Stable cyclodextrin-free carfilzomib formulations

Abstract

To provide a stable cyclodextrin-free chlorobutanol 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.SOLUTION: A cyclodextrin-free pharmaceutical composition comprising: (i) carfilzomib, or a pharmaceutically acceptable salt thereof; (ii) a solvent system comprising a pharmaceutically acceptable organic solvent suitable for injection which is a mixture of DMSO and chlorobutanol to completely dissolve the carfilzomib; and (iii) a bulking agent and optionally an excipient; wherein the composition is a ready-to-use injection or a pre-lyophilized formulation; wherein the injection is administered intravenously or subcutaneously. Such formulations, kits and methods substantially improve the solubility and stability of carfilzomib in aqueous solutions and facilitate both their manufacture and administration.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 959,829, filed January 10, 2020, which is incorporated herein by reference in its entirety.

[0002] The present disclosure provides stable cyclodextrin-free chlorobutanol 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 improve the solubility and stability of carfilzomib in aqueous solutions, facilitating both their manufacture and administration. [Background technology]

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

[0004] Carfilzomib is commercially available under the name Kyprolis® in single-dose vials containing either 30 mg or 60 mg of the active ingredient. In addition to lyophilized carfilzomib, each vial also contains sulfobutyl ether beta-cyclodextrin, citric acid, and sodium hydroxide for pH adjustment (target pH 3.5).

[0005] Efforts have been made to obtain improved carfilzomib compositions. For example, substituted cyclodextrin additives have been explored to promote the solubility of the active ingredient. However, the high cost and limited availability of substituted cyclodextrins limit their use in pharmaceutical compositions. Summary of the Invention [Problem to be solved by the invention]

[0006] Carfilzomib has very poor water solubility, is pH and concentration sensitive, and has an epoxide ring that is sensitive to nucleophilic attack, all of which present many challenges to preparing stable formulations of carfilzomib without the use of cyclodextrins. There remains a need for improved formulations of carfilzomib that have improved ease of manufacturing, means of administration, and stability over time. There remains a need for formulations that are easy for healthcare providers to prepare and administer. There remains a need for cyclodextrin-free carfilzomib formulations that have improved stability over time, especially when stored under ambient conditions.

[0007] It is an object of the present invention to provide a stable, ready-to-use or ready-to-dilute cyclodextrin-free carfilzomib formulation.

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

[0009] Another object of the present invention is to provide a process for the preparation of stable ready-to-use or ready-to-dilute 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 that is suitable for injection, which may be administered intravenously or subcutaneously.

[0011] It is yet another object of the present invention to provide a method for treating patients with multiple myeloma by administering a stable, ready-to-use or ready-to-dilute cyclodextrin-free carfilzomib formulation. [Means for solving the problem]

[0012] In one embodiment, the present invention provides a cyclodextrin-free pharmaceutical composition, which comprises: (i)Chemical structure: [ka] Carfilzomib with or a pharmaceutically acceptable salt thereof; (ii) a solvent system comprising a pharmaceutically acceptable organic solvent suitable for injection, which is a mixture of DMSO and chlorobutanol, to thoroughly dissolve carfilzomib; (iii) fillers and optionally excipients; Including, The composition is a ready-to-use injection or a lyophilized preformulation; the injection is administered intravenously or subcutaneously.

[0013] In embodiment 2, the present invention provides a pre-lyophilized formulation wherein said DMSO and chloro-butanol are present in a mixing ratio of 60:40 w / w, respectively.

[0014] In embodiment 3, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 or 2, which is a pre-lyophilized formulation comprising 48% chloro-butanol and 32% DMSO.

[0015] In embodiment 4, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 3, wherein the bulking agent is a sugar acid.

[0016] In embodiment 5, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 4, wherein the sugar acid is mannitol, glycine, lactic acid, or a combination thereof.

[0017] In embodiment 6, the present invention provides the cyclodextrin-free pharmaceutical composition of any one of embodiments 1 to 5, wherein the concentration of mannitol is 100 mM to 400 mM. Preferably, the concentration of mannitol is 220 mM.

[0018] In embodiment 7, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 6, which is a pre-lyophilized formulation comprising 48% chloro-butanol and 32% DMSO; and 220 nM mannitol.

[0019] In embodiment 8, the present invention provides a cyclodextrin-free pharmaceutical composition of any one of embodiments 1 to 7, which is a pre-lyophilized formulation comprising 48% chlorobutanol and 32% DMSO; 220 nM mannitol; and 0.01% polysorbate 80.

[0020] In embodiment 9, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 8, wherein the pH of the pre-lyophilized formulation is about 5-6.

[0021] In embodiment 10, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 9, wherein the pH of the solution mixture obtained after the lyophilization step is 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0.

[0022] In embodiment 11, the present invention provides a cyclodextrin-free pharmaceutical composition of any one of embodiments 1 to 10, wherein the optional excipient is selected from citrate, polysorbate 80, arginine, or any combination thereof.

[0023] In embodiment 12, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 11, wherein the optional excipient is absent.

[0024] In embodiment 13, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 12, wherein the carfilzomib concentration is 2 mg / mL.

[0025] In embodiment 14, the present invention provides the cyclodextrin-free pharmaceutical composition of any one of embodiments 1 to 13, wherein the injection is administered intravenously.

[0026] In embodiment 15, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 14, wherein the injection is administered subcutaneously.

[0027] In embodiment 16, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 15, wherein the composition is a ready-to-use injection.

[0028] In embodiment 17, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 16, obtained as a lyophilized powder or cake.

[0029] In embodiment 18, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 17, wherein the lyophilized powder or cake can be reconstituted in less than 5 minutes.

[0030] In embodiment 19, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 18, having a solution osmolality of 200 mOsmo to 600 mOsmo.

[0031] In embodiment 20, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 19, having a solution osmolality of 250 mOsmo to 400 mOsmo.

[0032] In embodiment 21, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 20, having a solution osmolality of 280 mOsmo to 320 mOsmo.

[0033] In embodiment 22, the present invention provides a cyclodextrin-free pharmaceutical composition according to any one of embodiments 1 to 21, having a solution osmolality of 280, 290, 300, 310 or 320 mOsmo.

[0034] In embodiment 23, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (i) a. combining DMSO and chloro-butanol to form a clear solution mixture and adjusting the temperature of the mixture to its freezing point; b. melting the mixture and adding a filler and optionally excipients; c. adding the carfilzomib to reach a clear solution; d. freeze-drying the solution obtained in step (c). a product vial containing a stable lyophilized powder or cake prepared by a method comprising: (ii) a reconstitution vial composition comprising sterile water; and a cyclodextrin-free carfilzomib kit suitable for injection comprising: The injections are administered intravenously or subcutaneously.

[0035] In embodiment 24, the present invention provides a carfilzomib injection kit according to embodiment 23, wherein the DMSO and chlorobutanol are present in a mixing ratio of 60:40 w / w, respectively.

[0036] In embodiment 25, the present invention provides a carfilzomib injection kit according to embodiment 23, wherein the filler is a sugar acid.

[0037] In embodiment 26, the present invention provides the carfilzomib injection kit of embodiment 23, wherein the sugar acid is mannitol or glycine, or a combination thereof.

[0038] In embodiment 27, the present invention relates to a method for manufacturing a medicament for the treatment of a pulmonary arthritis, comprising the steps of: o 25. A carfilzomib injection kit according to embodiment 24, wherein the kit is thawed at 25°C.

[0039] In embodiment 28, the present invention provides the carfilzomib injection kit of embodiment 26, wherein the concentration of the mannitol in the solution mixture in step (c) is 100 mM to 400 mM.

[0040] In embodiment 29, the present invention provides the carfilzomib injection kit of embodiment 26, wherein the concentrations of DMSO and chlorobutanol in the solution mixture in step (c) are 48% and 32%, respectively.

[0041] In embodiment 30, the present invention provides the carfilzomib injection kit of embodiment 26, wherein the concentrations of DMSO and chlorobutanol in the solution mixture in step (c) are 48% and 32%, respectively; and the concentration of mannitol in the solution mixture in step (c) is 220 mM.

[0042] In embodiment 31, the present invention provides the carfilzomib injection kit of embodiment 26, wherein the solution mixture obtained in step (c) has a pH of about 5 to 6.

[0043] In embodiment 32, the present invention provides the carfilzomib injection kit of embodiment 26, wherein the pH of the solution mixture obtained in step (d) is about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0.

[0044] In embodiment 33, the present invention provides a carfilzomib injection kit according to embodiment 26, further comprising filtering the solution obtained in step (c) in a sterile environment.

[0045] In embodiment 34, the present invention provides the carfilzomib injection kit of embodiment 24, wherein the optional excipient is selected from citrate, polysorbate 80, arginine, lactic acid, or any combination thereof.

[0046] In embodiment 35, the present invention provides a carfilzomib injection kit according to embodiment 24, wherein the optional excipient is absent.

[0047] In embodiment 36, the present invention provides the carfilzomib injection kit of embodiment 24, wherein the concentration of carfilzomib in the clear solution is 2 mg / mL.

[0048] In embodiment 37, the present invention provides a carfilzomib injection kit according to embodiment 24, wherein the injection is administered intravenously.

[0049] In embodiment 38, the present invention provides a carfilzomib injection kit according to embodiment 24, wherein the injection is administered subcutaneously.

[0050] In embodiment 39, the present invention provides the carfilzomib injection kit of embodiment 24, wherein the solution formed in step (b) has a solution osmolality of 200 mOsmo to 600 mOsmo.

[0051] In embodiment 40, the present invention provides the carfilzomib injection kit of embodiment 24, wherein the solution formed in step (b) has a solution osmolality of 250 mOsmo to 400 mOsmo.

[0052] In embodiment 41, the present invention provides the carfilzomib injection kit of embodiment 24, wherein the solution formed in step (b) has a solution osmolality of 280 mOsmo to 320 mOsmo.

[0053] In embodiment 42, there is provided a carfilzomib injection kit according to embodiment 24, wherein the solution formed in step (b) has a solution osmolality of 280, 290, 300, 310, or 320 mOsmo.

[0054] In embodiment 43, the present invention provides the carfilzomib injection kit of embodiment 24, wherein in step (b), the concentration of carfilzomib or the salt thereof is 2 mg / mL.

[0055] In embodiment 44, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising (a) combining DMSO and chlorobutanol to form a clear solution mixture and adjusting the temperature of the mixture to its freezing point; (b) melting the mixture and adding a filler and optionally excipients; (c) adding said carfilzomib while stirring to reach a clear solution; (d) freeze-drying the solution obtained in step (c). The present invention provides a method for the preparation of a cyclodextrin-free carfilzomib lyophilized powder or cake that is suitable for injection upon reconstitution, comprising:

[0056] In embodiment 45, the present invention provides the method of embodiment 44, further comprising filtering the solution obtained in step (c) in a sterile environment.

[0057] In embodiment 46, the present invention provides the method of embodiment 44, wherein the DMSO and chlorobutanol are present in a mixture ratio of 60:40 w / w, respectively.

[0058] In embodiment 47, the invention provides the method of embodiment 44, wherein the filler is a sugar acid.

[0059] In embodiment 48, the invention provides the method of embodiment 44, wherein the sugar acid is mannitol or glycine or a combination thereof.

[0060] In embodiment 49, the invention provides the method of embodiment 44, wherein the excipient is selected from citrate, polysorbate 80, arginine, lactic acid, or any combination thereof.

[0061] In embodiment 50, the present invention provides the method of embodiment 44, wherein the optional excipient is absent.

[0062] In embodiment 51, the present invention relates to a method for preparing a medicament for use in a pharmaceutical composition comprising administering to a subject the medicament ... for use in a pharmaceutical composition comprising administering to a subject the medicament for use in a pharmaceutical composition comprising o 45. The method of embodiment 44, wherein the mixture is melted at 250° C.

