Stable cyclodextrin-free carfilzomib formulation

Abstract

The present disclosure provides stable cyclodextrin-free carfilzomib formulations in aqueous solutions suitable for injection, kits containing the cyclodextrin-free carfilzomib formulations, and methods for preparing the cyclodextrin-free carfilzomib. Such formulations, kits, and methods substantially increase the solubility and stability of carfilzomib in aqueous solutions, facilitating both its manufacture and administration.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 959,833, filed January 10, 2020, which is incorporated in its entirety by reference.

[0002] This disclosure provides a stable cyclodextrin-free carfilzomib formulation in an aqueous solution suitable for injection, a kit containing the cyclodextrin-free carfilzomib formulation, and a method for preparing the cyclodextrin-free carfilzomib. Such formulations, kits, and methods substantially increase the solubility and stability of carfilzomib in aqueous solutions and facilitate both its manufacture and administration. [Background technology]

[0003] Carfilzomib is a selective proteasome inhibitor approved for the treatment of multiple myeloma. Carfilzomib is a tetrapeptide epoxyketone proteasome inhibitor with the following chemical structure: [ka] This irreversibly binds to the N-terminal threonine-containing active site of the 20S proteasome, which is the proteolytic core particle within the 26S proteasome. Carfilzomib has in vitro antiproliferative and pro-apoptotic activity in solid and hematopoietic tumor cells. In animals, carfilzomib inhibited proteasome activity in blood and tissues and delayed tumor growth in models of multiple myeloma, hematopoietic tumors, and solid tumors.

[0004] Carfilzomib is marketed under the name Kyprolis® in single-dose vials containing 10 mg, 30 mg, or 60 mg of the active ingredient. Each vial contains lyophilized carfilzomib, as well as sulfobutyl ether-β-cyclodextrin, citrate, and sodium hydroxide (target pH 3.5) for pH adjustment.

[0005] Efforts have been made to obtain improved carfilzomib compositions. For example, substituted cyclodextrin additives have been studied to enhance the solubility of the active ingredient. However, because substituted cyclodextrins are expensive and have limited availability, their use in pharmaceutical compositions is limited. [Overview of the Initiative] [Means for solving the problem]

[0006] Carfilzomib has extremely low water solubility, is sensitive to pH and concentration, and possesses an epoxide ring with poor resistance to nucleophilic attack. All of these factors present numerous challenges in preparing a stable carfilzomib formulation without cyclodextrin. There remains a need for an improved carfilzomib formulation with improved ease of manufacture, means of administration, and stability over time. There remains a need for a formulation that is easy for healthcare providers to prepare and administer. In particular, there remains a need for a cyclodextrin-free carfilzomib formulation with improved stability over time, especially when stored under ambient conditions.

[0007] The object of the present invention is to provide a stable, readily usable, or readily dilutable cyclodextrin-free carfilzomib formulation.

[0008] Another object of the present invention is to provide a kit containing a stable, ready-to-use or readily dilutable cyclodextrin-free carfilzomib formulation, such as a freeze-dried powder or cake.

[0009] Another object of the present invention is to provide a process for preparing stable, readily usable, or readily dilutable cyclodextrin-free carfilzomib formulations.

[0010] Another object of the present invention is to provide a stable, ready-to-use or ready-to-dilute cyclodextrin-free carfilzomib formulation suitable for intravenous or subcutaneous administration by injection.

[0011] Still another object of the present invention is to provide a method for treating a patient suffering from multiple myeloma by administering a stable, ready-to-use or ready-to-dilute cyclodextrin-free carfilzomib formulation.

[0012] In one embodiment, the present invention provides: (i)

Chemical Formula

[0013] In embodiment 2, the present invention provides the cyclodextrin-free pharmaceutical composition according to embodiment 1, wherein the solvent system is dimethyl sulfoxide, N-methyl-2-pyrrolidone, or dimethylacetamide.

[0014] In Embodiment 3, the present invention provides a cyclodextrin-free pharmaceutical composition according to either Embodiment 1 or 2, wherein the cosolvent system is optionally a mixture of ethanol and polyethylene glycol, or a mixture of tert-butyl alcohol and polyethylene glycol, in the presence of a first cosolubilizer.

[0015] In Embodiment 4, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the cosolvent system is 75% to 92% PEG400:ethanol (1:1, w / w) and the first cosolubilizer is absent.

[0016] In Embodiment 5, the present invention provides a cosolvent system that is an acid, ester, organic salt, organic base, or C 1~4 The present invention provides a cyclodextrin-free pharmaceutical composition, one of the above embodiments, which is a mixture of ethanol and PEG400 in the presence of a first co-solubilizing agent selected from alkyl alcohols.

[0017] In Embodiment 6, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the first co-solubilizing agent is an acid or ester selected from lactic acid, maleic acid, citric acid, benzoic acid, benzenesulfonic acid, acetic acid, or coconut fatty acid sucrose.

[0018] Embodiment 7 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the cosolvent system is an organic salt selected from benzalkonium chloride or protamine sulfate.

[0019] Embodiment 8 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the first co-solubilizing agent is ethanolamine or isopropyl alcohol.

[0020] In Embodiment 9, the present invention provides a cosolvent system comprising: 75%-92% PEG400:ethanol (1:1, w / w); 1.2%-5% lactic acid in PEG400:ethanol; 1.2%-5% maleic acid in PEG400:ethanol; 4.6% benzalkonium chloride in PEG400:ethanol; 1%-3.3% protamine sulfate in PEG400:ethanol; and 28-30% HS Solutol in PEG400:ethanol. The present invention provides a cyclodextrin-free pharmaceutical composition selected from the group consisting of 15%; 32% coconut fatty acid sucrose, PEG400:ethanol, 5% benzoic acid, PEG400:ethanol, 5% benzenesulfonic acid, PEG400:ethanol, 10% isopropyl alcohol, PEG400:ethanol, 1% to 5% citric acid in PEG400:ethanol, 1.2% to 5% acetic acid in PEG400:ethanol, or 5% ethanolamine in PEG400:ethanol, one of the above embodiments.

[0021] Embodiment 10 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the cosolvent system is PEG400:1.2% to 5% lactic acid in ethanol.

[0022] Embodiment 11 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the ratio of lactic acid to carfilzomib is 1.5:2 by weight.

[0023] Embodiment 12 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the ratio of lactic acid to carfilzomib is 0.4:2 by weight.

[0024] Embodiment 13 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the final maximum lactic acid concentration is 0.15%.

[0025] Embodiment 14 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the aqueous solution has a pH of 3.0 to 3.5 in the absence of a second co-solubilizing agent.

[0026] Embodiment 15 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the aqueous solution has a pH of 3.0 to 3.5.

[0027] Embodiment 16 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the aqueous solution has a pH of 3.0 to 3.5, and the second co-solubilizing agent is selected from organic sugars, water-soluble polymers, acids, amino acids, or any combination thereof.

[0028] Embodiment 17 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the second co-solubilizing agent is dextrose, mannitol, glycine, N-vinylpyrrolidone polymer, butyric acid, adipic acid, phenylalanine, arginine HCl, tryptophan, or N-acetyltryptophan, or any combination thereof.

[0029] Embodiment 18 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the second co-solubilizing agent is N-vinylpyrrolidone polymer, mannitol, or glycine, or any combination thereof.

[0030] Embodiment 19 provides a cyclodextrin-free pharmaceutical composition of any one of the above embodiments, wherein the second co-solubilizing agent is 1-ethenylpyrrolidine-2-one (PVP, also known as polyvinylpyrrolidone), mannitol, or glycine, or any combination thereof. Various PVPs are known to those skilled in the art; see, for example, https: / / www.brenntag.com / media / documents / bsi / product_data_sheets / material_science / ashland_polymers / pvp_polymers_brochure.pdf. PVPs are available in several grades of molecular weight and K value (viscosity of 1% solution), such as PVP K-12, K-15, K-17, K-30, K-60, K-90, or K-120.

[0031] Embodiment 20 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the PVP has a molecular weight range of 3,000 MW to 40,000 MW.

[0032] Embodiment 21 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the PVP has a molecular weight range of 10,000 MW to 17,000 MW.

[0033] Embodiment 22 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the PVP has a molecular weight range of 10,000 MW.

[0034] Embodiment 23 provides a cyclodextrin-free pharmaceutical composition from any one of the above embodiments, wherein the PVP is selected from the group consisting of 24% PVP 10,000 MW, 29% PVP 10,000 MW, 10% PVP 12,000 MW, 20% PVP 12,000 MW, 24% PVP 12,000 MW, or 24% PVP 17,000 MW.

[0035] Embodiment 24 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the composition comprises 0.75% to 1% dimethyl sulfoxide, 1.0% to 1.8% PEG400, 1.0% to 1.8% ethanol, 0.10% to 0.25% lactic acid, and 20% to 30% PVP 10,000 MW, and the concentration of carfilzomib is 2 mg / ml.

[0036] Embodiment 25 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the composition comprises 0.2% to 2% dimethyl sulfoxide, 0.5% to 2.5% PEG400, 0.5% to 2.5% ethanol, 0.05% to 0.5% lactic acid, and 10% to 40% PVP 10,000 MW, and the concentration of carfilzomib is 2 mg / ml.

[0037] In Embodiment 26, the present invention provides a composition comprising: 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, or 1.2% dimethyl sulfoxide; 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6% PEG400; 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6% ethanol; 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, and 0.35% lactic acid; and approximately 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, and 33% PVP. The present invention provides a cyclodextrin-free pharmaceutical composition according to any one of the aforementioned embodiments, containing 10,000 MW and having a carfilzomib concentration of 2 mg / ml.

[0038] Embodiment 27 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the composition comprises about 0.85% dimethyl sulfoxide, about 1.4% PEG400, about 1.4% ethanol, about 0.15% lactic acid, about 28.8% PVP 10,000 MW, and the concentration of carfilzomib is 2 mg / ml.

[0039] In Embodiment 28, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the composition has a solution osmolality of 200 mOsmo to 600 mOsmo.

[0040] In Embodiment 29, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the composition has a solution osmolality of 250 mOsmo to 400 mOsmo.

[0041] In Embodiment 30, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the composition has a solution osmolality of 280 mOsmo to 320 mOsmo.

[0042] In Embodiment 31, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the composition has a solution osmolality of 280, 290, 300, 310, or 320 mOsmo.

[0043] Embodiment 32 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the composition is a ready-to-use injectable preparation.

[0044] Embodiment 33 provides a cyclodextrin-free pharmaceutical composition according to any one of the above embodiments, wherein the composition is obtained as a freeze-dried powder or cake.

[0045] In Embodiment 34, the present invention provides the cyclodextrin-free pharmaceutical composition of Embodiment 33, wherein the freeze-dried powder or cake can be reconstituted in less than 5 minutes.

[0046] In Embodiment 35, the present invention is (i) (a) The above carfilzomib or a pharmaceutically acceptable salt thereof, C 1~4A mixture of alkyl alcohol and polyethylene glycol is dissolved in lactic acid to form a solution, wherein the concentration of carfilzomib or its salt is in the range of 20 mg / ml to 50 mg / ml. (b) A step of diluting the above carfilzomib solution with an acidic aqueous solution of a water-soluble polymer and sugar mixture having a pH of 2.5 to 4.5 to form a solution, wherein the concentration of carfilzomib or its salt is in the range of 1 mg / ml to 3 mg / ml. (c) A step of freeze-drying the solution obtained in step (b), A product vial pharmaceutical composition comprising a stable lyophilized powder or cake prepared by a process including, and (ii) A carfilzomib injection kit comprising a reconstituted vial composition containing sterile water, The present invention provides a kit in which the above-mentioned pharmaceutical composition does not contain cyclodextrin, and the injectable preparation is administered intravenously or subcutaneously.

[0047] Embodiment 36 provides the carfilzomib injection kit of Embodiment 35, wherein the water-soluble polymer is 1-ethenylpyrrolidine-2-one (PVP) and the sugar is mannitol or glycine or a combination thereof.

[0048] Embodiment 37 provides the carfilzomib injection kit of Embodiment 36, wherein the water-soluble polymer is a PVP having a molecular weight in the range of 3,000 MW to 40,000 MW, 10,000 MW to 17,000 MW, or 10,000 MW, and the sugar is mannitol or glycine.

[0049] Embodiment 38 provides the carfilzomib injection kit of Embodiment 36, wherein the water-soluble polymer is 24% PVP 10,000 MW, 29% PVP 10,000 MW, 10% PVP 12,000 MW, 20% PVP 12,000 MW, 24% PVP 12,000 MW, or 24% PVP 17,000 MW, and the sugar is mannitol.

[0050] Embodiment 39 provides the carfilzomib injection kit of Embodiment 36, wherein the water-soluble polymer is 20% PVP 12,000 MW or 24% PVP 12,000 MW, and the sugar is mannitol.

[0051] Embodiment 40 provides the carfilzomib injection kit of Embodiment 36, wherein the water-soluble polymer is 20% PVP 12,000 MW and the sugar is mannitol.

[0052] In Embodiment 41, the present invention provides the carfilzomib injection kit of Embodiment 36, wherein the pH of the acidic aqueous solution in step (b) is in the range of 3.0 to 3.5.

[0053] Embodiment 42 provides a carfilzomib injection kit of Embodiment 36, wherein the solution formed in step (b) contains 0.75% to 1% dimethyl sulfoxide, 1.0% to 1.8% PEG400, 1.0% to 1.8% ethanol, 0.10% to 0.25% lactic acid, and 20% to 30% PVP 10,000 MW, and the concentration of carfilzomib is 2 mg / ml.

[0054] Embodiment 43 provides a carfilzomib injection kit of Embodiment 36, wherein the solution formed in step (b) contains 0.2% to 2% dimethyl sulfoxide, 0.5% to 2.5% PEG400, 0.5% to 2.5% ethanol, 0.05% to 0.5% lactic acid, and 10% to 40% PVP 10,000 MW, and the concentration of carfilzomib is 2 mg / ml.

[0055] In Embodiment 44, the present invention provides a solution formed in step (b) containing 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, or 1.2% dimethyl sulfoxide; 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6% PEG400; 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6% ethanol; 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, and 0.35% lactic acid; and approximately 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, and 33% PVP. The present invention provides a carfilzomib injection kit according to Embodiment 36, which contains 10,000 MW and has a carfilzomib concentration of 2 mg / ml.

[0056] Embodiment 45 provides the carfilzomib injection kit of Embodiment 36, wherein the solution formed in step (b) contains about 0.85% dimethyl sulfoxide, about 1.4% PEG400, about 1.4% ethanol, about 0.15% lactic acid, and about 28.8% PVP 10,000 MW, and the concentration of carfilzomib is 2 mg / ml.

[0057] In Embodiment 46, the present invention provides the carfilzomib injection kit of Embodiment 36, wherein the solution formed in step (b) has a solution osmolality of 200 mOsmo to 600 mOsmo.

[0058] In Embodiment 47, the present invention provides the carfilzomib injection kit of Embodiment 36, wherein the solution formed in step (b) has a solution osmolality of 250 mOsmo to 400 mOsmo.

[0059] In Embodiment 48, the present invention provides the carfilzomib injection kit of Embodiment 36, wherein the solution formed in step (b) has a solution osmolality of 280 mOsmo to 320 mOsmo.

[0060] In Embodiment 49, the present invention provides a carfilzomib injection kit of Embodiment 36, wherein the solution formed in step (b) has a solution osmolality of 280, 290, 300, 310, or 320 mOsmo.

[0061] In Embodiment 50, the present invention provides the carfilzomib injection kit of Embodiment 36, wherein in step (b), the concentration of carfilzomib or the salt thereof is 2 mg / ml.

[0062] In Embodiment 51, the present invention provides the carfilzomib injection kit of Embodiment 36, wherein the ratio of lactate to carfilzomib is 1.5:2 by weight.

[0063] In Embodiment 52, the present invention provides the carfilzomib injection kit of Embodiment 36, wherein the ratio of lactate to carfilzomib is 0.4:2 by weight.

[0064] In Embodiment 53, the present invention provides the carfilzomib injection kit of Embodiment 36, in which the injection agent is administered intravenously.

[0065] In Embodiment 54, the present invention provides the carfilzomib injection kit of Embodiment 36, in which the injection agent is administered subcutaneously.

[0066] Embodiment 55 provides a process for preparing a cyclodextrin-free carfilzomib lyophilized powder or cake that is suitable for injection when reconstituted, (a) Carfilzomib or a pharmaceutically acceptable salt thereof, dimethyl sulfoxide, C 1~4 A mixture of alkyl alcohol and polyethylene glycol is dissolved in lactic acid to form a solution, wherein the concentration of carfilzomib or its salt is in the range of 20 mg / ml to 50 mg / ml. (b) A step of diluting a carfilzomib solution with an acidic aqueous solution of a water-soluble polymer and sugar mixture having a pH of 2.5 to 4.5 to form a solution, wherein the concentration of carfilzomib or its salt is in the range of 1 mg / ml to 3 mg / ml. The present invention provides a process comprising (c) a step of freeze-drying the solution obtained in step (b).

[0067] Embodiment 56 provides the process of Embodiment 55, wherein the water-soluble polymer is 1-ethenylpyrrolidine-2-one (PVP) and the sugar is mannitol or glycine or a combination thereof.

[0068] Embodiment 57 provides the process of Embodiment 55, wherein the water-soluble polymer is a PVP having a molecular weight in the range of 3,000 MW to 40,000 MW, 10,000 MW to 17,000 MW, or 10,000 MW, and the sugar is mannitol or glycine.

