EA040138B1 - IMMUNOPROTEASOME INHIBITOR BASED ON CRYSTAL SALTS OF PEPTIDE EPOXYKEtoneS - Google Patents

Description

State of the art Field of invention

This disclosure relates to the novel crystalline salts of (2S,3R)-N-[(2S)-3-(cyclopent-1-en-1ul)-1-[(2R)-2-methyloxiran-2-yl]-1- oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2(morpholin-4-yl)acetamido]propanamido]propanamide or salt hydrates, their pharmaceutical compositions, how to obtain them and how to use them.

Description of related technologies

Compound (2S,3R)-N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2ul] -3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide (Compound G) is useful as an immunoproteasome inhibitor

In eukaryotes, protein degradation is predominantly mediated through the ubiquitin metabolic pathway, in which proteins targeted for degradation are ligated to amino acid 76 of the ubiquitin polypeptide. Then, once targeted, ubiquitin proteins serve as substrates for the 26S proteasome, a multicatalytic protease that cleaves proteins into short peptides through its three major proteolytic activities. In addition to having a major function in intracellular protein metabolism, proteasome-mediated degradation also plays a key role in many processes, namely major histocompatibility complex (MHC) class I antigen presentation, apoptosis, cell growth regulation, NF-κB activation, antigen processing, and proinflammatory transduction. ny signals.

PCT Publication No. WO 2014/152134 describes tripeptide epoxy ketone proteasome inhibitors and methods of using these compounds to treat diseases and diseases associated with aberrant immunoproteasome activity. Because tripeptide epoxy ketone proteasome inhibitors, namely Compound G, are useful in the treatment of disease and disease in a patient, there is a need for readily soluble and stable forms of these compounds for their manufacture, delivery, storage and administration.

The essence of the invention

In one aspect, the disclosure provides a crystalline salt having the structure

where X- is a counterion. In some embodiments, X- is maleate, fumarate, oxalate, malate, sulfate, methanesulfonate, 2-naphthalenesulfonate, phosphate, halide, tartrate, citrate, tosylate, propionate, and/or benzoate. In various cases, the salt is a hydrate of the salt.

In some cases, X- is a maleate. For example, the crystalline salt may be a monomaleate.

Form A In some embodiments, the crystalline monomaleate is Form A characterized by (a) an X-ray powder diffraction pattern (XRPD) including peaks at about 6.9, 17.3, and 17.8±0.2° 2θ, using Cu Ka radiation, or (b) an XRPD pattern including peaks at about 6.9, 17.3, 17.8, 4.9, 6.8, 6.9, 7.7, 17.2, and 17.6 ±0.2° 2θ, using Cu Ka radiation, or (c) XRPD pattern including peaks at about 6.9, 17.3 17.8, 4.9, 6.8, 6.9, 7.7 , 17.2, 17.6, 10.9, 12.4, 13.5, 14.2, 16.1, 16.4, 18.5, 21.0, 22.0, 23.4, 23 .7, 24.5, and 25.2±0.2° 2θ using Cu Ka radiation, or (d) XRPD diffraction pattern essentially depicted in FIG. 1, or (e) a differential scanning calorimetry (DSC) thermogram essentially depicted in FIG. 2.

Form B In some embodiments, the crystalline monomaleate is Form B characterized by (a) an XRPD pattern including peaks at about 7.2, 18.4, and 22.0±0.2° 2θ using Cu Ka radiation, or (b) an XRPD pattern including peaks at about 6.8, 7.2, 18.4, 6.6, 13.6, 22.0, 17.4, 14.5, 18.0 and 5.0 ±0.2° 2θ using Cu Ka radiation, or (c) an XRPD diffraction pattern essentially depicted in FIG. 13, or (d) a DSC thermogram, essentially

- 1 040138 shown in FIG. 17.

Form C In some embodiments, the crystalline monomaleate is Form C characterized by (a) an XRPD pattern including peaks at about 7.4, 13.2, and 20.1±0.2° 2θ using Cu Ka radiation, or (b) XRPD pattern including peaks at about 6.6, 13.2, 7.4, 20.1, 13.6, 6.9, 16.9, 3.7, 17.9, and 19.9 ± 0.2° 2θ using Cu Ka radiation, or (c) an XRPD diffraction pattern essentially depicted in FIG. 7, or (d) DSC thermogram. essentially shown in Fig. 8.

Form D In some embodiments, the crystalline monomaleate is in form D characterized by (a) an XRPD pattern including peaks at about 4.9, 7.7, 10.9, 12.4, 13.6, and 15.3±0 .2° 2θ using Cu Ka radiation, or (b) an XRPD pattern including peaks at about 6.8, 4.9, 17.4, 15.3, 7.7, 3.4, 17.7, 13.6, 12.4, and 10.9±0.2° 2θ using Cu Ka radiation, or (c) an XRPD diffraction pattern essentially depicted in FIG. 9, or (d) a DSC thermogram essentially depicted in FIG. 10.

Form E In some embodiments, the crystalline monomaleate is Form E characterized by (a) an XRPD pattern including peaks at about 6.4, 7.3, and 19.8±0.2° 2θ using Cu Ka radiation, or (b) XRPD pattern including peaks at about 6.5, 3.3, 7.3, 19.8, 6.8, 16.5, 12.1, 21.5, 4.0, and 13.0± 0.2° 2θ using Cu Ka radiation, or (c) an XRPD diffraction pattern essentially depicted in FIG. 11, or (d) a DSC thermogram essentially depicted in FIG. 12.

Form F. In some embodiments, the crystalline monomaleate is Form F characterized by (a) an XRPD pattern including peaks at about 6.3, 19.0, and 19.6±0.2° 2θ using Cu Ka radiation, or (b) XRPD pattern including peaks at about 6.3, 7.1, 19.0, 17.5, 19.6, 17.9, 22.0, 13.5, 18.2, and 15.5± 0.2° 2θ using Cu Ka radiation, or (c) an XRPD diffraction pattern essentially depicted in FIG. 19, or (d) a DSC thermogram essentially depicted in FIG. 20.

In some cases, X ^- is a fumarate. For example, the crystalline salt is monofumarate.

Form G In some embodiments, the crystalline monofumarate is in Form G characterized by (a) an XRPD pattern including peaks at about 6.4, 7.2, 13.8, 16.0, 17.4, 18.5, 18 .7, 20.0, 20.9, 21.9, 24.5, and 25.8±0.2° 2θ using Cu Ka radiation, or (b) an XRPD diffraction pattern essentially depicted in FIG. 21, or (c) a DSC thermogram essentially depicted in FIG. 22. In some cases, the monofumarate is the hydrate of the monofumarate and may be a mixture of the hydrated and non-hydrated form (or anhydrite).

In some embodiments of the invention X-is an oxalate. In various embodiments of the invention X ^- represents malate. In some cases, X-is a sulfate. In various cases, X ^- represents methanesulfonate. In some embodiments of the invention, X ^- represents 2-naphthalenesulfonate. In various embodiments of the invention X ^- represents a phosphate. In some cases, a halide (eg, chloride, bromide, iodide). In various cases, X ^- is a tartrate. In some embodiments of the invention X ^- represents a citrate. In various embodiments of the invention, X-is a tosylate. In some cases, X ^- is a propionate. In various cases, X ^- is a benzoate. In any of these cases, the salt is in the form of a hydrate or a mixture of hydrated and non-hydrated forms (or anhydrite).

In another aspect, the disclosure provides a process for preparing the crystalline salt disclosed herein by mixing (a) Compound G, (b) maleic acid, and (c) a solvent to form a slurry.

In some embodiments, the molar ratio of compound G to maleic acid ranges from about 1:0.5 to 1:2, or about 1:1. In various cases, the solvent is selected from the group consisting of methanol (MeOH), ethanol (EtOH), isopropanol (IPA), ethyl acetate (EtOAc), isopropyl acetate (IPAc), tetrahydrofuran (THF), methyl tert-butyl ether (MTBE), acetone/n-heptane, acetone, diethyl ether (Et ₂ O)/EtOAc, hexane/EtOAc, MTBE/EtOAc,

- 2 040138 toluene, 1,4-dioxane, acetonitrile (ACN), 1-butanol, aqueous mixtures of the above and combinations thereof. For example, the solvent is EtOAc, IPAc, EtOH, aqueous mixtures thereof, or combinations thereof. In some embodiments of the invention, the mixing occurs at a temperature in the range from 0 to 80°C or at a temperature in the range from 40 to 60°C. Mixing occurs for up to 6 hours. In various embodiments of the invention, the method optionally includes cooling the suspension to 0°C. In some cases, the method optionally includes filtering the slurry to form a precipitate. In various cases, the method optionally includes washing, drying, or both washing and drying the precipitate. The method further includes recrystallization of the precipitate. Additionally or alternatively, the method further comprises (1) recovering compound G from the precipitate; and (2) mixing reduced compound G, maleic acid and a solvent to form a crystalline salt.

The disclosure further provides a formulation comprising the crystalline salts disclosed herein and one or more excipients. In some embodiments of the invention, the composition is a liquid dosage form.

In some cases, the dosage form is a lyophilized dosage form, and the lyophilized dosage form is restored to a liquid form. In some instances, the crystalline salt is in a liquid or reconstituted lyophilized dosage form at a concentration in the range of about 1 to about 150 mg/mL, or about 10 to about 70 mg/mL, or about 30 to about 50 mg/mL, by weight. free base crystalline salt.

In some embodiments of the invention, one or more excipients in the formulation are selected from the group consisting of a surfactant, a tonicity regulator, a buffer, and combinations thereof. In some cases, the lyophilized dosage form optionally contains a cryoprotectant, a bulking agent, or both. In various embodiments, the surfactant is a polysorbate, castor oil polyoxyl, poly(alkylene) glycol, caprylocaproyl polyoxyl glyceride, polyoxyalkylene block copolymers, and combinations thereof. In various cases, the tonicity regulator is a salt, a polyhydric alcohol, or combinations thereof. In some cases, the liquid dosage form or reconstituted lyophilized dosage form is isotonic. In some embodiments, the buffer is selected from the group consisting of citrate, phosphate, histidine, succinate, acetate, maleate, gluconate, and combinations thereof. In various instances, the liquid dosage form or reconstituted lyophilized dosage form has a pH in the range of about 3.0 to about 8.0, or about 4.0 to about 6.5. In various embodiments of the invention, the liquid dosage form or reconstituted lyophilized dosage form is suitable for parenteral administration to a subject (eg, a human). In some cases, parenteral administration is intravenous, intramuscular, intraperitoneal, or subcutaneous. For example, parenteral administration is subcutaneous. In some embodiments of the invention, the composition has a bioavailability of at least 55%, or at least 60%, or at least 65%.

Another aspect of the disclosure provides a method for inhibiting the immunoproteasome of a cell, comprising contacting the cell with a crystalline salt or formulation disclosed herein. In some embodiments, the LMP7 immunoproteasome is inhibited. In some cases, contact occurs in vivo. In various embodiments of the invention, contacting comprises administering to a subject suffering from a disorder associated with aberrant immunoproteasome activity. In some embodiments, the disorder is an autoimmune disease or inflammation. In some cases, this disease is psoriasis, dermatitis, systemic scleroderma, sclerosis, Crohn's disease, ulcerative colitis; respiratory failure syndrome, meningitis; encephalitis; uveitis; colitis; glomerulonephritis; eczema, asthma, chronic inflammation; atherosclerosis; lack of adhesion of leukocytes; rheumatoid arthritis; lupus erythematosus system (SLE); diabetes; multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; tuberculosis, sarcoidosis, polymyositis, granulomatosis, vasculitis; pernicious anemia (Addison's disease); disease, including diapedesis of leukocytes; inflammatory disease of the central nervous system (CNS); multiple organ failure syndrome; hemolytic anemia; myasthenia gravis; a disease mediated by an antigen-antibody complex; pulmonary-renal syndrome; antiphospholipid syndrome; allergic neuritis; Graves' disease; myasthenic Lambert-Eaton syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; muscle stiffness syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; polyneuropathy IgM; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia. In various cases, the disorder is lupus, lupus nephritis, rheumatoid arthritis, diabetes, scleroderma, ankylosing spondylitis, psoriasis, multiple sclerosis, Hashimoto's disease, meningitis, or inflammatory bowel disease.

Additional aspects and advantages will be apparent to those skilled in the art from an overview of the following detailed description. While the methods described herein are capable of being implemented in various forms, the description hereinafter includes specific embodiments with the understanding that the description is illustrative and does not limit the invention to the particular embodiments described herein.

Brief description of the drawings

In FIG. 1 shows an X-ray powder diffraction pattern (XRPD) for form A (compound G monomaleate prepared in ethyl acetate).

In FIG. 2 is a differential scanning calorimetry (DSC) thermogram for form A (compound G monomaleate prepared in ethyl acetate).

In FIG. 3 shows the thermogravimetric analysis (TGA) curve for form A (compound G monomaleate prepared in ethyl acetate).

In FIG. 4 is an isothermal plot of EAF for form A (40 to 95% relative humidity).

In FIG. 5 shows the XRPD pattern for Form B (compound G monomaleate hydrate form prepared in 95% ethanol).

In FIG. 6 is a DSP thermogram for form B (compound G monomaleate hydrate form prepared in 95% ethanol).