[0063] In embodiment 52, the present invention provides the method of embodiment 44, wherein the concentration of mannitol in the solution mixture in step (c) is 100 mM to 400 mM.

[0064] In embodiment 53, the present invention provides the method of embodiment 44, wherein the concentrations of DMSO and chlorobutanol in the solution mixture in step (c) are 48% and 32%, respectively.

[0065] In embodiment 54, the present invention provides the method of embodiment 44, wherein the concentrations of DMSO and chlorobutanol in the solution mixture in step (c) are 48% and 32%, respectively; and the concentration of mannitol in the solution mixture in step (c) is 220 mM.

[0066] In embodiment 55, the present invention provides a method according to embodiment 44, wherein the pH of the solution mixture obtained in step (c) is about 5-6.

[0067] In embodiment 56, the present invention provides the method of embodiment 44, wherein the pH of the solution mixture obtained in step (d) is about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.0.

[0068] In embodiment 57, the invention provides the method of embodiment 44, wherein the optional excipients are citrate and polysorbate 80.

[0069] In embodiment 58, the present invention provides the method of embodiment 44, wherein the concentration of carfilzomib in the clear solution is 2 mg / mL.

[0070] In embodiment 59, the present invention provides the method of embodiment 44, wherein the solution formed in step (b) has a solution osmolality of 200 mOsmo to 600 mOsmo.

[0071] In embodiment 60, the present invention provides the method of embodiment 44, wherein the solution formed in step (b) has a solution osmolality of 250 mOsmo to 400 mOsmo.

[0072] In embodiment 61, the present invention provides the method of embodiment 44, wherein the solution formed in step (b) has a solution osmolality of 280 mOsmo to 320 mOsmo.

[0073] In embodiment 62, the present invention provides the method of embodiment 44, wherein the solution formed in step (b) has a solution osmolality of 280, 290, 300, 310, or 320 mOsmo.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in this disclosure; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are merely illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0075] Other features and advantages of the present disclosure will be apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0076] [Figure 1] FIG. 1 illustrates (A) the lyophilized cake obtained from a 48% chlorobutanol, 32% DMSO, 220 mM mannitol formulation and (B) the resulting solution after reconstitution with sterile water to yield approximately 2 mg / mL CFZ. [Figure 2] FIG. 2 illustrates a visual comparison of carfilzomib, cyclodextrin, and chlorobutanol cyclodextrin-free formulations in water. [Figure 3] Figure 3 illustrates the lyophilization cycle and 25°C lyophilized cake CFZ stability in a 2 mg / mL formulation. DETAILED DESCRIPTION OF THE INVENTION

[0077] definition "C x-y The term "alkyl" refers to an unsubstituted saturated hydrocarbon group, including straight-chain and branched-chain alkyl groups containing x to y carbons in the chain.

[0078] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and their salts, such as those of the general formula: [ka] (In the formula, R 9 , R 10 and R 10’ are each independently hydrogen, alkyl, alkenyl, -(CH2) m -R 8 or R 9 and R 10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure; R 8 represents aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is 0 or an integer from 1 to 8. In some embodiments, R 9 or R 10 Only one of the groups is a carbonyl, e.g., R 9 , R 10 and nitrogen together do not form an imide. In some embodiments, R 9 and R 10 (and optionally R 10’ ) are each independently hydrogen, alkyl, alkenyl, or -(CH2) m -R 8 In certain embodiments, the amino group is basic, meaning that its protonated form has a pKa greater than 7.00.

[0079] The term "buffer" refers to a substance whose presence in a solution increases the amount of acid or alkali that must be added to produce a unit change in pH. Thus, a buffer is a substance that aids in adjusting the pH of a composition. Generally, a buffer is selected based on the desired pH and compatibility with the other components of the composition. Generally, a buffer has a pKa that is no more than one unit below or more than one unit above the desired pH of the composition (or the composition that it produces upon dissolution).

[0080] The term "CFZ" or "CFZ-API" refers to carfilzomib, which is an inhibitor of the proteasome and the active ingredient in KYPROLIS®.

[0081] The term "water," as used herein, refers to a liquid solution of H2O having a pH of approximately 7.0.

[0082] "C x-y The term "alkyl alcohol" refers to C alkyl alcohols substituted with hydroxy groups. x-y Refers to an alkyl group.

[0083] The term "substituted" refers to moieties having substituents replacing hydrogen on one or more non-hydrogen atoms of a molecule. It will be understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is subject to the permissible valences of the substituted atom and substituent, and that the substitution results in a stable compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic substituents, branched and unbranched substituents, carbocyclic and heterocyclic substituents, and aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and can be the same or different for appropriate organic compounds. For purposes of this disclosure, heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatoms. Substituents may include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that moieties substituted on the hydrocarbon chain can themselves be substituted, if desired.

[0084] As used herein, the term "peptide" refers to a chain of amino acids that is from about 2 to about 10 amino acids in length.

[0085] As used herein, the terms "natural" or "naturally occurring" amino acid refer to one of the 20 most commonly occurring amino acids. Natural amino acids are referred to by their standard one-letter or three-letter abbreviations.

[0086] The term "unnatural amino acid" or "unnatural" refers to any derivative or structural analog of a natural amino acid, including D-forms and β- and γ-amino acid derivatives. It is known that certain amino acids classified herein as unnatural amino acids, such as hydroxyproline, can be found naturally in certain organisms or proteins. Non-limiting examples of unnatural amino acids include β-alanine (β-Ala), γ-aminobutyric acid (GABA), 2-aminobutyric acid (2-Abu), α,β-dehydro-2-aminobutyric acid (Δ-Abu), 1-aminocyclopropane-1-carboxylic acid (ACPC), aminoisobutyric acid (Aib), 2-amino-thiazoline-4-carboxylic acid, 5-aminovaleric acid (5-Ava), 6-aminohexanoic acid (6-Ahx), 8-aminooctadecanoic acid (8-Ahx), 1-aminobutanoic acid (1 ... carboxylic acid (8-Aoc), 11-aminoundecanoic acid (11-Aun), 12-aminododecanoic acid (12-Ado), 2-aminobenzoic acid (2-Abz), 3-aminobenzoic acid (3-Abz), 4-aminobenzoic acid (4-Abz), 4-amino-3-hydroxy-6-methylheptanoic acid (statin, Sta), aminooxyacetic acid (Aoa), 2-aminotetralin-2-carboxylic acid (Atc), 4-amino-5-cyclohexyl Hexyl-3-hydroxypentanoic acid (ACHPA), para-aminophenylalanine (4-NH2-Phe), biphenylalanine (Bip), para-bromophenylalanine (4-Br-Phe), ortho-chlorophenylalanine (2-Cl-Phe), meta-chlorophenylalanine (3-Cl-Phe), para-chlorophenylalanine (4-Cl-Phe), meta-chlorotyrosine (3-Cl-Tyr ), para-benzoylphenylalanine (Bpa), tert-butylglycine (Tle), cyclohexylalanine (Cha), cyclohexylglycine (Chg), 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutyric acid (Dbu), 3,4-dichlorophenylalanine (3,4-Cl2-Phe), 3,4-difluorophenylalanine (3,4-F2-Phe), 3,5-diiodotyrosine (3,5-12-Tyr), ortho-fluorophenylalanine (2-F-Phe), meta-fluorophenylalanine (3-F-Phe), para-fluorophenylalanine (4-F-Phe), meta-fluorotyrosine (3-F-Tyr), homoserine (Hse), homophenylalanine (Hfe), homotyrosine (Htyr), 5-hydroxytryptophan (5-OH-Trp), hydroxyproline (Hyp), para-iodophenylalanine (4-1-Phe), 3-iodotyrosine (3-I-Tyr), indoline-2-carboxylic acid (Idc), isonipecotic acid (Inp), meta-methyltyrosine (3-Me-Tyr), I-naphthylalanine (I-Nal), 2-naphthylalanine Examples of amino acids include 2-Nal, para-nitrophenylalanine (4-NO2-Phe), 3-nitrotyrosine (3-NO2-Tyr), norleucine (Nle), norvaline (Nva), ornithine (Omithine) (Orn), ortho-phosphotyrosine (H2PO3-Tyr), octahydroindole-2-carboxylic acid (Oic), penicillamine (Pen), pentafluorophenylalanine (F5-Phe), phenylglycine (Phg), pipecolic acid (Pip), propargylglycine (Pra), pyroglutamic acid (pGlu), sarcosine (Sar), tetrahydroisoquinoline-3-carboxylic acid (Tic), and thiazolidine-4-carboxylic acid (thioproline, Th). The stereochemistry of an amino acid may be designated by prefixing the name or abbreviation with the designation "D" or "d" or "L" or "l" where appropriate. Alternatively, the asymmetric center may be designated by the conventional (S)- or (R)- designation. Additionally, αN-alkylated amino acids may be used, as may amino acids with amine-containing side chains (such as Lys and Orn) in which the amine is acylated or alkylated. See, for example, "Peptides and Mimics," by Hruby and Boteju, in Molecular Biology and Biotechnology: A Comprehensive Desk Reference, ed. Robert A. Meyers, VCH Publishers (1995), pp. 658-664.See "Conformationally Constrained Design," which is incorporated herein by reference.

[0087] The terms "prophylactic" or "therapeutic" treatment are art-recognized and include administration of one or more of the present compositions to a host. If it is administered prior to clinical manifestation of an undesired condition (e.g., a disease or other undesired condition in a host animal), the treatment is prophylactic (i.e., it protects the host from developing the undesired condition), whereas if it is administered after clinical manifestation of the undesired condition, the treatment is therapeutic (i.e., intended to reduce, ameliorate, or stabilize an existing undesired condition or its side effects).

[0088] The term "proteasome," as used herein, is intended to include immune and constitutive proteasomes.

[0089] As used herein, the term "inhibitor" describes a compound that blocks or reduces the activity of an enzyme or enzyme system, receptor, or other pharmacological target (e.g., inhibition of proteolytic cleavage of standard fluorogenic peptide substrates such as suc-LLVY-AMC, Box-LLR-AMC, and Z-LLE-AMC, inhibition of various catalytic activities of the 20S proteasome). Inhibitors may act by competitive, non-competitive, or uncompetitive inhibition. Inhibitors may bind reversibly or irreversibly; thus, the term includes compounds that are suicide substrates for the enzyme. Inhibitors may modify one or more sites on or near the active site of the enzyme, or they may cause a conformational change elsewhere in the enzyme. The term "inhibitor" is used more broadly herein than in the scientific literature, and also encompasses other classes of pharmaceutically or therapeutically useful agents, such as agonists, antagonists, stimulators, cofactors, etc.

[0090] As used herein, "low solubility" refers to, for example, sparingly soluble, sparingly soluble, very sparingly soluble, almost insoluble, or no solubility in water or another solution (e.g., the first combination); the terms "sparely soluble, sparingly soluble, very sparingly soluble, almost insoluble, or no solubility" correspond in meaning to the United States Pharmacopeia (USP) general terms for expressing approximate solubility. See, for example, DeLuca and Boylan, Pharmaceutical Dosage Forms: Parenteral Medications, vol. 1, Avis, K.E., Lackman, L., and Lieberman, H.A., eds.; Marcel Dekkar: 1084, pages 141-142.

[0091] [Table 1]

[0092] "Heterogeneous," as used herein, refers to a solution having a non-uniform (multi-phase) composition. For example, a heterogeneous solution can include a suspension (e.g., a slurry) of solid particles in a liquid.

[0093] "Homogeneous," as used herein, refers to a solution that is consistent or uniform throughout its volume (observed as a single-phase, clear solution).

