TW202430158A - Solid dispersion comprising amorphous 2-[3-[4-(1h-indazol-5-ylamino)quinazolin-2-yl]phenoxy]-n-propan-2-yl-acetamide - Google Patents

Abstract

The present disclosure relates to a solid dispersion comprising amorphous belumosudil. The present disclosure also provides pharmaceutical dosage forms comprising the solid dispersion comprising amorphous belumosudil as an active pharmaceutical ingredient and methods of using the pharmaceutical dosage forms to treat diseases or disorders regulated by ROCK.

Description

Solid dispersion containing amorphous 2-[3-[4-(1H-indazol-5-ylamino)quinazolin-2-yl]phenoxy]-N-propan-2-yl-acetamide

The present disclosure relates to solid dispersions comprising amorphous 2-{3-[4-( 1H -indazol-5-ylamino)-2-quinazolinyl]phenoxy} -N- (propan-2-yl)acetamide (also known as belumosudil and KD025). The present disclosure further relates to methods of preparing solid dispersions comprising amorphous belumosudil as described herein, pharmaceutical compositions comprising one or more of the solid dispersions, and methods of using the pharmaceutical compositions to treat diseases and disorders regulated by Rho-associated coiled-coil protein kinase (ROCK).

Belusudil, chemically known as 2-{3-[4-( 1H -indazol-5-ylamino)-2-quinazolinyl]phenoxy} -N- (propan-2-yl)acetamide, is represented by the following formula I: 2-(3-(4-( 1H -indazol-5-ylamino)quinazolin-2-yl)phenoxy) -N -isopropylacetamide (I)

Belusudil (also known as KD025) is an inhibitor of Rho-associated coiled-coil protein kinase (ROCK). Belusudil binds to and inhibits the serine/threonine kinase activity of ROCK1 and ROCK2 and is therefore useful for treating diseases, disorders and conditions regulated by ROCK, including autoimmune and fibrotic disorders, acute and chronic graft-versus-host disease (GVHD), idiopathic pulmonary fibrosis, and moderate to severe psoriasis, among other indications. The mesylate salt of belusudil 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 GVHD (cGVHD), in some cases after failure of at least two prior lines of systemic therapy.

In U.S. Patent 8,357,693 (the '693 patent), a method for preparing belusudil is disclosed, specifically in Example 82 thereof. The method disclosed in the '693 patent provides belusudil as a crude product, which is purified by high performance liquid chromatography (HPLC). Belusudil and methods for preparing the compound are also described in U.S. Patent Nos. 9,815,820, 10,183,931, and 10,696,660.

Berushedil is a weakly alkaline compound that is almost insoluble in water. Current methods of administering berushedil include formulating the mesylate salt of berushedil into pharmaceutically acceptable capsules and tablets for oral administration. Given the low water solubility of berushedil, the compound tends to precipitate out when transitioning from the acidic gastric environment of the stomach and early digestive pathways to the more neutral pH of the intestinal environment. Therefore, the low solubility of berushedil may affect the manner and timing of its bioabsorption and pharmacokinetic properties. In addition, the low solubility of berushedil may limit the use of traditional excipients and wet granulation methods.

The low solubility and variable pharmacokinetic properties of berusudil present challenges for the development of alternative formulations. To illustrate, a formulation of berusudil with enhanced solubility would provide greater flexibility and a wider range of options in developing different dosing regimens, formulations, and modes for delivering the compound to a subject.

Therefore, there remains a need for formulations containing berushedil that address these challenges.

In one aspect, the present disclosure provides a solid dispersion comprising an amorphous form of berusudil. In some embodiments, the amorphous form of berusudil is placed in a solid dispersion comprising a matrix carrier material. The use of amorphous berusudil in a solid dispersion enhances the solubility of berusudil and improves its biological properties, thereby providing an expanded method for formulating and delivering drugs.

In some embodiments, the present disclosure provides an amorphous solid dispersion comprising berusudil, optionally prepared using a spray drying technique.

In some embodiments, the amorphous form of berusudil is provided as an amorphous solid dispersion of berusudil formulated with at least one polymer (optionally, for example, with a pharmaceutically acceptable polymer).

Another aspect of the present disclosure provides a method for preparing a solid dispersion containing amorphous berusudil. A solvent evaporation method (such as spray drying) can be used to prepare a solid dispersion containing amorphous berusudil. A solid dispersion containing amorphous berusudil can be prepared by: dissolving berusudil in a suitable solvent; adding one or more carrier matrix materials; and removing the solvent by spray drying to provide a solid dispersion containing amorphous berusudil in the carrier matrix material.

Solid dispersions containing amorphous berusudil can be used to prepare solid pharmaceutical dosage forms, such as tablets and capsules. Pharmaceutical compositions containing an effective amount of amorphous berusudil can be used to treat diseases, disorders and conditions regulated by ROCK as further described herein.

The present disclosure provides solid dispersions comprising an amorphous form of berusudil that increases the solubility of the compound and improves the biological properties of the compound. Reliable dosing and absorption of berusudil are important to ensure sustained systemic exposure. Therefore, the development of reproducible drug delivery systems and characterization of their associated dissolution profiles provide advantages in ensuring sustained and effective dosing of berusudil.

Solvent evaporation methods such as spray drying can be used to prepare a solid dispersion containing amorphous berusudil. This technique involves dissolving or suspending berusudil in a polymer matrix carrier and then spraying the mixed stream. Spray drying removes the solvent to obtain a solid dispersion containing amorphous berusudil dispersed in a polymer matrix carrier.

Using spray drying technology, berushedil is converted into an amorphous state dispersed in a polymer matrix carrier. Amorphous berushedil prepared by the method disclosed herein enhances dissolution by reducing fineness and removing the lattice. The lack of crystallinity causes berushedil to dissolve without overcoming the lattice energy. The carrier material can additionally assist dissolution by improving the wettability, solubility and stability characteristics of berushedil in a supersaturated solution. The spray-dried solid dispersion containing amorphous berushedil provides good physicochemical properties (such as controlled fineness and fluidity), and the solid dispersion can be used for downstream processing, such as tablet compression.

Additionally, pharmaceutical compositions comprising solid dispersions containing amorphous berusudil exhibit increased dissolution rates compared to compositions comprising a crystalline form of berusudil; in some embodiments, a significant increase in dissolution rate is obtained with the solid dispersions herein. The increased solubility of solid dispersions containing amorphous berusudil provides advantages and greater flexibility in formulation development and drug delivery. Definition

As used herein, the term berusudil (or KD025) refers to 2-{3-[4-( 1H -indazol-5-ylamino)-2-quinazolinyl]phenoxy} -N- (propan-2-yl)acetamide represented by the following formula I: (I)

In some embodiments, amorphous berusudil is in free base form.

When the term "berushedil" is used herein, it should be understood that the term covers any form of the compound berushedil as well as its pharmaceutically acceptable salts, unless the context clearly indicates otherwise. The term "berushedil" refers to both the compound berushedil (e.g., free base form, amorphous form, or crystalline form), to a pharmaceutically acceptable salt of berushedil (e.g., the mesylate form as used in REZUROCK ^™ ), and to any form of berushedil that can be used in a formulation or pharmaceutical composition for administration of the compound to a patient.

The term "pharmaceutically acceptable salt" refers to non-toxic, inorganic acid and organic acid addition salts of berusudil. In some embodiments, the pharmaceutically acceptable salt of berusudil herein is a methanesulfonate salt.

As used herein, "about" includes the exact amount modified by the term about as well as amounts expected to be within experimental error (e.g., within 15%, 10%, or 5%). For example, "about 5 mg" means "5 mg" as well as mg ranges within experimental error (e.g., plus or minus 15%, 10%, or 5% of 5 mg). As used herein, the term "about" can be used to modify ranges as well as specific values.