In FIG. 7 shows the XRPD pattern for Form C (compound G monomaleate prepared in acetone).

In FIG. 8 shows the TGA curve (upper curve) and the DSP thermogram (lower curve) for form C (compound G monomaleate prepared in acetone).

In FIG. 9 shows the XRPD pattern for Form D (compound G monomaleate prepared in acetonitrile).

In FIG. 10 shows the TGA curve (top curve) and the DSP thermogram (bottom curve) for Form D (compound G monomaleate prepared in acetonitrile).

In FIG. 11 shows the XRPD pattern for Form E (compound G monomaleate prepared in isopropyl alcohol).

In FIG. 12 shows the TGA curve (upper trace) and the DSP thermogram (lower trace) for form E (compound G monomaleate prepared in isopropyl alcohol).

In FIG. 13 shows the XRPD pattern for Form B (compound G monomaleate hydrate prepared in 3% water/acetone).

In FIG. 14 shows XRPD patterns for Form B (compound G monomaleate hydrate prepared in 3% water/acetone) under the indicated drying conditions.

In FIG. 15 shows XRPD patterns for Form B (compound G monomaleate hydrate prepared in 3% water/acetone) under the indicated drying conditions.

In FIG. 16 shows the TGA curve (upper trace) and the DSP thermogram (lower trace) for Form B (compound G monomaleate hydrate prepared in 3% water/acetone) after drying at room temperature overnight.

In FIG. 17 shows the TGA curve (top curve) and the DSP thermogram (bottom curve) for form B (compound G monomaleate hydrate prepared in 3% water/acetone) after drying at 30° C. overnight.

In FIG. 18 is an isothermal dynamic vapor sorption (VVA) plot for Form B (compound G monomaleate hydrate prepared in 3% water/acetone).

In FIG. 19 shows the XRPD pattern for Form F (compound G monomaleate prepared in MeOH/MTBE).

In FIG. 20 shows the TGA curve (top curve) and the DSP thermogram (bottom curve) for form F (compound G monomaleate prepared in MeOH/MTBE).

In FIG. 21 shows the XRPD pattern for form G (compound G monofumarate).

In FIG. 22 shows the TGA curve (top curve) and the DSP thermogram (bottom curve) for form G (compound G monofumarate).

In FIG. 23 shows XRPD patterns of Compound G monomaleate prepared in the indicated solvents (Forms A and B) using the indicated ratios to maleic acid and dried in vacuo at room temperature.

In FIG. 24 shows XRPD patterns for form F (compound G monomaleate prepared in MBTE) after vacuum drying and heating to 100°C compared to form A.

In FIG. 25 shows XRPD patterns for Form C (compound G monomaleate prepared in acetone) after vacuum drying and heating to 100°C.

In FIG. 26 shows XRPD patterns for Form A (compound G monomaleate prepared in EtOAc) after drying under the indicated conditions.

In FIG. 27 shows the XRPD patterns for Form A (compound G monomaleate prepared in EtOAc) before and after the DSP test.

Detailed description

This document proposes new crystalline salt forms and their hydrates (2S,3R)-N- 4 040138

[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3- (4methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide (compound G), useful as a proteasome inhibitor

The crystalline form of the salt of Compound G described herein is soluble and stable in solution even at high concentrations. As such, the crystalline salt form of Compound G is suitable for pharmaceutical dosage forms suitable, for example, for parenteral administration. Compound G salt hydrates are also suitable for pharmaceutical dosage forms.

As used herein, the term crystalline refers to a solid in which the constituent atoms, molecules or ions are arranged in a regularly ordered repeating pattern in three dimensions.

As used herein, the term hydrate refers to the form of a substance that contains a bond between a substance and water. The hydrate is crystalline. As used herein, the term monohydrate refers to a hydrate that contains one molecule of water per molecule of substrate.

The term prophylactic or therapeutic treatment is recognized in the art and includes the administration to the host of one or more of the compositions in question. If the composition in question is administered prior to the clinical manifestation of the undesirable condition (e.g., a disease or other undesirable condition in the host animal), then the treatment is prophylactic (i.e., it protects the host from the development of the undesirable condition), and if the composition in question is administered after the manifestation of the undesirable condition, that treatment is therapeutic (i.e., it is intended to reduce, improve, or stabilize an existing undesirable condition or its side effects).

A therapeutically effective amount of a compound, with respect to the treatment in question, refers to the amount of the compound(s) in the formulation which, when administered as part of a desired dosing regimen (to a patient, e.g., a human), alleviates a symptom, ameliorates, or delays the onset of disease states, in accordance with the clinical acceptable standards for the disorder or disease being treated, or for cosmetic purposes, for example, with an appropriate benefit/risk ratio applicable to any medical treatment.

As used herein, the term treatment includes changing, reducing, or stopping the symptoms, clinical signs, and underlying disease pathology in order to improve or stabilize the patient's condition.

The compounds described in this text may be identified either by their chemical structure and/or chemical name in this document. When a chemical structure and a chemical name conflict, the chemical structure is decisive for the identification of the compound.

Unless otherwise indicated, the terms and abbreviations used in this description include the normal and traditional meaning in the relevant field.

Since the contribution of the present description is not limited to the specific implementation options or aspects described in this document, the description provides a person skilled in the art with additional implementation options, including changes and modifications to adapt to different applications and conditions. For example, changes and modifications to materials, synthetic methods, or techniques described herein will be apparent to those skilled in the art.

The ranges used herein for physical properties, namely molecular weight, or chemical properties, namely chemical formulas, all combinations and subcombinations of the ranges, and specific embodiments therein, are intended to be included.

Crystalline salts of compound G and their hydrates

In one aspect, the disclosure provides crystalline salts of compound G having the structure

- 5 040138 where X- is a counterion. Examples of X- include, for example,

fat), (methanesulfonate), (fumarate),

(oxalate),

or

(malate), SO ₄ ^2- (sul (maleate), (2-naphthalenesulfonate), PO ₄ ^3- (phosphate), haloOH O no. D. A genide

O (for example, chloride, bromide, iodide, fluorine),

BUT'

OH (citrate), ^Hi

(tosylate),

(propionate),

T°'

OH (tartrate),

or (benzoate). In some embodiments of the invention, X is a divalent anion (X ^2- ). In these embodiments, the bridging salt is formed with one X ^2- molecule, forming an ionic bond with each of the two molecules of compound G

In another aspect, the disclosure provides hydrates of compound G, namely compound G monohydrates or hydrates of salts of compound G.

Monomaleates and hydrates of compound G

In some embodiments of the invention X-is a maleate. In these embodiments, the crystalline salt of Compound G is a monomaleate (shown below). Compound G monomaleate has a molecular weight of 586.7 g/mol, pK _a 5, and is a white to yellow solid. Compound G monomaleate has a high solubility in water, which exceeds 100 mg/ml. This high solubility is advantageous as it allows Form A to be used in parenteral pharmaceutical compositions at high concentrations.

The formation of monomaleate was unexpected because maleic acid has two acid protons, each of which can form an ionic bond with a morpholino group in compound G to form a bridged maleate (shown below). However, mono-maleate forms are not formed, but rather mono-maleate forms, regardless of whether a molar ratio of maleic acid to compound G of 0.5:1 or a molar ratio of 1:1 is used in their preparation. Therefore, monomaleate does crystallize during production, regardless of the ratio of starting maleic acid used, and despite the heterogeneity of the reaction mixture that is formed when maleic acid is added to Compound G during its preparation.

- 6 040138

Monomaleate (formed)

Bridged maleate (not formed)

Compound G monomaleate (crystalline) is more preferred than Compound G (amorphous), not only because of its crystallinity, but also because it has increased solubility in water. For example, Compound G monomaleate has an aqueous solubility greater than 100 mg/mL at ambient temperature (eg, 20 to 25°C). In contrast, the solubility of compound G in water is only 8.9 mg/ml. See table. 1 below for additional solubility data for Compound G, and Table. 2 below for additional solubility data for Compound G monomaleate.

Table 1. Solubility of compound G (amorphous)

Solvent pH Solubility (mg/ml) Solvent pH Solubility (mg/ml) Water 7.4 8.9 PG 100% >100 0.9% saline 7, 6 9, 4 PG, 67% >100 BBS 7.2 7b PG, 33% 20.5 25 mM citrate sodium 4, 9 46.0 PG, 10% 12.1

- 7 040138

25 mM sodium citrate 5, 1 32.0 PEG400, 100% >50 25 mM sodium citrate 5, 2 24.8 PEG400, 67% >50 25 mM sodium citrate 5, 4 19.5 PEG 400, 33% 17.4 25 mM sodium citrate 5, 8 11.2 PEG400 10% 11, 8 25 mM sodium citrate 6, 3 9, 8 Glycerin, 100% He soluble 25 mM sodium citrate 6, 8 8.8 Glycerin, 67% 5, 4 NMP 100% >100 Glycerin, 33% 5, 8 NMP, 67% >100 Glycerin, 10% 8, 6 NMP, 33% >100 EtOH 100% >100 NMP, 10% 28.8 EtOH, 67% >100 EtOH, 33% 20.0 EtOH, 10% 13.7

Table 2 Solubility Data for Compound G Monomaleate (crystalline)

Solvent Solubility (mg/ml) Solvent Solubility (mg/ml) Acetonitrile ("ACN") 1.30 2- methyltetrahydrofuran ("THE") 1.76 Acetone 3, 19 Methyl tert-butyl ether ("MTVE") 0.17 Dichloromethane ("DCM") 0.21 Isopropanol ("ΙΡΑ") 3.28 ethyl acetate ("EA") 0.47 Isopropyl acetate ("1PAc") 0.13 ethanol ("ETOH") 6.97 Tetrahydrofuran ("THE") 1.96 methanol ("MeON") 42.13 Toluene 0.02

The high solubility of compound G monomaleate in water is unexpected, since the crystalline salt is more thermodynamically stable than the amorphous form (compound G) and hence is expected to be less soluble in water. Additionally, maleates of known compounds (eg, alprenolol and prazosin) have reduced solubility compared to other counterions, namely fumarate. See, for example, Olovson et al., Acta Pharmacol Toxicol 58 (1): 55-60 (1986) and Kumar et al., AAPS PHarmSciTech 14 (1): 141-150 (2013).

Compound G monomaleate is crystallized, for example, from ethyl acetate (Form A), 95% ethanol or

- 8 040138

3% water/acetone to produce monohydrate (Form B), acetone (Form C), acetonitrile (Form D), isopropyl alcohol (Form E), or MeOH/MTBE (Form F). Each of these forms can be characterized by the parameters described below. Each form can be characterized by X-ray powder diffractometry (XRPD), differential scanning calorimetry (DSC), or thermogravimetric analysis (TGA), each of which is described in the Methods section below. The dehydration of crystalline forms, which occurs during both DSC and TGA, is a kinetic process, which is influenced by the experimental parameters.

Form A (crystallized from ethyl acetate)

Form A is characterized by an XRPD pattern obtained as described in the Methods section, having peaks at about 6.9, 17.3 and 17.8±0.2° 2θ using Cu Ka radiation. Form A is also characterized by an XRPD pattern having peaks at about 4.9, 6.8, 6.9, 7.7, 17.2 and 17.6±0.2° 2θ using Cu Ka radiation. Form A is optionally further characterized by an X-ray diffraction pattern for the powder product having additional peaks at about 10.9, 12.4, 13.5, 14.2, 16.1, 16.4, 18.5, 21.0, 22, 0, 23.4, 23.7, 24.5, and 25.2 ±0.2° 2θ using Cu Ka radiation. In some embodiments, Form A is characterized by an X-ray diffraction pattern for the powder product, essentially depicted in FIG. 1.

Additionally or alternatively, form A is characterized by DSC obtained as described in the Methods section. Form A is characterized by a DSC thermogram having a dehydration endotherm beginning at about 135° C. to about 150° C. when Form A (crystallized from ethyl acetate) is heated in an aluminum crucible. For example, in embodiments where Form A is heated from about 30°C at a rate of about 10°C/min, Form A is characterized by a DSC thermogram depicting a melting process beginning at about 148°C and peaking at about 152°C as shown in Fig. 2 (crystallized from ethyl acetate). In some embodiments, form A is characterized by a DSC thermogram, essentially depicted in FIG. 2 (crystallized from ethyl acetate).

Additionally or alternatively, form A is characterized using TGA, obtained as described in the methods section. Form A is characterized by a weight loss in the range of about 1.5 to about 2.5% at an onset temperature in the range of about 10 to about 30°C. For example, Form A (crystallized from ethyl acetate) has a weight loss of about 0.8% starting at about 34°C as depicted in FIG. 3. In some embodiments, Form A (crystallized from ethyl acetate) has a TGA curve essentially depicted in FIG. 3.

Additionally or alternatively, form A is characterized by dynamic vapor sorption (DSP). For example, when exposed to chipboard as described in the Methods section, Form A has an overall weight gain of about 3.5% by weight at about 40% to about 95% relative humidity, as depicted in FIG. 4. Based on the absorption of about 1 mole of water per mole of Form A over the entire humidity range, the reversibility of this process during dehydration, the low degree of hysteresis, and the presence of a dehydration endotherm in FIG. 6 in temperature ranges below the melting process, but not in FIG. 2, Form A is considered to readily transition from an anhydrous to a hydrated version of Form A, and vice versa, depending on moisture conditions. The anhydrous form is crystallized using a solvent of poor miscibility with water (namely, for example, ethyl acetate). The hydrated form is crystallized using a solvent containing water (namely, for example, 95% ethanol/5% water or 3% acetone/water). Mutual transition between forms is carried out after crystallization by controlled exposure to humidity.