[0094] A "therapeutically effective amount" of a compound for the treatment methods of the invention refers to the amount of compound in a preparation that, when administered as part of a desired dosing regimen (to a patient, e.g., a human), reduces the symptoms, ameliorates the condition, or delays the onset of a disease state according to clinically acceptable criteria for the disorder or condition being treated or for cosmetic purposes, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0095] As used herein, the term "treat" or "treatment" includes reversing, alleviating, or arresting the symptoms, clinical signs, and underlying pathologies of a condition so as to improve or stabilize the patient's condition.

[0096] The solubility of many small organic compound drugs is pH dependent. The pH range appropriate for drug administration (e.g., by injection, where the tolerable pH range for intravenous administration is generally considered to be pH 3 to pH 10.5) is often not the same pH (e.g., pH 2 or less) at which sufficient solubility of the drug may be found in aqueous solution. To allow for pharmaceutically useful concentration levels of the drug in solution in a pH range that is acceptable and tolerable for administration (e.g., by injection), the order of solvent addition and pH adjustment when aqueous solutions are introduced is a useful consideration for formulations such as those claimed herein.

[0097] For basic drug molecules, solubility typically increases with lower pH, and in some cases, stability and shelf life issues exist without the use of cyclodextrins. For example, sufficient solubility can be achieved by lowering the pH of the solution with acid, but such a decrease in pH can lead to degradation reactions from the acidic conditions. See Table 1 for specific water solubility data for carfilzomib, which shows a somewhat modest increase in solubility with decreasing pH.

[0098] [Table 2]

[0099] Many acid-mediated degradation pathways exist for small molecule drugs and biomolecules, such as hydrolysis of amides or hydrolytic cleavage of functional epoxide moieties into smaller, inactive peptide fragments. The products of acid-mediated degradation can result in loss of pharmacological activity and can be toxic or genotoxic compounds even at trace levels. Therefore, it is beneficial to completely dissolve the CFZ-API 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 at the appropriate pH.

[0100] The flow scheme for the multi-step preparation of chlorobutanol cyclodextrin-free lyophilized drug product is as follows:

number

[0101] In step 1 of the flow scheme preparation, DMSO and chlorobutanol were mixed at a weight-to-weight ratio of 60:40%, which resulted in a clear, liquid solution of the two solvents. The mixture, which demonstrated solvent miscibility, was allowed to cool and then placed at room temperature for 2 hours. The mixture was then refrigerated at 4°C for 24 hours to ensure complete solidification, then released to room temperature and subsequently thawed at 37°C. In step 2, mannitol was added to the DMSO / chlorobutanol mixture from stock to a final concentration of 220 mM. Next, in step 3, CFZ API powder was added to the DMSO / chlorobutanol / mannitol mixture to a final concentration of 2 mg / ml. After stirring for 5 minutes at room temperature, CFZ dissolution was complete. A series of additional excipients (Table 6) were added to the mixture to improve the solubility of the lyophilized cake. The solution was then filtered using a 0.22 μm PES syringe filter attached to a NORMJECT® (silicone-free) syringe. The resulting filtrate was then examined for CFZ-API solubility recovery and stability by reverse-phase high-performance liquid chromatography (RP-HPLC). RP-HPLC was used to examine peak resolution and CFZ-API recovery through the use of a 3- to 5-point reference standard curve. The Onyx method (TM-0009) was used. Peak integrations of the standards were taken against the standard buffer, 50% acetonitrile in water; whereas, peak integrations of the formulation samples were taken against the formulation buffer. In step 4 (final) of the diagram, the filtrate underwent a lyophilization step. Reconstitution of the lyophilized product with water for injection (WFI) resulted in a CFZ-API solubility of approximately 2 mg / mL in the preferred formulation of the present invention.

[0102] In addition to increasing the solubility of carfilzomib in solution, the formulations prepared by the methods provided herein result in pharmaceutical solutions with surprising stability. While the high concentrations of proteasome inhibitor achieved by the processing methods provided herein would not be expected to be thermodynamically stable, the solutions have been shown to be unaffected by storage temperatures (e.g., the solutions may be stable at 2°C to 25°C) and by lyophilization and reconstitution. The stability of the cyclodextrin-free carfilzomib formulations of the present invention is sufficient to withstand adjustments to pH after the non-aqueous phase with little or no precipitation. This solution stability allows for the use of the CFZ-API in a pH range acceptable for injection, product stability, and other pharmaceutical purposes. Thus, pharmaceutical compositions prepared by the methods provided herein may be considered, for pharmaceutical use, supersaturated solutions that do not precipitate or lose concentration to a significant extent during use in any number of medical applications (e.g., the final pharmaceutical composition may be stable for at least 1 to 5 days, and potentially longer).

[0103] In some embodiments, the first combination is substantially free of organic solvents. For example, the water in the first combination can be water for injection (WFI). In some embodiments, the first combination is substantially free of buffers (e.g., the first combination lacks a buffer acid or a buffer base).

[0104] Pharmaceutical compositions obtained as sterile products using the procedures described herein are generally manufactured using aseptic techniques and sterile filtration, followed by filling into primary packaging units (e.g., glass vials), unless the preparation does not involve a sterilization step and contamination has occurred prior to use.

[0105] Carfilzomib compositions dissolved in aqueous buffer or solution can be, for example, sterile filtered, optionally lyophilized (in a contaminant-free, preserved container), and reconstituted with an appropriate aqueous diluent immediately prior to use. In certain embodiments, a lyophilized pharmaceutical composition as provided herein, for example, contains carfilzomib, e.g., Kyprolis, which contains 60 mg carfilzomib, 3000 mg sulfobutyl ether beta-cyclodextrin, 57.7 mg citric acid, and sodium hydroxide for pH adjustment (target pH 3.5). 220 mM mannitol, 20 mM citrate.

[0106] In some embodiments, the diluent is sterile water for injection (WFI). In some embodiments, the diluent is a sterile buffer (e.g., a citrate buffer). In some embodiments, the diluent comprises citric acid. In certain embodiments, reconstitution may be performed according to the following protocol (e.g., to achieve a carfilzomib concentration of 2 mg / mL): 1. Remove the vial from the refrigerator immediately before use. 2. Aseptically reconstitute each vial by slowly injecting 29 mL of Sterile Water for Injection, USP, allowing the solution to run directly down the interior wall of the vial to minimize foaming. 3. Slowly and gently swirl and / or invert the vial for approximately 1 minute, or until all cake or powder is completely dissolved. Do not shake to avoid foam formation. If foam forms, allow the solution to rest in the vial for approximately 2-5 minutes until the bubbling subsides. 4. After reconstitution, Kyprolis is ready for intravenous administration. The reconstituted product should be a clear, colorless solution. If any discoloration or particulate matter is observed, do not use the reconstituted product. 5. If administered in an intravenous bag, withdraw the calculated dose from the vial and dilute in 50 mL of 5% dextrose, USP, intravenous bag. 6. Immediately discard any vials containing unused material.

[0107] In the compositions provided herein, one source of pH control is a buffering agent. Generally, buffering agents are present as acids or bases, their conjugate bases or acids, respectively. In one embodiment, the buffer salt ranges from 1 to 100 mM. For example, the buffer salt range can be 5 to 50 mM, e.g., about 10 mM (in solid formulations, the amount of buffering agent is selected to produce this concentration after reconstitution / dilution). The concentration of the buffering agent and the pH of the solution can be selected to provide an optimal balance of solubility and stability.

[0108] Examples of suitable buffers include mixtures of a weak acid and an alkali metal salt (e.g., sodium, potassium) of the conjugate base of the weak acid, such as sodium tartrate and sodium citrate, etc. In some embodiments, the buffer is sodium citrate / citric acid.

[0109] In addition to producing stable, highly concentrated solutions of peptide proteasome inhibitors, formulations prepared by the methods provided herein can be achieved without the chemical degradation and stability limitations of other methods of complexation and formulation. For example, the methods provided herein avoid the use of strong acids (e.g., HCl) to lower the pH during complexation. While lowering the formulation pH to a value below 2 may promote carfilzomib solubility and produce a homogeneous solution prior to complexation, the acidity of the solution may cause degradation of the peptide proteasome inhibitor. Furthermore, carfilzomib contains a ketoepoxide functional group, making the inhibitor susceptible to hydrolysis by strong nucleophilic ions, such as chloride ions. Hydrolysis of the epoxide ring and acid-catalyzed nucleophilic cleavage of the epoxide moiety are pathways for compound degradation. For example, degradation of the compound of formula (5) results in the formation of a chlorohydrin degradation product (CDP) impurity. Based on its structure, this degradant is classified as an alkylating agent and, therefore, is considered a potentially genotoxic impurity by global regulatory agencies. Furthermore, in some embodiments, chloride ions can also decompose epoxides, resulting in the formation of chlorohydrin adducts. As shown in Example 2, reducing chloride ion levels in formulations of a compound of Formula (5) can minimize or eliminate such hydrolysis pathways, thereby improving product stability and quality. However, using the methods provided herein, such strong acids and nucleophilic ions are avoided, and thus, the degradation of carfilzomib to such degradation products can be significantly reduced, and in some cases even eliminated.

[0110] Pharmaceutical compositions suitable for injection may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include sterile water for injection, sterile buffers such as citrate buffer, bacteriostatic water, and Cremophor EL™ (BASF, Parsippany, NJ). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. The composition should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0111] Sterile injection solution can be prepared by incorporating the required amount of active compound in suitable solvent with one or combination of the above-listed components, and then optionally sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and other components required from the above-listed components.For the preparation of sterile powder for sterile injection solution, the preferred method is freeze-drying (lyophilization), which obtains powder of active ingredient and any other desired components from the solution that has been previously sterilized and filtered.

[0112] How to use The biological applications of proteasome inhibition are numerous. Proteasome inhibition has been suggested as a preventative and / or treatment for many diseases, including, but not limited to, proliferative diseases, neurotoxic / degenerative diseases, Alzheimer's disease, ischemic conditions, inflammation, autoimmune diseases, HIV, cancer, organ transplant rejection, septic shock, inhibition of antigen presentation, reduction of viral gene expression, parasitic infections, acidosis-related conditions, macular degeneration, pulmonary conditions, muscle wasting diseases, fibrotic diseases, bone and hair growth disorders. Thus, pharmaceutical formulations of highly potent proteasome-specific compounds, such as molecules of the epoxyketone class, provide a means of administering drugs to patients to treat these conditions.

[0113] At the cellular level, the accumulation of polyubiquitinated proteins, cell morphological changes, and apoptosis have been reported when cells are treated with various proteasome inhibitors. Proteasome inhibition has also been suggested as a possible antitumor therapeutic strategy. The fact that epoxomicin was first identified during screening for antitumor compounds confirms the proteasome as an antitumor chemotherapy target. Therefore, these compositions are useful for the treatment of cancer.

[0114] Both in vitro and in vivo models have shown that malignant cells are generally susceptible to proteasome inhibition. Indeed, proteasome inhibition has already been validated as a therapeutic strategy for the treatment of multiple myeloma. This may be due, in part, to the dependence of highly proliferative malignant cells on the proteasome system for rapid protein clearance (Rolfe et al., J. Mol. Med. (1997) 75:5-17; Adams, Nature (2004) 4:349-360). Thus, provided herein is a method for treating cancer, comprising administering to a patient in need thereof a therapeutically effective amount of carfilzomib as provided herein.

