Crystal Forms of ALK2 Inhibitors

Abstract

The present invention relates to a crystalline form of an ALK2 inhibitor, a method for preparing the same, and a related pharmaceutical preparation. The present invention also relates to the pharmaceutical use of the crystalline form.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of provisional application No. 62 / 751,255, filed on October 26, 2018, under 35 USC 119, the entire content of which is incorporated herein by reference. Background of the invention

[0003] The BMP signaling family is various subsets of the TGF - β superfamily. More than twenty known BMP ligands are recognized by three different type II receptors (BMPRII, ActRIIa, and ActRIIb) and at least four type I receptors (ALK1, ALK2, ALK3, and ALK6). Dimeric ligands facilitate the assembly of receptor heteromers, allowing constitutively active type II receptor serine / threonine kinases to phosphorylate type I receptor serine / threonine kinases. The activated type I receptor phosphorylates BMP - responsive (BR - ) SMAD effectors (SMAD1, SMAD5, and SMAD8) to facilitate nuclear translocation in a complex with SMAD4 (a co - SMAD that also facilitates TGF signaling). In addition, BMP signals can activate intracellular effectors (such as MAPKp38) in a SMAD - independent manner. Soluble BMP inhibitors (such as noggin, chordin, gremlin, and follistatin) limit BMP signaling by ligand chelation.

[0004] The role of BMP signaling in regulating the expression of hepcidin, a peptide hormone and central regulator of systemic iron balance, has also been proposed. Hepcidin binds to ferroportin, the only iron exporter in vertebrates, and promotes the degradation of ferroportin. The loss of ferroportin activity prevents the transfer of iron from intracellular stores in enterocytes, macrophages, and hepatocytes to the bloodstream. The link between BMP signaling and iron metabolism represents a potential therapeutic target.

[0005] Given the vast structural diversity of the BMP and TGF-β superfamilies at the levels of ligands (currently >25 different ligands) and receptors (four type I receptors and three type II receptors that recognize BMPs), traditional methods for inhibiting BMP signaling via soluble receptors, endogenous inhibitors, or neutralizing antibodies are impractical or ineffective. Endogenous inhibitors such as noggin and follistatin have limited specificity for ligand subclasses. Individual receptors have limited affinity for ligands, whereas receptor heterotetramers exhibit higher specificity for specific ligands. Neutralizing antibodies specific for a particular ligand or receptor have been previously described and are also limited by the structural diversity of this signaling system. Accordingly, there is a need in the art for agents (such as those listed above) that specifically antagonize the BMP signaling pathway and can be used to manipulate these pathways in therapeutic or experimental applications. SUMMARY OF THE INVENTION

[0007] One aspect of the invention relates to a crystalline compound having the structure of formula (I),

[0008] (I) and its salts.

[0009] Another aspect of the invention relates to a method for preparing the crystalline compound of formula (I).

[0010] In certain embodiments, the invention provides a pharmaceutical formulation suitable for a subject, comprising the crystalline compound of formula (I) and one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical formulation can be used to treat or prevent the conditions or diseases described herein.

[0011] DETAILED DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The XRPD pattern of form A of the monosuccinate salt of the compound of formula (I) is shown.

[0013] Figure 2 The XRPD pattern of form B of the monosuccinate salt of the compound of formula (I) is shown.

[0014] Figure 3 The XRPD pattern of the free base of the compound of formula (I) is shown.

[0015] Figure 4 The differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) patterns of the free base of the compound of formula (I) are shown.

[0016] Figure 5 The dynamic vapor sorption profile (DVS) of the free base of the compound of formula (I) is shown.

[0017] Figure 6Shows the XRPD patterns of the reference sample of the monosuccinate form A of the compound of formula (I) and form A prepared in THF.

[0018] Figure 7 Shows the 1 1H-NMR spectrum of the monosuccinate form A of the compound of formula (I).

[0019] Figure 8 Shows the XRPD patterns of the reference sample of the monosuccinate form A of the compound of formula (I) and form A prepared in EtOH.

[0020] Figure 9 Shows the DVS of the monosuccinate form A of the compound of formula (I) prepared in EtOH.

[0021] Figure 10 Shows the overlay of the XRPD spectra of the monosuccinate form A of the compound of formula (I) before and after DVS analysis, and the monosuccinate form C of the compound of formula (I).

[0022] Figure 11 Shows the overlay of the XRPD spectra of the monosuccinate form A of the compound of formula (I), the reference sample of the monosuccinate form A of the compound of formula (I), and the monosuccinate form B of the compound of formula (I) prepared in EtOH.

[0023] Figure 12 Shows the XRPD spectra of the monosuccinate form A of the compound of formula (I) and succinic acid.

[0024] Figure 13 Shows the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) graphs of the monosuccinate form A of the compound of formula (I).

[0025] Figure 14 Shows the XRPD spectra of the monosuccinate form A and form B of the compound of formula (I).

[0026] Figure 15 Shows the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) graphs of the monosuccinate form B of the compound of formula (I).

[0027] Figure 16 Shows the DVS of the monosuccinate form B of the compound of formula (I).

[0028] Figure 17 Shows the overlay of the XRPD spectra of the monosuccinate form B of the compound of formula (I) before and after DVS analysis.

[0029] Figure 18 Shows the 1 1H-NMR spectrum of the monosuccinate form B of the compound of formula (I).

[0030] Figure 19 Shows the superposition of the XRPD spectra of the mono-succinate forms A, B, and C of the compound of formula (I).

[0031] Figure 20 Shows the differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) graphs of the mono-succinate form C of the compound of formula (I).

[0032] Figure 21 Shows the 1 1H-NMR spectrum of the mono-succinate form C of the compound of formula (I).

[0033] Figure 22 Shows the superposition of the XRPD spectra of the mono-succinate forms A, B, and D of the compound of formula (I).

[0034] Figure 23 Shows the differential scanning calorimetry (DSC) of a mixture of the mono-succinate forms A and D of the compound of formula (I).

[0035] Figure 24 Shows the 1 1H-NMR spectrum of the mono-succinate form D of the compound of formula (I).

[0036] Figure 25 Shows the serum iron concentration caused by administration of form A.

[0037] Figure 26 Shows the percentage of transferrin saturation caused by administration of form A. DETAILED DESCRIPTION OF THE INVENTION

[0039] In certain embodiments, the present invention provides a crystalline compound having the structure of formula (I),

[0040] (I) and its salts.

[0041] In certain embodiments, the crystalline compound of formula (I) is not solvated (e.g., the crystal lattice does not contain solvent molecules). In certain such embodiments, the crystalline compound of formula (I) is anhydrous or substantially anhydrous.

[0042] In certain embodiments, the compound of formula (I) is in the form of a salt formed with an anion selected from chloride, bromide, succinate, cinchophenate, citrate, malate, semi-malate, tartrate, malonate, mesylate, phosphate, tosylate, sulfate, and bisulfate. In a preferred embodiment, the compound of formula (I) is in the form of a succinate, such as mono-succinate.

[0043] Any crystalline compound described herein can be used in the preparation of a medicament for treating any disease or condition disclosed herein.

[0044] The monosuccinate salt of the compound of formula (I) exists in at least the forms of "Form A", "Form B", "Form C" and "Form D", as detailed below. These different forms are understood herein as "polymorphs".

[0045] Polymorphs of a crystalline compound can be characterized by powder X-ray diffraction (XRPD). θ represents the diffraction angle, in degrees. In certain embodiments, the diffractometer used in XRPD measures the diffraction angle as twice the diffraction angle θ. Thus, in certain embodiments, the diffraction patterns described herein refer to the X-ray intensity measured relative to the angle 2θ.

[0046] In certain embodiments, the first anhydrous crystalline form of the monosuccinate salt of the compound of formula (I) has 2θ values of about 7.05 ± 0.2, 15.16 ± 0.2, 21.05 ± 0.2, 21.26 ± 0.2, and 24.47 ± 0.2. In further embodiments, the anhydrous crystal of the monosuccinate salt of the compound of formula (I) has 2θ values of about 3.58 ± 0.2, 7.05 ± 0.2, 13.8 ± 0.2, 14.16 ± 0.2, 15.16 ± 0.2, 16.18 ± 0.2, 16.80 ± 0.2, 17.15 ± 0.2, 17.69 ± 0.2, 18.29 ± 0.2, 18.84 ± 0.2, 20.29 ± 0.2, 21.05 ± 0.2, 21.26 ± 0.2, 22.68 ± 0.2, 23.84 ± 0.2, 24.47 ± 0.2, 24.84 ± 0.2, and 28.47 ± 0.2. In still further embodiments, the anhydrous crystal of the monosuccinate salt of the compound of formula (I) has 2θ values of about 3.58 ± 0.2, 7.05 ± 0.2, 10.59 ± 0.2, 10.75 ± 0.2, 13.80 ± 0.2, 14.16 ± 0.2, 15.16 ± 0.2, 15.68 ± 0.2, 16.18 ± 0.2, 16.80 ± 0.2, 17.15 ± 0.2, 17.69 ± 0.2, 17.97 ± 0.2, 18.29 ± 0.2, 18.59 ± 0.2, 18.84 ± 0.2, 19.27 ± 0.2, 20.29 ± 0.2, 21.05 ± 0.2, 21.26 ± 0.2, 21.56 ± 0.2, 21.78 ± 0.2, 22.68 ± 0.2, 23.84 ± 0.2, 24.47 ± 0.2, 24.84 ± 0.2, 25.15 ± 0.2, 26.10 ± 0.2, 27.12 ± 0.2, 27.78 ± 0.2, 28.47 ± 0.2, and 29.06 ± 0.2.

[0047] In certain embodiments, the anhydrous crystalline form of the monosuccinate salt of the compound of formula (I) has an XRPD pattern substantially as Figure 1 shown, labeled Form A.

[0048] In certain embodiments, the second anhydrous crystalline form of the monosuccinate salt of the compound of formula (I) has 2θ values of about 9.79 ± 0.2, 13.05 ± 0.2, 22.91 ± 0.2, 23.60 ± 0.2, and 26.25 ± 0.2. In further embodiments, the anhydrous crystal of the monosuccinate salt of the compound of formula (I) has 2θ values of about 3.25 ± 0.2, 9.79 ± 0.2, 13.05 ± 0.2, 16.75 ± 0.2, 19.50 ± 0.2, 22.91 ± 0.2, 23.60 ± 0.2, and 26.25 ± 0.2. In additional embodiments, the anhydrous crystal of the monosuccinate salt of the compound of formula (I) has 2θ values of about 3.25 ± 0.2, 9.79 ± 0.2, 13.05 ± 0.2, 13.61 ± 0.2, 14.39 ± 0.2, 16.75 ± 0.2, 18.50 ± 0.2, 19.50 ± 0.2, 22.91 ± 0.2, 23.60 ± 0.2, and 26.25 ± 0.2.

[0049] In certain embodiments, the anhydrous crystalline form of the monosuccinate salt of the compound of formula (I) has an XRPD pattern substantially as Figure 2 shown, labeled Form B.

[0050] In certain embodiments, the third anhydrous crystalline form of the free base of the compound of formula (I) has 2θ values of about 6.00 ± 0.2, 12.00 ± 0.2, 16.14 ± 0.2, 17.72 ± 0.2, 18.00 ± 0.2, 18.64 ± 0.2, and 23.50 ± 0.2. In certain embodiments, the anhydrous crystalline form of the monosuccinate salt of the compound of formula (I) has an XRPD pattern substantially as Figure 3 shown, labeled free base.

[0051] In certain embodiments, the present invention relates to a pharmaceutical composition comprising a crystalline compound of the monosuccinate salt of formula (I) and one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical composition is selected from tablets, capsules, and suspensions.

[0052] As used herein, the term "substantially pure" refers to a crystalline polymorph having a purity greater than 90%, meaning containing less than 10% of any other compound, including the corresponding amorphous compound or alternative polymorphs of the crystalline salt. Preferably, the crystalline polymorph is greater than 95% pure, or even greater than 98% pure.

[0053] Method for Preparing the Crystalline Form of the Compound of Formula (I)

[0054] In certain embodiments, the present invention relates to a method for preparing a crystalline compound having the structure of formula (I), comprising

[0055] a) providing a compound of formula (I);

[0056] b) adding an acid to form a mixture; and

[0057] c) crystallizing the compound of formula (I) from the mixture comprising the compound of formula (I).

[0058] In certain embodiments, the crystalline compound prepared by the method of the present invention is anhydrous. In other embodiments, the crystalline compound prepared by the method of the present invention is a hydrate.

[0059] In certain embodiments, the compound of formula (I) is present in at least one solvent. In certain embodiments, the acid is present in at least one solvent.

[0060] In certain embodiments, the compound of formula (I) and at least one solvent form a solution. In certain embodiments, the compound of formula (I) and at least one solvent form a slurry or suspension. In certain embodiments, the acid and at least one solvent form a solution. In certain embodiments, the acid and at least one solvent form a slurry or suspension.

[0061] In certain embodiments, the acid is selected from HCl, HBr, succinic acid, 1-OH-2-naphthoic acid, citric acid, malic acid, tartaric acid, malonic acid, methanesulfonic acid, phosphoric acid, toluenesulfonic acid, and sulfuric acid. In a preferred embodiment, the acid is succinic acid.

[0062] In certain embodiments, the solvent comprises acetone, acetonitrile, N,N-dimethylacetamide (DMA), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dioxane, ethanol, ethyl acetate, heptane, hexane, isopropyl acetate, methanol, methyl ethyl ketone, N-methyl-2-pyrrolidone (NMP), tetrahydrofuran (THF), toluene, 2-propanol, 1-butanol, water, or any combination thereof. In certain preferred embodiments, for example to obtain Form A, the solvent is THF, ethanol, or a mixture thereof. In other preferred embodiments, for example to obtain Form B, the solvent is ethanol.

[0063] In certain embodiments, the acid is present in at least one solvent and added to the compound of formula (I) to form a slurry, and the step of crystallizing the compound from the slurry comprises precipitating the compound from the slurry.

[0064] In certain embodiments, the compound of formula (I), the acid, and at least one solvent form a solution, and the step of crystallizing the compound from the mixture comprises bringing the solution to a supersaturated state to cause the compound of formula (I) to precipitate out of the solution.

[0065] In certain embodiments, supersaturating a mixture comprising a compound of formula (I) includes slowly adding an anti-solvent, such as heptane, hexane, ethanol, or another polar or non-polar liquid miscible with the organic solvent, allowing the solution to cool (with or without a seeding solution), reducing the volume of the solution, or any combination thereof. In certain embodiments, supersaturating a mixture comprising a compound of formula (I) includes adding an anti-solvent, cooling the solution to ambient temperature or lower, and reducing the volume of the solution, such as by evaporating the solvent from the solution. In certain embodiments, cooling the solution can be passive (e.g., allowing the solution to stand at ambient temperature) or active (e.g., cooling the solution in an ice bath or freezer).

[0066] In certain embodiments, the preparation method further includes separating the crystals, such as by filtering the crystals, by decanting the fluid from the crystals, or by any other suitable separation technique. In a further embodiment, the preparation method further includes washing the crystals.

[0067] In certain embodiments, the mixture comprising the compound of formula (I) is a slurry, and the step of crystallizing the compound from the mixture includes precipitating the compound from the slurry. In some embodiments, the crystallized compound is separated by centrifugation.

[0068] In certain embodiments, the preparation method further includes inducing crystallization. The method may also include drying the crystals, such as under reduced pressure. In certain embodiments, inducing precipitation or crystallization includes secondary nucleation, where nucleation occurs in the presence of a seed crystal or in interaction with the environment (crystallizer wall, stirrer impeller, ultrasonic instrument, etc.).

[0069] In certain embodiments, washing the crystals includes washing with a liquid selected from an anti-solvent, acetonitrile, ethanol, heptane, hexane, methanol, tetrahydrofuran, toluene, water, or a combination thereof. As used herein, "anti-solvent" refers to a solvent in which the salt crystals are insoluble, sparingly soluble, or partially soluble. In fact, adding an anti-solvent to a solution in which salt crystals are dissolved reduces the solubility of the salt crystals in the solution, thus promoting the precipitation of the salt. In certain embodiments, the crystals are washed with a combination of an anti-solvent and an organic solvent. In certain embodiments, the anti-solvent is water, while in other embodiments it is an alkane solvent, such as hexane or pentane, or an aromatic solvent, such as benzene, toluene, or xylene. In certain embodiments, the anti-solvent is methanol.

[0070] In certain embodiments, washing the crystals includes washing the crystallized compound of formula (I) with the above solvents or a mixture of one or more solvents. In certain embodiments, the solvent or solvent mixture is cooled before washing.

[0071] Use of the Crystalline Form of the Compound of Formula (I)

[0072] In various embodiments, the present invention provides compounds that inhibit the BMP signaling pathway, and methods of treating or preventing a disease or condition in a subject that would benefit from inhibition of BMP signaling. In various embodiments, the compounds of the present invention include compounds of formula (I) as disclosed herein and salts thereof (including pharmaceutically acceptable salts).

[0073] All references cited herein are incorporated by reference as if fully set forth herein. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Definitions of common terms in molecular biology can be found in: Benjamin Lewin, Genes V , published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology , published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference , published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). The following provides general guidance to those skilled in the art on many of the terms used in this application: Allen et al., Remington: The Science and Practice of Pharmacy 22nd Edition, Pharmaceutical Press (September 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology , CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 3rd Edition Revised, J. Wiley & Sons (New York, NY 2006); Smith, March’s Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th Edition, J. Wiley & Sons (New York, NY 2013); Singleton, Dictionary of DNA and Genome Technology 3rd Edition, Wiley-Blackwell (November 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual4th Edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012). For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2nd Edition, ColdSpring Harbor Press (Cold Spring Harbor NY, 2013); Köhler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion , Eur. J. Immunol. July 1976, 6(7):511-9; Queen and Selick, Humanized immunoglobulins , U.S. Patent No. 5,585,089 (December 1996); and Riechmann et al., Reshaping human antibodies for therapy , Nature March 24, 1988, 332(6162):323-7.

[0074] Those skilled in the art will recognize many methods or materials similar or equivalent to those described herein that can be used to practice the present invention. In light of the following detailed description, taken in conjunction with the accompanying drawings that illustrate, by way of example, various features of embodiments of the present invention, other features and advantages of the present invention will become apparent. In fact, the present invention is in no way limited to the methods and materials described. For convenience, certain terms employed in the specification, examples, and appended claims herein are collected here.

[0075] Unless otherwise noted or implicit from the context, the following terms and phrases have the meanings provided below. Unless otherwise expressly noted or obvious from the context, the following terms and phrases do not exclude meanings that the term or phrase may have acquired in the art to which it pertains. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that the present invention is not limited to the specific methods, protocols, reagents, etc. described herein and can therefore vary. The definitions and terms used herein are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, as the scope of the present invention is limited only by the claims.

[0076] Unless otherwise specified, the terms "a", "an", and "the" and similar references used in the context of describing one or more embodiments of the present application (especially in the context of the claims) may be construed to cover both the singular and the plural. The recitation of numerical ranges herein is merely intended to be a simplified method of referring separately to each individual numerical value falling within the range. Unless otherwise indicated herein, each individual numerical value is incorporated into the specification as if it were recited individually herein. Unless otherwise indicated herein or otherwise clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any examples or exemplary language provided herein for certain embodiments and all examples or exemplary language (e.g., "such as") is merely intended to better illustrate the present application and does not limit the scope of the present application as otherwise claimed. The abbreviation "e.g." is derived from the Latin exempli gratia and is used herein to denote non-limiting examples. Thus, the abbreviation "e.g." is synonymous with the term "for example". No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present application.

[0077] As used herein, the term "soft tissue" is used to refer to tissues that connect, support, or surround other structures and organs of the body. The term "soft tissue" may refer to muscle, ligament, tendon, fascia, skin, fibrous tissue, fat, synovium, nerve, and / or blood vessel.

[0078] As used herein, the term "abnormal bone formation" refers to the generation of bone in areas where bone is not normally present, such as soft tissue.

[0079] The terms "patient", "subject", and "individual" are used interchangeably herein and refer to an animal (especially a human) to whom treatment (including prophylactic treatment of a disease) is provided. As used herein, the term "subject" refers to humans and non-human animals. The terms "non-human animal" and "non-human mammal" are used interchangeably herein and include all vertebrates (e.g., mammals (such as non-human primates (especially higher primates), sheep, dog, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows) and non-mammals (such as chickens, amphibians, reptiles), etc.). In some embodiments, the subject is a human. In another embodiment, the subject is an experimental animal or an animal substitute as a disease model. In another embodiment, the subject is a domestic animal including companion animals (e.g., dogs, cats, rats, guinea pigs, hamsters, etc.).

[0080] As used herein, the term "at risk of having abnormal bone formation" refers to a subject who has been exposed to a condition known to cause abnormal bone formation in a population of subjects. Although not every subject exposed to such a condition will develop abnormal bone formation, all subjects exposed to these conditions can be considered "at risk". Such conditions typically include trauma (e.g., musculoskeletal trauma, central nervous system injury, or spinal cord injury).

[0081] As used herein, a therapeutic agent that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control sample.

[0082] The term "treatment" includes prophylactic treatment and / or therapeutic treatment. The term "prophylactic or therapeutic" treatment is well recognized in the art and includes administering to a host one or more of the subject compositions. If administered prior to the clinical manifestation of a deleterious condition (e.g., a disease or other deleterious state of a host animal), the treatment is prophylactic (i.e., it protects the host from developing the deleterious condition), while if administered after the manifestation of the deleterious condition, the treatment is therapeutic (i.e., it is intended to reduce, mitigate, or stabilize an existing deleterious condition or its side effects).

[0083] The terms "decrease", "reduced", "reduction" or "inhibit" as used herein all mean a statistically significant decrease or diminution in a property, level or other parameter. In some embodiments, "reduce", "reduction" or "decrease" or "inhibit" generally means a reduction of at least 10% compared to a reference level (e.g., absence of a given treatment), and can include (e.g.) at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more reduction. As used herein, "reduction" or "inhibition" compared to a reference level does not include complete inhibition or reduction. "Complete inhibition" is 100% inhibition compared to a reference level. The reduction can preferably be reduced to a level acceptable within the normal range for an individual not suffering from a particular disorder.

[0084] The terms "increased", "increase", "enhance" or "activate" as used herein all generally mean a statistically significant increase in a property, level or other parameter; for the avoidance of any doubt, the terms "increased", "increase", "enhance" or "activate" mean an increase of at least 10% compared to a reference level (e.g., an increase of at least about 20% or at least about 30% or at least about 40% or at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90% compared to a reference level or up to and including a 100% increase or any increase between 10 - 100%) or an increase of at least about 2-fold or at least about 3-fold or at least about 4-fold or at least about 5-fold or at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 1000-fold or more compared to a reference level.

[0085] The term "pharmaceutically acceptable" can refer to compounds and compositions that can be administered to a subject (e.g., a mammal or a human) without undue toxicity.

[0086] As used herein, the term "pharmaceutically acceptable carrier" can include any material or substance that, when combined with an active ingredient, allows the ingredient to retain its biological activity and is non-reactive with the immune system of a subject. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as phosphate buffered saline solution, water, emulsions (such as oil / water emulsions), and various types of wetting agents. The term "pharmaceutically acceptable carrier" excludes tissue culture medium.

[0087] The phrase "activity of ALK2" means ALK-2 enzyme activity (e.g., such as kinase activity; the ability of ALK-2 to phosphorylate BMP-responsive SMAD proteins) and / or ALK-2-mediated signal transduction (e.g., such as the ability of ALK-2 to mediate downstream signal transduction and transcriptional activity after activation of ALK-2 by binding to a BMP ligand). In some embodiments, "activity of ALK2" means ALK2-mediated BMP signal transduction. In some embodiments, "activity of ALK2" means ALK2-mediated BMP-responsive gene transcription (e.g., transcriptional activity mediated by BMP / ALK2 signal transduction).

[0088] The phrase "activity of ALK5" means ALK-5 enzyme activity (e.g., such as kinase activity; the ability of ALK-5 to phosphorylate TGF-β-responsive SMAD proteins; the ability of ALK-5 to phosphorylate SMAD2 or SMAD3) and / or ALK-5-mediated signal transduction (e.g., such as the ability of ALK-5 to mediate downstream signal transduction and transcriptional activity after activation of ALK-5 by binding to a TGF-β ligand). In some embodiments, "activity of ALK5" means ALK5-mediated TGF-β signal transduction. In some embodiments, "activity of ALK5" means ALK5-mediated TGF-β-responsive gene transcription (e.g., transcriptional activity mediated by TGFβ / ALK5 signal transduction).