Form B (hydrate form of compound G monomaleate crystallized from 95% ethanol)

In some embodiments, Form B (crystallized from 95% ethanol) has an XRPD pattern having peaks at about 6.1, 6.6, 7.2, 7.7, 9.4, 9.9, 10.8, 12.8, 14.5, 16.0, 16.4, 17.0, 17.4, 18.4 18.8, 19.8, 20.6, 21.8, 23.4, 26.6 , 27.0 and 42.0 ±0.2° 2θ, using Cu Ka radiation. In some cases, Form B (crystallized from 95% ethanol) has an X-ray diffraction pattern of the powder product essentially shown in FIG. 5. Additionally or alternatively, Form B (crystallized from 95% ethanol) is characterized by DSC as outlined in the Methods section. For example, in embodiments in which Form B (crystallized from 95% ethanol) is heated from about 30°C at a rate of about 10°C/min, Form B (crystallized from 95% ethanol) has a DSC thermogram depicting a melting process with beginning at about 148°C and peaking at about 152°C, as depicted in FIG. 6. More specifically, Form B (crystallized from 95% ethanol) has a DSC thermogram essentially depicted in FIG. 6. Form B, Compound G monomaleate hydrate, was also crystallized from 3% water/acetone. In these embodiments, form B is characterized by an XRPD pattern obtained as described in the Methods section having peaks at about 7.2, 18.4, and 22.0±0.2° 2θ using Cu Ka radiation. Form B (crystallized from 3% water/acetone) also has an XRPD pattern having peaks at about 6.8, 7.2, 18.4, 6.6, 13.6, 22.0, 17.4, 14, 5, 18.0 and 5.0 ±0.2° 2θ using Cu Ka radiation. In some embodiments of the invention

- 9 040138 Form B (crystallized from 3% water/acetone) has an X-ray diffraction pattern for the powder product essentially shown in FIG. 13. In some embodiments, Form B (crystallized from 3% water/acetone) is further processed and dried to obtain the residue described in Example 9. As depicted in FIG. 14 and 15, drying conditions do not affect the diffractogram. Additionally or alternatively, Form B (crystallized from 3% water/acetone) is characterized by DSC as outlined in the Methods section. For example, in embodiments in which Form B (crystallized from 3% water/acetone) is heated from about 30°C at a rate of about 10°C/min, Form B (crystallized from 3% water/acetone) is characterized by DSC , TGA and DSP as described in Example 9 and shown in FIG. 16, 17 and 18, respectively. In some embodiments, Form B (crystallized from 3% water/acetone) has a DSC thermogram essentially depicted in FIG. 17. Additionally or alternatively, Form B (crystallized from 3% water/acetone) is characterized by TGA as described in the Methods section. In some embodiments, Form B (crystallized from 3% water/acetone) has a TGA curve essentially depicted in FIG. 17.

Form C (crystallized from acetone)

Form C is characterized by an XRPD pattern obtained as described in the methods section, having peaks at about 7.4, 13.2 and 20.1 ± 0.2° 2θ using Cu Ka radiation. Form C is also characterized by an XRPD pattern having peaks at about 6.6, 13.2, 7.4, 20.1, 13.6, 6.9, 16.9, 3.7, 17.9, and 19.9 ±0.2° 2θ, using Cu Ka radiation. In some embodiments, Form C is characterized by an X-ray diffraction pattern for the powder product, essentially depicted in FIG. 7. Additionally or alternatively, form C is characterized by DSC as described in the Methods section. For example, in embodiments where Form C is heated from about 30°C at a rate of about 10°C/min, Form C has a DSC thermogram depicting a melting process beginning at about 142°C and peaking at about 159°C, as shown in FIG. 8. More specifically, form C is characterized by a DSC thermogram essentially depicted in FIG. 8. Additionally or alternatively, Form C is characterized by TGA as described in the Methods section. Therefore, Form C has a weight loss of about 6.0% at about 29 to 130°C, as depicted in FIG. 8. In some embodiments, form C is characterized by a TGA curve essentially depicted in FIG. 8.

Form D (crystallized from acetonitrile)

Form D is characterized by an XRPD pattern obtained as described in the methods section, having peaks at about 4.9, 7.7, 10.9, 12.4, 13.6, and 15.3±0.2° 2θ using Cu Ka radiation. Form D is also characterized by an XRPD pattern having peaks at about 6.8, 4.9, 17.4, 15.3, 7.7, 3.4, 17.7, 13.6, 12.4, and 10.9 ±0.2° 2θ, using Cu Ka radiation. In some embodiments, Form D is characterized by an X-ray diffraction pattern for the powder product, essentially depicted in FIG. 9. Additionally or alternatively, Form D is characterized by DSC as described in the Methods section. For example, in embodiments where Form D is heated from about 30°C at a rate of about 10°C/min, Form D has a DSC thermogram depicting a melting process beginning at about 149°C and peaking at about 152°C, as shown in FIG. 10. More specifically, form D is characterized by a DSC thermogram, essentially depicted in FIG. 10. Additionally or alternatively, Form D is characterized by TGA as described in the Methods section. Therefore, Form D has a weight loss of about 0.3% at about 27 to 130°C, as depicted in FIG. 10. In some embodiments, Form D is characterized by a TGA curve essentially depicted in FIG. 10.

Form E (crystallized from isopropyl alcohol)

Form E is characterized by an XRPD pattern obtained as described in the methods section, having peaks at about 6.4, 7.3 and 19.8±0.2° 2θ using Cu Ka radiation. Form E is also characterized by an XRPD pattern having peaks at about 6.5, 3.3, 7.3, 19.8, 6.8, 16.5, 12.1, 21.5, 4.0, and 13.0 ±0.2° 2θ using Cu Ka radiation. In some embodiments, Form E is characterized by an X-ray diffraction pattern for the powder product, essentially depicted in FIG. 11. Additionally or alternatively, Form E is characterized by DSC as outlined in the Methods section. For example, in embodiments where Form E is heated from about 30°C at a rate of about 10°C/min, Form E has a DSC thermogram depicting a melting process beginning at about 138°C and peaking at about 148°C, as shown in FIG. 12. More specifically, form E is characterized by a DSC thermogram essentially depicted in FIG. 12. Additionally or alternatively, Form E is characterized by TGA as described in the Methods section. Therefore, Form E has a weight loss of about 0.9% at about 32 to 99°C, as shown in FIG. 12. In some embodiments, Form E is characterized by a TGA curve essentially depicted in FIG. 12.

Form F (crystallized from MeOH/MTBE)

Form F is characterized by an XRPD pattern obtained as described in the Methods section.

- 10 040138 having peaks at about 6.3, 19.0 and 19.6 ±0.2° 2θ using Cu Κα radiation. Form F is also characterized by an XRPD pattern having peaks at about 6.3, 7.1, 19.0, 17.5, 19.6, 17.9, 22.0, 13.5, 18.2, and 15.5 ±0.2° 2θ, using Cu Ka radiation. In some embodiments, Form F is characterized by an X-ray diffraction pattern for the powder product, essentially depicted in FIG. 19. Additionally or alternatively, Form F is characterized by DSC as described in the Methods section. For example, in embodiments in which Form F is heated from about 30°C at a rate of about 10°C/min, Form F has a DSC thermogram depicting a melting process beginning at about 128°C and peaking at about 135°C, as shown in FIG. 20. More specifically, form F is characterized by a DSC thermogram essentially depicted in FIG. 20. Additionally or alternatively, Form F is characterized by TGA as described in the Methods section. Therefore, Form F has a weight loss of about 1.4% at about 32 to 99°C, as depicted in FIG. 20. In some embodiments, Form F is characterized by a TGA curve essentially depicted in FIG. 20.

Compound G monofumarates

In some embodiments of the invention X-is a fumarate. In these embodiments, the crystalline salt of Compound G is monofumarate (shown below). A specific crystalline form of compound G monofurmarate is form G.

Monofumarate

Form G is characterized by one or more of the parameters described below.

Form G is characterized by an XRPD pattern obtained as described in the Methods section, having peaks at about 6.4, 7.2, 13.8, 16.0, 17.4, 18.5, 18.7, 20.0, 20.9, 21.9, 24.5 and 25.8 ±0.2° 2θ using Cu Ka radiation. In embodiments of the invention Form G is characterized by an X-ray diffraction pattern for the powder product, essentially depicted in FIG. 21.

Additionally or alternatively, the G form is characterized by DSC. DSC thermograms are obtained as described in the Methods section. Form G dehydration is a kinetic process influenced by experimental parameters. Therefore, form G is characterized by a DSC thermogram having a dehydration endotherm with an onset in the range of about 75 to about 90° C. when form G is heated in a sealed aluminum crucible. For example, in embodiments in which Form G is heated from about 25°C at a rate of about 10°C/min, Form G is characterized by a DSC thermogram having a dehydration endotherm beginning at about 82°C and peaking at about 101°C, as shown in FIG. 22. In some embodiments, form G is characterized by a DSC thermogram, essentially depicted in FIG. 22.

Compound G oxalates

In some embodiments of the invention X-is an oxalate. In some cases, the crystalline salt of compound G is a monooxalate. In various cases, oxalate reacts with morpholino groups on two different molecules of compound G to form a bridging salt. Compound G oxalate is characterized by one or more of the parameters described below in the Methods section (eg, XRPD, DSC, TGA, and/or DSP).

Compound G malates

In some embodiments of the invention X-is a malate. In some instances, the crystalline salt of Compound G is a monomalate. In various cases, malate reacts with morpholino groups on two different molecules of compound G to form a bridging salt. Compound G malate is characterized by one or more of the parameters described below in the methods section (eg, XRPD, DSC, TGA, and/or DSP).

Compound G sulfates

In some embodiments of the invention X ^- represents a sulfate. Compound G sulfate may be characterized by one or more of the parameters described below in the methods section (eg, XRPD, DSC, TGA, and/or DSP).

Compound G methanesulfonates

In some embodiments of the invention, X ^- is a methanesulfonate. The methanesulfonate of compound G is characterized by one or more of the parameters described below in the section

- 11 040138 methods (eg XRPD, DSC, TGA and/or DSP).

2-Naphthalenesulfonates of compound G

In some embodiments of the invention X ^- represents 2-naphthalenesulfonate. The 2-naphthalenesulfonate compound G is characterized by one or more of the parameters described below in the methods section (eg, XRPD, DSC, TGA and/or DSP).

Compound G phosphates

In some embodiments of the invention X ^- represents a phosphate. The compound G phosphate is characterized by one or more of the parameters described below in the methods section (eg, XRPD, DSC, TGA, and/or DSP).

Compound G halides

In some embodiments of the invention, X ^- is a halide (eg, chloride, bromide, iodide, fluoride). Compound G halide is characterized by one or more of the parameters described below in the methods section (eg, XRPD, DSC, TGA, and/or DSP).

Compound G tartrates

In some embodiments of the invention, X ^- is a tartrate. Compound G tartrate is characterized by one or more of the parameters described below in the methods section (eg, XRPD, DSC, TGA, and/or DSP).

Compound G citrates

In some embodiments of the invention X ^- represents a citrate. Compound G citrate is characterized by one or more of the parameters described below in the Methods section (eg, XRPD, DSC, TGA, and/or DSP).

Compound G tosylates

In some embodiments of the invention, X ^- is a tosylate. Compound G tosylate is characterized by one or more of the parameters described below in the Methods section (eg, XRPD, DSC, TGA, and/or DSP).

Compound G Propionates

In some embodiments of the invention X ^- represents a propionate. Compound G propionate is characterized by one or more of the parameters described below in the Methods section (eg, XRPD, DSC, TGA, and/or DSP).

Compound G benzoates

In some embodiments of the invention, X ^- is a benzoate. Compound G benzoate is characterized by one or more of the parameters described below in the Methods section (eg, XRPD, DSC, TGA, and/or DSP).

Hydrates of salts of compound G

In some embodiments, the disclosure provides a salt hydrate of Compound G or a free base monohydrate of Compound G. The salt hydrate or free base monohydrate of Compound G is crystalline and has one or more of the parameters described below in the Methods section (e.g., XRPD, DSC , TGA and/or chipboard).

Methods for the preparation of crystalline salts, hydrates and hydrates of salts of compound G

The crystalline salts, hydrates and hydrates of the salts of Compound G are prepared by various methods known in the crystalline art. The following discussion of crystalline salts of compound G applies to the formation of hydrates of crystalline salts and to the free base crystalline hydrates of compound G.

In some embodiments, compound G (amorphous form) is mixed with an appropriate acid with an X ^- counterion, HX (e.g., maleic acid, fumaric acid, hydrochloric acid, oxalic acid, sulfuric acid, phosphoric acid, malic acid, methanesulfonic acid, 2- naphthalenesulfonic acid, tartaric acid, citric acid, toluenesulfonic acid, propionic acid, benzoic acid) in a solvent to form a suspension. The molar ratio of compound G to HX ranges from about 1:05 to about 1:2, namely about 1:1.