[0115] As used herein, the term "cancer" includes, but is not limited to, hematologic and solid tumors. Cancer refers to diseases of the blood, bone, organs, skin tissue, and vascular system, including, but not limited to, cancer of the bladder, blood, bone, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidney, liver, lung, lymph node, oral cavity, cervix, ovary, pancreas, prostate, rectum, kidney, skin, stomach, testicles, throat, and uterus. Specific cancers include leukemia (acute lymphocytic 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 prolymphocytic leukemia, lymphoplasmacytic lymphoma (Waldenstrom's macroglobulinemia, etc.), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin G, etc.), and leukemia. B-cell lymphoma, cytoplasmic lymphoma, thymic lymphoma, thyroid lymphoma, thyroid cancer ... cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathic T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides (Sézary syndrome), primary cutaneous anaplastic large cell lymphoma, lymphoma Tumor papulopathy, angioimmunoblastic T-cell lymphoma, unspecified peripheral T-cell lymphoma, and undifferentiated large cell lymphoma), Hodgkin lymphoma (nodular sclerosing, mixed cell type, lymphocyte-rich, lymphopenic or non-lymphopenic, nodular lymphocyte-predominant) myeloma (multiple myeloma, low-grade myeloma, smoldering myeloma), chronic myeloproliferative disorders, myelodysplastic / myeloproliferative disorders, myelodysplastic syndromes, immunodeficiency-associated lymphoproliferative disorders, histiocytic and dendritic cell neoplasms, mastocytosis, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant giant cell tumor, myeloma-related bone disease, osteosarcoma, breast cancer (hormone-dependent, hormone-independent), gynecological cancer (cervix, endometrium, fallopian tube, gestational trophoblastic disease, ovary, 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, dysembryoplastic neuroepithelial tumor, oligodendroglioma, ependymoma, glioblastoma multiforme, mixed glioma, oligoastrocytoma, medulloblastoma, retinoblastoma, neuroblastoma, germinoma, teratoma, malignant mesothelioma (peritoneal mesothelioma, pericardial mesothelioma, pleural mesothelioma), gastro-entero-pancreatic or gastro-enteropancreatic neuroendocrine tumor (GEP-NET), carcinoid, pancreatic endocrine tumor (PET), colorectal adenocarcinoma, colorectal carcinoma, aggressive neuroendocrine tumor, leiomyosarcoma These include, but are not limited to, mucinous carcinoma, signet ring cell adenocarcinoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, hemangioma, hepatic adenoma, focal nodular hyperplasia (nodular regenerative hyperplasia, hamartoma), non-small cell lung carcinoma (NSCLC) (squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma), small cell lung carcinoma, thyroid carcinoma, prostate cancer (hormone-refractory, androgen-independent, androgen-dependent, hormone-insensitive), and soft tissue sarcomas (fibrosarcoma, malignant fibrous histiocytoma, dermatofibrosarcoma, liposarcoma, rhabdomyosarcoma / leiomyosarcoma, angiosarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor / neurofibrosarcoma, extraskeletal osteosarcoma).

[0116] In some embodiments, carfilzomib or a pharmaceutical composition comprising same as provided herein can be administered to treat multiple myeloma in a patient. For example, multiple myeloma can include refractory myeloma and / or refractory multiple myeloma.

[0117] Many tumors of hematopoietic and lymphoid tissues are characterized by increased cell proliferation or an increase in specific cell types. Chronic myeloproliferative disorders (CMPDs) are clonal hematopoietic stem cell disorders characterized by proliferation in one or more myeloid lineages in the bone marrow, resulting in increased numbers of granulocytes, red blood cells, and / or platelets in the peripheral blood. As such, the use of proteasome inhibitors for the treatment of such diseases is attractive and under investigation (Cilloni et al., Haematologica (2007) 92:1124-1229). CMPDs may include chronic myeloid leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, polycythemia vera, chronic idiopathic myelofibrosis, essential thrombocythemia, and unclassifiable chronic myeloproliferative disorder. Provided herein are methods for treating CMPDs, comprising administering to a patient in need thereof an effective amount of a proteasome inhibitor compound disclosed herein.

[0118] Myelodysplastic / myeloproliferative disorders, such as chronic myelomonocytic leukemia, atypical chronic myeloid leukemia, juvenile myelomonocytic leukemia, and unclassifiable myelodysplastic / myeloproliferative disorders, are characterized by cellular hyperplasia of the bone marrow due to proliferation of one or more of the myeloid lineages. Inhibition of the proteasome with the compositions described herein can be useful for treating these myelodysplastic / myeloproliferative disorders by providing an effective amount of the compositions to patients in need of such treatment.

[0119] Myelodysplastic syndromes (MDS) refer to a group of hematopoietic stem cell disorders characterized by dysplasia and ineffective hematopoietic development in one or more of the major myeloid cell lineages. Targeting NF-kB with proteasome inhibitors in these hematologic malignancies induces apoptosis, thereby killing malignant cells (Braun et al. Cell Death and Differentiation (2006) 13:748-758). Further provided herein are methods for treating MDS, including administering an effective amount of a compound provided herein to a patient in need of treatment for MDS. MDS include refractory anemia, refractory anemia with ringed sideroblasts, refractory cytopenias with multilineage dysplasia, refractory anemia with excess blasts, unclassifiable myelodysplastic syndrome, and myelodysplastic syndrome with isolated del(5q) chromosomal abnormality.

[0120] Mastocytosis is the proliferation of mast cells and their subsequent accumulation in one or more organ systems. Mastocytosis includes, but is not limited to, cutaneous mastocytosis, indolent systemic mastocytosis (ISM), systemic mastocytosis with clonal hematologic non-mast cell lineage disease (SM-AHNMD), aggressive systemic mastocytosis (ASM), mast cell leukemia (MCL), mast cell sarcoma (MCS), and extradermal mastocytoma. Further provided herein is a method for treating mastocytosis, comprising administering an effective amount of a compound disclosed herein to a patient diagnosed with mastocytosis.

[0121] 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 many cytokine genes encoding, for example, IL-2, IL-6, granulocyte colony-stimulating factor, and IFN-β (Palombella et al., Cell (1994) 78:773-785). Thus, provided herein are methods for affecting the expression levels of IL-2, MHC-I, IL-6, TNFα, IFN-β, or any of the other previously mentioned proteins, each method comprising administering to a patient an effective amount of a proteasome inhibitor composition disclosed herein.

[0122] Also provided herein are methods of treating an autoimmune disease in a patient, comprising administering a therapeutically effective amount of a compound described herein. An "autoimmune disease," as used herein, is a disease or disorder arising from and directed against an individual's own tissues. Examples of autoimmune diseases or disorders include inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); systemic sclerosis and sclerosis; responses involving inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions, such as eczema and asthma, and other conditions involving T-cell infiltration and chronic inflammatory responses; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile-onset diabetes; and tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis in general. These include, but are not limited to, immune responses involving acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, as seen in the immune system; pernicious anemia (Addison's disease); diseases involving leukocyte extravasation; central nervous system (CNS) inflammatory disorders; multiple organ injury syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Coombs' positive anemia); myasthenia gravis; antigen-antibody complex-mediated diseases; antiglomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathy; Reiter's disease; stiff-person syndrome; Beheet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.

[0123] The immune system screens autologous cells that are virally infected, undergo oncogenic transformation, or present unfamiliar peptides on their surface. Intracellular proteolysis generates small peptides for presentation to T-lymphocytes to induce MHC class I-mediated immune responses. Accordingly, provided herein are methods of using the proteasome inhibitors provided herein as immunomodulators to inhibit or alter antigen presentation in cells, comprising exposing the cells (or administering to a patient) to a compound described herein. Certain embodiments include methods of treating a transplant- or transplant-related disease, such as graft-versus-host disease or host-versus-graft disease, in a patient, comprising administering a therapeutically effective amount of a compound described herein. The term "graft," as used herein, refers to biological material derived from a donor for transplant into a recipient. Grafts include a variety of materials, such as isolated cells, e.g., pancreatic islet cells; tissues, such as neonatal amniotic membrane, bone marrow, blood progenitor cells, and ocular tissue, e.g., corneal tissue; and organs, such as skin, heart, liver, spleen, pancreas, thyroid lobe, lung, kidney, and tubular organs (e.g., intestine, blood vessels, or esophagus). Tubular organs can be used to replace damaged portions of the esophagus, blood vessels, or bile duct. Skin grafts can be used for burns, as well as for dressings for damaged intestine or to close certain defects, such as diaphragmatic hernias. Grafts can be from any mammalian source, including humans, whether from a cadaveric or living donor. In some cases, the donor and recipient are the same patient. In some embodiments, the graft is an organ, such as bone marrow or heart, and the graft donor and host are matched for HLA class II antigens.

[0124] Histiocytic and dendritic cell neoplasms are derived from phagocytes and accessory cells, which play a major role in processing and presenting antigens to lymphocytes. Depleting proteasome content in dendritic cells has been shown to alter their antigen-induced responses (Chapatte et al. Cancer Res. (2006) 66:5461-5468).

[0125] In some embodiments, a cyclodextrin-free pharmaceutical formulation or kit as provided herein can be administered to a patient with histiocytic or dendritic cell neoplasms, including histiocytic sarcoma, Langerhans cell histiocytosis, Langerhans cell sarcoma, interdigitating dendritic cell sarcoma / tumor, follicular dendritic cell sarcoma / tumor, and nonspecific dendritic cell sarcoma.

[0126] Inhibition of the proteasome has been shown to be beneficial for treating diseases whereby cell types are proliferating and immune disorders; thus, in some embodiments, there is provided a treatment for lymphoproliferative disorders (LPDs) associated with primary immune disorders (PIDs) comprising administering an effective amount of a disclosed compound to a patient in need thereof. The most common clinical settings of immune deficiency associated with an increased incidence of lymphoproliferative disorders, including B-cell and T-cell neoplasms and lymphomas, are primary immune deficiency syndromes and other primary immune disorders, human immunodeficiency virus (HIV) infection, iatrogenic immunosuppression in patients receiving solid organ or bone marrow allografts, and iatrogenic immunosuppression associated with methotrexate treatment. Other PIDs commonly associated with LPD include, but are not limited to, ataxia-telangiectasia (AT), Wiskott-Aldrich syndrome (WAS), common variable primary immunodeficiency (CVID), severe combined immunodeficiency (SCID), X-linked lymphoproliferative disorder (XLP), Nijmegen chromosomal break syndrome (NBS), hyper-IgM syndrome, and autoimmune lymphoproliferative syndrome (ALPS).

[0127] Proteasome inhibition has also been associated with the inhibition of NF-κB activation and stabilization of p53 levels. Therefore, the compositions provided herein can also be used to inhibit NF-κB activation and stabilize p53 levels in cell culture. NF-κB is a key regulator of inflammation and is therefore an attractive target for anti-inflammatory therapeutic intervention. Therefore, the compositions provided herein can be useful for treating conditions associated with inflammation, including, but not limited to, COPD, psoriasis, asthma, bronchitis, emphysema, and cystic fibrosis.

[0128] The disclosed compositions can be used to treat conditions directly mediated by the proteolytic function of the proteasome, such as muscle wasting, or indirectly mediated through proteins processed by the proteasome, such as NF-κB. The proteasome contributes to the rapid excretion 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 protein and regulatory proteins, such as cyclins and the transcription factor NF-κB.

[0129] In some embodiments, the compositions provided herein are intended to treat stroke, ischemic injury to the nervous system, neurotrauma (e.g., percussive brain injury, spinal cord injury, and traumatic injury to the nervous system), multiple sclerosis and other immune-mediated neuropathies (e.g., Guillain-Barré syndrome and its variants, acute motor axonal neuropathy, acute inflammatory demyelinating polyneuropathy, and Fisher syndrome), HIV / AIDS dementia complex, axonomy, diabetic neuropathy, Parkinson's disease, , Huntington's disease, multiple sclerosis, bacterial, parasitic, fungal and viral meningitis, encephalitis, vascular dementia, multi-infarct dementia, Lewy body dementia, frontal lobe dementia such as Pick's disease, subcortical dementia (such as Huntington's or progressive supranuclear palsy), focal cortical atrophy syndromes (such as primary aphasia), metabolic toxic dementia (such as chronic hypothyroidism or B12 deficiency), and dementia caused by infection (such as syphilis or chronic meningitis).