[0069] Embodiment 58 provides the process of Embodiment 55, wherein the water-soluble polymer is 24% PVP 10,000 MW, 29% PVP 10,000 MW, 10% PVP 12,000 MW, 20% PVP 12,000 MW, 24% PVP 12,000 MW, or 24% PVP 17,000 MW, and the sugar is mannitol.

[0070] Embodiment 59 provides the process of Embodiment 55, wherein the water-soluble polymer is 20% PVP 12,000 MW or 24% PVP 12,000 MW, and the sugar is mannitol.

[0071] Embodiment 60 provides the process of Embodiment 55, wherein the water-soluble polymer is 20% PVP 12,000 MW and the sugar is mannitol.

[0072] In Embodiment 61, the present invention provides the process of Embodiment 55, wherein the pH of the acidic aqueous solution in step (b) is in the range of 3.0 to 3.5.

[0073] Embodiment 62 provides the process of Embodiment 55, wherein the solution formed in step (b) contains 0.75% to 1% dimethyl sulfoxide, 1.0% to 1.8% PEG400, 1.0% to 1.8% ethanol, 0.10% to 0.25% lactic acid, and 20% to 30% PVP 10,000 MW, and the concentration of carfilzomib is 2 mg / ml.

[0074] Embodiment 63 provides the process of Embodiment 55, wherein the solution formed in step (b) contains 0.2% to 2% dimethyl sulfoxide, 0.5% to 2.5% PEG400, 0.5% to 2.5% ethanol, 0.05% to 0.5% lactic acid, and 10% to 40% PVP 10,000 MW, with a carfilzomib concentration of 2 mg / ml.

[0075] In Embodiment 64, the present invention provides a solution formed in step (b) containing 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, or 1.2% dimethyl sulfoxide; 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6% PEG400; 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6% ethanol; 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, and 0.35% lactic acid; and approximately 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, and 33% PVP. The present invention provides a process according to Embodiment 55, which contains 10,000 MW and has a carfilzomib concentration of 2 mg / ml.

[0076] In Embodiment 65, the present invention provides the process of Embodiment 55, wherein the solution formed in step (b) contains about 0.85% dimethyl sulfoxide, about 1.4% PEG400, about 1.4% ethanol, about 0.15% lactic acid, about 28.8% PVP 10,000 MW, and the concentration of carfilzomib is 2 mg / ml.

[0077] In Embodiment 66, the present invention provides the process of Embodiment 55, wherein the solution formed in step (b) has a solution osmolality of 200 mOsmo to 600 mOsmo.

[0078] In Embodiment 67, the present invention provides the process of Embodiment 55, wherein the solution formed in step (b) has a solution osmolality of 250 mOsmo to 400 mOsmo.

[0079] In Embodiment 68, the present invention provides the process of Embodiment 55, wherein the solution formed in step (b) has a solution osmolality of 280 mOsmo to 320 mOsmo.

[0080] In Embodiment 69, the present invention provides the process of Embodiment 55, wherein the solution formed in step (b) has a solution osmolality of 280, 290, 300, 310, or 320 mOsmo.

[0081] In Embodiment 70, the present invention provides the process of Embodiment 55, wherein in step (b), the concentration of carfilzomib or the salt thereof is 2 mg / ml.

[0082] In Embodiment 71, the present invention provides the process of Embodiment 55, wherein in step (b), the ratio of lactic acid to carfilzomib is 1.5:2 by weight.

[0083] In Embodiment 72, the present invention provides the process of Embodiment 55, wherein in step (b), the ratio of lactic acid to carfilzomib is 0.4:2 by weight.

[0084] In Embodiment 73, the present invention provides the process of Embodiment 55, wherein the injectable agent is administered intravenously.

[0085] In Embodiment 74, the present invention provides the process of Embodiment 55, wherein the injectable agent is administered subcutaneously.

[0086] Embodiment 75 provides a method for treating multiple myeloma in a subject requiring treatment, comprising administering a therapeutically effective amount of any one cyclodextrin-free pharmaceutical composition from Embodiments 1 to 34, or any one kit from Embodiments 35 to 54.

[0087] Embodiment 76 provides the method of Embodiment 75, further comprising simultaneous, sequential, or individual administration of therapeutically effective amounts of chemotherapeutic agents.

[0088] Embodiment 77 provides a method for treating a solid tumor in a subject requiring treatment, comprising administering a therapeutically effective amount of any one cyclodextrin-free pharmaceutical composition from Embodiments 1 to 34, or any one kit from Embodiments 35 to 54.

[0089] Embodiment 78 provides the method of Embodiment 77, further comprising simultaneous, sequential, or individual administration of therapeutically effective amounts of chemotherapeutic agents.

[0090] In Embodiment 79, the present invention provides a carfilzomib injection kit comprising (a) a stable frozen carfilzomib or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, and (b) a soluble pharmaceutical composition, wherein the kit is (i) A step of dissolving the above-mentioned carfilzomib or a pharmaceutically acceptable salt thereof in dimethyl sulfoxide (DMSO) to form a DMSO solution, wherein the concentration of the carfilzomib or the above-mentioned salt thereof is in the range of 200 mg / ml to 250 mg / ml; (ii) A step of freezing the above DMSO solution at 2°C to 8°C to form the above frozen carfilzomib pharmaceutical composition, and optionally storing the frozen composition at 2°C to 8°C, (iii) The frozen carfilzomib pharmaceutical composition is thawed at the DMSO melting point, preferably at least 18°C, to form a thawed carfilzomib composition; the liquid carfilzomib composition is mixed with the dissolved pharmaceutical composition to form a solution, wherein the carfilzomib concentration is in the range of 1 mg / ml to 3 mg / ml; and the preparation is carried out by a process comprising these steps. The present invention provides a kit in which the above-mentioned pharmaceutical composition does not contain cyclodextrin, and the injectable preparation is administered intravenously or subcutaneously.

[0091] Embodiment 80 provides a kit of Embodiment 79, comprising: a co-solvent vial capable of dissolving the thawed carfilzomib pharmaceutical composition to form a solution, wherein the carfilzomib concentration is in the range of 20 mg / ml to 50 mg / ml; and an additional excipient vial capable of dissolving the thawed carfilzomib pharmaceutical composition to form a solution, wherein the carfilzomib concentration is in the range of 1 mg / ml to 3 mg / ml.

[0092] In Embodiment 81, the present invention provides a kit of Embodiment 80 in which the frozen carfilzomib composition is stored at 2°C to 8°C and the dilution step (iii) is performed in a clinic facility.

[0093] In Embodiment 82, the present invention provides the kit of Embodiment 81, wherein the frozen carfilzomib composition is stored in a moisture-free storage container or apparatus.

[0094] Embodiment 83 provides the kit of Embodiment 82, wherein the water-free container is a 0.5 mL microcentrifuge Eppendorf tube and a 3 cc glass Schott 1A vial equipped with a freeze-drying stopper and a crimp seal.

[0095] In Embodiment 84, the present invention relates to the cosolvent vial being optionally configured in the presence of a first cosolubilizer, C 1~4 The kit of Embodiment 80 is provided, comprising a cosolvent system selected from alkyl alcohols, polyethylene glycol, or combinations thereof.

[0096] Embodiment 85 provides the kit of Embodiment 80, wherein the cosolvent vial contains a cosolvent system selected from a combination of ethanol and PEG in the presence of a first cosolubilizing agent, which is lactic acid.

[0097] In Embodiment 86, the present invention provides a kit of Embodiment 80, wherein the additional excipient vial optionally contains an aqueous solution having a pH of 2.5 to 4.5 in the presence of a second co-solubilizer, which is a water-soluble polymer.

[0098] Embodiment 87 provides the kit of Embodiment 86, wherein the water-soluble polymer is 1-ethenylpyrrolidine-2-one (PVP).

[0099] Embodiment 88 provides the kit of Embodiment 87, wherein the water-soluble polymer is a PVP having a molecular weight in the range of 3,000 MW to 40,000 MW, 10,000 MW to 17,000 MW, or 10,000 MW.

[0100] Embodiment 89 provides the kit of Embodiment 87, wherein the water-soluble polymer is 24% PVP 10,000 MW, 29% PVP 10,000 MW, 10% PVP 12,000 MW, 20% PVP 12,000 MW, 24% PVP 12,000 MW, or 24% PVP 17,000 MW.

[0101] In Embodiment 90, the present invention provides the kit of Embodiment 87, wherein the water-soluble polymer is 20% PVP 12,000 MW or 24% PVP 12,000 MW.

[0102] Embodiment 91 provides the kit of Embodiment 87, wherein the water-soluble polymer is 20% PVP 12,000 MW.

[0103] In Embodiment 92, the present invention provides the kit of Embodiment 86, wherein the pH of the aqueous solution is in the range of 3.0 to 3.5.

[0104] Embodiment 93 provides the kit of Embodiment 87, wherein the solution formed in step (iii) contains 0.75% to 1% dimethyl sulfoxide, 1.0% to 1.8% PEG400, 1.0% to 1.8% ethanol, 0.10% to 0.25% lactic acid, and 20% to 30% PVP 10,000 MW, with a carfilzomib concentration of 2 mg / ml.

[0105] Embodiment 94 provides the kit of Embodiment 87, wherein the solution formed in step (iii) contains 0.2% to 2% dimethyl sulfoxide, 0.5% to 2.5% PEG400, 0.5% to 2.5% ethanol, 0.05% to 0.5% lactic acid, and 10% to 40% PVP 10,000 MW, with a carfilzomib concentration of 2 mg / ml.

[0106] In Embodiment 95, the present invention provides a solution formed in step (iii) containing 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, or 1.2% dimethyl sulfoxide; 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6% PEG400; 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6% ethanol; 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, and 0.35% lactic acid; and approximately 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, and 33% PVP. The kit of Embodiment 87 is provided, containing 10,000 MW and having a carfilzomib concentration of 2 mg / ml.

[0107] In Embodiment 96, the present invention provides the kit of Embodiment 87, wherein the solution formed in step (iii) contains about 0.85% dimethyl sulfoxide, about 1.4% PEG400, about 1.4% ethanol, about 0.15% lactic acid, about 28.8% PVP 10,000 MW, and the concentration of carfilzomib is 2 mg / ml.

[0108] In Embodiment 97, the present invention provides the kit of Embodiment 87, wherein the solution formed in step (iii) has a solution osmolality of 200 mOsmo to 600 mOsmo.

[0109] Embodiment 98 provides the kit of Embodiment 87, wherein the solution formed in step (iii) has a solution osmolality of 250 mOsmo to 400 mOsmo.

[0110] In Embodiment 99, the present invention provides the kit of Embodiment 87, wherein the solution formed in step (iii) has a solution osmolality of 280 mOsmo to 320 mOsmo.

[0111] In Embodiment 100, the present invention provides a kit of Embodiment 87, wherein the solution formed in step (iii) has a solution osmolality of 280, 290, 300, 310, or 320 mOsmo.

[0112] Embodiment 101 provides the kit of Embodiment 87, wherein the solution formed in step (iii) has a concentration of 2 mg / ml of carfilzomib or its salt.

[0113] Embodiment 102 provides the kit of Embodiment 87, wherein the ratio of lactate to carfilzomib is 1.5:2 by weight.

[0114] Embodiment 103 provides the kit of Embodiment 87, wherein the ratio of lactate to carfilzomib is 0.4:2 by weight.

[0115] Embodiment 104 provides the kit of Embodiment 87, in which the injectable agent is administered intravenously.

[0116] Embodiment 105 provides the kit of Embodiment 87, in which the injectable agent is administered subcutaneously.

[0117] Embodiment 106 is a process for preparing a cyclodextrin-free frozen carfilzomib composition, (i) A step of dissolving the above-mentioned carfilzomib or a pharmaceutically acceptable salt thereof in dimethyl sulfoxide (DMSO) to form a DMSO solution, wherein the concentration of the carfilzomib or the above-mentioned salt thereof is in the range of 200 mg / ml to 250 mg / ml; (ii) A process is provided which includes the step of freezing the above DMSO solution at 2°C to 8°C to form the above frozen carfilzomib pharmaceutical composition, and optionally storing the frozen composition at 2°C to 8°C.

[0118] Embodiment 107 provides the process of Embodiment 106, further comprising the steps of: thawing the frozen carfilzomib pharmaceutical composition at the DMSO melting point, preferably at least 18°C, to form a thawed carfilzomib composition; mixing the liquid carfilzomib composition with a dissolved pharmaceutical composition to form a solution, wherein the carfilzomib concentration is in the range of 1 mg / ml to 3 mg / ml, and the pharmaceutical composition is cyclodextrin-free and suitable for injection.

[0119] In Embodiment 108, the present invention relates to the above-mentioned soluble pharmaceutical composition in the presence of lactic acid. 1~4 The present invention provides a process of Embodiment 107, in which a solution is formed comprising a mixture of an alkyl alcohol and polyethylene glycol, wherein the concentration of carfilzomib or its salt is in the range of 20 mg / ml to 50 mg / ml.

[0120] Embodiment 109 provides the process of Embodiment 107, further comprising a second vial containing an acidic aqueous solution of a water-soluble polymer having a pH of 2.5 to 4.5, which can be further diluted to form a more diluted solution, wherein the concentration of carfilzomib or its salt is in the range of 1 mg / ml to 3 mg / ml.

[0121] In Embodiment 110, the present invention provides the process of Embodiment 107, wherein the water-soluble polymer is 1-ethenylpyrrolidine-2-one (PVP).

[0122] In Embodiment 111, the present invention provides the process of Embodiment 107, wherein the water-soluble polymer is a PVP having a molecular weight in the range of 3,000 MW to 40,000 MW, 10,000 MW to 17,000 MW, or 10,000 MW.

[0123] In Embodiment 112, the present invention provides the process of Embodiment 107, wherein the water-soluble polymer is 24% PVP 10,000 MW, 29% PVP 10,000 MW, 10% PVP 12,000 MW, 20% PVP 12,000 MW, 24% PVP 12,000 MW, or 24% PVP 17,000 MW.

[0124] In Embodiment 113, the present invention provides the process of Embodiment 107, wherein the water-soluble polymer is 20% PVP 12,000 MW or 24% PVP 12,000 MW.

[0125] In Embodiment 114, the present invention provides the process of Embodiment 109, wherein the water-soluble polymer is 20% PVP 12,000 MW.

[0126] Embodiment 115 provides the process of Embodiment 109, wherein the pH of the acidic aqueous solution is in the range of 3.0 to 3.5.

[0127] Embodiment 116 provides the process of Embodiment 109, wherein the more diluted solution contains 0.75% to 1% dimethyl sulfoxide, 1.0% to 1.8% PEG400, 1.0% to 1.8% ethanol, 0.10% to 0.25% lactic acid, and 20% to 30% PVP 10,000 MW, with a carfilzomib concentration of 2 mg / ml.

[0128] Embodiment 117 provides the process of Embodiment 109, wherein the more diluted solution contains 0.2% to 2% dimethyl sulfoxide, 0.5% to 2.5% PEG400, 0.5% to 2.5% ethanol, 0.05% to 0.5% lactic acid, and 10% to 40% PVP 10,000 MW, with a carfilzomib concentration of 2 mg / ml.

[0129] In Embodiment 118, the present invention provides a more diluted solution containing 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, or 1.2% dimethyl sulfoxide; 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6% PEG400; 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6% ethanol; 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, and 0.35% lactic acid; and approximately 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, and 33% PVP. The present invention provides a process according to Embodiment 109, which contains 10,000 MW and has a carfilzomib concentration of 2 mg / ml.

[0130] In Embodiment 119, the present invention provides the process of Embodiment 109, wherein the more diluted solution comprises about 0.85% dimethyl sulfoxide, about 1.4% PEG400, about 1.4% ethanol, about 0.15% lactic acid, and about 28.8% PVP 10,000 MW, with a carfilzomib concentration of 2 mg / ml.

[0131] In Embodiment 120, the present invention provides the process of Embodiment 109, wherein the more diluted solution has a solution osmolality of 200 mOsmo to 600 mOsmo.

[0132] In Embodiment 121, the present invention provides the process of Embodiment 109, wherein the more diluted solution has a solution osmolality of 250 mOsmo to 400 mOsmo.

[0133] In Embodiment 122, the present invention provides the process of Embodiment 109, wherein the more diluted solution has a solution osmolality of 280 mOsmo to 320 mOsmo.

[0134] In Embodiment 123, the present invention provides the process of Embodiment 109, wherein the more diluted solution has a solution osmolality of 280, 290, 300, 310, or 320 mOsmo.

[0135] In Embodiment 124, the present invention provides the process of Embodiment 109, wherein the concentration of carfilzomib or its salt in the diluted solution is 2 mg / ml.

[0136] Embodiment 125 provides the process of Embodiment 109, wherein the ratio of lactic acid to carfilzomib in the diluted solution is 1.5:2 by weight.

[0137] In Embodiment 126, the present invention provides the process of Embodiment 109, wherein the ratio of lactic acid to carfilzomib in the diluted solution is 0.4:2 by weight.

[0138] In Embodiment 127, the present invention provides the process of Embodiment 109 in which an injectable agent is administered intravenously.

[0139] In Embodiment 128, the present invention provides the process of Embodiment 109 in which an injectable agent is administered subcutaneously.

[0140] Embodiment 129 provides a method for treating multiple myeloma in a patient requiring treatment, comprising administering a therapeutically effective amount of carfilzomib solution obtained from any one of the injection kits of Embodiments 79 to 105.

[0141] Embodiment 130 provides the method of Embodiment 129, further comprising simultaneous, sequential, or individual administration of therapeutically effective amounts of chemotherapeutic agents.