The solvent which is added to the compound G and HX is any solvent in which the desired crystalline salts can be formed. Suitable solvents include, but are not limited to, methanol (MeOH), ethanol (EtOH), isopropanol (IPA), ethyl acetate (EtOAc), isopropyl acetate (IPAc), tetrahydrofuran (THF), methyl tert-butyl ether (MTBE), acetone /n-heptane, acetone, diethyl ether (Et ₂ O)/EtOAc, hexane/EtOAc, MTBE/EtOAc, toluene, 1,4dioxane, acetonitrile (ACN), 1-butanol, aqueous mixtures of the above and combinations thereof. In some embodiments, the solvent is EtOAc, IPAc, EtOH, aqueous mixtures thereof, or combinations thereof. For example, the solvent is EtOAc.

The mixing step occurs at a temperature in the range of about 0 to 80°C, or about 30 to 70°C, or about 40 to 60°C (for example, about 0, 5, 10 15, 20, 25, 30, 35 , 40, 45, 50, 55, 60, 65, 70, 75 or 80°C). In some cases, the mixing step occurs at 50°C.

The mixing step lasts for a period of time up to about 6 hours, or up to about 5 hours, or up to about 4 hours,

- 12 040138 or up to about 3 hours, or up to about 2 hours, or up to about 1 hour. In some embodiments, the mixing step occurs for at least 15 minutes, or at least 30 minutes, or at least 45 minutes , or at least 1 hour, or at least 2 hours, or at least 3 hours, or at least 4 hours, or at least 5 hours. In various cases, the mixing step occurs for from about 1 hour to about 6 hours, or from about 4 hours to about 6 hours, or from about 3 hours to about 5 hours.

The crystalline salt of Compound G is isolated from the slurry by cooling the slurry to about -10 to about 10°C, or about -5 to about 5°C, or to about 0°C. In some embodiments of the invention, the cooled suspension is filtered to obtain a precipitate. The precipitate is then optionally washed, dried, or both.

In some cases, the crystalline salt of compound G is purified by recrystallization. In various cases, the crystalline salt of Compound G is purified by (1) recovering Compound G from the precipitate and (2) mixing the reduced Compound G with HX and a solvent to recover the crystalline salt of Compound G.

As shown in the Examples section below, a variety of solvents have been found suitable for the preparation of crystalline salts of Compound G, namely Compound G monomaleates, in good yield and purity.

Pharmaceutical Compositions and Administration of Compound G Crystalline Salts

Another aspect of the disclosure provides pharmaceutical compositions (alternatively referred to throughout the text as formulations) that contain the crystalline salts described herein and one or more pharmaceutically acceptable excipients. The phrase pharmaceutically acceptable is used herein to refer to those ligands, materials, compositions and/or dosage forms that are medically suitable for use in contact with human and animal tissues without causing undue toxicity, irritation, allergic reaction or other problems, or complications commensurate with a reasonable benefit/risk ratio. The compositions described herein are formulated into any dosage form.

In some embodiments of the invention, the formulations are for parenteral administration. The phrases parenteral administration and parenterally administered as used herein mean methods of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraocular, intracardiac, intradermal, intraperitoneal , transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, injection and infusion. For example, parenteral administration includes intravenous, intramuscular, intraperitoneal, or subcutaneous injection.

Parenteral pharmaceutical compositions are liquid dosage forms or lyophilized dosage forms that are reconstituted into a liquid for parenteral injection.

The high solubility of the crystalline salts of Compound G described herein makes the salts suitable for subcutaneous administration. Subcutaneous administration is the preferred form of administration because these formulations can be self-administered at home (rather than traveling to a medical facility for an infusion), which is convenient for patients, and they also have fewer side effects (eg, less pain at the site of injection and bruising) than with other forms of fluid administration (eg, intravenous or intramuscular). Both the convenience and reduced side effects of subcutaneous formulations result in better patient compliance. However, subcutaneous administration has a practical injection volume limit of about 0.3 to about 1.5 ml, such as about 1.0 ml. Therefore, it is often necessary to include inactive ingredients in subcutaneous formulations having high concentrations of drug in order to deliver a therapeutically effective amount of drug. For example, delivery of about 10 to about 100 mg of Compound G for the treatment of autoimmune disorders implies a subcutaneous injection concentration of about 6 to about 100 mg/mL. Accordingly, the high solubility of the crystalline salts of compound G described herein (in excess of 100 mg/ml) makes them suitable for subcutaneous administration.

Therefore, in some embodiments of the invention, the pharmaceutical composition contains a crystalline salt of compound G in a concentration in the range from about 0.1 to about 200 mg/ml, or from about 1 to about 150 mg/ml, or from about 10 to about 70 mg/ml. ml, or about 30 to about 50 mg/ml, or about 100 to about 200 mg/ml, or about 75 to about 125 mg/ml. For example, the concentration is about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mg/mL.

In some embodiments of the invention, one or more excipients include a surfactant, a tonicity regulator, a buffer, or combinations thereof. In embodiments of the invention in which the composition is a lyophilized dosage form, one or more excipients further include a cryoprotectant, a bulking agent, or both. Suitable cryoprotectants include, but are not limited to, glucose, sucrose, trehalose, lactose, mannitol, sorbitol, colloidal silicon dioxide, maltose, poly(vinyl pyrrolidone), fructose,

- 13 040138 dextran, glycerin, polyvinyl alcohol, glycine, hydroxypropyl-beta-cyclodextrin and gelatin.

Suitable bulking agents include, but are not limited to, sugars, namely mannitol, lactose, sucrose, trehalose, sorbitol, glucose, and raffinose; amino acids, namely arginine, glycine and histidine; and polymers, namely dextran and polyethylene glycol.

One or more excipients include a surfactant, namely a nonionic surfactant. Nonionic surfactants are useful in stabilizing the composition against degradation due to shipping and storage stress. Suitable surfactants for inclusion in pharmaceutical compositions include, but are not limited to, polysorbates and polyethers. For example, the surfactant is a polysorbate (eg, polysorbate 80 or polysorbate 20), castor oil polyoxyl, poly(alkylene) glycol (eg, polyethylene glycol, polypropylene glycol), caprylocaproyl polyoxyl glyceride, polyoxyalkylene block copolymer (eg, polyoxyethylene polyoxypropylene), and combinations thereof. . In some embodiments of the invention, the surfactant is additionally a co-solvent, namely N-methyl-2-pyrrolidone (NMP).

One or more excipients include a tonicity regulator (sometimes referred to as an isotonic agent). Tonicity regulators are added to subcutaneous formulations to ensure that the formulation has an osmolality that is appropriate for the patient's cells (eg, 250 to 350 mosm) to minimize or prevent tissue damage at the injection site. Tonicity regulators include salts and polyols (eg sugars, namely non-reducing sugars, sugar alcohols and sugar acids). Specifically contemplated tonicity regulators include, but are not limited to, NaCl, KCl, glucose, fructose, sucrose, maltose, lactose, sucrose, mannose, raffinose, mannitol, xylitol, galactitol, glucositol, inositol, sorbitol, trehalose, and glycerol. Accordingly, this document also provides pharmaceutical compositions that are isotonic.

One or more excipients include a buffer. Pharmaceutically acceptable buffers include, but are not limited to, citrate, phosphate, histidine, succinate, acetate, maleate, gluconate, and combinations thereof. In some embodiments of the invention, the pH of the composition is in the range from about 3.0 to 8.0, or from about 4.0 to 7.0, or from about 4.0 to 6.5.

The crystalline salt formulations disclosed herein are administered to a subject, namely a human subject or an animal subject. In some embodiments, these formulations have a bioavailability of at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%. In some instances, the formulations disclosed herein have a bioavailability of up to about 90%, or up to about 85%, or up to about 80%, or up to about 75%, or up to about 65%, or up to about 60%. For example, the formulations exhibit a bioavailability of about 45% to about 90%, or about 50% to about 70%, or about 50% to about 65%.

The crystalline salts disclosed herein are also formulated into pharmaceutical compositions having the forms and containing the excipients detailed below.

In some embodiments of the invention, the pharmaceutical compositions contain a pharmaceutically acceptable carrier. The phrase pharmaceutically acceptable carrier as used herein means a pharmaceutically acceptable material, composition, or excipient, namely a liquid or solid excipient, diluent, excipient, solvent, or encapsulating material. As used herein, the formulation of a pharmaceutically acceptable carrier includes a buffer, sterile water for injection, diluents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Each carrier must be acceptable in terms of compatibility with the other ingredients of the formulation and not cause harm to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include (1) sugars, namely lactose, glucose and sucrose; (2) starches, namely corn starch, potato starch and substituted or unsubstituted β-cyclodextrin; (3) cellulose and its derivatives, namely sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, namely cocoa butter and suppository waxes; (9) oils, namely peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, namely propylene glycol; (11) polyhydric alcohols, namely glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, namely ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, namely magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations. In certain embodiments of the invention, the pharmaceutical compositions provided herein are non-pyrogenic, that is, they do not cause a significant increase in temperature when administered to a patient.

- 14 040138

As excipients in the compositions are also wetting agents, emulsifiers and lubricants, namely sodium lauryl sulfate and magnesium stearate, as well as dyes, anti-adhesives, coating agents, sweeteners, flavors and fragrances, preservatives and antioxidants.

Examples of pharmaceutically acceptable antioxidant excipients include (1) water-soluble antioxidants, namely ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) fat-soluble antioxidants, namely ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, namely citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The pharmaceutical composition also contains excipients, namely preservatives, wetting agents, emulsifiers and dispersants. Prevention of the influence of microorganisms is provided by the addition of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It is also desirable to include tonicity regulating agents such as sugars and the like in the compositions. Additionally, prolonged absorption of the injectable pharmaceutical form is achieved by the addition of agents that delay absorption, namely aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of one or more of the compounds provided herein, it is desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. For example, delayed absorption of a parenterally administered compound is achieved by dissolving or suspending the compound in an oil vehicle.

The composition must be stable under the conditions of manufacture and storage and must be protected from the contaminating effects of microorganisms, namely bacteria and fungi. Prevention of exposure to microorganisms is achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents in the composition, for example, sugars, polyhydric alcohols, namely mannitol, sorbitol, and sodium chloride. Delayed absorption of injectable compositions is achieved through the use of agents that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by adding, if necessary, the active compound in the required amount in a suitable solvent with one or a combination of the ingredients listed above, followed by filtered sterilization. Typically, dispersions are prepared by incorporating the active ingredient into a sterile vehicle which contains the basic dispersion medium and the other necessary ingredients listed above. In the case of sterile powders for the preparation of injectable sterile solutions, the methods of preparation are freeze-drying (lyophilization), which results in a powder of the active ingredient, plus any additional target ingredient from its source solution, sterilized by filtration.

Injectable depot forms are prepared by forming microencapsulated or nanoencapsulated matrices of the compound provided herein in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot formulations are also prepared by administering the drug in liposomes, microemulsions or nanoemulsions that are compatible with body tissues.

In one embodiment, the therapeutic crystalline salts are formulated with carriers that protect the therapeutic compounds from rapid excretion from the body, namely controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, namely ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Such formulations are obtained using standard methods or commercially, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) are also used as pharmaceutically acceptable carriers. They are prepared according to methods known to those skilled in the art, such as those described in US Pat. No. 4,522,811, which is incorporated herein by reference in its entirety.

Pharmaceutical compositions are packaged in a container, pack or tablet box, along with instructions for use.

Methods of using crystalline salts of compound G

The crystalline salts disclosed herein act as immunoproteasome (IP) inhibitors. In some instances, the crystalline salts disclosed herein inhibit the LMP7 subunit of the PI. LMP7 activity can be inhibited by at least 10%, according to

- 15 040138 at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% , as measured in the proteasome subunit assay described in the examples below. The crystalline salt described herein inhibits one or more additional IP subunits, namely LMP2, MECL-1, β 1, β2 and β5. In various embodiments, the crystalline salt disclosed herein inhibits LMP7 and one or both of LMP2 and MECL-1. The compounds disclosed herein reduce the activity or expression of cytokines, for example one or more of IL-2, MHC-I, IL-6, TNFa, and IFN-β. Therefore, methods are provided in which a compound disclosed herein inhibits the expression or activity of one or more of IL-2, MHC-I, IL-6, TNFa, and IFN-β by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%.

This document further provides methods for inhibiting an immunoproteasome in a cell by contacting the cell with one or more of the crystalline salts or compositions thereof described herein. In some embodiments, the LMP7 immunoproteasome subunit is inhibited. The contacting step described herein occurs in vivo or in vitro.

There are numerous biological consequences of proteasome inhibition. Proteasome inhibition has been suggested as a preventive and/or treatment for a variety of diseases, including, but not limited to, neurotoxic/degenerative diseases, Alzheimer's disease, ischemic diseases, inflammation, autoimmune diseases, HIV, transplant organ rejection, septic shock, inhibition of antigen presentation, reduced viral gene expression, parasitic infections, diseases associated with acidosis, macular degeneration, pulmonary diseases, muscle atrophy, fibrotic diseases, and dysplasia of bones and hair. Therefore, the pharmaceutical compositions containing crystalline salts described herein allow the administration of salts to a patient for the treatment of these diseases.

