33, further comprising, after step (i), (j) leaving the suspension until any bubbles or foam have disappeared, and (k) dispensing the liquid formulation containing bermosudil into a container.

36. 34. The process of claim 33, further comprising visually inspecting the contents of the container and / or the suspension after one or more of steps (c)-(i).

37. 34. The process of claim 33, further comprising visually inspecting the suspension for foaming and / or precipitation after steps (c), (e), (f), and (j).

38. 35. The process of claim 34, wherein the bermosudil, sodium benzoate, sucralose, silicon dioxide, and povidone are added to the container in the following weight percentages, calculated taking into account a predetermined volume: about 3 to 5 weight percent bermosudil, 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.

39. 39. The process according to any one of claims 33 to 38, wherein the beromosudil added to the formulation is jet-milled beromosudil mesylate.

40. 33. The process of claim 32, (a) dispensing a first portion of the diluent into a container; (b) dispensing a preservative, a sweetener, the suspending agent, the thickener, an antifoaming agent, and a quantity of the vermosudil into the container; (c) mixing the contents of the vessel with a homogenizer equipped with a high shear screen to form a suspension; (d) measuring the pH of the suspension; (e) adding to the suspension, while mixing, a sufficient amount of a pH adjuster to obtain a pH of about 2.5 to about 4.0; and (f) adding a second portion of the diluent to the container to achieve a predetermined volume to provide the liquid formulation comprising bermosudil.

41. 41. The process of claim 40, wherein the mixing with a homogenizer is performed at a rotation speed of about 4000 to 6000 rpm.

42. A liquid formulation comprising bermosudil produced by the process of any one of claims 32 to 41.

43. A kit for preparing a liquid formulation of belmosudil, comprising: (a) a quantity of belmosudil in a first container; and (b) a reconstitution vehicle comprising a suspending agent and a thickening agent in a diluent in a second container.

44. 44. The kit of claim 43, wherein: (a) the bermosudil in the first container is in powder form; and (b) the suspending agent is silicon dioxide and the thickening agent is povidone.

45. 45. The kit of claim 43 or 44, further comprising a sweetener in the reconstitution vehicle.

46. The liquid formulation according to any one of claims 1 to 20 or the pharmaceutical composition according to claim 21, wherein bermosudil is at a concentration of 40 mg / ml.

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