The acronym API refers to "active pharmaceutical ingredient" and, as used herein, is synonymous with the definition of berusudil (or KD025) and its pharmaceutically acceptable salt (optionally, the mesylate salt of berusudil).

As used herein, "administering" or "administering to" refers to the act of prescribing one or more drugs containing the API for a subject to take during treatment, the act of dispensing the one or more drugs to the subject, and/or the act of receiving or ingesting the one or more drugs into the body. Thus, the API (Berushedil) can be "administered" by a physician or other medical professional who writes a prescription for the one or more drugs; and/or "administered" by a pharmacist who fills the prescription and/or dispenses the one or more drugs to the subject; and/or "administered" by a patient or subject who ingests the drug and/or his or her partner or caregiver who provides the drug to the subject.

As used herein, the term "solid dispersion" refers to a system in which the API is dispersed throughout a solid carrier (in some embodiments, a solid matrix carrier). In some embodiments, the carrier comprises small molecules and/or polymers or copolymers, optionally polymers. Thus, a solid dispersion will include at least two components, one of which is the API and the other is the carrier. Additional additives (e.g., surfactants) may optionally be included. Optionally, in a solid dispersion, the API is homogenously or uniformly dispersed throughout the carrier matrix.

The term "amorphous solid dispersion" as used herein refers to a single-phase amorphous system in which the API is molecularly dispersed or dissolved in a carrier matrix (optionally, a polymer matrix).

In some embodiments herein, the ratio of berusudil to one or more carrier matrix materials in the solid dispersion may be about 10:90 to 90:10 by weight; or about 20:80 to 80:20 by weight; or about 25:75 to 75:25 by weight; or 40:60 to 60:40 by weight.

Unless otherwise indicated, the term "amorphous" or "amorphous form" means that a substance or component is not substantially crystalline, and is a disordered solid form, i.e., a solid form that substantially lacks long range crystalline order as determined by XRPD data. The substantially amorphous state includes APIs that are at least about 50% by weight, optionally at least about 60% by weight, optionally at least about 70% by weight, optionally at least about 80% by weight, optionally at least about 90% by weight, optionally at least about 95% by weight, or optionally at least 99% by weight in amorphous form compared to other forms of the substance or component.

In some embodiments, amorphous berusudil is a solid state form of berusudil that is substantially amorphous; in some embodiments, it is in the form of berusudil that includes at least about 95% amorphous form; in some embodiments, it is in the form of berusudil that is at least 98% amorphous form. Whether berusudil is in amorphous form can be characterized, for example, by XRPD techniques as described herein or by another technique known to those skilled in the art.

As used herein, "carrier matrix" or "carrier matrix material" refers to a component that stabilizes, suspends and/or transports the amorphous form of berushedil in the solid state. Optionally, the carrier matrix material is an amorphous polymer material. The polymer selection of the solid dispersion may play an important role in the overall final product properties. The polymer used as the carrier matrix material can include polyvidone derivatives, such as polyvinyl pyrrolidone (PVP) and polyvinyl pyrrolidone-vinyl acetate copolymer (PVPVA) (such as those sold under the trade name Kollidon VA 64®); polymethacrylate derivatives (such as the Eudragit® series); polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymers (PPPEG) (such as those currently sold under the trade name Soluplus®); hydroxypropyl methylcellulose (HPMC) and hydroxypropyl methylcellulose acetate succinate (HPMCAS). The optional carrier matrix materials herein are PVPVA and PPPEG.

The term "effective amount" used in conjunction with amorphous berusudil means an amount capable of treating or preventing a disorder, disease or condition as disclosed herein, or a symptom thereof. For example, in a pharmaceutical composition, an effective amount of amorphous berusudil may be at a level that will provide the desired effect; for example, in a unit dose for oral administration, about 0.5 to 15 mg/kg subject weight, optionally about 1 to 5 mg/kg subject weight, optionally about 3 mg/kg patient weight. For example, the dosage of berusudil may be the currently administered therapeutic dose of 200 mg per day, or alternatively, a dose in the range of 10 mg up to 1000 mg, optionally in the range of 100 mg to 400 mg for adult patients; optionally, in the range of 100 mg to 200 mg.

In addition, optionally, for pediatric patients, the dosage of berushedil can be in the range of 10 mg to 200 mg. The dosage of berushedil can be adjusted according to the patient's weight. For example, for pediatric patients with a weight in the range of about 6 kg to less than 20 kg, the dosage can be in the range of about 10 to 50 mg, administered once a day; in another embodiment, for pediatric patients with a weight in the range of about 10 kg to less than 20 kg, the dosage can be about 50 mg, administered once a day; for pediatric patients with a weight in the range of about 20 kg to less than 40 kg, the dosage can be about 100 mg, once a day; and for pediatric patients with a weight equal to or greater than 40 kg, the dosage can be 200 mg, once a day.

It will be apparent to those skilled in the art that the effective amount of the amorphous form of berusudil disclosed herein is expected to vary depending on the severity of the indication being treated, the route of administration, and/or other drugs (e.g., proton pump inhibitors or CYP3A inducers) administered to the subject in consideration of drug-drug interactions.

As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium or zinc stearate, or stearic acid). Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation (e.g., including the API, so as to avoid slow crystallization over time) and maintaining the stability of the other ingredients, and not injurious to the patient.

As used herein, "suitable solvent", e.g., as used to dissolve berusudil in a solvent system with a carrier matrix material, means a solvent or a mixture of one or more solvents that is compatible with berusudil and one or more carrier matrix materials and is capable of sufficiently dissolving berusudil and the one or more carrier matrix materials to enable the use of spray drying techniques. The term "suitable solvent" may include a mixture of solvents and is therefore interchangeable with "suitable solvent system". The solubility of the API and/or the carrier matrix material may be confirmed by filtration and HPLC analysis or by visual inspection (e.g., producing a clear or substantially clear solution upon visual inspection). Solvent "suitable" also means that the solvent(s) do not present unacceptable toxicity or environmental hazards and are acceptably used in the manufacture of a pharmaceutical for human consumption.

Unless the context requires otherwise, "or" is used in an inclusive sense (equivalent to "and/or"). General Preparation and Use

A solid dispersion comprising amorphous berusudil can be prepared by dissolving berusudil in a suitable solvent; adding one or more carrier matrix materials to the berusudil solution; and removing the solvent, or substantially removing the solvent, to provide amorphous berusudil dispersed in the carrier matrix.

In the first step of the method involving adding berusudil to a suitable solvent, berusudil can be in various forms, for example, any polymorphic crystalline form or solvate. Berusudil can be in salt form or can be in free base form. If berusudil is in the form of an acid addition salt and amorphous berusudil in free base form is desired, sufficient base can be added to the solvent to form berusudil free base. The base can be an inorganic base, such as an alkaline metal hydroxide, or the base can be an amine, such as diethylamine, triethylamine, etc.

In the context of this method, the choice of solvent is an important consideration for preparing amorphous solid dispersions, and a combination of solvents can be used to obtain the desired solvent parameters. As described in Example 1 herein, a large number of solvent screening experiments were conducted to obtain a solvent system that can be used to dissolve belusudil and carrier matrix materials, thereby enabling the use of spray drying techniques. After these solubility experiments, it was found that a suitable solvent system for preparing amorphous belusudil via spray drying contained a mixture of triethylamine (TEA) and acetone. In contrast, solvent systems comprising acetone, ethyl acetate, acetonitrile (ACN), tetrahydrofuran, methanol, dichloromethane (DCM), dimethylformamide (DMF), isopropyl alcohol (IPA), methyl ethyl ketone, methyl isobutyl ketone (MIBK), methyl tertiary butyl ether (MTBD), n-heptane, toluene, a mixture of DCM and methanol, a mixture of ethanol and hexane, and a mixture of an aqueous solution and acetone or ACN were unable to effectively dissolve belusudil, and/or belusudil was determined to be almost insoluble or slightly soluble in one or more of these solvents, such that these solvents and/or solvent systems are not "suitable solvents" as defined herein.