Accordingly, the contacting step in the methods disclosed herein comprises administering one or more of the crystalline salts or compositions thereof described herein to a subject who suffers from a disorder associated with aberrant immunoproteasome activity. As described in more detail below, the disorder is an autoimmune disease or inflammation. In some embodiments of the invention, the disease is psoriasis, dermatitis, systemic scleroderma, sclerosis, Crohn's disease, ulcerative colitis; acute respiratory failure syndrome, meningitis; encephalitis; uveitis; colitis; glomerulonephritis; eczema, asthma, chronic inflammation; atherosclerosis; lack of adhesion of leukocytes; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes; multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; tuberculosis, sarcoidosis, polymyositis, granulomatosis, vasculitis; pernicious anemia (Addison's disease); disease, including diapedesis of leukocytes; inflammatory disease of the central nervous system (CNS); multiple organ failure syndrome; hemolytic anemia; myasthenia gravis; a disease mediated by an antigen-antibody complex; pulmonary-renal syndrome; antiphospholipid syndrome; allergic neuritis; Graves' disease; myasthenic Lambert-Eaton syndrome; bullous pymphegoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; muscle stiffness syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; polyneuropathy IgM; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia. In some cases, the disorder is lupus, lupus nephritis, rheumatoid arthritis, diabetes, scleroderma, ankylosing spondylitis, psoriasis, multiple sclerosis, Hashimoto's disease, meningitis, or inflammatory bowel disease.

The proteasome regulates NF-κB, which in turn regulates genes involved in the immune and inflammatory response. For example, NF-κB is required for the expression of the immunoglobulin light chain gene, the IL-2 receptor α-chain gene, the class I major histocompatibility complex gene, and a number of cytokine genes encoding, for example, IL-2, IL-6, granulocyte colony stimulating factor, and IFN-β (Palombella et al., Cell (1994) 78: 773-785). Therefore, provided herein are methods of influencing the expression level of IL-2, MHC-I, IL-6, TNFa, IFN-β, or any of the other previously mentioned proteins, each method comprising administering to a patient a therapeutically effective amount of a crystalline salt or composition, disclosed in this document.

Also provided herein is a method for treating an autoimmune disease in a patient, comprising administering a therapeutically effective amount of a crystalline salt as described herein. An autoimmune disease as used herein is a disease or disorder that occurs and is directed against a person's own tissues. Examples of autoimmune diseases include, but are not limited to, inflammatory responses such as inflammatory skin diseases including psoriasis and dermatitis (eg, atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel disease

- 16 040138 nickname (namely Crohn's disease and ulcerative colitis); acute lung failure syndrome (including adult acute lung failure syndrome (ARDS)); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions, namely eczema and asthma, and other conditions including T-cell infiltration and chronic inflammatory responses; atherosclerosis; lack of adhesion of leukocytes; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes mellitus (eg, type I diabetes mellitus or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes commonly found in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; pernicious anemia (Addison's disease); diseases associated with leukocyte diapedesis; inflammatory disease of the central nervous system (CNS); multiple organ failure syndrome; hemolytic anemia (including, but not limited to, cryoglobinemia or anemia with a positive Coombs test); myasthenia gravis; diseases mediated by antigen-antibody complex; pulmonary-renal syndrome; antiphospholipid syndrome; allergic neuritis; Graves' disease; myasthenic Lambert-Eaton syndrome; bullous pymphegoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; muscle stiffness syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; polyneuropathy IgM; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

The immune system looks for autologous cells that are infected with a virus, have undergone an oncogenic transformation, or have unfamiliar peptides on their surface. Intracellular proteolysis generates small peptides for presentation to T lymphocytes to induce MHC class I mediated immune responses. Therefore, this document provides a method of using a crystalline salt or a composition as provided herein as an immunomodulatory agent to inhibit or alter antigen presentation in cell, including exposing the cell to (or administering to the patient) a compound described herein. Specific embodiments of the invention include a method of treating rejection or transplant related diseases, namely graft versus host disease or host versus graft disease, comprising administering a therapeutically effective amount of a compound described herein. As used herein, the term graft refers to biological material obtained from a donor for transplantation into a recipient. Grafts include such diverse material as, for example, isolated cells, namely islet cells; tissues, namely the amniotic membrane of the newborn; Bone marrow; hematopoietic precursor cells; eye tissue, namely corneal tissue; and organs, namely the skin, heart, liver, spleen, pancreas, thyroid lobes, lungs, kidneys, and tubular organs (eg, intestines, blood vessels, or esophagus). Tubular organs are used to replace damaged parts of the esophagus, blood vessels, or bile ducts. Skin grafts are used not only for burns, but also as a bandage for a damaged intestine or to close certain defects, namely a diaphragmatic hernia. The transplant is obtained from any mammal, including humans, from cadavers or living donors. In some cases, the donor and recipient are the same patient. In some embodiments, the graft is a bone marrow or organ, namely a heart, and the graft donor and host match HLA class II antigens.

Proteasome inhibition has also been associated with inhibition of NF-κB activation and stabilization of p53 levels. Therefore, the crystalline salts and compositions thereof provided herein are also useful in inhibiting NF-κB activation and stabilizing p53 levels in cell culture. Because NF-κB is a key regulator of inflammation, it is an attractive target for anti-inflammatory therapeutic intervention.

Therefore, the crystalline salts and compositions thereof provided herein are useful in the treatment of diseases associated with inflammation, including, but not limited to, COPD, psoriasis, asthma, bronchitis, emphysema, and cystic fibrosis.

The disclosed crystalline salts and compositions thereof are used to treat diseases mediated directly by the proteolytic function of the proteasome, namely muscle atrophy, or mediated indirectly through proteins that are processed by the proteasome, namely NF-κB. The proteasome is involved in the rapid clearance and post-translational processing of proteins (eg, enzymes) involved in cellular regulation (eg, cell cycle, gene transcription, and metabolic pathway), cell-to-cell communication, and immune response (eg, antigen presentation). Specific examples discussed below include β-amyloid protein and regulatory proteins, namely cyclins and the transcription factor NF-kB.

In some embodiments, a crystalline salt or a composition thereof provided herein is useful in the treatment of neurodegenerative diseases and diseases, including, but not limited to, stroke, ischemic damage to the nervous system, neuronal

- 17 040138 trauma (for example, stroke brain injury, spinal cord injury and traumatic injury of the nervous system), multiple sclerosis and other immune-mediated neuropathies (for example, Guillain-Barré syndrome and its variants, acute motor axonal neuropathy, acute inflammatory demyelinating polyneuropathy and syndrome Fisher), HIV/AIDS-dementia complex, axonomia, diabetic neuropathy, Parkinson's disease, Huntington's disease, bacterial, parasitic, fungal and viral meningitis, encephalitis, vascular dementia, multi-infarct dementia, Lewy body dementia, dementia of the frontal lobe, namely Pick's disease , subcortical dementia, namely Huntington's disease or progressive supranuclear palsy), focal cortical atrophy syndromes (namely primary aphasia), metabolic-toxic dementias (namely chronic hypothyroidism or B12 deficiency), and dementias caused by infections (namely syphilis or chronic meningitis ).

Alzheimer's disease is characterized by extracellular deposits of β-amyloid protein (β-AR) in senile plaques and brain vessels. β-AP is a 39 to 42 amino acid peptide fragment derived from the parent amyloid protein (APP). At least three isoforms of APP are known (695, 751 and 770 amino acids). Alternative mRNA splicing produces isoforms; normal processing affects part of the β-AR sequence, thereby preventing the formation of βAR. Abnormal protein processing by the proteasome is believed to promote the spread of β-AR in the brain in Alzheimer's disease. The APP-processing enzyme in rats contains about ten different subunits (from 22 kDa to 32 kDa). The 25 kDa subunit has the N-terminal sequence XGln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which is identical to the β-subunit of the human multicatalytic protease (Kojima S. et al., Fed. Eur. Biochem. Soc. (1992) 304: 57-60). The APP-processing enzyme is cleaved at the Glnl5 - Lys16 bond; in the presence of a calcium ion, the enzyme is also cleaved at the Met-1 - Asp1 bond, and the Asp1 - Ala2 bonds release the extracellular domain of β-AR.

Therefore, provided herein is a method for treating Alzheimer's disease comprising administering to a patient a therapeutically effective amount of a crystalline salt or a composition thereof disclosed herein. Such treatment includes decreasing the rate of βAR processing, decreasing the rate of β-AR plaque formation, decreasing the rate of β-AR formation, and reducing the clinical signs of Alzheimer's disease.

This document also provides methods for treating cachexia and muscle atrophy. The proteasome cleaves many proteins in maturing reticulocytes and growing fibroblasts. In cells deprived of insulin or serum, the rate of proteolysis nearly doubles. Inhibition of the proteasome reduces proteolysis, thereby reducing both muscle protein loss and nitrogen load on the kidneys or liver. Peptide proteasome inhibitors (for example, a compound or composition provided herein) are useful in the treatment of diseases, namely chronic infectious disease, fever, muscle dysfunction (atrophy) and denervation, nerve damage, starvation, kidney failure associated with acidosis, kidney disease and liver failure. See, for example, Goldberg, US Pat. No. 5,340,736, which is incorporated herein by reference in its entirety. Treatments include reducing the rate of degradation of muscle protein in the cell, reducing the rate of degradation of intracellular protein, and reducing the rate of degradation of p53 protein in the cell. Each of these methods includes contacting a cell (in vivo or in vitro, e.g. a muscle in a patient) with an effective amount of a pharmaceutical composition disclosed herein to reduce the rate of degradation of muscle protein in the cell, reduce the rate of intracellular protein degradation in the cell, and/or decrease in the rate of degradation of the p53 protein in the cell. In some embodiments, the methods include administering to a patient a therapeutically effective amount of a crystalline salt, or a pharmaceutical composition thereof, as disclosed herein.

Fibrosis is the excessive and permanent formation of scar tissue as a result of hyperproliferative growth of fibroblasts and is associated with activation of the TGF-β signaling pathway. Fibrosis involves extensive deposition of extracellular matrix and can occur in virtually any tissue or in several different tissues. Normally, the level of intracellular signaling protein (Smad), which activates the transcription of target genes upon stimulation with TGF-β, is regulated by proteasome activity. However, under conditions of hyperproliferation, accelerated degradation of the signaling components of TGF-β is observed. Therefore, in some embodiments of the invention, a method is provided for the treatment of hyperproliferative diseases, namely diabetic retinopathy, macular degeneration, diabetic nephropathy, glomerulosclerosis, IgA nephropathy, cirrhosis, biliary atresia, congestive heart failure, scleroderma, radiation-induced fibrosis and pulmonary fibrosis ( idiopathic pulmonary fibrosis, connective tissue disease, sarcoidosis, interstitial lung disease, and external lung disease). Treatment of burn victims is often hampered by fibrosis, therefore, in some embodiments of the invention, the compound provided herein is administered by topical or systemic administration for the treatment of burns. Wound closure after surgery is often associated with disfiguring scarring, which can be prevented by inhibiting fibrosis. Therefore, in certain embodiments, this document provides a method for preventing or reducing scarring by administering a crystalline salt or composition thereof disclosed herein.

Another protein that is processed by the proteasome is NF-κB, a member of the Rel protein family. The Rel family of transcriptional activator proteins is divided into two groups. The first group requires proteolytic processing and includes p50 (NF-kB1, 105 kDa) and p52 (NF-k2, 100 kDa). The second group does not require proteolytic processing and includes p65 (RelA, Rel (c-Rel) and RelB). Both homo- and heterodimers can be formed by members of the Rel family; NFkB, for example, is a p50-p65 heterodimer. After phosphorylation and ubiquitination of IkB and p105, both proteins are degraded and processed, respectively, with the formation of active NF-kB, which moves from the cytoplasm to the nucleus. Ubiquitinated p105 is also processed by purified proteasomes (Palombella et al., Cell (1994) 78: 773-785). Active NF-kB forms a stereospecific enhancing complex with other transcription activators and, for example, HMG I (Y), inducing selective expression of a particular gene.

NF-kB regulates genes involved in the immune and inflammatory response and mitotic processes. For example, NF-kB is required for the expression of an immunoglobulin light chain gene, an IL-2 receptor α-chain gene, a major histocompatibility complex class I gene, and a number of cytokine genes encoding, for example, IL-2, IL-6, granulocyte colony stimulating factor, and IFN-β (Palombella et al., Cell (1994) 78: 773-785). Some embodiments of the invention include methods of influencing the expression level of IL-2, MHC-I, IL-6, TNFa, IFN-β, or any other previously mentioned proteins, each method comprising administering to the patient a therapeutically effective amount of a crystalline salt or a composition thereof, disclosed in this document. Complexes comprising p50 are rapid response neurotransmitters to acute inflammatory and immune responses (Thanos D. and Maniatis T., Cell (1995) 80: 529-532).

NF-kB is also involved in the expression of cell adhesion genes that encode for E-selectin, Pselectin, ICAM and VCAM-1 (Collins, T., Lab. Invest. (1993) 68:499-508). In some embodiments, a method for inhibiting cell adhesion (e.g., cell adhesion mediated by E-selectin, P-selectin, ICAM, or VCAM-1) is provided, comprising contacting a cell with an effective amount of a crystalline salt or a pharmaceutical composition thereof disclosed herein. . In some embodiments, a method for inhibiting cell adhesion (e.g., E-selectin, P-selectin, ICAM, or VCAM-1 mediated cell adhesion) is provided, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition disclosed herein.