[0130] Alzheimer's disease is characterized by the extracellular deposition of β-amyloid protein (β-AP) in senile plaques and cerebral blood vessels. β-AP is a peptide fragment of 39–42 amino acids derived from the amyloid protein precursor (APP). At least three isoforms of APP are known (695, 751, and 770 amino acids). These isoforms arise through alternative splicing of mRNA; normal processing affects a portion of the β-AP sequence, thereby preventing the generation of β-AP. Aberrant protein processing by the proteasome is thought to contribute to the abundance of β-AP in Alzheimer's brains. In rats, the APP-processing enzyme contains approximately 10 distinct subunits (22–32 kDa). The 25 kDa subunit has an N-terminal sequence of X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which is identical to the β-subunit of human macrophages (Kojima, S. et al., Fed. Eur. Biochem. Soc., (1992) 304:57-60). The APP processing enzyme cleaves the Gln15-Lys16 bond; in the presence of calcium ions, this enzyme also cleaves the Met1-Asp1 and Asp1-Ala2 bonds, releasing the extracellular domain of β-AP.

[0131] Accordingly, one embodiment is a method of treating Alzheimer's disease comprising administering to a patient an effective amount of a composition provided herein, including 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.

[0132] Also provided herein are methods for treating cachexia and muscle-wasting disorders. The proteasome degrades many proteins in maturing reticulocytes and proliferating fibroblasts. In insulin- or serum-deprived cells, the rate of protein degradation nearly doubles. Inhibiting the proteasome reduces protein degradation, thereby reducing both muscle protein loss and nitrogen overload in the kidney or liver. Peptide proteasome inhibitors such as those provided herein are useful for treating conditions such as renal and hepatic failure associated with cancer, chronic infectious diseases, fever, muscle wasting (atrophy) and denervation, nerve injury, starvation, and acidosis. See, e.g., Goldberg, U.S. Pat. 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 p53 protein degradation in cells; and inhibiting the growth of p53-associated cancers. Each of these methods involves contacting a cell (in vivo or in vitro, e.g., muscle of a patient) with an effective amount of a pharmaceutical composition disclosed herein.

[0133] Fibrosis is the excessive and persistent formation of scar tissue resulting from the hyperproliferative growth of fibroblasts and is associated with activation of the TGF-β signaling pathway. Fibrosis involves the extensive deposition of extracellular matrix and can occur in virtually any tissue or across several different tissues. Normally, the levels of intracellular signaling proteins (Smads), which 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. Accordingly, in certain embodiments, methods are provided for treating hyperproliferative conditions such as diabetic retinopathy, macular degeneration, diabetic nephropathy, glomerulosclerosis, IgA nephropathy, liver cirrhosis, biliary atresia, congestive heart failure, scleroderma, radiation-induced fibrosis, and pulmonary fibrosis (idiopathic pulmonary fibrosis, collagen vascular disease, sarcoidosis, interstitial lung disease, and extrinsic lung injury). Treatment of burn victims is often hindered by fibrosis, and therefore, in some embodiments, the inhibitors provided herein can be administered locally or systemically to treat burns. Post-surgical wound closure is often accompanied by disfiguring scars, which can be prevented by inhibiting fibrosis. Thus, in certain embodiments, methods are provided herein for preventing or reducing scarring.

[0134] Another protein processed by the proteasome is NF-κB, a member of the Rel protein family. The Rel family of transcriptional activator proteins can be divided into two groups. The first group requires proteolytic processing and includes p50 (NF-κB1, 105 kDa) and p52 (NF-κB2, 100 kDa). The second group does not require proteolytic processing and includes p65 (RelA, Rel (c-Rel), and RelB). Both homo- and heterodimers can be formed by Rel family members; NF-κB, for example, is a p50-p65 heterodimer. After phosphorylation and ubiquitination of IκB and p105, these two proteins are degraded and processed, respectively, to generate active NF-κB, which translocates from the cytoplasm to the nucleus. Ubiquitinated p105 is also processed by purified proteasomes (Palombella et al., Cell (1994) 78:773-785). Active NF-κB forms stereospecific enhancer complexes with other transcriptional activators and, for example, HMG I(Y), which induce the selective expression of specific genes.

[0135] NF-κB regulates genes involved in immune and inflammatory responses and 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 many cytokine genes encoding, for example, IL-2, IL-6, granulocyte colony-stimulating factor, and IFN-β (Palombella et al., Cell (1994) 78:773-785). Some embodiments include methods of affecting the level of expression of IL-2, MHC-I, IL-6, TNFα, IFN-β, or any of the other previously mentioned proteins, each method comprising administering to a patient an effective amount of a composition disclosed herein. p50-containing complexes are rapid mediators of acute inflammation and immune responses (Thanos, D. and Maniatis, T., Cell (1995) 80:529-532).

[0136] NF-κB also contributes to 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 a cell with (or administering to a patient) an effective amount of a pharmaceutical composition disclosed herein.

[0137] Ischemia and reperfusion injury result in hypoxia, a condition in which there is a lack of oxygen reaching the body's tissues. This condition increases the degradation of Iκ-Bα, which in turn leads to the activation of NF-κB. It has been shown that the severity of the damage resulting from hypoxia can be reduced by administering a proteasome inhibitor. Thus, provided herein are methods for treating ischemic conditions or reperfusion injury, comprising administering to a patient in need thereof an effective amount of a compound disclosed herein. Examples of such conditions or injuries include, but are not limited to, acute coronary syndrome (vulnerable plaque), arterial occlusive disease (occlusion of cardiac, cerebral, peripheral arteries, and blood vessels), atherosclerosis (coronary artery sclerosis, coronary artery disease), infarction, heart failure, pancreatitis, myocardial hypertrophy, stenosis, and restenosis.

[0138] NF-κB also specifically binds to the HIV enhancer / promoter. Compared to Nef in mac239, the HIV regulatory protein Nef in pbj14 differs by two amino acids in the region controlling protein kinase binding. Protein kinases are thought to signal the phosphorylation of IκB, triggering its degradation through the ubiquitin-proteasome pathway. After degradation, NF-κB is released into the nucleus, thus promoting HIV transcription (Cohen, J., Science, (1995) 267:960). Provided herein are methods for inhibiting or reducing HIV infection and methods for reducing viral gene expression levels in a patient, each method comprising administering to the patient an effective amount of a composition disclosed herein.

[0139] Viral infection contributes to the pathology of many diseases. Cardiac conditions such as ongoing myocarditis and dilated cardiomyopathy have been linked to Coxsackievirus B3. In a comparative genome-wide microarray analysis of infected mouse hearts, specific proteasome subunits were uniformly upregulated in the hearts of mice that developed chronic myocarditis (Szalay et al., Am J Pathol 168:1542-52, 2006). Several viruses utilize the ubiquitin-proteasome system during the viral entry step, which involves release of the virus from the endosome into the cytoplasm. Mouse hepatitis virus (MHV) belongs to the coronavirus 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 a proteasome inhibitor resulted in decreased viral replication, which correlated with lower viral titers compared to untreated cells. Human hepatitis B virus (HBV), a member of the Hepadnaviridae virus family, similarly requires virally encoded envelope proteins for proliferation. Inhibition of the proteasome degradation pathway significantly reduces 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 pathogenesis. Thus, in certain embodiments, methods are provided for treating viral infections, such as SARS or hepatitis A, B, C, D, and E, comprising contacting cells (or administering to a patient) an effective amount of a compound disclosed herein.

[0140] The overproduction of lipopolysaccharide (LPS)-induced cytokines, such as TNFα, is thought to be central to the processes associated with septic shock. Furthermore, it is generally accepted that the first step in cellular activation by LPS 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). Thus, in certain embodiments, compositions as provided herein may be used for the inhibition of TNFα to prevent and / or treat septic shock.

[0141] Intracellular proteolysis generates small peptides for presentation to T-lymphocytes to induce MHC class I-mediated immune responses. The immune system screens for autologous cells that are virally infected or have undergone oncogenic transformation. One embodiment is a method for inhibiting antigen presentation in a cell, comprising exposing the cell to a composition described herein. A further embodiment is a method for suppressing a patient's immune system (e.g., inhibiting transplant rejection, allergies, asthma), comprising administering to the patient an effective amount of a composition described herein. The compositions provided herein can also be used to treat autoimmune diseases such as lupus, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel diseases, e.g., ulcerative colitis and Crohn's disease.

[0142] Another embodiment is a method for altering the repertoire of antigenic peptides produced by the proteasome or other Ntns with multiple catalytic activities. For example, if the PGPH activity of the 20S proteasome is selectively inhibited, a different set of antigenic peptides will be produced by the proteasome and presented in MHC molecules on the cell surface than would be produced and presented either without enzyme inhibition or, for example, with selective inhibition of the chymotrypsin-like activity of the proteasome.

[0143] 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 a cell with a composition described herein. A further embodiment is a method for reducing the cellular content of NF-κB in a cell, muscle, organ, or patient, comprising contacting the cell, muscle, organ, or patient with a composition described herein.

[0144] Other eukaryotic transcription factors that require proteolytic processing include general transcription factor TFIIA, herpes simplex virus VP16 accessory protein (host cell factor), virus-inducible IFN regulatory factor 2 protein, and membrane-associated sterol regulatory element-binding protein 1.

[0145] Further provided herein are methods for affecting the cyclin-dependent eukaryotic cell cycle, comprising exposing cells (in vitro or in vivo) to the compositions disclosed herein. Cyclins are proteins involved in cell cycle control. The proteasome is responsible for the degradation of cyclins. Examples of cyclins include mitotic cyclins, G1 cyclins, and cyclin B. Degradation of cyclins allows cells to exit one cell cycle stage (e.g., mitosis) and enter another (e.g., division). All cyclins are thought to associate with p34cdc2 protein kinase or related kinases. The proteolytic targeting signal is located at amino acids 42-RAALGNISEN-50 (destruction box). There is evidence that cyclins are converted to forms vulnerable to ubiquitin ligase or that cyclin-specific ligases are activated during mitosis (Ciechanover, A., Cell, (1994) 79:13-21). Inhibition of the proteasome inhibits cyclin degradation and thus inhibits cell proliferation, for example, in cyclin-associated cancers (Kumatori et al., Proc. Natl. Acad. Sci. USA (1990) 87:7071-7075). Provided herein are methods for treating a proliferative disease (e.g., cancer, psoriasis, or restenosis) in a patient, comprising administering to the patient an effective amount of a composition disclosed herein. Also provided herein are methods for treating cyclin-associated inflammation in a patient, comprising administering to the patient a therapeutically effective amount of a composition described herein.

[0146] Further embodiments include methods for affecting proteasome-dependent regulation of oncoproteins and methods for treating or inhibiting cancer growth, each comprising exposing cells (in vivo, e.g., in a patient, or in vitro) to a composition disclosed herein. The E6 proteins from HPV-16 and HPV-18 stimulate the ATP- and ubiquitin-dependent conjugation and degradation of p53 in crude reticulocyte lysates. Recessive oncogene p53, along with mutated thermolabile E1, has been shown to accumulate at nonpermissive temperatures in cell lines. Increased levels of p53 can lead to apoptosis. Examples of proto-oncoproteins degraded by the ubiquitin system include c-Mos, c-Fos, and c-Jun. One embodiment is a method for treating p53-associated apoptosis, comprising administering to a patient an effective amount of a composition disclosed herein.