For example, WO 2021/129589 A1 reportedly identifies a solvent that is claimed to be a "good solvent" for dissolving berushedil to produce its amorphous form. WO 2021/129589 A1 provides a working example (Example 22 thereof) involving the preparation of amorphous KD025, which includes the use of DMF as a solvent. However, the applicant has found that berushedil is only slightly soluble in DMF, and therefore DMF is not a suitable solvent. WO 2021/129589 A1 further identifies a solvent recommended for preparing an amorphous form of belusudil, which is said to be selected from one or more of methanol, acetone, methyl ethyl ketone, DMF, dimethyl sulfoxide (DMSO), n-methylpyrrolidone and ethylene glycol dimethyl ether. However, WO 2021/129589 A1 does not provide working examples describing the use of these solvents. The applicant has found through practical working examples as described herein that the solvents identified in WO 2021/129589 A1 are not suitable solvents as defined herein due to the low solubility of berusudil and/or the carrier matrix in the solvent and/or their incompatibility during spray drying and/or drug development.

The amorphous solid dispersion containing berusudil can be used to prepare solid pharmaceutical dosage forms such as tablets and capsules.

In one aspect, the present disclosure provides a pharmaceutically acceptable composition comprising a therapeutically effective amount of amorphous berusudil formulated with one or more drug excipients. The pharmaceutical composition can be specifically formulated for administration in solid form and is suitable for administration to a patient via a method suitable for use of solid form APIs (e.g., via oral administration, e.g., tablets or capsules).

In some embodiments, the present disclosure provides a solid pharmaceutical dosage form (capsule, tablet, pill, powder, granule, etc.) for oral administration comprising an amorphous solid dispersion of berushedil, wherein the solid pharmaceutical dosage form is mixed with one or more pharmaceutically acceptable excipients (including pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate) and/or any one of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders, such as carboxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and/or gum arabic; (3) wetting agents, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retardants, such as wax; (6) absorption accelerators, such as quaternary ammonium compounds and surfactants, such as poloxamers and sodium lauryl sulfate; (7) wetting agents, such as, for example, cetyl alcohol, glyceryl monostearate, and nonionic surfactants; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (10) coloring agents; and (11) Controlled release agents, such as crospovidone or ethylcellulose. In the case of capsules, tablets and pills, the pharmaceutical composition may also contain a buffer. Similar types of solid compositions may also use such excipients as lactose or milk sugar and high molecular weight polyethylene glycols as fillers in soft and hard shell gelatin capsules.

Tablets can be made by compression or molding (optionally with one or more auxiliary ingredients). Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surfactant or a dispersant. Molded tablets can be prepared by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets and other solid dosage forms (such as capsules, pills and granules) of the pharmaceutical composition of this disclosure can be optionally scored or prepared with coatings and shells (such as other coatings known in the field of enteric coatings and drug formulations). They can also be formulated into active ingredients therein that can be provided slowly or controlled release, for example, using hydroxypropylmethylcellulose, other polymer bases, liposomes and/or microspheres of different ratios that provide a desired release curve. They can be formulated into for rapid release, such as freeze drying. These compositions can also optionally contain sunscreens, and can have such a composition that they only release one or more active ingredients, or optionally, in a certain part of the gastrointestinal tract, release in a delayed manner. The example of the embedding composition that can be used includes polymeric substances and waxes. If appropriate, the active ingredient may also be in microencapsulated form using one or more of the above-mentioned excipients.

Besides inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, coloring agents, aroma agents and preservatives.

Pharmaceutical compositions comprising an effective amount of amorphous berusudil can be used to inhibit ROCK1 and ROCK2, preferentially inhibiting ROCK2, and are therefore useful for treating diseases regulated by ROCK enzymes, such as autoimmune disorders and/or fibrotic disorders, including, inter alia, 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.

The compositions provided herein are further useful for treating bronchiolitis obstructive syndrome (BOS), a potentially serious complication following lung transplantation or allogeneic hematopoietic stem cell transplantation (allogeneic HSCT).

In view of the public text of this article, the following abbreviations may be used for reference. Abbreviations: ACN Acetonitrile API Active pharmaceutical ingredient (here KD025) DCM Dichloromethane DSC/TGA Differential Scanning Calorimetry/Thermogravimetric Analysis FaSSIF Fasting state analog bowel fluid GC-HS Gas chromatography-headspace HPLC HPLC HPMC Hydroxypropyl methylcellulose HPMCAS Hydroxypropyl Methylcellulose Acetate Succinate h/hr Hours MDSC Modulation Differential Scanning Calorimetry MEK Methyl Ethyl Ketone PK Pharmacokinetics PLM Polarized light microscopy PMA Polymethacrylate PO Oral PPPEG Polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol PSD Microscopic distribution PVP Polyvinylpyrrolidone PVPVA Polyvinylpyrrolidone-vinyl acetate RCS Refrigeration system RH Relative humidity RRT Relative retention time RT Room temperature SDD Spray drying dispersion SEM Scanning electron microscopy TEA Triethylamine Temp. temperature THF Tetrahydrofuran TFA Trifluoroacetic acid XRPD X-ray powder diffraction Instruments:

Unless other instrument details are described in the following examples, the following instruments and procedures can be used to collect data as described in the examples herein. It will be appreciated by those skilled in the art that alternative instruments and procedures can be optionally used to collect characterization data, such as PLM, XRPD, TGA, DSC/TGA, and PSD.

XRPD, PSD and MDSC data were collected using the following instruments and procedures as described in Table 1, Table 2 and Table 3, respectively: Table 1 XRPD parameters Instruments: Rigaku Miniflex 6G Radiation source: Cu-Kα (1.5406 Å) Scan mode: Coupling 2ϴ/ϴ Scan range: 5°-40° Scanning speed: 5°/min Step increment: 0.0005° Voltage: 40 kV Current: 15 mA Rotation: 30 rpm Diverging Slits: 0.625 mm Sample holder: Zero Background Cup Table 2 PSD parameters Instruments: Malvern Mastersizer 3000 Dispersion device: Aero S(Dry) Hopper height: 1.5 mm Dispersed gas: Nitrogen Air Pressure: 1.5 bar Feed rate: 20%-40% Shading Limitation: 0.1%-10% Refractive Index Name: Drug and polymer dispersions Refractive Index: 1.6 density 0.5 g/mL Measurement duration: 30 s Measurement Repeat n = 3 Table 3 MDSC parameters Instruments: TA Discovery DSC2500 with RCS90 Cooler Scan mode: Modulation Temperature range: 0°C-240°C Heating rate: 2.0°C/min Modulation time period: 60 s Modulation amplitude: ± 1.0°C Pot/lid type Non-sealed Repeat n = 3 Example 1 1.1 Solvent screening

The purpose of the experiments described in this example is to explore the use of spray drying technology to enhance the solubility of berusudil and improve its biological properties of amorphous solid dispersions. However, spray drying technology requires a suitable solvent system that is compatible with berusudil and polymer matrix materials.