Ischemia and reperfusion injury result in hypoxia, a condition in which there is a lack of oxygen reaching body tissues. This condition causes increased degradation of IK-Βα, leading to the activation of NF-kB. It has been shown that the severity of injury leading to hypoxia is reduced by the administration of a proteasome inhibitor. Therefore, provided herein is a method for treating ischemic disease or reperfusion injury, comprising administering to a patient in need of such treatment a therapeutically effective amount of a crystalline salt or composition thereof provided herein. Examples of such diseases or injuries include, but are not limited to, acute coronary syndrome (vulnerable plaques), arterial occlusion (occlusion of the heart, brain, peripheral arteries, and vessels), atherosclerosis (coronary sclerosis, coronary artery disease), heart attacks, heart failure, pancreatitis, myocardial hypertrophy, stenosis and restenosis.

NF-kB also binds specifically to the HIV enhancer/promoter. Compared to Nef mac239, the HIV regulatory protein Nef pbj14 differs by two amino acids in the region that controls protein kinase binding. The protein kinase is thought to signal phosphorylation of IkB, causing degradation of IkB via the ubiquitin-proteasome pathway. After degradation, NF-kB is released into the nucleus, thereby enhancing HIV transcription (Cohen, J., Science, (1995) 267:960). Provided herein is a method for inhibiting or attenuating HIV infection in a patient and a method for reducing viral gene expression, each method comprising administering to the patient a therapeutically effective amount of a crystalline salt or composition thereof disclosed herein.

Viral infections contribute to the pathology of many diseases. Heart disease, namely ongoing myocarditis and dilated cardiomyopathy, have been associated with the Coxsackie B3 virus. In a comparative genome-wide microarray analysis of infected mouse hearts, specific proteasome subunits were uniformly upregulated in the hearts of mice that developed chronic myocarditis (Szalay et al., Am J. Pathol 168:1542-52, 2006). Some viruses use the ubiquitin-proteasome system during the viral entry stage, where the virus is released from the endosome into the cytosol. Mouse hepatitis virus (MHV) belongs to the Coronaviridae family, which also includes the severe acute respiratory syndrome (SARS) corona virus. Yu and Lai (J Virol 79:644648, 2005) showed that treatment of MHV-infected cells with a proteasome inhibitor resulted in

- 19 040138 decrease in virus replication, which correlates with a reduced virus titer compared to that of untreated cells. The human hepatitis B virus (HBV), a member of the Hepadnaviridae family of viruses, also requires virus-encoded envelope proteins to reproduce. Inhibition of the proteasome biodegradation pathway causes a significant decrease in the amount of secreted envelope proteins (Simsek et al., J Virol 79:12914-12920, 2005). In addition to HBV, other hepatitis viruses (A, C, D, and E) can also use the ubiquitin-proteasome biodegradation pathway for secretion, morphogenesis, and pathogenesis. Accordingly, in certain embodiments, the invention provides a method of treating a viral infection, namely SARS or hepatitis A, B, C, D, and E, comprising contacting a cell with an effective amount of a crystalline salt or a composition thereof disclosed herein. In some embodiments, the invention provides a method of treating a viral infection, namely SARS or hepatitis A, B, C, D, and E, comprising administering to a patient a therapeutically effective amount of a crystalline salt or a composition thereof disclosed herein.

Increased production of lipopolysaccharide (LPS)-induced cytokines, namely TNFa, is considered central to the processes associated with septic shock. Additionally, it is generally accepted that the first step in LPS cell activation is LPS binding to specific membrane receptors, and the α- and β-subunits of the 20S proteasome complex have been identified as LPS-binding proteins, suggesting that LPS-induced signal transduction may be an important therapeutic target. in the treatment or prevention of sepsis (Qureshi, N. et al. J. Immun. (2003) 171: 1515-1525). Therefore, in certain embodiments, the crystalline salts and compositions thereof provided herein are used to inhibit TNFa to prevent and/or treat septic shock.

Intracellular proteolysis produces small peptides for presentation to T lymphocytes to elicit MHC class I mediated immune responses. The immune system checks for autologous cells that are infected with a virus or have undergone oncogenic transformation. One embodiment of the invention is a method for inhibiting antigen presentation in a cell, comprising exposing the cell to a composition described herein. In some embodiments, the cell is contacted with an effective amount of a compound or composition provided herein to inhibit antigen presentation in the cell. An additional embodiment of the invention is a method of suppressing a patient's immune system (eg, inhibiting transplant rejection, allergies, asthma) comprising administering to the patient a therapeutically effective amount of a composition described herein. The crystalline salts and compositions provided herein are also used to treat autoimmune diseases, namely lupus, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel diseases, namely ulcerative colitis and Crohn's disease.

Another embodiment of the invention is a method of modifying the spectrum of antigenic peptides produced by a proteasome or other Ntn with multicatalytic activity. For example, if the peptidylglutamyl peptide hydrolyzing activity of the 20S proteasome is selectively inhibited, the proteasome produces a different set of antigenic peptides that are found in MHC molecules on cell surfaces than it would have been formed and found either without any enzyme inhibition or with, for example, selective inhibition of chymotrypsin-like proteasome activity.

Certain proteasome inhibitors block both biodegradation and processing of ubiquitinated NF-κB in vitro and in vivo. Proteasome inhibitors also block IKb-a biodegradation and NF-kB activation (Palombella, et al. Cell (1994) 78:773-785; and Traenckner, et al., EMBO J. (1994) 13:54335441). In some embodiments of the invention, a method for inhibiting IκB-α biodegradation is provided, comprising contacting a cell with a crystalline salt or a composition thereof described herein. In some embodiments, the cell is contacted with an effective amount of a crystalline salt or composition thereof to inhibit the biodegradation of IκB-a. An additional embodiment of the invention is a method for reducing the cellular concentration of NF-κB in a cell, muscle, organ, or patient, comprising contacting the cell, muscle, organ, or patient with a crystalline salt or composition thereof described herein. In some embodiments of the invention, the cell is contacted with an effective amount of the composition to reduce the cellular concentration of NF-κB in the cell.

Other eukaryotic transcription factors that require proteolytic processing include the general transcription factor TFIIA, the herpes simplex virus accessory protein VP16 (host cell factor), the virus-inducible protein regulatory factor IFN 2, and protein 1 that binds sterol-regulating elements to the membrane.

Additionally, this document provides methods for influencing cyclin-dependent eukaryotic cell cycles, including exposing the cell (in vitro or in vivo) to the composition disclosed herein. Cyclins are proteins involved in cell cycle control. The proteasome is involved in the biodegradation of cyclins. Examples of cyclins include mitotic cyclins, cyclins G1, and cyclin B. Biodegradation of cyclins allows the cell to exit one cellular stage.

- 20 040138 exact cycle (eg mitosis) and move to another (eg fission). All cyclins are believed to be associated with p34cdc2 protein kinase or related kinases. The targeting signal for proteolysis is localized to amino acids 42-RAALGNISEN-50 (destruction box). There is evidence that cyclin is converted to a form unprotected by ubiquitin ligase, or that cyclin-specific ligase is activated during mitosis (Ciechanover A., Cell, (1994) 79:13-21). Inhibition of the proteasome inhibits the biodegradation of cyclin and therefore inhibits cell proliferation (Kumatori et al., Proc. Natl. Acad. Sci. USA (1990) 87:7071-7075). Provided herein is a method for treating a proliferative disease in a patient (eg, psoriasis or restenosis) comprising administering to the patient a therapeutically effective amount of a crystalline salt or composition thereof disclosed herein. Also provided herein is a method for treating cyclin-associated inflammation in a patient, comprising administering to the patient a therapeutically effective amount of a crystalline salt or composition thereof as described herein.

In another embodiment of the invention, the disclosed compositions are useful for the treatment of a parasitic infection, namely infections caused by protozoan parasites. It is believed that the proteasome of these parasites is mainly involved in the operations of cell differentiation and replication (Paugam et al., Trends Parasitol. 2003, 19 (2): 55-59). Additionally, entameba individuals have been shown to lose their ability to encapsulate when exposed to proteasome inhibitors (Gonzales et al., Arch. Med. Res. 1997, 28, Spec no: 139-140). In certain such embodiments, the disclosed crystalline salts and compositions thereof are useful in the treatment of parasitic infections in humans caused by a protozoan parasite selected from the Plasmodium species (including P. falciparum, P. vivax, P. malariae, and P. ovale, which cause malaria). ), Trypanosoma spp. (including T. cruzi, which causes Chagas disease, and T. brucei, which causes African sleeping sickness), Leishmania spp. (including L. amazonesis, L. donovani, L. infantum, L. mexicana, and others), Pneumocystis carinii (a protozoan known to cause pneumonia in AIDS patients and other immunosuppressed patients), Toxoplasma gondii, Entamoeba histolytica, Entamoeba invadens, and Giardia lamblia. In certain embodiments, the disclosed crystalline salts and compositions thereof are useful in the treatment of parasitic infections in animals and livestock caused by a protozoan parasite selected from Plasmodium hermani, Cryptosporidium sp., Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona, and Neurospora crassa. Other compounds useful as proteasome inhibitors in the treatment of parasitic diseases are described in WO 98/10779, which is incorporated herein in its entirety.

In certain embodiments of the invention, the disclosed crystalline salts and compositions thereof irreversibly inhibit proteasome activity in a parasite. This irreversible inhibition has been shown to cause erythrocyte and leukocyte enzymatic activity to cease without recovery. In certain such embodiments of the invention, the long half-life of the cellular elements of the blood provides long-term protection against therapy against repeated exposure to parasites. In certain embodiments of the invention, the long half-life of blood cells provides long-term protection against chemoprophylaxis against future infection.

Prokaryotes have what is equivalent to the eukaryotic 20S proteasome particle. Although the composition of the prokaryotic 20S particle subunit is simpler than that of eukaryotes, it has the ability to hydrolyze peptide bonds in a similar manner. For example, a nucleophilic attack on a peptide bond occurs via a threonine residue at the N-terminus of the β-subunits. In some embodiments of the invention, a method for treating prokaryotic infections is provided, comprising administering to a patient a therapeutically effective amount of a crystalline salt or a composition thereof provided herein. Prokaryotic infections include diseases caused by mycobacteria (namely tuberculosis, leprosy or Buruli ulcer) or archaebacteria.

Inhibitors that bind to the 20S proteasome have also been shown to stimulate bone formation in bone organ cultures. Additionally, when such inhibitors were administered systemically to mice, certain proteasome inhibitors increased bone volume and bone formation rate by more than 70% (Garrett, I. R. et al., J. Clin. Invest. (2003) 111: 1771-1782), hence that the ubiquitin-proteasome mechanism regulates osteoblast differentiation and bone formation. Therefore, the disclosed crystalline salts and compositions thereof are useful in the treatment and/or prevention of diseases associated with bone loss, namely osteoporosis.

Provided herein is a method of treating a disease or disease selected from an autoimmune disease, a transplant or transplant related disease, a neurodegenerative disease, a fibrosis related disease, an ischemia related disease, an infection (viral, parasitic or prokaryotic), and a disease related to with loss of bone tissue, including the introduction of a crystalline salt or a composition from it, proposed in this document.

Bone tissue is an excellent source of factors that can stimulate bone cells. Therefore, bovine bone extracts contain not only structural proteins that are responsible for maintaining the structural integrity of the bone, but also biologically active factors.

- 21 040138 bone growth, which can stimulate the proliferation of bone cells. Among these latter factors is a recently described family of proteins called bone morphogenetic proteins (BMPs). All of these growth factors affect other cell types as well as bone cells, including what Hardy, M. H., et al., Trans Genet (1992) 8:55-61 describes evidence that bone morphogenetic proteins (BMPs) differentially expressed in hair follicles during development. Harris, S. E., et al., J. Bone Miner Res (1994) 9:855-863 describes the effect of TGF-β on the expression of BMP-2 and other substances in bone cells. Expression of BMP-2 in mature follicles also occurs during maturation and after a period of cell proliferation (Hardy, et al. (1992, supra). Therefore, the crystalline salts and formulations thereof provided herein are also useful for promoting hair growth. follicle.

This document also provides a method for treating lysosomal storage disease by administering a compound disclosed herein. Lysosomal storage diseases are a group of diseases resulting from abnormal metabolism of various substrates, including glycosphingolipids, glycogen, mucopolysaccharides, and glycoproteins. Metabolism of exogenous and endogenous macromolecular compounds usually occurs in lysosomes, and this process is usually regulated stepwise by degradation enzymes. Therefore, insufficient activity of one enzyme can disrupt the process, leading to the accumulation of certain substrates. Proteasome inhibition has been shown to improve the function of certain substrates in patients suffering from lysosomal storage diseases (Y. Shimada et al. Biochem. Biophys. Res. Commun. (2011) 415(2):274-8). Most of these diseases are clinically classified into subtypes: 1) infantile form; 2) youthful form or 3) age form. The infantile forms are often the most severe, usually without residual enzyme activity. Age-related forms are often less severe with low but often detectable residual enzymatic activity. The severity of youthful forms is between infantile forms and age-related forms. Non-limiting examples of such disorders include Pompe disease, Gaucher disease, Fabry disease, GM1 gangliosidosis, Tay-Sachs disease, Sandhoff disease, Niemann-Pick disease, Krabbe disease, Farber disease, metachromatic leukodystrophy, Hurler-Scheie disease, Hunter disease, Sanfilipo A disease, Sanfilippo B disease, Sanfilippo C disease, Sanfilippo D disease, Morquio A disease, Morquio B disease, Marothmo-Lami disease, Sly disease, α-mannosidosis, β-mannosidosis, fucosidosis, sialidosis, and Schindler-Kanzaki disease. Therefore, one embodiment of the invention is a method of treating Pompe's disease, comprising administering to a patient a therapeutically effective amount of a crystalline salt or a composition thereof provided herein.