[0142] Embodiment 131 provides a method for treating a solid tumor in a subject requiring treatment, comprising administering a therapeutically effective amount of carfilzomib solution obtained from any one of the injection kits of Embodiments 79 to 105.

[0143] Embodiment 132 provides the method of Embodiment 131, further comprising simultaneous, sequential, or individual administration of therapeutically effective amounts of chemotherapeutic agents.

[0144] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs. Methods and materials are described herein for use in this disclosure; other preferred methods and materials known in the art may also be used. Materials, methods and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification, including definitions, shall prevail.

[0145] Other features and advantages of this disclosure will become apparent from the detailed description and drawings below, as well as from the claims. [Brief explanation of the drawing]

[0146] [Figure 1] (a) Water, (b) Cyclodextrin (CAPTISOL®), and the cyclodextrin-free formulation of the present invention are shown in a visual comparison of the carfilzomib active ingredient. [Figure 2] This shows a 3D solubility plot of solvent spheres used to solubilize CFZ-API. [Figure 3] A 2D solubility plot comparing various solubility parameters for solubilizing CFZ-API is shown. [Figure 4-1]This is a list of solvents generated by the Hansen Solubility Parameter software, with δD=16-19.5; δP=5-18; and δH=7-19.6. [Figure 4-2] This is a list of solvents generated by the Hansen Solubility Parameter software, with δD=16-19.5; δP=5-18; and δH=7-19.6. [Figure 4-3] This is a list of solvents generated by the Hansen Solubility Parameter software, with δD=16-19.5; δP=5-18; and δH=7-19.6. [Figure 4-4] This is a list of solvents generated by the Hansen Solubility Parameter software, with δD=16-19.5; δP=5-18; and δH=7-19.6. [Figure 4-5] This is a list of solvents generated by the Hansen Solubility Parameter software, with δD=16-19.5; δP=5-18; and δH=7-19.6. [Figure 5] This lists solvents generated by Hansen Solubility Parameter software with δD=16~24; δP=8~14; and δH=17~24. [Figure 6A] The results show no significant percentage loss of the main peak when measured by RP-HPLC of the liquid CAPTISOL®-free formulation at temperatures of 2-8°C (A) and 25°C (B). [Figure 6B] The results show no significant percentage loss of the main peak when measured by RP-HPLC of the liquid CAPTISOL®-free formulation at temperatures of 2-8°C (A) and 25°C (B). [Figure 7] This shows the visual difference between frozen carfilzomib formulations in containers with and without crimp seals after 4 weeks of storage at 2°C to 8°C. [Figure 8]Shows frozen CFZ-API at 2 to 8°C. High-concentration CFZ-API in DMSO had no significant loss of percentage main peak at the 4th week when measured by RP-HPLC. [Figure 9] Shows the proteasome activity of a carfilzomib formulation without CAPTISOL® administered subcutaneously to mice. [Figure 10] Shows the proteasome activity of a carfilzomib formulation without CAPTISOL® administered intravenously to mice. DETAILED DESCRIPTION OF THE INVENTION

[0147] Definitions The term "C x~y alkyl" refers to an unsubstituted saturated hydrocarbon group, including straight-chain alkyl groups and branched-chain alkyl groups containing x to y carbon atoms in the chain.

[0148] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, which is, for example, a moiety that can be represented by the following general formula:

Chemical Formula

[0149] The term "buffer" refers to a substance whose presence in a solution increases the amount of acid or alkali that needs to be added to produce a unit change in pH. Therefore, a buffer is a substance that helps adjust the pH of a composition. Typically, buffers are selected based on the desired pH and their compatibility with the other components of the composition. Generally, a buffer has a pKa (or the pKa produced when the composition dissolves) that is one unit less or one unit more than the desired pH of the composition.

[0150] As used herein, the term "water" refers to a solution of H2O with a pH of approximately 7.0.

[0151] The term “C x~y "Alkyl alcohol" is a C substituted with a hydroxyl group. x~y This refers to an alkyl group.

[0152] The term “substituted” refers to a portion of a molecule having substituents that substitute hydrogen on one or more non-hydrogen atoms. “Substituting” or “substituted with ~” should be understood to include the implicit condition that such substitution results in a stable compound that does not spontaneously undergo changes such as rearrangement, cyclization, or elimination, according to the allowable valencies of the substituted atom and substituent. As used herein, the term “substituted” includes all allowable substituents of an organic compound. In a broader embodiment, allowable substituents include acyclic and cyclic substituents, branched and unbranched substituents, carbocyclic and heterocyclic substituents, and aromatic and non-aromatic substituents of an organic compound. There may be one or more allowable substituents, which may be the same or different for a given organic compound. For the purposes of this disclosure, heteroatoms such as nitrogen may have any allowable substituents of the organic compounds described herein that satisfy the hydrogen substituent and / or the valency of the heteroatom. Examples of substituents include halogens, hydroxyls, carbonyls (such as carboxyls, alkoxycarbonyls, formyls, or acyls), thiocarbonyls (such as thioesters, thioacetates, or thioformates), alkoxyls, phosphoryls, phosphates, phosphonates, phosphinates, aminos, amides, amidines, imines, cyanos, nitros, azides, sulfhydryls, alkylthios, sulfates, sulfonates, sulfamoyls, sulfonamides, sulfonyls, heterocyclyls, aralkyls, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that a substituted moiety on a hydrocarbon chain may, where appropriate, be substituted itself.

[0153] As used herein, the term "peptide" refers to a chain of amino acids with a length of approximately 2 to 10 amino acids.

[0154] As used herein, the terms “natural” or “naturally occurring” amino acids refer to one of the 20 most common naturally occurring amino acids. Natural amino acids are referred to by their standard one- or three-letter abbreviations.

[0155] The term “preventive or therapeutic” treatment is recognized in the art and involves administering one or more of the compositions to a host. If it is administered before clinical signs of an undesirable condition (e.g., disease or other undesirable state in the host animal), the treatment is preventive (i.e., it protects the host from the development of the undesirable condition), whereas if it is administered after signs of an undesirable condition, the treatment is therapeutic (i.e., it is intended to reduce, improve or stabilize an existing undesirable condition or its side effects).

[0156] As used herein, the term “proteasome” is intended to include immunoproteasomes and constitutive proteasomes.

[0157] As used herein, the term “inhibitor” is intended to describe a compound that blocks or reduces the activity of an enzyme, or a system of enzymes, receptors, or other pharmacological targets (e.g., inhibition of proteolytic cleavage of standard fluorescent peptide substrates such as suc-LLVY-AMC, Box-LLR-AMC, and Z-LLE-AMC; inhibition of various catalytic activities of the 20S proteasome). Inhibitors can act in a competitive, uncompetitive, or noncompetitive manner. Because inhibitors can bind reversibly or irreversibly, this term includes compounds that are suicide substrates of enzymes. Inhibitors can also modify one or more sites on or near the active site of an enzyme, or they can cause conformational changes at other locations on the enzyme. In this specification, the term "inhibitor" is used more broadly than in scientific literature to include other classifications of pharmacologically or therapeutically useful agents, such as agonists, antagonists, stimulants, and cofactors.

[0158] As used herein, “low solubility” refers to being slightly soluble, sparingly soluble, very sparingly soluble, nearly soluble, or insoluble in, for example, water or another solution (e.g., the first combination). The terms “slightly soluble, sparingly soluble, very sparingly soluble, nearly soluble, or insoluble” correspond to the general meanings of the approximate solubility expressions in the United States Pharmacopeia (USP). For example, see DeLuca and Boylan in Pharmaceutical Dosage Forms: Parenteral Medications, vol. 1, Avis, KE, Lackman, L. and Lieberman, HA, eds; Marcel Dekkar: 1084, pages 141-142.

[0159] [Table 1]

[0160] As used herein, "heterogeneous" refers to a solution having a non-homogeneous (multiphase) composition. For example, heterogeneous solutions may include suspensions of solid particles in a liquid (e.g., slurries).

[0161] As used herein, “homogeneous” means a solution that is constant or homogeneous throughout its entire volume (observed as a single-phase, clear solution).

[0162] The “therapeutic effective dose” of a compound in this treatment method refers to the amount of the compound in a preparation that, when administered as part of a desired drug regimen (for a patient, e.g., a human), alleviates symptoms, improves a condition, or delays the onset of a disease or condition, or a condition, based on clinically acceptable criteria for cosmetic purposes, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0163] As used herein, the term “to treat” or “treatment” includes methods of improving or stabilizing a patient’s condition, including reversing, reducing, or cessating the symptoms, clinical signs, and underlying pathology of the condition.

[0164] Many small molecule organic compound drugs have pH-dependent solubility. The pH range suitable for drug administration (for intravenous administration, a tolerable pH range of pH 3 to pH 10.5 is generally considered, as is the case for injections, etc.) is often not the same pH at which sufficient solubility of the drug is observed in aqueous solution (e.g., at pH 2 or below). In order to achieve a pharmaceutically useful concentration level of the drug in the solution within a pH range that is acceptable and tolerable for administration (e.g., by injection), the order of solvent addition and pH adjustment when introducing the aqueous solution is a useful consideration for the formulations of the present invention claimed herein.

[0165] For basic drug molecules, solubility is usually enhanced at lower pH levels, but this can sometimes lead to stability and shelf-life issues when used without cyclodextrin. For example, sufficient solubility can be achieved by lowering the pH of the solution with an acid, but such a decrease in pH can lead to decomposition reactions due to acidic conditions. See Table 1 for the intrinsic water solubility data of carfilzomib, which shows a somewhat moderate increase in solubility as the pH decreases.

[0166] [Table 2]

[0167] Small molecule drugs and biomolecules are subject to numerous acid-mediated degradation pathways, including hydrolysis of amides in small inert peptide fragments or hydrolysis and ring-opening of functional epoxide moieties. Products of acid-mediated degradation may lack pharmacological activity and may be toxic or genotoxic compounds, even at trace levels. Therefore, it is beneficial that CFZ-API is completely dissolved in a solvent such as N-methyl-2-pyrrolidone (NMP) or dimethyl sulfoxide (DMSO) and the co-solvent mixture of the present invention before introducing an aqueous solution with an appropriate pH.

[0168] To balance the conflicting need to avoid the side reaction of acid-mediated decomposition that occurs at low pH, the inventors discovered specific pH conditions combined with the addition of a soluble polymer cosolubilizer. Surprisingly, the pH of the aqueous solution was achieved by adding a specific concentration of acid, such as methanesulfonic acid (around pH 3.0-3.5), in the presence of a polymer containing a similar structure, preferably a pyrrolidone ring or a polyvinylpyrrolidone (PVP) ring, preferably 10,000 MW of PVP, in the presence of a soluble polymer cosolubilizer that solubilizes 90% or more of the CFZ-API. Other cosolubilizers containing pyrrolidone rings can also act to solubilize and stabilize CFZ. Some examples of pyrrolidone agents useful for solubilizing CFZ include methylpyrrolidone (e.g., n-vinyl 3-methyl 2-pyrrolidone, n-vinyl 4-methyl 2-pyrrolidone, and n-vinyl 5-methylpyrrolidone) as a solvent, monomer, or polymer.

[0169] Minimizing the number of steps required to dissolve CFZ-API in solution helps facilitate manufacturing and clinical handling. To simplify the multi-step procedure, various combinations of water-miscible solvents, co-solvents, acids, and aqueous solutions were mixed. From the formulation test samples 1-3 discussed above, combinations of water-miscible solvents and co-solvents were able to yield similar solubility of CFZ-API at ≥20 mg / ml. Further screening revealed that the introduction of an aqueous solvent step to further dilute CFZ-API to ≥2 mg / ml could not be combined with a water-miscible solvent and / or co-solvent step because CFZ-API would not dissolve. To maximize the solubility of CFZ-API, it was found that each solvent mixture must be added in a multi-step pattern before the final option for formulation presentation, which may be a ready-to-use or lyophilized formulation. A flow scheme for the multi-step preparation of a cyclodextrin-free lyophilized formulation is described below.

number

[0170] In step 1 of the flow scheme preparation, due to its very low water solubility, a non-aqueous solvent consisting of a water-miscible organic solvent and a cosolvent system is first added to dissolve the CFZ-API solid. In step 2 of the flow scheme preparation, the non-aqueous CFZ-API solution is then introduced into an acidic aqueous environment with a final pH of 3.0-3.5 to obtain maximum CFZ-API solubility. Subsequently, the solution is filtered using a 0.22 μm PES syringe filter equipped on a NORMJECT® (silicone-free) syringe. The obtained filtrate is then inspected for the recovery and stability of CFZ-API solubility using reversed-phase high-performance liquid chromatography (RP-HPLC). Using RP-HPLC, peak decomposition and CFZ-API recovery were determined using a 3-5 point reference standard curve. Standard peak integration was taken against standard buffer (50% acetonitrile in water), while peak integration of the formulation sample was taken against the formulation buffer. In step 3 of the figure, a freeze-drying step was performed on the filtrate. When the lyophilized product was reconstituted with water for injection (WFI), a CFZ-API solubility of approximately 1.5 mg / ml to 5 mg / ml was obtained in the preferred formulation of the present invention.

[0171] To maximize the solubility of CFZ-API, a multi-step solvent addition was used, including individual steps to acidify the solution to obtain the target concentration and pH of CFZ-API. Cyclodextrin-free CFZ-API formulations can be prepared using the following two-step or three-step scheme.

number

[0172] The first step may consist of dissolving CFZ-API in an organic mixture to a concentration of approximately 20-50 mg / ml. This organic mixture may consist of DMSO, PEG400, ethanol, and lactic acid. The second step may involve adding an acidic solubilizer-containing solution in water to achieve a final CFZ-API concentration of 2 mg / ml. This mixture may consist of povidone, water, and MSA, and may have a pH of approximately 2.9. When this mixture is added to the first step containing CFZ-API, it should partially precipitate into a clear solution at approximately pH 3. If step 2 does not achieve the target pH, an additional step may be required to achieve the target pH of 3 with the minimum amount of MSA or MEA. Once the pH is achieved, filtration is required to remove any excess CFZ-API that has not been solubilized. The results from these experiments suggest that the order in which these excipients are introduced to CFZ-API is important to obtain maximum CFZ-API solubility. For example, aqueous mixtures containing povidone do not dissolve in CFZ-API, and therefore cannot be added to CFZ-API before organic mixtures.

[0173] Apart from lyophilized and injectable emulsions, another embodiment of the cyclodextrin-free CFZ formulation presented is a frozen form stored at 2°C–8°C. This option is advantageous because it eliminates the need for a lyophilizer or freezer to maintain the high solubility and stability of CFZ. The composition can be prepared by dissolving CFZ API in DMSO at ≥200 mg / ml and storing it at 2°C–8°C to obtain a frozen product. This frozen state at high temperatures is due to the high melting temperature of DMSO, which is 19°C. The pre- and post-preparation flow scheme for the preparation of a frozen cyclodextrin-free CFZ-API formulation is described below. The first step is carried out by preparation. Storage and transport at 2°C–8°C maintain the frozen state of the formulation. The clinic receives the formulation solution from steps 2 and 3, or a combination thereof, and adds it to the thawed formulation at the time of clinical administration.

number

[0174] How to use The biological applications of proteasome inhibition are numerous. Proteasome inhibition has been proposed as a preventive and / or therapeutic method for a wide range of diseases, including, but not limited to, proliferative disorders, neurotoxic / degenerative diseases, Alzheimer's disease, ischemia, inflammation, autoimmune diseases, HIV, cancer, organ graft rejection, septic shock, antigen presentation inhibition, decreased viral gene expression, parasitic infections, symptoms associated with acidosis, macular degeneration, pulmonary diseases, muscle wasting diseases, fibrosis, and bone and hair growth disorders. Therefore, pharmaceutical formulations for highly potent proteasome-specific compounds, such as epoxy ketone class molecules, provide a means of administering drugs to patients and treating these conditions.

[0175] At the cellular level, accumulation of polyubiquitinated proteins, changes in cell morphology, and apoptosis have been reported upon treatment of cells with various proteasome inhibitors. Proteasome inhibition has also been proposed as a possible antitumor therapeutic strategy. The fact that epixomicin was the first to be identified in the examination of antitumor compounds demonstrates the proteasome as an antitumor chemotherapy target. Therefore, these compositions are useful for the treatment of cancer.

[0176] In both in vitro and in vivo models, malignant cells have been shown to be generally susceptible to proteasome inhibition. Indeed, proteasome inhibition has already been demonstrated as a therapeutic strategy for the treatment of multiple myeloma. This may be partly due to the fact that highly proliferative malignant cells rely on the proteasome system for rapid protein removal (Rolfe et al., J.Mol.Med. (1997) 75:5-17; Adams, Nature (2004) 4:349-360). Therefore, a method for treating cancer is provided herein, comprising administering a therapeutically effective amount of the peptide proteasome inhibitor provided herein to patients requiring such treatment.