[0089] The phrase "activity of ALK1" means ALK-1 enzyme activity (e.g., such as kinase activity; the ability of ALK-1 to phosphorylate BMP-responsive SMAD proteins) and / or ALK-1-mediated signal transduction (e.g., such as the ability of ALK-1 to mediate downstream signal transduction and transcriptional activity after activation of ALK-1 by binding to a BMP ligand). In some embodiments, "activity of ALK1" means ALK1-mediated BMP signal transduction. In some embodiments, "activity of ALK1" means ALK1-mediated BMP-responsive gene transcription (e.g., transcriptional activity mediated by BMP / ALK1 signal transduction).

[0090] The phrase "activity of ALK4" means ALK-4 enzymatic activity (e.g., kinase activity; the ability of ALK-4 to phosphorylate activin-responsive SMAD proteins; the ability of ALK-4 to phosphorylate SMAD 2 or SMAD 3) and / or ALK-4-mediated signal transduction (e.g., the ability of ALK-4 to mediate downstream signal transduction and transcriptional activity after activation of ALK-4 by binding to an activin ligand). In some embodiments, "activity of ALK4" means ALK4-mediated activin signal transduction. In some embodiments, "activity of ALK4" means ALK4-mediated activin-responsive gene transcription (e.g., transcriptional activity mediated by activin / ALK4 signal transduction).

[0091] The phrase "activity of ALK6" means ALK-6 enzymatic activity (e.g., kinase activity; the ability of ALK-6 to phosphorylate BMP-responsive SMAD proteins) and / or ALK-6-mediated signal transduction (e.g., the ability of ALK-6 to mediate downstream signal transduction and transcriptional activity after activation of ALK-6 by binding to a BMP ligand). In some embodiments, "activity of ALK6" means ALK6-mediated BMP signal transduction. In some embodiments, "activity of ALK6" means ALK6-mediated GDF5 signal transduction. In some embodiments, "activity of ALK6" means ALK6-mediated BMP-responsive gene transcription (e.g., transcriptional activity mediated by BMP / ALK6 signal transduction).

[0092] Human ALK2 is a 509-amino acid protein. The protein sequence is published, for example, as GenBank accession number NP_001104537.1 (with the corresponding nucleotide sequence of NM_001111067.2), UniProt entry Q04771.

[0093] Human ALK5 has at least two isoforms: a 503-amino acid protein (isoform 1) and a 426-amino acid protein. The protein sequence of human ALK5 isoform 1 is published, for example, as GenBank accession number NP_004603.1 (with the corresponding nucleotide sequence of NM_004612.2). The protein sequence of the 426-amino acid isoform is published, for example, as GenBank accession number NP_001124388.1 (with the corresponding nucleotide sequence of NM_001130916.1). Information on the two isoforms is also published as UniProt entry P36897.

[0094] Human ALK1 is a 503 - amino acid protein. The protein sequence is published, for example, as GenBank accession number NP_001070869.1 (with the corresponding nucleotide sequence of NM_001077401.1; transcript variant 2) and NP_000011.2 (with the corresponding nucleotide sequence of NM_000020.2; transcript variant 1), UniProt entry P37023.

[0095] Human ALK3 is a 532 - amino acid protein. The protein sequence is published, for example, as GenBank accession number NP_004320 (with the corresponding nucleotide sequence of NM_004329.2), UniProt entry P36894.

[0096] Human ALK4 has at least three isoforms. Isoform a is a 505 - amino acid protein. The protein sequence is published, for example, as GenBank accession number NP_004293 (with the corresponding nucleotide sequence of NM_004302), UniProt entry P36896.

[0097] Isoform a of human ALK6 is a 532 - amino acid protein, while isoform b is a 502 - amino acid protein. The protein sequence of human ALK6 isoform a is published, for example, as GenBank accession number NP_001243722 (with the corresponding nucleotide sequence of NM_001256793.1). The protein sequence of human ALK6 isoform b is published, for example, as GenBank accession number NP_001194 (with the corresponding nucleotide sequence of NM_01203.2).

[0098] Note that each of the above - mentioned proteins is further processed in vivo (such as by cleavage of the signal sequence) to produce a mature form.

[0099] As used herein, the term "comprising" means that other elements may be present in addition to the recited defined elements. The use of "comprising" indicates inclusion rather than limitation.

[0100] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. This term permits the presence of additional elements that do not materially affect one or more of the basic and novel or functional characteristics of this aspect of the invention.

[0101] The term "consisting of" refers to the compositions, methods, and their respective components as described herein, which do not include any element not recited in the description of the embodiment.

[0102] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, such that the description includes both the case where the circumstance occurs and the case where the circumstance does not occur.

[0103] Change in expression: A change in expression refers to a change in the level of a gene transcript (e.g., mRNA) or gene product (e.g., protein) that is detectable in a biological sample (e.g., a sample from a patient with Sjogren's syndrome, such as in a salivary gland biopsy) relative to a control (e.g., a healthy subject). A "change" in expression includes an increase (upregulation) or a decrease (downregulation) in expression.

[0104] Bone morphogenetic protein 6 (BMP6): A member of the TGF-β superfamily of growth factors. Expression of BMP6 has been detected in several different mammalian tissues and cell types, including smooth muscle cells, growth plate chondrocytes, bronchiolar epithelial cells, cornea, epidermis, salivary glands, and cells of the nervous system (Blessing et al., J Cell Biol 135(1):227-239, 1996). In vitro, BMP6 has been shown to inhibit cell division, promote terminal epithelial differentiation, and induce endochondral bone formation, osteoblast differentiation, and neuronal maturation (Heikinheimo et al., Cancer Res 59:5815-5821, 1999). BMP6 is also known as plant-associated growth factor (TGFB-related), VGR, VGR1, and VG-1-related protein. Genomic sequences, mRNA sequences, and protein sequences of BMP6 from many different species are publicly available (e.g., from the GenBank database of the National Center for Biotechnology Information).

[0105] Control: "Control" refers to a sample or standard (e.g., a salivary gland sample obtained from a patient with Sjogren's syndrome) that is used for comparison with an experimental sample. In some embodiments, the control is a sample obtained from a healthy volunteer (also referred to herein as a "normal" control). In some embodiments, the control is a historical control or a standard value (i.e., a previously tested control sample or group of samples that represents a baseline or normal value).

[0106] Diagnosis: The process of identifying a disease based on the signs, symptoms, and / or various test results of the disease. The conclusion obtained through this process is also referred to as a "diagnosis". Forms of tests that are commonly performed include physical examination, blood tests, medical imaging, genetic analysis, urine analysis, and biopsy.

[0107] Diagnostically significant amount: In some embodiments, a "diagnostically significant amount" refers to an increase or decrease in the level of BMP6 (or any other gene or protein) in a biological sample, the increase or decrease being sufficient to permit the distinction of one patient population from another (such as a population of Sjogren's syndrome patients from a group of healthy individuals). In some embodiments, the diagnostically significant increase or decrease is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold or at least 40-fold. RT-PCR is provided herein as an example of how BMP6 expression can be detected. Immunoassays (such as ELISA) are another example of methods for detecting the expression of BMP6. However, those skilled in the art will recognize that there are other methods for measuring gene expression and that depending on the method used, variations in the detected expression levels may occur. Thus, the diagnostically significant amount may vary if another detection method is used. In other embodiments, a "diagnostically significant amount" refers to an increase or decrease in the electrical potential of the salivary gland, the increase or decrease being sufficient to permit the distinction of one patient population from another (such as a population of Sjogren's syndrome patients from a group of healthy controls). In some embodiments, the diagnostically significant increase or decrease is about 10%, about 20%, about 30%, about 40% or about 50%.

[0108] Immunosuppressive drug: Any agent or compound having the ability to reduce the body's immune system response. In some embodiments, the immunosuppressive drug is a corticosteroid. In other embodiments, the immunosuppressive drug is a small molecule (such as cyclosporine) or a monoclonal antibody (such as a cytokine blocker).

[0109] Inhibitor: Any chemical compound, nucleic acid molecule, small molecule, peptide or polypeptide (such as an antibody) that can reduce the activity of a gene product or interfere with gene expression. In some embodiments, the inhibitor can directly or indirectly reduce or inhibit the activity of a protein encoded by a gene. Direct inhibition can be achieved, for example, by binding to the protein and thereby preventing the protein from binding to its intended target (such as a receptor). Indirect inhibition can be achieved, for example, by binding to the intended target of the protein (such as a receptor or binding partner) and thereby blocking or reducing the activity of the protein. In some embodiments, the inhibitors of the present disclosure can inhibit a gene by reducing or inhibiting the expression of the gene (especially by interfering with gene expression (transcription, processing, translation, post-translational modification) (e.g., by interfering with the mRNA of the gene and blocking the translation of the gene product or by post-translational modification of the gene product or by causing a change in the intracellular localization)). In various embodiments of the invention, the inhibitor is one or more compounds of formula (I).

[0110] Inhibition of expression or activity: As used herein, an agent that inhibits the expression or activity of a gene (such as BMP6) is an agent that reduces the level of mRNA or protein expressed by the gene (such as BMP6) in a cell or tissue or reduces (including eliminating) one or more activities of the gene or the encoded protein (such as BMP6). Similarly, an agent that inhibits BMP signaling is any compound that inhibits, blocks, or prevents signaling events in the BMP signaling pathway (such as phosphorylation of downstream targets, e.g., phosphorylation of SMAD1 / 5 / 8).

[0111] Measuring expression levels: Quantifying the amount of a gene product present in a sample. The quantification can be numerical or relative. Detection of the expression of a gene product (such as BMP6 mRNA or protein) can be achieved using any method known in the art or described herein (such as by RT-PCR, antibody binding (e.g., ELISA), or immunohistochemical methods). In some embodiments, the detected change is an increase or decrease in expression compared to a control. In some examples, the detected increase or decrease is at least two-fold, at least three-fold, or at least four-fold increase or decrease compared to a control. In other embodiments of the method, the increase or decrease is a diagnostically significant amount, and a diagnostically significant amount refers to a change large enough to provide a statistical probability of diagnosis.

[0112] Methyl-CpG-binding protein 2 (MECP2): DNA methylation is a major modification of the eukaryotic genome and plays an important role in mammalian development. The human proteins MECP2, MBD1, MBD2, MBD3, and MBD4 comprise a family of nuclear proteins related to the presence of a methyl-CpG-binding domain (MBD) in each. With the exception of MBD3, each of these proteins is capable of binding specifically to methylated DNA. MECP2, MBD1, and MBD2 can also repress transcription of methylated gene promoters. Compared to other MBD family members, MECP2 is X-linked and subject to X inactivation. MECP2 is non-essential in stem cells but is essential for embryonic development. Mutations in MECP2 are the cause of most cases of Rett syndrome, a progressive neurodevelopmental disorder, and one of the most common causes of mental retardation in females. MECP2 is also known as RS; RTS; RTT; PPMX; MRX16; MRX79; MRXSL; AUTSX3; MRXS13; and DKFZp686A24160. The genomic sequence, mRNA sequence, and protein sequence of MECP2 are publicly available (such as from the GenBank database of the National Center for Biotechnology Information).

[0113] Noggin (NOG): A secreted protein that binds to and inactivates members of the transforming growth factor-β (TGF-β) superfamily of signaling proteins (such as BMP4 and BMP6). By diffusing more effectively through the extracellular matrix than members of the TGF-β superfamily, this protein can play a major role in creating a morphogenic gradient. The protein appears to have pleiotropic effects both early and late in development. The nucleotide and amino acid sequences of noggin are publicly available (e.g., in the GenBank database (for human noggin, see NCBI Gene ID 9241)).

[0114] Nonsteroidal anti-inflammatory drug (NSAID): A type of anti-inflammatory agent that acts by inhibiting the production of prostaglandins. NSAIDs exert anti-inflammatory, analgesic, and antipyretic effects. Examples of NSAIDs include ibuprofen, ketoprofen, piroxicam, naproxen, sulindac, aspirin, choline salicylate, diflunisal, fenoprofen, indomethacin, meclofenamic acid, salsalate, tolmetin, and magnesium salicylate.

[0115] Restoring salivary flow (or increasing salivary flow): The process of increasing saliva production in a subject having reduced salivary flow (such as may be caused by Sjogren's syndrome and / or increased BMP6 expression). An increase in salivary flow can be indicated, for example, by an increase in salivary flow rate and / or an increase in the volume of salivary flow. In some embodiments, restoring salivary flow can be achieved by administering a therapeutic agent. In some examples, the therapeutic agent is a drug (such as pilocarpine (Salagen TM ) or cevimeline (Evoxac TM ). In other examples, the therapeutic agent is an inhibitor of BMP6 expression or activity.

[0116] Restoring tear production: The process of increasing tear production in a subject having reduced tearing (such as may be caused by Sjogren's syndrome). In some embodiments, restoring tear production can be achieved by administering a therapeutic agent. In certain examples, the therapeutic agent is an inhibitor of BMP6 expression or activity.

[0117] Salivary gland: An exocrine gland that produces saliva. As used herein, "salivary gland" includes any salivary gland in a human subject (including, for example, the parotid gland, minor salivary glands, submandibular gland, sublingual gland, and von Ebner's glands). There are over 600 minor salivary glands throughout the oral cavity.

[0118] Sjögren's syndrome (SS): An autoimmune disorder characterized by immune cells that attack and destroy the glands that produce tears and saliva. Sjögren's syndrome is not life-threatening or life-shortening, but can significantly reduce quality of life. The hallmark symptoms of the disorder are dry mouth and dry eyes. Sjögren's syndrome can also cause dry skin, dry nose, and dry vagina, and can affect other organs of the body (including the kidneys, blood vessels, lungs, liver, pancreas, and brain). Sjögren's syndrome affects 1 - 4 million people in the United States, and women are nine times more likely to develop the disease. At the time of diagnosis, most people with Sjögren's are at least 40 years old.

[0119] Many different criteria can be used to identify subjects with Sjögren's syndrome and include one or more of the following: (i) ocular symptoms (e.g., persistent dry eyes and / or recurrent sensations of sand or grit in the eyes); (ii) oral symptoms (e.g., daily sensation of dry mouth, persistently swollen salivary glands, and / or drinking liquids to swallow dry foods); (iii) objective evidence of ocular involvement, which is defined as a positive result on the Schirmer's test (≤5 mm in 5 minutes) and / or a Rose bengal score or other ocular surface staining score (≥4 according to the van Bijsterveld's scoring system) performed without anesthesia; (iv) histopathology of the minor salivary glands (measuring the focus score or Tarpley score); (v) salivary gland involvement demonstrated by objective evidence of the following: a positive result based on unstimulated whole saliva flow (≤1.5 ml in 15 minutes), parotid sialography showing the presence of diffuse sialectasis (punctate, cavitary, or destructive) without signs of obstruction in the main ducts, and / or salivary scintigraphy showing delayed uptake, decreased concentration, and / or delayed excretion of the tracer; or (vi) autoantibodies (antibodies to the Ro (SSA) antigen or La (SSB) antigen or both present in the serum). Thus, in some embodiments, subjects exhibiting one or more of the above signs or symptoms are selected for treatment according to the methods disclosed herein.

[0120] In the absence of another connective tissue disease, the presence of sicca (dryness) symptoms (sicca symptomology) is named "primary Sjögren's syndrome". Primary Sjögren's syndrome may also be characterized in that a subject has a positive result for any four of the six criteria listed above (as long as the histopathology (item iv) or serology (item vi) is positive) or the presence of any three of the four objective criteria listed above (i.e., items iii, iv, v, and vi). In the presence of item i or ii listed above, plus any two of the criteria of items iii, iv, and v, a patient with an autoimmune process (such as rheumatoid arthritis, systemic lupus erythematosus, progressive systemic sclerosis, scleroderma, or polymyositis) is said to have "secondary Sjögren's syndrome".

[0121] Therapeutically effective amount: The amount of a specified drug or therapeutic agent sufficient to achieve a desired effect in a subject or cell being treated with the agent. The effective amount of the agent will depend on several factors (including but not limited to the subject or cell being treated and the mode of administration of the therapeutic composition).

[0122] X-inactive specific transcript (non-protein coding) (XIST): X inactivation is an early developmental process in female mammals that silences one of a pair of X chromosomes by transcription, thereby equalizing the dosage between males and females. This process is regulated by several factors, including a region of the X chromosome called the X inactivation center (XIC). The XIST gene is only expressed from the XIC of the inactivated X chromosome. The transcript is spliced but does not code for a protein. The transcript remains in the nucleus, where it coats the inactivated X chromosome. XIST is also known as XCE, XIC, and SXI1. The genomic sequence and RNA sequence of XIST are publicly available (such as from the GenBank database of the National Center for Biotechnology Information).

[0123] Abbreviations

[0124] AAV Adeno-associated virus

[0125] ATP Adenosine triphosphate

[0126] BMP6 Bone morphogenetic protein 6

[0127] BSA Bovine serum albumin

[0128] BW Body weight

[0129] CGH Comparative genomic hybridization

[0130] ELISA Enzyme-linked immunosorbent assay

[0131] EP electric potential

[0132] FS lesion score

[0133] HIF-1 alpha hypoxia-inducible factor 1-alpha

[0134] HO heterotopic ossification

[0135] HTS hypotonic solution

[0136] HV healthy volunteer

[0137] IFN interferon

[0138] IL interleukin

[0139] IM intramuscular

[0140] IPA Ingenuity Pathway Analysis

[0141] MECP2 methyl-CpG-binding protein 2

[0142] MyD88 myeloid differentiation primary response gene 88

[0143] NOD non-obese diabetic

[0144] OD optical density

[0145] O / N overnight

[0146] PDGF platelet-derived growth factor

[0147] pSS primary Sjögren's syndrome

[0148] qPCR quantitative polymerase chain reaction

[0149] RIN RNA integrity number

[0150] RT room temperature

[0151] RT-PCR reverse transcriptase polymerase chain reaction

[0152] Runx2 runt-related transcription factor 2

[0153] RVD regulatory volume decrease

[0154] SFR salivary flow rate

[0155] SG salivary gland

[0156] SMG submandibular gland

[0157] SS Sjögren's syndrome

[0158] TEER Trans - epithelial electrical resistance

[0159] TGF Transforming growth factor

[0160] TRIF TIR - domain - containing adapter - inducing interferon - β

[0161] WT Wild - type

[0162] XIST X - inactive specific transcript (non - protein - coding)

[0163] In addition, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

[0164] It should be understood that the present invention is not limited to the specific methods, protocols, reagents, etc. described herein and may thus vary therefrom. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which is defined only by the claims.

[0165] The methods and compositions provided herein are based in part on the discovery that one or more of the crystalline compounds or compositions described herein act as BMP inhibitors by inhibiting signaling through the BMP type I receptor, ALK2. In addition, one or more of the crystalline compounds or compositions described herein are shown herein to be effective in treating and / or preventing abnormal bone formation in soft tissues. Accordingly, methods and compositions for treating abnormal bone formation in soft tissues are provided, including treatment with one or more of the crystalline compounds or compositions described herein.

[0166] In various embodiments, the present invention provides methods for treating abnormal bone formation in the soft tissues of a subject, the methods comprising: administering a therapeutically effective amount of one or more of the crystalline compounds or compositions described herein. In some embodiments, the subject is determined to have abnormal bone formation or be at risk of having abnormal bone formation prior to treatment. In some embodiments, the subject has undergone musculoskeletal trauma, spinal cord injury, or central nervous system injury. In some embodiments, the abnormal bone formation is associated with heterotopic ossification disease. In some embodiments, the heterotopic ossification disease is selected from the group consisting of: acquired heterotopic ossification, fibrodysplasia ossificans progressiva, ankylosing spondylitis, traumatic heterotopic ossification, burn-associated heterotopic ossification or blast injury-associated heterotopic ossification, and joint replacement-associated heterotopic ossification. In some embodiments, the soft tissues include muscle, tendon, ligament, and / or fascia. In some embodiments, at least one additional agent is administered to the subject. In some embodiments, the at least one additional agent includes a corticosteroid, a non-steroidal anti-inflammatory drug (NSAID), a lipoxygenase inhibitor, a leukotriene inhibitor, a mast cell stabilizer, an antihistamine, a TNF inhibitor, an IL-23 blocker, or an IL-1 signaling inhibitor. In some embodiments, the therapeutically effective amount of one or more of the crystalline compounds or compositions described herein includes a dose in the range of 5 mg / kg to 250 mg / kg. In some embodiments, the therapeutically effective amount of one or more of the crystalline compounds or compositions described herein does not cause a weight loss greater than 20% of the total body mass.

[0167] In various embodiments, the present invention provides methods for treating abnormal bone formation in the soft tissues of a subject, the methods comprising: administering to the subject a therapeutically effective amount of an inhibitor of a BMP type I serine-threonine kinase receptor, wherein the inhibitor of the BMP type I serine-threonine kinase receptor is one or more of the crystalline compounds or compositions described herein. In some embodiments, the BMP type I serine-threonine receptor is ALK2, ALK3, or ALK6. In some embodiments, the BMP type I serine-threonine receptor is ALK2 or ALK3.

[0168] In various embodiments, the present invention provides methods for treating abnormal bone formation in the soft tissues of a subject, the methods comprising: administering to the subject a therapeutically effective amount of an inhibitor of a BMP type II serine-threonine kinase receptor, wherein the inhibitor of the BMP type II serine-threonine kinase receptor is one or more of the crystalline compounds or compositions described herein. In some embodiments, the BMP type II serine-threonine receptor is ACVR2A, ACVR2B, BMPR2, or TGFβR2.

[0169] In various embodiments, the present invention provides methods for inhibiting serine-threonine kinase receptors in a subject, the methods comprising: administering to the subject an inhibitor of a serine-threonine kinase receptor under conditions effective to inhibit the serine-threonine kinase receptor, wherein the inhibitor of the serine-threonine kinase receptor is one or more of the crystalline compounds or compositions described herein. In some embodiments, the serine-threonine kinase receptor is a BMP type I receptor, a BMP type II receptor, or a TGF-β type I receptor. In some embodiments, the serine-threonine kinase receptor is a BMP type I receptor. In some embodiments, the BMP type I receptor is ALK2, ALK3, or ALK6. In some embodiments, the BMP type I receptor is ALK2 or ALK3. In some embodiments, the serine-threonine kinase receptor is a BMP type II receptor. In some embodiments, the BMP type II receptor is ACVR2A, ACVR2B, BMPR2, or TGFβR2. In some embodiments, the serine-threonine kinase receptor is a TGF-β type I receptor. In some embodiments, the TGF-β type I receptor is ALK5.

[0170] In various embodiments, the present invention provides methods for identifying one or more compounds for inhibiting serine-threonine kinase receptors, the methods comprising: a) providing a sample comprising a serine-threonine kinase receptor; b) contacting the sample with one or more of the crystalline compounds or compositions described herein; and c) performing an assay to identify one or more compounds that inhibit the serine-threonine kinase receptor, wherein the assay is an in vitro assay, an in vivo assay, or an ex vivo assay. In some embodiments, the serine-threonine kinase receptor is a BMP type I receptor, a BMP type II receptor, or a TGF-β type I receptor. In some embodiments, the assay is an in vitro assay.

[0171] In various embodiments, the present invention provides methods for treating a subject having Sjögren's syndrome, the methods comprising: a) selecting a subject having increased BMP6 expression in the subject's salivary gland relative to a control; and b) administering to the subject a therapeutically effective amount of an agent that inhibits the expression or activity of BMP6, thereby treating the subject having Sjögren's syndrome, wherein the agent that inhibits the expression or activity of BMP6 is one or more of the crystalline compounds or compositions described herein. In some embodiments, the salivary gland is a minor labial salivary gland, a parotid gland, or a submandibular gland.

[0172] In various embodiments, the present invention provides methods for treating a subject having diffuse intrinsic pontine glioma (DIPG), the methods comprising selecting a subject having diffuse intrinsic pontine glioma (DIPG), and administering to the subject a therapeutically effective amount of one or more of the crystalline compounds or compositions described herein, thereby treating the subject having diffuse intrinsic pontine glioma (DIPG).