[0147] In another embodiment, the disclosed compositions are useful for treating parasitic infections, such as those caused by protozoan parasites. The proteasomes of these parasites are believed to be primarily involved in cell differentiation and replication (Paugam et al., Trends Parasitol. 2003, 19(2):55-59). Furthermore, Entamoeba species have been shown to lose their ability to form encystments when exposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res. 1997, 28, Spec No: 139-140). In certain such embodiments, the disclosed compositions are directed against Plasmodium species (including P. falciparum, P. vivax, P. malariae, and P. ovale, which cause malaria), Trypanosoma species (including T. cruzi, which causes Chagas disease, and T. brucei, which causes African sleeping sickness), Leishmania species (including L. amazonesis, L. donovani, L. infantum, L. mexicana, etc.), Pneumocystis carinii, The present invention is useful for treating parasitic infections in humans caused by protozoan parasites selected from Toxoplasma gondii, Entamoeba histolytica, Entamoeba invadens, and Giardia lamblia.In certain embodiments, the disclosed compositions are useful for treating parasitic infections in animals and livestock caused by protozoan parasites selected from Plasmodium hermani, Cryptosporidium spp., Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona, and Neurospora crassa. Other compounds useful as proteasome inhibitors in the treatment of parasitic diseases are described in WO 98 / 10779, which is incorporated herein in its entirety.

[0148] In certain embodiments, the disclosed compositions irreversibly inhibit proteasome activity in parasites. Such irreversible inhibition has been shown to induce a shutdown of enzymatic activity in red and white blood cells without recovery. In certain such embodiments, the long half-life of blood cells may provide long-term protection for treatment against repeated exposure to the parasite. In certain embodiments, the long half-life of blood cells may provide long-term protection for chemoprophylaxis against future infection.

[0149] Prokaryotes have what is equivalent to the eukaryotic 20S proteasome particle. Although the subunit composition of the prokaryotic 20S particle is simpler than that of eukaryotes, it also has the ability to hydrolyze peptide bonds. For example, nucleophilic attack at the peptide bond occurs through a threonine residue at the N-terminus of the β-subunit. In some embodiments, a method of treating a prokaryotic infection is provided, comprising administering to a patient an effective amount of a proteasome inhibitor composition disclosed herein. Prokaryotic infections can include diseases caused by either mycobacteria (such as tuberculosis, leprosy, or Buruli ulcer) or archaea.

[0150] It has also been shown that inhibitors that bind to the 20S proteasome stimulate bone formation in bone organ cultures.Furthermore, when such inhibitors are systemically administered to mice, certain proteasome inhibitors increase bone volume and bone formation rate by more than 70% (Garrett, IR et al., J. Clin. Invest. (2003) 111:1771-1782), thus suggesting that the ubiquitin-proteasome mechanism regulates osteoblast differentiation and bone formation.Therefore, the disclosed compositions may be useful in the treatment and / or prevention of diseases associated with bone loss, such as osteoporosis.

[0151] Provided herein are methods for treating a disease or condition selected from cancer, autoimmune diseases, graft or transplant-related conditions, neurodegenerative diseases, fibrosis-related conditions, ischemia-related conditions, infections (viral, parasitic, or prokaryotic), and diseases associated with bone loss, comprising administering a proteasome inhibitor as provided herein, e.g., a compound of Formula (5).

[0152] Bone tissue is an excellent source of factors capable of stimulating bone cells. Thus, extracts of bovine bone tissue contain not only structural proteins that contribute to maintaining the structural integrity of bone, but also biologically active bone growth factors that can stimulate bone cells to proliferate. Among these latter factors, a family of proteins called bone morphogenetic proteins (BMPs) has recently been described. All of these growth factors have effects on other cell types as well as bone cells, including Hardy, MH, et al., Trans Genet (1992) 8:55-61, which describes evidence that bone morphogenetic proteins (BMPs) are differentially expressed in hair follicles during development. Harris, SE, et al., J Bone Miner Res (1994) 9:855-863, describes the effects of TGF-β and other substances on the expression of BMP-2 in bone cells. BMP-2 expression also occurs in mature follicles during maturation and after the cell proliferation phase (Hardy, et al. (1992) supra). Thus, the compounds provided herein may also be useful in stimulating hair follicle growth.

[0153] Finally, the disclosed compositions are also useful as diagnostic agents for screening for proteins (e.g., enzymes, transcription factors) processed by Ntn hydrolases, including the proteasome (e.g., for use in diagnostic kits or clinical tests). The disclosed compositions are also useful as test reagents for specifically binding to and inhibiting the proteolytic activity associated with the X / MB1 subunit or α-chain. For example, the activity of (and specific inhibitors of) other subunits of the proteasome can be determined.

[0154] Most cellular proteins are subject to proteolytic processing during maturation or activation. The enzyme inhibitors disclosed herein can be used to determine whether a cellular, developmental, or physiological process or outcome is regulated by the proteolytic activity of a particular Ntn hydrolase. One such method includes obtaining an organism, an intact cell preparation, or a cell extract; exposing the organism, cell preparation, or cell extract to a composition disclosed herein; exposing the compound-exposed organism, cell preparation, or cell extract to a signal; and monitoring the process or outcome. The high selectivity of the compounds disclosed herein allows for rapid and precise export or implication of Ntn (e.g., the 20S proteasome) in certain cellular, developmental, or physiological processes.

[0155] Administration The compositions prepared as described herein can be administered in various forms, depending on the disorder to be treated and the age, condition, and weight of the patient, as is well known in the art. For example, if the compositions are to be administered orally, they can be formulated as tablets, capsules, granules, powders, or syrups; or, for parenteral administration, they can be formulated as injections (intravenous, intramuscular, or subcutaneous), infusion preparations, or suppositories. For application via the ocular mucosa, they can be formulated as eye drops or eye ointments. These formulations can be prepared by conventional means in combination with the methods described herein, and the active ingredient can be mixed with any conventional additives or excipients, such as binders, disintegrants, lubricants, correctives, solubilizers, suspending aids, emulsifiers, or coating agents, in addition to cyclodextrins and buffers, as needed. Although the dosage varies depending on the patient's condition, age, and weight, the nature and severity of the disorder to be treated or prevented, the route of administration, and the drug form, a daily dosage of 0.01 to 2000 mg of the compound is generally recommended for an adult patient, which can be administered in a single dose or in divided doses. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is generally that amount of compound that produces a therapeutic effect. In general, compositions intended for parenteral use (e.g., intravenous, subcutaneous injection) contain a substituted cyclodextrin. Compositions administered via other routes, particularly oral routes, contain a substituted or unsubstituted cyclodextrin.

[0156] The exact administration time and / or amount of the composition that will achieve the most effective results in terms of efficacy of treatment in a given patient will depend on the activity, pharmacokinetics and bioavailability of the particular compound, the physiological condition of the patient (including age, sex, type and stage of disease, general health, responsiveness to a given dosage and type of medication), route of administration, etc. However, the above guidelines can be used as a basis for fine-tuning the treatment, e.g., determining the optimal time and / or amount of administration, with no more than routine experimentation consisting of monitoring the patient and adjusting the dosage and / or timing.

[0157] The phrase "pharmaceutically acceptable" is used herein to refer to ligands, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0158] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid injectable, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can act as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch, potato starch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, (10) oils such as olive 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) buffers such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water; (17) isotonic saline, (18) Ringer's solution; (19) ethyl alcohol; (20) 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 increase in body temperature when administered to a patient.

[0159] The term "pharmaceutically acceptable salts" refers to relatively non-toxic inorganic and organic acid addition salts of the inhibitors. These salts can be prepared in situ during the final isolation and purification of the inhibitors, or by separately reacting purified carfilzomib in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and lauryl sulfonate salts and amino acid salts. (See, e.g., Berge et al. (1977) "Pharmaceutical Salts," J. Pharm. Sci. 66:1-19.)

[0160] In some embodiments, the peptide proteasome inhibitors provided herein may contain one or more acidic functional groups and thus can form pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these examples refers to the relatively non-toxic inorganic and organic base addition salts of the inhibitor. These salts can also be prepared in situ during the final isolation and purification of the inhibitor, or by separately reacting the purified inhibitor in its free acid form with a suitable base, such as a hydroxide, carbonate, or bicarbonate salt of a pharmaceutically acceptable metal cation, ammonia, or a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts. Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (e.g., Berge et al., supra).

[0161] Wetting agents, emulsifying agents, and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0162] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0163] Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powder, granules, each containing a predetermined amount of the inhibitor(s) as the 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 troche (using an inert matrix, such as gelatin and glycerin or sucrose and acacia), and / or as a mouthwash, etc. The composition may also be administered as a bolus, electuary, or paste.

[0164] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, and the like), 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 fillers, such as starches, cyclodextrins, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents. (agents), 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) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be employed as injections in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar and high molecular weight polyethylene glycols.

[0165] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents, or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered inhibitor moistened with an inert liquid diluent.

[0166] Tablets and other solid dosage forms, such as dragees, capsules, pills, and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may also be formulated to provide slow or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release profile. They may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that releases the active ingredient only, or preferably in a certain part of the digestive tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if desired, with one or more of the above-mentioned excipients.

[0167] The liquid dosage form for oral administration includes pharmaceutically acceptable emulsion, microemulsion, solution, suspension, syrup and elixir.In addition to active ingredients, this liquid dosage form can contain inert diluents commonly used in the art, such as 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, oil (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 and their mixtures.

[0168] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0169] In addition to the active inhibitor, suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0170] Formulations for rectal or vaginal administration may be given as suppositories, which may be prepared by mixing one or more inhibitors with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active agent(s).

[0171] Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0172] Dosage forms for the topical or transdermal administration of the inhibitor(s) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and any preservatives, buffers, or propellants which may be required.

[0173] The ointments, pastes, creams and gels may contain, in addition to the inhibitor(s), excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide or mixtures thereof.

[0174] Powders and sprays can contain, in addition to an inhibitor(s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0175] Carfilzomib can be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation, or solid particles containing the composition. A non-aqueous (e.g., fluorocarbon propellant) suspension can be used. In some embodiments, sonic nebulizers are preferred because they minimize exposure of the drug to shear, which can cause degradation of the compound.

[0176] Typically, aqueous aerosols are prepared by formulating an aqueous solution or suspension of a drug with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary depending on the requirements of a particular composition, but generally include nonionic surfactants (Tweens, Pluronics, sorbitan esters, lecithin, Cremophor), pharmaceutically acceptable cosolvents, such as polyethylene glycol, innocuous proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars, or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

[0177] Transdermal patches have the added advantage of providing controlled delivery of the inhibitor to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the inhibitor across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the inhibitor in a polymer matrix or gel.

[0178] Pharmaceutical compositions suitable for parenteral administration comprise 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 immediately before use in sterile injectable solutions or dispersions, and may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0179] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions provided herein include water for injection (e.g., sterile water for injection), ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), buffers (such as citrate buffer), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate, etc. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0180] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes buffer solutions, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. In some embodiments, the pharmaceutically acceptable carrier is a buffer solution (e.g., citrate buffer). In some embodiments, the pharmaceutically acceptable carrier is sterile water for injection. In some embodiments, the pharmaceutically acceptable carrier includes citric acid.

[0181] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include an isotonicity adjusting agent, such as sugar, in the composition. Furthermore, agents that delay absorption, such as aluminum monostearate and gelatin, can be included to prolong the absorption of injectable medications.

[0182] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. For example, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0183] Injectable depot forms are made by forming microencapsule matrices of the inhibitor in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0184] Pharmaceutical preparations can be administered orally, parenterally, topically, or rectally. They are, of course, administered in a form appropriate for each administration route. For example, they are administered in tablet or capsule form, by injection, inhalation, eye lotion, ointment, suppository, infusion; topically by lotion or ointment; and rectally by suppository. In some embodiments, administration is oral.