[0177] As used herein, the term “cancer” includes, but is not limited to, cancers of the blood, bone, and solid tumors. Cancer refers to diseases of the blood, bone, organs, skin tissue, and vascular system, including, but is not limited to, cancers of the bladder, blood, bone, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidney, liver, lung, lymph nodes, mouth, neck, ovaries, pancreas, prostate, rectum, kidneys, skin, stomach, testes, throat, and uterus. Specific cancers include leukemia (acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia), mature B-cell neoplasms (small lymphocytic lymphoma, B-cell prelymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenström's macroglobulinemia), splenic marginal zone lymphoma, plasmacytoma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, extranodal peripheral zone B Cellular lymphoma (MALT lymphoma), peripheral zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse B-cell lymphoma, large mediastinal (thymic) B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma and Burkitt lymphoma / leukemia), mature T-cell and natural killer (NK) cell neoplasms (pre-lymphocytic T-cell leukemia, large granular T-cell lymphocytic leukemia, progressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal N K / T cell lymphoma, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoides (Sézary syndrome), primary cutaneous anaplastic large cell lymphoma, lymphomatoid papular dysplasia, angioimmunoblastic T cell lymphoma, unspecified peripheral T cell lymphoma and anaplastic large cell lymphoma), Hodgkin lymphoma (tubular sclerosis, mixed cell solid, lymphocyte-rich, lymphocyte-depleted or non-depleted, nodular lymphocyte-dominant), myeloma (multiple myeloma, painless myeloma, etc.) Myeloma (a type of bulri), chronic myeloproliferative disorders, spinal cord dysplasia / myeloproliferative disorders, spinal cord dysplasia syndromes, immunodeficiency-associated lymphoproliferative disorders, histiocytic and dendritic cell neoplasms, mastocytosis, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant giant cell tumor, myeloma bone disease, osteosarcoma, breast cancer (hormone-dependent and hormone-independent), gynecological cancers (cervix, endometrium, fallopian tubes, gestational trophoblastic disease, ovaries, peritoneum, uterus, vagina and vulva), basal cell carcinoma (BCC), squamous cell carcinoma (SCC),Malignant melanoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, Kaposi's sarcoma, astrocytoma, pilocytic astrocytoma, germ dysplastic neuroepithelial tumor, oligodendroglioma, ependymal cell tumor, pleomorphic gliablastoma, mixed glioma, oligoastrocytoma, medulloblastoma, retinoblastoma, neuroblastoma, germ cell tumor, teratoma, malignant mesothelioma (peritoneal mesothelioma, pericardial mesothelioma, pleural mesothelioma), gastrointestinal pancreatic or neuroendocrine tumors of the gastrointestinal pancreas (GEP-NET), carcinoid, pancreatic endocrine tumor (PET), colorectal adenocarcinoma, colorectal carcinoma, progressive neuroendocrine tumor, leiomyosarcoma, glial adenocarcinoma, signet ring cell carcinoma This includes, but is not limited to, cancer, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, hemangioma, hepatic adenoma, focal nodular hyperplasia (nodular regenerative hyperplasia, hamartoma), non-small cell lung cancer (NSCLC) (squamous cell lung cancer, adenocarcinoma, large cell lung cancer), small cell lung carcinoma, thyroid cancer, prostate cancer (hormone-resistant, androgen-independent, androgen-dependent, hormone-insensitive), and soft tissue sarcomas (fibrosarcoma, malignant fibrous histiocytoma, dermatofibrosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, hemangioendothelioma, synovial sarcoma, malignant peripheral nerve sheath tumor / neurofibrosarcoma, extraskeletal osteosarcoma).

[0178] In some embodiments, the peptide proteasome inhibitors provided herein, or pharmaceutical compositions comprising them, may be administered to treat multiple myeloma in patients. For example, multiple myeloma may include refractory and / or refractory multiple myeloma, or newly diagnosed multiple myeloma.

[0179] Many tumors of hematopoietic and lymphoid tissues are characterized by cell proliferation, or an increase in specific types of cells. Chronic myeloproliferative disorders (CMPDs) are clonal hematopoietic stem cell disorders characterized by proliferation of one or more myeloid cells of the myeloid cell lineage, resulting in an increase in the number of granulocytes, erythrocytes, and / or platelets in the peripheral blood. Therefore, the use of proteasome inhibitors for the treatment of these diseases is attractive and has been investigated (Cilloni et al., Haematologica (2007) 92:1124-1229). Examples of CMPDs include chronic myeloid leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, polycythemia vera, chronic idiopathic myelofibrosis, essential thrombocythemia, and unclassifiable chronic myeloproliferative disorders. A method for treating CMPDs is provided herein, comprising administering an effective amount of a proteasome inhibitor compound disclosed herein to a patient in need of such treatment.

[0180] Spinal malformations / myeloproliferative disorders, such as chronic myelomonocytic leukemia, atypical chronic myeloid leukemia, juvenile myelomonocytic leukemia, and unclassifiable spinal malformations / myeloproliferative disorders, are characterized by a cellular hyperplasia of the bone marrow resulting from the proliferation of one or more myeloid cell lineages. Inhibiting the proteasome with the compositions described herein may be helpful in treating these spinal malformations / myeloproliferative disorders by providing an effective amount of the compositions to patients requiring such treatment.

[0181] Dysplasia of the spinal cord (MDS) refers to a group of hematopoietic stem cell disorders characterized by dysplasia and ineffective hematopoiesis in one or more major myeloid cell lines. Targeting of NF-κB with proteasome inhibitors in these hematological malignancies induces apoptosis, thereby killing malignant cells (Braun et al. Cell Death and Differentiation (2006) 13:748-758). Methods for treating MDS are further provided herein, comprising administering an effective amount of the compound provided herein to a patient requiring such treatment. Examples of MDS include refractory anemia, refractory anemia with ring sideroblasts, refractory cytopenia with pluripotent dysplasia, refractory anemia with superblasts, unclassifiable dysplasia of the spinal cord, and dysplasia of the spinal cord with isolated deletion (5q) chromosomal abnormalities.

[0182] Mastosis is the proliferation and subsequent accumulation of mast cells in one or more organ systems. Examples of mastocytosis include, but are not limited to, cutaneous mastocytosis, painless systemic mastocytosis (ISM), systemic mastocytosis with associated clonal hematological non-mastocytosis lineage disease (SM-AHNMD), progressive systemic mastocytosis (ASM), mast cell leukemia (MCL), mast cell sarcoma (MCS), and extracutaneous mastocytoma. Furthermore, this specification provides a method for treating mastocytosis, comprising administering an effective amount of one of the compounds disclosed herein to a patient diagnosed with mastocytosis.

[0183] The proteasome regulates NF-κB, which in turn regulates genes involved in immune and inflammatory responses. For example, NF-κB is required for the expression of immunoglobulin light chain κ genes, IL-2 receptor α chain genes, class I major histocompatibility complex genes, and several 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, methods are provided herein for influencing the expression levels of any of the previously mentioned proteins, IL-2, MHC-I, IL-6, TNFα, IFN-β, or other such proteins, each method comprising administering an effective amount of a proteasome inhibitor composition disclosed herein to a patient.

[0184] A method for treating an autoimmune disease in a patient is also provided herein, comprising administering a therapeutically effective amount of a compound described herein. In this specification, “autoimmune disease” means a disease or disorder arising from or against the tissues of an individual. Examples of autoimmune diseases or disorders include inflammatory reactions such as inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); dyspnea syndromes (including adult dyspnea syndrome (ARDS)); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions such as eczema and asthma, and other conditions with T-cell infiltration and chronic inflammatory reactions; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes mellitus (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile-onset diabetes mellitus; and tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis. Typical cytokine and T lymphocyte-mediated acute and delayed hypersensitivity-related immune responses; pernicious anemia (Addison's disease); diseases involving extravasation of leukocytes; central nervous system (CNS) inflammatory disorders; multiple organ injury syndromes; hemolytic anemia (including, but not limited to, cryoglobulinemia or Coombs-positive anemia); myasthenia gravis, antigen-antibody complex-mediated diseases; anti-glomerular basement membrane diseases; antiphospholipid syndromes; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyglandular endocrine disorders; Reiter's disease; Stiffman syndrome; Behçet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP); or autoimmune thrombocytopenia.

[0185] The immune system screens autologous cells that are virally infected, undergoing oncogenic transformation, or presenting unknown peptides on their surface. Intracellular proteolysis generates small peptides for presentation to T lymphocytes, inducing an MHC class I-mediated immune response. Thus, methods of using proteasome inhibitors provided herein as immunomodulators for inhibiting or modifying antigen presentation in cells are provided herein, comprising exposing cells to (or administering to a patient) the compounds described herein. Certain embodiments include methods of treating graft or transplant-related diseases in a patient, such as graft-versus-host disease or host-versus-graft disease, comprising administering a therapeutically effective amount of the compounds described herein. As used herein, the term “graft” refers to biological material derived from a donor for transplantation into a recipient. Examples of grafts include isolated cells such as pancreatic islet cells; tissues such as neonatal amniotic membrane, bone marrow, hematopoietic progenitor cells, and eye tissue, such as corneal tissue; and a wide range of materials such as skin, heart, liver, spleen, pancreas, thyroid lobe, lung, kidney, and tubular organs (e.g., intestines, blood vessels, or esophagus). Tubular organs may be used to replace damaged portions of the esophagus, blood vessels, or bile ducts. Skin grafts may be used not only for burns but also as bandage material for damaged intestines or to close certain defects such as diaphragmatic hernias. Grafts are obtained from any mammalian source, including humans, whether from a cadaver or a living donor. In some cases, the donor and recipient are the same patient. In some embodiments, the graft is bone marrow or an organ, such as a heart, and the graft donor and host are HLA class II antigen matched.

[0186] Histiocytic and dendritic cell neoplasms are induced from phagocytic and accessory cells, which play a major role in the processing and presentation of antigens to lymphocytes. It has been shown that depleting the proteasome content in dendritic cells alters these antigen-induced responses (Chapatte, et al. Cancer Res. (2006) 66:5461-5468).

[0187] In some embodiments, cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered to patients with histiocytic cell and dendritic cell neoplasms. Histiocytic cell and dendritic cell neoplasms include histiocytic sarcoma, Langerhans cell histioproliferation, Langerhans cell sarcoma, finger-entrenched dendritic cell sarcoma / tumor, follicular dendritic cell sarcoma / tumor, and unspecified dendritic cell sarcoma.

[0188] Proteasome inhibition has been shown to be beneficial in treating cell type proliferation diseases and immunodeficiency disorders; therefore, in some embodiments, treatment for lymphoproliferative disorders (LPDs) associated with primary immunodeficiency disorders (PIDs) is provided, comprising administering an effective amount of the compounds of this disclosure to patients in need. The most common clinical backgrounds of immunodeficiency associated with the increased incidence of lymphoproliferative disorders, including B-cell and T-cell neoplasms and lymphomas, are primary immunodeficiency syndromes and other primary immune diseases, human immunodeficiency virus (HIV) infection, iatrogenic immunosuppression in patients who have undergone solid organ or bone marrow allogeneic transplantation, and iatrogenic immunosuppression associated with methotrexate treatment. Other PIDs commonly associated with LPD include, but are not limited to, ataxia telangiectasia (AT), Wiscott-Aldrich syndrome (WAS), unclassified immunodeficiency (CVID), severe combined immunodeficiency (SCID), X-linked lymphoproliferative disorder (XLP), Nijmiehen chromosome instability syndrome (NBS), hyper-IgM syndrome, and autoimmune lymphoproliferative syndrome (ALPS).

[0189] Proteasome inhibition is also associated with the inhibition of NF-κB activation and the stabilization of p53 levels. Therefore, the compositions provided herein may also be used to inhibit NF-κB activation and stabilize p53 levels in cell cultures. Since NF-κB is a major regulator of inflammation, it is an attractive target for anti-inflammatory therapeutic interventions. Therefore, the compositions provided herein may be useful in the treatment of inflammation-related conditions, including but not limited to COPD, psoriasis, asthma, bronchitis, emphysema, and cystic fibrosis.

[0190] The disclosed compositions may be used to treat conditions directly mediated by the proteasome's proteolytic function, such as muscle wasting, or conditions indirectly mediated through proteins processed by the proteasome, such as NF-κB. Proteasomes are involved in the rapid elimination and post-translational processing of proteins (e.g., enzymes) involved in cellular regulation (e.g., cell cycle, gene transcription, and metabolic pathways), intercellular communication, and immune responses (e.g., antigen presentation). Specific examples discussed below include β-amyloid proteins and regulatory proteins, such as cyclins and the transcription factor NF-κB.

[0191] In some embodiments, the compositions provided herein are used to treat stroke, ischemic injury to the nervous system, neurotrauma (e.g., impact-induced brain injury, spinal cord injury, and traumatic injury to the nervous system), multiple sclerosis and other immune-mediated neuropathy (e.g., Guillain-Barré syndrome and its variants, acute motor axonal neuropathy, acute inflammatory demyelinating polyneuropathy, and Fisher syndrome), HIV / AIDS dementia syndrome, axonomy, diabetic neuropathy, Parkinson's disease, Huntington's disease It is useful in treating neurodegenerative diseases and conditions, including, but not limited to, dementia caused by disease, multiple sclerosis, bacterial, parasitic, fungal, and viral meningitis, encephalitis, vascular dementia, multiple infarct dementia, Lewy body dementia, frontal lobe dementia such as Pix disease, subcortical dementia (e.g., Huntington's disease or progressive supranuclear palsy), focal cortical atrophy syndrome (e.g., primary aphasia), metabotoxic dementia (e.g., chronic hypothyroidism or B12 deficiency), and dementia caused by infection (e.g., syphilis or chronic meningitis).

[0192] Alzheimer's disease is characterized by extracellular deposits of β-amyloid protein (β-AP) in senile plaques and cerebral blood vessels. β-AP is a 39-42 amino acid peptide fragment derived from amyloid protein precursor (APP). At least three isoforms of APP are known (695, 751, and 770 amino acids). Alternative splicing of mRNA generates isoforms, and normal processing affects a portion of the β-AP sequence, thereby preventing β-AP production. Abnormal protein processing by the proteasome is thought to contribute to the large amount of β-AP in Alzheimer's brains. APP processing enzymes in rats contain approximately 10 different subunits (22kDa-32kDa). The 25 kDa subunit has the N-terminal sequence X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which is identical to the β-subunit of human macropain (Kojima, S. et al., Fed. Eur. Biochem. Soc., (1992) 304:57-60). The APP-treated enzyme cleaves at the Gln15--Lys16 bond, and in the presence of calcium ions, the enzyme also cleaves at the Met-1--Asp1 bond and the Asp1--Ala2 bond, releasing the extracellular domain of β-AP.

[0193] Accordingly, one embodiment is a method for treating Alzheimer's disease, comprising administering an effective amount of the composition provided herein to a patient. Such treatment includes reducing the rate of β-AP processing, reducing the rate of β-AP plaque formation, reducing the rate of β-AP production, and reducing the clinical signs of Alzheimer's disease.

[0194] Methods for treating cachexia and muscle wasting diseases are also provided herein. Proteasomes degrade many proteins in the maturation of reticulocytes and the growth of fibroblasts. In cells deficient in insulin or serum, the rate of protein degradation is nearly doubled. Inhibition of proteasomes reduces protein degradation, thereby reducing both muscle protein loss and nitrogen load on the kidneys or liver. The peptide proteasome inhibitors provided herein are useful in treating conditions such as cancer, chronic infectious diseases, fevers, muscle disuse (atrophy) and denervation, nerve injury, fasting, renal failure associated with acidosis, and hepatic failure. See, for example, Goldberg U.S. Patent No. 5,340,736. Methods of treatment include reducing the rate of muscle protein degradation in cells, reducing the rate of intracellular protein degradation, reducing the rate of intracellular p53 protein degradation, and inhibiting the growth of p53-related cancers. Each of these methods involves contacting cells (in vivo or in vitro, e.g., muscle in a patient) with an effective amount of the pharmaceutical composition disclosed herein.

[0195] Fibrosis is the excessive and persistent formation of scar tissue resulting from the hyperproliferative growth of fibroblasts and is associated with the activation of the TGF-β signaling pathway. Fibrosis is accompanied by extensive deposition of extracellular matrix and can occur in substantially any tissue or across several different tissues. Normally, the levels of intracellular signaling proteins (SMADs) that activate the transcription of target genes upon TGF-β stimulation are regulated by proteasome activity. However, accelerated degradation of TGF-β signaling components has been observed in cancer and other hyperproliferative conditions. Therefore, in certain embodiments, methods are provided for treating hyperproliferative conditions such as 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, collagen vascular disease, sarcoidosis, interstitial lung disease, and exogenous lung injury). Treatment of burn victims is often hindered by fibrosis; therefore, in some embodiments, inhibitors provided herein for the treatment of burns may be administered topically or systemically. Postoperative wound suturing often results in unsightly scarring, which can be prevented by inhibiting fibrosis. Accordingly, in certain embodiments, methods for preventing or reducing scarring are provided herein.

[0196] Another protein processed by the proteasome is NF-κB, a member of the Rel protein family. The Rel family of transcription activator proteins can be divided into two groups. The first group requires proteolytic treatment and includes p50 (NF-κB1, 105 kDa) and p52 (NF-κ2, 100 kDa). The second group does not require proteolytic treatment and includes p65 (RelA, Rel(c-Rel), and RelB). Both homodimers and heterodimers can be formed by Rel family members; for example, NF-κB is a p50-p65 heterodimer. After phosphorylation and ubiquitination of IκB and p105, the two proteins are degraded and processed, respectively, to produce active NF-κB that translocates from the cytoplasm to the nucleus. Ubiquitinated p105 can also be treated with purified proteasomes (Palombella et al., Cell (1994) 78:773-785). Active NF-κB forms stereospecific enhancer complexes with other transcription activators, such as HMG I(Y), to induce selective expression of specific genes.