[0173] In various embodiments, the present invention provides methods for treating abnormal bone formation in soft tissue of a subject, the methods comprising: administering to the subject a therapeutically effective amount of an inhibitor of the TGF-β type I receptor serine-threonine kinase receptor, wherein the inhibitor of the TGF-β type I serine-threonine kinase receptor is one or more of the crystalline compounds or compositions described herein. In some embodiments, the TGF-β type I receptor is ALK5.

[0174] In various embodiments, the present invention provides methods for inhibiting a serine-threonine kinase receptor in a subject, the methods comprising: administering to the subject an inhibitor of the serine-threonine kinase receptor under conditions effective to inhibit the serine-threonine kinase receptor, wherein the inhibitor of the serine-threonine kinase receptor is one or more of the crystalline compounds or compositions described herein. In some embodiments, the serine-threonine kinase receptor is a BMP type I receptor, a BMP type II receptor, or a TGF-β type I receptor. In some embodiments, the serine-threonine kinase receptor is a BMP type I receptor. In some embodiments, the BMP type I receptor is ALK2, ALK3, or ALK6. In some embodiments, the BMP type I receptor is ALK2 or ALK3. In some embodiments, the serine-threonine kinase receptor is a BMP type II receptor. In some embodiments, the BMP type II receptor is ACVR2A, ACVR2B, BMPR2, or TGFβR2. In some embodiments, the serine-threonine kinase receptor is a TGF-β type I receptor. In some embodiments, the TGF-β type I receptor is ALK5.

[0175] In various embodiments, the present invention provides methods for increasing saliva flow in a subject, the methods comprising: a) selecting a subject having increased BMP6 expression in the salivary glands of the subject relative to a control; and b) administering to the subject a therapeutically effective amount of an agent that inhibits the expression or activity of BMP6, thereby increasing saliva flow in the subject, wherein the agent that inhibits the expression or activity of BMP6 is one or more of the crystalline compounds or compositions described herein or a submandibular gland.

[0176] In various embodiments, the present invention provides methods of increasing salivary flow in a subject, the methods comprising: a) selecting a subject having increased BMP6 expression in the salivary glands of the subject relative to a control; and b) administering to the subject a therapeutically effective amount of an agent that inhibits BMP signaling, thereby increasing salivary flow in the subject, wherein the agent that inhibits BMP signaling is one or more of the crystalline compounds or compositions described herein. In some embodiments, the salivary gland is a minor labial salivary gland, a parotid gland, or a submandibular gland.

[0177] Heterotopic ossification disease

[0178] The term "heterotopic ossification" refers to abnormal bone formation in soft tissues where bone is not normally present. Acquired heterotopic ossification can occur substantially with any of the following: musculoskeletal trauma, spinal cord injury, central nervous system injury, head injury, cerebrovascular accident, sickle cell anemia, hemophilia, tetanus, poliomyelitis, multiple sclerosis, toxic epidermal necrolysis, and burns. Examples of musculoskeletal trauma include, but are not limited to, hip, knee, shoulder, or elbow arthroplasty; fractures; joint dislocations; or soft tissue trauma (including quadriceps and brachialis muscles). Acquired heterotopic ossification can also be associated with fever, swelling, and erythema (e.g., local patchy redness of the skin). In one embodiment, neurogenic heterotopic ossification is not associated with local trauma.

[0179] The genetic diseases fibrodysplasia ossificans progressiva (FOP) and progressive osseous heterplasia (POH) are the most severe manifestations of heterotopic bone formation. FOP occurs rarely and is the result of a mutation in ACVR1, which encodes a bone morphogenetic protein type I receptor. Patients with POH have an inactivating mutation in the GNAS gene, which can also cause Albright's hereditary osteodystrophy (AHO) when the mutation is inherited from the mother.

[0180] Myositis ossificans circumscripta is characterized by intramuscular proliferation of fibroblasts, new bone, and / or cartilage.

[0181] HO typically appears between 3 and 12 weeks after injury. Heterotopic ossification can be reliably diagnosed by computed tomography, bone scintigraphy, and ultrasonography. After two to six weeks, the abnormal bone formation has progressed to the point where it is detectable by radiography. Bone maturation typically occurs within six months.

[0182] Conventional Treatments for Heterotopic Ossification:Conventional treatments typically involve non-steroidal anti-inflammatory drugs (indomethacin, rofecoxib), or bisphosphonates (etidronate, pamidronate), warfarin sodium (Coumadin) / warfarin, salicylates, and / or local radiation may also be administered. Surgery is often the only option for treatment.

[0183] Treatment outcomes can be measured by a standard radiological grading system for HO, and treatment outcomes include measurements related to the following: changes in range of motion in the affected joint measured by goniometry, the mean duration of objective improvement in clinical symptoms or clinical signs related to HO, changes in standardized functional measurements or joint-specific measurements.

[0184] Applications

[0185] The BMP signaling pathway and the TGF-β signaling pathway are essential for normal organogenesis and patterning, as well as for normal remodeling and pathological remodeling of mature tissues. Defects in the BMP signaling pathway are associated with many congenital and acquired disease processes, including hereditary hemorrhagic telangiectasia syndrome, primary pulmonary hypertension, juvenile familial polyposis, and sporadic renal cell carcinoma and prostate cancer. It has been shown that in certain disease states associated with defective signaling components, attenuated BMP signaling may be the cause, while other findings have shown that in some cases, excessive BMP signaling may be pathogenic (Waite et al. Nat. Rev. Genet. 4:763-773, 2005; Yu et al. J. Biol. Chem. 280:24443-24450, 2003). The ability to experimentally modulate BMP signaling will provide a means for studying therapies and determining the underlying causes of these conditions. One or more of the crystalline compounds or compositions described herein are inhibitors of ALK2, a type I BMP receptor, and can be used to disrupt signaling through the BMP pathway.

[0186] Treatment of Anemia (Including Iron Deficiency Anemia and Anemia of Chronic Disease)

[0187] For a review, see Weiss et al. N. Engl. J. Med.352:1011-1023, 2005. Anemia of inflammation (also known as anemia of chronic disease) can be seen in patients with chronic infections, autoimmune diseases (such as systemic lupus erythematosus and rheumatoid arthritis and Castleman’s disease), inflammatory bowel disease, cancer (including multiple myeloma), and renal failure. Anemia of inflammation is often caused by the maladaptive expression of the peptide hormone hepcidin. Hepcidin causes the degradation of ferroportin, a key protein that enables the transport of iron from intracellular stores in macrophages and from enterocytes. Many patients with renal failure have a combination of erythropoietin deficiency and overexpression of hepcidin. BMP signaling induces the expression of hepcidin, and inhibition of hepcidin expression with a BMP antagonist increases iron levels. The compounds described herein can be used to treat anemia attributable to chronic disease or inflammation and the associated hyperhepcidinemic state.

[0188] Based on the elevation of IL-6 in anemia of inflammation of various etiologies, the effects of chronic IL-6 administration in vivo, and the protection against anemia in IL-6-deficient rodents, the inflammatory cytokine IL-6 is thought to be the major cause of the elevated hepcidin expression in the inflammatory state (Weiss et al. N. Engl. J. Med. 352:1011-1023, 2005). It has been shown that stimulation of hepatoma cell lines with IL-6 induces hepcidin expression, while treatment with a BMP antagonist abrogates IL-6-induced hepcidin expression (Yu et al. Nat. Chem. Biol. 4:33-41, 2008). In addition, BMP antagonists can inhibit hepcidin expression induced by injection of pathogens in vivo. It has also been shown that systemic iron administration in mice and zebrafish rapidly activates BMP-responsive SMADs and hepcidin expression in the liver, and BMP antagonism effectively blocks these responses (Yu et al. Nat. Chem. Biol. 4:33-41, 2008). Previous findings (BMP antagonists can inhibit hepcidin expression and increase serum iron levels in vivo (data not shown)) support the functional importance of BMP signaling in iron regulation. Collectively, these data suggest that iron-mediated and inflammation-mediated regulation of hepcidin and circulating iron levels require BMP signaling. Thus, one or more of the crystalline compounds or compositions described herein, which disrupt BMP signaling through ALK2, can be used to alter iron availability in various situations for therapeutic benefit.

[0189] The compounds and / or pharmaceutical compositions described herein can be used in anemic conditions to (i) enhance the efficacy of dietary or oral iron supplementation, which is safer than intravenous administration of iron, to increase serum iron concentration; (ii) enhance the accumulation of hemoglobin in the blood during a contemplated surgery or enable self - blood donation during a contemplated surgery; and (iii) potentiate the efficacy of erythropoietin and its analogs, such that lower doses of erythropoietin can be administered for anemia while minimizing the known toxicities and side effects of erythropoietin (i.e., hypertension, cardiovascular events, and tumor growth).

[0190] B. Treatment of Fibrodysplasia Ossificans Progressiva (FOP)

[0191] In various embodiments, the present disclosure relates to the treatment and / or prevention of a disease or disorder (including abnormal bone growth) in the soft tissue of a subject. Heterotopic ossification (HO) involves unwanted bone growth, characterized by the inappropriate differentiation of cells into osteoblasts. This condition results in bone formation (usually near joints), where bone formation often limits joint mobility. HO can occur following nerve injury and direct injury to the soft tissue around joints (such as muscle or connective tissue), where HO subsequently develops.

[0192] There are three recognized etiologies of HO: traumatic, neurogenic, and genetic. Traumatic HO typically occurs after fractures, dislocations, surgical procedures, and severe burns. Most commonly, HO is seen around the hip following a fracture and open reduction internal fixation (ORIF) procedure or total hip arthroplasty (THA). Similarly, HO is often associated with pathologies such as traumatic brain injury (TBI), spinal cord injury (SCI), central nervous system (CNS) infections, tumors, stroke, tetanus, poliomyelitis, tabes dorsalis, multiple sclerosis, and selective dorsal rhizotomy. The presence of idiopathic myospasm is also associated with the development of HO.

[0193] Bone morphogenetic proteins (BMPs) exhibit a broad spectrum of biological activities in various tissues, including bone, cartilage, blood vessels, heart, kidney, neurons, liver, and lung. BMPs are members of the transforming growth factor-β (TGF-β) family, which bind to type II serine-threonine kinase receptors and type I serine-threonine kinase receptors and transduce signals through Smad and non-Smad signaling pathways. Fibrodysplasia ossificans progressiva (FOP), a type of ectopic ossification disorder, is an autosomal dominant rare disease that affects one in 1-2 million people. It is characterized by malformation of the great toe during embryonic development and progressive heterotopic endochondral ossification (HEO) after birth, which leads to the formation of a second skeleton of ectopic bone. Individuals with classic features of FOP have the same heterozygous activating mutation (R206H) in the gene encoding activin A receptor type 1 (ACVR1), also known as ALK2, a BMP type I receptor. Currently, there is no effective treatment for this rare and devastating disease. Accordingly, there remains a need for compositions and methods for treating ectopic ossification and ectopic ossification diseases and conditions.

[0194] FOP is caused by the presence of constitutively activated mutant forms of ALK2 in affected individuals (Shore et al. Nat. Genet. 38:525-527, 2006). Specific inhibitors of BMP signaling, such as one or more of the crystalline compounds or compositions described herein, can be used to prevent excessive bone formation in response to trauma, musculoskeletal stress, or inflammation. Such compounds can also be used to aid in the regression of pathologic bone. One or more of the crystalline compounds or compositions described herein can be administered systemically or locally to concentrate or limit the effect to the area of trauma or inflammation.

[0195] One or more crystalline compounds or compositions (ALK2 inhibitors) described herein can be used as a chronic therapy to inhibit spontaneous bone formation in highly susceptible individuals. One or more crystalline compounds or compositions described herein can be used as a chronic therapy to inhibit spontaneous bone formation in highly susceptible individuals. Transient therapy can be used to prevent abnormal bone formation in FOP individuals by administering prior to, during, or even after a traumatic event, in which FOP individuals develop osteomas or pathologic bone most often associated with trauma. Transient treatment with a BMP inhibitor as described herein (e.g., one or more crystalline compounds or compositions described herein) can be used immediately before, during, or after a necessary or urgent medical or surgical procedure (even important immunizations and tooth extractions) in an individual with FOP to prevent pathologic calcification. Combination therapies with other bone inhibitors, immunomodulatory, or anti-inflammatory agents (such as NSAIDs, steroids, cyclosporine, cyclophosphamide, azathioprine, methotrexate, rituxumab, etanercept, or similar agents) can increase the effectiveness of BMP antagonists in inhibiting heterotopic bone formation in this disorder.

[0196] The present invention provides methods and compositions for the treatment and / or prevention of abnormal bone formation in soft tissue. In certain embodiments, the methods and compositions treat and / or prevent a disease or disorder, including abnormal bone formation in soft tissue. Exemplary diseases or disorders that can be treated with the methods and compositions described herein include, but are not limited to, heterotopic ossification diseases (such as fibrodysplasia ossificans progressiva, ankylosing spondylarthritis, traumatic heterotopic ossification, burn-related heterotopic ossification or blast injury-related heterotopic ossification, and joint replacement-related heterotopic ossification).

[0197] Thus, in one aspect, the present invention provides a method for treating and / or preventing abnormal bone formation in soft tissue of a subject, the method comprising: administering a therapeutically effective amount of a pharmaceutical composition comprising one or more crystalline compounds or compositions described herein.

[0198] C. Treatment of Cancer

[0199] Overactive BMP signaling (which can occur due to overexpression of BMP or, paradoxically, due to loss of BMP type II receptor expression) may contribute to tumorigenesis, growth, or metastasis of certain solid tumors, including breast cancer, prostate cancer, bone cancer, lung cancer, and renal cell carcinoma (Yu et al.). J. Biol. Chem.280:24443 - 24450, 2008; Waite et al. Nat. Rev. Genet. 4:763 - 773, 2003; Alarmo et al. Genes, Chromosomes Cancer 45:411 - 419, 2006; Kim et al. Cancer Res. 60:2840 - 2844, 2000; Kim et al. Clin. Cancer Res. 9:6046 - 6051, 2003; Kim et al. Oncogene 23:7651 - 7659, 2004). If increased BMP activity associated with BMP overexpression or BMP type II receptor deficiency contributes to the pathogenesis of the disease, then inhibiting BMP signaling activity using a compound as described herein at the level of the BMP type I receptor (downstream of both the ligand and the type II receptor) may be an effective means of normalizing BMP signaling activity and potentially inhibiting tumor growth or tumor metastasis.

[0200] It is contemplated herein that one or more of the crystalline compounds or compositions described herein be used as adjuvant chemotherapy or primary chemotherapy for treating cancer (e.g., they can be used to slow or prevent the growth or metastasis of such tumor cells (and other tumor - constituent cell types)) to obtain a clinical benefit. Additionally, a BMP inhibitor as described herein can be used to interfere with the bone - metastatic properties of certain types of cancer (e.g., adenocarcinomas such as prostate and breast cancer). Further, one or more of the crystalline compounds or compositions described herein as described herein can be used to inhibit osteoblast activity (as adjuvant chemotherapy or primary chemotherapy) in tumors that form bone or are bone - derived (such as osteosarcoma). Additionally, one or more of the crystalline compounds or compositions described herein as described herein can be used to inhibit osteoclast activity (also regulated by BMP through its target gene RANKL), which is pathologically increased in conditions such as multiple myeloma and other bone - targeted tumors. The use of BMP inhibitors in these conditions can reduce the presence of osteolytic lesions and fractures attributable to tumor involvement. In some embodiments, the cancer is diffuse intrinsic pontine glioma (DIPG).

[0201] D. Treatment of Pathological Bone Formation

[0202] Compositions comprising one or more of the crystalline compounds or compositions described herein, as described herein, can be used to treat or mitigate pathological bone formation / ossification in inflammatory conditions such as ankylosing spondylitis or other "seronegative" spondyloarthropathies, where autoimmunity and inflammation in such conditions appear to stimulate bone formation. One application of the compounds would be to prevent excessive bone formation following joint surgery, particularly in patients with ankylosing spondylitis or rheumatoid arthritis. Compositions comprising one or more of the crystalline compounds or compositions described herein, as described herein, can also be used to prevent calcinosis (dystrophic soft tissue calcification) in diseases such as systemic lupus erythematosus, scleroderma or dermatomyositis.

[0203] Blunt traumatic injury to muscle can cause abnormal bone formation within the muscle in certain individuals, resulting in a condition called myositis ossificans traumatica (Cushner et al. Orthop. Rev. 21:1319 - 1326, 1992.). Head trauma and burns can also induce ectopic bone formation, significantly impairing patient recovery and rehabilitation. Optionally, in addition to anti - inflammatory drugs (e.g., non - steroidal anti - inflammatory drugs such as indomethacin or ibuprofen) typically prescribed for such conditions, treatment with one or more of the crystalline compounds or compositions described herein, as described herein, can help prevent pathological bone formation in susceptible individuals or help reduce or regress lesions in recently affected or long - affected individuals. Few other muscles have been described as developing ossification in the presence of injury or trauma, including the myocardium, and similar treatment with BMP inhibitors as described herein may be helpful in those cases.

[0204] E. Treatment of heterotopic or maladaptive bone formation

[0205] BMP signaling and its transcriptional targets are associated with intimal and medial vascular remodeling and calcification in Monckeberg’s vascular calcification disease and in atherosclerotic vascular disease (Bostrom et al. J. Clin. Invest. 91:1800 - 1809, 1993; Tyson et al. Arterioscler. Thromb. Vasc. Biol. 23:489 - 494, 2003). BMP and BMP - induced bone differentiation are also associated with cardiac valve calcification. Native heart valves can calcify, particularly when they are already abnormal. A typical example is the mitral - aortic valve, which commonly becomes calcified, leading to stenosis. Patients with calcific aortic valve stenosis often require cardiac surgery for valve replacement. Abnormal calcification can adversely affect the function of prosthetic vascular grafts or heart valves. For example, prosthetic heart valves become calcified, resulting in narrowing and frequent leakage.

[0206] As described herein, one or more crystalline compounds or compositions described herein can be used to inhibit vascular calcification disease or valvular calcification disease alone, or in combination with atherosclerotic disease, renal disease, renal osteodystrophy, or parathyroid disease.

[0207] Pharmaceutical compositions comprising one or more compounds of Formula I as described herein can be used to inhibit vascular calcification disease or valvular calcification disease alone, or in combination with atherosclerotic disease, renal disease, renal osteodystrophy, or parathyroid disease.

[0208] One or more crystalline compounds or compositions described herein as described herein can be used to inhibit calcification of prosthetic vascular materials or prosthetic valve materials by systemic or local administration or direct incorporation into a prosthetic material or other implant (e.g., in a mixture with a polymer that coats or constitutes all or a portion of the implant or prosthesis).

[0209] Pharmaceutical compositions as described herein comprising one or more crystalline compounds or compositions described herein can be used to inhibit calcification of prosthetic vascular materials or prosthetic valve materials by systemic or local administration or incorporation directly into a prosthetic material or other implant (e.g., in a mixture with a polymer that coats or constitutes all or a portion of an implant or prosthesis).

[0210] In some cases, it is desirable to delay the fracture healing after fracture, or intentionally suppress the fracture healing in certain positions, to prevent functional damage due to maladaptive bone formation. For example, if fracture occurs and surgery cannot be performed immediately due to medical reasons or practical reasons, fracture healing can be temporarily "suspended" by using one or more crystalline compounds or compositions as described herein as described herein, until clear surgery or operation can be performed. For example, this can prevent the need for intentional refracture to ensure the correct juxtaposition of bone fragments. It is expected that if the treatment period is relatively short, then when the administration of one or more crystalline compounds or compositions as described herein is stopped, normal fracture healing process will occur immediately. In other cases, the new bone growth of any amount may impair function (such as when fracture directly affects joints). In these cases, the overall inhibition or local inhibition of BMP activity (by systemically delivering or locally delivering BMP antagonists as described herein via diffusion from local implants or matrix) can be used to inhibit fracture healing or prevent fracture callus at key areas.

[0211] F. Immunomodulation via BMP antagonists

[0212] BMP has been reported to attenuate the inflammatory or immune response (Choi et al. Nat. Immunol. 7:1057-1065, 2006; Kersten et al. BMC Immunol. 6:9, 2005), which can impair an individual's ability to resist infection (i.e., viral, bacterial, fungal, parasitic, or tuberculosis). One or more of the crystalline compounds or compositions (inhibitors of BMP signaling through ALK2) described herein can be used to enhance the inflammatory or immune response, enabling the individual to clear the infection more rapidly. One or more of the crystalline compounds or compositions described herein can be used to enhance the inflammatory or immune response, enabling the individual to clear the infection more rapidly.

[0213] Lymphocytes and other immune cells express BMP receptors on their cell surfaces, and there is increasing evidence that BMP regulates the development and maturation of various humoral and cellular immune compartments and regulates humoral and cellular immune responses in the mature organism. The effects of BMP signaling on immune cells may be context-specific (as is well known for many cytokines of immunological importance), and thus it must be determined empirically whether they enhance or reduce the development or function of specific lymphocyte populations. BMP antagonism using compounds such as those described herein can be an effective protocol for deliberately skewing the development of the cellular, innate, or humoral immune compartments for therapeutic purposes, or for therapeutic skewing of immune responses in the mature immune system. These protocols can target congenital disorders of cellular, innate, or humoral immunity, or disorders in which the immune response is inappropriately attenuated (e.g., as an adjuvant to facilitate successful antigen sensitization when immunization by other means is difficult or ineffective), or disorders in which the immune response is excessive or inappropriate (e.g., autoimmunity and auto-sensitization). In some cases, BMP antagonists as described herein may also be effective for the deliberate induction of immune tolerance (i.e., in allotransplantation or autoimmunity).

[0214] G. Treatment of skin diseases

[0215] Expansion of cultured keratinocytes- In vitro, BMP inhibits keratinocyte proliferation and promotes differentiation (reviewed in Botchkarev et al. Differentiation 72:512-526, 2004). In patients in need of skin grafts (e.g., after burns), skin grafts are made from cultured keratinocytes. Keratinocytes can be derived from other animals (xenografts), but these are only temporary as they are generally rejected by the immune system. Keratinocytes can be derived from the patient himself or herself and can be grown in the laboratory into cell layers (cultured epithelial autografts). A patient is unlikely to reject keratinocytes derived from his / her own body. Addition of a BMP antagonist as described herein to a keratinocyte culture can be used to promote keratinocyte proliferation, enabling the patient to receive the graft more quickly.

[0216] Improved epithelialization - BMP6 is highly expressed in skin injury and high levels of BMP6 have been detected in chronic human wounds of different etiologies (Kaiser et al. J. Invest. Dermatol. 111:1145-1152, 1998). In mice overexpressing BMP6 in the skin, epithelial reformation and skin wound healing are significantly delayed (Kaiser et al. J. Invest. Dermatol. 111:1145-1152, 1998). Improved epithelial formation can reduce scar formation. Local or systemic administration of one or more of the crystalline compounds or compositions described herein is contemplated herein to enhance epithelial formation of skin wounds, for example, in the treatment of pressure ulcers (bedsores) or non-healing or poorly healing skin ulcers (e.g., in patients with peripheral vascular disease, diabetes, venous insufficiency). The compounds are also expected to reduce scar formation.

[0217] Promotion of hair growth - The growth of hair follicles on the scalp is cyclic, having three phases: anagen (growth phase), catagen (degenerative phase), and telogen (resting phase). Recent evidence suggests that BMP signaling delays the transition from telogen to anagen (Plikus et al. Nature 451:340-344, 2008). Inhibition of BMP signaling using one or more of the crystalline compounds or compositions described herein can shorten the telogen phase and increase the number of hair follicles in the anagen phase. One or more of the crystalline compounds or compositions described herein can be used to treat conditions where there is a deficiency of hair follicles or where hair loss occurs more frequently than hair growth. These conditions include androgenetic alopecia (male pattern baldness), alopecia greata, and telogen effluvium.

[0218] Treatment of psoriasis — Psoriasis is an inflammatory skin disorder that can occur following skin trauma and subsequent repair and inflammation (Koebner phenomenon). BMPs can be involved in the repair and inflammatory mechanisms that give rise to psoriasis, since overexpression of BMP6 in mouse skin results in skin lesions similar to those observed in patients with psoriasis (Blessing et al. J. Cell. Biol. 135:227-239, 1996). One or more of the crystalline compounds or compositions described herein can be administered locally or systemically to treat established psoriasis or to prevent its development following skin injury.