To investigate suitable solvent systems for use with berusudil and polymer matrix materials, the organic solvent systems described in this section (and in Tables 4A, 4B, and 4C) were prepared and evaluated. A. Solubility Evaluation Using HPLC

The solubility (w/v) of berusudil in various solvents was determined using HPLC. As described in Table 4A below, a saturated solution of berusudil was prepared with approximately 99-105 mg of berusudil and 2.0 mL of solvent in a 5 mL amber vial. The solution was thoroughly vortexed and loaded onto a laboratory rotator and rotated constantly at 200 rpm for 24 h. The resulting sample was filtered through a 0.45 µm filter, and the filtrate was used to quantify berusudil using a linear method by HPLC. Samples of IPA, acetonitrile, ethyl acetate, DCM, toluene, MIBK, acetone, n-heptane, and methyl tertiary butyl ether (MTBE) were injected into the HPLC without any further dilution; samples of methanol, DMF, and DMSO were further diluted and then injected into the HPLC; for the methanol, DMF, and DMSO solutions, 0.1 mL of the filtered solution was transferred to a 100 mL volumetric flask and diluted to volume with diluent before injection. The solubility is reported in mg/mL in Table 4A. Table 4A : Results of Solubility Experiments Using HPLC Solvent Sample weight (mg/2 mL solvent ) Solubility (mg/mL) result water 101.75 0.01 Almost insoluble Methanol 102.46 3.85 Slightly soluble Acetonitrile 99.89 0.00 Almost insoluble Toluene 101.51 NA Almost insoluble Ethyl acetate 102.94 0.00 Almost insoluble DCM 100.81 0.00 Almost insoluble MIBK 102.74 0.02 Almost insoluble acetone 102.64 0.01 Almost insoluble MTBE 103.20 0.00 Almost insoluble IPA 103.44 0.03 Almost insoluble n-Heptane 101.05 NA Almost insoluble DMF 100.94 2.15 Slightly soluble DMSO 105.58 23.41 Soluble B. pH - based solubility assessment

The solubility of berushedil was examined in various pH buffers (i.e., in buffers of pH 1.2, 3.5, 4.5, 6.8, 7.4, and 10). To prepare the buffers, prepare stock solutions for pH adjustment as follows: prepare a 0.2 M HCl solution by transferring 17.0 mL of 35% HCl to 500 mL of water, mix thoroughly, and dilute the solution to 1000 mL with water; prepare a 2 M acetic acid solution by transferring 116.0 mL of acetic acid to 500 mL of water, mix thoroughly, and dilute to 1000 mL with water; prepare a 0.2 M NaOH solution by transferring 4.01492 g of sodium hydroxide pellets to 250 mL of water, dissolving, and diluting to 500 mL with water; prepare a phosphate buffer stock solution by transferring approximately 2.72 g of potassium dihydrogen phosphate buffer to a 100 mL volumetric flask, dissolving, and diluting to the stated volume with water.

Buffer solution containing sample (berushedil) was prepared as follows.

Sample of pH 1.2 buffer. Transfer 1.53145 g of potassium chloride to a 100 mL volumetric flask, dissolve and dilute to volume with water. Transfer 25.0 mL of this solution to a 100 mL volumetric flask; add 42.5 mL of 0.2 M hydrochloric acid solution and dilute the solution to volume with water. Add 2.0 mL of this pH 1.2 buffer to a 5 mL amber vial containing 100.15 mg of belusudil.

Sample of buffer at pH 3.5 . Transfer 4.11871 g of potassium phthalate to a 100 mL volumetric flask, dissolve and dilute to volume with water. Transfer 25.0 mL of this solution to a 100 mL volumetric flask, add 8.3 mL of 0.2 M hydrochloric acid, and dilute the solution to volume with water. Add 2.0 mL of this buffer to a 5 mL amber vial containing 100.52 mg of belusudil.

pH 4.5 buffer sample. Add 25.0 mL of the pH 3.5 buffer (from the previous section) to a 100 mL volumetric flask. Add an additional 10 mL of 0.2M sodium hydroxide and dilute the solution to volume with water. Add 2.0 mL of this pH 4.5 buffer to a 5 mL amber vial containing 101.53 mg of Belusudil.

pH 6.8 buffer sample. Transfer 25.0 mL of the phosphate buffer stock solution to a 100 mL volumetric flask; add 23 mL of 0.2 M sodium hydroxide and dilute the solution to volume with water. Add 2.0 mL of this pH 6.8 buffer to a 5 mL amber vial containing 100.18 mg of belusudil.

pH 7.4 buffer sample. Transfer 25.0 mL of the phosphate buffer stock solution to a 100 mL volumetric flask; add 41 mL of 0.2 M sodium hydroxide and dilute the solution to volume with water. Add 2.0 mL of this pH 7.4 buffer to a 5 mL amber vial containing 100.52 mg of belusudil.

Sample of pH 10.0 buffer. Add 1.52493 g potassium chloride and 1.21879 g boric acid to a 100 mL volumetric flask, dissolve and dilute to volume with water. Transfer 25.0 mL of this solution to a 100 mL volumetric flask; add 22 mL of 0.2 M sodium hydroxide and dilute the solution to volume with water. Add 2.0 mL of this pH 10.0 buffer to a 5 mL amber vial containing 103.8 mg belusudil.

The pH buffer samples prepared according to the previous paragraph were each vortexed thoroughly and loaded onto a laboratory rotator and rotated constantly at 200 rpm for 24 h. In each case, undissolved sample was observed in the vial after 24 h. The resulting sample solution was filtered through a 0.45 µ needle filter and the filtrate was collected and injected into the HPLC without any further dilution. The pH was observed after equilibration for 24 h at 25°C. The results are shown in Table 4B. Table 4B : Solubility of Belusudil in pH Buffer pH of the buffer used The pH observed after equilibration for 24 h at 25°C Solubility (mg/mL) result 1.2 1.42 NA Almost insoluble 3.5 3.22 NA Almost insoluble 4.5 4.32 NA Almost insoluble 6.8 4.45 NA Almost insoluble 7.4 4.84 NA Almost insoluble 10 5.64 NA Almost insoluble   C. Solubility Assessment - Visual Observation

Solubility evaluations were further performed using each of the solvent systems listed in Table 4C, using 25 mg of berushedil per sample. Table 4C : Solvent Systems Used in Solubility Experiments Solvent system acetone Tetrahydrofuran (THF) Methanol Dichloromethane (DCM) Methyl Ethyl Ketone (MEK) 90:10 DCM:Methanol 80:20 DCM:Methanol 50:50 DCM:Methanol 95:5 acetone:water Acetonitrile (ACN) 90:10 ACN: Water 50:50 ethanol:hexane

All solutions were prepared with 10 wt.% berusudil and then diluted to 1 wt.% berusudil. Under these conditions, berusudil was not soluble in any of the solvent systems listed in Table 4C. All solutions were then heated to 40°C, but no improvement was observed under heating. Therefore, the solvent systems listed in Table 4C are not suitable for dissolving berusudil and polymer carrier matrix materials to enable the use of techniques for preparing amorphous forms. After further experiments, a mixture of berusudil, triethylamine (TEA) and acetone (TEA: molar equivalents of acetone) in a 3:1 ratio provided a clear solution of 5 wt.% berusudil. Therefore, a mixture of TEA and acetone was specified as a solvent system for preparing the solid dispersions used in the following examples. 1.2 Polymer carrier matrix and ratio selection

The following three polymers were selected to prepare six formulations: (1) polyvinylpyrrolidone-vinyl acetate copolymer (PVPVA) (sold under the trade name Kollidon® VA 64); (2) polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG, or "PPPEG" herein) (sold under the trade name Soluplus® and available from BASF); and (3) hydroxypropyl methylcellulose acetate succinate (HPMCAS-M, or HPMC) (available from Shin Etsu Chemical Co.).

The ratios of berusudil to polymer carrier matrix (by weight) were selected for evaluation as follows: Table 5 : Berusudil (KD025): Polymer Ratios for Six Formulations Example No. Polymer carrier Ratio: API to polymer 1.1 HPMC 20:80 1.2 PVPVA 20:80 1.3 PPPEG 20:80 1.4 HPMC 40:60 1.5 PVPVA 40:60 1.6 PPPEG 40:60 1.3 Preparation of spray-dried dispersions

An acetone suspension of berusudil and TEA was first prepared, and then the three polymer carrier matrix materials in Table 5 were added using the berusudil:polymer ratios listed in Table 5. Spray drying of the suspension was then performed as follows.