[0197] NF-κB modulates genes involved in immune and inflammatory responses, as well as mitotic events. For example, NF-κB is required for the expression of immunoglobulin light chain κ genes, IL-2 receptor α chain genes, class I major histocompatibility complex genes, and several cytokine genes encoding, for example, IL-2, IL-6, granulocyte colony-stimulating factor, and IFN-β (Palombella et al., Cell (1994) 78:773-785). Several embodiments include methods for influencing the expression levels of any of IL-2, MHC-I, IL-6, TNFα, IFN-β, or other previously mentioned proteins, each method comprising administering an effective amount of the composition disclosed herein to a patient. Complexes containing p50 are rapid modulotropes of acute inflammation and immune responses (Thanos, D. and Maniatis, T., Cell (1995) 80:529-532).

[0198] NF-κB is also involved in the expression of cell adhesion genes encoding E-selectin, P-selectin, ICAM, and VCAM-1 (Collins, T., Lab. Invest. (1993) 68:499-508). In some embodiments, methods are provided for inhibiting cell adhesion (e.g., cell adhesion mediated by E-selectin, P-selectin, ICAM, or VCAM-1), comprising contacting cells with an effective amount of the pharmaceutical composition disclosed herein (or administering it to a patient).

[0199] Ischemia and reperfusion injury results in hypoxia, a condition in which oxygen is deficient in reaching body tissues. This condition leads to increased degradation of Iκ-Bα, thereby activating NF-κB. The severity of injuries resulting in hypoxia has been shown to be reduced by the administration of proteasome inhibitors. Accordingly, a method for treating ischemic conditions or reperfusion injury is provided herein, comprising administering an effective amount of the compounds disclosed herein to a patient requiring such treatment. Examples of such conditions or injuries include, but are not limited to, acute coronary insufficiency syndrome (unstable plaque), arterial occlusive diseases (cardiac, cerebral, peripheral artery and vascular occlusion), atherosclerosis (coronary artery disease), infarction, heart failure, pancreatitis, myocardial hypertrophy, stenosis, and restenosis.

[0200] NF-κB also specifically binds to the HIV enhancer / promoter. Compared to the Nef of mac239, the HIV regulatory protein Nef of pbj14 differs by two amino acids in the region that controls protein kinase binding. The protein kinase is thought to signal phosphorylation of IκB, triggering IκB degradation via the ubiquitin-proteasome pathway. After degradation, NF-κB is released into the nucleus, thus enhancing HIV transcription (Cohen, J., Science, (1995) 267:960). Methods for inhibiting or reducing HIV infection in a subject, and methods for reducing the level of viral gene expression are provided herein, each method comprising administering an effective amount of the composition disclosed herein to a patient.

[0201] Viral infections contribute to the pathology of many diseases. Cardiac conditions such as persistent myocarditis and dilated cardiomyopathy are associated with coxsackievirus B3. Comparative whole-genome microarray analysis of infected mouse hearts showed that 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 utilize the ubiquitin-proteasome system in their viral entry process, where the virus is released from endosomes into the cytosol. Mouse hepatitis virus (MHV) belongs to the Coronaviridae family, which also includes the severe acute respiratory syndrome (SARS) coronavirus. Yu and Lai (J Virol 79:644-648, 2005) demonstrated that treatment of MHV-infected cells with proteasome inhibitors resulted in reduced viral replication and correlated with reduced viral titers compared to untreated cells. Human hepatitis B virus (HBV), a member of the Hepadnaviridae virus family, similarly requires a virus-encoded envelope protein for transmission. Inhibition of the proteasome degradation pathway results in a significant reduction in the amount of secreted envelope protein (Simsek et al, J Virol 79:12914-12920, 2005). In addition to HBV, other hepatitis viruses (A, C, D, and E) may also utilize the ubiquitin-proteasome degradation pathway for secretion, morphogenesis, and disease onset. Accordingly, in certain embodiments, methods are provided for treating viral infections such as SARS or hepatitis A, B, C, D, and E, comprising contacting cells with an effective amount of a compound disclosed herein (or administering it to a patient).

[0202] Overproduction of lipopolysaccharide (LPS)-induced cytokines such as TNFα is considered central to the processes associated with septic shock. Furthermore, it is generally recognized that the first step in LPS-induced cell activation is the binding of LPS to specific membrane receptors. The α- and β-subunits of the 20S proteasome complex have been identified as LPS-binding proteins, suggesting that LPS-induced signaling 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 compositions provided herein may be used to inhibit TNFα for the prevention and / or treatment of septic shock.

[0203] Intracellular proteolysis generates small peptides for presentation to T lymphocytes, inducing an MHC class I-mediated immune response. The immune system screens for autologous cells that are infected with a virus or undergoing oncogenic transformation. One embodiment is a method for inhibiting antigen presentation in cells, comprising exposing cells to the composition described herein. A further embodiment is a method for suppressing a patient's immune system (e.g., inhibiting graft rejection, allergies, or asthma), comprising administering an effective amount of the composition described herein to the patient. The compositions provided herein may also be used to treat autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease.

[0204] Another embodiment is a method for modifying the repertoire of antigen peptides produced by the proteasome or other Ntn having multicatalytic activity. For example, if the PGPH activity of the 20S proteasome is selectively inhibited, a different set of antigen peptides from those produced and presented by the proteasome and presented in MHC molecules on the cell surface may be produced by the proteasome without the use of any enzyme inhibition, or, for example, by selective inhibition of the chymotrypsin-like activity of the proteasome.

[0205] Certain proteasome inhibitors block both the degradation and processing of ubiquitinated NF-κB in vitro and in vivo. Proteasome inhibitors also block IκB-α degradation and NF-κB activation (Palombella, et al. Cell (1994) 78:773-785, and Traenckner, et al., EMBO J. (1994) 13:5433-5441). In some embodiments, methods are provided for inhibiting IκB-α degradation, comprising contacting cells with the compositions described herein. Further embodiments are methods for reducing the intracellular content of NF-κB in cells, muscles, organs, or patients, comprising contacting cells, muscles, organs, or patients with the compositions described herein.

[0206] Other eukaryotic transcription factors that require proteolytic treatment include the basic transcription factor TFIIA, herpes simplex virus VP16 accessory protein (host cell factor), virus-induced IFN regulator 2 protein, and membrane-bound sterol regulator-binding protein 1.

[0207] Furthermore, methods for influencing the cyclin-dependent eukaryotic cell cycle are provided herein, comprising exposing cells (in vitro or in vivo) to compositions disclosed herein. Cyclins are proteins involved in the regulation of the cell cycle. Proteasomes are involved in the degradation of cyclins. Examples of cyclins include mitotic cyclins, G1 cyclins, and cyclin B. Cyclin degradation allows cells to exit one cell cycle stage (e.g., mitosis) and enter another stage (e.g., cell division). All cyclins are thought to be associated with the p34cdc2 protein kinase or related kinases. The proteolytic targeting signal is localized to amino acid 42-RAALGNISEN-50 (destruction box). There is evidence that cyclins are converted to a form susceptible to ubiquitin ligases, or that cyclin-specific ligases are activated during mitosis (Ciechanover, A., Cell, (1994) 79:13-21). Proteasome inhibition inhibits cyclin degradation and, therefore, inhibits cell proliferation, for example, in cyclin-related cancers (Kumatori et al., Proc. Natl. Acad. Sci. USA (1990) 87:7071-7075). A method for treating proliferative disorders in a patient (e.g., cancer, psoriasis, or restenosis) is provided herein, comprising administering to the patient an effective amount of the composition disclosed herein. A method for treating cyclin-related inflammation in a patient is also provided herein, comprising administering to the patient a therapeutically effective amount of the composition described herein.

[0208] Additional embodiments include methods for influencing the proteasome-dependent regulation of oncogeneic proteins and methods for treating or inhibiting cancer growth, each method comprising exposing cells (in vivo, e.g., in a patient, or in vitro) to the compositions disclosed herein. HPV-16 and HPV-18-induced E6 proteins stimulate ATP and ubiquitin-dependent conjugation, as well as the degradation of p53 in reticular erythrolysis. The recessive oncogene p53 has been shown to accumulate at non-permissible temperatures in cell lines with mutant thermal instability E1. Elevated levels of p53 can lead to apoptosis. Examples of proto-oncogeneic proteins degraded by the ubiquitin system include c-Mos, c-Fos, and c-Jun. One embodiment is a method for treating p53-related apoptosis, comprising administering an effective amount of the compositions disclosed herein to a patient.

[0209] In another embodiment, the compositions of this disclosure are useful for treating parasitic infections, such as infections caused by parasitic protozoa. The proteasomes of these parasites are thought to be primarily involved in cell differentiation and replication activity (Paugam et al., Trends Parasitol. 2003, 19(2):55-59). Furthermore, it has been shown that Entomoeba species lose their ability to form encapsulation when exposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res. 1997, 28, Spec No: 139-140). In certain embodiments of this disclosure, the compositions of this disclosure include Plasmodium sps (including P. falciparum, P. vivax, P. malariae, and P. ovale, which cause malaria), Trypanosoma sps (including T. cruzi, which causes Chagas disease, and T. brucei, which causes African sleeping sickness), Leishmania sps (including L. amazonesis, L. donovani, L. infantum, L. mexicana, etc.), and Pneumocystis carinii. It is useful for treating parasitic infections in humans caused by parasitic protozoa selected from among Toxoplasma carinii (a protozoan known to cause pneumonia in patients with AIDS and other immunosuppressive conditions), Toxoplasma gondii, Entamoeba histolytica, Entamoeba invadens, and Giardia lamblia.In certain embodiments, the compositions of this disclosure are useful for treating parasitic infections in animals and livestock caused by parasitic protozoa selected from Plasmodium hermani, Cryptosporidium sps, Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona, and Neurospora crassa. Other compounds useful as proteasome inhibitors in the treatment of parasitic diseases are described in their entirety in International Publication No. 98 / 10779, which is incorporated herein by reference.

[0210] In certain embodiments, the compositions of this disclosure irreversibly inhibit proteasome activity within parasites. Such irreversible inhibition has been shown to induce a shutdown of enzyme activity in red and white blood cells without recovery. In certain embodiments, the long half-life of the blood cells can provide long-term protection in relation to treatment of repeated exposure to the parasite. In certain embodiments, the long half-life of the blood cells can provide long-term protection in relation to chemoprevention against future infections.

[0211] Prokaryotes possess proteasome particles equivalent to those of eukaryotes (20S). Although the subunit composition of prokaryotic 20S particles is simpler than that of eukaryotes, they similarly possess the ability to hydrolyze peptide bonds. For example, nucleophilic attack on peptide bonds occurs via a threonine residue on the N-terminus of the β-subunit. In some embodiments, methods are provided for treating prokaryotic infections, comprising administering to a patient an effective amount of a proteasome inhibitor composition disclosed herein. Prokaryotic infections may include diseases caused by either mycobacteria (e.g., tuberculosis, leprosy, or Buruli ulcer) or archaea.

[0212] Inhibitors that bind to the 20S proteasome have also been shown to stimulate osteogenicity in bone tissue culture. Furthermore, systemic administration of these inhibitors to mice resulted in a more than 70% increase in bone mass and osteogenicity rate with specific proteasome inhibitors (Garrett, IR et al., J. Clin. Invest. (2003) 111:1771-1782), suggesting that the ubiquitin-proteasome mechanism regulates osteoblast differentiation and osteogenicity. Therefore, the compositions of this disclosure may be useful in the treatment and / or prevention of bone loss-related diseases such as osteoporosis.

[0213] Methods for treating diseases or conditions selected from cancer, autoimmune diseases, graft or graft-related conditions, neurodegenerative diseases, fibrosis-related conditions, ischemia-related conditions, infections (viral, parasitic, or prokaryotic), and bone loss-related conditions are provided herein, comprising administering proteasome inhibitors provided herein. For example, compounds of formula (5).

[0214] Bone tissue is an excellent source of factors capable of stimulating osteocytes. Therefore, bovine bone tissue extracts contain not only structural proteins responsible for maintaining the structural integrity of bone, but also bioactive bone growth factors that can stimulate osteocyte proliferation. Among these latter factors is a recently described family of proteins called bone morphogenetic proteins (BMPs). All of these growth factors have effects on other types of cells, as well as osteocytes; for example, Hardy, MH, et al., Trans Genet (1992) 8:55-61 describes evidence of differential expression of bone morphogenetic proteins (BMPs) in developing hair follicles. Harris, SE, et al., J Bone Miner Res (1994) 9:855-863 explains the effects of TGF-β on the expression of BMP-2 and other substances in osteocytes. BMP-2 expression in mature hair follicles occurs during maturation and after the cell proliferation period (Hardy, et al. (1992, above)). Therefore, the compounds provided herein may also be useful for stimulating hair follicle growth.

[0215] Finally, the compositions of this disclosure are also useful as diagnostic reagents (e.g., in diagnostic kits or for use in clinical laboratories) for screening proteins (e.g., enzymes, transcription factors) that are processed by Ntn hydrolases containing proteasomes. The compositions of this disclosure are also useful as research reagents for specifically binding to the X / MB1 subunit or α chain and inhibiting associated proteolytic activity. For example, the activity of other subunits of the proteasome (and their specific inhibitors) can be determined.

[0216] Most cellular proteins are subjected to proteolytic processes during maturation or activation. Enzyme inhibitors disclosed herein can be used to determine whether a cell, development, or physiological process or output is regulated by the proteolytic activity of a particular Ntn hydrolase. One such method comprises obtaining an organism, an intact cell preparation, or a cell extract; exposing the organism, cell preparation, or cell extract to a composition disclosed herein; exposing the organism, cell preparation, or cell extract exposed to the compound to a signal; and monitoring the process or output. The high selectivity of the compounds disclosed herein allows for the rapid and precise removal or inclusion of Ntn (e.g., 20S proteasome) in a given cell, development, or physiological process.

[0217] Administration Compositions prepared as described herein can be administered in various forms depending on the disorder being treated, as well as the patient's age, condition, and weight, as is well known in the art. For example, when administered orally, the compositions may be formulated as tablets, capsules, granules, powders, or syrups; or, when administered parenterally, they may be formulated as (intravenous, intramuscular, or subcutaneous) injections, infusion preparations, or suppositories. When administered via the mucoocular route, they may be formulated as eye drops or ophthalmic ointments. These formulations may be prepared by conventional means in conjunction with the methods described herein, and, if desired, the active ingredient may be mixed with any conventional additives or excipients such as binders, disintegrants, lubricants, correctors, solubilizers, suspension aids, emulsifiers, or coatings, in addition to cyclodextrin and buffers. The dosage varies depending on the patient's symptoms, age and weight, the nature and severity of the disorder being treated or prevented, the route of administration, and the form of the drug. Generally, a daily dose of 0.01 to 2000 mg of this compound is recommended for adult patients, and this can be administered as a single dose or in divided doses. The amount of the active ingredient that can be combined with the carrier material to produce a single-dose formulation is generally the amount of the compound that produces the therapeutic effect. Generally, compositions intended for parenteral use (e.g., intravenous, subcutaneous injection) contain substituted cyclodextrins. Compositions administered via other routes, particularly the oral route, contain substituted or unsubstituted cyclodextrins.

[0218] The precise timing and / or dosage of a composition that yields the most effective results in terms of treatment efficacy in a given patient will vary depending on the activity, pharmacokinetics, and bioavailability of the particular compound, the patient's physiological conditions (e.g., age, sex, type and stage of disease, overall physical condition, response to a given dose, and type of drug), and the route of administration. However, the above guidelines may be used as a standard for fine-tuning the treatment (e.g., to determine the optimal timing and / or dosage), which may only require routine experimentation consisting of monitoring the patient and adjusting the dosage and / or timing.

[0219] The term "pharmaceutically acceptable" is used herein to mean a ligand, material, composition and / or dosage form that, within the bounds of sound medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or difficulties, and that is commensurate with a reasonable benefit / risk ratio.

[0220] As used herein, the phrase “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating agent. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient. Some examples of materials that may function as a pharmaceutically acceptable carrier include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch, potato starch, and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, etc. (10) Oils such as leaf oil, corn oil and soybean oil; (11) Glycols such as propylene glycol; (12) Polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; (13) Esters such as ethyl oleate and ethyl laurate; (14) Agar; (15) Buffers such as magnesium hydroxide and aluminum hydroxide; (16) Alginic acid; (17) Water from which pyrogenic substances have been removed; (18) Isotonic saline solution; (19) Ringer's solution; (10) Ethyl alcohol; (11) Phosphate buffer; and (21) Other non-toxic compatible substances used in pharmaceutical formulations. In certain embodiments, the pharmaceutical compositions provided herein are non-pyrogenic, i.e., do not induce a significant rise in temperature when administered to a patient.

[0221] The term "pharmaceutically acceptable salt" refers to inorganic and organic acid addition salts of inhibitors that have relatively low toxicity. These salts can be prepared in situ during the final isolation and purification of the inhibitor, or by reacting purified peptide proteasome inhibitors in free base form with a suitable organic or inorganic acid separately, and then isolating the resulting salt. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, lauryl sulfonate, and amino acid salts. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).

[0222] In some embodiments, the peptide proteasome inhibitors provided herein may contain one or more acidic functional groups, and thus it is possible to form pharmaceutically acceptable salts having pharmaceutically acceptable bases. In such cases, the term “pharmaceutically acceptable salt” refers to inorganic and organic base addition salts of the inhibitor that have relatively low toxicity. These salts may also be prepared in situ during the final isolation and purification of the inhibitor, or they may be prepared by separately reacting the purified inhibitor in its free acid form with a suitable base such as a pharmaceutically acceptable metal cation hydroxide, carbonate, or bicarbonate, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Typical alkali salts or alkaline earth salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, and aluminum salts. Typical organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, and piperazine (see, for example, Berge et al. above).