[0219] Treatment of corneal scarring — BMP6 expression is associated with conjunctival scarring (Andreev et al. Exp. Eye Res. 83:1162-1170, 2006). One or more of the crystalline compounds or compositions described herein can be used to prevent or treat corneal scarring and resulting blindness 。

[0220] H. Treatment of systemic hypertension

[0221] Infusion of BMP4 induces systemic hypertension in mice (Miriyala et al. Circulation 113:2818-2825, 2006). Vascular smooth muscle cells express multiple BMP ligands. BMPs increase the expression of voltage-gated potassium channels and thereby increase the contraction of vascular smooth muscle (Fantozzi et al. Am. J. Physiol. Lung Cell. Mol. Physiol. 291:L993-1004, 2006). Accordingly, it is contemplated herein that one or more of the crystalline compounds or compositions described herein inhibit BMP signaling, which can be used to lower blood pressure. A sustained reduction in blood pressure in patients with hypertension is expected to prevent myocardial infarction, congestive heart failure, cerebrovascular accident, and renal failure. Treatment as described herein can be used to target hypertension in specific vascular beds (such as in pulmonary hypertension) via local delivery (e.g., via an aerosol).

[0222] Treatment of pulmonary hypertension

[0223] BMP signaling contributes to the pathogenesis of pulmonary hypertension. For example, mice with reduced BMP4 levels are protected from pulmonary hypertension and pulmonary vascular remodeling induced by long-term breathing of low oxygen concentration (Frank et al. Circ. Res. 97:496-504, 2005). In addition, mutations in the gene encoding type II BMP receptor (BMPRII) are frequently found in patients with sporadic and familial pulmonary arterial hypertension. It is expected that reduced BMP signaling may cause pulmonary hypertension. However, Yu and colleagues (Yu et al. J. Biol. Chem. 280:24443-24450, 2008) reported that BMPRII deficiency anomalously increases BMP signaling through subtypes of BMP ligands, and thus increased BMP signaling may actually contribute to the development of pulmonary hypertension.

[0224] One or more of the crystalline compounds or compositions described herein can be used to prevent the development of pulmonary arterial hypertension in patients at risk of the disease (e.g., patients with BMPRII mutations), or to treat patients with idiopathic pulmonary arterial hypertension or acquired pulmonary hypertension. Reduced pulmonary hypertension in the individuals treated as described herein is expected to have reduced tachypnea, right ventricular hypertrophy, and right ventricular failure.

[0225] A pharmaceutical composition comprising one or more of the crystalline compounds or compositions described herein can be used to prevent the development of pulmonary arterial hypertension in patients at risk of the disease (e.g., patients with BMPRII mutations), or to treat patients with idiopathic pulmonary arterial hypertension or acquired pulmonary hypertension. Reduced pulmonary hypertension in the individuals treated as described herein is expected to have reduced tachypnea, right ventricular hypertrophy, and right ventricular failure.

[0226] Treatment of ventricular hypertrophy

[0227] BMP-10 levels are increased in the hypertrophied ventricles of rats with hypertension, and this BMP ligand induces hypertrophy in cultured neonatal rat ventricular myocytes (Nakano et al. Am. J. Physiol. Heart. Circ. Physiol. 293:H3396-3403, 2007). Inhibition of BMP-10 signaling can be used to prevent / treat ventricular hypertrophy. Ventricular hypertrophy can lead to congestive heart failure due to diastolic dysfunction. A pharmaceutical composition comprising one or more of the crystalline compounds or compositions described herein can prevent / treat congestive heart failure.

[0228] Treatment of neurological disorders

[0229] Treatment of spinal cord injuries and neuropathies- BMP is an effective inhibitor of axonal regeneration in the adult spinal cord after spinal cord injury (Matsuura et al., J. Neurochem. 2008). It has been reported that after spinal cord contusion, BMP expression is elevated in oligodendrocytes and astrocytes around the injury site. Intrathecal administration of noggin, a BMP inhibitor, results in enhanced motor activity and significant regrowth of the corticospinal tract after spinal cord contusion.

[0230] RGMa inhibits axonal growth, axonal recovery, and synapse reformation after spinal cord injury (effects blocked by antibodies against RGMa) (Hata et al., J. Cell. Biol. 173:47 - 58, 2006; Kyoto et al., Brain Res. 1186:74 - 86, 2007). RGMa enhances BMP signaling (Babitt et al., J. Biol. Chem. 280:29820 - 29827, 2005), suggesting that BMP signaling may be responsible for preventing axonal growth and axonal recovery.

[0231] Based on these considerations, it would be expected that treatment with one or more of the crystalline compounds or compositions described herein would increase axonal growth and axonal recovery after spinal cord injury. It would be expected that treatment as described herein would prevent / treat neuropathy associated with a broad spectrum of disorders, including diabetes. In addition, treatment with one or more of the crystalline compounds or compositions described herein as described herein can be used to treat pain and motor dysfunction associated with neuropathy.

[0232] Treatment of neurological disorders associated with central nervous system inflammation - BMP4 and BMP5 have been detected in multiple sclerosis and Creutzfeldt - Jakob disease lesions (Deininger et al., Acta Neuropathol. 90:76 - 79, 1995). BMP has also been detected in mice with experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (Ara et al., J. Neurosci. Res. 86:125 - 135, 2008). Treatment as described herein can be used to prevent or treat multiple sclerosis, as well as other neurological disorders associated with central nervous system inflammation or maladaptive injury repair processes mediated by BMP signaling.

[0233] Treatment of dementia- Inhibitors of BMP signaling can promote neurogenesis in murine neural progenitor cells (Koike et al., J. Biol. Chem. 282: 15843-15850, 2007). Treatment with one or more of the crystalline compounds or compositions described herein as described herein can be used to enhance neurogenesis in a variety of neurological disorders associated with accelerated loss of neurons, including cerebrovascular accidents and Alzheimer's disease, as well as other dementias.

[0234] Altered memory and learning - BMP signaling plays an important role in the development and maintenance of neurons involved in memory and cognitive behavior. For example, in a novel environment, mice lacking the BMP antagonist chordin have enhanced spatial learning but less exploratory activity (Sun et al., J. Neurosci. 27:7740-7750, 2007). Treatment with one or more of the crystalline compounds or compositions described herein as described herein can be used to alter or prevent memory or learning (e.g., induce amnesia due to anesthesia) or to prevent post-traumatic stress disorder conditions in other situations that may cause distress.

[0235] Treatment of atherosclerosis

[0236] Substantial evidence indicates that BMP ligands are pro-inflammatory and pro-atherogenic in the vessel wall (Chang et al., Circulation 116:1258-1266, 2007). Knockdown of BMP4 expression reduces inflammatory signals, while knockdown of BMP antagonists (e.g., follistatin or noggin) increases inflammatory signals. Treatment with one or more of the crystalline compounds or compositions described herein as described herein can be used to reduce vascular inflammation associated with atherosclerosis, autoimmune diseases, and other vasculitides. By reducing atherosclerosis, the treatment described herein will reduce acute coronary syndromes (angina and heart attacks), transient ischemic attacks, strokes, peripheral vascular disease, and other vascular ischemic events. In addition, to the extent that atherosclerosis contributes to the pathogenesis of aneurysm formation, the compounds described herein can be used to slow the progression of aneurysm formation, reduce the frequency of aneurysm structures, and the need for vascular surgery.

[0237] Since many of the BMPs and BMP-induced gene products that affect matrix remodeling are overexpressed in early atherosclerotic lesions, BMP signaling can promote plaque formation and progression (Bostrom et al., J Clin Invest. 91: 1800-1809, 1993; Dhore et al., Arterioscler Thromb Vasc Biol. 21: 1998-2003, 2001). Thus, BMP signaling activity in atherosclerotic plaques can represent a form of maladaptive injury-repair or can contribute to inflammation. Over time, BMP signaling can also induce populations of resident vascular cells or neovascular cells to differentiate into osteoblast-like cells, leading to intimal and medial calcification of blood vessels (Hruska et al., Circ Res. 97: 105-112, 2005). Calcific vascular disease or arteriosclerosis is associated with reduced vascular distensibility, as well as an increased risk of cardiovascular events and mortality, and is particularly problematic when associated with underlying atherosclerotic disease (Bostrom et al., Crit Rev Eukaryot Gene Expr. 10: 151-158, 2000). However, if signals that contribute to the progression of both atherosclerotic and calcific lesions can be intercepted, both can regress (Sano et al., Circulation. 103: 2955-2960, 2001). In some aspects, treatment with one or more of the crystalline compounds or compositions described herein, as described herein, can be used to limit the progression of atherosclerotic plaques and vascular calcification in vivo.

[0238] Treatment of Sjögren's syndrome

[0239] Sjögren's syndrome is an autoimmune disorder in which immune cells attack and damage the glands that produce tears and saliva. Sjögren's syndrome is considered a rheumatic disorder, meaning that it causes inflammation in joints, muscles, skin, and / or other organs. The hallmark symptoms of the disorder are dry mouth and dry eyes. Sjögren's syndrome can also cause dry skin, dry nose, and dry vagina, and can affect other organs of the body (including the kidneys, blood vessels, lungs, liver, pancreas, and brain). Sjögren's syndrome affects 1-4 million people in the United States and is currently the second most common autoimmune rheumatic disease in the United States. At the time of diagnosis, most people with Sjögren's are at least 40 years old, and women are nine times more likely to develop the disease. Sjögren's syndrome can occur as a primary rheumatic condition or as a secondary condition associated with other rheumatic diseases such as systemic lupus erythematosus ("lupus"), sclerosing cholangitis, or rheumatoid arthritis.

[0240] Sjögren's syndrome can damage vital organs of the body, with symptoms that can remain stable, worsen, or go into remission. Some patients only experience mild symptoms of dry eyes and dry mouth, while other patients experience cycles of good health followed by severe illness. Although many patients are able to manage problems based on symptoms, other patients suffer from blurred vision, persistent eye discomfort, recurrent oral infections, swollen parotid glands, hoarseness, and difficulty swallowing and eating. Debilitating fatigue and joint pain can severely impair quality of life.

[0241] There is currently no known cure for Sjögren's syndrome, nor is there a specific treatment to restore glandular secretions. Treatment is usually symptomatic and supportive (including moisture replacement therapies to relieve symptoms of dry eyes and dry mouth). Non-steroidal anti-inflammatory drugs can be used to treat musculoskeletal symptoms. For individuals with severe complications, corticosteroids or immunosuppressive drugs are often prescribed. These drugs can have serious side effects. In addition, the diagnosis of the disease is currently based on a combination of indications (such as objective and subjective dryness, autoantibodies, and mononuclear infiltration), and is mainly a process of ruling out other known diseases to achieve a diagnosis of Sjögren's syndrome. Therefore, not only is it necessary to accurately diagnose patients with Sjögren's syndrome, but it is also necessary to identify viable therapeutic targets for the treatment of the disease.

[0242] Bone morphogenetic protein 6 (BMP6) is a member of the TGF-β superfamily of growth factors. Expression of BMP6 has been detected in several different mammalian tissues and cell types (including smooth muscle cells, growth plate chondrocytes, bronchiolar epithelial cells, cornea, epidermis, salivary glands, and nervous system cells) (Blessing et al., J Cell Biol 135(1):227-239, 1996). In vitro, BMP6 has been shown to inhibit cell division, promote terminal epithelial differentiation, and induce endochondral bone formation, osteoblast differentiation, and neuronal maturation (Heikinheimo et al., Cancer Res 59:5815-5821, 1999).

[0243] DNA methylation is a major modification of the eukaryotic genome and plays an important role in mammalian development. The human proteins MECP2, MBD1, MBD2, MBD3, and MBD4 comprise a family of nuclear proteins related to the presence of a methyl-CpG binding domain (MBD) in each. With the exception of MBD3, each of these proteins is capable of binding specifically to methylated DNA. MECP2, MBD1, and MBD2 can also repress transcription of methylated gene promoters. Compared to other MBD family members, MECP2 is X-linked and subject to X inactivation.

[0244] X inactivation is an early developmental process in female mammals that silences one of a pair of X chromosomes by transcription, equalizing dosage between males and females. This process is regulated by several factors, including a region of the X chromosome called the X inactivation center (XIC). The XIST gene (X-inactive specific transcript, non-protein coding) is expressed only from the XIC of the inactive X chromosome. The transcript is spliced but does not encode a protein. The transcript remains in the nucleus where it coats the inactive X chromosome.

[0245] Also provided herein are methods of treating a subject having Sjögren's syndrome by selecting a subject having increased BMP6 expression and administering to the subject a therapeutically effective amount of an agent that inhibits the expression or activity of BMP6, wherein the agent is one or more of the crystalline compounds or compositions described herein. Also provided herein are methods of treating a subject having Sjögren's syndrome, the method comprising administering to the subject a therapeutically effective amount of one or more of the crystalline compounds or compositions described herein, thereby treating the subject.

[0246] Male patients with Sjögren's syndrome express XIST, a non-coding RNA that is not normally expressed in males. Also described is the finding that male patients with Sjögren's syndrome downregulate MECP2 and other proteins involved in DNA methylation. In some embodiments, the biological sample is a salivary gland (such as a minor salivary gland).

[0247] In a subset of male patients with Sjögren's syndrome, Y chromosome gene expression is downregulated, as are the expressions of ribosomal proteins that regulate RNA processing and viral replication and proteins that regulate DNA methylation. These findings provide additional markers that can be used for the diagnosis and treatment of Sjögren's syndrome.

[0248] Also provided are methods of treating a male subject having Sjögren's syndrome by selecting a male subject having increased XIST expression and administering to the subject a therapeutically effective amount of an agent that inhibits XIST expression. Also provided are methods of treating a male subject having Sjögren's syndrome by selecting a male subject having increased XIST expression and administering to the subject a therapeutically effective amount of one or more of the crystalline compounds or compositions described herein. Also provided are methods of treating a male subject having Sjögren's syndrome by selecting a male subject having decreased MECP2 expression and administering to the subject a therapeutically effective amount of one or more of the crystalline compounds or compositions described herein. Also provided herein are methods of treating a male subject having Sjögren's syndrome, the method comprising administering to the male subject a therapeutically effective amount of one or more of the crystalline compounds or compositions described herein, thereby treating the male subject.

[0249] Compared to healthy control subjects, patients with Sjogren's syndrome exhibit a statistically significant increase in BMP6 expression in the salivary glands. Overexpression of BMP6 in the salivary glands increases the electrical potential in the salivary glands. The present disclosure provides the discovery that administration of an inhibitor of BMP6 signaling increases salivary flow in the salivary glands, wherein the inhibitor is one or more of the crystalline compounds or compositions described herein.

[0250] The present disclosure provides methods for increasing salivary flow in a subject. In some embodiments, the method comprises administering to the subject an inhibitor of BMP6 signal transduction, wherein the inhibitor is one or more of the crystalline compounds or compositions described herein. In other embodiments, the method comprises selecting a subject having increased BMP6 expression in the salivary glands of the subject relative to a control, and administering to the subject an inhibitor of BMP6 signaling, wherein the inhibitor is one or more of the crystalline compounds or compositions described herein. In some cases, the subject has Sjogren's syndrome.

[0251] In some embodiments, the salivary glands exhibiting increased BMP6 expression are the minor labial salivary glands, parotid glands, or submandibular glands.

[0252] In some embodiments, an inhibitor of BMP6 is administered locally to the salivary glands, wherein the inhibitor is one or more of the crystalline compounds or compositions described herein.

[0253] In some embodiments, the inhibitor of BMP6 signaling inhibits BMP type I receptor ALK2 and / or BMP type I receptor ALK3, wherein the inhibitor is one or more of the crystalline compounds or compositions described herein.

[0254] In some embodiments, the biological sample is a tissue sample (such as salivary gland tissue (e.g., tissue obtained by biopsy of the salivary gland)). In some examples, the salivary gland is the minor labial salivary gland, parotid gland, or submandibular gland. In other embodiments, the biological sample is a body fluid sample (such as a saliva, tear, blood, or serum sample).

[0255] In some embodiments, the disclosed method further comprises providing appropriate therapy to a subject diagnosed with Sjogren's syndrome. In some examples, appropriate therapy includes administering an agent that promotes saliva production (e.g., one or more of the crystalline compounds or compositions described herein), administering a corticosteroid, administering an immunosuppressive drug, administering a non-steroidal anti-inflammatory drug, administering an agent that inhibits BMP6 expression or activity, administering an agent that inhibits BMP signaling (e.g., one or more of the crystalline compounds or compositions described herein), or any combination thereof.

[0256] Also provided is a method of increasing salivary flow in a subject by selecting a subject having increased BMP6 expression in a salivary gland and administering to the subject a therapeutically effective amount of an agent that inhibits the expression or activity of BMP6. In some embodiments, the agent is one or more of the crystalline compounds or compositions described herein. In some embodiments, the salivary gland is a minor labial salivary gland, a parotid gland, or a submandibular gland.

[0257] In some embodiments, the agent that inhibits BMP signaling is one or more of the crystalline compounds or compositions described herein.

[0258] In some embodiments, the agent that inhibits the expression or activity of BMP6 or the agent that inhibits BMP signaling is administered locally to the salivary gland, wherein the agent is one or more of the crystalline compounds or compositions described herein. In other embodiments, the agent that inhibits the expression or activity of BMP6 or the agent that inhibits BMP signaling is administered systemically, wherein the agent is one or more of the crystalline compounds or compositions described herein.

[0259] In some embodiments, the method further comprises providing appropriate therapy to a subject diagnosed with Sjögren's syndrome. In some embodiments, the appropriate therapy includes administering an agent that promotes saliva production (e.g., one or more of the crystalline compounds or compositions described herein), administering a corticosteroid, administering an immunosuppressive drug, administering a non-steroidal anti-inflammatory drug, administering an agent that inhibits the expression or activity of BMP6, administering an agent that inhibits BMP signaling (e.g., one or more compounds of formula I), or any combination thereof.

[0260] In some embodiments, the disclosed method further comprises providing appropriate therapy to a male subject diagnosed with Sjögren's syndrome. In some embodiments, the appropriate therapy includes administering an agent that promotes saliva production (e.g., one or more of the crystalline compounds or compositions described herein), administering a corticosteroid, administering an immunosuppressive drug, administering a non-steroidal anti-inflammatory drug, administering an agent that inhibits the expression or activity of BMP6, administering an agent that inhibits BMP signaling (e.g., one or more of the crystalline compounds or compositions described herein), administering an agent that inhibits XIST expression, administering a nucleic acid molecule encoding MECP2, or any combination thereof.

[0261] Also provided is a method of treating a male subject with Sjögren's syndrome by selecting a male subject having increased XIST expression and / or decreased MECP2 expression and (i) administering to the subject a therapeutically effective amount of an agent that inhibits XIST expression or (ii) administering to the subject a therapeutically effective amount of a nucleic acid molecule encoding MECP2 or both (i) and (ii).

[0262] There is also provided a method of increasing salivary flow in male subjects by selecting a subject having increased XIST expression and / or decreased MECP2 expression and administering to the subject (i) a pharmaceutically effective amount of an agent that inhibits XIST expression or (ii) a pharmaceutically effective amount of a nucleic acid molecule encoding MECP2 (such as a vector encoding MECP2) or both (i) and (ii).

[0263] Exemplary XIST inhibitors include, for example, antisense oligonucleotides or siRNA molecules that specifically hybridize to an XIST nucleic acid molecule. The XIST nucleic acid sequence is publicly available (such as the human XIST RNA sequence deposited under GenBank TM accession number NR-001564). Appropriate antisense oligonucleotides or siRNAs targeting XIST can be designed by those skilled in the art using the publicly available XIST sequence. The XIST antisense transcript Tsix is a known inhibitor of XIST that can be used in conjunction with the disclosed methods (Senner and Brockdorff, Curr Opin Genet Dev 19(2):122-126, 2009; Stavropoulos et al., Proc Natl Acad Sci USA 98(18):10232-10237,2001).

[0264] As described herein, significant alterations in sex chromosome gene expression (including increased XIST expression, decreased MECP2 expression, and marked silencing of Y chromosome gene expression) were identified in male SS patients. This gene expression pattern (termed autoimmune Xist Y chromosome inactivation syndrome (AXYIS)) was also identified in affected tissues from males diagnosed with autoimmune diseases associated with pSS (including rheumatoid arthritis, type II diabetes, systemic sclerosis, and lymphoma).

[0265] In particular, the following findings are described herein: in a subset of male Sjogren's syndrome patients, Y chromosome gene expression is downregulated (e.g., the expression of genes RPS4Y1, RPS4Y2, JAR1D1D, CYORF15B, and CYORF14 is downregulated), as are the expressions of ribosomal proteins that regulate RNA processing and viral replication (such as RPS4Y1, RPS4Y2, and RPS4X) and proteins that regulate DNA methylation (such as MDB6 and NASP). In addition, numerous duplications and / or deletions were identified in the opsins (OPN1LW, OPN1MW, and OPN1MW2) and the tex28 region of the X chromosome in male patients with Sjogren's syndrome. These findings provide additional markers that can be used for the diagnosis and treatment of Sjogren's syndrome in males.

[0266] The present invention provides a method for treating Sjogren's syndrome in a subject in need thereof (such as a subject having increased BMP6 expression in the salivary gland) by administering to the subject an agent that inhibits BMP6 (such as a compound that inhibits the expression (mRNA expression or protein expression) or at least one biological activity of BMP6), wherein the agent or compound is one or more of the crystalline compounds or compositions described herein. The agent or compound can also be an agent or compound that inhibits BMP signaling (such as one or more of the crystalline compounds or compositions described herein).

[0267] In various embodiments, the present invention provides a method for treating a subject having Sjogren's syndrome, or for increasing salivary flow in a subject, the method comprising selecting a subject having increased BMP6 expression in the salivary gland of the subject relative to a control, and administering to the subject a therapeutically effective amount of an agent that inhibits the expression or activity of BMP6 or an agent that inhibits BMP signaling, thereby treating a subject having Sjogren's syndrome or increasing salivary flow in the subject, wherein the agent is one or more of the crystalline compounds or compositions described herein. In some embodiments, the salivary gland is the minor labial salivary gland, the parotid gland, or the submandibular gland. In some embodiments, the agent that inhibits the expression or activity of BMP6 is one or more of the crystalline compounds or compositions described herein.

[0268] Treatment of diffuse intrinsic pontine glioma (DIPG)

[0269] Diffuse intrinsic pontine glioma (DIPG) (also known as diffuse intrinsic pontine glioma) is a tumor located in the pons (midbrain) of the brainstem. The brainstem is the lowest part of the brain, connecting the brain to the spinal cord. Diffuse intrinsic pontine glioma has been associated with a gain-of-function mutation in ACVR1 that is the same as that in progressive osseous heteroplasia.

[0270] In various embodiments, the present invention provides a method for treating a subject having diffuse intrinsic pontine glioma (DIPG), the method comprising: selecting a subject having diffuse intrinsic pontine glioma (DIPG), and administering to the subject a therapeutically effective amount of one or more of the crystalline compounds or compositions described herein, thereby treating a subject having diffuse intrinsic pontine glioma (DIPG).

[0271] Proliferation, transplantation and differentiation of progenitor cells (including embryonic and adult stem cells) in vitro and in vivo

[0272] For modulating the differentiation and regeneration of precursor cell populations and stem cell populations, and in some cases preventing (although in other cases directing) the differentiation of tissues towards lineages, BMP signaling is important. Treatment with one or more of the crystalline compounds or compositions described herein as described herein can be used for (i) maintaining a pluripotent state in a stem cell population or a pluripotent cell population in vivo or in vitro; (ii) expanding a stem cell population or a pluripotent cell population in vivo or in vitro; (iii) directly differentiating a stem cell population or a pluripotent cell population in vivo or in vitro; (iv) manipulating or directing the differentiation of a stem cell population or a pluripotent cell population in vivo or in vitro alone or in combination with other treatments or sequentially with other treatments; and (v) regulating a differentiated cell population to de-differentiate into a pluripotent population or a progenitor cell population.