[0185] The phrases "parenteral administration" and "administered parenterally", as used herein, mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

[0186] The phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally," as used herein, refer to the administration of a ligand, drug, or other substance other than directly to the central nervous system, e.g., subcutaneous administration, so that it enters the patient's system and is therefore subject to metabolism and other similar processes.

[0187] The peptide proteasome inhibitors described herein may be administered to humans and animals for therapy by any suitable route of administration, including orally, nasally, as by powder, ointment or drop, including buccal and sublingually, as by spray, rectal, vaginal, parenteral, intracisternal and topical administration.

[0188] Regardless of the route of administration selected, the peptide proteasome inhibitors and / or pharmaceutical compositions provided herein, which may be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0189] Actual dosage levels of the active ingredients in the pharmaceutical compositions provided herein may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and route of administration without toxicity to the patient.

[0190] The concentration of the disclosed compounds in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the compound to be administered, the pharmacokinetic characteristics of the compound used, and the route of administration. Generally, the compositions provided herein may be provided in an aqueous solution containing, among other substances, about 0.1 to 10% w / v of a compound disclosed herein for parenteral administration. A typical dosage range is about 0.01 to about 50 mg / kg body weight / day, given in one to four divided doses. Each divided dose may contain the same or different compounds. The dosage will be effective, depending on several factors, including the patient's overall health and the formulation and route of administration of the compound selected.

[0191] In another embodiment, the pharmaceutical composition is an oral or parenteral solution. Another embodiment is a lyophilized preparation that can be reconstituted prior to administration. As a solid, the formulation can also include tablets, capsules, or powders.

[0192] Also provided herein are combination therapies in which one or more other therapeutic agents are administered together with a pharmaceutical composition comprising carfilzomib or a peptide proteasome inhibitor. Such combination treatment may be achieved by the simultaneous, sequential, or separate administration of the individual components of the treatment.

[0193] In certain embodiments, the cyclodextrin-free pharmaceutical formulations or kits as provided herein may be administered in conjunction with one or more other proteasome inhibitors.

[0194] In certain embodiments, a cyclodextrin-free pharmaceutical formulation or kit as provided herein can be administered in conjunction with one or more chemotherapeutic agents. Suitable chemotherapeutic agents include natural products such as vinca alkaloids (i.e., vinblastine, vincristine, and vinorelbine), taxanes (e.g., docetaxel, paclitaxel, e.g., docetaxel), epidipodophyllotoxins (i.e., etoposide, teniposide), antibiotics (dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin; e.g., doxorubicin), anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin, enzymes (L-asparaginase, which metabolizes L-asparagine systemically and depletes cells that are unable to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, ifosfamide, cyclophosphamide, and analogs, melphalan, chlorambucil, e.g. ... methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates (busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazine (DTIC), etc.; antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate), pyrimidine analogs (fluorouracil, floxuridine, and cytarabine), purine analogs and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine); aromatase inhibitors (anastrozole, exemestane, and letrozole); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; DNA binding / cytotoxic agents (e.g., xalipsis);Histone deacetylase (HDAC) inhibitors (e.g., trichostatin, sodium butyrate, apicidan, suberoyl anilide hydroxamic acid) acid) (SAHA (vorinostat)), trichostatin A, depsipeptide, apicidin, A-161906, scriptaid, PXD-101, CHAP, butyric acid, depudecin, oxamflatin, phenylbutyrate, valproic acid, MS275 (N-(2-aminophenyl)-4-[N-(pyridin-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, leuprorelin, and triptorelin). Other chemotherapeutic agents may include mechlorethamine, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine, or any analogue or derived variant thereof;

[0195] In certain embodiments, the cyclodextrin-free pharmaceutical formulations or kits as provided herein contain one or more histone deacetylase (HDAC) inhibitors (e.g., trichostatin, sodium butyrate, apicidan, suberoyl anilide hydroxamic acid, acid) (“SAHA” (vorinostat)), trichostatin A, depsipeptide, apicidin, A-161906, scriptaid, PXD-101, CHAP, butyric acid, depudecin, oxamflatin, phenylbutyrate, valproic acid, MS275 (N-(2-aminophenyl)-4-[N-(pyridin-3-ylmethoxy-carbonyl)aminomethyl]benzamide), LAQ824 / LBH589, CI994, MGCD0103, ACY-1215, panobinostat; e.g., SAHA, ACY-1215, panobinostat).

[0196] In certain embodiments, the cyclodextrin-free pharmaceutical formulations or kits as provided herein may be administered in conjunction with one or more nitrogen mustards (mechlorethamine, ifosfamide, cyclophosphamide and analogs, melphalan, chlorambucil, e.g., melphalan).

[0197] In certain embodiments, a cyclodextrin-free pharmaceutical formulation or kit as provided herein may be administered in conjunction with one or more DNA binding / cytotoxic agents (eg, Zalipsis).

[0198] In certain embodiments, a cyclodextrin-free pharmaceutical formulation or kit as provided herein may be administered in conjunction with one or more taxanes (eg, docetaxel, paclitaxel, eg, docetaxel).

[0199] In certain embodiments, the cyclodextrin-free pharmaceutical formulations or kits as provided herein may be administered in conjunction with one or more antibiotics (dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin; e.g., doxorubicin).

[0200] In some embodiments, a cyclodextrin-free pharmaceutical formulation or kit as provided herein may be administered in conjunction with one or more cytokines, including, but not limited to, interferon-γ, -α, and -β, interleukins 1-8, 10, and 12, granulocyte-monocyte colony-stimulating factor (GM-CSF), TNF-α and -β, and TGF-β.

[0201] In some embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein can be administered in conjunction with one or more steroids. Suitable steroids include 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, and fluticasone propionate. These may include, but are not limited to, acetaminophen, formocortal, halcinonide, halobetasol propionate, halometasone, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide and salts and / or derivatives thereof (e.g., hydrocortisone, dexamethasone, methylprednisolone and prednisolone; e.g., dexamethasone).

[0202] In certain embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein can be administered in conjunction with dexamethasone. In certain embodiments, the combination therapy includes the dosing regimen provided on the Kyprolis label, e.g., 1. Kyprolis is administered intravenously over 2 to 10 minutes on 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 is considered one treatment cycle (Table A).

[0203] In cycle 1, 20 mg / m 2 If tolerated during Cycle 1, the dose will be increased to 27 mg / m at the start of Cycle 2. 2 to 27 mg / m in subsequent cycles. 2 Treatment may continue until disease progression or unacceptable toxicity occurs.

[0204] Calculate the dose using the patient's actual body surface area at baseline. 2 Patients larger than 2.2m 2 Patients should receive a dose based on their body surface area. No dose adjustment is required for weight changes of 20% or less.

[0205] [Table 3]

[0206] 2. Hydrate patients to reduce the risk of nephrotoxicity and tumor lysis syndrome (TLS) with Kyprolis treatment. Maintain adequate fluid volume status throughout treatment and closely monitor blood chemistries. Administer 250 mL to 500 mL of intravenous saline or other appropriate intravenous fluid before each dose in Cycle 1. Administer an additional 250 mL to 500 mL of intravenous fluid as needed following Kyprolis administration. Continue intravenous hydration as needed during subsequent cycles. Also monitor patients during this period for fluid overload.

[0207] 3. Administer 27 mg / m KYPROLIS before all doses during Cycle 1 to reduce the incidence and severity of infusion reactions. 2 Premedicate with 4 mg of dexamethasone orally or intravenously before every Kyprolis dose during the first cycle of dose escalation to 10 mg. If these symptoms occur or reappear during the next cycle, resume premedication with dexamethasone (4 mg orally or intravenously).

[0208] In some embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered in conjunction with one or more immunotherapeutic agents. Suitable immunotherapeutic agents may include, but are not limited to, MDR modulators (verapamil, valspodar, biricodar, tariquidar, laniquidar), cyclosporine, pomalidomide, thalidomide, CC-4047 (Actimid), lenalidomide (Revlimid), and monoclonal antibodies. The monoclonal antibodies may be either naked or conjugated, such as rituximab, tositumomab, alemtuzumab, epratuzumab, ibritumomab tiuxetan, gemtuzumab ozogamicin, bevacizumab, cetuximab, erlotinib, and trastuzumab. In certain embodiments, the pharmaceutical compositions provided herein are administered in combination with lenalidomide (Revlimid).

[0209] In some embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein (e.g., pharmaceutical compositions including carfilzomib) can be administered in conjunction with: (i) One or more of the following: one or more second chemotherapeutic agents (e.g., one or more HDAC inhibitors, e.g., SAHA, ACY-1215, panobinostat; one or more nitrogen mustards, e.g., melphalan; one or more DNA-binding / cytotoxic agents, e.g., dylapsin; one or more taxanes, e.g., docetaxel; one or more antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin; e.g., doxorubicin); one or more other proteasome inhibitors (e.g., other compounds of formulae (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., Array 520); ·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 multikinase inhibitors (e.g., TG02); ·One or more PI3K delta inhibitors (e.g., TGR-1202); and (ii) one or more steroids (e.g., dexamethasone);

[0210] In other embodiments, the cyclodextrin-free pharmaceutical formulations or kits provided herein may be administered in conjunction with: (i) One of the following: one or more second chemotherapeutic agents (e.g., one or more HDAC inhibitors, e.g., SAHA, ACY-1215, panobinostat; one or more nitrogen mustards, e.g., melphalan; one or more DNA-binding / cytotoxic agents, e.g., dylapsin; one or more taxanes, e.g., docetaxel; one or more antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin; e.g., doxorubicin); one or more other proteasome inhibitors (e.g., other compounds of formulae (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., Array 520); ·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 multikinase inhibitors (e.g., TG02); ·One or more PI3K delta inhibitors (e.g., TGR-1202); and (i) Dexamethasone.

[0211] For drug products intended for subcutaneous injection, the main potential adverse effects were identified as promotion of site pain, local irritation, and possible tissue injury. It was recommended that the upper formulation osmolality for drug products intended for subcutaneous injection should be 600 mOsmo / kg (Wang, International Journal of Pharmaceutics, Vol. 490, Issues 1-2, 25 July 2015, Pages 308-315).

[0212] The osmolality of a pharmaceutical composition is preferably adjusted to maximize the stability of the active ingredient and minimize discomfort to the patient upon administration. It is generally preferred that the pharmaceutical composition be isotonic with serum, i.e., have the same or similar osmolality, which is achieved by adding an osmolality-adjusting agent. Serum is approximately 300 + / - 50 mOsmoles per kilogram; therefore, the osmolality of an isotonic pharmaceutical composition would be considered to be about 180 to about 420 mOsmoles. In some embodiments, this range is about 250 to about 350 mOsmoles. An osmolality-adjusting agent is understood to be a molecule that contributes to the osmolality of a solution. Examples of osmolality-adjusting agents suitable for adjusting osmolality include, but are not limited to, amino acids (e.g., arginine, cysteine, histidine, and glycine), salts (e.g., sodium chloride, potassium chloride, and sodium citrate), and / or sugars (e.g., sucrose, glucose, and mannitol). The concentration of the osmotic agent in the formulation is preferably about 1 mM to 1 M, more preferably about 10 mM to about 200 mM. In some embodiments, the concentrations of NaCl and sucrose are adjusted to produce an isotonic pharmaceutical composition. In some embodiments exemplified below, the pharmaceutical composition contains about 40 to 100 mg / mL etanercept, about 120 mM NaCl, about 25 mM arginine, about 1% sucrose, and water. In particular, the pharmaceutical composition may consist essentially of about 50 to 100 mg / mL etanercept, about 120 mM NaCl, about 25 mM arginine, about 1% sucrose, about 0.01% polysorbate 20, and water. [Example]

[0213] Example 1: Excipient screening Table 1: Excipient screening It was found that 220 mM mannitol was the most preferred excipient in the DMSO / chlorobutanol mixture for dissolving the CFZ API. In anticipation of improving the dissolution of the lyophilized cake, a series of additional excipients, as listed in Table 1, were screened against the CFZ-API in the chlorobutanol and DMSO solution mixture. Table 1 lists the various excipients that were screened. Because the CFZ-API contains an epoxyketone moiety that is susceptible to nucleophilic attack, non-nucleophilic excipients were carefully selected for screening. Additionally, 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.