[0223] Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the composition.

[0224] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, e.g., ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite; (2) oil-soluble antioxidants, e.g., ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol; and (3) metal chelating agents, e.g., citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid.

[0225] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (flavored base ingredients, usually sucrose and acacia or tragacanth), powders, granules, each containing a predetermined amount of the inhibitor as an active ingredient, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a lozenge (using an inactive matrix, e.g., gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash. The composition may also be administered as a bolus, lick, or paste.

[0226] In solid dosage forms for oral administration (capsules, tablets, pills, sugar-coated pills, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or bulking agents such as starch, cyclodextrin, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) binders such as glycerol. (4) Humectants; disintegrants such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution retarders such as paraffin; (6) absorption enhancers such as quaternary ammonium compounds; (7) wetting agents such as acetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) colorants. In the case of capsules, tablets, and pills, the pharmaceutical composition may also include buffers. Similar types of solid compositions may also be used as fillers in soft and hard filled gelatin capsules with excipients such as lactose and high molecular weight polyethylene glycol.

[0227] Tablets may be prepared by compression or molding, using one or more auxiliary components as needed. Compressed tablets may be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked carboxymethylcellulose sodium), a surfactant, or a dispersant. Molded tablets may be prepared by molding a mixture of powdering inhibitors moistened with an inert liquid diluent in a suitable machine.

[0228] Tablets and other solid dosage forms, such as sugar-coated pills, capsules, tablets, and granules, may be notched as needed, or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation field. These may also be formulated to provide sustained or controlled release of the active ingredient within them, for example, using various proportions of hydroxypropyl methylcellulose, other polymer matrices, liposomes, and / or microspheres to provide a desired release profile. These may be sterilized, for example, by filtration using a bacterial-retaining filter, or by incorporating a sterilizer in the form of a sterile solid composition that can be dissolved immediately before use in sterile water or any other injectable sterile medium. These compositions may also optionally contain opacifiers and may optionally release only the active ingredient to a specific portion of the gastrointestinal tract in a delayed manner, or preferentially release the active ingredient. Examples of embedding compositions that may be used include polymer substances and waxes. The active ingredient may also be in microencapsulated form, where appropriate, containing one or more of the above excipients.

[0229] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may contain, for example, water or other solvents, solubilizers, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, as well as mixtures thereof, which are commonly used in the art.

[0230] In addition to inert diluents, oral compositions may also contain auxiliary agents such as humectants, emulsifiers and suspending agents, sweeteners, flavoring agents, colorants, fragrances, and preservatives.

[0231] In addition to the activity inhibitor, the suspension may contain suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

[0232] Formulations for rectal or vaginal administration may be provided as suppositories, which can be prepared by mixing one or more inhibitors with one or more suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol, suppository wax, or salicylates, which are solid at room temperature but liquid at body temperature and therefore melt in the rectal or vaginal cavity to release the activator.

[0233] Suitable formulations for vaginal administration include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing carriers known to be suitable in the art.

[0234] Dosage forms for topical or transdermal administration of inhibitors include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active ingredient may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives, buffers, or propellants.

[0235] In addition to inhibitors, ointments, pastes, creams, and gels may contain excipients such as animal fats and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0236] Powders and sprays may contain, in addition to the inhibitor, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures thereof. Sprays may further contain conventional propellants such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons such as butane and propane.

[0237] Peptide proteasome inhibitors can be administered by aerosol. This can be achieved by preparing an aqueous aerosol, liposome preparation, or solid particles containing the composition. Non-aqueous (e.g., fluorocarbon propellant) suspensions may be used. In some embodiments, sonic sprayers are preferred because they minimize the exposure of the drug to shear that can lead to the degradation of the compound.

[0238] Typically, aqueous aerosols are prepared by compounding an aqueous solution or suspension of a drug with conventionally pharmaceutically acceptable carriers and stabilizers. These carriers and stabilizers vary depending on the requirements of the specific composition, but typically include nonionic surfactants (Tween, Pluronic, sorbitan esters, lecithin, Cremophor), pharmaceutically acceptable cosolvents such as polyethylene glycol, harmless proteins such as serum albumin, amino acids such as sorbitan esters, oleic acid, lecithin, and glycine, buffers, salts, sugars, or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

[0239] Transdermal patches offer the additional advantage of providing controlled delivery of inhibitors to the body. These dosage forms can be prepared by dissolving or dispersing the drug in a suitable medium. Absorption enhancers can also be used to increase the flow of inhibitors across the skin. The rate of this flow can be controlled either by providing a rate-controlled membrane or by dispersing the inhibitor in a polymer matrix or gel.

[0240] A pharmaceutical composition suitable for parenteral administration includes one or more peptide proteasome inhibitors in combination with one or more pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostatic agents, solutes that make the formulation isotonic with the blood of the target recipient, or suspensions or thickeners.

[0241] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions provided herein include water for injection (e.g., sterile water for injection), ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), buffers (e.g., citrate buffers), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0242] Pharmaceutical compositions typically contain a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes buffers, sterile water for injection, solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are suitable for drug administration. In some embodiments, the pharmaceutically acceptable carrier is a buffer (e.g., citrate buffer). In some embodiments, the pharmaceutically acceptable carrier is sterile water for injection. In some embodiments, the pharmaceutically acceptable carrier contains citrate.

[0243] These compositions may also contain auxiliary agents such as preservatives, humectants, emulsifiers, and dispersants. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, and phenol sorbate, may be included to ensure prevention of microbial activity. It is also desirable to include tonicity modifiers such as sugars in the composition. Furthermore, absorption-delaying agents such as aluminum monostearate and gelatin may be included to prolong the absorption of the injectable drug.

[0244] In some cases, it is desirable to delay the absorption of a drug via subcutaneous or intramuscular injection in order to prolong its effects. For example, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0245] Injectable depot formulations can be prepared by forming a microcapsule matrix of the inhibitor within a biodegradable polymer such as polylactide-polyglycolide. The rate of drug release can be adjusted depending on the ratio of drug to polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydrous). Injectable depot formulations can also be prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues.

[0246] Drug preparations can be administered orally, parenterally, topically, or rectally. Naturally, these are administered in forms suitable for each route of administration. For example, they can be administered by injection, inhalation, eye drops, ointments, suppositories, or infusions in the form of tablets or capsules; topically by lotions or ointments; or rectally by suppositories. In some embodiments, administration is orally.

[0247] As used herein, the phrases “parenteral administration” and “administered parenterally” mean administration by means of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous injection, intramuscular injection, intra-arterial injection, intrathecal injection, intracapsular injection, intraorbital injection, intracardiac injection, intradermal injection, intraperitoneal injection, transtracheal injection, subcutaneous injection, subepidermal injection, intra-articular injection, subcapsular injection, subarachnoid injection, intraspinal injection, and intrasternal injection, as well as infusion.

[0248] As used herein, the phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “peripherally administered” mean the administration of a ligand, drug, or other substance by means other than direct administration to the central nervous system, such as subcutaneous administration, so that the ligand, drug, or other substance enters the patient’s system and is subjected to metabolism and other similar processes.

[0249] The peptide proteasome inhibitors described herein may be administered to humans and other animals for therapeutic purposes by any suitable route of administration, including oral administration, intranasal administration by spray, rectal administration, vaginal administration, parenteral administration, intracisional administration, and topical administration including buccal and sublingual administration in the form of powder, ointment, or drops.

[0250] Regardless of the route of administration selected, peptide proteasome inhibitors and / or pharmaceutical compositions provided herein, which can be used in an appropriate hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0251] The actual dosage levels of the active ingredients in the pharmaceutical compositions provided herein can be modified to obtain an amount of the active ingredient effective in achieving a desired therapeutic response for a particular patient, composition, and method of administration without causing toxicity to the patient.

[0252] The concentration of the disclosed compound in a pharmaceutically acceptable mixture varies depending on several factors including the dosage of the administered compound, the pharmacokinetic characteristics of the compound used, and the route of administration. In general, the compositions provided herein may be provided for parenteral administration as an aqueous solution containing from about 0.1% to 10% w / v of the compound disclosed herein, among other materials. A typical dosage range is from about 0.01 to about 50 mg / kg of body weight per day, administered in 1 to 4 divided doses. Each divided dose may contain the same or different compounds. The dosage will be an effective amount depending on several factors including the overall health of the patient, and the formulation of the selected compound and the route of administration.

[0253] In another embodiment, the pharmaceutical composition is an oral solution or a parenteral solution. In another embodiment, it is a freeze-dried preparation that can be reconstituted before administration. As a solid, the formulation may also include tablets, capsules, or powders.

[0254] Conjoint therapy, in which one or more other therapeutic agents are administered together with a peptide proteasome inhibitor or a pharmaceutical composition comprising a peptide proteasome inhibitor, is also provided herein. Such combined treatment can be achieved by simultaneous, sequential, or separate administration of the individual components of the treatment.

[0255] In certain embodiments, the cyclodextrin-free pharmaceutical formulation or kit provided herein can be administered in combination with one or more other proteasome inhibitors.

[0256] In certain embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered in combination with one or more chemotherapeutic agents. Suitable chemotherapeutic agents include natural products, e.g., vinca alkaloids (i.e., vinblastine, vincristine, and vinorelbine), taxanes (e.g., docetaxel, paclitaxel, e.g., docetaxel), epidipodophilotoxins (i.e., etoposide, teniposide), antibiotics (dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin; e.g., doxorubicin), anthracyclines, mitoxantrone, bleomycin, plicamycin (mitramycin), and mitomycin, enzymes (L-asparaginase, which systemically metabolizes L-asparagine and depletes cells that lack the ability to synthesize asparagine itself); antiplatelet agents; antiproliferative / antimitotic alkylating agents, e.g., nitrogen mustard (mechloretamine, ifosfamide, cyclophosphamide and its analogs, melpha) Lan, chlorambucil (e.g., melphalan), ethyleneimine and methylmelamine (hexamethylmelamine and thiotepa), alkyl sulfonates (busulfan), nitrosourea (carmustine (BCNU) and its analogs, streptozocin), trazene-dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites, e.g., folate analogs (methotrexate), pyrimidine analogs (fluorouracil, phloxuridine, and cytarabine), purine analogs and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine); aromatase inhibitors (anastrozole, exemestane, and letrozole); platinum-coordinate complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; DNA binding / cytotoxic agents (e.g., Zalypsis);Histone deacetylase (HDAC) inhibitors (e.g., trichostatin, sodium butyrate, apicidan, suberoyl anilide hydroxamate (SAHA (vorinostat)), trichostatin A, depsipeptide, apicidine, A-161906, ScriptAid, PXD-101, CHAP, butyrate, depdesin, oxamfratin, phenylbutyrate, valproic acid, MS275 (N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide), LAQ824 / LBH589, CI994, MGCD0103, ACY-1215, panobinostat); hormones (i.e., estrogens) and hormone agonists, such as luteinizing hormone-releasing hormone (LHRH) agonists (goserelin, leuprolide, and triptorelin). Other chemotherapeutic agents may include mechloretamine, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine, or any of the aforementioned analogs or derivative variants.

[0257] In certain embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered in combination with one or more histone deacetylase (HDAC) inhibitors (e.g., trichostatin, sodium butyrate, apicidan, suberoylanilide hydroxamate ("SAHA" (vorinostat)), trichostatin A, depsipeptide, apicidine, A-161906, ScriptAid, PXD-101, CHAP, butyrate, depdesin, oxamfratin, phenylbutyrate, valproic acid, MS275 (N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide), LAQ824 / LBH589, CI994, MGCD0103, ACY-1215, panobinostat; e.g., SAHA, ACY-1215, panobinostat).

[0258] In certain embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered in combination with one or more nitrogen mustards (mechloretamine, ifosfamide, cyclophosphamide and its analogues, melphalan, chlorambucil, e.g., melphalan).

[0259] In certain embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered in combination with one or more DNA-binding / cytotoxic agents (e.g., Zalypsis).

[0260] In certain embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered in combination with one or more taxanes (e.g., docetaxel, paclitaxel, e.g., docetaxel).

[0261] In certain embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered in combination with one or more antibiotics (dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin; for example, doxorubicin).

[0262] In some embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered in combination with one or more cytokines. Examples of cytokines include, but are not limited to, interferon-γ, -α, and -β, interleukin 1-8, 10, and 12, granulocyte-monocyte colony-stimulating factor (GM-CSF), TNF-α and -β, and TGF-β.

[0263] In some embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered in combination with one or more steroids. Suitable steroids include 21-acetoxypregnenolone, alclomethasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, crocortol, cloprednol, corticosterone, cortisone, cortivazole, deflazacort, desonide, desoxymethasone, dexamethasone, diflorasone, diflucortol, difluprednate, enoxolone, fluazacort, fluchloronide, flumethasone, flunisolid, fluocinolone acetonide, fluocinonide, fluocortin butyl, flucortolone, fluorometholone, fluperolone acetate, flupredniden acetate, fluprednisolone, flulandrenolide, fluticasone propionate, formocortal, and Examples of rucinonides include, but are not limited to, halobetazole propionate, halomethasone, hydrocortisone, loteprednol etavonate, mazipredone, medrisone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylaminoacetate, sodium prednisolone phosphate, prednisone, prednival, prednylidene, rimexolone, thixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexaacetonide, and their salts and / or derivatives (e.g., hydrocortisone, dexamethasone, methylprednisolone, and prednisolone; e.g., dexamethasone).

[0264] In certain embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered in combination with dexamethasone. In certain embodiments, the combination therapy includes, for example, the drug regimen provided on the following KYPROLIS label.

[0265] 1. KYPROLIS is administered intravenously for 2-10 minutes for two consecutive days each week for three weeks (days 1, 2, 8, 9, 15, and 16), followed by a 12-day rest period (days 17-28). Each 28-day period constitutes one treatment cycle (Table A).

[0266] In cycle 1, KYPROLIS was administered at 20 mg / m². 2 Administer at the dose indicated. If tolerated in cycle 1, increase the dose to 27 mg / m² in cycle 2. 2 The dosage was gradually increased to 27 mg / m² in subsequent cycles. 2 This should be maintained. Treatment may be continued until the disease progresses or unacceptable toxicity occurs.

[0267] The dosage is calculated using the patient's actual body surface area at baseline. 2.2m² 2 For patients with a body surface area exceeding 2.2m², 2 The dosage should be administered based on the body surface area. If the weight change is 20% or less, dose adjustment is not necessary.

[0268] [Table 3]

[0269] 2. Hydrate the patient to reduce the risk of nephrotoxicity and oncolytic syndrome (TLS) from KYPROLIS treatment. Maintain appropriate fluid volume levels throughout the treatment and closely monitor blood chemistry. Administer 250 mL–500 mL of normal saline or other suitable intravenous fluid before each dose of Cycle 1. Administer an additional 250 mL–500 mL of intravenous fluid as needed after KYPROLIS administration. Continue intravenous hydration as needed in subsequent cycles. Monitor the patient for fluid overload during this period.

[0270] 3. Before the full dose of KYPROLIS in Cycle 1, and at 27 mg / m² 2Prior to all KYPROLIS doses in the first cycle of dose increase, administer 4 mg of dexamethasone orally or intravenously as a pre-administration to reduce the occurrence and severity of infusion reactions. If these symptoms occur or recur in subsequent cycles, resume pre-administration of dexamethasone (4 mg orally or intravenously).

[0271] In some embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered in combination with one or more immunotherapeutic agents. Suitable immunotherapeutic agents may include, but are not limited to, MDR modifiers (verapamil, valspodar, biricodal, thaliquidal, ranikidal), cyclosporine, pomalidomide, thalidomide, CC-4047 (Actimid), lenalidomide (Revlimid), and monoclonal antibodies. Monoclonal antibodies may be unprotected or conjugated with rituximab, tositumomab, alemtuzumab, epratuzumab, ibritumomab tiuxetan, gemtuzumab ozogamisin, bevacizumab, cetuximab, erlotinib, and trastuzumab, etc. In certain embodiments, the pharmaceutical compositions provided herein are administered in combination with lenalidomide (Revlimid).

[0272] In some embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein (e.g., pharmaceutical compositions containing carfilzomib) may be administered in combination with the following: (i) One or more of the following: • One or more secondary chemotherapeutic agents (e.g., one or more HDAC inhibitors, e.g., SAHA, ACY-1215, panobinostat; one or more nitrogenous mustards, e.g., melphalan; one or more DNA-binding / cytotoxic agents, e.g., Zylapsis; one or more taxanes, e.g., docetaxel; one or more antibiotics (dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin; e.g., doxorubicin); • One or more other proteasome inhibitors (e.g., another compound of formulas (1) to (5)); one or more cytokines; one or more immunotherapeutic agents (e.g., Revlimid); one or more topoisomerase inhibitors; one or more m-TOR inhibitors; one or more protein kinase inhibitors (e.g., sorafenib); one or more CDK inhibitors (e.g., Dinaciclib); one or more KSP (Eg5) inhibitors (e.g., Array520); one or more PI13 delta inhibitors (e.g., GS-1101 PI3K); one or more dual inhibitors: PI3K delta and gamma inhibitors (e.g., CAL-130); one or more multi-kinase inhibitors (e.g., TG02); one or more PI3K delta inhibitors (e.g., TGR-1202); and (ii) one or more steroids (e.g., dexamethasone).