[0273] Many stem cell lineages and precursor lineages require BMP signaling in order to determine whether they will expand, differentiate towards a particular tissue lineage, commit to a particular tissue type and integrate with or undergo programmed cell death in a particular tissue type. BMP signaling often interacts with signals provided by growth factors (bFGF, PDGF, VEGF, HBEGF, PIGF, etc.), Sonic Hedgehog (SHH), the Notch signaling pathway and the Wnt signaling pathway to effect these changes (Okita et al. Curr. Stem Cell Res. Ther. 1:103-111, 2006). Treatment with one or more of the crystalline compounds or compositions described herein as described herein can be used to direct the differentiation of stem cells (e.g., embryonic stem cells) or tissue progenitor cells towards a particular lineage for therapeutic applications (Park et al. Development 131:2749-2762, 2004; Pashmforoush et al. Cell 117:373-386, 2004). Alternatively, for certain cell populations, BMP inhibitors as described herein can be effective in preventing differentiation and promoting expansion in order to generate a sufficient number of cells effective for clinical applications. The exact dosage and / or combination of one or more of the crystalline compounds or compositions described herein and other BMP antagonists or one or more growth factors or one or more signaling molecules can be highly specific for each cell type and tissue type.

[0274] For example, certain embryonic stem cell lines require co-culture with leukemia inhibitory factor (LIF) to inhibit differentiation and maintain the pluripotency of certain cultured embryonic stem cell lines (Okita et al. Curr. Stein Cell Res. Ther. 1:103-111, 2006). Use of one or more of the crystalline compounds or compositions described herein can be used to maintain pluripotency in the absence of LIF. Other ES cell lines require co-culture with a specific feeder cell layer in order to maintain pluripotency. When concerns about contamination of the feeder cell layer or its DNA or protein components would complicate or preclude the use of the cells for human therapy applications, use of one or more of the crystalline compounds or compositions described herein (alone or in combination with other agents) can be effective in maintaining pluripotency.

[0275] In another embodiment, in some cases, antagonizing BMP signaling with a protein (such as noggin) shortly before withdrawal of LIF from culture can induce differentiation into the cardiomyocyte lineage (Yuasa et al. Nat. Biotechnol. 23:607-611, 2005). Use of a pharmacological BMP antagonist (such as one or more of the crystalline compounds or compositions described herein) can achieve a similar effect, if not a more effective one. Such differentiated cells can be therapeutically introduced into diseased myocardium. Alternatively, such treatment may actually be more effective for transplanted progenitor cells that have engrafted into diseased myocardium. Systemic therapy with a protein antagonist of BMP (such as noggin) would be extremely costly and would require complex dosing. Systemic or local delivery of a BMP antagonist as described herein can bias such progenitor cells towards in situ differentiation into functional cardiomyocytes.

[0276] Use of compounds in mammals

[0277] By using a dosage and administration regimen determined to be appropriate by those skilled in the art, a pharmaceutical composition comprising one or more crystalline compounds or compositions as described herein can be used to treat a subject (e.g., a human, domestic pet, livestock, or other animal), and these parameters can vary depending on, for example, the type and severity of the condition being treated, the overall health of the subject, the therapeutic index of the compound, and the route of administration. Standard clinical trials can be used to optimize the dosage and dosing frequency of any particular pharmaceutical composition comprising one or more crystalline compounds or compositions as described herein. Exemplary routes of administration that can be used include oral, parenteral, intravenous, intraarterial, subcutaneous, intramuscular, topical, intracranial, intraorbital, ocular, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or administration by suppository. Methods for making formulations that can be used with the methods and compositions described herein are well known in the art and can be found, for example, in Remington: The Science and Practice of Pharmacy (20th Edition, A. R. Gennaro), Lippincott Williams & Wilkins, 2000.

[0278] Inhibition of BMP signaling in insects

[0279] Compared to the BMP receptors of chordates, one or more crystalline compounds or compositions as described herein can be active, and possibly even selective, for the BMP receptors of arthropods. Inhibiting BMP signaling in arthropod larvae or eggs can cause severe developmental abnormalities and, when this pathway is inhibited, can impair their reproductive capacity, for example, via the same dorsalization observed in zebrafish and Drosophila. A BMP antagonist that is highly selective for arthropod BMP receptors compared to human BMP receptors can be used as an insecticide or pest control agent, which is apparently less toxic and more environmentally friendly than current strategies.

[0280] Ex vivo applications

[0281] In addition to administration to a patient in a therapeutic method, one or more crystalline compounds or compositions as described herein can also be used for ex vivo treatment of cells and tissues, as well as structural materials to be implanted into a patient (see above). For example, one or more crystalline compounds or compositions as described herein can be used to treat explant tissues that can be used, for example, in transplantation.

[0282] Treatment of hypercholesterolemia or hyperlipoproteinemia

[0283] Treatment with small molecule or recombinant BMP inhibitors reduces vascular inflammation (via macrophage accumulation and cathepsin activity), atherogenesis, and vascular calcification in mice lacking low density lipoprotein receptor (LDLR- / -). Without wishing to be bound by theory, as a potential explanation for the effect on vascular inflammation, it has been found that oxidized LDL (oxLDL) increases BMP2 expression and induces the production of reactive oxygen species (ROS) in human aortic endothelial cells. Based on the inhibition by small molecule or recombinant BMP inhibitors, the ROS production induced by oxLDL appears to require BMP signaling. Treatment with small molecule BMP inhibitors reduces plasma low density lipoprotein levels without inhibiting HMG-CoA reductase activity, indicating a role for BMP signaling in the regulation of LDL cholesterol biosynthesis. It has also been found that small molecule BMP inhibitors inhibit hepatosteatosis observed in LDLR-deficient mice fed a high fat diet. Small molecule or recombinant BMP inhibitors inhibit the synthesis of ApoB-100 in hepatoma cells in vitro. These findings implicate BMP signaling in vascular calcification and atherogenesis and provide at least two novel mechanisms by which BMP signaling may contribute to the pathogenesis of atherosclerosis. These studies highlight the BMP signaling pathway as a therapeutic target in the treatment of atherosclerosis while identifying several novel functions of BMP signaling in the regulation of vascular oxidative stress, inflammation, and lipid metabolism.

[0284] In various embodiments, one or more of the crystalline compounds or compositions described herein as described herein can be used to reduce the circulating levels of ApoB-100 in a patient. In various embodiments, one or more of the crystalline compounds or compositions described herein as described herein can be used to reduce the circulating levels of LDL in a patient. In various embodiments, one or more of the crystalline compounds or compositions described herein as described herein can be used to treat hypercholesterolemia, hyperlipidemia, or hyperlipoproteinemia (including congenital or acquired hypercholesterolemia, hyperlipidemia, or hyperlipoproteinemia). In some embodiments, the congenital hypercholesterolemia, hyperlipidemia, or hyperlipoproteinemia is autosomal dominant hypercholesterolemia (ADH), familial hypercholesterolemia (FH), polygenic hypercholesterolemia, familial combined hyperlipidemia (FCHL), hyperapobetalipoproteinemia, or small, dense LDL syndrome (LDL phenotype B).

[0285] In some embodiments, acquired hypercholesterolemia, acquired hyperlipidemia, or acquired hyperlipoproteinemia is associated with: diabetes, a high-fat diet and / or sedentary lifestyle, obesity, metabolic syndrome, endogenous or secondary liver disease, primary biliary cirrhosis or other cholestatic disorders, alcoholism, pancreatitis, nephrotic syndrome, end-stage renal disease, hypothyroidism, and iatrogenesis attributed to the administration of thiazides, beta-blockers, retinoids, highly active antiretroviral agents, estrogens, progesterones, or glucocorticoids. In various embodiments, one or more crystalline compounds or compositions as described herein can be used to treat diseases, disorders, or syndromes associated with defects in lipid absorption or lipid metabolism (such as sitosterolemia, cerebrotendinous xanthomatosis, or familial hypobetalipoproteinemia).

[0286] In various embodiments, one or more crystalline compounds or compositions as described herein can be used to treat diseases, disorders, or syndromes caused by hyperlipidemia (such as coronary artery disease and its manifestations (e.g., myocardial infarction; angina; acute coronary syndrome (such as unstable angina); cardiac dysfunction (such as congestive heart failure caused by myocardial infarction); or arrhythmias associated with myocardial ischemia / myocardial infarction), stroke attributed to occlusion of a portion of the cerebral arterial supply, intracerebral hemorrhage, peripheral arterial disease (e.g., mesenteric ischemia); renal artery stenosis; limb ischemia and claudication; subclavian steal syndrome; abdominal aortic aneurysm; thoracic aortic aneurysm, pseudoaneurysm, intramural hematoma; or penetrating aortic ulcer, dissecting aneurysm, aortic stenosis, vascular calcification, xanthomas (such as xanthomas affecting tendons or xanthelasmas of the sclera and skin), xanthomata, or hepatic steatosis).

[0287] In various embodiments, one or more crystalline compounds or compositions as described herein, or combinations thereof, can be used to treat the above diseases, disorders, or syndromes in individuals exhibiting normal circulating lipid levels or metabolism, regardless of circulating lipid levels.

[0288] In various embodiments, one or more crystalline compounds or compositions as described herein can be used to reduce secondary cardiovascular events caused by coronary artery vascular disease, cerebrovascular disease, or peripheral vascular disease. In various embodiments, one or more crystalline compounds or compositions as described herein can be used to treat an individual regardless of lipid level, e.g., to treat an individual exhibiting normal circulating cholesterol levels and circulating lipid levels. In various embodiments, one or more crystalline compounds or compositions as described herein can be co-administered with an HMG-CoA reductase inhibitor.

[0289] In various embodiments, one or more of the crystalline compounds or compositions described herein can be used for the prevention of cardiovascular disease in individuals having elevated cardiovascular risk markers (e.g., C-reactive protein) or, for example, an elevated Framingham Risk Score. In various embodiments, one or more of the crystalline compounds or compositions described herein can be used for the prevention of cardiovascular disease in individuals exhibiting normal circulating cholesterol levels and circulating lipid levels.

[0290] In various embodiments, one or more of the crystalline compounds or compositions described herein are used for the treatment or prevention of the foregoing diseases, conditions or syndromes, and the patient being treated is not diagnosed with and / or does not have one or more of the following conditions: vascular inflammation associated with atherosclerosis, autoimmune diseases and other vasculitides; atherosclerotic diseases, atherosclerotic plaques and / or vascular calcification; aneurysms and / or aneurysm formation; acute coronary syndromes (angina and heart attacks), transient ischemic attacks, strokes, peripheral vascular disease or other vascular ischemic events.

[0291] In various embodiments, one or more of the crystalline compounds or compositions described herein are used for the treatment or prevention of the foregoing diseases, conditions or syndromes (e.g., for reducing the circulating levels of ApoB-100 and / or LDL in a patient; for treating hypercholesterolemia, hyperlipidemia or hyperlipoproteinemia (including congenital or acquired hypercholesterolemia, hyperlipidemia or hyperlipoproteinemia); for treating diseases, conditions or syndromes associated with defects in lipid absorption or lipid metabolism; for treating diseases, conditions or syndromes caused by hyperlipidemia; for reducing secondary cardiovascular events caused by coronary vascular disease, cerebrovascular disease or peripheral vascular disease; or for reducing secondary cardiovascular events caused by coronary vascular disease, cerebrovascular disease or peripheral vascular disease), and the patient being treated is also diagnosed with and / or also has one or more of the following conditions: vascular inflammation associated with atherosclerosis, autoimmune diseases and other vasculitides; atherosclerotic diseases, atherosclerotic plaques and / or vascular calcification; aneurysms and / or aneurysm formation; acute coronary syndromes (angina and heart attacks), transient ischemic attacks, strokes, peripheral vascular disease or other vascular ischemic events.

[0292] T. Treatment of cartilage defects

[0293] Selective inhibition of specific BMP receptors enables chondrogenesis by preventing calcification and mineralization of scaffolds produced by mesenchymal stem cells (Hellingman et al., Tissue Eng Part A. 2011 Apr;17(7-8):1157-67. Epub Jan 17, 2011). Thus, in some embodiments, the compounds of the invention as described herein may be useful for promoting cartilage repair / regeneration in patients with cartilage injury or cartilage defect, and for generating cartilage tissue (e.g., for implantation) ex vivo or in vitro from suitable cells such as mesenchymal stem cells.

[0294] U. Use of compounds with varying degrees of selectivity: compounds that inhibit BMP signaling via specific BMP type I receptors, or compounds that also affect signaling via TGF-β, activin, AMP kinase or VEGF receptors Conduction, or compounds that also affect signaling via TGF-β, activin, AMP kinase or VEGF receptors.

[0295] In various embodiments, several of the compounds of the invention as described herein will have relatively greater selectivity for specific BMP type I receptors. The pathogenesis of certain diseases may be attributed to abnormal signaling of a specific receptor. For example, fibrodysplasia ossificans progressiva is a disease caused by abnormal (constitutively active) ALK2 function (Yu et al., Nat. Chem. Biol. 4:33-41, 2008). In such cases, in various embodiments, the compounds of the invention as described herein that specifically antagonize the function of BMP type I receptor subtypes may have the advantages of reduced toxicity or side effects or greater efficacy or both.

[0296] In some embodiments, the compounds of the invention as described herein may have a high selectivity for BMP signaling compared to TGF-β signaling, activin signaling, AMP kinase signaling, and VEGF receptor signaling. Other compounds may have less specificity and may target other pathways in addition to BMP signaling. For example, in the treatment of tumors, when the molecular phenotype of a tumor in a particular patient reveals dysregulation of multiple pathways, an agent that inhibits BMP signaling as well as one or more of the above pathways may have a beneficial effect (e.g., reducing tumor size).

[0297] In some embodiments, the compounds of the invention as described herein (e.g., one or more crystalline compounds or compositions described herein) are highly selective for ALK2 compared to ALK1 or ALK3 or ALK4 or ALK5 or ALK6. Selective inhibition of ALK2 compared to ALK1 or ALK3 or ALK4 or ALK5 or ALK6 can minimize adverse effects or toxicity. Due to the known importance of intestinal crypt stem cell recycling and the significance of ALK3 function in juvenile polyposis, chronic ALK3 inhibition may impair normal mucosal epithelial cell renewal. ALK1 inhibition may impair normal vascular remodeling and result in complications similar to human hereditary hemorrhagic telangiectasia type 2 (HHT2) such as capillary leakage, arteriovenous malformations, and bleeding. Thus, compounds that selectively inhibit ALK2 relative to ALK3 and ALK1 can help avoid this type of toxicity that may be encountered with the use of non-selective inhibitors.

[0298] In certain embodiments, the invention provides methods of inhibiting ALK2 activity in a human, the methods comprising administering to the human one or more crystalline compounds or compositions described herein, wherein the one or more crystalline compounds or compositions described herein selectively inhibit the activity of human ALK2 relative to the activity of human ALK1. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is about 1 / 2 of its IC 50 for inhibiting the activity of human ALK1. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 5 of its IC 50 for inhibiting the activity of human ALK1. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 10 of its IC 50 for inhibiting the activity of human ALK1. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 15 or 1 / 20 or 1 / 30 or 1 / 40 or 1 / 50 or 1 / 100 or 1 / 200 or 1 / 300 or 1 / 400 or 1 / 500 or 1 / 600 or 1 / 800 or 1 / 1000 or 1 / 1500 or 1 / 2000 or 1 / 5000 or 1 / 10000 or 1 / 15000 or 1 / 20000 or 1 / 40000 or 1 / 50000 or 1 / 60000 or 1 / 70000 or 1 / 80000 or 1 / 90000 or 1 / 100000 of its IC 50 for inhibiting the activity of human ALK1.

[0299] In certain embodiments, the present invention provides a method of inhibiting ALK2 activity in a human, the method comprising administering to the human one or more of the crystalline compounds or compositions described herein, wherein the one or more crystalline compounds or compositions described herein selectively inhibit the activity of human ALK2 relative to the activity of human ALK3. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 15 of its IC 50 for inhibiting the activity of human ALK3. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 20 of its IC 50 for inhibiting the activity of human ALK3. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 30 of its IC 50 for inhibiting the activity of human ALK3. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 50 or 1 / 100 or 1 / 200 or 1 / 300 or 1 / 400 or 1 / 500 or 1 / 600 or 1 / 800 or 1 / 1000 or 1 / 1500 or 1 / 2000 or 1 / 5000 or 1 / 10000 or 1 / 15000 or 1 / 20000 or 1 / 40000 or 1 / 60000 or 1 / 70000 or 1 / 80000 or 1 / 90000 or 1 / 100000 of its IC 50 for inhibiting the activity of human ALK3.

[0300] In certain embodiments, the present invention provides a method of inhibiting ALK2 activity in a human, the method comprising administering to the human one or more of the crystalline compounds or compositions described herein, wherein the one or more crystalline compounds or compositions described herein selectively inhibit the activity of human ALK2 relative to the activity of human ALK4. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 1000 of its IC 50 for inhibiting the activity of human ALK4. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 2000 of its IC 50 for inhibiting the activity of human ALK4. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC50 is 1 / 3000 of its IC 50 for inhibiting human ALK4 activity. In some such embodiments, one or more of the crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 3000 of its IC 50 for inhibiting human ALK4 activity. Or 1 / 4000 or 1 / 5000 or 1 / 6000 or 1 / 7000 or 1 / 8000 or 1 / 9000 or 1 / 10000 or 1 / 12000 or 1 / 14000 or 1 / 16000 or 1 / 18000 or 1 / 20000 or 1 / 25000 or 1 / 30000 or 1 / 40000 or 1 / 50000 or 1 / 60000 or 1 / 70000 or 1 / 80000 or 1 / 90000 or 100000.

[0301] In certain embodiments, the present invention provides a method for inhibiting ALK2 activity in a human, the method comprising administering to the human one or more of the crystalline compounds or compositions described herein, and relative to the activity of human ALK6, one or more of the crystalline compounds or compositions described herein selectively inhibit the activity of human ALK2. In some such embodiments, one or more of the crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 2 of its IC 50 for inhibiting human ALK6 activity. In some such embodiments, one or more of the crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 5 of its IC 50 for inhibiting human ALK6 activity. In some such embodiments, one or more of the crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 10 of its IC 50 for inhibiting human ALK6 activity. In some such embodiments, one or more of the crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 15 or 1 / 20 or 1 / 30 or 1 / 40 or 1 / 50 or 1 / 100 or 1 / 200 or 1 / 300 or 1 / 400 or 1 / 500 or 1 / 600 or 1 / 800 or 1 / 1000 or 1 / 1500 or 1 / 2000 or 1 / 5000 or 1 / 10000 or 1 / 15000 or 1 / 20000 or 1 / 40000 or 1 / 50000 or 1 / 60000 or 1 / 70000 or 1 / 80000 or 1 / 90000 or 1 / 100000 of its IC 50 for inhibiting human ALK6 activity.

[0302] In one aspect, the present invention provides a method of inhibiting ALK2 activity in a human, the method comprising administering to the human one or more of the crystalline compounds or compositions described herein, wherein the one or more crystalline compounds or compositions described herein selectively inhibit the activity of human ALK2 relative to the activity of human ALK5. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 1000 of its IC 50 for inhibiting the activity of human ALK5. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 2000 of its IC 50 for inhibiting the activity of human ALK5. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 3000 of its IC 50 for inhibiting the activity of human ALK5. In some such embodiments, the one or more crystalline compounds or compositions described herein inhibit the activity of human ALK2, and its IC 50 is 1 / 4000 or 1 / 5000 or 1 / 6000 or 1 / 7000 or 1 / 8000 or 1 / 9000 or 1 / 10000 or 1 / 12000 or 1 / 14000 or 1 / 16000 or 1 / 18000 or 1 / 20000 or 1 / 25000 or 1 / 30000 or 1 / 40000 or 1 / 50000 or 1 / 60000 or 1 / 70000 or 1 / 80000 or 1 / 90000 or 1 / 100000 of its IC 50 for inhibiting the activity of human ALK5.

[0303] As used herein, a therapeutic agent that "prevents" a disease or condition refers to a compound that reduces the occurrence or frequency of the disease or condition in a treated sample relative to an untreated control sample in a statistical sample, or delays the onset or reduces the severity of one or more symptoms of the disease or condition relative to an untreated control sample. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable cancerous growths in a treated population relative to an untreated control population, e.g., by a statistically and / or clinically significant amount. Prevention of infection includes, for example, reducing the number of diagnoses of infection in a treated population relative to an untreated control population, and / or delaying the onset of infection symptoms in a treated population compared to an untreated control population. Prevention of pain includes, for example, reducing the degree or delaying the sensation of pain experienced by subjects in a treated population relative to an untreated control population.

[0304] The term "treatment" includes prophylactic and / or therapeutic treatment. The term "prophylactic or therapeutic" treatment is well recognized in the art and includes administering to a host one or more of the subject compositions. If administered before the clinical manifestation of an undesired condition (e.g., a disease or other undesired state of a host animal), then the treatment is prophylactic (i.e., it protects the host from developing the unwanted condition), whereas if administered after the manifestation of an undesired condition, the treatment is therapeutic (i.e., it is intended to reduce, ameliorate, or stabilize an existing undesired condition or its side effects).

[0305] Combination therapies

[0306] In certain cases, one or more of the crystalline compounds or compositions described herein can be used in combination with other current or future pharmaceutical therapies, because the effect of inhibiting BMP alone may not be optimal per se, and / or in combination with therapies that act on different pathways that functionally interact with BMP signaling or therapies that act on the BMP pathway itself, may have a synergistic effect or be more effective. In certain cases, co-administration of a BMP inhibitor (e.g., one or more of the crystalline compounds or compositions described herein) with an additional pharmaceutical therapy reduces the dose of the additional pharmaceutical therapy such that it is less than the amount required to achieve a therapeutic effect when used as a single therapy (e.g., in the absence of a BMP inhibitor as described herein).

[0307] Some non-limiting examples of combination therapies can include the following.

[0308] Co-administration of erythropoietin (Epogen) and a BMP antagonist as described herein can be particularly effective for certain types of anemia of inflammation as described above, particularly in diseases in which both chronic inflammation and erythropoietin deficiency contribute to promoting anemia, such as end-stage renal disease.

[0309] In certain embodiments, a BMP inhibitor as described herein (e.g., one or more of the crystalline compounds or compositions described herein) can be administered in combination with other antihyperlipidemic or antilipemic agents, including but not limited to HMG-CoA reductase inhibitors (e.g., atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, or simvastatin), fibrates (e.g., bezafibrate, ciprofibrate, clofibrate, gemfibrozil, or fenofibrate), ezetimibe, niacin, cholesterol ester transfer protein (CETP) inhibitors (e.g., torcetrapib, anacetrapib, or dalcetrapib), cholestyramine, colestipol, probucol, dextrothyroxine, bile acid sequestrants, or combinations thereof.

[0310] In certain embodiments, a BMP inhibitor as described herein (e.g., one or more of the crystalline compounds or compositions described herein) can be administered in combination with the treatment of diabetes, including but not limited to sulfonylureas (e.g., chlorpropamide, tolbutamide, glyburide, glipizide, or glimepiride), drugs that reduce the amount of glucose produced by the liver (e.g., metformin), meglitinides (e.g., repaglinide or nateglinide), drugs that reduce the absorption of carbohydrates from the intestine (e.g., α-glucosidase inhibitors such as acarbose), drugs that affect blood glucose control (e.g., pramlintide or exenatide), DPP-IV inhibitors (e.g., sitagliptin), insulin therapy, thiazolidinones (e.g., troglitazone, ciglitazone, pioglitazone, or rosiglitazone), oxadiazolidinediones, α-glucosidase inhibitors (e.g., miglitol or acarbose), agents that act on the ATP-dependent potassium channels of β-cells (e.g., tolbutamide, glyburide, glipizide, glicazide, or repaglinide), nateglinide, glucagon inhibitors, inhibitors of hepatic enzymes involved in the stimulation of gluconeogenesis and / or glycogenolysis, or combinations thereof.

[0311] In certain embodiments, a BMP inhibitor as described herein (e.g., one or more of the crystalline compounds or compositions described herein) can be co-administered in combination with a treatment for obesity, which includes but is not limited to orlistat, sibutramine, phentermine, phenylbutazone, diethylpropion, benzphetamine, clobenzorex, dextroamphetamine, rimonabant, cetilistat, GT 389-255, APD356, pramlintide / AC137, PYY3-36, AC 162352 / PYY3-36, oxyntomodulin, TM 30338, AOD 9604, oleoyl-estrone, bromocriptine, ephedrine, leptin, pseudoephedrine, or a pharmaceutically acceptable salt thereof, or a combination of the foregoing.