[0214] [Table 4]

[0215] method: pH was measured using a Mettler Toledo SEVENEASY™ pH meter in combination with a Mettler Inlab® MicroProbe pH electrode. Samples were warmed to room temperature prior to measurement. Lyophilization was performed in a VirTis® Freeze Dryer (SP Scientific). Osmolality was measured using an Advanced Osmometer Model 3900. Each measurement was performed using 250 μL of sample, and a 290 osmolality standard was tested to ensure the system was operating properly. Reverse-phase HPLC was performed on an Agilent 1100 HPLC using Chromeleon 7.2 software.

[0216] Example 2: Freeze-drying screening Preparation of pre-lyophilized formulation: DMSO and chlorobutanol were mixed in a 60:40% weight-to-weight ratio, resulting in a clear liquid solution of the two solvents (Tesconi et al., Journal of Pharmaceutical Sciences Vol. 88, No. 5, May 1999). Once the mixture was solvent miscible, it was allowed to cool and then placed at room temperature for 2 hours. The mixture was then refrigerated at 4°C for 24 hours to ensure complete solidification, then allowed to warm to room temperature and subsequently melted at 37°C. Mannitol was added from a stock solution to the DMSO / chlorobutanol mixture to a final concentration of 220 mM. CFZ-API powder was then added to the DMSO / chlorobutanol / mannitol mixture to a final concentration of 2 mg / ml. After stirring for 5 minutes at room temperature, dissolution of the CFZ-API was complete.

[0217] After compounding, the samples were filter-sterilized using a DMSO PAL membrane, and 1 mL of the solution was aseptically filled into 3 cc vials. The vials containing the CFZ solution were frozen to -45°C at a rate of 1°C / min and held at -45°C for 1 hour. The samples were subjected to an annealing step utilizing a temperature of -12°C at a rate of 0.5°C / min; after annealing, the samples were returned to -45°C at a rate of 0.5°C / min and held there for 2 hours. Primary drying was performed at -25°C for 6 hours. Secondary drying of the CFZ solution was performed at 40°C for 6 hours, followed by 50°C for 6 hours. The elevated temperature secondary drying step was designed to remove chlorobutanol and DMSO from the lyophilized cake. Vacuum was maintained at 150 mTorr and 50 mTorr during primary and secondary drying, respectively. After lyophilization, the glass vials were sealed and stored at 2-8°C until further analysis. In a search for optimal cake appearance, a series of variations on the freeze-drying cycle described above were tried. The inventors found that the presence of a freezing step and a mannitol annealing step contributed to a refined cake appearance. This is in contrast to the freeze-drying procedure taught by Tesconi et al., which uses a cycle in which the freezing and annealing steps are omitted. Surprisingly, the inventors found that when both steps were omitted, complete cake collapse occurred.

[0218] Example 3: Reconstitution Solvent Screening After lyophilization, the samples were dissolved in a series of reconstitution solutions, including water for injection (WFI). For preferred formulation sample No. 3 (48% chlorobutanol, 32% DMSO, and 220 mM mannitol), the formulation cake exhibited the best reconstitution properties when dissolved in sterile water, although a small amount of visible particulates was observed. We found that the addition of organic solvents and acids to the reconstitution solution did not improve reconstitution efficiency or the presence of particles. Osmolality measurements of the reconstituted cake confirmed the sublimation of DMSO and chlorobutanol during the lyophilization cycle, as shown in Table 2. Table 2 also summarizes the pH of the reconstituted samples, which ranged from 2.6 to 5.2. When the reconstituted samples were introduced into a 5% dextrose solution using a 100 ml bag to mimic clinical IV bag administration, the resulting sample pH was 3.8 for all samples tested. Final CFZ concentrations were tested in 5% dextrose solution from 0.1 to 1 mg / mL to mimic current administration targets.

[0219] [Table 5]

[0220] The most refined cake was achieved when the initial formulation conditions were 48% chlorobutanol, 32% DMSO, 220 mM mannitol, + / - citrate buffer, and + / - polysorbate 80, and when the sample was frozen and an annealing step was performed during the lyophilization cycle. Figure 1 shows photographs of (A) the lyophilized cake obtained from 48% chlorobutanol, 32% DMSO, 220 mM mannitol formulation Sample No. 3 in Table 2 and (B) the clear solution with observed particles obtained after reconstitution with sterile water to obtain approximately 2 mg / mL CFZ-API.

[0221] Example 4: Visual Observation of Chlorobutanol Cyclodextrin-Free CFZ-API Sample Formulations Carfilzomib (CFZ) is a proteasome inhibitor and the active ingredient in Kyprolis®, a lyophilized drug product for the treatment of multiple myeloma. The current commercial formulation of Kyprolis contains Captisol®, a cyclodextrin used to aid in the dissolution of the CFZ-API. The present invention provides a stable, cyclodextrin-free formulation of the CFZ-API in aqueous solution suitable for injection. Figure 2 illustrates (a) a water-insoluble CFZ-API (b) a current commercial Kyprolis formulation containing Captisol (center vial); and (c) a cyclodextrin-free chlorobutanol formulation of the present invention (right vial). Each sample contains a CFZ-API concentration of 2 mg / mL.

[0222] [Table 6]

[0223] Example 5: Stability testing and analysis Analytical Testing: twenty five o The CFZ-API in the chlorobutanol formulations prepared above was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) to accurately quantify the CFZ-API concentration after two weeks of storage at C. Tested before and after lyophilization, two formulation solution samples were compared and analyzed for stability: (i) Sample Formulation No. 2 in Table 2 (48% chlorobutanol, 32% DMSO, 220 mM mannitol, and 20 mM citrate); and (ii) Sample Formulation No. 3 in Table 2 (48% chlorobutanol, 32% DMSO, and 220 mM mannitol).

[0224] The CFZ sample stability data are summarized in Figure 3. No loss of the main peak was observed during the lyophilization cycle, while the final sample concentration was calculated to be 1.9 mg / ml. Furthermore, after 2 weeks of exposure to 25°C, no loss of the main peak was observed in the lyophilized cakes of either Samples 2 or 3.

[0225] Other embodiments While the present disclosure is to be read in conjunction with the detailed description thereof, it should be understood that the foregoing description is illustrative, but not limiting, of the scope of the disclosure, as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. (i) Carfilzomib having the following chemical structure: 【Chemistry 1】 ; or a pharmaceutically acceptable salt thereof; (ii) A solvent system comprising a pharmaceutically acceptable organic solvent suitable for injection, which is a mixture of DMSO and chlorobutanol for completely dissolving the carfilzomib; (iii) Fillers and optionally excipients, Includes, A cyclodextrin-free pharmaceutical composition which is an immediate-use injection or a pre-lyophilized formulation; the injection is administered intravenously or subcutaneously.

2. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the DMSO and chlorobutanol are present in a pre-freeze-dried formulation in a mixed ratio of 60 to 40 w / w.

3. The cyclodextrin-free pharmaceutical composition according to claim 1, which is a pre-lyophilized formulation containing 48% chlorobutanol and 32% DMSO.

4. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the filler is mannitol, glycine, lactic acid, or a combination thereof.

5. The cyclodextrin-free pharmaceutical composition according to claim 4, wherein the concentration of mannitol is 100 mM to 400 mM.

6. The cyclodextrin-free pharmaceutical composition according to claim 1, which is a pre-lyophilized formulation containing 48% chlorobutanol and 32% DMSO; and 220 nM mannitol.

7. The cyclodextrin-free pharmaceutical composition according to claim 1, which is a pre-lyophilized formulation containing 48% chlorobutanol and 32% DMSO; 220 nM mannitol; and 0.01% polysorbate 80.

8. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the aforementioned optional excipient is absent.

9. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the pH of the formulation before freeze-drying is approximately 5 to 6.

10. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the pH of the solution mixture obtained after the freeze-drying step is 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.

0.

11. A cyclodextrin-free pharmaceutical composition according to claim 1, having a solution osmotic pressure of 200 mOsmo to 600 mOsmo.

12. The cyclodextrin-free pharmaceutical composition according to claim 1, wherein the carfilzomib concentration is 2 mg / mL.

13. A cyclodextrin-free carfilzomib kit suitable for injection, (i) (a) A step of combining DMSO and chlorobutanol to form a clear solution mixture and adjusting the temperature of the mixture to its freezing point; (b) The step of melting the mixture and adding a filler and optionally an excipient; (c) Adding carfilzomib to achieve a clear solution; (d) A step of freeze-drying the solution obtained in step (c); A product vial pharmaceutical composition comprising a stable freeze-dried powder or cake prepared by a method including; (ii) A reconstituted vial composition containing sterile water, Includes, A cyclodextrin-free carfilzomib kit, administered intravenously or subcutaneously.

14. The cyclodextrin-free kit according to claim 13, wherein the DMSO and chlorobutanol are present in a mixed ratio of 60 to 40 w / w, respectively.

15. The cyclodextrin-free kit according to claim 13, wherein the filler is mannitol, glycine, or a combination thereof.

16. The cyclodextrin-free kit according to claim 13, wherein the concentrations of DMSO and chlorobutanol in the solution mixture in step (c) are 48% and 32%, respectively, and the concentration of mannitol in the solution mixture in step (c) is 220 mM.

17. The cyclodextrin-free kit according to claim 13, wherein the pH of the solution mixture obtained in step (c) is about 5 to 6.

18. The cyclodextrin-free kit according to claim 13, wherein the pH of the solution mixture obtained in step (d) is approximately 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and 3.

0.

19. A method for preparing cyclodextrin-free carfilzomib freeze-dried powder or cake, (a) A step of combining DMSO and chlorobutanol to form a clear solution mixture and adjusting the temperature of the mixture to its freezing point; (b) The step of melting the mixture and adding a filler and optionally an excipient; (c) Adding the carfilzomib while stirring to achieve a clear solution; (d) A step of freeze-drying the solution obtained in step (c). Methods that include...

20. The method according to claim 19, wherein the DMSO and chlorobutanol are present in a mixed ratio of 60 to 40 w / w, respectively.

21. The method according to claim 19, wherein the filler is mannitol, glycine, or a combination thereof.

22. The method according to claim 19, wherein the excipient is selected from citrate, polysorbate 80, arginine, lactic acid, or any combination thereof.

23. The method according to claim 19, wherein the aforementioned optional excipient is absent.

24. The method according to claim 21, wherein the concentration of mannitol in the solution mixture in step (c) is 100 mM to 400 mM.

25. The method according to claim 19, wherein the concentrations of DMSO and chlorobutanol in the solution mixture in step (c) are 48% and 32%, respectively.

26. The method according to claim 21, wherein the concentrations of DMSO and chlorobutanol in the solution mixture in step (c) are 48% and 32%, respectively; and the concentration of mannitol in the solution mixture in step (c) is 220 mM.

27. The method according to claim 19, wherein the pH of the solution mixture obtained in step (c) is about 5 to 6.

28. The method according to claim 19, wherein the pH of the solution mixture obtained in step (d) is about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.

0.

29. The method according to claim 19, wherein the concentration of carfilzomib in the clear solution is 2 mg / mL.

30. The method according to claim 19, wherein the solution formed in step (b) has a solution osmotic pressure of 280 mOsmo to 320 mOsmo.

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