[0273] In other embodiments, the cyclodextrin-free pharmaceutical formulation or kit provided herein may be administered in combination with the following. (i) one of the following: one or more second chemotherapeutic agents (e.g., one or more HDAC inhibitors, such as SAHA, ACY-1215, panobinostat; one or more nitrogen mustards, such as melphalan; one or more DNA-binding / cytotoxic agents, such as Zylapsis; one or more taxanes, such as docetaxel; one or more antibiotics (dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin; e.g., doxorubicin); one or more other proteasome inhibitors (e.g., another compound of formulas (1) to (5)); one or more cytokines; one or more immunotherapeutic agents (e.g., Revlimid); one or more topoisomerase inhibitors; one or more m-TOR inhibitors; • One or more protein kinase inhibitors (e.g., sorafenib); • One or more CDK inhibitors (e.g., Dinaciclib); • One or more KSP(Eg5) inhibitors (e.g., Array520); • One or more PI13 delta inhibitors (e.g., GS-1101 PI3K); • One or more dual inhibitors: PI3K delta and gamma inhibitors (e.g., CAL-130); • One or more multi-kinase inhibitors (e.g., TG02); • One or more PI3K delta inhibitors (e.g., TGR-1202); and (ii) Dexamethasone. [Examples]

[0274] Example 1: Screening of non-aqueous and aqueous solvents In the initial solvent screening, we investigated the solubility profiles using three target CFZ-API concentrations as follows: Non-aqueous phase: (a) CFZ-API target concentration of 200 mg / ml to 250 mg / ml for screening water-miscible solvents, and (b) CFZ-API target concentration of 20 to 50 mg / ml for screening co-solvents, Aqueous phase: Target CFZ-API final concentration of approximately 2 mg / ml.

[0275] Example 1A. Screening of non-aqueous solvents a. Screening of organic water miscible solvents. Since CFZ-API is highly insoluble in water, various organic water-miscible solvents were initially screened to dissolve the CFZ-API formulation raw material powder. Table 1 shows the visual observation of solubility patterns satisfying initial CFZ-API concentrations of 200 mg / ml to 250 mg / ml in various water-miscible solvents. As shown in Table 1, test solutions 7 to 11 resulted in insolubility of CFZ-API, so only test solutions 1 to 6 proceeded to co-solvent screening.

[0276] [Table 4]

[0277] b. Screening of cosolvents Co-solvents were screened for further dilution of CFZ-API to a second target concentration of 20–50 mg / ml in the non-aqueous, water-miscible solutions identified above. Based on the results in Table 1 above, DMSO or NMP were selected as preferred water-miscible solvents. Table 2 lists the various co-solvents that were further screened for diluting CFZ-API pre-dissolved in DMSO or NMP water-miscible solvents to meet the target CFZ-API concentration of 20–50 mg / ml. Since CFZ-API contains an epoxy ketone moiety sensitive to nucleophilic attack, non-nucleophilic solvents and / or excipients were carefully selected for screening. Furthermore, solvents / excipients approved for parenteral use for intravenous (IV) and subcutaneous (SC) injection at acceptable concentrations based on FDA injection limits were also carefully selected for screening.

[0278] [Table 5]

[0279] Sample formulations were expected to exhibit lower CFZ-API recovery compared to those in which precipitated CFZ-API did not precipitate after filtration. Using placebo buffer under each condition, CFZ-API precipitated from the solution, rather than any other substance, was determined. Visual observation of samples 14–15 and 21–34 in Table 2 indicated that the presence of water resulted in CFZ-API precipitation within the target concentration range of 20 mg / ml–50 mg / ml. Samples 14–34 in Table 2 resulted in CFZ-API insolubility, and only samples 1–13 were further investigated in the subsequent aqueous solvent screening.

[0280] When diluted to 2 mg / ml with povidone and water and adjusted to pH 3, visual observation revealed that lactic acid and maleic acid combined with PEG400 and ethanol yielded the greatest solubility, which will be discussed in the next section. Furthermore, compared to maleic acid containing the formulation that precipitated after 3 days of storage at 2°C to 8°C, lactic acid containing the formulation was found to have longer-lasting CFZ-API solubility at 2°C to 8°C. The absence of lactic acid in the formulation resulted in precipitation of CFZ-API at pH 3, indicating that lactic acid not only lowers the pH but also acts as a co-solubilizer for CFZ-API.

[0281] Example 1B: Screening of aqueous solvents Water was introduced into CFZ-API in the presence of one or more solubilizers to ensure better dispersion of the compound in solution. Precipitation was observed in all samples during this step, but pH adjustment was performed using methanesulfonic acid to increase solubility and achieve a pH range of 3.0–3.1. Since most samples remained turbid after pH adjustment, the solutions were filtered through a 0.22 μm PES membrane filter, and the solubility of CFZ-API was examined on RP-HPLC. Table 3 shows the pH adjustment, visual observation after filtration, and CFZ-API solubility for various extenders and solubilizers, including water, used with CFZ-API in DMSO, PEG400, ethanol, and lactic acid to reach a CFZ-API concentration of 2 mg / ml. The results conclude that all PVP-containing formulations (conditions 3–8 in Table 3) yielded higher CFZ-API solubility compared to other formulations. Based on evidence of solubility from NMP and PVP, the inventors hypothesize that a pyrrolidone ring or other molecule having a similar structure may solubilize CFZ-API.

[0282] [Table 6]

[0283] As shown in Table 4, acids, amino acids, and other excipients including Pluronic solubilized or partially solubilized CFZ-API at a concentration of 2 mg / ml. Visual observations were performed when the pH was adjusted to approximately 3.0 with HCl, triethanolamine (TEA), or monoethanolamine (MEA). The recovery and stability of CFZ-API with these excipients are still under investigation.

[0284] [Table 7]

[0285] Example 2. Visual observation of cyclodextrin-free CFZ-API sample formulations. Carfilzomib (CFZ) is a proteasome inhibitor and active ingredient in KYPROLIS®, a lyophilized formulation for the treatment of multiple myeloma. The currently marketed formulations of KYPROLIS contain CAPTISOL®, a cyclodextrin used to enhance the solubility of CFZ-API. This invention provides a stable, cyclodextrin-free formulation for CFZ-API in aqueous solution, suitable for injection. Figure 1 shows (a) water-insoluble CFZ-API in the presence of phosphate buffer (PBS) (left vial), (b) the currently marketed KYPROLIS formulation containing CAPTISOL (center vial), and (c) the cyclodextrin-free formulation of the present invention (right vial). Each sample contains a CFZ-API concentration of 2 mg / ml. The yellow color of the cyclodextrin-free polyvinylpyrrolidone (PVP) formulation sample is partly due to the PVP cosolubilizer, while the clear solution indicates that CFZ-API dissolves in the cyclodextrin-free formulation.

[0286] [Table 8]

[0287] Example 3: Solubility modeling for defining the solvent space As shown in Tables 1, 2, and 5, CFZ-API can be dissolved in highly concentrated water-miscible solvents such as DMSO or NMP. In an effort to further understand the solvation properties of CFZ, the solvent space of solvents that can potentially solubilize CFZ was expanded beyond those tested in Table 1 using solubility parameter evaluation. In this approach, solubility (originally defined by Hildebrandt as the square root of the total cohesive energy) was calculated using the software package (Hansen Solubility Parameter / HSP), where the total parameter δ is divided into contributions from dispersibility force (δD), polarity force (δP), and hydrogen bonding force (δH) according to the following equation: δ 2 =δD 2 +δP 2 +δH 2 See https: / / www.hansen-solubility.com / HSP-science / basics.php. Using the solubility of CFZ-API in several solvents, experimentally measured with the results shown in Table 1, and from previously unpublished results, the information shown in Table 5 below was generated, identifying suitable solvents (CFZ solubility > 200 mg / ml) with a score of 1 and unsuitable solvents (CFZ solubility < 1 mg / ml) with a score of 0. Subsequently, a 3D solubility sphere was obtained, where suitable solvents are inside the sphere and unsuitable solvents are outside the sphere. Table 5 also shows the solubility parameters obtained from the HSP software, which are identified as δD (dispersibility), δP (polarity), and δH (hydrogen bond) force. The Relative Energy Distance (RED) term in the table simply refers to the ratio of the distance to the center of the sphere for each solvent divided by the radius of the sphere from the center point. The distance is obtained from the following known equation defined by Hansen. Ra 2 =4(δD1-δD2) 2 +(δP1-δP2) 2 +(δH1-δH2) 2

[0288] All solvents with a RED score close to 0 allow for good solubility with CFZ. Any number greater than 1 is considered unsuitable or less suitable for CFZ.

[0289] [Table 9]

[0290] Figure 2 shows a 3D solubility plot of solvent spheres. As shown, circles indicate solvents that are suitable or more suitable for CFZ-API as determined throughout this experiment, and squares indicate unsuitable solvents. To study the respective contributions of the dispersibility (D), polarity (P), and H-bonding (H) parameters to the overall solubility, 2D plots were drawn and the solubility parameters δD, δP, and δH were compared, as shown in Figure 3. Figure 3 shows that, based on the experimental data, δP vs. δH and δH vs. δD successfully differentiated suitable solvents from unsuitable ones, but the correlation was weaker for the comparison of δP vs. δD. This suggests that the H-bonding parameter was the major factor contributing to the overall solubility pattern of CFZ, while the dispersibility and polarity parameters played a smaller role.

[0291] Table 5 shows that the RED scores for NMP and DMSO are close to 1, which reflects the expectation that CFZ-API would have lower solubility in these solvents. However, we were surprised to find that CFZ-API was highly soluble in these solvents. This finding suggests that H-bonding force, compared to dispersibility and polarity, plays a major role in determining the solubility of CFZ-API and, consequently, in the selection of solvents.

[0292] Based on the information shown in Table 5 and the plots in Figures 2 and 3, the inventors can carefully select potential solvents that may be effective for CFZ by selecting molecules within the HSP database that fall within the selected solubility limits. The selected limits based on the inventors' experimental results are as follows: δD = 16~19.5; δP = 5~18; and δH = 7~19.6. Using HSP software, a list of molecules falling within the specified range shown in Figure 4 was generated. This list identifies each molecule by name, CAS number, δD, δP, δH values, δHD / A term (hydrogen bonding parameter subdivided into donor and acceptor contributions), and boiling point. Based on the details in Figure 4, the inventors can select various suitable solvents for CFZ-API within the HSP database. Furthermore, molecules not in the HSP database but with the specified δD, δP, and δH values ​​within the above ranges may be expected to provide a suitable or better solubility profile for CFZ-API.

[0293] The inventors also found that PVP assisted in the solubilization of CFZ-API when diluted from higher to lower concentrations. Using HSP software, the δD, δP, and δH values ​​of PVP were calculated as δD=21.4, δP=11.6, and δH=21.6. Since the molecular weight of a polymer can affect solubility, and molecular weights are not enumerated in this source, a wide range of δD, δP, and δH values ​​is not unexpected. Figure 5 lists molecules from a solubility database that are expected to be soluble with PVP for each of the above conditions using the following ranges: Hansen source: δD=16~24; δP=8~14; and H=17~24. Molecules with solubility parameters within the ranges specified herein are expected to solubilize PVP, but since the molecular weight of PVP also affects the solubility profile, identifying suitable molecular weights of PVP suitable for dissolving CFZ-API is also useful in selecting a suitable PVP.

[0294] Example 4: Freeze-drying cycle process, bulking agent screening, and results To achieve long-term stability, we investigated the lyophilization of the cyclodextrin-free liquid formulation of CFZ-API.

[0295] Freeze-drying process: A 1 mL fill of the CFZ-API liquid formulation prepared in a 3 mL Schott 1A glass vial as described above was loaded into a VirTis Genesis 12 EL lyophilizer (TS Systems LyoStar®-3, SP Scientific, Warminster, PA). The lyophilization cycle used for screening the extender consisted of maintaining the shelf temperature at 4°C for 30 minutes, followed by cooling the shelf to -45°C at 0.2°C / min. An annealing step was then performed from -45°C to -12°C at 0.3°C / min. The shelf temperatures for primary and secondary drying were -25°C and 25°C, respectively. The heating rates applied to primary and secondary drying were 0.2°C / min and 0.1°C / min, respectively.

[0296] Table 5 describes the results of initial freeze-drying studies of formulations containing PVP 12,000 MW, mannitol, or a bulking agent such as glycine, before and after freeze-drying. PVP was selected as a candidate co-solubilizer due to the high solubility of CFZ-API in PVP before and after freeze-drying, as described above. Since PVP is available on the market in various molecular weights and concentrations, various PVPs with 10,000 MW, 12,000 MW, and 17,000 MW, as well as concentrations ranging from 10% to 40%, were tested. The inventors discovered two solubility trends influenced by the molecular weight and concentration of PVP. First, CFZ-API was found to be more soluble in lower MW PVP. In this case, 10,000 MW PVP provided the best solubility, followed by 12,000 MW PVP, and then 17,000 MW PVP. Second, higher concentrations of PVP provided higher solubility of CFZ-API. The combination of these two trends revealed that high concentrations of low molecular weight PVP can be a suitable option for achieving maximum CFZ-API solubility. It was found that 29% 10,000 MW PVP was suitable for dissolving 2.2 mg / ml of CFZ-API, while a lower concentration of 20% 12,000 MW PVP was suitable for dissolving 2 mg / ml of CFZ-API.

[0297] [Table 10]

[0298] Results from screening of lyophilization and extenders mostly resulted in low-quality lyophilized cakes, although two selected extenders provided more visually appealing cakes in the overall formulation presentation. These two cakes (20% PVP 12,000 MW formulation and 200 mM mannitol formulation) were reconstituted with water for injection (WFI). The 20% PVP 12,000 MW formulation was reconstituted into a clear solution within 5 minutes (shown in the second column of Table 5). The recovery of CFZ-API to 1.8 mg / ml was lower than the expected 2 mg / ml, which is likely due to other components of the powder absorbing water and becoming more dilute than theoretically measured. The observed decrease in osmolality levels from before to after lyophilization is due to ethanol evaporation, as confirmed by an ethanol quantification assay. Investigating the swapping of ethanol with tert-butyl alcohol (TBA) and optimizing the freeze-drying cycle may resolve the observed trace amounts of cake disintegration.

[0299] Example 5: Stability Test and Analysis Analytical testing: The CFZ-AP in the PVP formulations prepared above was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) to accurately quantify the concentration of CFZ-API over (A) storage at 2°C to 8°C for 1 day, 2 days, and 1 week, and (B) storage at 25°C for 8 hours and 1 day. Samples of three formulation solutions, namely (i) KYPROLIS® (non-human / NHU), (ii) 2 mg / ml CAPTISOL®-free CFZ-API (consisting of 0.85% DMSO, 1.4% PEG400, 1.4% ethanol, 0.15% lactic acid, and 28.8% PVP 10K), and (iii) 2 mg / ml CAPTISOL® CFZ-API (consisting of 0.85% NMP, 1.4% PEG400, 1.4% ethanol, 0.15% lactic acid, and 28.8% PVP 10K), were compared and analyzed for stability testing.

[0300] Figures 6A and 6B show the percentage principal peaks over storage times at 2–8°C and 25°C, respectively. While there was no significant loss of the principal peak in CAPTISOL®-free CFZ-API in NMP, DMSO was found to be preferable due to lower toxicity concerns.

[0301] Example 6 - Stability analysis of a carfilzomib formulation without frozen cyclodextrin Because DMSO is highly hygroscopic, careful handling of samples is required under low humidity conditions. Furthermore, moisture-free storage containers and / or apparatus are useful for maintaining the frozen state and achieving high CFZ stability at 2°C to 8°C. Containers such as 0.5 mL microcentrifuge Eppendorf tubes and 3 cc glass Schott 1A vials equipped with lyophilization stoppers and crimp seals were investigated for the stability of CFZ in DMSO. Figure 7 shows the visual difference between frozen carfilzomib formulations in containers with and without crimp seals after 4 weeks of storage at 2°C to 8°C.

[0302] Crimp-sealed containers produced a more crystalline frozen solid formulation compared to the uncrimp-sealed frozen formulation. Eppendorf tubes kept CFZ in DMSO frozen for up to 3 weeks, after which it became liquid. Formulation stability after 4 weeks of storage at 2°C–8°C was measured by RP-HPLC, as shown in Figure 8. There was no significant loss of the percentage principal peak over the 4 weeks, suggesting that the frozen state maintained short-term stability.

[0303] Example 7: Preliminary single-dose local tolerance study in male BALB / c mice A study was conducted to measure the proteasome activity of the cyclodextrin-free 2 mg / ml CFZ-API formulation of the present invention administered subcutaneously (A) and intravenously (B) to male BALB / c mice. This study was conducted at the Charles River Laboratory, Inc. testing facility (Spencerville, OH). Both subcutaneous and intravenous exposure routes were selected because they are potential routes of human exposure.

[0304] Proteasome activity of CFZ-API administered subcutaneously to mice CFZ-AP in the PVP formulation prepared above was administered subcutaneously to mice. The test substance and control substance were administered to appropriate animals by a single subcutaneous injection into the lower flank (caudodorsal back) region on day 1. The dose volume for each animal was based on the most recent body weight measurement. Animals were temporarily restrained for dose administration and no sedatives were administered. Doses were given using a syringe fitted with a needle. The first day of administration was designated as day 1. The flank region of the animals was shaved to remove hair before the first dose. Care was taken to avoid skin abrasions during the shaving procedure. The contour of the injection site (2 cm × 2 cm) was drawn with an indestructible marker and then remarked as needed. To accurately quantify the proteasome activity (chymotrypsin-like percent (%CT-L) activity) of the formulation at 5, 10, 15, and 20 hours post-administration, the results were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). Three formulation solutions, namely (i) 2 mg / ml CAPTISOL®-free CFZ-API (consisting of 0.85% DMSO, 1.4% PEG400, 1.4% ethanol, 0.15% lactic acid, and 28.8% PVP 10K), (ii) 5 mg / ml KYPROLIS® NHU, and (iii) 16.7 mg / ml KYPROLIS® NHU, were compared and analyzed for pharmacodynamic testing.