[0312] In certain embodiments, a BMP inhibitor as described herein (e.g., one or more of the crystalline compounds or compositions described herein) can be co-administered in combination with an anti-hypertensive agent, which includes but is not limited to beta-blockers (e.g., alprenolol, atenolol, timolol, pindolol, propranolol, and metoprolol), ACE (angiotensin-converting enzyme) inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, quinapril, and ramipril), calcium channel blockers (e.g., nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem, and verapamil), and alpha-blockers (e.g., doxazosin, urapidil, prazosin, and terazosin), or a combination of the foregoing. In certain embodiments, a BMP inhibitor as described herein can be co-administered in combination with a treatment for anemia (e.g., anemia associated with renal failure and hemodialysis), which includes but is not limited to erythropoiesis-stimulating agents (e.g., erythropoietin).

[0313] Tyrosine kinase receptor inhibitors (such as SU-5416) and BMP inhibitors as described herein (e.g., one or more crystalline compounds or compositions described herein) may have a synergistic effect in inhibiting angiogenesis, particularly for anti-angiogenic therapy against tumors. BMP signaling (BMP-4) is thought to be critical for the commitment of stem or progenitor cells to hematopoietic / endothelial common progenitors and can promote the proliferation, survival, and migration of mature endothelial cells required for angiogenesis (Park et al. Development 131:2749-2762, 2004). Thus, antagonism of BMP signaling using compounds as described herein can provide additional inhibition of angiogenesis at the endothelial precursor and cell levels. Similarly, co-treatment with BMP inhibitors as described herein (e.g., one or more crystalline compounds or compositions described herein) and other tyrosine kinase receptor inhibitors (such as imatinib (Gleevec)) can be used to inhibit vascular remodeling and angiogenesis in certain tumors.

[0314] A combination of a Sonic Hedgehog agonist and a BMP inhibitor as described herein (e.g., one or more crystalline compounds or compositions described herein) may be particularly useful for promoting hair growth, as SHH activity is known to stimulate the transition of hair follicles from the telogen (resting) phase of hair growth (Paladini et al. J. Invest. Dermatol. 125:638-646, 2005), while inhibition of the BMP pathway shortens the telogen phase of hair growth (Plikus et al. Nature 451:340-344, 2008). It would be expected that the use of both would result in a relatively increased duration of the anagen or growth phase of hair growth.

[0315] In applications aimed at inhibiting bone differentiation, the combined use of a Notch regulator (e.g., a γ-secretase inhibitor) and a BMP antagonist as described herein (e.g., one or more crystalline compounds or compositions described herein) may be more effective than either agent alone, as increasing evidence suggests that the two pathways act together to influence cell differentiation (Kluppel et al. Bioessays 27:115-118, 2005). These therapies may have a synergistic effect in the treatment of tumors in which one or both pathways are disrupted (Katoh, Stem Cell Rev . 3:30-38, 2007).

[0316] The combined use of Indian hedgehog factor (IHH) antagonists and BMP antagonists (e.g., one or more crystalline compounds or compositions as described herein) can inhibit pathological bone formation. IHH is responsible for the commitment of bone precursors to chondrocytes or chondrogenic cells. Endochondral bone formation involves the coordinated activities of both chondrogenesis (facilitated by BMP and IHH signals) and its subsequent calcification due to the mineralization program initiated by BMP signals (Seki et al. J. Biol. Chem. 279:18544-18549, 2004; Minina et al. Development 128:4523-4534, 2001). Thus, co-administration of an IHH antagonist with one or more crystalline compounds or compositions as described herein can more effectively inhibit pathological bone growth (such as FOP) attributable to overactive BMP signaling or any of the inflammatory or traumatic conditions of pathological bone formation described above.

[0317] There is strong experimental evidence for the efficacy of both Smo antagonism and BMP antagonism in the treatment of glioblastoma. Compounds as described herein (e.g., one or more crystalline compounds or compositions as described herein) can be used in combination with Smo antagonists to treat glioblastoma.

[0318] In some embodiments, one or more crystalline compounds or compositions as described herein are co-administered with an agent selected from the group consisting of: corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), lipoxygenase inhibitors, leukotriene inhibitors, mast cell stabilizers, antihistamine drugs, tumor necrosis factor (TNF) inhibitors, IL-23 blockers, IL1-RA therapy, cytotoxic therapy, bisphosphonates, antirheumatic drugs, CTA4-Ig therapy, anti-growth factor therapy, and inhibitors of interleukin-1 signaling.

[0319] Exemplary corticosteroids for use in combination with one or more crystalline compounds or compositions as described herein include, but are not limited to, prednisone, cortisol, and hydrocortisone. In one embodiment, the corticosteroid is prednisone.

[0320] Exemplary NSAIDs for use in combination with one or more crystalline compounds or compositions as described herein include, but are not limited to, naproxen, ibuprofen, meloxicam, diclofenac, aspirin, piroxicam, sulindac, meclofenamic acid, and indomethacin.

[0321] In some embodiments, one or more of the crystalline compounds or compositions described herein are administered in combination with a lipoxygenase inhibitor such as sodium meclofenamate or zileuton.

[0322] Exemplary leukotriene inhibitors for use in combination with one or more of the crystalline compounds or compositions described herein include, for example, montelukast, zafirlukast, and pranlukast.

[0323] Non-limiting examples of mast cell stabilizers for use in combination with one or more of the crystalline compounds or compositions described herein include, but are not limited to, sodium cromoglycate, cromolyn acid, ketotifen, olopatadine, omalizumab, pemirolast, quercetin, theophylline, caffeine, paraxanthine, aminophylline, and theobromine.

[0324] In some embodiments, one or more of the crystalline compounds or compositions described herein are administered in combination with an antihistamine drug (e.g., diphenhydramine, cetirizine, ranitidine, famotidine, chlorpheniramine, chlorodiphenhydramine, and fexofenidine, etc.).

[0325] Exemplary anti-tumor necrosis factor (anti-TNF) drugs expected to be used in combination with one or more of the crystalline compounds or compositions described herein include, but are not limited to, infliximab, etanercept, adalimumab, certolizumab, bupropion, and golimumab.

[0326] Exemplary inhibitors of interleukin-23 (IL-23) signaling expected to be used in combination with one or more of the crystalline compounds or compositions described herein include, but are not limited to, ustekinumab and BI-855066.

[0327] Exemplary inhibitors of interleukin-1 (IL-1) signaling or IL-1RA therapy expected to be used in combination with one or more of the crystalline compounds or compositions described herein include, but are not limited to, anakinra, canakinumab, and rilonacept.

[0328] Exemplary cytotoxic therapies for use in combination with one or more of the crystalline compounds or compositions described herein include, but are not limited to, methotrexate, cyclophosphamide, 5-fluorouracil, doxorubicin, vincristine, bleomycin, procarbazine, prednisilone, dacarbazine, etoposide, cisplatin, oxaliplatin, and the like.

[0329] Exemplary bisphosphonates for use in combination with one or more of the crystalline compounds or compositions described herein include, but are not limited to, alendronate (FOSAMAX TM ), ibandronate (BONIVA TM ), risedronate (ACTONEL TM , ATELVIA TM ), and zoledronic acid (RECLAST TM ).

[0330] Exemplary anti-growth factor therapies for use in combination with one or more of the crystalline compounds or compositions described herein include, but are not limited to, anti-PDGF therapy, anti-FGF therapy, and anti-VEGF therapy.

[0331] Exemplary disease modifying anti-rheumatic drugs for use in combination with one or more of the crystalline compounds or compositions described herein include, but are not limited to, azathioprine (IMURAN TM ), cyclophosphamide (CYTOXAN TM ), cyclosporine (NEORAL TM ), hydroxychloroquine (PLAQUENIL TM ), leflunomide (ARAVA TM ), methotrexate (RHEUMATREX TM , TREXALL TM ), sulfasalazine (AZULFIDINE TM ), and tofacitinib (XELJANZ TM ), and the like.

[0332] In a further embodiment, one or more of the crystalline compounds or compositions described herein can be administered in combination with cyclosporine, mycophenylate mofetil, and the like.

[0333] In some embodiments, one or more of the crystalline compounds or compositions described herein are administered in combination with at least one additional agent, wherein the at least one additional agent comprises an anti-inflammatory agent. Exemplary anti-inflammatory agents include, but are not limited to, inhibitors of the activity of substance P; inhibitors of the secretion of substance P; inhibitors of the effects of substance P; inhibitors of the activity of histamine; inhibitors of the secretion of histamine; inhibitors of the effects of histamine; inhibitors of mast cell function; inhibitors of Toll-like receptor signaling; inhibitors of MyD88; inhibitors of TRIF; apyrase; and agents that catalyze the hydrolysis of ATP.

[0334] In some embodiments, one or more of the crystalline compounds or compositions described herein are administered in combination with at least one additional agent, wherein the at least one additional agent comprises an anti-growth factor agent. Exemplary anti-growth factor agents include, but are not limited to, inhibitors of PDGF ligands; inhibitors of PDGF-AA; inhibitors of PDGF-BB; inhibitors of PDGFR-α receptor function; inhibitors of PDGFR-β receptor function; neutralizing antibodies against activin A; neutralizing antibodies against activin B; neutralizing antibodies against activin A ligands; neutralizing antibodies against activin B ligands; neutralizing antibodies against heterodimeric ligands containing inhibin bA subunits encoded by INHBA; neutralizing antibodies against heterodimeric ligands containing inhibin bB subunits encoded by the INHBB gene; ligand traps for BMP ligands; ligand traps for activin ligands; ligand traps for the soluble extracellular domain of type II activin receptor ActRIIA; ligand traps for the soluble extracellular domain of type II activin receptor ActRIIB; ligand traps for the soluble extracellular domain of BMP type I receptor ALK2; ligand traps for the soluble extracellular domain of BMP type I receptor ALK3; and ligand traps for the soluble extracellular domain of BMP type I receptor ALK6.

[0335] In some embodiments, one or more of the crystalline compounds or compositions described herein are administered in combination with at least one additional agent, wherein the at least one additional agent comprises an anti-osteogenic signaling agent or an anti-chondrogenic signaling agent. Exemplary anti-osteogenic signaling agents or anti-chondrogenic signaling agents include, but are not limited to, RAR-γ agonists; non-selective RAR agonists; agents that inhibit the activity of the osteogenic transcription factor Runx2; agents that inhibit the expression of the osteogenic transcription factor Runx2; agents that promote the degradation of the osteogenic transcription factor Runx2; agents that inhibit the activity of the chondrogenic transcription factor Sox9; agents that inhibit the expression of the chondrogenic transcription factor Sox9; agents that promote the degradation of the chondrogenic transcription factor Sox9; inhibitors of HIF-1α activity; and inhibitors of HIF-1α expression.

[0336] In certain embodiments, the compounds of the invention can be used alone or administered in combination with another type of therapeutic agent. As used herein, the phrase "administered in combination" refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered when the previously administered therapeutic compound is still active in the body (e.g., the two compounds are concurrently active in the subject, which can include a synergistic effect of the two compounds). For example, the different therapeutic compounds can be administered concomitantly or sequentially in the same dosage form or in separate dosage forms. In certain embodiments, the different therapeutic compounds can be administered to each other within 1 hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or one week. In some embodiments, an additional therapeutic compound is administered within about 5 minutes to about 168 hours before or after administering a compound of formula (I), a compound of formula (II), or a compound of formula (III). Thus, a subject receiving such treatment can benefit from the combined effects of the different therapeutic compounds.

[0337] In certain embodiments, the combined administration of a compound of the invention with one or more additional therapeutic agents (e.g., one or more additional chemotherapeutic agents) provides improved efficacy relative to the separate administration of each of the compound of the invention or the one or more additional therapeutic agents. In certain such embodiments, the combined administration provides an additive effect, where an additive effect refers to the sum of the effects of the separate administrations of the compound of the invention and each of the one or more additional therapeutic agents.

[0338] When used in combination, one or more of the crystalline compounds or compositions described herein can be administered alone or in a formulation different from that of at least one additional agent as described herein, or can be administered in a single formulation comprising one or more of the crystalline compounds or compositions described herein and the additional agent. One or more of the crystalline compounds or compositions described herein can be administered simultaneously or concurrently with at least one additional agent. The one or more of the crystalline compounds or compositions described herein can be administered using the same or different modes of administration (e.g., oral, intravenous, injection, etc.). Administration of one or more of the crystalline compounds or compositions described herein and at least one additional agent can occur simultaneously within 15 min, within 30 min, or can be separated by at least 1 hour (e.g., at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, or more). Those skilled in the art can readily determine an appropriate dosing regimen for combination therapy comprising one or more of the crystalline compounds or compositions described herein and at least one additional agent, e.g., to reduce side effects, to prevent metabolic interference from one of the agents, to enhance the activity of one or more of the crystalline compounds or compositions described herein, or to otherwise improve pharmacodynamic or pharmacokinetic factors.

[0339] It is contemplated herein that the combination of at least one additional agent as described above with one or more of the crystalline compounds or compositions described herein can produce a synergistic effect that is greater than the sum of the effects of each agent administered alone. In such an embodiment, it is contemplated that a lower dose of one or more of the crystalline compounds or compositions described herein will be administered in combination with a second agent compared to the dose required for a therapeutic effect when one or more of the crystalline compounds or compositions described herein are administered alone.

[0340] Dose and administration

[0341] In one aspect, the methods described herein provide methods for treating a disease or disorder (including abnormal bone formation) (e.g., heterotopic ossification disease) in a subject. In one embodiment, the subject can be a mammal. In another embodiment, the mammal can be a human, although the methods are effective for all mammals. The methods comprise administering to the subject an effective amount of a pharmaceutical composition comprising one or more of the crystalline compounds or compositions described herein.

[0342] The dosage range of the medicament depends on the potency and includes an amount large enough to produce the desired effect (e.g., a reduction in at least one symptom of abnormal bone formation). The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary with the type of inhibitor (e.g., antibody or fragment, small molecule, siRNA, etc.) and the age, condition, and sex of the patient. The dosage can be determined by those skilled in the art and can also be adjusted by an individual physician in the event of any complications. Generally, the dosage varies from 0.1 mg / kg body weight to 1 g / kg body weight. In some embodiments, the dosage range is from 0.1 mg / kg body weight to 1 g / kg body weight, from 0.1 mg / kg body weight to 500 mg / kg body weight, from 0.1 mg / kg body weight to 250 mg / kg body weight, from 0.1 mg / kg body weight to 100 mg / kg body weight, from 0.1 mg / kg body weight to 50 mg / kg body weight, from 0.1 mg / kg body weight to 10 mg / kg body weight, from 10 mg / kg to 100 mg / kg, from 15 mg / kg to 100 mg / kg, from 20 mg / kg to 100 mg / kg, from 25 mg / kg to 100 mg / kg, from 30 mg / kg to 100 mg / kg, from 40 mg / kg to 100 mg / kg, from 50 mg / kg to 100 mg / kg, from 60 mg / kg to 100 mg / kg, from 70 mg / kg to 100 mg / kg, from 75 mg / kg to 100 mg / kg, from 25 mg / kg to 50 mg / kg, from 50 mg / kg to 200 mg / kg, from 75 mg / kg to 250 mg / kg, from 100 mg / kg to 300 mg / kg, from 100 mg / kg to 200 mg / kg, from 100 mg / kg to 400 mg / kg, from 100 mg / kg to 500 mg / kg, from 100 mg / kg to 750 mg / kg from 200 mg / kg to 1000 mg / kg, from 300 mg / kg to 1000 mg / kg, from 400 mg / kg to 1000 mg / kg, from 500 mg / kg to 1000 mg / kg, from 600 mg / kg to 1000 mg / kg, from 700 mg / kg to 1000 mg / kg, from 800 mg / kg to 1000 mg / kg, from 900 mg / kg to 1000 mg / kg, from 250 mg / kg to 750 mg / kg, from 300 mg / kg to 600 mg / kg, or any range therebetween.

[0343] In certain embodiments, the dosage of the agent is at least 10 mg / kg / day; in other embodiments, the dosage of the agent is at least 20 mg / kg / day, at least 25 mg / kg / day, at least 30 mg / kg / day, at least 40 mg / kg / day, at least 50 mg / kg / day, at least 60 mg / kg / day, at least 70 mg / kg / day, at least 80 mg / kg / day, at least 90 mg / kg / day, at least 100 mg / kg / day, at least 125 mg / kg / day, at least 150 mg / kg / day, at least 175 mg / kg / day, at least 200 mg / kg / day, at least 250 mg / kg / day, at least 300 mg / kg / day, at least 400 mg / kg / day, at least 500 mg / kg / day or more.

[0344] In some embodiments, the dosage range of the agent for use in human subjects is from 10 mg / day to 250 mg / day, from 15 mg / day to 200 mg / day, from 20 mg / day to 200 mg / day, from 25 mg / day to 200 mg / day, from 25 mg / day to 175 mg / day, from 25 mg / day to 150 mg / day, from 25 mg / day to 125 mg / day, from 25 mg / day to 100 mg / day, from 25 mg / day to 75 mg / day, from 25 mg / day to 50 mg / day, from 50 mg / day to 200 mg / day, from 75 mg / day to 200 mg / day, from 100 mg / day to 200 mg / day, from 125 mg / day to 200 mg / day, from 150 mg / day to 200 mg / day, from 175 mg / day to 200 mg / day, from 50 mg / day to 200 mg / day, from 50 mg / day to 175 mg / day, from 50 mg / day to 150 mg / day, from 50 mg / day to 100 mg / day, from 50 mg / day to 75 mg / day, from 75 mg / day to 200 mg / day, from 75 mg / day to 175 mg / day, from 75 mg / day to 150 mg / day, from 75 mg / day to 125 mg / day, from 75 mg / day to 100 mg / day, from 100 mg / day to 200 mg / day, from 100 mg / day to 175 mg / day, from 100 mg / day to 125 mg / day, from 125 mg / day to 200 mg / day, from 125 mg / day to 175 mg / day, from 125 mg / day to 150 mg / day, from 150 mg / day to 200 mg / day, from 150 mg / day to 175 mg / day, from 175 mg / day to 200 mg / day, or any range therebetween.

[0345] In one embodiment, the dose of one or more of the crystalline compounds or compositions described herein for the treatment of abnormal bone formation in soft tissue in humans is less than the dose of one or more of the crystalline compounds or compositions described herein typically used in the treatment of tumor diseases and cancers.

[0346] The administration of the above dose can be repeated over a limited period of time. In some embodiments, the dose is administered once a day or multiple times a day (e.g., but not limited to, three times a day). In another embodiment, the above dose is administered daily for several weeks or months. The duration of treatment depends on the clinical progress of the subject and the responsiveness to treatment. After an initial higher treatment dose, a subsequent relatively low maintenance dose is considered.

[0347] A therapeutically effective amount is the amount of a medicament sufficient to produce a statistically significant and measurable change in at least one symptom of cancer (see “Efficacy Measurement” below). Such effective amounts can be measured in clinical trials as well as in animal studies of a given medicament.

[0348] A medicament useful in the methods and compositions described herein can be administered systemically or orally. Also contemplated herein is that the medicament can also be administered intravenously (by bolus infusion or continuous infusion), by inhalation, intranasally, intraperitoneally, intramuscularly, subcutaneously, intracavity delivery, and can be delivered by peristaltic means (if desired) or by other means known to those skilled in the art.

[0349] In some embodiments, a pharmaceutically acceptable formulation for administering an active compound provides sustained delivery to the subject (such as “slow release” of the active compound). For example, after administering a pharmaceutically acceptable formulation to a subject, the formulation can deliver the medicament or composition for at least one week, two weeks, three weeks, or four weeks. Preferably, a subject to be treated according to the methods described herein is treated with the active composition for at least 30 days (by repeated administration or by using a sustained delivery system or both).

[0350] As used herein, the term “sustained delivery” is intended to encompass the sustained delivery of a composition in the body over a period of time after administration (preferably at least several days, one week, several weeks, one month, or longer). The sustained delivery of an active compound can be demonstrated, for example, by the continuous therapeutic effect of the composition over time (such as the sustained delivery of a medicament can be demonstrated by the continuous improvement or maintained improvement of cancer symptoms in a subject).

[0351] A therapeutic composition containing at least one agent can be administered routinely in unit doses. As used in reference to a therapeutic composition, the term "unit dose" refers to a physically discrete unit suitable as a unit dose for a subject, each unit containing a predetermined quantity of the active material calculated to produce the desired therapeutic effect, together with the required physiologically acceptable diluent (i.e., carrier or vehicle).

[0352] The composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The amount to be administered and the timing depend on the subject to be treated, the subject's system's ability to utilize the active ingredient, and the degree of the desired therapeutic effect. The agent can be targeted by means of a targeting moiety (such as, for example, an antibody) or targeted liposome technology. In some embodiments, the agent can be targeted to a tissue by using, for example, a bispecific antibody generated by the chemical conjugation of an anti-ligand antibody (Ab) with an Ab against a specific target. To avoid the limitations of chemical conjugates, molecular conjugates of antibodies can be used to generate recombinant bispecific single-chain Abs against ligands and / or chimeric inhibitors at cell surface molecules. Adding an antibody to the agent allows the agent to accumulate cumulatively at the desired target site (e.g., the tumor site). Antibody-based targeting moieties or non-antibody-based targeting moieties can be employed to deliver a ligand or inhibitor to the target site. Preferably, a natural binder for an unregulated antigen or a disease-related antigen is used for this purpose.

[0353] The precise amount of the active ingredient to be administered depends on the judgment of the practicing physician and is specific for each individual. However, dosage ranges suitable for systemic application are disclosed herein and depend on the route of administration. The dosage regimen suitable for administration is also variable but is characterized by an initial administration followed by repeated dosing at one or more intervals by subsequent injection or other means of administration. Alternatively, continuous intravenous infusion is contemplated that is sufficient to maintain the concentration in the blood or skeletal muscle tissue within the range specified for in vivo therapy.

[0354] Efficacy measurement

[0355] The efficacy of a given treatment for a disorder including abnormal bone growth, as described herein, can be determined by a skilled clinician. However, if any one or all of the signs or symptoms of a disease or disorder are altered in a beneficial manner (e.g., reduced ossification, regression of abnormal bone growth, reduced pain, increased range of motion, etc.) after treatment with an agent comprising one or more of the crystalline compounds or compositions described herein, and other clinically acceptable symptoms or markers of the disease are improved or even reduced by, for example, at least 10%, the treatment is considered to be an “effective treatment” (as the term is used herein). Efficacy can also be measured by the inability of an individual to deteriorate (as evaluated by stabilization of the disease or disorder, hospitalization, or need for medical intervention) (i.e., the progression of the disease stops or at least slows down). Methods for measuring these metrics are known to those of skill in the art and / or are described herein. Any treatment for a disease in an individual or animal (some non-limiting examples include humans or mammals) and includes: (1) inhibiting the disease (e.g., preventing or slowing the progression of abnormal bone growth); or (2) alleviating the disease (e.g., causing regression of symptoms); and (3) preventing or reducing the likelihood of disease development (e.g., post-traumatic ossification).

[0356] An effective amount for treating a disease means an amount sufficient to produce an effective treatment (as defined herein) for the disease when administered to a mammalian subject in need thereof. The efficacy of an agent can be determined by evaluating physical metrics of abnormal bone growth (e.g., reduced size of abnormal bone growth, slowed abnormal bone deposition, regression of bone growth, improved mobility, etc.).

[0357] Pharmaceutical compositions

[0358] In certain embodiments, the present invention relates to pharmaceutical compositions comprising a crystalline compound of formula (I) and one or more pharmaceutically acceptable excipients, and formulations prepared using such crystalline compounds and one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical formulations can be used to treat or prevent the conditions or diseases described herein. In certain embodiments, the pharmaceutical formulations have a sufficiently low pyrogenic activity to be suitable for intravenous use in human patients. In certain embodiments, the present invention also relates to formulations suitable for nutraceutical, veterinary, and agriculture-related uses.

[0359] Exemplary pharmaceutically acceptable excipients are provided herein, including, for example, binders, disintegrants, lubricants, flavorants, solubilizers, suspending aids, emulsifiers, coating agents, cyclodextrins, and / or buffering agents. Although the dosage may vary depending on the patient's symptoms, age and weight, the nature and severity of the condition to be treated or prevented, the route of administration, and the form of the drug, a daily dosage of 0.01 to 3000 mg of the compound is generally recommended for adult human patients, and this may be administered in a single dose or divided doses. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form is generally the amount of the compound that produces a therapeutic effect.