A Buchi B-290 spray dryer was used in this study. Nitrogen was used as the drying gas. The solution feed rate (mL/min) and the atomization pressure (psi) were adjusted to 17.5 and 26, respectively. The inlet temperature ranged between 84ºC-101ºC, and the outlet temperature was adjusted to range between 49ºC-51ºC. The spray dried formulation was oven dried using a convection plate dryer at 50ºC for 24 h to remove residual solvents. Example 2

As described in this Example 2, all six formulations of Example 1 were characterized for surface morphology, crystallinity, and dissolution. All six formulations produced solid products that were considered suitable for further evaluation based on their morphology and amorphous state. However, after analysis and characterization of surface morphology, crystallinity, and dissolution as described in Sections 2.1-2.3 below, Example 1.2 (20:80 KD025:PVPVA), Example 1.3 (20:80 KD025:PPPEG), and Example 1.6 (40:60 KD025:PPPEG) were selected as preferred candidates for further evaluation as described in Examples 3 to 6. 2.1 Scanning Electron Microscopy

The dispersions of Examples 1.1, 1.2 and 1.3 were selected for visual observation using scanning electron microscopy (SEM). The samples were prepared by dispensing the samples onto adhesive carbon coated sample posts and coating a thin gold conductive layer using a Cressington 108 Auto. The samples were analyzed using a FEI Quanta 200 SEM equipped with an Everhart-Thornley (secondary electron) detector operating in high vacuum mode. The results are shown in Figure 1 herein. Typical morphological features of solid dispersions were observed, consisting of intact and collapsed spheres with smooth surfaces. However, based on this evaluation, the use of a polymer carrier matrix containing PVPVA and PPPEG was advantageous over HPMC because the dispersion containing HPMC showed some filaments, whereas the dispersion containing PVPVA and PPPEG produced a more homogenous matrix of belusudil suspended in the polymer matrix. 2.2 X- ray powder diffraction

The crystalline form of the six dispersions of Example 1 was evaluated using X-ray powder diffraction (XRPD) (using a Rigaku Miniflex 6G X-ray diffraction instrument). The samples were irradiated with monochromatic Cu Kα radiation and analyzed in continuous scanning mode between 5° and 40°. The samples were rotated during analysis to minimize preferential orientation effects. Figure 2 shows the XRPD results for each of Examples 1.1 to 1.6. These results confirm that each of the solid dispersions of Example 1 exists in an amorphous state, as reflected by the lack of crystalline peaks. 2.3 Dissolution Evaluation

The initial step in the dissolution evaluation was to directly determine the solubility of berusudil in biorelevant media (0.1N HCl or FaSSIF), which was found to be >1000 µg/mL and 5 µg/mL, respectively. The dissolution properties of the six dispersions and the crystalline mesylate form of berusudil from Example 1 were tested by non-sink dissolution (results are shown in Figure 3). The dissolution test was used to measure the solubility increment above the solubility of bulk crystalline berusudil in the biorelevant FaSSIF medium after exposure to a low pH environment for 30 minutes. During the test, the samples were shifted from 0.1N HCl [theoretical Cmax = 1000 μgA/mL] to FaSSIF [theoretical Cmax = 500 μgA/mL] by dilution. The drug concentration measured in this test is a combination of free drug in SDI, drug in micelles, and drug in drug-polymer colloids. The purpose of this experiment was to rank and select lead formulations. The overall two-phase dissolution data are summarized in Table 6. Table 6 : Two-phase dissolution data example Formulation ratio API: Carrier/Carrier C _max μgA/mL in SGF C _max after transfer to FaSSIF (μgA/mL) Drug concentration at C ₂₁₀ (μgA/mL) AUC _35-210 FaSSIF (min*μgA/mL) Increased AUC of _formulation compared to AUC _API 1.1 20:80/HPMC 1028 10.2 7.0 1300 1.9 1.2 20:80/PVPVA 899 99.9 20.8 5200 7.4 1.3 20:80/PPPEG 932 369.6 369.6 58000 82.9 1.4 40:60/HPMC 1015 22.6 6.6 1400 2.0 1.5 40:60/PVPVA 990 15.3 11.3 1900 2.7 1.6 40:60/PPPEG 977 79.7 50.4 9800 14.0 API 932 6.1 6.1 700 NA

In general, for most formulations, the Cmax at low pH in the gastric environment was much higher than that at high pH. However, as shown in Table 6, Examples 1.3 and 1.6 (two PPPEG dispersions, 20:80 and 40:60) and Example 1.2 (20:80 KD025:PVPVA dispersion) had Cmax of 369.6, 79.7, and 99.9 μgA/mL, respectively, which were superior to the other three formulations. Furthermore, these formulations had higher drug concentrations at the end of the dissolution process (210 min) compared to crystalline KD025, with reported values of 20.8 μgA/mL (Example 1.2 [20:80 KD025:PVPVA]), 369.6 μgA/mL (Example 1.3 [20:80 KD025: PPPEG]), and 50.4 μgA/mL (Example 1.6 [40:60 KD025: PPPEG]) compared to 6.1 for crystalline KD025. In summary, an increase in the area under the curve (AUC) was observed for Example 1.2 (20:80 KD025:PVPVA), Example 1.3 (20:80 KD025:PPPEG), and Example 1.6 (40:60 KD025:PPPEG) compared to belusudil (KD025). Example 3

Based on the surface morphology, crystallinity and dissolution studies of Example 2, three formulations were selected for additional characterization and in vivo evaluation and experiments, namely: Example 1.2 (20:80 KD025:PVPVA), Example 1.3 (20:80 KD025:PPPEG) and Example 1.6 (40:60 KD025:PPPEG). For ease of reference, these selected formulations are identified below as: Formulation 1 [ F1 ]: 20:80 KD025:PPPEG (Example 1.3); Formulation 2 [ F2] : 20:80 KD025:PVPVA (Example 1.2); and Formulation 3 [ F3 ]: 40:60 KD025:PPPEG (Example 1.6).

For this example, F1, F2 and F3 were prepared according to the processing conditions listed in Table 7 below and then analyzed for their amorphous state, surface morphology, residual solvents, microstructure distribution and thermal evaluation. Table 7 : Methods used to prepare amorphous dispersion formulations F1 , F2 and F3 Formulation (ratio API:vehicle/vehicle) Processing conditions F1 20:80/PPPEG F2 20:80/PVPVA F3 40:60/PPPEG Batch size, solid (g) 71.65 71.65 34.15 Spray solvent Acetone:TEA Acetone:TEA Acetone:TEA Spray solution (wt% total solids) 7.4% 7.4% 3.5% Nozzle Type Dual Fluid - 1.5 mm air cap, 0.7 mm fluid tip Dry gas mode Recycle Cyclone separator type High efficiency cyclone separator Solution flow rate (mL/min) About 17.5 About 17.5 About 17.5 Atomization pressure (psi) 26 26 26 Inlet temperature(°C) 93-97 84-86 86-101 Outlet temperature(°C) 49-50 50-51 49-51 Condenser temperature(°C) (-17) - (-22) (-17) - (-22) (-17) - (-22) Secondary drying temperature/time 50 50 50 Wet SDD yield (%) 83.9 90.3 71.2 Dry SDD yield (%) 81.5 90.3 70.6 Residual solvent (wt%) Acetone: ND TEA: < LOQ Acetone: ND TEA: 1631 Acetone: ND TEA: < LOQ Appearance Yellow powder Off-white powder Bright yellow powder 3.1 Amorphous dispersion

Using the instrumental conditions described in Table 1 above, XRPD results were obtained for the three formulations produced according to the procedure described in Table 7. The results are shown in Figure 5. All solid dispersions were confirmed to be amorphous (Figure 5). SEM images of F1, F2 and F3 were also obtained at 1500x and 5000x magnifications (following the procedure in Example 2.1) and the results are shown in Figure 6. No crystals were observed in any of the three solid dispersions. Formulations F1 and F2 (20:80 dispersions) were both expanded and collapsed spheres, and F3 formed expanded spheres that were mostly fused in clusters. 3.2 Evaluation of Residual Solvent Content

The residual acetone and TEA remaining in the three solid dispersions (F1, F2, and F3) were measured using GC-HS after the secondary drying steps listed in Table 7. The measurements were performed using an HP 6890 Series GC equipped with an Agilent 7697A headspace sampler. The tests were performed using a 30 m x 0.32 mm x 1.8 μ capillary column with a 6% cyanopropylphenyl, 94% dimethylpolysiloxane GC column.