The disclosed crystalline salts and compositions thereof are also useful as diagnostics (eg, in diagnostic kits or for use in clinical laboratories) for screening proteins (eg, enzymes, transcription factors) treated with Ntn hydrolases, including the proteasome. The disclosed crystalline salts and compositions thereof are also useful as research reagents for specific binding of the X/MB1 subunit or α chain and inhibition of associated proteolytic activities. For example, the activity (and specific inhibitors) of other proteasome subunits can be determined.

Most cellular proteins undergo proteolytic processing during maturation or activation. The enzymatic inhibitors disclosed herein are used to determine whether a cellular, physiological, or developmental process or outcome is regulated by the proteolytic activity of a particular Ntn hydrolase. One such method includes obtaining an organism, an intact cell preparation, or a cell extract; exposure to an organism, cell preparation, or cell extract of the composition disclosed herein; the effect of the signal on the organism, cell preparation or cell extract exposed to the compound; and monitoring the process or result. The high selectivity of the compounds disclosed herein allows for the rapid and precise exclusion or recruitment of Ntn (eg, the 20S proteasome) in a given cellular, developmental, or physiological process.

Examples

The following examples are provided for illustration and are not intended to limit the scope of the invention.

Example 1. Methods of characterization.

Powder X-ray diffractometry (XRPD) data were obtained on a PANalytical X'Pert3 X-ray (Figs. 1, 21 and 22-27), Shimadzu XRD-7000 (Fig. 5) or on a Bruker D8Advance X-ray powder diffractometer (Figs. 1, 9, 11 , 13-15 and 19). The samples were scanned continuously from 4 to 40° (2θ) with a step size of 0.02° at 40 kV and 40 mA with CuKa radiation (1.54 A) (FIG. 1). The samples were scanned continuously from 3 to 40° (2θ) with a step size of 0.013° at 45 kV and 40 mA with CuKa radiation (1.54 A) (FIGS. 21 and 23-27). The samples were scanned continuously from 5 to 70° (2θ) with a step size of 0.02° at 40 kV and 35 mA with CuKa radiation (1.54 A) (FIG. 5). The samples were scanned continuously from 3 to 40° (2θ) with a step size of 0.02° at 40 kV and 40 mA with CuKa radiation (1.54

- 22 040138

A) (Fig. 1, 9, 11, 13-15 and 19).

Differential scanning calorimetry (DSC) was performed on a TA Instruments Q2000 calorimeter in an aluminum crucible (Fig. 22), TA Q20 DSC in a small-sized Tzero crucible (Fig. 2), or TA Instruments Q20 in a Tzero aluminum crucible (Fig. 6) or in the system dynamic vapor sorption using a rolled aluminum crucible (Fig. 8, 10, 12, 16, 17 and 20) in an atmosphere of dry nitrogen.

Thermogravimetric analysis (TGA) was performed on a TA Instruments Q500 (FIG. 22) or NETZSCH TG209 F1 analyzer (FIG. 3) in a platinum crucible (FIG. 22) or a Tzero aluminum crucible (FIG. 3) under dry nitrogen.

Moisture sorption data were collected using SMS (surface roughness measurement systems) DVSIntrinsic (FIG. 4) or dynamic vapor sorption systems (FIG. 18). The equilibrium criteria were ±0.002% weight change (FIG. 4) per 10 minutes with a maximum equilibration time of 180 minutes.

¹ H-NMR was performed on a Varian 400 MHz instrument. Solid samples were dissolved in DMSO-d ₆ and transferred to NMR tubes for analysis.

Example 2 Screening out the salt of compound G.

Compound G is reacted with six different acids, each in six different solvent systems (total of 36 screening experiments), to determine if a crystalline salt of Compound G can be formed.

More specifically, about 15 mg of Compound G and an equivalent molar amount of acid are mixed in a 2.0 ml glass tube. About 1.0 ml of the appropriate solvent system is added to the tube. The resulting suspensions are stirred at about 600 rpm at room temperature for about two days. The suspensions are then centrifuged to isolate solids for XRPD analysis. The results of screening experiments are presented in table. 3. Of the six acids tested, two lead to the formation of a crystalline salt of Compound G: maleic acid and fumaric acid.

Table 3. Screening results of Compound G salt

Solvent acids IPA EtOAc THF MTVE Acetone/n-heptane EtOH/ _H2O 1 HC1 transparent transparent transparent us transparent transparent transparent 2 H ₃ RO ₄ transparent transparent transparent transparent transparent transparent 3 maleic acid crystal ly maleate crystal ly maleate crystal ly maleate crystal ly maleate crystal s maleate transparent 4 fumaric acid transparent fumarate crystals Form G transparent crystals fumarate a Form G crystal s Fumarate Form G transparent 5 L-tartaric acid transparent transparent transparent transparent transparent transparent 6 lemon acid transparent transparent transparent transparent transparent transparent

Transparent: no or limited solids

Example 3 Additional Screening of Compound G Salt

To Compound G (20 mg, pre-dissolved in 140 µl solvent) was added 1 equivalent of acid (pre-dissolved in 40 to 240 µl solvent) and the mixtures were allowed to stand for 96 hours in a sealed tube. The following solvents are used: toluene, ethanol, methanol, isopropanol, hexane/ethyl acetate (1:1), 1,4-dioxane, acetonitrile, 1-butanol, ethyl acetate, acetone, MTBE/ethyl acetate (1:1) and diethyl ether/ethyl acetate (1:1). The following acids are used: sulfuric, methanesulfonic, tosyl (monohydrate), 2-naphthalenesulfonic, L-malic, propionic, benzoic, oxalic and phosphoric. A solid precipitate is observed with the combinations presented in table. 4.

- 23 040138

Table 4. Salt Screening Variables

Acid Solvent system Sulfuric acid _Et2O /EtOAc 1-naphthalenesulfonic acid Hexane/EtOAc MTBE/EtOAc Oxalic acid Toluene Hexane/EtOAc MTBE/EtOAc Et ₂ O/EtOAc Phosphoric acid Hexane/EtOAc MTBE/EtOAc Et ₂ O/EtOAc L-malic acid Hexane/EtOAc

Example 4 Linear increase of compound G maleate.

The production of the monomaleate form of compound G is linearly increased as follows. Compound G (about 200 mg) is reacted with maleic acid at a molar ratio of 1:1 or 1:2 by weighing both starting materials into a glass tube. A volume of MTBE or acetone is added to each glass tube and the resulting suspension is stirred on a magnetic plate. The suspension is then dried under vacuum at room temperature to give Form B.

Compound G (about 200 mg) was also reacted with maleic acid (about 20 mg; 1:0.5 molar ratio) using EtOAc as solvent. The resulting suspension is stirred on a magnetic plate at a speed of about 600 rpm at room temperature. If a white solid separates after stirring, about 9.0 ml of EtOAc is added to the suspension. The suspension is stirred for two days and then isolated by centrifugation. The isolated solids are dried in air or at 50° C. under vacuum overnight to give form A.

A brief description of the maleates of compound G, obtained in linearly increased experiments, is presented in table. 5.

Table 5. Experimental data on linear increase for compound G maleate

Acid consumption (compound G: acid) MTVE Acetone 1 Maleic acid (1:1) Form A maleate mixed with amorphous Maleate Form B 2 Maleic acid (1:2) Form A maleate mixed with amorphous Maleate Form B Acid consumption (compound G:acid) EtOAc 3 Maleic acid (1:0.5) Maleate Form A 4 Maleic acid (1:0.5) Maleate Form A 5 Maleic acid (1:0.5) Maleate Form A

The XRPD results from the maleate ramp experiments are shown in FIG. 23. Consistent diffractograms are observed for maleate from the same solvent. The maleate from MTBE (Form A) shows little crystallinity, and the maleate from acetone (Form B) shows a slightly different XRPD from the XRPD for crystallized in EtOAc (Form A). However, after heat treatment to 100° C. in TGA, as shown in FIG. 24 and 25, the XRPD of compound G monomaleate crystallized from MTBE (form A) and acetone (form B) is in good agreement with the XRPD of monomaleate crystallized from EtOAc (form A).

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Example 5 Further Processing and Characterization of Form A

Various drying conditions (air drying, vacuum drying, moisture cycling) were used to process Form A (crystallized from EtOAc). The resulting crystalline salts characterize XRPD (FIGS. 25 and 26), DSC and TGA. Vacuum dried maleates are also characterized by 1H-NMR to determine free base/maleic acid stoichiometry. The XRPD results in FIG. 26 show that all samples under different drying conditions have the same diffraction pattern. The Dynamic Vapor Sorption (VVA) test is also used to characterize Form A from EtOAc, as depicted in FIG. 4. Characteristics for these experiments are given in table. 6.

Table 6. Characteristics of form A (from EtOAc) at a ratio of 1:0.5

Drying conditions Mass loss at TGA (% wt.) Endotherm on DSC (°C) NMR stoichiometry (free form/acid) humidity cycle * 18 138.8 / CT vacuum 2.5 92.1, 140.7 1.0:1.0 air 2.5 145.1 / 50°C vacuum 0.4 147.6 1.0:1.0 air 2.3 144.5 / 50°C vacuum 2.1 145.5 1.0:1.1

*Humidity cycle (HMC) is 40% RH-95% RH-0% RH-95% in air

Example 6 Synthetic Methods

Method 1 Preparation of compound G monomaleate with isopropyl acetate/ethanol.

EtOH (11.5 kg) is added to compound G (3.6 kg in 37.88 kg IPAc). The resulting solution is heated to 50° C. and after 15 min maleic acid (1.62 kg of a 12.4 wt% solution in EtOH) is added, followed by a seed (18.0 g) of the title compound. The suspension is stirred for 0.5 h at 50° C. and maleic acid (4.90 kg of a 13.4 wt% solution in EtOH) is added over 3 hours. The mixture is stirred at 50°C for 4 hours, cooled to -3°C for 9.5 hours, kept at -2-3°C for 2 hours, filtered and washed with IPAc/EtOH (2:1, 12, 0 kg) at -5-5°C. The filter cake was dried in vacuo at 4045° C. for 17 hours to give Compound G monomaleate (3.86 kg, 99.0% pure).

Method 2: Decomposition of the salt to form Form A with ethyl acetate.

IPAc (37.8 kg) was added to Form A (3.56 kg) at 15-25°C followed by 3.5% NaHCO ₃ (37.8 kg) and the resulting suspension was stirred for 1 hour to obtain a solution . The aqueous layer is removed and the organic layer is washed with 5% Na ₂ SO ₄ (aqueous, 36.9 kg) at 15-25°C. The aqueous layer is removed and the organic layer is concentrated to 4-7 L at below 45°C. The organic layer is extracted three times with ethyl acetate (32.0 kg) at 15-25°C and the solution is concentrated to about 7-11 L at below 45°C. Then add ethyl acetate (28.8 kg) and the solution is heated to 45-55°C. Maleic acid (720 g) is dissolved in 19.4 kg of ethyl acetate and 1/10 of this solution is added over 30 minutes at 45-55°C. A seed (9.09 g) was added at 45-55° C. and the mixture was stirred for 30 minutes. The rest of the maleic acid solution is added at 45-55°C for 1 hour. The mixture is stirred for an additional 2 hours at 45-55°C, then cooled to 1°C for 8 hours. The mixture is stirred for 1 hour at 5 -5°C, then filtered, washed with ethyl acetate (13.0 kg) and dried at 40-50°C in vacuo for 2628 h to obtain 3.42 kg of maleate (99.1% pure) as a colorless solid. The XRPD pattern is shown in Fig. 1, the DSC characteristics are depicted in FIG. 2, the TGA data is depicted in FIG. 3.

Method 3 Preparation of compound G monomaleate using 0.5 eq. maleic acid (form A).

Maleic acid (0.085 mmol, 9.9 mg in THF (0.5 ml)) was added to compound G (100 mg, 0.170 mmol) in THF (0.5 ml). The mixture was allowed to stand overnight and filtered to give compound G monomaleate (50.5 mg) as a colorless solid.

Method 4: Recrystallization of compound G monomaleate.

Ethanol (0.5 ml) was added to form A (0.05 g, 0.0852 mmol), the solution was refluxed for 5 minutes and allowed to cool to 20° C. overnight. The purified compound is isolated as a colorless solid (42 mg).

A similar recrystallization method was carried out using the following solvents to prepare compound G monomaleate: THF, iPrOH-EtOAc (1:1), iPrOH, iPrOH-toluene (1:1), dioxane and acetonitrile.