[0360] The precise dosing time and / or amount of the composition that produces the most effective result in terms of therapeutic efficacy in a given patient will depend on the activity, pharmacokinetics, and bioavailability of the particular compound, the patient's physiological condition (including age, gender, disease type and stage, general physical condition, response to a given dose, and type of drug), the route of administration, etc. However, the above guidelines can be used as a basis for fine-tuning the treatment, for example, determining the optimal time and / or amount of administration, which only requires routine experimentation consisting of monitoring the subject and adjusting the dose and / or timing.

[0361] In certain embodiments, the individual to whom the composition is administered is a mammal, such as a human or non-human mammal. When administered to an animal such as a human, the composition or compound is preferably administered as a pharmaceutical composition, which comprises, for example, a compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiological buffered saline, or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In a preferred embodiment, when such a pharmaceutical composition is used for human administration, particularly for invasive routes of administration (i.e., routes that avoid transport or diffusion across an epithelial barrier, such as injection or implantation), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients may be selected, for example, to achieve delayed release of the medicament or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in dosage unit form, such as tablets, capsules (including dispersed capsules and gelatin capsules), granules, lyophilizates for reconstitution, powders, solutions, syrups, suppositories, injections, etc. The composition may also be present in a transdermal delivery system, such as a skin patch. The composition may also be present in a solution suitable for topical administration by ocular mucosa, such as eye drops.

[0362] A pharmaceutically acceptable carrier may contain physiologically acceptable agents which are used, for example, to stabilize a compound, increase the solubility of a compound or increase the absorption of a compound, such as a compound of the present invention. These physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of the pharmaceutically acceptable carrier (including the physiologically acceptable agents) depends, for example, on the route of administration of the composition. The formulation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (formulation) may also be a liposome or other polymeric matrix which may contain, for example, a compound of the present invention therein. For example, liposomes containing phospholipids or other lipids are relatively simple, non-toxic, physiologically acceptable and metabolizable carriers to prepare and administer.

[0363] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0364] The phrase "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations. In certain embodiments, the pharmaceutical compositions of the present invention are pyrogen-free, i.e., do not cause a significant temperature increase when administered to a patient.

[0365] The term "pharmaceutically acceptable salts" refers to relatively non-toxic inorganic and organic acid addition salts of a compound. These salts can be prepared in situ during the final isolation and purification of the compound, or separately by reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, dodecylsulfate, and amino acid salts, etc. The preparation of crystalline salts is described in detail in the examples below (see, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19).

[0366] In other cases, the compounds useful in the methods of the present invention may contain one or more acidic functional groups and thus be capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. In such cases, the term "pharmaceutically acceptable salts" refers to relatively non-toxic inorganic and organic base addition salts of the compound. These salts can likewise be prepared in situ during the final isolation and purification of the compound, or separately by reacting the purified compound in its free acid form with a suitable base (e.g., a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation), with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali metal or alkaline earth metal salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts and aluminum salts, etc. Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc. (see, e.g., Berge et al., supra).

[0367] The pharmaceutical composition (preparation) can be administered to a subject by any of a variety of administration routes, including, for example, oral (e.g., infusion in an aqueous or non-aqueous solution or suspension, tablets, capsules (including dispersed capsules and gelatin capsules), boluses, powders, granules, pastes applied to the tongue); by absorption through the oral mucosa (e.g., sublingual); anal, rectal or vaginal (e.g., as suppositories, creams or foams); parenteral (including intramuscular, intravenous, subcutaneous or intrathecal, e.g., as a sterile solution or suspension); nasal; intraperitoneal; subcutaneous; transdermal (e.g., as a patch applied to the skin); and topical (e.g., as a cream, ointment or spray applied to the skin, or as eye drops). The compound can also be formulated for inhalation. In certain embodiments, the compound can simply be dissolved or suspended in sterile water. Details of suitable administration routes and compositions suitable for that administration route can be found, for example, in U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896 and the patents cited therein.

[0368] The preparation can conveniently be presented in unit dosage form and can be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host to be treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally the amount of the compound that produces a therapeutic effect. Generally, on a percentage basis, the amount is from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0369] The methods of preparing these preparations or compositions include the step of bringing the active compound (e.g., the compound of the present invention) into association with a carrier and optionally one or more accessory ingredients. Generally, the preparation is made by uniformly and intimately bringing the compound of the present invention into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

[0370] The preparations of the present invention suitable for oral administration can be in the form of capsules (including dispersed capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and gum arabic or tragacanth), lyophilizates, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and gum arabic), and / or as mouthwashes, etc., each containing a predetermined amount of the compound of the present invention as an active ingredient. The composition or compound can also be administered in the form of boluses, troches or pastes.

[0371] For the preparation of solid dosage forms for oral administration, such as capsules (including dispersed capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc., the active ingredient is mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dibasic calcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including dispersed capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also contain buffering agents. Solid compositions of a similar type can also be used as fillers in soft and hard gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, etc.

[0372] Tablets can be made by compression or molding, optionally containing one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or croscarmellose sodium), surfactants, or dispersing agents. Molded tablets can be prepared by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine.

[0373] Tablets and other solid dosage forms of the pharmaceutical composition, such as dragees, capsules (including dispersed capsules and gelatin capsules), pills, and granules, may optionally be scored or prepared with coatings and shells (such as enteric coatings and other coatings well-known in the pharmaceutical formulation art). They may also be formulated to provide for slow or controlled release of the active ingredient therein, for example, using different proportions of hydroxypropyl methylcellulose to provide the desired release profile, other polymeric matrices, liposomes, and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter or by inclusion of a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be compositions that release the active ingredient only, or preferentially, in a portion of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. If appropriate, the active ingredient may also be in the form of microcapsules with one or more of the above excipients.

[0374] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, lyophilized products for reconstitution, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrins and their derivatives, solubilizing agents, and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0375] In addition to the inert diluent, the compositions of the present invention may also include adjuvants such as wetting agents, lubricants, emulsifying agents, and suspending agents, such as sodium lauryl sulfate and magnesium stearate, or sweetening agents, flavoring agents, coloring agents, fragrances, preservatives, or antioxidants.

[0376] In addition to the active compound, the suspension may contain suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, and sorbitan esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, and tragacanth, and mixtures thereof.

[0377] Preparations of pharmaceutical compositions for rectal, vaginal, or urethral administration may be provided in the form of suppositories, which may be prepared by mixing one or more active compounds with one or more suitable non-irritating excipients or carriers, including, for example, cocoa butter, polyethylene glycol, suppository wax, or salicylates, which are solid at room temperature but liquid at body temperature and thus melt in the rectal or vaginal cavity and release the active compound.

[0378] Formulations of the pharmaceutical composition for oral administration can be provided in the form of mouthwash, oral spray or oral ointment.

[0379] Optionally or additionally, the composition can be formulated for delivery via a catheter, stent, wire or other intracavitary device. Delivery by such a device may be particularly useful for delivery to the bladder, urethra, ureter, rectum or intestine.

[0380] Formulations suitable for vaginal administration also include vaginal suppositories, tampons, creams, gels, pastes, foams or spray formulations, containing suitable carriers known in the art.

[0381] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound can be mixed with a pharmaceutically acceptable carrier under sterile conditions and with any preservatives, buffers or propellants that may be required.

[0382] In addition to the active compound, ointments, pastes, creams and gels may also contain excipients such as animal and vegetable fats, oils, waxes, paraffin wax, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0383] In addition to the active compound, powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0384] Alternatively, the compounds described herein can also be administered by aerosol. This is achieved by preparing an aqueous aerosol, a liposomal formulation or solid particles containing the composition. Non-aqueous (e.g., fluorocarbon propellant) suspensions can be used. Sonic nebulizers are preferred because they minimize the exposure of the medicament to shear that can cause degradation of the compound.

[0385] Generally, aqueous aerosols are prepared by formulating an aqueous solution or suspension of the medicament with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular composition but generally include nonionic surfactants (Tweens, Pluronics, sorbitan esters, lecithin, Cremophors), pharmaceutically acceptable co-solvents such as polyethylene glycol, innocuous proteins such as serum albumin, oleic acid, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols are usually prepared from isotonic solutions.

[0386] Transdermal patches have the additional advantage of providing controlled delivery of the compounds of the present invention to the body. These dosage forms can be prepared by dissolving or dispersing the active compound in a suitable medium. Penetration enhancers can also be used to increase the flux of the compound through the skin. The rate of this flux can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymeric matrix or gel.

[0387] Ophthalmic formulations, ophthalmic ointments, powders, solutions, etc. are also included within the scope of the present invention. Exemplary ophthalmic formulations are described in U.S. Publication Nos. 2005 / 0080056, 2005 / 0059744, 2005 / 0031697, and 2005 / 004074, and U.S. Patent No. 6,583,124, the contents of which are incorporated herein by reference. If desired, liquid ophthalmic formulations have properties similar to or are compatible with tears, aqueous humor, or vitreous humor. The preferred route of administration is local administration (e.g., topical administration, such as eye drops, or administration via an implant).

[0388] As used herein, the phrases "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders, which can be reconstituted immediately prior to use into sterile injectable solutions or dispersions, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0389] As used herein, the phrases "systemic administration", "administered systemically", "peripheral administration", and "administered peripherally" refer to the administration of a ligand, drug, or other substance other than direct entry into the central nervous system, such that it enters the patient's system and thus undergoes metabolism and other similar processes, such as subcutaneous administration.

[0390] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. For example, appropriate fluidity can be maintained by using coating materials such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants.

[0391] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. By including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc., the action of microorganisms can be ensured to be prevented. It may also be necessary to include isotonic agents such as sugars, sodium chloride, etc. in the composition. In addition, the absorption of injectable pharmaceutical forms can be prolonged by including agents that delay absorption such as aluminum monostearate and gelatin.

[0392] In certain cases, in order to prolong the action of a drug, it is desirable to slow down the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of poorly water-soluble crystalline or amorphous materials. Then the absorption rate of the drug depends on its dissolution rate, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered pharmaceutical forms is achieved by dissolving or suspending the drug in an oily vehicle.

[0393] Injectable long-lasting forms of the drug are prepared by forming a microencapsulation matrix of the compounds of the present invention in biodegradable polymers such as poly(lactide-co-glycolide). Depending on the ratio of the drug to the polymer and the nature of the specific polymer used, the drug release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Long-lasting injectable formulations of the drug are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0394] The pharmaceutical formulations of the drug can be administered orally, parenterally, topically, or rectally. Of course, they are provided in forms suitable for each route of administration. For example, they are in the form of tablets or capsules and are administered by injection, inhalation, eye lotion, ointment, suppository, infusion; topically administered with lotions or ointments; and rectally administered by suppositories. Oral administration is preferred.

[0395] For use in the methods of the present invention, the active compound can be administered per se or as a pharmaceutical composition that contains, for example, 0.1 - 99.5% (more preferably 0.5 - 90%) of the active ingredient and a pharmaceutically acceptable carrier.

[0396] The introduction method can also be provided by refillable or biodegradable devices. In recent years, various sustained-release polymer devices have been developed and tested in vivo for the controlled delivery of drugs, including protein biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including biodegradable and non-biodegradable polymers, can be used to form implants for the sustained release of compounds at specific target sites.

[0397] These compounds can be administered to humans and other animals for treatment by any suitable route of administration, including orally, nasally (e.g., by spray), rectally, intravaginally, parenterally, intracranially, and topically, such as by powder, ointment, or drops, including orally and sublingually.

[0398] Regardless of the chosen route of administration, the compounds of the present invention (which may be used in a suitable hydrated form) and / or the pharmaceutical compositions are formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.

[0399] The actual dosage level of the active ingredient in the pharmaceutical composition can vary so as to obtain an amount of the active ingredient that effectively achieves the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0400] The selected dosage level depends on various factors, including the activity of the particular compound or combination of compounds or their esters, salts, or amides used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular compound, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts. Generally, the compositions of the present invention can be provided in an aqueous solution containing about 0.1 - 30% w / v of the compounds disclosed herein, and further, for parenteral administration. Typical dosage ranges are from about 0.01 to about 50 mg / kg body weight per day, provided in a single dose or in 2 - 4 divided doses. Each divided dose can contain the same or different compounds of the present invention.

[0401] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe a therapeutically effective amount of the desired pharmaceutical composition. For example, the physician or veterinarian can start administration of the pharmaceutical composition or compound at a level below the dose required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is reached. For the methods of treatment of the present invention, a "therapeutically effective amount" of a compound means an amount of the compound in a formulation that, when administered as part of a desired dosage regimen (administered to a mammal, preferably a human), alleviates symptoms, improves the condition, or slows the onset of the disease condition according to clinically acceptable criteria for the disorder or condition being treated or for cosmetic purposes, e.g., with a reasonable benefit / risk ratio applicable to any medical treatment. It is generally understood that the effective amount of a compound will vary depending on the body weight, sex, age, and medical history of the subject. Other factors that can affect the effective amount can include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another class of therapeutic agents administered in combination with the compounds of the present invention. A greater total dose can be delivered by administering the agent in multiple doses. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison’s Principles of Internal Medicine 13th Edition, 1814 - 1882, incorporated herein by reference).

[0402] Generally, the suitable daily dose of the active compound used in the compositions and methods of the present invention will be the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such effective dose generally depends on the factors described above.

[0403] If desired, the effective daily dose of the active compound can be administered as one, two, three, four, five, six or more sub-doses, administered at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound can be administered two or three times a day. In a preferred embodiment, the active compound is administered once a day.

[0404] The patient to receive such treatment is any animal in need, including primates, particularly humans, and other mammals such as horses, cows, pigs and sheep; and generally poultry and pets.

[0405] In certain embodiments, the compounds of the present invention can be used alone or co-administered with another type of therapeutic agent. As used herein, the phrase "co-administered" refers to any form of administration of two or more different therapeutic compounds such that the previously administered therapeutic compound remains effective in the body while the second compound is being administered (e.g., both compounds are effective in the patient simultaneously, which can include a synergistic effect of the two compounds). For example, the different therapeutic compounds can be administered in the same formulation or in separate formulations simultaneously or sequentially. In certain embodiments, the different therapeutic compounds can be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours or one week of each other. Thus, an individual receiving such treatment can benefit from the combined effects of the different therapeutic compounds.

[0406] The present invention includes the use of pharmaceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. In certain embodiments, the expected salts of the present invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetraalkyl ammonium salts. In certain embodiments, the expected salts of the present invention include, but are not limited to, L-arginine, benenthamine, benzathine penicillin, betaine, calcium hydroxide, choline, dimethylethanolamine, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine and zinc salts. In certain embodiments, the expected salts of the present invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts.

[0407] Pharmaceutically acceptable acid addition salts may also exist as various solvates, such as solvates with water, methanol, ethanol, dimethylformamide, dimethyl sulfoxide, etc. Mixtures of these solvates can also be prepared. The source of such solvates can be from the crystallization solvent, inherent in the solvent used for preparation or crystallization, or foreign to such a solvent. In some embodiments, the solvate of the disclosed compound can be a dimethyl sulfoxide solvate.

[0408] Wetting agents, emulsifying agents and lubricants, such as sodium lauryl sulfate and magnesium stearate, and coloring agents, release agents, coating agents, sweetening agents, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition.

[0409] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0410] The present invention has been generally described above, and the present invention will be more readily understood by reference to the following examples, which are for the purpose of illustrating certain aspects and embodiments of the present invention only and are not intended to limit the present invention. Examples

[0411] Example 1: Synthesis of the free base of the compound of formula I

[0412] Step 1: Synthesis of Compound 4

[0413]

[0414] To a solution of 2-bromomalondialdehyde (700 g, 4.64 mol) in DMSO (2100 mL, 3 vol) and DIEA (889 mL, 5.1 mol) was added a solution of 1-methyl-4-(piperidin-4-yl)piperazine (1020 g, 5.56 mol) in DMSO (1050 mL, 1.5 vol). The reaction was heated to 50 °C and stirred overnight. To the resulting solution was added a 40 °C solution of 3-aminopyrazole (385.2 g, 4.64 mol) in DMSO (350 mL, 0.5 vol). Glacial acetic acid (1.30 L, 22.4 mol) was added via an addition funnel. An exotherm from 50.8 °C to 63.6 °C was observed during the addition. The reaction was heated to 95 °C for 6 h, cooled to room temperature and stirred overnight. The resulting solid was removed by filtration, washed with DMSO (2 x 350 mL), and transferred to a 3 L 4-neck RBF. MTBE (3000 mL) was added and the slurry was stirred for 3 h. The solid was filtered, washed with MTBE (2 x 700 mL), and dried in vacuo at 55 °C for 2 h. 505.0 g of Compound 4 was isolated. Yield = 36.3%

[0415] 1 H NMR (400 MHz, chloroform-d) δ = 8.43 (d, J = 2.5 Hz, 1H), 8.09 (d, J =2.7 Hz, 1H), 7.94 (d, J = 2.3 Hz, 1H), 6.57 (s, 1H), 3.55 (br d, J = 12.3 Hz,2H), 2.73 - 2.53 (m, 7H), 2.47 (br s, 3H), 2.40 - 2.25 (m, 5H), 1.99 (br d, J= 12.7 Hz, 2H), 1.73 (dq, J = 4.0, 12.0 Hz, 3H).

[0416] Step 2: Synthesis of Compound 5

[0417]

[0418] To a solution of 6-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine (800 g, 2.66 mol), chloroform (9600 mL), and THF (2400 mL) at 0 - 5 °C was added portionwise solid NBS (477 g, 2.66 mol) over 3 h while maintaining the internal temperature < 5 °C. The resulting slurry was warmed to room temperature while stirring overnight. The reaction was filtered and the unwanted solid was rinsed with chloroform (2 × 800 mL). The filtrate was transferred to a separatory funnel and washed with saturated NaHCO3 (6000 mL, 7.5 vol). The layers were separated and the aqueous layer was back-extracted with chloroform (1600 mL, 2 vol). The bottom organic layer was combined and washed with brine (5000 mL). The top aqueous layer was back-extracted with chloroform (1600 mL, 2 vol). The bottom organic layer was concentrated until ~4 vol remained. The concentrated slurry was solvent-exchanged with heptane (3 x 2400 mL), concentrating back to 4 volumes each time and the resulting slurry was stirred overnight at ambient temperature. The slurry was filtered, washed with MTBE (2 x 1600 mL), and dried in vacuo at 40 °C overnight to give 889 g of a tan solid, Compound 5. Yield = 85.6%

[0419] 1 H NMR (400 MHz, chloroform-d) δ = 8.48 (d, J = 2.5 Hz, 1H), 8.03 (d, J = 2.7 Hz, 1H), 7.92 (s, 1H), 3.55 (br d, J = 12.3 Hz, 2H), 3.07 - 2.81 (m, 1H), 2.76 - 2.65 (m, 4H), 2.62 (br s, 4H), 2.50 - 2.27 (m, 8H), 1.98 (br d, J = 12.7 Hz, 2H), 1.73 (dq, J = 3.8, 12.0 Hz, 3H).

[0420] Step 3: Synthesis of the free base of the compound of formula I

[0421]

[0422] 3-Bromo-6-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidine (Compound 5, 500 g, 1.32 mol), 7-fluoro-6-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)quinoline (420 g, 1.05 eq), 2-MeTHF (7.5 L, 1 volume), and 3 M K3PO4 (1.32 L, 2.64 volume, 3eq) in a flask were degassed with a nitrogen stream for 30 min at room temperature. PdCl2(dppf)DCM (75.4 g) was added to the degassed slurry, and the resulting mixture was degassed with nitrogen for an additional 30 min. The reaction was heated to 60 °C and stirred overnight at 60 °C. The resulting mixture was cooled and concentrated to a minimum volume. CHCl3 (7 L, 14 volume) and water (3.5 L, 7 volume) were added to the concentrate, and the layers were separated after stirring for 30 min. The aqueous layer (upper layer) was extracted with 2 X 5 L (2 X 10 volume) of CHCl3. The combined organic layers were concentrated to a minimum volume and absorbed into 8.5 L (17 volume) of 1:1 MeOH: CHCl3. 250 g of Nexagen thiopropyl silica gel and 250 g of Norit CA1 carbon were added to the resulting solution. The resulting slurry was heated to 50 °C overnight. The slurry was filtered at room temperature to remove the solids and the resulting filtrate was concentrated to a minimum volume. The thick slurry was absorbed into 8.5 L (17 volume) of DCM, and 250 g of Nexagen thiopropyl silica gel and 250 g of Norit CA1 carbon were added to the resulting solution, and the resulting slurry was heated to 35 °C overnight. The slurry was filtered at room temperature to remove the solids. The filtrate was placed in a separatory funnel and the aqueous layer was removed. The resulting solution was solvent-exchanged with EtOAc, where a solid formed. The resulting slurry was diluted with EtOAc to a total volume of 5 L (10 volume). MTBE (2.5 L) was added and the resulting slurry was stirred overnight at room temperature. The solid was filtered and washed with 2 X 2.5 L (2 X 5 volume) of MTBE to give 430 g of the title compound. (Total yield 68.5%)

[0423] 11H NMR (400 MHz, chloroform-d) δ = 8.83 (d, J = 4.5 Hz, 1H), 8.57 (d, J = 2.7 Hz, 1H), 8.31 (s, 1H), 8.22 (d, J = 2.7 Hz, 1H), 7.80 (d, J = 12.0 Hz, 1H), 7.60 - 7.53 (m, 2H), 3.90 (s, 3H), 3.75 - 3.55 (m, 3H), 2.89 - 2.73 (m, 2H), 2.68 (br s, 4H), 2.60 - 2.39 (m, 5H), 2.33 (s, 3H), 2.16 - 1.98 (m, 2H), 1.79 (dq, J = 3.7, 11.9 Hz, 2H).

[0424] Example 2A: Synthesis of the amorphous compound of the monosuccinate of formula I

[0425]

[0426] 7-Fluoro-6-methoxy-4-(6-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline (701.93 g, 1.48 mol) and ethanol (14 L, 200 Proof) were stirred at 70 °C for 2 h. A solution of succinic acid (176 g, 1.49 mol) and ethanol (5 L, 200 Proof) was added slowly, and the contents were stirred at 75 °C for 8 h to form the salt completely. The resulting slurry was concentrated in vacuo to 5 L while maintaining the internal temperature above 20 °C during the concentration. Heptane was added to a total volume of 14 L, and the slurry was concentrated again while maintaining the internal temperature above 20 °C. The above concentration / reconstitution was carried out 6 times to achieve a solvent ratio of ethanol in heptane of less than 5 Wt.%, as determined by proton NMR (CDCl3). The final slurry was cooled to 20 °C, and the solid was filtered without washing. The solid was placed in a vacuum oven at 45 °C to constant weight to give the title compound as a yellow solid (864.1 g, 98.6% yield).

[0427] 11H NMR (400 MHz, DMSO-d6) δ = 8.83 (d, J = 2.6 Hz, 1H), 8.79 (d, J = 4.6 Hz, 1H), 8.66 (d, J = 2.7 Hz, 1H), 8.61 (s, 1H), 7.83 (d, J = 12.3 Hz, 1H), 7.70 (s, 1H), 7.68 (d, J = 4.6 Hz, 1H), 3.90 (s, 3H), 3.74 (br d, J = 12.6 Hz, 2H), 2.78 - 2.62 (m, 3H), 2.61 - 2.52 (m, 3H), 2.47 - 2.33 (m, 9H), 2.23 (s, 3H), 1.90 (br d, J = 11.7 Hz, 2H), 1.65 - 1.55 (m, 2H).

[0428] Example 2B: Synthesis of the amorphous compound of the monosuccinate of formula I

[0429]

[0430] 7-Fluoro-6-methoxy-4-(6-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline (16 g, 0.336 mol) and ethanol (360 mL, 200 Proof) were stirred at 70 °C for 2 h. A solution of succinic acid (4.0 g, 0.339 mol) and ethanol (160 mL, 200 Proof) was added slowly, and the contents were stirred at 75 °C for 8 h to form the salt completely. The resulting slurry was concentrated in vacuo to 160 mL while maintaining the internal temperature above 20 °C (important for the correct polymorph). Heptane was added to a total volume of 160 mL, and the slurry was stirred at >20 °C for 8 h. The final slurry was cooled to 20 °C, the solid was filtered and washed with 2 x 23 mL of 1:1 ethanol:heptane. The solid was placed in a vacuum oven at 45 °C and dried to constant weight to afford the title compound as a solid. (18.26 g, 91% yield)

[0431] 11H NMR (400 MHz, DMSO-d6) δ = 8.83 (d, J = 2.6 Hz, 1H), 8.79 (d, J =4.6 Hz, 1H), 8.66 (d, J = 2.7 Hz, 1H), 8.61 (s, 1H), 7.83 (d, J = 12.3 Hz,1H), 7.70 (s, 1H), 7.68 (d, J = 4.6 Hz, 1H), 3.90 (s, 3H), 3.74 (br d, J =12.6 Hz, 2H), 2.78 - 2.62 (m, 3H), 2.61 - 2.52 (m, 3H), 2.47 - 2.33 (m, 9H),2.23 (s, 3H), 1.90 (br d, J = 11.7 Hz, 2H), 1.65 - 1.55 (m, 2H).