[0305] (B) Proteasome activity of CFZ-API administered intravenously to mice CFZ-API in the PVP formulation prepared above was administered intravenously to mice, and CFZ-API in the PVP formulation prepared above was administered subcutaneously to mice. The test substance and control substance were administered to appropriate animals by a single intravenous (slow bolus) injection into the tail vein on day 1. The dose volume for each animal was based on the most recent body weight measurement. The animals were temporarily restrained for dose administration, and no sedatives were administered. Doses were given using a syringe fitted with a needle. Day 1 of administration was designated as day 1. To accurately quantify the proteasome activity (%CT-L activity) of the formulation at 5, 10, 15, and 20 hours post-administration, the results were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). Three comparative studies were conducted as follows.

[0306] [Table 11]

[0307] Figure 9 shows the proteasome activity results of the CAPTISOL®-free CFZ-API formulation and the KYPROLIS® NHU formulation of the present invention administered intravenously to mice at 2 mg / ml. For clarity, the 1.7 mg / mL CFZ-API sample was a liquid version, which was lyophilized. The lyophilized product was reconstituted to 2 mg / mL and used as a separate sample injection. It was shown that lower doses of cyclodextrin-free CFZ-API achieved the same potency as CAPTISOL®-containing CFZ-API (KYPROLIS NHU) when administered subcutaneously. This reduction in dose can be interpreted as a reduction in the toxicity exerted by CFZ-API. The CAPTISOL®-free formulation administered subcutaneously was a liquid DP containing a 10,000 mw PVP formulation. This resulted in lower percentage proteasome activity or higher proteasome inhibition compared to KYPROLIS® NHU containing and without hyaluronidase.

[0308] Figure 10 shows the proteasome activity results of CAPTISOL®-free CFZ-API and KYPROLIS® NHU administered intravenously to mice at 2 mg / ml. Compared to KYPROLIS® NHU, the CAPTISOL®-free formulation containing 28.8% PVP 10,000 mw showed the highest proteasome inhibition. The lyophilized PVP 12,000 mW formulation showed slightly lower proteasome inhibition, but this is still within an acceptable range and within the standard deviation of currently marketed KYPROLIS®.

[0309] Animal studies observed significantly higher pharmacodynamics in mice derived from the cyclodextrin-free formulation of the present invention compared to the current CAPTISOL®-containing formulation. It can be hypothesized that because CAPTISOL® encapsulates CFZ-API, its bioavailability characteristics are impaired or less optimal than those of cyclodextrin-free CFZ-API. Similarly, additives or co-solubilizers (e.g., PVP, DMSO, NMP, etc.) may improve the bioavailability of CFZ-API in cyclodextrin-free formulations.

[0310] Other Embodiments This disclosure should be read in conjunction with its detailed description, but it should be understood that the foregoing description is intended to be illustrative and not to limit the scope of this disclosure as defined by the attached claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. (i) 【Chemistry 1】 Carfilzomib, which has the chemical structure of ; or a pharmaceutically acceptable salt thereof; (ii) A solvent system comprising a pharmaceutically acceptable solvent suitable for injection, selected from the group consisting of dimethyl sulfoxide, N-methyl-2-pyrrolidone, dimethylacetamide, or ethyl lactate; optionally in the presence of a first co-solubilizer, C １～４ A cosolvent system comprising an alkyl alcohol and polyethylene glycol; and an aqueous solution having a pH of 3.0 to 3.5 in the presence of a second cosolubilizer comprising polyvinylpyrrolidone (PVP), mannitol, or glycine, or any combination thereof; A cyclodextrin-free pharmaceutical composition containing, If the second co-solubilizing agent contains PVP, the PVP has a molecular weight of 17,000 MW or less and is present at a concentration of 20% or more. The composition is in the form of a ready-to-use injectable preparation, or a composition for preparing a freeze-dried powder or cake; The aforementioned injectable agent is a composition administered intravenously or subcutaneously.

2. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the solvent is dimethyl sulfoxide, N-methyl-2-pyrrolidone, or dimethylacetamide.

3. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the cosolvent system is optionally a mixture of ethanol and polyethylene glycol, or a mixture of tert-butyl alcohol and polyethylene glycol, in the presence of the first cosolubilizer.

4. The cyclodextrin-free pharmaceutical composition according to claim 3, wherein the cosolvent system is 75% to 92% PEG400:ethanol (1:1, w / w) in the absence of the first cosolubilizer.

5. The aforementioned cosolvent system may be an acid, ester, organic salt, organic base, or C １～４ The cyclodextrin-free pharmaceutical composition according to claim 3, which is a mixture of ethanol and PEG400 in the presence of the first co-solubilizing agent selected from alkyl alcohols.

6. The cyclodextrin-free pharmaceutical composition according to claim 3, wherein the first co-solubilizing agent is an acid or ester selected from lactic acid, maleic acid, citric acid, benzoic acid, benzenesulfonic acid, acetic acid, or coconut fatty acid sucrose.

7. The cyclodextrin-free pharmaceutical composition according to claim 3, wherein the cosolvent system comprises an organic salt selected from benzalkonium chloride or protamine sulfate.

8. The cyclodextrin-free pharmaceutical composition according to claim 3, wherein the first co-solubilizing agent is ethanolamine or isopropyl alcohol.

9. The aforementioned cosolvent system is 75%–92% PEG400: Ethanol (1:1, w / w); PEG400: 1.2% to 5% lactic acid in ethanol; PEG400: 1.2% to 5% maleic acid in ethanol; PEG400: 4.6% benzalkonium chloride in ethanol; PEG400: 1% to 3.3% protamine sulfate in ethanol; PEG400: 28%–30% HS Solutol 15 in ethanol; PEG400: 32% coconut fatty acid sucrose in ethanol; PEG400: 5% benzoic acid in ethanol; PEG400: 5% benzenesulfonic acid in ethanol; PEG400: 10% isopropyl alcohol in ethanol; PEG400: 1% to 5% citric acid in ethanol; PEG400: 1.2% to 5% acetic acid in ethanol; and PEG400: 5% ethanolamine in ethanol A cyclodextrin-free pharmaceutical composition according to claim 3, selected from the group consisting of the following.

10. The cyclodextrin-free pharmaceutical composition according to claim 3, wherein the cosolvent system is PEG400: 1.2% to 5% lactic acid in ethanol.

11. The cyclodextrin-free pharmaceutical composition according to claim 6, wherein the ratio of lactic acid to carfilzomib is 1.5:2 by weight.

12. The cyclodextrin-free pharmaceutical composition according to claim 6, wherein the ratio of lactic acid to carfilzomib is 0.4:2 by weight.

13. The cyclodextrin-free pharmaceutical composition according to claim 10, wherein the final maximum lactic acid concentration is 0.15%.

14. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the second co-solubilizing agent is polyvinylpyrrolidone (PVP).

15. The cyclodextrin-free pharmaceutical composition according to claim 14, wherein the PVP has a molecular weight range of 3,000 MW to 17,000 MW.

16. The cyclodextrin-free pharmaceutical composition according to claim 15, wherein the PVP has a molecular weight range of 10,000 MW to 17,000 MW.

17. The cyclodextrin-free pharmaceutical composition according to claim 14, wherein the PVP has a molecular weight of 10,000 MW.

18. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the PVP is selected from the group consisting of 10,000 MW of 24% PVP, 10,000 MW of 29% PVP, 12,000 MW of 20% PVP, 12,000 MW of 24% PVP, or 17,000 MW of 24% PVP.

19. A cyclodextrin-free pharmaceutical composition according to claim 1, comprising 0.75% to 1% dimethyl sulfoxide, 1.0% to 1.8% PEG400, 1.0% to 1.8% ethanol, 0.10% to 0.25% lactic acid, and 20% to 30% PVP 10,000 MW, wherein the concentration of carfilzomib is 2 mg / ml.

20. A cyclodextrin-free pharmaceutical composition according to claim 1, comprising 0.2% to 2% dimethyl sulfoxide, 0.5% to 2.5% PEG400, 0.5% to 2.5% ethanol, 0.05% to 0.5% lactic acid, and 20% to 40% PVP 10,000 MW, wherein the concentration of carfilzomib is 2 mg / ml.

21. Dimethyl sulfoxide in concentrations of 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, or 1.2%; PEG400 in concentrations of 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6%; Ethanol in concentrations of 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6%; 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, and 0.35% lactic acid; The cyclodextrin-free pharmaceutical composition according to claim 1, comprising approximately 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, and 33% of PVP 10,000 MW, wherein the concentration of carfilzomib is 2 mg / ml.

22. A cyclodextrin-free pharmaceutical composition according to claim 1, comprising approximately 0.85% dimethyl sulfoxide, approximately 1.4% PEG400, approximately 1.4% ethanol, approximately 0.15% lactic acid, and approximately 28.8% PVP 10,000 MW, wherein the concentration of carfilzomib is 2 mg / ml.

23. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the composition has a solution osmolality of 200 mOsmo to 600 mOsmo.

24. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the composition has a solution osmolality of 250 mOsmo to 400 mOsmo.

25. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the composition has a solution osmolality of 280 mOsmo to 320 mOsmo.

26. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the composition has a solution osmolality of 280, 290, 300, 310, or 320 mOsmo.

27. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the composition is in the form of a ready-to-use injectable preparation.

28. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the composition is a composition for preparing freeze-dried powder or cake.

29. The cyclodextrin-free pharmaceutical composition according to claim 28, wherein the freeze-dried powder or cake can be reconstituted in less than 5 minutes.

30. (i) (a) Carfilzomib or its salt (b) Dimethyl sulfoxide, C １～４ Alkyl alcohols, polyethylene glycol, and lactic acid, (c) Polyvinylpyrrolidone (PVP) or mannitol A product vial pharmaceutical composition obtained as a stable freeze-dried powder or cake, and (ii) Reconstituted vial composition containing sterile water A carfilzomib injection kit containing, If the product vial pharmaceutical composition contains PVP, the PVP has a molecular weight of 17,000 MW or less, and the product vial pharmaceutical composition contains 20% or more PVP. A kit wherein the pharmaceutical composition is cyclodextrin-free, and the injectable preparation is administered intravenously or subcutaneously.

31. The kit according to claim 30, wherein the PVP has a molecular weight in the range of 3,000 MW to 17,000 MW.

32. The kit according to claim 30, wherein the PVP is 24% PVP 10,000 MW, 29% PVP 10,000 MW, 20% PVP 12,000 MW, 24% PVP 12,000 MW, or 24% PVP 17,000 MW.

33. The kit according to claim 30, wherein the PVP is 20% PVP 12,000 MW or 24% PVP 12,000 MW.

34. The kit according to claim 30, wherein the PVP is 20% PVP 12,000 MW.

35. The kit according to claim 30, wherein the ratio of lactic acid to carfilzomib is 1.5:2 by weight.

36. The kit according to claim 30, wherein the ratio of lactic acid to carfilzomib is 0.4:2 by weight.

37. The kit according to claim 30, wherein the injectable agent is administered intravenously.

38. The kit according to claim 30, wherein the injectable agent is administered subcutaneously.

39. A process for preparing cyclodextrin-free carfilzomib lyophilized powder or cake that is suitable for injection when reconstituted, (a) The carfilzomib or a pharmaceutically acceptable salt thereof is dimethyl sulfoxide, C １～４ A step comprising dissolving a mixture of alkyl alcohol and polyethylene glycol in lactic acid to form a solution, wherein the concentration of carfilzomib or its salt is in the range of 20 mg / ml to 50 mg / ml, (b) A step of diluting the carfilzomib solution with an acidic aqueous solution of polyvinylpyrrolidone (PVP) or mannitol having a pH of 2.5 to 4.5 to form a solution, wherein the concentration of the carfilzomib or its salt is in the range of 1 mg / ml to 3 mg / ml, and if the acidic aqueous solution contains PVP, the PVP has a molecular weight of 17,000 MW or less, and the solution formed in step (b) contains 20% or more of the PVP. A process comprising (c) freeze-drying the solution obtained in step (b).

40. The process for preparing cyclodextrin-free calfilzomib freeze-dried powder or cake according to claim 39, wherein the PVP has a molecular weight in the range of 3,000 MW to 17,000 MW.

41. A process for preparing cyclodextrin-free calfilzomib freeze-dried powder or cake according to claim 39, wherein the PVP is 10,000 MW of 24% PVP, 10,000 MW of 29% PVP, 12,000 MW of 20% PVP, 12,000 MW of 24% PVP, or 17,000 MW of 24% PVP.

42. A process for preparing cyclodextrin-free calfilzomib freeze-dried powder or cake according to claim 39, wherein the PVP is 20% PVP 12,000 MW or 24% PVP 12,000 MW.

43. A process for preparing cyclodextrin-free calfilzomib freeze-dried powder or cake according to claim 39, wherein the PVP is 20% PVP 12,000 MW.

44. A process for preparing cyclodextrin-free calfilzomib freeze-dried powder or cake according to claim 39, wherein the pH of the acidic aqueous solution in step (b) is in the range of 3.0 to 3.

5.

45. A process for preparing cyclodextrin-free carfilzomib freeze-dried powder or cake according to claim 39, wherein the solution formed in step (b) contains 0.75% to 1% dimethyl sulfoxide, 1.0% to 1.8% PEG400, 1.0% to 1.8% ethanol, 0.10% to 0.25% lactic acid, and 20% to 30% PVP 10,000 MW, and the concentration of carfilzomib is 2 mg / ml.

46. A process for preparing cyclodextrin-free carfilzomib freeze-dried powder or cake according to claim 39, wherein the solution formed in step (b) contains 0.2% to 2% dimethyl sulfoxide, 0.5% to 2.5% PEG400, 0.5% to 2.5% ethanol, 0.05% to 0.5% lactic acid, and 20% to 40% PVP 10,000 MW, and the concentration of carfilzomib is 2 mg / ml.

47. The solution formed in step (b) contains 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, 1.15%, or 1.2% dimethyl sulfoxide; PEG400 in concentrations of 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6%; Ethanol in concentrations of 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, and 1.6%; 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, and 0.35% lactic acid; A process for preparing cyclodextrin-free carfilzomib lyophilized powder or cake according to claim 39, comprising approximately 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, and 33% of PVP 10,000 MW, wherein the concentration of carfilzomib is 2 mg / ml.

48. A process for preparing cyclodextrin-free carfilzomib lyophilized powder or cake according to claim 39, wherein the solution formed in step (b) contains about 0.85% dimethyl sulfoxide, about 1.4% PEG400, about 1.4% ethanol, about 0.15% lactic acid, and about 28.8% PVP 10,000 MW, and the concentration of carfilzomib is 2 mg / ml.

49. The process for preparing cyclodextrin-free calfilzomib freeze-dried powder or cake according to claim 39, wherein the solution formed in step (b) has a solution osmolality of 200 mOsmo to 600 mOsmo.

50. The process for preparing cyclodextrin-free calfilzomib freeze-dried powder or cake according to claim 39, wherein the solution formed in step (b) has a solution osmolality of 250 mOsmo to 400 mOsmo.

51. The process for preparing cyclodextrin-free calfilzomib freeze-dried powder or cake according to claim 39, wherein the solution formed in step (b) has a solution osmolality of 280 mOsmo to 320 mOsmo.

52. The process for preparing cyclodextrin-free calfilzomib freeze-dried powder or cake according to claim 39, wherein the solution formed in step (b) has a solution osmolality of 280, 290, 300, 310, or 320 mOsmo.

53. A process for preparing cyclodextrin-free carfilzomib freeze-dried powder or cake according to claim 39, wherein in step (b), the concentration of the carfilzomib or the salt thereof is 2 mg / ml.

54. A process for preparing cyclodextrin-free carfilzomib freeze-dried powder or cake according to claim 39, wherein in step (b), the ratio of lactic acid to carfilzomib is 1.5:2 by weight.

55. A process for preparing cyclodextrin-free carfilzomib freeze-dried powder or cake according to claim 39, wherein in step (b), the ratio of lactic acid to carfilzomib is 0.4:2 by weight.

56. A process for preparing cyclodextrin-free carfilzomib lyophilized powder or cake according to claim 39, which is suitable for intravenous administration when reconstituted.

57. A process for preparing cyclodextrin-free carfilzomib lyophilized powder or cake according to claim 39, which is suitable for subcutaneous administration when reconstituted.

58. A cyclodextrin-free pharmaceutical composition according to any one of claims 1 to 29, for treating multiple myeloma in subjects requiring treatment for multiple myeloma.

59. A kit according to any one of claims 30 to 38 for treating multiple myeloma in a patient requiring treatment for multiple myeloma.

60. A cyclodextrin-free pharmaceutical composition according to any one of claims 1 to 29 for treating solid tumors in subjects requiring treatment of solid tumors.

61. A kit according to any one of claims 30 to 38 for treating solid tumors in subjects requiring treatment of solid tumors.

62. The cyclodextrin-free pharmaceutical composition according to claim 58 or 60, wherein the treatment further comprises simultaneous, sequential, or individual administration of a therapeutically effective amount of a chemotherapeutic agent.

63. The kit according to claim 59 or 61, wherein the treatment further comprises simultaneous, sequential, or individual administration of therapeutically effective amounts of chemotherapeutic agents.

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