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Example 7 PK Study Using Form A

Form A is for subcutaneous administration at a concentration of 45 mg/ml, as described in table. 7.

The bioavailability of form A, expressed as a percentage (% F), when each formulation is administered as a single subcutaneous dose of about 3 mg/kg to cynomolgus monkeys (3 males/dose) is also shown in Table 1. 7.

Table 7. Compositions used for FA studies in macaques

Compound pH %F (macaque) 10% polysorbate 80 (water.) 4.5 59.0±10.1 10% KOLLIPHORE EL (water.) 4.5 62.3 ± 17.8 10% polysorbate 80/10% K-methyl-2pyrrolidone (water.) 5 76.2 ± 7.8 10% KOLLIPHORE EL/10% N-methyl-2-pyrrolidone (water.) 5 68.3 ± 8.1 water 3, 6 70.4±15.1 1:1 (v/v) LABRASOL/propylene glycol no response 45.2 + 3.3

Example 8 Polymorphism Control.

Method A. About 30 mg of compound G is added to the solvent listed in table. 8, then shaken at 50° C. at 700 rpm. Residual compounds separated by centrifugation (5 min at 9000 rpm) and examined using XRPD, DSC and TGA after 7 days, as shown in table. 8 and in FIG. 7-13.

Method B To 50 mg of compound G was added methanol (1.0 ml) followed by MTBE (0.5 ml). After keeping the mixture overnight, the precipitate is collected and examined using XRPD, DSC and TGA, which is shown in table. 8 and in FIG. 19 and 20.

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Table 8. Polymorphism control

Form Way Crystallization / solvent sludge XRPD (20 ± 0.2°) TGA DSC IN A 3% H ₂ 0 / acetone 6.8, 7.2, 18.4, 6.6, 13.6, 22.0, 17.4, 14.5, 18.0 and 5.0 (Fig. 13) WITH A Acetone 6.6, 13.2, 7.4, 20.1, 13.6, 6.9, 16.9, 3.7, 17.9 and 19.9 (Fig. 7) A loss of 6.0% of the mass is observed from 29.2 to 130.0°C (Fig. 8) Standard : 67.2 J/g Start: 141.9°C Peak: 148.63°C (Fig. 8) D A Acetonitrile 6.8, 4.9, 17.4, 15.3, 7.7, 3.4, 17.7, 13.6, 12.4 and 10.9 (Fig. 9) A loss of 0.3% of the mass is observed from 26.8 to 130.0°C (Fig. 10) Standard : 86.20 j/g Start: 14 8.53°C Peak: 151.75°С (Fig. 10) E A isopropyl alcohol 6.5, 3.3, 7.3, 19.8, 6.8, 16.5, 12.1, 21.5, 4.0 and 13.0 (Fig. 11) A loss of 0.9% of the mass is observed from 32.5 to 99.3°C (Fig. 12) Standard : 57.8 J/g Start: 138.2°C Peak: 147.61°С (Fig. 12) F IN Meon/MTBE 6.3, 7.1, 19.0, 17.5, 19.6, 17.9, 22.0, 13.5, 18.2 and 15.5 (Fig. 19) A loss of 1.4% of the mass is observed from 31.9 to 99.4°C (Fig. 20) Standard : 71.2 J/g Start: 128.05°С Peak: 135.46°С (Fig. 20)

Example 9 Further Processing and Characterization of Form B

Method: 2 g of compound G is added to 3% water in acetone (20 ml), then shaken at 50° C. at 700 rpm overnight. The remainder is examined using XRPD, DSC and TGA.

The residue is dried under vacuum at room temperature or 30° C. for 1, 4 or 24 hours. Results

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XRPD in Fig. 14-15 show that all samples under different drying conditions have the same diffraction pattern. The DSC/TGA results for a sample dried at room temperature overnight are shown in FIG. 16 and dried overnight at 30°C are shown in FIG. 17. The Dynamic Vapor Sorption (VVA) test is also used to characterize Form F from 3% H ₂ O/acetone as depicted in FIG. 18. After drying overnight at room temperature, the Karl Fischer test shows that the water content of Form F is 2.51%.

Example 10 Characteristic Peaks for Forms A and C-G.

In table. 9 below shows the XRPD peaks that are unique to each polymorph.

Table 9. Polymorphic screening

Form Unique XRPD peaks (20 ± 0.2°) Height A 6, 9 6261 17.3 1237 17, 8 1030 IN 7.2 7829 18.4 4173 22.0 1251 WITH 7.4 2147 13.2 2206 20, 1 1386 D 4.9 2818 7.7 1637 10, 9 1033 12.4 1259 13, 6 1445 15, 3 1975 E 6, 4 17796 7.3 1906 19, 8 1831 F 6, 3 6553 19.0 1690 19.6 1155

Example 11 Form A Stability

The stability of form A and its free base is tested at ambient conditions (25°C and 40% relative humidity, RH) and at elevated temperature and humidity (40°C and 75% RH) for more than one month. The free base form decomposes rapidly at elevated temperature and humidity. However, under the same conditions and over the same period of time, no changes are observed in the form A. See table. 10 below. Hence Form A exhibits greater stability than its free base.

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Table 10 Stability Comparison of Form A and Its Free Base

Form Conditions Lot No. % Purity at t=0 % Purity For 1 month % expansions free base 25°C/40% RH 1 95.1 93.2 1.9 25°C/40% RH 2 85.6 84.0 16 25°C/40% RH 3 93.7 92.3 1.4 40°C/75% RH 1 95.1 92.3 67.5 40°C/75% RH 2 85.6 92.3 28.8 40°C/75% RH 3 93.7 72.5 21.1 Maleate 25°C/40% RH 4 99.1 99.1 0, 0 25°C/40% RH 5 99.4 99.3 0, 1 40°C/75% RH 4 99.1 98.9 0.2 40°C/75% RH 5 99.4 99.3 0, 1

The above description is provided for clarity of understanding only, and is not intended to be undue restrictive, as those skilled in the art will recognize various modifications within the scope of this invention.

In the document of this specification and the following claims, unless the context otherwise requires, the term contain and its variants, such as contains and containing, should be understood as including the specified element or step, or group of elements or steps, but not as excluding any other an element or step, or a group of elements or steps.

In the specification document, in which the compositions are described as containing components or materials, it is provided that the compositions also consist essentially of or consist of any combination of the listed components or materials, unless otherwise indicated. Similarly, if the methods are described as including certain steps, it is contemplated that the methods also consist essentially of or consist of any combination of the steps listed, unless otherwise noted. The invention illustratively described herein can be properly practiced in the absence of any element or step that is not explicitly described in this document.

The practical implementation of the method disclosed in this document, and its individual steps can be implemented manually and/or using automatic devices provided by electronic equipment. While the methods are described with reference to specific implementations, those skilled in the art will recognize that other methods of performing the actions associated with said methods may be used. For example, the order of the various steps can be changed without departing from the scope or spirit of the method, unless otherwise described. Additionally, some of the individual steps may be combined, omitted, or further subdivided into additional steps.

All patents, publications, and references cited in this document are incorporated herein by reference in their entirety. In the event of any conflict between this disclosure and the incorporated patents, publications, and references, this disclosure shall govern.

Claims (34)

CLAIM 1. Crystalline salt having a structure where X ^- is a maleate and the crystalline salt is a monomaleate salt having a powder X-ray diffraction pattern (XRPD) including peaks at 6.9, 17.3 and 17.8±0.2° 2θ using Cu radiation Ka (form A). (2) mixing the reduced compound G, maleic acid, and a solvent to form a crystalline salt. 2. Crystalline salt according to claim 1, further including peaks at 4.9, 6.8 and 7.7±0.2° 2θ using Cu Ka radiation 3. The method of obtaining a crystalline salt according to claim 1, including mixing: (a) connections G (b) maleic acid; and (c) a solvent for making a suspension. 4. The method according to claim 3, characterized in that the molar ratio of compound G to maleic acid is in the range from about 1:0.5 to 1:2. 5. Method according to claim 4, characterized in that the molar ratio is 1:1. 6. Process according to any one of claims 3 to 5, characterized in that the solvent is selected from the group consisting of methanol (MeOH), ethanol (EtOH), isopropanol (IPA), ethyl acetate (EtOAc), isopropyl acetate (IPAc), tetrahydrofuran ( THF), methyl tert-butyl ether (MTBE), acetone/n-heptane, acetone, diethyl ether (Et ₂ O)/EtOAc, hexane/EtOAc, MTBE/EtOAc, toluene, 1,4-dioxane, acetonitrile (ACN ), 1-butanol, aqueous mixtures of the above, and combinations thereof. 7. Method according to any one of claims 3 to 6, characterized in that the mixing takes place at a temperature in the range from 0 to 80°C. 8. A method according to any one of claims 3 to 7, characterized in that the mixing takes place for up to about 6 hours. 9. The method according to any one of claims 3 to 8, further comprising cooling the suspension to 0°C. 10. The method of claim 9 further comprising filtering the slurry to form a precipitate. 11. The method of claim 10, further comprising washing, drying, or both washing and drying the precipitate. 12. The method of claim 11 further comprising recrystallizing the precipitate. 13. The method of claim 11, further comprising (1) recovery of compound G from the precipitate; 14. A pharmaceutical composition containing a crystalline salt according to any one of claims 1, 2 and one or more excipients. 15. Pharmaceutical composition according to claim 14 in the form of a liquid dosage form. 16. The pharmaceutical composition according to claim 14 in the form of a lyophilized dosage form, characterized in that the lyophilized dosage form is restored to a liquid dosage form. 17. A pharmaceutical composition according to claim 15 or 16, wherein the crystalline salt has a concentration in the range of about 1 to about 150 mg/ml in liquid dosage form or reconstituted lyophilized dosage form, based on the weight of the free base of the crystalline salt. 18. Pharmaceutical composition according to claim 17, characterized in that the concentration is in the range from about 10 to about 70 mg/ml. 19. Pharmaceutical composition according to any one of claims 14-18, characterized in that one or more excipients are selected from the group consisting of a surfactant, a tonicity regulator, a buffer, and combinations thereof. 20. The pharmaceutical composition of claim 19, wherein the lyophilized dosage form further comprises a cryoprotectant, a bulking agent, or both. 21. The pharmaceutical composition of claim 19, wherein the surfactant is polysorbate, castor oil polyoxyl, poly(alkylene) glycol, caprylocaproyl polyoxylglyceride, polyoxyalkylene block copolymers, and combinations thereof. 22. A pharmaceutical composition according to any one of claims 14 to 21, characterized in that the liquid or reconstituted lyophilized dosage form is isotonic. 23. Pharmaceutical composition according to claim 19, characterized in that the buffer is selected from the group consisting of citrate, phosphate, histidine, succinate, acetate, maleate, gluconate, and combinations thereof. 24. A pharmaceutical composition according to any one of claims 14-21, characterized in that the liquid dosage form or reconstituted lyophilized dosage form has a pH in the range from about 3.0 to about 8.0. 25. Pharmaceutical composition according to any one of claims 15-24, characterized in that the liquid medicinal 26. Pharmaceutical composition according to claim 25, characterized in that the parenteral administration is intravenous, intramuscular, intraperitoneal or subcutaneous. 27. Pharmaceutical composition according to any one of claims 14 to 26, characterized in that the composition exhibits a bioavailability of at least 55%. 28. A method for inhibiting the immunoproteasome of a cell, comprising contacting the cell with a crystalline salt according to any one of claims 1, 2 or a composition according to any one of claims 14-27. 29. The method of claim 28, wherein the LMP7 immunoproteasome is inhibited. - 29 040138 30. The method of claim 28 wherein the contacting comprises administering to a subject suffering from a disorder associated with aberrant immunoproteasome activity. - 30 040138 form or reconstituted lyophilized dosage form suitable for parenteral administration to a subject. 31. The method of claim 30 wherein the disorder is an autoimmune disease or inflammation. 32. The method of claim 31, wherein the disorder is psoriasis, dermatitis, systemic scleroderma, sclerosis, Crohn's disease, inflammatory bowel disease, ulcerative colitis; respiratory failure syndrome, meningitis; encephalitis; uveitis; colitis; glomerulonephritis; eczema, asthma, chronic inflammation; atherosclerosis; lack of adhesion of leukocytes; rheumatoid arthritis; ankylosing spondylitis; systemic lupus erythematosus (SLE); diabetes; multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's disease; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; tuberculosis, sarcoidosis, polymyositis, granulomatosis, vasculitis; pernicious anemia (Addison's disease); disease, including diapedesis of leukocytes; inflammatory disease of the central nervous system (CNS); multiple organ failure syndrome; hemolytic anemia; myasthenia gravis; a disease mediated by an antigen-antibody complex; pulmonary-renal syndrome; antiphospholipid syndrome; allergic neuritis; Graves' disease; myasthenic Lambert-Eaton syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; muscle stiffness syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; polyneuropathy IgM; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia. 33. The use of a crystalline salt according to any one of claims 1, 2 or a composition according to any one of claims 14 to 27 for the manufacture of a medicament for the treatment of an autoimmune disease or inflammation. 34. The use of a crystalline salt according to any one of claims 1, 2 or a composition according to any one of claims 14 to 27 for the treatment of an autoimmune disease or inflammation.