[0432] Analytical methods for Examples 3 - 7

[0433] X-ray powder diffraction

[0434] X-ray powder diffraction (XRPD) patterns were collected on a Bruker D8 diffractometer using Cu Kα radiation (40 kV, 40 mA), a θ-2θ goniometer, and divergence and receiving slits of 0.6 mm, and a Lynxeye detector. The instrument was performance-checked using a certified corundum standard (NIST 1976). The software used for data collection was Diffrac Plus XRDCommander v2.6.1, and the data were analyzed and presented using Materials Data Jade Software v 9.7.0. Samples were prepared by transferring 2 to 50 mg of the sample to a zero-background holder coated with a thin layer of petroleum jelly and leveled with a glass plate and analyzed at ambient temperature. During the analysis, the sample was rotated in its own plane. The instrument parameters are provided in Table 1 below.

[0435]

[0436] Differential scanning calorimetry

[0437] Differential scanning calorimetry (DSC) analysis was performed using a TA Instruments Q1000 DSC, s / n 1000-0189. Samples were weighed into aluminum hermetic pans and lids with manual pinholes. The pans were crimped using a Tzero press. Samples were analyzed from 25 to 350 °C at 10 °C / minute. Data were processed using Universal Analysis 2000, v 4.5A.

[0438] Thermogravimetric analysis

[0439] Thermogravimetric analysis (TGA) was performed using a TA Instruments Q50 TGA, s / n 50-0180. Samples were loaded into tared platinum TGA pans and analyzed from 25 to 350 °C at 10 °C / minute. Data were processed using UniversalAnalysis 2000, v 4.5A.

[0440] Dynamic vapor sorption / desorption

[0441] Dynamic vapor sorption / desorption (DVS) analysis was performed using a Hiden Isochema, IGAsorp, s / n IGSA 126. Samples were loaded into tared baskets on the instrument and dried at 40 °C for 1 hour. Samples were then analyzed at 25 °C in the following percentage relative humidity (%RH) steps: adsorption (%RH): 0.0, 10.0, 20.0, 30.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 95.0, desorption (%RH): 90.0, 80.0, 70.0, 60.0, 50.0, 40.0, 30.0, 20.0, 10.0, 0.0.

[0442] Example 3A: Characterization of the free base of the compound of formula I

[0443] Using the XRPD technique detailed above, the free base of the compound of Formula I is crystalline and exhibits the following peaks: Page 4 Figure 3 . Additionally, the thermal data show that the free base is anhydrous and non-solvated ( Figure 4 ). DSC shows a sharp endotherm with an onset temperature of 238.6 °C, indicating melting. A minimal weight loss (0.4%) was observed in TGA until decomposition at 266.3 °C. DVS data indicate that the material is non-hygroscopic with a weight gain and loss of 0.5% and a slight hysteresis upon desorption ( Figure 5 ). No changes were observed in XRPD after DVS analysis.

[0444] Example 4A: Synthesis of the crystalline compound of the monosuccinate of formula I, form A

[0445] 2 g of the free base of the compound of Formula I was added to a 60 mL container. 0.5 g of succinic acid was dissolved in 30 mL of THF, and then the acid solution was added to the free base solid and stirred overnight at ambient temperature. The solid was centrifuged using a 0.45 µm nylon filter tube and dried under vacuum for approximately 2 hours before analysis by XRPD ( Figure 6 ) and NMR ( Figure 7 ). Both analyses indicated that Form A was isolated from the scale-up. The peak at 2.6 ppm was attributed to one mole of succinic acid. The yield of the scale-up was 88%.

[0446] Example 4B: Synthesis of the crystalline compounds of the monosuccinate of formula I, forms A and C

[0447] 2 g of the free base of the compound of Formula I was added to a 20 mL scintillation vial. 0.5 g of succinic acid was dissolved in 20 mL of EtOH, and then the acid solution was added to the free base solid and stirred overnight at ambient temperature. The solid was centrifuged using a 0.45 µm nylon filter tube and dried under ambient conditions before XRPD analysis ( Figure 8 ), which indicated that Form A was isolated from the scale-up. The yield of the scale-up was 79%. The material was also analyzed by DVS ( Figure 9 ). DVS indicated that the material was hygroscopic, with a weight gain and loss of 38% and a distinct hysteresis upon desorption. This was similar to the product of Example 3A. The material was not hygroscopic below 80% RH. XRPD analysis after DVS ( Figure 10 ) indicated that the material became highly disordered and transformed into the hydrate Form C.

[0448] Example 4C: Synthesis of the crystalline compounds of the monosuccinate of formula I, forms A and B

[0449] 100 mg of the free base of the compound of Formula I was added to a dram vial. 25 mg of succinic acid was dissolved in 1 mL of EtOH, and then the acid solution was added to the free base solid and slurried for 1 day. The solid was centrifuged using a 0.45 µm nylon filter tube and dried under ambient conditions before XRPD analysis, which indicated that the material was a mixture of Form A and Form B ( Figure 11 ).

[0450] Example 5: Polymorph solvent screening study

[0451] Thirty experiments were initially conducted as part of the screening for stable polymorphs of the monosuccinate salt of the compound of formula I and are detailed in Table 2. Table 3 presents nineteen experiments starting from the monosuccinate salt of the compound of formula I and using crystallization techniques that promote the formation of more stable polymorphs, such as slow cooling, slow evaporation, vapor diffusion, and slurrying over a long period. Table 4.3 presents eleven experiments studying the formation of the succinate salts of the compound of formula I starting from the free base and different ratios of succinic acid to determine if any other stoichiometries exist.

[0452] The stable polymorph and salt formation experiments yielded Form A, Form B, Form C, or a mixture of forms. Form C can be a hydrate as it was produced from systems containing water or solvents known to contain water (i.e., ethanol or THF). Form B was characterized as an anhydrous polymorph that resulted from various conditions and solvent systems during the screening process. All polymorphs were confirmed to be 1:1 stoichiometric salts of the free base of the compound of formula I and succinic acid. No other stoichiometries of the succinate salts were identified.

[0453] Table 2: Polymorph Screening Study of the Monosuccinate Salt of the Compound of Formula (I)

[0454] Solvent Experimental conditions Observation results XRPD results <![CDATA[CHCl3]]> Attempted slow cooling from 55 °C to room temperature Thin slurry Form B Dioxane Slow cooling from 65 °C to room temperature Clear solution then precipitation Form A + form B EtOAc Attempted slow cooling from 65 °C to room temperature Thin slurry Form A + form B EtOH Slurried, RT, 6 d Yellow slurry Form A EtOH Vapor diffusion using heptane Yellow slurry Form A IPA Attempted slow cooling from 65 °C to room temperature Thin slurry Form A MeOH Slurried, RT, 6 d Yellow slurry Form A MeOH Vapor diffusion using MTBE Yellow slurry Form A THF Slurried, RT, 6 d Yellow slurry Form A Water Cool slowly from 55 °C to freezing and then evaporate rapidly Thick viscous gel, free of solids - Water Slurry at RT Thick viscous gel, free of solids - - 97% RH Stress, RT Dark yellow solid Form C, disordered <![CDATA[20:80 THF / H2O (a w =0.96)]]> Slurry, RT, 6 d Viscous solid, not separable - <![CDATA[50:50 EtOH / H2O(a w =0.84)]]> Cool slowly from 55 °C to freezing Clear solution then viscous solid, not separable - <![CDATA[90:10 acetone / H2O(a w =0.71)]]> Slurry, RT, 6 d Yellow slurry Form B + Form C <![CDATA[90:10 acetone / H2O(a w =0.71)]]> Cool slowly from 55 °C to RT Clear solution then precipitate Form C, disordered <![CDATA[90:10 EtOH / H2O(a w =0.53)]]> Slurry, RT, 6 d Yellow slurry Form B + Form C <![CDATA[90:10 EtOH / H2O(a w =0.53)]]> Cool slowly from 55 °C to freezing Clear solution then precipitate Form B <![CDATA[98:2 IPA / H2O (a w =0.28)]]> Slurry, RT, 6 d Yellow slurry Form A

[0455] Table 3: Salt Formation Experiments of the Free Base of the Compound of Formula (I) and Succinic Acid

[0456] Solvent Experimental conditions Observation results XRPD results Acetone 1:1 KER-047 / succinic acid, heated to 55 °C, cooled to room temperature, solid separated, dried under vacuum at room temperature Dilute slurry Form A ACN 1:1 KER-047 / succinic acid, heated to 55 °C, cooled to room temperature, solid separated, dried under vacuum at room temperature Dilute slurry Form A EtOH 1:1 KER-047 / succinic acid, heated to 55 °C, cooled to room temperature, solid separated, dried under vacuum at room temperature Clear solution then precipitate Form A + small amount of Form C EtOH 1:2 KER-047 / succinic acid, heated to 65 °C, cooled to room temperature, solid separated Clear solution then precipitate Form A EtOH 2:1 KER-047 / succinic acid, heated to 65 °C, cooled to room temperature, solid separated Clear solution then precipitate Form A + small amount of Form C EtOH 1:6 KER-047 / succinic acid, heated to 65 °C, cooled to room temperature, solid separated Dilute slurry Form B EtOH 1:1 KER-047 / succinic acid, 100 mg API heated in 5 mL EtOH to 65 °C, 25 mg acid in 5 mL EtOH added at 65 °C, seed of Form A added, SC to RT Clear solution then precipitate Form A EtOH 1:1 KER-047 / succinic acid, 100 mg API heated in 5 mL EtOH to 65 °C, 25 mg acid in 5 mL EtOH added at 65 °C, seed of Form B added, SC to RT Clear solution then precipitate Form A THF 1:2 KER-047 / succinic acid, heated to 65 °C, cooled to room temperature, solid separated, dried under vacuum at room temperature Thick slurry Form B THF 2:1 KER-047 / succinic acid, heated to 65 °C, cooled to room temperature, solid separated, dried under vacuum at room temperature Dilute slurry Form A + small amount of Form C EtOH 500 mg free base in 10 mL EtOH, stirred at 70 °C for 3.5 h, 125 mg succinic acid in 4 mL EtOH, heated at 75 °C for 6 h, quenched to freezing Yellow slurry then after adding acid, the solution becomes clear and then yellow precipitate starts to form, yellow solid after separation Form A

[0457] Example 6: Solvent Screening Study of Stable Polymorphs

[0458] Using Form A of the compound of formula (I) as the starting material, the experiments in Table 4 below determined the differences between Form A and Form B as both are anhydrous. The ratios of ethanol and heptane and the temperature conditions of these solvents were explored as these were selected as the final crystallization conditions. Additional experiments were conducted using crystallization techniques that promote the formation of more stable polymorphs, such as slow cooling and slurrying over a long period. Other experiments were also conducted on metastable forms, such as crash precipitation, crash cool, and rapid evaporation techniques.

[0459] In addition to Forms A, B, and C, a new polymorph in the mixture was identified as Form D. Based on the characterization data, this substance may be a hydrate. The disordered XRPD pattern of Form C is different from that of the more crystalline Form D.

[0460] Table 4: Polymorph Screening Study Starting from Form A of the Monosuccinate Salt of the Compound of Formula (I)

[0461] Solvent Experimental Conditions Observation Results XRPD Results <![CDATA[CHCl3]]> Slurried at 55°C and cooled to freezing Yellow slurry Form B <![CDATA[CHCl3]]> Heat the slurry to 55°C and isolate the heat Yellow slurry Form B <![CDATA[CHCl3]]> Slurried at RT for 3 days Yellow slurry Form B EtOAc Quenched from 70°C to dry ice / IPA temperature and the solid was separated Clear solution, yellow solid broken, then sticky yellow solid Stop EtOAc Quenched from 75°C to freezing and then rapidly evaporated, cycle 1X Clear solution then yellow solid Form A EtOH Slurried below ambient temperature for 3 days Yellow slurry Form A EtOH Attempted rapid precipitation with heptane and then frozen Clear solution then yellow precipitate Form A + Form D EtOH Slurried at ambient temperature for 3 d Yellow slurry Form A EtOH Slurried at 40°C for 3 d Yellow slurry Form A EtOH Heated to 70°C, filtered, added heptane in a 1:1 ratio, frozen, and then rapidly evaporated Clear solution then yellow precipitate Form A + Form B EtOH Heated to 70°C, filtered, added heptane in a 1:5 ratio, frozen, and then rapidly evaporated Clear solution then yellow precipitate Form A + Form B EtOH Heated to 70°C, filtered, added heptane in a 1:10 ratio, frozen, and then rapidly evaporated Clear solution then yellow precipitate Form B + Form D EtOH Heated to 70°C, filtered, added heptane in a 1:20 ratio, frozen, and the solid was separated Clear solution then yellow precipitate, sticky yellow solid when separated Stop EtOH Heated to 70°C, filtered, added heptane in a 1:5 ratio, and frozen Clear solution then yellow precipitate Form A EtOH Heated to 70°C, filtered, added heptane in a 1:5 ratio, and at room temperature Clear solution then yellow precipitate Form A MeOH Slurried below ambient temperature for 3 days Yellow slurry Form A + Form B MeOH Slurried at ambient temperature for 3 d Yellow slurry Form A + Form B MeOH Slurried at 40°C for 3 d Yellow slurry Form A + Form B THF Quenched from 60°C to freezing and then rapidly evaporated, cycle 1X Clear solution then yellow solid Form A + a small amount of Form B

[0462] Form A is an anhydrous and non-solvated crystalline solid ( Figure 12 ). DSC shows a sharp endotherm with an onset temperature of 184.3 °C ( Figure 13 ). A minimal weight loss (0.5%) was observed in TGA until decomposition at 201.1 °C.

[0463] Form B is a poorly crystalline solid, anhydrous and unsolvated ( Figure 14 ). DSC shows a small endotherm at 138.6 °C, followed by a sharp endotherm with an onset temperature of 169.6 °C. A hot stage microscope was required to determine the nature of the first small endothermic event. A minimal weight loss (0.9%) was observed in TGA until decomposition at 178.0 °C ( Figure 15 ). DVS data indicate that the material is slightly hygroscopic, with a weight increase and decrease of 12% and a slight hysteresis during desorption ( Figure 16 ). Although form B absorbs less moisture overall, the moisture increase in form B starts immediately even at lower relative humidities. XRPD analysis after DVS is also consistent with form B ( Figure 17 ). Chemical shifts in proton NMR indicate that the material is consistent with the chemical structure and there is no residual solvent ( Figure 18 ). The peak at 2.6 ppm is attributed to one mole of succinic acid.

[0464] Form C is a hydrated or solvated disordered material ( Figure 19 ). DSC shows an initial broad endotherm with a maximum at 83.7 °C, followed by another endotherm with a maximum at 120.8 °C, and then an exotherm with a maximum at 134.8 °C. A final sharp endotherm with an onset temperature of 181.8 °C was observed. This suggests that form C may desolvate and recrystallize to form A, which has a melting temperature of 184 °C ( Figure 20 ). Chemical shifts in proton NMR are consistent with the chemical structure and there is no residual solvent, indicating that form C is indeed a hydrate ( Figure 21 ). The peak at 2.6 ppm is attributed to one mole of succinic acid.

[0465] Form D was obtained as described above as a mixture with form A or form B, as indicated by the arrows in the comparative XRPD spectra shown in Figure 22 . Form D was obtained using ethanol or heptane. Thermal data show that form D is hydrated or solvated ( Figure 23)。The DSC shows an initial broad endothermic peak with a maximum at 49.8 °C, followed by a small exothermic peak with a maximum at 132.3 °C, and then an exothermic peak with a maximum at 134.8 °C. A final sharp endotherm is observed starting at 182.7 °C. Thus, Form D may be desolvated and recrystallized to Form A, which has a melting temperature of 184 °C. Since the sample also contains a mixture of Form A, there are Form A seeds that may facilitate recrystallization. The chemical shifts in the proton NMR are consistent with the chemical structure, and there is little residual solvent (1.5% EtOH by weight), indicating that Form D may be a hydrate( Figure 24 )。The peak at 2.6 ppm is attributed to one mole of succinic acid.

[0466] Example 7: Competitive Slurrying of Form A and Form B of the Compound of Formula I

[0467] To determine the thermodynamically stable polymorph between anhydrous Form A and Form B, competitive slurries were prepared in three different solvent systems: ethanol, tetrahydrofuran, and 2-butanone (MEK) / water (95:5). Each solvent system was evaluated at three different temperatures: 5 °C, room temperature (RT), and 40 °C, as shown in Table 5. The selection of ethanol and tetrahydrofuran was based on the solubility of the material. MEK was chosen as a potential process solvent, and water was added to improve solubility.

[0468] Table 5: Competitive Slurries of Form A and Form B - Part 1

[0469] Solvent Experimental Conditions Observation Results XRPD Results EtOH At 5°C, slurried for 5 days Yellow slurry Form A + Form B THF At 5°C, slurried for 5 days Yellow slurry Form A + Form B MEK / H2O 95:5 At 5°C, slurried for 5 days Yellow slurry Form C + Form B, highly disordered EtOH At RT, slurried for 5 days Yellow slurry Form A + Form B THF At RT, slurried for 5 days Yellow slurry Form A + Form B MEK / H2O 95:5 At RT, slurried for 5 days Yellow slurry Form C + Form B, highly disordered EtOH At 40°C, slurried for 5 days Yellow slurry Form A THF At 40°C, slurried for 5 days Yellow slurry Form A + Form B + a small amount of Form C MEK / H2O 95:5 At 40 °C, slurried for 5 days Yellow slurry Form C + Form B, highly disordered

[0470] The addition of 5% water resulted in partial conversion to the hydrate Form C. The use of a known water-containing solvent, such as THF, also resulted in partial conversion to Form C. Therefore, additional competitive slurries were carried out in three additional solvent systems: ethanol / heptane (1:5), ethyl acetate / water (97:3), and isopropanol / water (99:1). The ethanol / heptane system was evaluated at -15 °C, 5 °C, room temperature (RT), and 40 °C. The water-containing systems were evaluated only at room temperature. The results are shown in Table 6 below. The slurries were carried out for 7 days instead of 5 days, as done in the above slurries. Form D was also included in the water-containing competitive slurries.

[0471] Table 6: Competitive Slurries of Form A and Form B - Part 2

[0472]

[0473] The above results indicate that over time, Form A is the most stable polymorph as it decreases in the presence of Form B compared to slurrying carried out over 5 days. A wide temperature range (~40 °C to -15 °C) was designed for the study to determine if there is a critical transition temperature between Form A and Form B. Although Form A has a higher endothermic melting event, the heat of fusion of Form B is higher, so the thermodynamic stability may depend on Form A.

[0474] Slurrying with 97:3 EtOAc / water led to complete conversion to Form C, whereas slurrying with 99:1 IPA / water led to Form A. These data suggest that at low water activity, the anhydrous Form A is the most thermodynamically stable. At high water activity, the hydrate Form C is more dominant.

[0475] Example 8: Serum iron level

[0476] This study evaluated the heme iron parameters after administration of Form A. Healthy male Sprague Dawley rats at 8 weeks of age were dosed daily by oral gavage with vehicle or Form A at doses of 1 and 2.5 mg. After 90 days of dosing, the study was terminated and serum samples were collected for analysis of iron parameters. The collected blood was allowed to clot at room temperature and then centrifuged in a centrifuge and the serum was aliquoted. Serum iron and unsaturated iron binding capacity (UIBC) were determined using a Cobas 6000 (Roche) blood chemistry analyzer. Transferrin saturation was calculated as the percentage of total iron binding capacity to total serum iron level.

[0477] Example 9: Effect of Form A on iron-refractory iron deficiency anemia

[0478] The effect of Form A on iron-refractory iron-deficiency anemia (IRIDA) was determined using an IRIDA mouse model. Briefly, 8-week-old C57BL / 6 mice were intravenously injected once every 3 days with lipid-encapsulated siRNA targeting luciferase (control) or TMPRSS6 (0.75 mg / kg). The mice received siRNA treatment until the cohort receiving TMPRSS6 siRNA had increased hepcidin and decreased serum hemoglobin and serum iron. At this time, the mice were further randomized to receive vehicle or Form A. The mice were euthanized 12 days after the first siRNA administration. Whole blood was collected and hematological parameters were determined. In addition, serum was collected and hepcidin concentration was determined by ELISA and total iron content was determined by colorimetric assay.

[0479] Incorporate For reference

[0480] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0481] Equivalent

[0482] Although specific embodiments of the invention have been discussed, the foregoing description is illustrative rather than restrictive. After reading this specification and the following claims, many variations of the invention will become apparent to those skilled in the art. The full scope of the invention should be determined by reference to the full scope of the claims and their equivalents, the specification, and these variations.

Claims

1. A crystalline compound having the structure of formula (I), monosuccinate, wherein the crystalline compound has 2θ values of 3.58 ± 0.2, 7.05 ± 0.2, 10.59 ± 0.2, 10.75 ± 0.2, 13.80 ± 0.2, 14.16 ± 0.2, 15.16 ± 0.2, 15.68 ± 0.2, 16.18 ± 0.2, 16.80 ± 0.2, 17.15 ± 0.2, 17.69 ± 0.2, 17.97 ± 0.2, 18.29 ± 0.2, 18.59 ± 0.2, 18.84 ± 0.2, 19.27 ± 0.2, 20.29 ± 0.2, 24.47 ± 0.2, 24.84 ± 0.2, and 28.47 ± 0.

2.

2. The crystalline compound of claim 1, wherein the compound is anhydrous.

3. The crystalline compound of claim 1, which has 2θ values of 3.58 ± 0.2, 7.05 ± 0.2, 10.59 ± 0.2, 10.75 ± 0.2, 13.80 ± 0.2, 14.16 ± 0.2, 15.16 ± 0.2, 15.68 ± 0.2, 16.18 ± 0.2, 16.80 ± 0.2, 17.15 ± 0.2, 17.69 ± 0.2, 17.97 ± 0.2, 18.29 ± 0.2, 18.59 ± 0.2, 18.84 ± 0.2, 19.27 ± 0.2, 20.29 ± 0.2, 21.05 ± 0.2, 21.26 ± 0.2, 21.56 ± 0.2, 21.78 ± 0.2, 22.68 ± 0.2, 23.84 ± 0.2, 24.47 ± 0.2, 24.84 ± 0.2, 25.15 ± 0.2, 26.10 ± 0.2, 27.12 ± 0.2, 27.78 ± 0.2, 28.47 ± 0.2, and 29.06 ± 0.

2.

4. The crystalline compound of claim 1, which has an XRPD pattern substantially as shown in Figure 1 and is designated Form A.

5. A pharmaceutical composition comprising the crystalline compound of any one of claims 1 - 4 and one or more pharmaceutically acceptable excipients.

6. A method for preparing a crystalline compound having the structure of formula (I): Comprising: a) Providing a compound of formula (I); b) Adding succinic acid to form a mixture; and c) Crystallizing the compound of formula (I) from the mixture comprising the compound of formula (I), wherein the crystalline compound is the mono-succinate salt, and wherein the crystalline compound has 2θ values of 3.58 ± 0.2, 7.05 ± 0.2, 10.59 ± 0.2, 10.75 ± 0.2, 13.80 ± 0.2, 14.16 ± 0.2, 15.16 ± 0.2, 15.68 ± 0.2, 16.18 ± 0.2, 16.80 ± 0.2, 17.15 ± 0.2, 17.69 ± 0.2, 17.97 ± 0.2, 18.29 ± 0.2, 18.59 ± 0.2, 18.84 ± 0.2, 19.27 ± 0.2, 20.29 ± 0.2, 24.47 ± 0.2, 24.84 ± 0.2 and 28.47 ± 0.

2.

7. Use of a crystalline compound according to any one of claims 1 - 4 for the manufacture of a medicament for the treatment of a subject suffering from anemia.

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