The residual solvents detected in all formulations are reported in Table 8 below. For all three formulations (F1, F2, and F3), no acetone levels were detected. For F1 and F3 (PPPEG formulations), TEA levels were below the limit of quantification (LOQ), and for F2 (PVPVA formulation), TEA levels were 1631 ppm. These levels are below the International Conference on Harmonization (ICH) specified limits for acetone and TEA (5000 ppm). Table 8 : Evaluation of Residual Solvents for F1 , F2 , and F3 Preparation Residual acetone (ppm) Residual TEA(ppm) Measured Tg ºC F1 ND ＜LOQ 73 F2 ND 1631 90 F3 ND ＜LOQ 67 ND = Not Detected 3.3 Fine distribution

The fineness distribution (PSD) of F1, F2 and F3 was measured using the parameters summarized in Table 2 and the laser diffraction method (Mastersizer 3000 with Aero S device). 200 mg of sample was added to a standard venture disperser with a hopper gap of 1.5 mm and then fed into the dispersion system. The feed rate was adjusted (20%-40%) to keep the laser obscuration level between 0.1% and 15%. The sample particles were transported and suspended through the optical cell using compressed air at 1.5 bar. A measurement time of 10 seconds was used and a background measurement was performed with air for 10 seconds. The Dv10, Dv50 and Dv90 diameters were used to characterize the fineness distribution of the powders.

The results of the PSD studies are shown in Table 9 and Figure 7. F1 and F2 (20:80 Berushedil dispersions) showed very similar fineness with Dv50 less than 10 µm and a narrow range of fineness for both formulations (Dv10-Dv90 values of about 3 to about 28 for F1 and about 2 to about 17 for F2). F3 (40% Berushedil dispersion) was found to have a bimodal distribution with larger total particles (Dv10 = 6.54, Dv90 = 187). These results are consistent with the observations made by SEM (Figure 6), which showed that the particles of F3 were fused together in clusters. Table 9 : Fineness data for F1 , F2 and F3 Preparation Dx(10)(µm) Dx(50)(µm) Dx(90)(µm) F1 3.01 8.32 27.9 F2 1.88 4.56 17.2 F3 6.54 32.9 187 3.4 Differential Scanning Calorimetry

DSC of F1, F2 and F3 was performed using the instrumentation listed above in Table 3. The samples were placed in a non-sealed aluminum pan and heated at a constant rate of 2.0°C/min through a temperature range of 0°C to 240°C. The system was purged by a nitrogen flow of 50 mL/min to ensure an inert atmosphere throughout the measurement.

The results of the thermal analysis (mDSC) are shown in Figure 8. All three formulations F1, F2 and F3 have a single Tg (Tg (°C) of 73, 90 and 67 for F1, F2 and F3, respectively), indicating good homogeneity. F3 (40% KD025 dispersion) shows a broad Tg with an unclear onset, indicating that the molecular mobility of the dispersion is high during spray drying. Example 4 4.1 In vitro drug release

In vitro drug release evaluation of F1, F2 and F3 was performed using a USP Type II Distek 2100 dissolution apparatus. A two-stage dissolution test was performed. Pre-weighed SDI powder was briefly suspended in the medium and transferred to a 50 mL volume of pre-warmed (37ºC) 0.1N simulated gastric fluid (SGF) (pH approximately 1.0, without pepsin or bile salts) with a stirring paddle at 100 rpm. After 30 minutes of gastric pH exposure, 2x concentrated (FaSSIF) was added to SGF, resulting in a final pH of 6.8 in a total volume of 100 mL of FaSSIF (100 mM PBS containing 2.24 mg/mL SIF powder (original) (Biorelevant Inc)). At predetermined time points, 1.0 mL samples were taken and analyzed using an appropriate HPLC method.

The results are shown in Table 10 and Figure 9. As reflected in Figure 9, the in vitro performance results indicate that F1, F2 and F3 exhibit improved dissolution properties compared to the crystalline mesylate salt of berusudil. The data indicate that F1, F2 and F3 are viable options for achieving high relative solubility while maintaining acceptable chemical and physical stability. Table 10 : Non-sink dissolution data for F1 , F2 , F3 Preparation Total drug C _maxGB (µgA/mL) Total drug C _maxFaSSIF (µgA/mL) Total drug AUC _35-210FaSSIF (min*µgA/mL) Total drug C ₂₁₀ (µgA/mL) F1 940 356.6 57800 314.3 F2 929 100.4 9400 28.1 F3 894 79.2 6000 29 Methanesulfonate/API 932 6.1 700 6.1 4.2 Measurement and evaluation

Reverse phase high performance liquid chromatography (RPHPLC) (Agilent 1200 series LC (1220 and 1260)) was used to determine and analyze impurities in F1, F2, and F3 during the treatment period compared to berusudil mesylate. The HPLC was equipped with a diode array detector. A gradient method with a Zorbax SB-CN column was used. The mobile phase consisted of (A) 50 mM potassium phosphate buffer and (B) acetonitrile, pumped at a flow rate of 1.4 mL/min at ambient temperature, with detection at 250 nm. The mobile phase gradient was maintained as follows (min, %B): (0, 20.0); (20.0, 30.0); (30.0, 60); (40.0, 60).

The results of this assay are reported in Table 11 below and shown in Figure 10. The impurity spectrum was similar to that of the mesylate salt form of berusudil. No degradation was observed during processing. Table 11 : Assay Evaluation and Impurity Spectrum Preparation RRT KD025 Methanesulfonate F1 20:80(PPPEG) F2 20:80 (PVPVA) F3 40:60(PPPEG) 0.61 0.62 0.66 0.82 1.09 1.17 1.37 1.44 0.10% detected, < 0.05% detected, < 0.05% 0.32% 0.34% 0.09% detected, < 0.05% detected, < 0.05% detected, < 0.05% 0.31% 0.29% 0.10% detected, < 0.05% 0.06% 0.28% 0.27% detected, < 0.05% 0.10% detected, < 0.05% detected, < 0.05% 0.08% 0.31% 0.29% 0.07% Total impurities determination (wt%) 0.75% -- 0.69% 19.9% 0.72% 19.1% 0.85% 40.0% Example 5 5.1 Stability Assessment

To evaluate the physical and chemical stability of formulations F1, F2, and F3, the three formulations were aged for up to 8 weeks at 25°C/60% relative humidity (RH) in open packaging and at 40°C/75% RH in open and closed packaging. The physical and chemical stability of F1, F2, and F3 were evaluated by appearance and XRPD. The collected XRPD patterns are shown in Figures 11A (F1), 11B (F2), and 11C (F3).

Overall, even with the observed physical changes shown in Figure 11B, F2 (20:80 KD025:PVPVA) formed a hard solid over eight weeks under all conditions and formed a dispersion that remained amorphous under all conditions. As shown in Figure 11A, F1 (20:80 PPPEG dispersion) remained amorphous at 25ºC/60% RH, open conditions and at 40ºC/75% RH, closed conditions. At 40ºC/75% RH, open conditions, F1 formed a hard mass. As shown in Figure 11C, F3 remained amorphous at 40ºC/75% RH, closed conditions and formed crystals at 25ºC/60% RH, open conditions and 40ºC/75% RH, open conditions. This study provides information on the time and storage conditions required to pre-manufacture the final dosage form (i.e. tablet compression, suspension, etc.). Example 6

As described in this Example 6, the pharmacokinetics (PK) of F1, F2, and F3 were evaluated in male beagle dogs after oral (PO) administration. 6.1 Suspension Formulations for In Vivo Administration

For the PK dog model, suspension formulations for administration of F1, F2, and F3 were developed. Suspensions of 25 mgA/mL were prepared with each of F1, F2, and F3 in 0.5 wt.% Methocel A4M and evaluated for visual appearance, syringeability, and crystallinity by PLM. Methocel A4M was added to purified, preheated water (65ºC ± 5ºC) until it was fully dispersed in the water. The mixture was then cooled to room temperature with continuous mixing. Based on the desired dosage, the amount of solid dispersion (F1, F2, F3) powder was slowly added. A wet paste was initially formed and became a suspension as mixing continued. The suspension was evaluated using PLM (5X magnification) at T = 0, T = 1 h, and T = 2.5 h; images are shown in Figure 12.

The F1 dispersion (20:80 KD025:PPPEG) was homogenous, free of clumps or crystals, and remained unchanged for 2.5 hours. The F1 formulation remained stable as a suspension for at least 4.5 h at room temperature with stirring. Both F2 (20:80 KD025:PVPVA) and F3 (40:60 KD025:PPPEG) showed clumps by PLM at T = 0 that grew over time; however, no crystals were observed and the suspension remained injectable (injection via a 20-gauge feeding tube).

The suspension formulation was successfully developed and selected for ease of administration in the dog PK model; however, solid dosage forms may also be considered. Solid dosage forms (such as tablets) are relatively easy to manufacture, package, and transport, are more stable than liquids, and can be formulated with coatings and shaped to facilitate swallowing. Therefore, one skilled in the art may consider using solid dosage forms as an alternative to the liquid suspension used in this dog study. 6.2 Canine In Vivo PK Evaluation

Male beagle dogs were selected for the bioperformance evaluation of F1, F2 and F3. The studies were conducted according to the protocol approved by the Pharmaron Institutional Animal Care and Use Committee. Twenty male beagle dogs aged 1-1.5 years were assigned dosage and feeding conditions, wherein their body weight was maintained between 11 and 12 kg during the course of the study. All animals were housed in an environment with a 12-hour light/dark cycle. F1, F2 and F3 were tested under fasting and fed conditions and compared with belusudil free base powder and immediate release (IR) tablets containing the crystalline mesylate salt of belusudil used as a control. For the fasting group, the dogs were fasted overnight and the drugs were administered to the dogs in the morning at the same time in the fasting state. Food was returned to the dogs after plasma collection 4 h post-dose. For the fed group, dogs were fasted overnight and fed 1 h prior to dosing. All dogs had access to water throughout the study. All samples were dosed orally (PO); tablets were administered as is. Following tablet administration, 5 mL of vehicle was used to assist the dogs in swallowing the tablet. Formulations 1, 2, 3, and free base samples were dosed via oral vigor using a suspension formulation of 0.5 wt% Methocel A4M. Following suspension administration, dogs were given 5 mL of vehicle to ensure that all suspension was flushed into the stomach. 6.3 Study Design

A total of twenty beagles were dosed via the dosing regimen summarized in Table 12. Four dogs were assigned to each group AJ. Groups A/F, B/G, C/H, D/I, and E/J shared the same animals; animals in groups AE were dosed first. After a seven-day washout period, animals in groups FJ were dosed. Dosing in each case was via oral administration. Four males in each group were dosed. Table 12 : Beagle PK Dosing Information for F1 , F2 , and F3 and API Control Group handle Dosage (approximately 1000 mg KD025 equivalent) Food Control A KD025 tablets 1000 mg (5 x 200 mg tablets) fasting B KD025 - Free Alkali Powder 1000 mg fasting C F1 (20:80 KD025/PPPEG) 5025 mg of F1 SDD fasting D F2 (20:80 KD025/PVPVA) 5235 mg of F2 SDD fasting E F3 (40:60 KD025/PPPEG) 2500 mg of F3 SDD fasting F KD025 - Tablets 1000 mg (5 x 200 mg tablets) Eating G KD025 - Free Alkali Powder 1000 mg Eating H F1 (20:80 KD025/PPPEG) 5025 mg of F1 SDD Eating I F2 (20:80 KD025/PVPVA) 5235 mg of F2 SDD Eating J F3 (40:60 KD025/PPPEG) 2500 mg of F3 SDD Eating 6.4 Sample collection

Blood samples were collected from each animal at predetermined time points before dosing and at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 30, and 36 hours after dosing. Blood samples (1 mL) were collected from each animal via jugular vein. These blood samples were placed in tubes containing dipotassium ethylenediaminetetraacetic acid and then centrifuged at 2000 g for 10 minutes at 2ºC to 8ºC to obtain plasma. 6.5 LC/MS Conditions

KD025 in dog plasma samples was evaluated using an LC-MS/MS system consisting of two Shimadzu LC-30AD pumps, a DGU-20A5R© degasser, a Rack changer II, and an AB Sciex Triple Quads 5500 LC/MS/MS mass spectrometer. Analytical separations were performed on an Agilent ZORBAX XDB-Phenyl 5 µm (50 × 2.1 mm) column at room temperature. The mobile phase consisted of: A: 5% acetonitrile (0.1% formic acid) in water; B: 95% acetonitrile (0.1% formic acid) in water. The flow rate was 0.6 mL/min. The injection volume was 2 μL, and the lower limit of quantification (LLOQ) was 10 ng/mL. 6.6 Data Collection and Statistical Analysis

Data acquisition was performed using Sciex Analyst 1.6.3 software (AB Sciex, Foster City, CA). Pharmacokinetic parameters such as area under the curve (AUC0-36h), maximum plasma concentration (Cmax), and time to reach Cmax (Tmax) were calculated by noncompartmental analysis (Phoenix™ WinNonlin™ 6.1). AUC calculations were performed using the linear trapezoidal algorithm. Data statistics and plasma profile analysis (profile) were performed using Excel 2010 software.

Table 13 summarizes the mean pharmacokinetic parameters following administration of the test formulations to male beagles (n = 4) at a dose of 40 mg/kg. Figure 13 plots the plasma concentration of KD025 versus time over the twenty-four data collection period for the following dog groups: Group A = fasted, tablet formulation; Group D = fasted, F2 formulation (20:80 KD025:PVPVA); Group F = fed, tablet formulation; and Group I = fed, F2 (20:80 KD025:PVPVA). Table 13 : Mean Plasma Pharmacokinetic Parameters Following Oral Administration at 1000 mg/ Dog in Beagles AUC (0-36h) [h*ng/mL] Cmax [ng/mL] T _max [h] Relative bioavailability Food Effect condition Preparation average value Standard Deviation %CV average value Standard Deviation %CV _Tmax AUC ratio of formulation to tablet AUC ratio between fed and fasted states fasting Tablets 32276 31304 97.0 2842 2358 83.0 8.0 ± 4.9 N/A N/A fasting Free base 5699 5347 93.8 683 642 94.0 8.0 ± 6.7 0.18 N/A fasting F1 10794 5537 51.3 1883 1032 54.8 5.50 ± 1.00 0.33 N/A fasting F2 19548 6465 33.1 2915 843 28.9 6.00 ± 0.00 0.61 N/A fasting F3 5841 5418 92.8 809 556 68.7 5.50 ± 2.52 0.18 N/A Eating Tablets 43340 13176 30.4 4773 1571 32.9 6.50 ± 1.00 N/A 1.34 Eating Free base 18805 12064 64.2 2103 592 28.2 8.0 ± 6.7 0.43 3.30 Eating F1 29331 5373 18.3 2883 237 8.2 5.00 ±1.15 0.68 2.72 Eating F2 36342 12177 33.5 3100 231 7.5 10.0 ± 6.3 0.84 1.86 Eating F3 27908 4021 14.4 2465 522 21.2 4.50 ± 1.00 0.64 4.78

When solid dispersion formulations F1, F2, and F3 were compared to the tablets, it was surprising to find that F2 (20:80 KD025:PVPVA) performed better than the other (PPPEG) formulations. For F2, the mean Cmax was found to be similar for the fasted and fed groups, i.e., 2915.0 ng/mL for the fasted group and 3100 ng/mL for the fed group. In contrast, the mean Cmax for the reference tablets in the fasted state was significantly different from the fed state, i.e., 2842 ng/mL for the fasted group and 4773 ng/mL for the fed group. This improved control of Cmax by amorphous berushedil may be advantageous from a pharmacokinetic perspective.

Additionally, the F2 (KD025:PVPVA) test set exhibited lower variability compared to the reference tablet, as characterized by %CV (100x standard deviation/mean). Under fasting conditions, the %CV for the F2 formulation was found to be 28.9, while the %CV for the tablet was 83.0. When compared to fasting conditions, the percentage variability for the F2 formulation was even lower at 7.5 %CV, and the reference tablet had a % variability of 32.9 %CV. In summary, the solid dispersion formulation F2 (20:80 KD025:PVPVA) reduced the inter-subject variability and minimized the food effect in terms of Cmax. The AUC observed with the F2 formulation was slightly lower than the actual reference tablet; however, the variability was better controlled. Under fasting conditions, using %CV, AUC variability was improved for F2 (20:80 KD025:PVPVA) (33.1) compared to the tablet (97.0). In summary, the use of the solid dispersion formulation F2 was surprisingly effective in achieving minimal variability, and the food effect of F2 was lower than that of the free base tablet. During the study in this example, no adverse reactions to the animals were observed during or after the study.

without

FIG. 1 shows scanning electron microscopy (SEM) images of the solid dispersion of Example 1 (specifically, Nos. 1.1, 1.2, and 1.3 in Table 5) captured at 1500x and 5000x magnifications.

FIG2 shows the XRPD results of six spray dried formulations prepared as in Example 1.

FIG3 shows the two-phase dissolution data of six spray-dried berusudil formulations of Example 1 compared to the crystalline mesylate form of berusudil.

FIG. 4 is an expanded view of a portion of the data of FIG. 3 .

FIG5 shows the XRPD results of three solid dispersions of Example 3.

FIG. 6 shows SEM images of three solid dispersions of Example 3 (Formulations F1, F2, and F3) captured at 1500x and 5000x magnification.

FIG7 shows the microscopic data of three solid dispersions of Example 3 (Formulations F1, F2, and F3).

FIG8 shows the modulated differential scanning calorimetry (mDSC) evaluation of the Tg for homogeneity determination after one cooling cycle (upper profile) and one heating cycle (lower profile) of the three solid dispersions of Example 3 (Formulations F1, F2, and F3).

FIG. 9 shows the non-sink dissolution data of the three solid dispersions of Example 3 (Formulations F1, F2 and F3) compared to the crystalline mesylate form of berusudil.

FIG. 10 shows the assay and impurity data of the three solid dispersions (Formulations F1, F2 and F3) of Example 3 compared to the crystalline mesylate form of berusudil and the dilution described in Example 4.

11A-11C show the XRPD diffraction patterns of the three solid dispersions of Example 3 after an 8-week stability study as described in Example 5 ( FIG. 11A : F1 [20:80 KD025:PPPEG]; FIG. 11B : F2 [20:80 KD025:PVPVA]; and FIG. 11C : F3 [40:60 KD025:PPPEG]).

FIG. 12 shows polarized light microscopy images (5X magnification) of solid dispersions of Example 3 (F1, F2, and F3) in the form of a 25 mgA/mL suspension in 0.5 wt.% Methocel A4M (aqueous).

Figure 13 shows the in vivo plasma concentration (ng/mL) of berusudil versus time curves after administration of berusudil as described in Example 6: (a) fasting, tablet formulation; (b) fasting, F2 (20:80 KD025:PVPVA); (c) fed, tablet formulation; and (d) fed, F2 (20:80 KD025:PVPVA).

without

Claims (23)

A solid dispersion comprising substantially amorphous 2-{3-[4-( 1H -indazol-5-ylamino)-2-quinazolinyl]phenoxy} -N- (propan-2-yl)acetamide or a pharmaceutically acceptable salt thereof (berushedil) and one or more carrier materials. A solid dispersion as described in claim 1, wherein the one or more carrier materials are selected from polymers. A solid dispersion as described in claim 1, wherein the one or more carrier materials are selected from polyvinyl pyrrolidone-vinyl acetate copolymer, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer and hydroxypropyl methylcellulose acetate succinate. The solid dispersion of claim 1, 2 or 3, wherein the ratio of berusudil to the one or more carrier materials is about 10:90 to 90:10 by weight; or about 20:80 to 80:20 by weight; or about 25:75 to 75:25 by weight; or about 40:60 to 60:40 by weight. The solid dispersion as described in any one of claims 1 to 4, wherein the carrier material is a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer. A solid dispersion as described in any one of claims 1 to 4, wherein the carrier material is vinylpyrrolidone-vinyl acetate copolymer. A solid dispersion as described in any one of claims 1 to 3 or claim 5 or 6, wherein the ratio of berusudil to the carrier material is about 20:80 by weight. The solid dispersion of any one of claims 1 to 7, wherein at least about 95% of berusudil in the dispersion is in amorphous form. The solid dispersion of any one of claims 1 to 7, wherein at least about 99% of berusudil in the dispersion is in amorphous form. The solid dispersion as described in claim 8 or 9 is characterized in that the solid dispersion contains solid particles with a fine diameter of less than 10 µm. A pharmaceutical formulation comprising a therapeutically effective amount of the solid dispersion as described in any one of claims 1 to 10. The pharmaceutical formulation of claim 11, wherein the pharmaceutical formulation is a tablet or a capsule. A method for treating a disease or disorder regulated by ROCK, the method comprising administering to a subject in need of treatment the solid dispersion of any one of claims 1 to 10 or the pharmaceutical formulation of claim 11 or 12. The method of claim 13, wherein the disease or disorder is graft-versus-host disease (GVHD). The method of claim 14, wherein the GVHD is chronic or acute. The method of claim 13, wherein the disease or disorder is an autoimmune or fibrotic disorder. The method of claim 16, wherein the autoimmune or fibrotic disorder is pulmonary fibrosis; idiopathic pulmonary fibrosis; cystic fibrosis; radiation-induced fibrosis; arterial, cardiac, endomyocardial, renal or hepatic fibrosis; moderate to severe psoriasis; rheumatoid arthritis; multiple sclerosis; systemic lupus erythematosus (SLE); Crohn's disease; dermatitis; or eczema. The method of claim 13, wherein the disease or disorder is bronchiolitis obliterans syndrome (BOS). The method of claim 18, wherein the BOS is BOS after lung transplantation or after allogeneic hematopoietic stem cell transplantation (allogeneic HSCT). A method for preparing an amorphous form of berusudil, the method comprising dissolving berusudil and one or more carrier materials in a suitable solvent to form a solution. A method as described in claim 20, wherein the suitable solvent comprises a mixture of triethylamine and acetone. The method of claim 20 or 21, further comprising spray drying the solution of berusudil and one or more carrier materials to remove the suitable solvent. A method as described in any one of claims 20 to 22, wherein the carrier material is selected from vinyl pyrrolidone-vinyl acetate copolymer and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer.