METHOD FOR PREPARING A LOW CHLORIDE PHARMACEUTICAL COMPOSITION

专利摘要:
cyclodextrin complexation methods for the formulation of peptide proteasome inhibitors. the present disclosure provides methods for formulating compositions comprising one or more peptide proteasome inhibitors and a cyclodextrin, particularly a substituted cyclodextrin. these methods substantially increase the solubility and stability of these proteasome inhibitors and facilitate both their manufacture and administration.
公开号:BR112012028726B1
申请号:R112012028726-5
申请日:2012-09-13
公开日:2021-07-13
发明作者:Evan Lewis；Peter Shwonek；Sean Dalziel；Mouhannad Jumaa
申请人:Onyx Therapeutics, Inc.；
IPC主号:

专利说明:

Reference to Related Order
[001] This application claims the benefit of United States Provisional Patent Application No. 61/644,122, filed May 8, 2012, which is incorporated herein by reference in its entirety. Technical Field
[002] This disclosure provides cyclodextrin complexation methods for formulating compositions comprising one or more peptide proteasome inhibitors and a cyclodextrin, or a mixture of cyclodextrins, particularly a substituted cyclodextrin(s). Such methods substantially increase the solubility and stability of these proteasome inhibitors and facilitate both their manufacture and administration. Background
[003] The proteasome has been validated as a therapeutic target, as demonstrated by the FDA approval of bortezomib, a boronic acid proteasome inhibitor, for the treatment of various cancer indications, including multiple myeloma. Nevertheless, other highly more specific proteasome inhibitors that could have less toxic side effects have recently been described. These compounds include peptide epoxide ketones, such as epoxomicin, described in US Patent No. 6,831,099, the contents of which are incorporated herein by reference, and those described in US Patent No. 7,232,818, the contents of which are incorporated herein by reference. Nevertheless, the low aqueous solubility of some of these compounds makes it difficult to formulate compositions at a sufficiently high concentration to allow the practice to be administered with antineoplastic or other desired pharmacological effects. Therefore, additional methods for the formulation of peptide epoxide ketones are needed. summary
Provided herein are methods of complexing cyclodextrin for formulating a peptide proteasome inhibitor (e.g. a compound of the formula (1) - (5) or a pharmaceutically acceptable salt thereof) with a cyclodextrin. Many peptide proteasome inhibitors have been exposed to have low water solubility. This low solubility can be overcome by complexing the compound with a cyclodextrin using the methods provided herein. For example, homogeneous solutions of a compound of formula (5) (Carfilzomib) can be obtained at a pharmaceutically useful pH (eg about 3.5 mg/ml) and at higher concentrations (eg about 5 mg µg/ml) than could be obtained without cyclodextrin and the complexation processes between the compound and the cyclodextrin provided in this context. In addition to increasing the solubility of a peptide proteasome inhibitor in solution, formulations prepared by the methods provided herein result in pharmaceutical solutions which are endowed with surprising stability. The stability of a complexed inhibitor is reflected in the absence of precipitation from the homogeneous complexed inhibitor solution over prolonged periods of time and thermal stresses. For example, the complexed inhibitor can remain soluble for periods of time and under thermal stresses that exceed those typical for the practical use of injectable pharmaceuticals manufactured aseptically. Although the high concentrations achieved by the processing methods provided in this context may not be expected to be thermodynamically stable, the physical stability of the solutions has been shown not to be affected by the storage temperature (for example, solutions may be stable from -20°C to 25°C °C), freeze-thaw cycles, and lyophilization and reconstitution. The stability of supersaturated solutions of complexed peptide proteasome inhibitor and cyclodextrin is sufficient to tolerate pH adjustments following complexation without precipitation. For example, performing complexation in the pH range 2.5 - 3, then pH titration with sodium hydroxide solution to pH 3.5. This physical stability of the solution allows the use of the complexed material in an acceptable pH range for injection and other pharmaceutical purposes, as well as display stability in a pH range where chemical stability and adequate shelf life is achieved. For that reason, pharmaceutical compositions prepared by the methods provided herein may be supersaturated solutions that do not precipitate or decrease in concentration to a significant extent during their use in any number of medicinal applications (e.g., a bulk solution during manufacturing of sterile product may not precipitate for several days after sterile filtration while being held in a sterile vial-fill containment tank. Similarly, the final reconstituted pharmaceutical compositions may be stable for a range of hours to days facilitating their use as medicinal agents).
[005] In addition to stable production, highly concentrated solutions of a peptide proteasome inhibitor, formulations prepared by the complexation methods provided in this context can be achieved without the chemical degradation and stability limitations of other formulation methods. For example, the methods provided in this context avoid the use of strong acids (eg HCl) to lower the pH during complexation. Although lowering the pH of the formulation to a value less than 2 can facilitate dissolution of the peptide proteasome inhibitor and produce a homogeneous solution prior to complexation, the acidity of the solution can result in degradation of the peptide proteasome inhibitor. For example, in the case of the peptide proteasome inhibitor carfilzomib, the use of a strong acid such as HCl can result in hydrolysis of the pharmacological epoxide, and through nucleophilic attack with chloride ions, result in the formation of a degrading chlorohydrin adduct (CDP ):

[006] Based on its structure, the degradant is classified as an alkylating agent, which is a class of compound considered by the FDA to be a potentially genotoxic impurity. Importantly, from a regulated product safety point of view, the use of the methods provided in this context avoids these strong acids and for that reason the protease inhibitor peptide degradation reactions for these compounds can be reduced so significant and, in some cases, may even be eliminated.
[007] In one aspect, methods for preparing a pharmaceutical composition are presented which include: (i) providing a first combination that includes: (a) one (or more) peptide proteasome inhibitors (e.g., a compound of the formula (1) - (5) or a pharmaceutically acceptable salt thereof); (b) one or more cyclodextrins (“CDs”); and (c) water; wherein the first combination is heterogeneous and the compound or salt has a low solubility in the first combination; and (ii) contacting the first combination with an acid to form a second combination, wherein the compound is more soluble in the second combination than in the first combination.
[008] In another aspect, methods for preparing a pharmaceutical composition are presented, which include: (i) providing a first combination that includes: (a) a compound:
or a pharmaceutically acceptable salt thereof; (b) one or more cyclodextrins (“CDs”); and (c) water; wherein the first combination is heterogeneous and the compound or salt has a low solubility in the first combination; and (ii) contacting the first combination with an acid to form a second combination, wherein the compound is more soluble in the second combination than in the first combination.
[009] In a further aspect, methods for preparing a pharmaceutical composition are set forth which include: (i) providing a first combination which includes: (a) a compound:
or a pharmaceutically acceptable salt thereof; (b) SBECD; and (c) water for injection; wherein the first combination is heterogeneous and the compound or salt has a low solubility in the first combination; and (ii) contacting the first combination with an aqueous citric acid solution to form a second combination, wherein the compound is more soluble in the second combination than in the first combination.
[010] In one aspect, pharmaceutical compositions are presented, which are prepared by any of the methods described in this context.
[011] In one aspect, methods are presented for treating cancer (e.g., multiple myeloma, e.g., multiple myeloma that is relapsed and/or refractory) in a patient, which include administering to the patient a therapeutically effective amount of a pharmaceutical composition prepared by any of the methods described in this context.
[012] In another aspect, methods for treating autoimmune disease in a patient are disclosed, which include administering to the patient a therapeutically effective amount of a pharmaceutical composition prepared by any of the methods described herein.
[013] In another aspect, methods for treating a graft or transplant-related condition in a patient are disclosed, which include administering to the patient a therapeutically effective amount of a pharmaceutical composition prepared by any of the methods described herein.
[014] In another aspect, methods for treating neurodegenerative disease in a patient are disclosed, which include administering to the patient a therapeutically effective amount of a pharmaceutical composition that is prepared by any of the methods described in this context.
[015] In another aspect, methods for treating an associated fibrotic condition in a patient are disclosed, which include administering to the patient a therapeutically effective amount of a pharmaceutical composition prepared by any of the methods described herein.
[016] In another aspect, methods for treating an associated fibrotic condition in a patient are disclosed, which include administering to the patient a therapeutically effective amount of a pharmaceutical composition prepared by any of the methods described herein.
[017] In another aspect, methods for treating a related ischemic condition in a patient are disclosed, which include administering to the patient a therapeutically effective amount of a pharmaceutical composition prepared by any of the methods described herein.
[018] In another aspect, methods for treating an infection in a patient are disclosed, which include administering to the patient a therapeutically effective amount of a pharmaceutical composition prepared by any of the methods described herein.
[019] In another aspect, methods for treating an infection in a patient are disclosed, which include administering to the patient a therapeutically effective amount of a pharmaceutical composition prepared by any of the methods described herein.
[020] In another aspect, methods for treating disease associated with bone loss in a patient are disclosed, which include administering to the patient a therapeutically effective amount of a pharmaceutical composition prepared by any of the methods described herein.
[021] In another aspect, methods for treating an infection in a patient are disclosed, which include administering to the patient a therapeutically effective amount of a pharmaceutical composition prepared by any of the methods described herein.
[022] Embodiments may include one or more of the following aspects.
[023] The first combination does not include appreciable amounts of any organic solvent(s). In some embodiments, the first combination does not include any amount or kind of organic solvent(s) described in US patent 7,232,818 and/or 7,417,042 and/or 7,737,112 and/or US-2009-0105156 and /or US-2011 0236428, each of which is incorporated herein by reference. In some embodiments, the first blend is free of any organic solvent (e.g., contains less than 5%, less than 4%, less than 3%, less than 2%, less than 1% (w/ w or w/v) of any organic solvent). In some embodiments, the first blend is substantially free of any organic solvent (e.g., contains less than 0.5%, less than 0.2, less than 0.1, less than 0.05% (p /p or w/v) of any organic solvent). In certain embodiments, the first combination does not include a detectable amount of any organic solvent.
[024] The first combination does not include appreciable amounts of any buffer. In some embodiments, the first combination does not include any amount or kind of any buffer(s) described in US patent 7,232,818 and/or 7,417,042 and/or 7,737,112 and/or US-2009-0105156 and /or US-2011-0236428, each of which is incorporated herein by reference. In some embodiments, the first blend is free of any buffer(s) (e.g., contains less than 5%, less than 4%, less than 3%, less than 2%, less than 1% ( w/w or w/v) of any buffer). In some embodiments, the first blend is substantially free of any buffer(s) (e.g., contains less than 0.5%, less than 0.2, less than 0.1, less than 0.05% (p/p or p/v) of any buffer(s)). In some embodiments, the first combination does not include a detected amount of any buffer(s).
[025] The second combination includes a complex of the compound and one or more cyclodextrins.
[026] The acid is added in the form of an aqueous solution.
[027] At least one or more cyclodextrins is HPBCD or SBECD (eg SBECD).
[028] The inventors have found that it may be advantageous to minimize the amount of chloride ion (or other nucleophilic anions) in the methods and pharmaceutical compositions described in this context.
[029] In some embodiments, at least one or more cyclodextrins (added to the first combination) is a low chloride cyclodextrin. As used in this context, a "low chloride cyclodextrin" refers to a cyclodextrin that is endowed with less than or equal to 0.05% w/w sodium chloride, or if present( s) a source(s) of chloride other than (or in addition to) sodium chloride, a "low chloride cyclodextrin" refers to a cyclodextrin that is endowed with a chloride ion content that is less than or equal to the amount of chloride that would be present in a cyclodextrin that is endowed with 0.05% w/w sodium chloride. In some embodiments, the low chloride cyclodextrin is a low chloride SBECD. The determination of chloride concentration can be carried out by a variety of methods known in the art (for example for cyclodextran obtained commercially from the manufacturer's product specification, for example by gravimetric techniques, for example by potentiometric techniques).
[030] In some embodiments, the amount of chloride ions present (for example, the molar ratio of chloride ions to compound) is sufficiently low so as to provide a 2 year shelf life when stored at 2-8°C.
[031] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 2.0.
[032] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 1.5.
[033] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 1.2.
[034] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 1.0.
[035] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 0.9.
[036] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 0.8.
[037] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 0.7.
[038] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 0.6.
[039] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 0.5.
[040] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 0.4.
[041] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 0.3.
[042] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 0.2.
[043] In some embodiments, the molar ratio of chloride ions to the compound in the first combination is not greater than 0.1.
[044] In some embodiments, the molar ratio of chloride ions to the compound in the first combination ranges from 0.2 to 1.2 (e.g., 0.3 to 1.2, e.g., 0.2 to 0.4 , eg 0.3 to 0.4, eg 0.32).
[045] In embodiments, the molar ratios of chloride ions to the compound described in this context may also be present in the second and/or third combinations.
[046] By way of example, the molar ratio of chloride ions to the compound in the first combination can be calculated as illustrated below using a vial of dry carfilzomib powder ("CFZ") as the basis for the calculation: Mass of content vial = 3.212 g Mass of CFZ = 61.8 mg Maximum mass of chloride (under 0.03% w/w chloride ions) = 0.0009636 g Maximum molar mass of chloride = 2.714 x 10-5 (atomic mass of Cl = 35.5) Molar mass of CFZ = 8.584 x 10-5 (MW CFZ = 719.9)
[047] Molar ratio of solid state Cl/CFZ in a flask = 0.32 This calculation can also be determined for the first combination using, for example, the chloride content of the cyclodextrin (and any other source of chloride ion ) and the mass of compost that are added to prepare the first blend.
[048] As one skilled in the art can appreciate, it is to be expected that this ratio is the same in the bulk precursor solution used to fill the vial (pre-lyophilization) as when the content of the dry powder vial is reconstituted in sterile water for patient administration.
[049] The provision of a first combination (step (i)) includes adding the compound to a solution of one or more cyclodextrins and water.
[050] The compound is a crystalline solid. In embodiments, the crystalline form of the compound has an X-ray powder diffraction pattern comprising from 2 to 8 characteristic peaks expressed in degrees 2θ under 6.10; 9.32; 10.10; 12.14; 13.94; 18.44; 20.38; and 23.30.
[051] The method further includes mixing the first combination before contacting the first combination with an acid.
[052] Steps (i) and (ii) are both performed in a single vessel.
[053] The method further includes mixing the second blend for a time sufficient to achieve a homogeneous third blend.
[054] The dissolved and complexed concentration of the compound in the third combination ranges from 1 mg/ml to 20 mg/ml.
[055] The dissolved and complexed concentration of the compound in the third combination ranges from 4 to 8 mg/ml.
[056] The pH of the third combination ranges from 2 to 4.
[057] The method further includes filtering the third combination.
[058] The method further comprises lyophilizing the third combination to provide a lyophilizate.
[059] The method further comprises mixing the lyophilizate with a pharmaceutically acceptable carrier.
[060] The pharmaceutically acceptable carrier comprises sterile water for injection. In embodiments, the pharmaceutically acceptable carrier further includes citric acid.
[061] Unless otherwise defined, all technical and scientific terms used in this context have the same meaning as is commonly understood by one normally versed in the art to which the present description relates. Methods and materials are described in this context for use in this exhibition; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned in this context are incorporated by reference in their entirety. In case of divergence, this report, including definitions, will control.
[062] Other essential aspects and advantages of the exhibition will be evident from the following detailed description and figures, and from the claims. Description of Drawings
[063] Figure 1 is a line graph that shows the complexation of CFZ-API by SBECD over time.
[064] Figure 2 illustrates the independence of pharmaceutical compositions prepared in this context on the physiochemical properties (e.g., particle size) of the proteasome inhibitor.
[065] Figure 3 is a line graph that shows an increase in CFZ-API solubilization with increasing SBECD concentration.
[066] Figure 4 illustrates the independence of the solubility of the CFZ-API/SBECD complex at the processing or storage temperature.
[067] Figure 5 illustrates the correlation between the levels of the chlorohydrin degradation product (CDP) and the interaction of two factors of water and chloride content under pH 3.5.
[068] Figure 6 illustrates the solubility of carfilzomib in SBECD under pH 1.5 and pH 3.5, 25°C and 5°C, (5.9 mg/ml citric acid). Detailed Description
[069] Provided herein are methods of cyclodextrin complexing the formulation of a peptide proteasome inhibitor (for example a compound of the formula (1) - (5) or a pharmaceutically acceptable salt thereof) with a cyclodextrin. Also provided in this context are pharmaceutical compositions comprising a peptide proteasome inhibitor and a cyclodextrin, wherein the composition has a chloride ion as described above in this context (e.g., the composition is prepared using a low content cyclodextrin of chloride; for example, a molar ratio of chloride ions to the compound is 0.32). In some embodiments, formulations that are endowed with low chloride ions content as described in this context can result in decreased formation of undesirable degradation products. Definitions
[070] The term "Cx-y alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight chain alkyl groups and branched chain alkyl groups containing from x to y carbons in the chain, including groups haloalkyl such as trifluoromethyl and 2,2,2-trifluoroethyl, and the like. The terms "C2-y alkenyl" and "C2-y alkynyl" refer to substituted or unsubstituted saturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one double or triple bond, respectively.
[071] The term "alkoxy" refers to an alkyl group that is endowed with an oxygen bonded thereto. Alkoxy groups that are representative include methoxy, ethoxy, propoxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. For this reason, the substituent of an alkyl that makes that alkyl an ether is or resembles an alkoxy.
[072] The term "C1-6 alkoxyalkyl" refers to a C1-6 alkyl group substituted with an alkoxy group, thereby forming an ether.
[073] The term "C1-6 aralkyl" as used in this context refers to a C1-6 alkyl group substituted with an aryl group.
[074] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and their salts, for example, a radical that can be represented by the general formulas:
where R9, R10and R10' each independently represents a hydrogen, an alkyl, an alkenyl, -(CH2)m—R8, or R9and R10 taken together with the N atom to which they are attached complete a heterocycle which is provided with 4 up to 8 atoms in the ring structure; R8 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocyclyl or a polycyclyl; and em is zero or an integer from 1 to 8. In some embodiments, only one of R9 or R10 is a carbonyl, eg, R9, R10, and the nitrogen together do not form an imide. In some embodiments, R9 and R10(and optionally R10^ each independently represents a hydrogen, an alkyl, an alkenyl, or -(CH2)m—R8. In certain embodiments, an amino group is basic, meaning that its protonated form has a pKa above 7.00.
[075] The terms "amide" and "starch" are art-recognized as an amino-substituted carbonyl and include a radical which can be represented by the general formula:
where R9, R10 are as defined above. In some embodiments, the amide will not include imides that may be unstable.
[076] The term "aryl" as used in this context includes substituted or unsubstituted single ring aromatic groups of 5, 6, and 7 elements wherein each ring atom is carbon. The term "aryl" also includes polycyclic ring systems that are provided with two or more cyclic rings where two or more carbons are common to two adjacent rings where at least one of the rings is aromatic, for example the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkenyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.
[077] The term “buffer” is a substance that by its presence in the solution increases the amount of acid or alkali that must be added to cause a unit change in pH. Thus, a buffer is a substance that helps to regulate the composition's pH. Typically, a buffer is chosen based on the desired pH and compatibility with other components of the composition. In general, a buffer has a pKa that is no more than 1 unit less than or greater than the desired pH of the composition (or that the composition will produce on dissolution).
[078] The term "water" as used in this context refers to a liquid solution of H2O that is endowed with a pH of approximately 7.0.
[079] The terms "carbocycle" and "carbocyclyl", as used in this context, refer to a non-aromatic ring, substituted or unsubstituted, in which each atom of the ring is constituted by carbon. The terms "carbocycle" and "carbocyclic" also include polycyclic ring systems that are provided with two or more cyclic rings where two or more carbons are common to two adjacent rings where at least one of the rings is carbocyclic, e.g. the other two rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
[080] The term "carbonyl" is art-recognized and includes such radicals as they may be represented by the general formulas:
wherein X is a bond or represents an oxygen or a sulfur, and R11 represents a hydrogen, an alkyl, an alkenyl, -(CH2)m—R8or a pharmaceutically acceptable salt, R11' represents a hydrogen, an alkyl, an alkenyl or —(CH2)m—R8, where m and R8 are as defined above. Where X is an oxygen and R11or R11' is not hydrogen, the formula represents an "ester". Where X is an oxygen, and R11 is a hydrogen, the formula represents a “carboxylic acid”.
[081] The term "C1-6 heteroaralkyl" as used in this context refers to a C1-6 alkyl group substituted with a heteroaryl group.
[082] The term "heteroaryl" includes ring structures of 5 to 7 substituted or unsubstituted aromatic elements, for example, 5 to 6 element rings, which ring structures include from one to four heteroatoms. The term "heteroaryl" also includes polycyclic ring systems that are provided with two or more cyclic rings where two or more carbons are common to two adjacent rings where at least one of the rings is heteroaromatic, for example the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
[083] The term "heteroatom" as used in this context means an atom of any element other than carbon or hydrogen. For example, heteroatoms include nitrogen, oxygen, phosphorus, and sulfur.
[084] The term "heterocyclyl" or "heterocyclic group" refers to substituted or unsubstituted 3 to 10 non-aromatic ring structures, e.g., 3 to 7 element rings, which ring structures include from one to four heteroatoms. The term "heterocyclyl" or "heterocyclic group" also includes polycyclic ring systems that are provided with two or more cyclic rings where two or more carbons are common to two adjacent rings where at least one of the rings is heterocyclic, for example, of the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.
[085] The term "C1-6 hydroxyalkyl" refers to a C1-6 alkyl group substituted with a hydroxy group.
[086] The term "thioether" refers to an alkyl group, as defined above, which is endowed with a sulfur radical attached thereto. In some embodiments, the "thioether" is represented by -S-alkyl. Representative thioether groups include methylthio, ethylthio, and the like.
[087] The term "substituted" refers to radicals that are provided with substituents that replace a hydrogen on one or more non-hydrogen atoms of the molecule. It will be understood that "substitution" or "substituted with" includes the implied condition that such substitution is in accordance with the allowable valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g. spontaneously transforming, such as by rearrangement, cyclization, elimination, etc. As used in this context, the term "substituted" is considered to include all permissible substituents of organic compounds. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Permissible substituents may be one or more and the same or different for the appropriate organic compounds. For the purposes of this exposition, heteroatoms such as nitrogen may be provided with hydrogen substituents and/or any permissible substituents of the organic compounds described in this context that satisfy the valences of the heteroatoms. Substituents may include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), a alkoxy, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, a starch, an amidine, an imine, a cyano, a nitro, an azide, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic radical. It will be understood by those skilled in the art that the substituted radicals in the hydrocarbon chain may themselves be substituted, if appropriate.
[088] In some embodiments, the compounds provided herein, or salts thereof, are substantially isolated or purified. By "substantially isolated" is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compounds provided herein. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99%, by weight of the compounds, or their salt. Methods for isolating the compounds and their salts are routine in the art.
[089] As used in this context, the term "peptide" refers to a chain of amino acids that is about two to about ten amino acids in length.
[090] As used in this context, the term "natural" or "naturally occurring" amino acid refers to one of the twenty most commonly occurring amino acids. Natural amino acids are referred to by their standard one- or three-letter abbreviations.
[091] The term "unnatural amino acid" or "unnatural" refers to any derivative or structural analog of a natural amino acid that includes the D-forms, and derivatives of β and Y amino acids. amino acids, for example hydroxyproline, which are classified as an unnatural amino acid in this context, can be found in nature within a particular organism or a specific protein. Non-limiting examples of unnatural amino acids include: β-Alanine (β-Ala), y-Aminobutyric acid (GABA), 2-Aminobutyric acid (2-Abu), α,β-Dehydrodro-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-Aminooctanoic acid (8-Aoc), 11-Aminobenzoic 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 (Statina, Sta), Aminooxyacetic acid (Aoa), 2-Aminotetralin-2-carboxylic acid (Atc), 4- Amino-5-cyclohexyl-3-hydroxypentanoic (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-Benzo ylphenylalanine (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), Homoserin (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 (1-Nal), 2 Naphthylalanine (2-Nal), para-Nitrophenylalanine (4-NO2-Phe), 3-Nitrotyrosine (3-NO2-Tyr), Norleucine (Nle), Norvaline (Nva), Omitine (Orn), Ortho-Phosphotyrosine (H2PO3-Tyr), Octahydroindole-2-carboxylic acid (Oic), Penicillamine (Pen), Pentafluorophenyl Alanine (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). Amino acid stereochemistry may be designated by the precedence of the name or abbreviation with the designation "D" or "d" or "L" or "l" as appropriate. Alternatively, chiral centers can be represented with the conventional (S)-, or (R)- designations. Furthermore, αN-alkylated amino acids can be employed, in the same way as amino acids that are provided with side chains that contain amines (such as Lys and Orn) in which the amine has been acylated or alkylated. See, for example, "Peptides and Mimics, Design of Conformationally Constrained" by Hruby and Boteju, in Molecular Biology and Biotechnology: A Comprehensive Desk Reference, ed. Robert A. Meyers, VCH Publishers (1995), pp. 658-664, which is incorporated herein by reference.
[092] The term "complexation" as used in this context refers to the formation of an intermolecular inclusion complex, or an intermolecular association, in solution and between one or more peptide proteasome inhibitors and one or more cyclodextrin molecules. Inclusion and/or association provides utility as a mechanism that substantially increases the concentration of inhibitor(s) that can be achieved in aqueous solution compared to aqueous phase dissolution at a similar pH range without the complexing agent (i.e., one or more cyclodextrin molecules).
[093] The term "prophylactic or therapeutic" treatment is art-recognized and includes administering to the host one or more subject compositions. If administered prior to the clinical manifestation of the undesirable condition (eg, disease or other undesirable condition of the host animal), then the treatment is prophylactic (ie, it protects the host against the development of the undesirable condition), whereas if it is is administered after the manifestation of the undesirable condition, the treatment is therapeutic, (ie, it is intended to lessen, ameliorate, or stabilize the existing undesirable condition or its side effects).
[094] The term "proteasome" as used in this context is understood to include both immunological and constitutive proteasomes.
[095] As used in this context, the term "inhibitor" is intended to describe a compound that blocks or reduces an activity of an enzyme or enzyme system, receptors, or other pharmacological target (eg, 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). An inhibitor can act with competitive inhibition, non-competitive, or incapable of being competitive. An inhibitor can bind reversibly or irreversibly, and for that reason the term includes compounds that are suicidal substrates for an enzyme. An inhibitor can modify one or more sites in or near the active site of the enzyme, or it can cause a conformational change elsewhere in the enzyme. The term inhibitor is used more widely in this context than in the scientific literature so as to also encompass other classes of pharmacologically or therapeutically useful agents, such as agonists, antagonists, stimulants, cofactors, and the like.
[096] As used in this context, "low solubility" refers to being moderately soluble, slightly soluble, very slightly soluble, practically insoluble, or insoluble in, for example, water or another solution (eg, a first combination); the terms "moderately soluble, slightly soluble, very slightly soluble, practically insoluble, or insoluble" correspond in meaning to the general terms of the United States Pharmacopeia (USP) for the expression of approximate solubility. See, for example, DeLuca and Boylan in Pharmaceutical Dosage Forms: Parenteral Medications, vol. 1, Avis, KE, Lackman, L. and Lieberman, HA, eds; Marcel Dekkar: 1084, pages 141-142:

[097] “Heterogeneous”, as used in this context, refers to a solution that is endowed with a non-uniform composition (multiphase). For example, a heterogeneous solution might include a suspension of solid particles in a liquid (eg a paste).
[098] "Homogeneous", as used in this context, refers to a solution that is consistent or uniform throughout its volume (single phase, observed as a clear solution).
[099] A "therapeutically effective amount" of a compound, with respect to the method of treatment under consideration, refers to an amount of the compound(s) in a preparation which, when administered as part of a dosing regimen desired (for a patient, eg, a human), alleviates a symptom, ameliorates a condition, or delays the onset of disease conditions, in accordance with clinically acceptable standards for the disorder or condition being treated or for cosmetic purposes, for example, under a reasonable risk/benefit ratio applicable to any medical treatment.
[0100] As used in this context, the term "treat" or "treatment" includes reversing, reducing, or controlling the symptoms, clinical signs, and underlying pathology of a condition in a manner to ameliorate or stabilize a patient's condition. Compounds
[0101] Provided in this context are methods for preparing formulations of peptide proteasome inhibitors that have low water solubility characteristics. Peptide proteasome inhibitors comprise an epoxide-containing or aziridine-containing moiety that contains groups close to three-element rings that contain heteroatoms in such a way that a ring-opening reaction of the three-element ring containing heteroatoms is facilitated. Such groups include, for example, electron-withdrawing groups such as a carbonyl. In some embodiments, a peptide proteasome inhibitor is an epoxide peptide proteasome inhibitor. As used in this context, the "peptide epoxide proteasome inhibitor" comprises a ketone radical which is provided with an epoxy group on one side of the ketone with a peptide on the other.
[0102] The peptide of a proteasome inhibitor peptide includes from 2 to 10 amino acids. For example, the peptide can have from 2 to 8 amino acids; 2 to 6 amino acids; 2 to 5 amino acids; 2 to 4 amino acids; 3 to 10 amino acids; 4 to 10 amino acids; 6 to 10 amino acids; 8 to 10 amino acids; 3 to 4 amino acids; 3 to 5 amino acids; and 4 to 6 amino acids. In some embodiments, the peptide is 3 or 4 amino acids long.
[0103] In some embodiments, a peptide proteasome inhibitor is a compound of the formula (1):
where: X is oxygen, NH or N(C1-6 alkyl); W is a peptide comprising from two to ten amino acids, where the amino acids can be natural, unnatural, or a combination thereof; and R is a hydrogen atom or a C 1-4 alkyl group, which may be substituted with one or more of a hydroxy, halogen, amino, carboxy, carbonyl, thio, sulfide, ester, amide or ether functionality; or a pharmaceutically acceptable salt thereof.
[0104] In some embodiments, X is configured to facilitate interaction with an N-terminal nucleophilic group in an Ntn hydrolase. For example, irreversible interactions of enzyme inhibitors with the β5/Pre2 subunit of the 20S proteasome leading to inhibition appear to be facilitated by the configuration illustrated above. In the case of other Ntn hydrolases, the opposite stereochemistry of the α-carbon of peptide epoxides or peptide aziridines may be of use. In some embodiments, X is oxygen.
[0105] The stereochemistry of the α‘ carbon (that carbon that forms a part of the epoxide or aziridine ring) can be (R) or (S). Note that a compound may have a number of stereocenters which are endowed with the indicated up-down (or β-α, where β as drawn in this context is above the plane of the page) or (R)-(S) (or that is, it is not necessary for each stereocenter in the compound to conform to established preferences). In some embodiments, the stereochemistry of the α‘ carbon is (R), that is, the X atom is β, or above the plane of the molecule, when plotted as in formula (1).
[0106] In the case of a compound of formula (1), the β' carbon is replaced with two hydrogen atoms. With respect to stereochemistry, the chiral α‘ carbon is denoted with a star, and the Cahn-Ingold-Prelog rules are followed for determining the absolute stereochemistry. These rules are described, for example, in Organic Chemistry, Fox and Whitesell; Jones and Bartlett Publishers, Boston, Mass. (1994); Section 5-6, pp 177-178, which section is incorporated into this context by reference. The stereochemistry of the α‘ carbon is (R) when oxygen or nitrogen has the highest priority, the peptide ketone group has the second highest priority, and the group —CH2—X— has the third highest priority. If the relative priorities of the peptide-ketone, —CH2—X—, and R groups change, the nominal stereochemistry may change, but the essential configuration of the groups may remain the same, for some embodiments. That is, with reference to the general structure immediately above, the peptide ketone is joined to the a' chiral carbon from the left, R is joined to the chiral a' carbon from the right, and the atom(s) X projects from the plane of the page. The nitrogen atom of the aziridine ring can also, in principle, be chiral, as discussed in March, Advanced Organic Chemistry, 4th Ed. (1992) Wiley-Interscience, New York, pp. 98-100, which pages are incorporated herein by reference.
[0107] W is a peptide comprising from two to ten amino acids, where the amino acids can be natural, unnatural, or a combination thereof. For example, the peptide can have from 2 to 8 amino acids; from 2 to 6 amino acids; from 2 to 5 amino acids; from 2 to 4 amino acids; from 3 to 10 amino acids; from 4 to 10 amino acids; from 6 to 10 amino acids; from 8 to 10 amino acids; from 3 to 4 amino acids; from 3 to 5 amino acids; and from 4 to 6 amino acids. In some embodiments, the peptide is 3 or 4 amino acids long. In some embodiments of utility for inhibiting the chymotrypsin-like (CT-L) activity of the proteasome, between four and eight amino acids are present, and in some embodiments for inhibiting CT-L, between four and six amino acids are present. . In other embodiments of utility for inhibiting the PGPH activity of the proteasome, between two and eight amino acids are present, and in accordance with some embodiments for inhibiting PGPH, between three and six amino acids are present. The link between W and the ketone moiety in formula (1) can be made between either terminus of the peptide. For example, according to some embodiments, the ketone is attached to the carboxy terminus of the peptide. Alternatively, the ketone can be attached to the amino terminus of the peptide. In some embodiments, the ketone can be attached to a side chain of the peptide.
[0108] Examples of a compound of formula (1) can be found in U.S. Patent No. 7,737,112, which is incorporated by reference in its entirety in this context. In some embodiments, a compound of formula (1) has low water solubility.
[0109] The peptide proteasome inhibitor for the inhibition of chymotrypsin-like (CT-L) activity of Ntn can include a peptide that has at least four amino acids. In some CT-L inhibitor embodiments, the inhibitor has a peptide that is endowed with at least four amino acids and an α',β'-epoxy ketone or α',β'-aziridine ketone moiety (tetrapeptide epoxy ketones or ketones tetrapeptide aziridine).
[0110] In some embodiments, a peptide proteasome inhibitor that is endowed with low water solubility can be a compound of formula (II):
where: each A is independently selected from C=O, C=S, and SO2; or A is optionally a covalent bond when adjacent to an occurrence of Z; L is absent or selected from C=O, C=S, and SO2; M is absent or is C1-12 alkyl; Q is absent or is selected from O, NH and N(C1-6 alkyl); X is selected from O, NH and N(C1-6 alkyl); Y is absent or is selected from O, NH, N(C1-6 alkyl), S, SO, SO2, CHOR10, and CHCO2R10; each Z is independently selected from O, S, NH and N(C16 alkyl); or Z is optionally a covalent bond when adjacent to an occurrence of A; R 1 , R 2 , R 3 , and R 4 are each independently selected from C 1-6 alkyl, hydroxyl C 1-6 alkyl, C 1-6 alkoxyalkyl, aryl, and C 1-6 aralkyl, any of which is optionally substituted with one or more substituents of amide, amine, carboxylic acid (or a salt thereof), ester, thiol, or thioether; R5is N(R6)LQR7; R6 is selected from hydrogen, OH, and C1-6 alkyl; R7 is selected from hydrogen, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, aryl, C1-6 aralkyl, heteroaryl, C1-6 heteroaralkyl, R8ZAZ-C1-8 alkyl-, R11Z-C1-8 alkyl -, (R8O)(R9O)P(=O)O-C1-8 alkyl-ZAZ-C1-6 alkyl-, R8ZAZ-C1-8 alkyl- ZAZ-C1-8 alkyl-, heterocyclylMZAZ-C1-8 alkyl- , (R8O)(R9O)P(=O)O—C1-8 alkyl-, (R10)2N—C1-12 alkyl, (R10)3N+-C1-12 alkyl, heterocyclylM-, carbocyclylM-, R11SO2C1 alkyl -8-, and R11SO2NH; or R6and R7together are C1-6 alkyl-Y-C1-6 alkyl, C1-6 alkyl-ZAZ-C1-6 alkyl, ZAZ-C1-6 alkyl-ZAZ-C1-6 alkyl, ZAZ-C1-6 alkyl- ZAZ, or C1-6 alkyl-A, thereby forming a ring; R8 and R9 are independently selected from hydrogen, metal cation, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, aryl, heteroaryl, C1-6 aralkyl, and C1-6 heteroaralkyl, or R8and R9 together are C1 alkyl -6, thereby forming a ring; each R10 is independently selected from hydrogen and C16 alkyl; and R11 is independently selected from hydrogen, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, C1-6 aralkyl, and C1-6 heteroaralkyl, provided from the time when when R6is H or CH3and Q is absent, LR7 is not hydrogen, unsubstituted C1-6 alkylC=O, an additional chain of amino acids, t-butoxycarbonyl (Boc), benzoyl (Bz), fluoren-9-ylmethoxycarbonyl (Fmoc), triphenylmethyl (trityl), benzyloxycarbonyl (Cbz), trichloroethoxycarbonyl (Troc); or substituted or unsubstituted aryl or heteroaryl; and in any occurrence of the ZAZ sequence, at least one element of the sequence must be other than a covalent bond; or a pharmaceutically acceptable salt thereof.
[0111] According to certain embodiments, when R6 is H, L is C=O, and Q is absent, R7 is not hydrogen, C1-6 alkyl, or substituted or unsubstituted aryl or heteroaryl. In accordance with certain embodiments, when R6 is H and Q is absent, R7 is not a protecting group such as those described in Greene, TW and Wuts, PGM, "Protective Groups in Organic Synthesis", John Wiley & Sons, 1999 or Kocienfski, PJ, “Protecting Groups”, Georg Thieme Verlag, 1994.
[0112] According to some embodiments, R1, R2, R3, and R4 are selected from C1-6 alkyl or C1-6 aralkyl. For example, R2 and R4 are C1-6 alkyl and R1 and R3 are C1-6 aralkyl. According to some embodiments, R2 and R4 are isobutyl, R1 is 2-phenylethyl, and R3 is phenylmethyl.
[0113] According to some embodiments, L and Q are absent and R7 is selected from C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, C1-6 aralkyl, and C1-6 heteroaralkyl. For example, R6 is C1-6 alkyl and R7 is selected from butyl, allyl, propargyl, phenylmethyl, 2-pyridyl, 3-pyridyl, and 4-pyridyl.
[0114] According to some embodiments, L is SO2, Q is absent, and R7 is selected from C1-6 alkyl and aryl. For example, R7 can be selected from methyl and phenyl.
[0115] According to some embodiments, L is C=O and R7 is selected from C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, aryl, C1-6 aralkyl, heteroaryl, C1-6heteroaralkyl, R8ZA -C1-8 alkyl-R11Z-C1-8 alkyl-, (R8O)(R9O)P(=O)O-C1-8 alkyl-, (R8O)(R9O)P(=O)O—C1-8 alkyl -ZAZ-C1-8 alkyl-, (R8O)(R9O)P(=O)O-C1-8 alkyl-Z-C1-8alkyl-, R8ZA-C1-8 alkyl-ZAZ-C1-8alkyl-, heterocyclyl MZAZ -C1-8 alkyl-, (R10)2N—C1-8 alkyl-, (R10)3N+—C1-8 alkyl-, heterocyclyl-M carbocyclyl M-, R11SO2C1-8 alkyl-, and R11SO2NH-, where each occurrence of Z and A is independently other than a covalent bond. According to some embodiments, L is comprised of C=O, Q is absent, and R7 is H.
[0116] According to some embodiments, R6 is C1-6 alkyl, R7 is C1-6 alkyl, Q is absent, and L is C=O. According to some of these embodiments, R7 is ethyl, isopropyl, 2,2,2-trifluoroethyl, or 2-(methylsulfonyl)ethyl.
[0117] According to some embodiments, L is C=O, Q is absent, and R7 is C1-6 aralkyl. For example, R7 can be selected from 2-phenylethyl, phenylmethyl, (4-methoxyphenyl)methyl, (4-chlorophenyl)methyl, and (4-fluorophenyl)methyl.
[0118] According to some embodiments, L is C=O, Q is absent, R6 is C1-6 alkyl, and R7 is aryl. For example, R7 can be a substituted or unsubstituted phenyl.
[0119] According to some embodiments, L is C=O, Q is absent or O, n is 0 or 1, and R7 is -(CH2)ncarbocyclyl. For example, R7 can be cyclopropyl or cyclohexyl.
[0120] According to some embodiments, L and A are C=O, Q is absent, Z is O, n is an integer ranging from 1 to 8 (eg 1), and R7 is selected from R8ZA- C1-8alkyl-, R11Z-C1-8alkyl-, R8ZA-C1-8alkyl-ZAZ-C1-8alkyl-, (R8O)(R9O)P(=O)O-C1-8alkyl-ZAZ-C1-8alkyl- -, (R8O)(R9O)P(=O)O—C1-8 alkyl-Z-C1-8 alkyl-, and heterocyclyl MZAZ-C1-8 alkyl-, where each occurrence of A is independently other than one covalent bond. For example, R7 can be heterocyclyl MZAZ-C1-8 alkyl- where heterocyclyl is a substituted or unsubstituted oxodioxolenyl or N(R12)(R13), where R12and R13 together are C1-6 alkyl-Y-C1-6 alkyl, such as C1-3 alkyl-Y-C1-3 alkyl, thereby forming a ring.
[0121] According to some embodiments, L is C=O, Q is absent, n is an integer ranging from 1 to 8, and R7 is selected from (R8O)(R9O)P(=O)O—Ci -8alkyl-, (R10)2NCi-8alkyl, (R10)3N+(CH2)n-, and heterocyclyl-M-. According to some such embodiments, R7 is -C1-8 alkyl N(R10)2 or -C1-8 alkyl N+(R10)3, wherein R10 is C1-6 alkyl. For example, R7 is heterocyclyl M-, where heterocyclyl is selected from morpholino, piperidino, piperazino and pyrrolidino.
[0122] According to some embodiments, L is C=O, R6 is C1-6 alkyl, Q is selected from O and NH and R7 is selected from C1-6alkyl, cycloalkyl-M, C1-6 aralkyl, and C1-6 heteroaralkyl. According to some embodiments, L is C=O, R6 is C1-6 alkyl, Q is selected from O and NH, and R7 is C1-6 alkyl, wherein C1-6 alkyl is selected from methyl, ethyl and isopropyl. According to some embodiments, L is C=O, R6 is C1-6 alkyl, Q is selected from O and NH, and R7 is C1-6 aralkyl, where aralkyl is phenylmethyl. According to some embodiments, L is C=O, R6 is C1-6 alkyl, Q is selected from O and NH, and R7 is C1-6 heteroaralkyl, where heteroaralkyl is (4-pyridyl)methyl.
[0123] According to some embodiments, L is absent or is C=O, and R6and R7 together are C1-6 alkyl-Y-C1-6 alkyl, C1-6 alkyl-ZA-C1-6 alkyl, or C1-6 alkyl -A, where each occurrence of Z and A is independently other than a covalent bond, thereby forming a ring. According to some embodiments, L is C=O, Q and Y are absent, and R6and R7 together are C1-3alkyl-Y-C1-3alkyl. According to some embodiments, L and Q are absent, and R6 and R7 together are C1-3 alkyl-Y-C1-3 alkyl. According to some embodiments, L is C=O, Q is absent, Y is selected from NH and N-C1-6 alkyl, and R6 and R7 together are C1-3 alkyl-Y-C1-3 alkyl. According to some embodiments, L is C=O, Y is absent, and R6and R7 together are C1-3alkyl-Y-C1-3alkyl. According to some embodiments, L and A are C=O, and R6and R7 together are C1-2alkyl-ZA-C1-2alkyl. According to some embodiments, L and A are C=O and R6and R7 together are C2-3alkyl-A.
[0124] A compound of the formula (2) may be endowed with the following stereochemistry:

[0125] Other non-limiting examples of a compound of formula (2) can be found, for example, in U.S. Patent No. 7,232,818, which is included by reference herein in its entirety. According to some embodiments, a compound of formula (2) is endowed with a low solubility in water.
[0126] According to some embodiments, a peptide proteasome inhibitor may be a compound of formula (3:
where: X is oxygen, NH or N(C 1-6 alkyl); Y is NH, N(C1-6 alkyl), O, or C(R9)2; Z is O or C(R9)2; R1, R2, R3, and R4 are all hydrogen; each R5, R6, R7, R8, and R9 is independently selected from hydrogen, C1-6 alkyl, C1-6 hydroxylalkyl, C1-6 alkoxyalkyl, aryl, and C1-6 aralkyl, each of which is optionally substituted with one or more of an alkyl, amide, amine, carboxylic acid or a pharmaceutically acceptable salt thereof, carboxyl ester, thiol, and thioether; m is an integer from 0 to 2; en is an integer from 0 to 2; or a pharmaceutically acceptable salt thereof.
[0127] According to some embodiments, X is O. According to some other embodiments, Y is N(C1-6 alkyl), O, or C(R9)2. According to some embodiments, Z is C(R9)2. According to some other embodiments, R5, R6, R7, and R8 are independently selected from C1-6 alkyl, C1-6 hydroxylalkyl, and C1-6 aralkyl and each R9 is comprised of hydrogen. For example, R6and R8are independently C1-6alkyl, R5and R7are independently C1-6aralkyl, and each R9is H. According to some embodiments, n is 0 or 1.
[0128] According to some embodiments, X is O and R5, R6, R7, and R8 are independently selected from C1-6 alkyl, C1-6 hydroxylalkyl, and C1-6 aralkyl. For example, R6and R8are independently C1-6 alkyl and R5and R7 are independently C1-6 aralkyl.
[0129] According to some embodiments, X is O, R6 and R8 are both isobutyl, R5 is phenylethyl, and R7 is phenylmethyl.
[0130] According to some embodiments, R5, R6, R7, and R8 are independently selected from hydrogen, C1-6 alkyl, C1-6 hydroxylalkyl, C1-6 alkoxyalkyl, aryl, and C1-6 aralkyl, each of which is optionally substituted with a group selected from alkyl, amide, amine, carboxylic acid or a pharmaceutically acceptable salt thereof, carboxylic ester, thiol, and thioether. According to some embodiments, at least one of R5 and R7 is C1-6 aralkyl substituted with alkyl such as perhaloalkyl. For example, R7 is C1-6 aralkyl substituted with trifluoromethyl.
[0131] According to some embodiments, Y is selected from N - alkyl, O, and CH2. According to some of those embodiments, Z is CH2, and m is both 0. According to some embodiments, Z is CH2, m is 0, and n is 2 or 3. According to some embodiments, Z is O, m is 1 , and it is 2.
[0132] According to some embodiments, a compound of the formula (3) is comprised of a compound of the formula (4:
wherein: X is O, NH, or N-alkyl, preferably O; R1, R2, R3, and R4 are all hydrogen; and R5, R6, R7, and R8 are independently selected from hydrogen, C1-6 alkyl, C1-6 hydroxylalkyl, C1-6 alkoxyalkyl, aryl, and C1-6 aralkyl, each of which is optionally substituted with a selected group from amide, amine, carboxylic acid or a pharmaceutically acceptable salt thereof, carboxylic ester, thiol, and thioether, or a pharmaceutically acceptable salt thereof.
[0133] According to some embodiments, R5, R6, R7, and R8 are independently selected from C1-6 alkyl, C1-6 hydroxylalkyl, and C1-6 aralkyl. For example, R6and R8are independently C1-6 alkyl and R5and R7 are independently C1-6 aralkyl.
[0134] According to some embodiments, X is O and R5, R6, R7, and R8 are independently selected from C1-6 alkyl, C1-6 hydroxylalkyl, and C1-6 aralkyl. For example, R6 and R8 are independently C1-6 alkyl and R5 and R7 are independently C1-6 aralkyl.
[0135] According to some embodiments, X is O, R6 and R8 are both isobutyl, R5 is phenylethyl, and R7 is phenylmethyl.
[0136] According to some embodiments, a compound of formula III is endowed with the following stereochemistry:

[0137] Non-limiting examples of a compound of formulas (3) and (4) can be found, for example, in U.S. Patent No. 7,417,042, which is incorporated herein in its entirety by reference. According to some embodiments, a compound of formulas (3) or (4) is endowed with a low solubility in water.
[0138] According to some embodiments, the peptide proteasome inhibitor is a compound of the formula (5):
or a pharmaceutically acceptable salt thereof. The compound of formula (5) is also known as carfilzomib.
[0139] Any of the compounds described in this context can be isolated in an amorphous condition or crystalline form. The preparation and purification of crystalline compounds such as those provided herein may be carried out as is known in the art, for example, as described in US publication no. 2009/0105156, which is incorporated herein by reference in its entirety.
[0140] According to some embodiments, a crystalline compound of formula (5) is substantially pure. According to some embodiments, the melting point of the crystalline compound of formula (5) is in the range of about 200°C to about 220°C, about 205°C to about 215°C, about 211 °C to about 213°C, or even about 212°C. According to some embodiments, a crystalline compound of formula (5) can have a melting point of about 205°C to about 215°C. For example, the compound can have a melting point of about 211°C to about 213°C. According to some embodiments, the DSC of a crystalline compound of formula (5) is endowed with an endothermic maximum temperature under about 212°C, for example, resulting from melting and decomposition of the crystalline form of the compound.
[0141] An X-ray powder diffraction pattern of a crystalline compound of formula (5) is endowed with characteristic diffraction peaks expressed in degrees 2 theta (2θ). For example, a crystalline compound of formula (5) may have a characteristic peak expressed in degrees 2θ under 6.10. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under 9.32. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under 10.10. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under 12.14. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under 13.94. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under 18.44. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under 20.38. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under 23.30. According to some embodiments, a crystalline compound of formula (5) is provided with an X-ray powder diffraction pattern comprising from 2 to 8 characteristic peaks expressed in degrees 2θ under 6.10; 9.32; 10.10; 12.14; 13.94; 18.44; 20.38; and 23.30. For example, a crystalline compound of formula (5) may be provided with an X-ray powder diffraction pattern comprising characteristic peaks expressed in degrees 2θ under 6.10; 9.32; 10.10; 12.14; 13.94; 18.44; 20.38; and 23.30.
[0142] According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under about 6.1. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ at about 9.3. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under about 10.1. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under about 12.1. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under about 13.9. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under about 18.4. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under about 20.4. According to some embodiments, a crystalline compound of formula (5) is endowed with a characteristic peak expressed in degrees 2θ under about 23.3. According to some embodiments, a crystalline compound of formula (5) is provided with an X-ray powder diffraction pattern comprising from 2 to 8 characteristic peaks expressed in degrees 2θ under about 6.1; 9.3; 10.1; 12.1; 13.9; 18.4; 20.4; and 23.3. According to some embodiments, a crystalline compound of formula (5) is provided with an X-ray powder diffraction pattern comprising characteristic peaks expressed in degrees 2θ under about 6.1; 9.3; 10.1; 12.1; 13.9; 18.4; 20.4; and 23.3.
[0143] According to some embodiments, a crystalline compound of formula (5) is provided with an X-ray powder diffraction pattern which is provided with characteristic peaks expressed in degrees 2θ under 6.10; 8.10; 9.32; 10.10; 11.00; 12.14; 12.50; 13.64; 13.94; 17.14; 17.52; 18.44; 20.38; 21.00; 22.26; 23.30; 24.66; 25.98; 26.02; 27.84; 28.00; 28.16; 29.98; 30.46; 32.98; 33.22; 34.52; and 39.46.
[0144] According to some embodiments, a crystalline compound of formula (5) is endowed with an X-ray powder diffraction pattern which is endowed with characteristic peaks expressed in degrees 2θ under 6.1; 8.1; 9.3; 10.1; 11.0; 12.1; 12.5; 13.6; 13.9; 17.1; 17.5; 18.4; 20.4; 21.0; 22.3; 23.3; 24.7; 25.9; 26.0; 27.8; 28.0; 28.2; 30.0; 30.5; 33.0; 33.2; 34.5; and 39.5.
[0145] The X-ray powder diffraction (XRPD) analysis was performed using a Shimadzu XRD-6000 X-ray powder diffractometer using Cu Kα radiation. The instrument is equipped with a long fine-focus X-ray tube. The tube voltage and amperage were set to 40 kV and 40 mA, respectively. The divergence and dispersion grooves were set under 1° and the receiving groove was set under 0.15 mm. Diffracted radiation was detected by means of a NAI scintillation detector. A continuous θ-2θ scan under 3°/min was used. (0.4 sec./0.02°) from 2.5 to 40° 2θ. A silicon pattern was analyzed to verify instrument alignment. Data were collected and analyzed using XRD-6100/7000 v.5.0. The samples were prepared for analysis by placing them in an aluminum support with a silicon insert.
[0146] According to some embodiments, a crystalline compound of formula (5) is a crystalline salt of a compound of formula (5). For example, a crystalline salt of a compound of formula (5) can be selected from the group consisting of: citrate, tartrate, trifluoroacetate, methanesulfonate, toluenesulfonate, hydrochloride, and hydrobromide salts. According to some embodiments, a crystalline salt of a compound of formula (5) is a citrate salt. According to some embodiments, the crystalline solid can exist in the form of a co-crystal.
[0147] According to some embodiments, a crystalline citrate salt of a compound of formula (5) is substantially pure. According to some embodiments, the melting point of the crystalline citrate salt of a compound of formula (5) is in the range of about 180 to about 190°C, for example, about 184 to about 188°C. . According to some embodiments, the DSC of a crystalline citrate salt of a compound of formula (5) is endowed with a sharp endothermic maximum at about 187°C, e.g., resulting from melting and decomposition of the crystalline form.
[0148] According to some embodiments, a crystalline compound of formula (5) is provided with an X-ray powder diffraction pattern comprising two or more characteristic peaks expressed in degrees 2θ under 4.40; 7.22; 9.12; 12.36; 13.35; 14.34; 15.54; 16.14; 16.54; 17.00; 18.24; 18.58; 19.70; 19.90; 20.30; 20.42; 21.84; 22.02; 23.34; 23.84; 24.04; 24.08; 24.48; 24.76; 25.48; 26.18; 28.14; 28.20; 28.64; 29.64; 31.04; 31.84; 33.00; 33.20; 34.06; 34.30; 34.50; 35.18; 37.48; 37.90; and 39.48. For example, a crystalline citrate salt of a compound of formula (5) may be provided with an X-ray powder diffraction pattern which is provided with characteristic peaks expressed in degrees 2θ under 4.40; 7.22; 9.12; 12.36; 13.35; 14.34; 15.54; 16.14; 16.54; 17.00; 18.24; 18.58; 19.70; 19.90; 20.30; 20.42; 21.84; 22.02; 23.34; 23.84; 24.04; 24.08; 24.48; 24.76; 25.48; 26.18; 28.14; 28.20; 28.64; 29.64; 31.04; 31.84; 33.00; 33.20; 34.06; 34.30; 34.50; 35.18; 37.48; 37.90; and 39.48. Pharmaceutical Compositions
[0149] The methods provided in this context include the manufacture and use of pharmaceutical compositions which include any of the compounds provided in this context. Also included are the pharmaceutical compositions themselves.
According to some embodiments, the compounds provided herein may be formulated as described in U.S. Patent No. 7,737,112.
[0151] Also provided in this context are cyclodextrin complexing methods for preparing a pharmaceutical composition of a peptide proteasome inhibitor (e.g. a compound of the formula (1) - (5) or a salt, solvate, hydrate, co-crystal, or pharmaceutically acceptable polymorph thereof). The method comprises providing a first combination that is provided with a protease inhibitor peptide, a cyclodextrin, and water, wherein the first combination is heterogeneous and the proteasome inhibitor peptide or salt has a low solubility in the first combination. The method further comprises altering the pH of the first combination to form a second combination, wherein the solubility of the peptide proteasome inhibitor in the second combination is greater than the solubility of the peptide proteasome inhibitor in the first combination. For example, the method can include contacting the first combination with an acid to form the second combination. The second combination may still be heterogeneous and may still facilitate a sufficient increase in solubility such that the complexation process can be initiated and progressed. This can allow a greater part of the inhibitor to be complexed while in a heterogeneous mixture form through partial complexation, or complete the complexation by forming a homogeneous solution. In the case of a heterogeneous complexed mixture, once a desired extent of solubilization and complexation has been achieved, excess solids can be removed by filtration to provide a homogeneous solution.
[0152] The term "complexation" as used in this context refers to the formation of an intermolecular inclusion complex, or an intermolecular association, in solution and between one or more peptide proteasome inhibitors and one or more molecules of cyclodextrin. The inclusion and or association provides utility as a mechanism to substantially increase the concentration of inhibitor(s) that can be achieved in the aqueous solution compared to aqueous phase dissolution at a similar pH range without the complexing agent (i.e., one or more cyclodextrin molecules).
[0153] A complexed or associated state is evidenced when a dissolved concentration of the inhibitor(s) is measurable, by means of a conventional analytical method such as HPLC, and the concentration substantially exceeds that which can be obtained by means of dissolution of the inhibitor(s) in water without the cyclodextrin(s) present. The complexed or associated solution of the inhibitor(s) and cyclodextrin(s) can be prepared in such a way as to exceed the concentration in the aqueous solution where the cyclodextrin(s) are absent that is useful for the formulation. of a medicinal compound of convenient injection volume and dispensed dose. Furthermore, the complexed or associated solution of inhibitor(s) exhibits physical stability (or otherwise described as metastability) in that the inhibitor remains in a homogeneous solution (no precipitation or crystallization of solid particles) for longer periods of time. than are typical of inhibitor solutions without the presence of a cyclodextrin. As a result of this prolonged duration of a clear remaining solution, no crystal nucleation and subsequent depletion of super saturation occur for all practical conditions of use as a medicinal formulation.
[0154] Many small molecule organic compounds drugs are endowed with pH-dependent solubility. Often an appropriate pH range for administering a drug (such as by injection where the tolerable pH range is generally considered to be 3 - 10.5 for intravenous administration) is not the same pH where sufficient solubility of the drug can be found in the aqueous solution (eg under pH 2 or less). To allow a pharmaceutically useful concentration level of drug in solution under an acceptable and tolerable pH range for administration (eg by injection), complexation or association of the drug with cyclodextrin(s) as claimed in this context constitutes a practical method. It can increase the concentration in the solution that can be reached within the tolerable pH range for administration. This increase could be, for example, from initially 1 - 100 micrograms per milliliter without cyclodextrin(s), increased to 500 - 10,000 micrograms per milliliter with cyclodextrin(s). Complexation or association thus constitutes a technology which allows an otherwise hardly water-soluble compound to be sufficiently solubilized and developed as a pharmaceutically useful compound. Those skilled in the art understand that the amount of cyclodextrin(s) required to achieve a desired concentration and state of physical stability can vary. For this reason, the amount of cyclodextrin can be determined on an individual combination basis using widely known methods.
[0155] For basic drug molecules, solubility is usually increased at lower pH. This also presents stability and shelf life challenges in some cases if used without complexing or associating agents such as cyclodextrin(s). For example, sufficient solubility can be achieved by lowering the pH of a solution with an acid, however such lowering of pH can lead to degradation reactions from acidic conditions. See Table 1 for intrinsic aqueous solubility data for carfilzomib, which shows some moderate increase in solubility with decreasing pH. Table 1: Aqueous solubility of carfilzomib as a function of pH, without cyclodextrins

[0156] There are numerous acid-mediated degradation reaction pathways for small molecule drugs and biological molecules, such as hydrolysis of amides into smaller inactive peptide fragments, or hydrolytic opening of functional epoxide halves. Acid-mediated degradation products may lack pharmacological activity, and may be toxic or genotoxic compounds even at residual levels. Complexing or associating compounds under pH conditions where significant degradation is avoided further expands the utility of cyclodextrins to facilitate the clinical and commercial development of compounds that are endowed with pH dependent stability characteristics.
[0157] In order to balance the conflicting needs to avoid acid-mediated degradation side reactions that occur under low pH with increasing rate of complexation by decreasing the pH, a unique pH condition was found. Surprisingly, the pH of an aqueous solution obtained by adding certain concentrations of acids, eg citric acid (around pH 2.5 to 3.0), was found to be sufficient to lower the pH to initiate complexation without initiating significant levels of degradation side reactions. In this state, the inhibitor was partially solubilized through the pH condition, but not entirely. As a result, there was a heterogeneous mixture (eg, a slurry) of the inhibitor partially dissolved in the aqueous solution of cyclodextrin and citric acid, and partially existing as solid particles (crystals) of the inhibitor. Over time (typically several hours to a day), the dissolved inhibitor fraction will become complex or be associated with the cyclodextrin. This process will allow more of the inhibitor's solid particles to dissolve and then become complexed. Over time, mass transfer can occur from the initially solid phase inhibitor, to the dissolved phase inhibitor, to a complexed dissolved state of the inhibitor - cyclodextrin. Most commonly, cyclodextrin complexation is achieved through the formation of a homogeneous solution of the compound to be complexed. For carfilzomib, the formation of a homogeneous solution will require a very low pH where degradation reactions will occur, such as those with strong acidic hydrogen chloride that form potential genotoxic impurities. In this case, it was practical and useful to carry out the complexation process in a heterogeneous state under the milder pH condition of 2.5 - 3.0 using citric acid, a weak carboxylic acid. Once the target concentration of complexed inhibitor is reached. The slurry complexation process was completed by filtering out any undissolved solid particles of the inhibitor. The resulting homogeneous solution can then be pH adjusted as needed to a suitable pH range for intravenous administration (eg, pH 3.5, using aqueous sodium hydroxide). In addition, the homogeneously pH adjusted complexed solution could be diluted with water to the exact concentration desired for the next step of product manufacturing and to ensure the label strength of the medicinal product was accurate.
[0158] The combined effect of cyclodextrin concentration and pH on complexation has a greater solubilization capacity than if each technique were used alone. The extents of solubilization are relatively independent of temperature which is convenient for the manufacture to maintain cold conditions most preferred for the manufacture of sterile product and to minimize any temperature-accelerated degradation reactions.
[0159] The second combination includes complexes of a peptide proteasome inhibitor and cyclodextrin(s). These complexes are endowed with improved water solubility over the proteasome inhibitor peptide alone. For example, homogeneous solutions of a compound of formula (5) (carfilzomib) can be obtained at a pharmaceutically useful pH (eg about 3.5) and at higher concentrations (eg about 5 mg/ml) than could be obtained without the cyclodextrin and the complexation processes between the compound and the cyclodextrin provided in this context.
[0160] In addition to increasing the solubility of a peptide proteasome inhibitor in solution, formulations prepared by the methods provided herein result in pharmaceutical solutions that are endowed with surprising stability. Even though the high concentrations of proteasome inhibitor achieved through the processing methods provided in this context cannot be expected to be thermodynamically stable, the solutions have been shown to be unaffected by storage temperature (eg solutions can be stable from -20° C to 25°C), freeze-thaw cycling, and lyophilization and reconstitution. The stability of the complexed peptide proteasome inhibitor and cyclodextrin complex is sufficient to tolerate adjustments to pH following complexation without precipitation. This solution stability allows the use of the complexed material over an acceptable pH range for injection, product stability and other pharmaceutical purposes. For this reason, pharmaceutical compositions prepared by the methods provided in this context may, for pharmaceutical uses, be considered supersaturated solutions that do not precipitate or decrease in concentration to a significant extent during their use in any number of medicinal applications (for example , a final pharmaceutical composition may be stable for a range of at least 1-5 days, and potentially longer).
[0161] A first combination can be prepared by adding a solid form of the peptide proteasome inhibitor to an aqueous solution of one or more cyclodextrins. According to some embodiments, when the proteasome inhibitor peptide is a compound of formula (5) or a pharmaceutically acceptable salt thereof, the concentration of the one or more cyclodextrins in the solution ranges from less than about 1% to potentially as much as the solubility limit of the cyclodextrin(s), for example, about 40%. According to some embodiments, for manufacturing purposes, the concentration of the one or more cyclodextrins in the solution ranges from about 15% to about 30%. According to some embodiments, for purposes of reconstituting the finished drug product as a solution for therapeutic administration or ready for further dilution prior to administration, the concentration of the one or more cyclodextrins in the solution ranges from about 5% to about 15% , for example, approximately 10%. Upon further dilution, this concentration may be further reduced as may be considered appropriate for injection or other drug delivery pathways. The molar ratio of the one or more cyclodextrins in solution to the compound of formula (5) ranges from about 0.5 to about 100. According to some embodiments, this ratio exists as a molar excess of cyclodextrin to shift the equilibrium of stability complexation to prefer the complexed state over the uncomplexed state. For example, the molar ratio (moles of cyclodextrin divided by moles of proteasome inhibitor) ranges from about 10 to about 20. According to some embodiments, the weight/weight ratio of cyclodextrin to proteasome inhibitor ranges from about 30 up to about 60. Excessive foaming of cyclodextrin solutions can be a complication for manufacturing processes. Surprisingly, the addition of the peptide proteasome inhibitor to the aqueous solution of cyclodextrin(s) can control the foaming of the solution in the first combination.
[0162] According to some embodiments, a first combination consists essentially of a peptide proteasome inhibitor, a cyclodextrin, and water.
[0163] The solid form of the peptide proteasome inhibitor added to the cyclodextrin and water solution may be a crystalline form of the compound as described in this context (for example, the compound may be polymorphic or a specific polymorph as described in this context). According to some embodiments, the solid form of the peptide proteasome inhibitor is amorphous.
[0164] The first combination is heterogeneous (eg a suspension or slurry). Such a solution can be characterized by the total weight percent solids and particle size distribution of the solution. For example, where the peptide proteasome inhibitor is a compound of formula (5) or a pharmaceutically acceptable salt thereof, the first combination may be provided with a total weight percent solids from about 1% to about 45% ( for example, from about 1% to about 40%; from about 1% to about 35%; from about 1% to about 30%; from about 1% to about 25%; from about 1% to about 20%; from about 1% to about 15%; from about 1% to about 10%; from about 5% to about 45%; from about 10% to about 45 %; from about 12% to about 45%; from about 15% to about 45%; from about 20% to about 45%; from about 25% to about 45%; from about 30 % to about 45%; from about 35% to about 45%; from about 5% to about 35%; from about 10% to about 40%; from about 15% to about 37% ; and from about 18% to about 36%). According to some embodiments, the first blend can be provided with a weight percent solids from about 20% to about 33%. According to some embodiments, the first blend can be provided with a weight percent solids from about 30% to about 33%. Over time of manufacture the ratio of solids that are dissolved versus the undissolved ratio can vary depending on solubility and extent of complexation. Initially, the one or more cyclodextrins are very soluble in water, and the inhibitor is sparingly soluble, thus remaining mostly as a heterogeneous mixture or slurry.
[0165] According to some embodiments, the first combination is provided with a particle size distribution with primary particles of varying diameter from less than about 1 micrometer to about 300 micrometers or more (e.g., from about 1 µm to about 200 µm; from about 1 µm to about 150 µm; from about 1 µm to about 125 µm; from about 1 µm to about 100 µm; from about 1 µm to about 50 µm ; from about 1 µm to about 10 µm; from about 5 µm to about 300 µm; from about 25 µm to about 300 µm; from about 50 µm to about 300 µm; from about 60 µm up to about 300 µm; from about 75 µm to about 300 µm; from about 100 µm to about 300 µm; from about 125 µm to about 300 µm; from about 150 µm to about 300 µm; from about 200 µm to about 300 µm; from about 225 µm to about 300 µm; from about 250 µm to about 300 µm; from about 5 µm to about of 150 µm; from about 25 µm to about 200 µm; from about 50 µm to about 125 µm; from about 10 µm to about 100 µm; from about 75 µm to about 225 µm; and from about 100 µm to about 200 µm). Primary particles can exist in the form of distinct particles or in the form of agglomerates comprised of one or many primary particles. Agglomerates of primary particles can be substantially larger in size than primary particles. It is therefore useful to include a high energy mixing device, such as a high shear mixer (often configured as a rotor stator mixer), in addition to a general suspension impeller mixer. The high energy mixer runs from about 5 minutes to about 90 minutes (for example, about 5 minutes to about 80 minutes; about 5 minutes to about 75 minutes; about 5 minutes to about about 60 minutes; about 5 minutes to about 45 minutes; about 5 minutes to about 30 minutes; about 10 minutes to about 90 minutes; about 15 minutes to about 90 minutes; about 30 minutes to about about 90 minutes; about 45 minutes to about 90 minutes; about 50 minutes to about 90 minutes; about 75 minutes to about 90 minutes; about 15 minutes to about 75 minutes; about 20 minutes to about about 70 minutes; about 30 minutes to about 70 minutes; about 45 minutes to about 75 minutes; and about 10 minutes to about 45 minutes), for example, in the course of time of about 60 minutes it will break large agglomerates into primary particles dispersed in the solution of cyclodextrin. Additional mixing can help break up primary particles into smaller fragments of primary particles. This process design facilitates a robust method in which the mixing system(s) achieve(s) essentially dispersed primary particles of varying size distribution from less than about 1 micrometer to about 30 micrometers, e.g. to about 10 micrometers regardless of the size distribution and degrees of agglomeration of the proteasome-inhibiting solids. For this reason the charge-to-charge variation of the proteasome inhibitor particle size distribution is not of great importance to process performance as the mixing system(s) reduces the agglomerates and primary particles typically within of the preferable particle size distribution range. For example, the first blend may have a particle size distribution initially less than about 1 micrometer to about 10,000 micrometers for a size distribution of less than about 1 micrometer to about 30 micrometers after application of the high energy mixing step.
[0166] According to some embodiments, the first combination is substantially free of organic solvent. For example, the water in the first combination could be water for injections (WFI). In accordance with some embodiments, the first combination is substantially buffer free (e.g., the first combination is devoid of a buffer acid or buffer base).
[0167] The method may further comprise mixing the first blend before changing the pH of the first blend such as by using a high shear mixer and a regular impeller. The general mixer can be operated, for example, at any speed of rotation sufficient to keep the suspension of particles out of the bottom of the mixing tank. Mixing speed is a function of tank and impeller geometry among other factors and is sufficiently determined by those skilled in the art through the visual appearance of the slurry or solution mixture. Similarly, the speed of the high-shear mixer is dependent, for example, on the diameter of the mixing element, the geometry of the stator, the width of the gap, and other factors. The energy input to the slurry can be determined through theoretical calculations or through empirical measurements. Alternatively, the high speed shear mixing speed and duration of high speed operation required can be determined by those skilled in the art by microscopic observation of the slurry samples following combinations of various mixing speeds and time. Once deagglomeration and primary particles have been reduced, excess speed and high speed shear mixing time can be applied without detriment to the process. For example, in accordance with some embodiments, mixing may include subjecting agitation to the first blend at a speed from about 500 rpm to about 10,000 rpm. For example, high shear mixing can be carried out at a speed of about 2000 rpm to about 3500 rpm. For smaller and larger tank and mixer diameters, the relevant speeds can vary significantly.
[0168] Mixing of the first combination can be carried out at a temperature from about 0°C to about 30°C (for example, from about 5°C to about 25°C; from about 10°C to about 10°C; about 30°C; from about 15°C to about 25°C; from about 5°C to about 20°C; from about 2°C to about 22°C; and from about 20 °C to about 30°C). According to some embodiments, mixing of the first blend is carried out for a time sufficient to achieve a variable particle size distribution from less than about 1 micrometer to about 30 micrometers in the first blend. Mixing of the first combination is carried out for a period of time varying from about 30 minutes to about 90 minutes, for example, 60 minutes.
[0169] Changing the pH of the first solution may include raising or lowering the pH of the first solution by adding an acid or a base. According to some embodiments, when the proteasome inhibitor peptide is comprised of a compound of formula (5) or a pharmaceutically acceptable salt thereof, the pH of the first combination ranges from about 4 to about 7. According to some embodiments , an acid is added to change the pH, such as an inorganic acid or an organic acid. Non-limiting examples of acids include lactic acid, acetic acid, formic acid, citric acid, oxalic acid, uric acid, succinic acid, maleic acid, fumaric acid, benzoic acid, tartaric acid, glycine hydrochloride, bisulfate (existing, for example, in the form of a sodium, potassium, or ammonium salt), and phosphoric acid or phosphate salts. According to some embodiments, the acid is comprised of an organic acid. According to some embodiments, the acid is comprised of citric acid. A suitable acid can have one or more pKa values, with a first pKa ranging from about -6 to about +5. For example, the acid has a first pKa in the range of about +1 to about +4.5. According to some embodiments, the acid has a first pKa in the range of about +1.5 to about +3.5. See, for example, Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Eds. P. Heinrich Stahl and Camille G. Wermuth, Verlag Helvetica Chimica Acta (Switzerland) 2002, 336-341, which is incorporated by reference in its entirety in this context.
[0170] According to some embodiments, for compounds in which the solubility and complexation is in effect increased by increasing the pH, the pH is changed by adding a base, for example, an inorganic or organic base. Non-limiting examples of inorganic bases include sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, magnesium hydroxide, and sodium, potassium or ammonium carbonate or bicarbonate salts. Non-limiting examples of organic bases include pyridine, methyl amine, triethyl amine, imidazole, benzimidazole, histidine, and a phosphazene base. An organic base can have a pKb or a first pKb ranging from about -6 to about +10. The relevant pKa or pKb of the acid or base respectively needs to be within a sufficient range to achieve a certain increase in inhibitor solubility. According to some embodiments, the acid or base is added in the form of an aqueous solution (for example, an aqueous solution of an acid).
[0171] Changing the pH of the first solution results in the formation of a second combination in which the peptide proteasome inhibitor is more soluble than in the first combination. For example, a peptide proteasome inhibitor can be at least about 10% more soluble (for example, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 400%, at least about 500%, at least about 1000%, at least about 1250%, at least about 1500%, at least about 2000%, at least about 2500%, at least at least about 3000%, at least about 4000%, at least about 5000%, at least about 5500%, at least about 6000%, at least about 7500%, at least about 8000%, at least about 9000%, and at least about 10,000% more soluble) in the second combination as compared to the solubility of the inhibitor in the first combination.
[0172] Without intending to be bound by theory, changing the pH of the first combination initiates the complexation of one or more cyclodextrins and the peptide proteasome inhibitor. Increased complexation alters the balance of the solution, causing further complexation and ultimately resulting in the solubilization of the peptide proteasome inhibitor. Following addition of the additive, the second combination can be mixed for a sufficient time to achieve either a sufficiently solubilized heterogeneous mixture with inhibitor that has been brought to the complex condition, or a third homogeneous combination in which the entire inhibitor has been complexed and none remain in the form of undissolved solids. For example, the concentration of the proteasome inhibitor in the third combination can range from about 1 to about 18 mg/ml, for example, about 2 to about 8 mg/ml, about 4 to about 6 mg/ml. ml, or about 5 to about 6 mg/ml. According to some embodiments, the pH of the third combination ranges from about 1.5 to about 4, for example, about 2 to about 3.5 or about 2.5 to about 3.5. Considering the cases where sufficient complexation can be achieved without necessarily dissolving and complexing the entire mass of inhibitor present as a slurry, it may be useful to terminate the complexation process once a desired concentration. In these cases, a homogeneous solution of the desired inhibitor concentration can be achieved by filtering the excess solids content of the inhibitor. This leaves the inhibitor that has been brought to the complex condition and the cyclodextrin(s) in the form of a functionally stable solution, even though the dynamic equilibrium of complexation and solubilization may imply a thermodynamically non-stable state.
[0173] The complexation of the peptide proteasome inhibitor in the third combination is at least about 50% (eg at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 94%, at least about 95 %, at least about 96%, at least about 97%, at least about 98%, at least about 99%). According to some embodiments, the complexation of the peptide proteasome inhibitor in the third combination is at least about 99%. Conceptually, for some combinations of cyclodextrin concentration, inhibitor concentration, pH, and complexation time, a 100% complex solution of the inhibitor can be prepared, in which the mixture becomes homogeneous.
[0174] According to some embodiments, the method described above is performed in a single vessel. For example, mixing of the complexation slurry in the method can be accomplished using a probe-type high-shear mixer (e.g., a homogenizer) within a jacketed, temperature-controlled mixing tank.
[0175] Provided herein is a method for preparing a pharmaceutical composition of a compound of the formula (5) or a pharmaceutically acceptable salt form thereof, the method comprising providing a first combination of a compound of the formula ( 5), a cyclodextrin, and water, wherein the first combination is heterogeneous and the compound or salt has a solubility that is low in the first combination. According to some embodiments, the cyclodextrin is SBECD and water is comprised of WFI. The method further comprises contacting the first combination with an acid to form a second combination, wherein the compound is more soluble in the second combination than in the first combination. According to some embodiments, the acid is comprised of a citric acid (for example, an aqueous citric acid solution).
[0176] A non-limiting example of the method includes providing a first combination that includes water (eg, WFI), SBECD, and the compound of formula (5) or a pharmaceutically acceptable salt thereof in a vessel. According to some embodiments, water and SBECD are mixed prior to addition to the compound. The first combination can be mixed until a heterogeneous solution is obtained (for example, from about 30 to about 90 minutes, from about 40 to about 80 minutes, and from about 50 to about 70 minutes). According to some embodiments, the first blend is blended for about 60 minutes. In the event that the compound agglomerates in the first combination, the particle size for any agglomerated compound may be reduced. Once a heterogeneous mixture (eg a slurry) is obtained, an acid (eg an organic acid such as citric acid) is added to the first blend to prepare the second blend. In some embodiments the acid is added in the form of an aqueous solution. Mixing can then be continued until a third homogeneous blend is prepared, or for shorter periods of time it remains in the form of a heterogeneous blend, with a desired extent of complexation and solubilization being obtained. In accordance with some embodiments, mixing of the second combination is conducted for a period of time varying from about 1 to about 48 hours, e.g., up to 18 hours. According to some embodiments, mixing of the second combination is conducted for about 12 hours. For example, mixing can be carried out for about six hours. According to some embodiments, the concentration of the compound in the third combination ranges from about 1 to about 15 mg/ml (for example, from about 3 to about 12 mg/ml, from about 4 to about 8 mg /ml, about 5 mg/ml). According to some embodiments, the method is used to prepare a solution of the compound for injection. According to other embodiments, the method is used to prepare a solution for lyophilization in the form of a finished aseptic pharmaceutical product that can be stored, transported, and reconstituted with water or other vehicle when ready for injection into a patient.
[0177] Pharmaceutical compositions obtained in the form of sterile products using the procedures described in this context are typically manufactured by applying aseptic techniques and sterile filtration before filling into the main packaging unit (for example, glass vials), to unless the preparation involved a sterilization step and no contamination occurs prior to use.
[0178] The peptide proteasome inhibitor composition dissolved in aqueous buffer or aqueous solution, for example, following sterile filtration may optionally be lyophilized (in a contaminant-free and contaminant-free container) and reconstituted in appropriate aqueous diluent exactly before use. In accordance with some embodiments, the diluent is comprised of sterile water for injection (WFI). According to some embodiments, the diluent is comprised of a sterile buffer (e.g., a citrate buffer). According to some embodiments, the diluent comprises citric acid.
[0179] In the compositions provided in this context, a source of pH control is comprised of a buffer. Typically, a buffer is present in the form of an acid or a base and its base or conjugate acid, respectively. According to one embodiment, the buffer salt range is 1100 mM. For example, the buffering salt range can be on the order of 5-50 mM (eg about 10 mM (in solid formulations, the amount of buffer is selected to produce this concentration after reconstitution / dilution)) . The buffer concentration and pH of the solution can be chosen to provide a balance of solubility and stability that is optimal.
[0180] Examples of buffers that are suitable include mixtures of weak acids and alkali metal salts (eg sodium, potassium) of the conjugate base of weak acids such as sodium tartrate and sodium citrate. According to some embodiments, the buffer is comprised of sodium citrate/citric acid.
[0181] The solubilization of poorly water-soluble drugs through cyclodextrin complexation has been extensively studied. Cyclodextrins are comprised of cyclic oligosaccharides that consist of 6, 7, or 8 glucose units (α-CD, β-CD, and Y—CD) joined via α-1,4 bonds. The inner diameters of α-CD, β-CD, and Y-CD are approximately 5A, 6A, and 8A, respectively. The inner cavity is relatively hydrophobic due to CH2 and ether groups, while the outer cavity, which consists of primary and secondary hydroxyl groups, is more polar. The water inside the cavity tends to be replaced by more non-polar molecules. The ability of cyclodextrins to form non-covalent inclusion complexes with molecules that partially fit within their non-polar cavity leads to drug solubilization.
[0182] Two water-soluble β-CD derivatives of pharmaceutical interest are sulfobutyl ether beta-cyclodextrin (SBECD) and hydroxypropyl beta-cyclodextrin (HPCD), which have been shown to be safe and well tolerated. Both SBECD (brand name Captisol®) and HPCD (brand name Kleptose®) are used in commercially available intravenous products.
[0183] Cyclodextrins, as provided herein, include alpha-, beta- and gamma-cyclodextrin. According to one embodiment, the one or more cyclodextrins are comprised of a substituted or unsubstituted β-cyclodextrin, present, for example, under from 5-35% (w/v). According to some embodiments, the amount of cyclodextrin is about 25% (w/v). according to a certain embodiment, the amount of a cyclodextrin in a formulation suitable for injection is about 10% (w/v). according to another embodiment, the one or more cyclodextrins are comprised of a substituted β-cyclodextrin. Substituted cyclodextrins increase the solubility of cyclodextrin and decrease the toxic effects associated with unsubstituted cyclodextrins. Examples of substituted β-cyclodextrins include those substituted with one or more hydrophilic groups, such as monosaccharides (eg, glycosyl, maltosyl), carboxyalkyl (eg, carboxylmethyl, carboxyethyl), hydroxyalkyl-substituted (eg, hydroxyethyl, 2-hydroxypropyl ) and sulfoalkyl ether substituted beta-cyclodextrin. Particularly beta-cyclodextrins that are suitable include hydroxypropyl beta-cyclodextrin (HPBCD) and sulfobutyl ether beta-cyclodextrin (SBECD). According to some embodiments, the cyclodextrin is SBECD. Nevertheless, it is understood that typically any substitution for the cyclodextrin, including substitution through hydrophobic groups such as alkyls, will improve its aqueous solubility by disrupting the hydrogen bonding network within the solid cyclodextrin crystal structure, lowering it. forms the structure energy of the solid. It is believed that the degree of substitution is not of great importance; nevertheless, according to some embodiments, the degree of substitution is at least 1% and typically 2% to 10%, such as 3% to 6%.
[0184] According to some embodiments, one or more cyclodextrins may be used. For example, a mixture of two or more cyclodextrins can be used to complex a peptide proteasome inhibitor provided herein. According to some embodiments, captisol and kleptose can be used to complex a peptide proteasome inhibitor such as carfilzomib.
[0185] The inventors have found that it may be advantageous to minimize the amount of chloride ion (or other nucleophilic anions) in the methods and pharmaceutical compositions described in this context.
[0186] According to some embodiments, at least one of the one or more cyclodextrins (added to the first combination) is comprised of a low chloride cyclodextrin. As used in this context, a "low chloride cyclodextrin" refers to a cyclodextrin that is endowed with less than or equal to .05% w/w sodium chloride, or if a source of chloride that unless (or in addition to) sodium chloride is/are present, a "low chloride cyclodextrin" refers to a cyclodextrin that is endowed with a sodium ion content that is less than or equal to an amount of chloride that would be present in a cyclodextrin that is endowed with 0.05% w/w sodium chloride. According to some embodiments, the low chloride cyclodextrin is a low chloride SBECD. The determination of chloride concentration can be carried out by a variety of methods that are known in the art (for example, for cyclodextran obtained commercially from the manufacturer's product specification, for example, by means of gravimetric techniques, for example, through potentiometric techniques).
[0187] According to some embodiments, the amount of chloride ion present is sufficiently low so as to provide a shelf life of 2 years when stored at 2-8 degrees C.
[0188] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 2.0.
[0189] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 1.5.
[0190] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 1.2.
[0191] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 1.0.
[0192] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 0.9.
[0193] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 0.8.
[0194] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 0.7.
[0195] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 0.6.
[0196] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 0.5.
[0197] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 0.4.
[0198] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 0.3.
[0199] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 0.2.
[0200] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination is no greater than 0.1.
[0201] According to some embodiments, the molar ratio of chloride ions to the compound in the first combination ranges from about 0.2 to 1.2 (for example, 0.3 to 1.2, for example 0, 2 to 0.4, eg 0.3 to 0.4, eg 0.32).
[0202] In embodiments, the molar ratios of chloride ion to compound described in this context can also be in the second and/or third combination.
[0203] In the methods described in this context, the compositions provided in this context (for example, cyclodextrin solutions, first combinations, second combinations, third combinations, and pharmaceutical compositions) are provided with low concentrations of any strong nucleophilic ion (for example, ion of chloride, bromide ion, fluoride ions, and iodide ion). For example, the solution can have a nucleophilic ion concentration of up to and including 8.5x10-3 M. According to some embodiments, solutions that are low in nucleophilic ion can be purchased commercially or can be prepared using technology that is known in the art, including, for example, nanofiltration, ultrafiltration, diafiltration, ion exchange chromatography, reverse osmosis, and electrolysis.
[0204] According to some embodiments, a pharmaceutical composition in the form that is provided herein comprises up to and including 8.5x10-3 M of a nucleophilic ion. According to some embodiments, the nucleophilic ion is present in the form of a salt, eg a sodium salt, the nucleophilic salt may exist in solution with other cations other than sodium (eg hydrogen cations, potassium, magnesium and calcium). According to some embodiments, the pharmaceutical composition as provided herein comprises up to 8.5x10-3M of a nucleophilic ion. For example, the pharmaceutical composition comprises less than 8.5x10-3M of a nucleophilic ion.
[0205] In the methods described in this context, the compositions that are provided in this context (for example, cyclodextrin solutions, first combinations, second combinations, third combinations, and pharmaceutical compositions) are provided with low concentrations of chloride ion. For example, a solution can be provided with a chloride ion concentration of up to and including 0.03% (w/v) (eg, 0 to 0.03%; 0.01 to 0.03%; 0.015 to 0.03%; 0.02 to 0.03%; 0.025 to 0.03%; 0 to 0.025%; 0 to 0.2%; 0 to 0.01%; 0.005% to 0.025%; and 0.015% to 0.025%). According to some embodiments, solutions that are endowed with low chloride ion content can be purchased commercially or can be prepared using technology that is known in the art, including, for example, nanofiltration, ultrafiltration, diafiltration, ion exchange chromatography , reverse osmosis, and electrolysis.
[0206] According to some embodiments, the pharmaceutical composition as provided herein comprises up to and including 0.03% (w/v) of a chloride ion. According to some embodiments, the chloride ion is present in the form of a salt, eg sodium chloride, the chloride salt may exist in solution with cations other than sodium (eg hydrogen cations , potassium, magnesium and calcium). According to some embodiments, the pharmaceutical composition as provided herein comprises up to 0.03% (w/v) of a chloride ion. For example, the pharmaceutical composition comprises less than 0.03% (w/v) of a chloride ion.
[0207] In the methods described in this context, the compositions that are provided in this context (for example, cyclodextrin solutions, first combinations, second combinations, third combinations, and pharmaceutical compositions) are provided with low concentrations of sodium chloride. For example, a solution may have a sodium chloride concentration of up to and including 0.05% (w/v) (eg, 0 to 0.05%; 0.01 to 0.05%; 0.015 to 0.05%; 0.02 to 0.05%; 0.025 to 0.05%; 0.03 to 0.05%; 0.04 to 0.05%; 0 to 0.045%; 0 to 0.04% ; 0 to 0.035%; 0 to 0.03%; 0 to 0.025%; 0 to 0.2%; 0 to 0.01%; 0.01% to 0.04%; 0.025% to 0.045%; and 0 .02% to 0.03%). According to some embodiments, solutions that are endowed with low sodium chloride content can be purchased commercially or can be prepared using technology that is known in the art, including, for example, nanofiltration, ultrafiltration, diafiltration, ion exchange chromatography , reverse osmosis, and electrolysis.
[0208] According to some embodiments, a pharmaceutical composition as provided herein comprises up to and including 0.05% (w/v) of sodium chloride. According to some embodiments, a pharmaceutical composition as provided herein comprises up to 0.5% (w/v) of sodium chloride. For example, a pharmaceutical composition comprises less than 0.05% (w/v) sodium chloride.
[0209] According to some embodiments, a solution of a cyclodextrin that is endowed with a low concentration of any strong nucleophilic ion (eg, chloride ion, bromide ion, fluoride ion, and iodide ion) is used to formulating a peptide proteasome inhibitor (for example a compound of formula (1) to (5) or a pharmaceutically acceptable salt thereof) provided herein. For example, cyclodextrin solutions used to formulate a peptide proteasome inhibitor can have a nucleophilic ion concentration of up to and including 8.5x10-3 M. Such solutions can be purchased commercially or can be prepared using technology that is known in the art, including, for example, nanofiltration, ultrafiltration, diafiltration, ion exchange chromatography, reverse osmosis, and electrolysis.
[0210] According to some embodiments, a solution of one or more cyclodextrins used to formulate a peptide proteasome inhibitor comprises up to and including 8.5x10-3 M of a nucleophilic ion. According to some embodiments, the nucleophilic ion is present in the form of a salt, eg a sodium salt, the nucleophilic salt may exist in solution with other non-sodium cations (eg hydrogen cations , potassium, magnesium and calcium). According to some embodiments, a pharmaceutical composition as provided herein comprises up to 8.5x10-3M of a nucleophilic ion. For example, a pharmaceutical composition comprises less than 8.5x10-3M of a nucleophilic ion.
[0211] According to some embodiments, a solution of a cyclodextrin that is endowed with a low concentration of chloride ion is used to formulate a peptide proteasome inhibitor (for example, a compound of formula (1) through (5) or a pharmaceutically acceptable salt thereof) provided herein. For example, cyclodextrin solutions used to formulate a peptide proteasome inhibitor can be provided with a chloride ion concentration of up to and including 0.03% (w/v) (eg 0 to 0.03%; 0, 01 to 0.03%; 0.015 to 0.03%; 0.02 to 0.03%; 0.025 to 0.03%; 0 to 0.025%; 0 to 0.2%; 0 to 0.01%; 0.005 % to 0.025%; and 0.015% to 0.025%). These solutions can be purchased commercially or can be prepared using technology that is known in the art, including, for example, nanofiltration, ultrafiltration, diafiltration, ion exchange chromatography, reverse osmosis, and electrolysis.
[0212] According to some embodiments, a solution of one or more cyclodextrins used to formulate a peptide proteasome inhibitor comprises up to and including 0.03% (w/v) of a chloride ion. According to some embodiments, the chloride ion is present in the form of a salt, e.g., sodium chloride, the chloride salt may exist in solution with other non-sodium cations (e.g., cations of hydrogen, potassium, magnesium and calcium). According to some embodiments, a pharmaceutical composition as provided herein comprises up to 0.03% (w/v) of a chloride ion. For example, a pharmaceutical composition comprises less than 0.03% (w/v) of a chloride ion.
[0213] According to some embodiments, a solution of a cyclodextrin that is endowed with a low concentration of sodium chloride is used to formulate a peptide proteasome inhibitor (for example, a compound of formula (1) through (5) or a pharmaceutically acceptable salt thereof) provided herein. For example, cyclodextrin solutions used to formulate a peptide proteasome inhibitor can be provided with a sodium chloride concentration of up to and including 0.05% (w/v) (eg, 0 to 0.05%; 0, 01 to 0.05%; 0.015 to 0.05%; 0.02 to 0.05%; 0.025 to 0.05%; 0.03 to 0.05%; 0.04 to 0.05%; 0 to 0.045%; 0 to 0.04%; 0 to 0.035%; 0 to 0.03%; 0 to 0.025%; 0 to 0.2%; 0 to 0.01%; 0.01% to 0.04% ; 0.025% to 0.045%; and 0.02% to 0.03%). These solutions can be purchased commercially or can be prepared using technology that is known in the art, including, for example, nanofiltration, ultrafiltration, diafiltration, ion exchange chromatography, reverse osmosis, and electrolysis.
[0214] According to some embodiments, a solution of one or more cyclodextrins used to formulate a peptide proteasome inhibitor comprises up to and including 0.05% (w/v) sodium chloride. According to some embodiments, a pharmaceutical composition as provided herein comprises up to 0.03% (w/v) of sodium chloride. For example, a pharmaceutical composition comprises less than 0.03% (w/v) sodium chloride.
[0215] In addition to the production of stable, highly concentrated solutions of a peptide proteasome inhibitor, formulations prepared by the methods provided in this context can be obtained without the chemical degradation and stability limitations of other complexation and formulation methods. For example, the methods provided in this context avoid the use of strong acids (eg HCl) to lower the pH during complexation. Although lowering the pH of the formulation to a value less than 2 can facilitate dissolution of the peptide proteasome inhibitor and produce a homogeneous solution prior to complexation, the acidity of the solution can result in degradation of the peptide proteasome inhibitor. In addition, the peptide proteasome inhibitor contains a keto epoxide functional group, and the inhibitor is susceptible to hydrolysis by strong nucleophilic ions, such as chloride ions. The hydrolysis of the epoxide ring and acid-catalyzed nucleophilic opening of the epoxide moiety is a compound degradation pathway. For example, degradation of a compound of formula (5) results in the formation of a chlorohydrin degradation product (CDP) impurity. Based on its structure, this degradation product is classified as an alkylator, therefore global regulatory authorities consider it to be a potentially genotoxic impurity. Furthermore, according to some embodiments, the chloride ion can also degrade the epoxide resulting in the formation of a chlorohydrin adduct. As illustrated in Example 2, the reduction of chloride ion levels in a formulation of a compound of formula (5) can minimize or eliminate these hydrolysis pathways resulting in improved stability and product quality. Nevertheless, in using the methods provided in this context, the use of such strong acids and nucleophilic ions is avoided and for that reason the degradation of the peptide proteasome inhibitor by these degradation products can be significantly reduced and, in some cases, may be even eliminated.
[0216] Pharmaceutical compositions that are 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 dispersions. For intravenous administration, carriers that are suitable 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 must be fluid to the extent that easy syringability exists. The composition must 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 that contains, 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, through the use of a coating such as lecithin, through maintenance of the required particle size in the case of dispersion, and through the use of surface-active agents. Preventing the action of microorganisms can be achieved through various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including an agent which delays absorption in the injectable solutions, for example, aluminum monostearate and gelatin.
[0217] Sterile injectable solutions can be prepared by including the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by including the active compound in a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation comprise freeze-drying or freeze-drying (lyophilization), which provides a powder of the active ingredient plus any additional desired ingredient from a previously filtered sterile solution thereof. .
[0218] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules, for example, gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, lozenges and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose and/or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
[0219] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.
[0220] Systemic administration of a therapeutic compound as described in this context may also be through transmucosal or transdermal means. For transmucosal or transdermal administration, penetration agents that are appropriate for the barrier to be permeated are used in the formulation. Such penetration agents are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, balms, gels, or creams as are generally known in the art.
[0221] Pharmaceutical compositions can also be prepared in the form of suppositories (for example, with normal suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
[0222] In addition, intranasal distribution is possible, as described, inter alia, in Hamajima et al., Clin. Immunol. Immunopathol., 88(2), 205-10 (1998). Liposomes (for example, as described in U.S. Patent No. 6,472,375) and microencapsulation can also be used. Biodegradable segmentable microparticle delivery systems (for example, such as described in U.S. Patent No. 6,471,996) can also be used.
[0223] According to one embodiment, therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid clearance from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biocompatible, biodegradable polymers can be used, such as vinyl acetate and ethylene, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. These formulations can be prepared using standard techniques, or obtained commercially, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, such as are described in U.S. Patent No. 4,522,811.
[0224] The pharmaceutical composition can be administered at once, or it can be divided into a number of smaller doses to be administered at time intervals. It is understood that the precise dosage and duration of treatment is a function of the ailment being treated and can be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data. It should be noted that concentrations and dosage values may also vary according to the severity of the condition to be alleviated. It should also be understood that for any particular patient, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person who administers or supervises the administration of the compositions, and that the concentration ranges exposed in this context they are merely exemplary and are not intended to limit the scope or practice of the claimed compositions.
[0225] Dosage forms or compositions containing a compound as described in this context in the range of 0.005% to 100% with the balance consisting of the non-toxic carrier can be prepared. Methods for preparing these compositions are known to those skilled in the art. The contemplated compositions may contain from 0.001%-100% active ingredient, according to one embodiment 0.1-95%, according to another embodiment 75-85%.
[0226] Pharmaceutical compositions may be included in a container, package, or dispenser together with instructions for administration. Methods of Use
[0227] The biological consequences of proteasome inhibition are numerous. Proteasome inhibition has been suggested as a prevention and/or treatment of a multitude of disorders including, but not limited to, proliferative disorders, neurotoxic/degenerative disorders, Alzheimer's disease, ischemic conditions, inflammation, autoimmune disorders. diseases, HIV, cancers, organ graft rejection, septic shock, antigen presentation inhibition, decreased viral gene expression, parasitic infections, conditions associated with acidosis, macular degeneration, pulmonary conditions, muscle wasting disorders, fibrotic disorders, bone and hair growth disorders. For that reason, pharmaceutical formulations for very potent proteasome-specific compounds, such as the epoxide ketone class of molecules, provide a means of administering a drug to a patient and treating these conditions.
[0228] Under the cellular level, accumulation of polyubiquitinated proteins, cellular morphological changes, and apoptosis have been reported in the treatment of cells with various proteasome inhibitors. Proteasome inhibition has also been suggested as a possible antitumor therapeutic strategy. The fact that epoxomycin was initially identified in a screen for antitumor compounds validates the proteasome as an antitumor chemotherapeutic target. For that reason, these compositions are useful for the treatment of cancer.
[0229] Both in vitro and in vivo models have shown that malignant cells, in general, are 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 proliferating malignant cells in the proteasome system to rapidly remove proteins (Rolfe et al., J. Mol. Med. (1997) 75:5-17; Adams, Nature (2004) 4:349-360). For that reason, there is provided herein a method of treating cancers which comprises administering to a patient in need of such treatment a therapeutically effective amount of a peptide proteasome inhibitor as provided herein.
[0230] As used in this context, the term "cancer" includes, but is not limited to, solid and blood-transmissible tumors. Cancer refers to diseases of the blood, bones, organs, skin tissue and the vascular system, which include, but are not limited to, cancers of the bladder, blood, bone, brain, breast, cervix, breast, colon. , endometrium, esophagus, eye, head, kidney, liver, lung, lymph nodes, mouth, neck, ovaries, pancreas, prostate, rectum, kidney, skin, stomach, testicles, throat, and uterus. Specific cancers include, but are not limited to, leukemia (acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, breast cancers. mature B cells, small cell lymphocytic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenstrom's macroglobulinemia), splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disorders, heavy chain diseases , extranodal marginal zone B cell lymphoma (MALT lymphoma), nodal marginal zone B cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse B cell lymphoma, mediastinal (thymic) large B cell lymphoma , intravascular large B-cell lymphoma, primary effusion lymphoma and Burkitt's lymphoma/leukemia, natural killer (NK) cell neoplasms and celiac disease. mature T squid, T cell prolymphocytic leukemia, large T cell granular lymphocytic leukemia, aggressive NK cell leukemia, adult T cell leukemia/lymphoma, extranodal T cell / NK lymphoma, enteropathy-type T cell lymphoma, lymphoma of hepatosplenic T cells, blastic NK cell lymphoma, fungoid mycosis (Sezary's syndrome), primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, unspecified peripheral T cell lymphoma and large cell anaplastic lymphoma, lymphoma Hodgkin's syndrome (nodular sclerosis, mixed cellularity, rich, depleted or non-depleted lymphocytes, nodular predominant lymphocytes), myeloma (multiple myeloma, indolent myeloma, latent myeloma), chronic myeloproliferative disease, myelodysplastic/myeloproliferative disease, myelodysplastic syndromes associated with lymphoproliferative disorders, immunodeficiency, histiocytic cell neoplasms as and dendritic, mastocytosis, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell malignant tumor, bone myeloma diseases, osteosarcoma, breast cancer (hormone-dependent, hormone-independent), gynecological cancers (cervical, endometrial, fallopian tube , gestational, ovarian, peritoneal, uterine, vaginal and vulvar trophoblastic disease), basal cell carcinoma (BCC), squamous cell carcinoma (SCC), malignant melanoma, bulging dermatofibrosarcoma, Merkel cell carcinoma, Kaposi's sarcoma, astrocytoma, pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor, oligodendrogliomas, ependymoma, glioblastoma multiforme, mixed gliomas, oligoastrocytomas, medulloblastoma, retinoblastoma, neuroblastoma, germinoma, teratoma, mesothelioma, mesothelioma, mesothelioma, mesothelioma, mesothelioma, mesothelioma, mesothelioma, mesothelioma, mesothelioma, mesothelioma, mesothelioma gastroenteropancreatic neuroendocrine (GEP-NET), carcinoid, endocrine tumor for Ncreatic (PET), colorectal adenocarcinoma, colorectal carcinoma, aggressive neuroendocrine tumor, mucinous adenocarcinoma leiomyosarcoma, Signet Ring adenocarcinoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, hemangioma, liver adenoma, focal nodular hyperplasia (regenerative lung cancer), 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, independent, androgen independent, androgen dependent, hormone-insensitive), and soft tissue sarcomas (fibrosarcoma, malignant fibrous histiocytoma, dermatofibrosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, hemangiosarcoma, synovial sarcoma, malignant peripheral nerve sheath tumor / extraskeletal nervesarcoma, osteofibrosarcoma).
[0231] According to some embodiments, a peptide proteasome inhibitor as provided herein, or pharmaceutical composition comprising the same, can be administered to treat multiple myeloma in a patient. For example, multiple myeloma can include refractory and/or refractory multiple myeloma.
[0232] Many tumors of hematopoietic and lymphoid tissues are characterized by an increase in the proliferation of cells, or of a particular cell type. Chronic myeloproliferative disorders (CMPDs) are hematopoietic stem cell disorders characterized by proliferation in the spinal cord of one or more of the myeloid lineages, resulting in increased numbers of granulocytes, red blood cells, and/or platelets in the peripheral blood. Thus, the use of a proteasome inhibitor to treat these disorders is attractive and is being examined (Cilloni et al., Haematologica (2007) 92: 1124-1229). DMARDs can include chronic myelogenous leukemia, chronic eosinophilic leukemia, polycythemia vera, chronic idiopathic myelofibrosis, essential thrombocythemia, and unclassifiable chronic myeloproliferative disorder. Provided herein is a method of treating CMPD which comprises administering to a patient in need of such treatment an effective amount of the proteasome inhibitor compound disclosed herein.
[0233] Myelodysplastic/myeloproliferative disorders, such as chronic myelomonocytic leukemia, atypical chronic myelogenous leukemia, juvenile myelomonocytic leukemia, and unclassifiable myelodysplastic/myeloproliferative disorder, are characterized by bone marrow hypercellularity due to proliferation in one or more of the myeloid lineages. Proteasome inhibition with a composition described in this context can serve to treat these myelodysplastic/myeloproliferating disorders by providing an effective amount of the composition to a patient in need of such treatment.
[0234] Myelodysplastic syndromes (MDS) refer to a group of hematopoietic stem cell disorders characterized by dysplasia and ineffective haematopoiesia in one or more of the major myeloid cell lines. Targeting NF-kB with a proteasome inhibitor in these hematologic malignancies induces apoptosis, thereby killing the malignant cell (Braun et al. Cell Death and Differentiation (2006) 13:748-758). Further provided in this context is provided for a method of treating MDS which comprises administering to a patient in need of such treatment an effective amount of a compound provided herein. MDS includes refractory anemia, refractory anemia with chained sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess blasts, unclassifiable myelodysplastic syndrome, and myelodysplastic syndrome associated with an isolated (5q) chromosome abnormality.
[0235] Mastocytosis is a 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 associated hematologic non-mast cell lineage disease (SM-AHNMD), aggressive systemic mastocytosis (ASM), mast cell leukemia ( MCL), mast cell sarcoma (MCS) and extra cutaneous mast cell tumor. Further provided in this context is a method of treating mastocytosis which comprises administering an effective amount of the compound disclosed herein to a patient diagnosed with mastocytosis.
[0236] The proteasome regulates NF-KB, which in turn regulates genes involved in the immune and inflammatory response. For example, NF-KB is required for expression of the immunoglobulin light chain K gene, the IL-2 receptor α chain gene, the class I major histocompatibility complex gene, and a number of cytokine genes that encode , for example, granulocyte colony-stimulating factor IL-2, IL-6, , and IFN-β (Palombella et al., Cell (1994) 78:773-785). Thus, provided in this context are methods for affecting the expression level of IL-2, MHC-I, IL-6, TNFα, IFN-β or any of the other aforementioned proteins, with each method comprising administering to a patient an effective amount of a proteasome inhibitory composition disclosed in this context.
[0237] Also provided in this context is provided for a method of treating an autoimmune disorder in a patient comprising administering a therapeutically effective amount of the compound described in this context. An “autoimmune disease” in this context is an illness or disorder that arises from and is directed against an individual's own tissue. Examples of autoimmune disorders or disorders include, but are not limited to, inflammatory responses such as inflammatory skin disorders which include psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; responses associated with inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveite; 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 mellitus (for example Type I diabetes mellitus or insulin dependent diabetes mellitus); multiple sclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjorgen's syndrome; juvenile diabetes; and immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes typically found in tuberculosis, sarcoidosis, polymyositis, granulomatosis, and vasculitis; pernicious anemia (Addison's disease); diseases involving leukocyte diapedesis; inflammatory disorder of the central nervous system (CNS); multiple organ injury syndrome; hemolytic anemia (which includes, but is not limited to, cryoglobinemia or Coombs positive anemia); myasthenia gravis; antigen-antibody complex-mediated diseases; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrinopathies; Reiter's disease; hard-man syndrome; Beheet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathies; immunological thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia.
[0238] The immune system sifts autologous cells that are virally infected, have undergone oncogenic transformation, or have foreign peptides on their surface. Intracellular proteolysis generates small peptides for presentation to T lymphocytes to induce MHC class I-mediated immune responses. Accordingly, provided in this context is a method of using a proteasome inhibitor that is provided as an immunomodulatory agent to inhibit or alter the presentation of antigens in a cell, which comprises exposing the cell (or administering to a patient) to compound described in this context. Specific embodiments include a method of treating graft or transplant related ailments, such as graft versus host disease or host versus graft disease in a patient, which comprises administering a therapeutically effective amount of the compound described in this context. The term “graft” as used in this context refers to biological material derived from a donor for transplantation into a recipient. Grafts include miscellaneous materials such as, for example, isolated cells, such as island cells; tissue such as the amniotic membrane of a newborn, bone marrow, hematopoietic precursor cells, and ocular tissue such as corneal tissue; and organs such as skin, heart, liver, spleen, pancreas, thyroid lobe, lung, kidney, tubular organs (eg, intestine, blood vessels, or esophagus). Tubular organs can be used to replace damaged parts of the esophagus, blood vessels, or bile duct. Skin grafts can be used not only for burns, but also as a dressing for an injured bowel or to close certain defects such as a herniated diaphragm. The graft is derived from any source originating from mammals, including humans, whether from cadavers or living donors. In some cases, the giver and recipient understand the same patient. According to some embodiments, the graft is comprised of bone marrow or an organ such as the heart and graft donor and host are matched for HLA class II antigens.
[0239] Histiocytic and dendritic cell neoplasms are derived from phagocytes and accessory cells, which have important roles in the processing and presentation of antigens to lymphocytes. Depletion of proteasome content in dendritic cells has been shown to alter their antigen-induced responses (Chapatte et al. Cancer Res. (2006) 66:5461-5468). According to some embodiments, the composition provided in this context can be administered to a patient with histiocytic or dendritic cell neoplasm. Histiocytic and dendritic cell neoplasms include histiocytic sarcoma, Langerhans cell histiocytosis, Langerhans cell sarcoma, interdigitating dendritic cell sarcoma/tumor, follicular dendritic cell sarcoma/tumor, and unspecified dendritic cell sarcoma.
[0240] Proteasome inhibition has been shown to be beneficial in treating diseases in which a cell type is proliferating and immunological disorders; therefore, according to some embodiments, treatment of lymphoproliferative disorders (LPD) associated with primary immunological disorders (PID) is provided which comprises administering an effective amount of the exposed compound to a patient in need thereof. The most common clinical definitions of immunodeficiency associated with an increasing incidence of lymphoproliferating disorders, including neoplasms and B-cell and T-cell lymphomas, are primary immunodeficiency syndromes and other primary immune disorders, human immunodeficiency virus (HIV) infection, iatrogenic immunosuppression in patients who received solid organ or bone marrow allografts, and iatrogenic immunosuppression associated with methotrexate treatment. Other PIDs commonly associated with, but not limited to, LPDs are ataxia telangiectasia (AT), Wiskott-Aldrich syndrome (WAS), common variable immunodeficiency (CVID), severe combined immunodeficiency (SCID), linked lymphoproliferative disorder to X (XLP), Nijmegen disruption syndrome (NBS), hyper IgM syndrome, and autoimmune lymphoproliferative syndrome (ALPS).
[0241] Proteasome inhibition has also been associated with inhibition of NF-KB activation and stabilization of p53 levels. In this way, compositions provided in this context can also be used to inhibit NF-kB activation, and stabilize p53 levels in cell culture. Since NF-kB is a key regulator of inflammation, it is an attractive target for anti-inflammatory therapeutic intervention. Thus, compositions provided herein may be useful for treating conditions associated with inflammation, which include, but are not limited to, COPD, psoriasis, asthma, bronchitis, emphysema, and cystic fibrosis.
[0242] The exposed compositions can be used to treat conditions mediated directly by proteolytic function of the proteasome, such as muscle wasting, or mediated indirectly through proteins that are processed through the proteasome, such as NF-KB. The proteasome participates in the rapid elimination and post-translational processing of proteins (eg, enzymes) involved in cellular regulation (eg, cell cycle, gene transcription, and metabolic pathways), intercellular communication, and the immune response (eg, , antigen presentation). Specific examples discussed below include the β-amyloid protein and regulatory proteins such as cyclins and NF-kB transcription factor.
[0243] According to some embodiments, a composition provided in this context is useful for the treatment of neurodegenerative disorders and conditions, including, but not limited to, stroke, ischemic nervous system injury, neural trauma ( for example, percussive brain injury, spinal cord injury, and traumatic injury to the nervous system), multiple sclerosis, and other immune-mediated neuropathies (eg, Guillain-Barre syndrome and its variants, acute motor axonal neuropathy, acute inflammatory demyelinating polyneuropathy , and Fisher's Syndrome), HIV/AIDS dementia complex, axonomiay, 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 dementias (such as Hunting supranuclear palsy ton or progressive), focal cortical atrophy syndromes (such as primary aphasdia), toxic metabolic dementias (such as chronic hypothyroidism or B12 deficiency), and dementias caused by infections (such as syphilis or chronic meningitis).
[0244] Alzheimer's disease is characterized by extracellular deposits of β-amyloid protein (β-AP) in senile plaques and cerebral vessels. β-AP is a 39- to 42-amino acid peptide fragment derived from an amyloid protein precursor (APP). At least three isoforms of APP are known (amino acids 695, 751, and 770). Alternative mRNA splicing generates the isoforms; normal processing affects a part of the β-AP sequence, thus preventing the generation of β-AP. Abnormal protein processing via the proteasome is believed to contribute to the abundance of β-AP in the Alzheimer's brain. The APP processing enzyme in rats contains about ten different subunits (22kDa-32kDa). The 25 kDa subunit is endowed with an N-terminal sequence of X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser, which is identical to the human macropain β-subunit (Kojima, S. et al. ., Fed. Eur. Biochem. Soc., (1992) 304:57-60). The APP processing enzyme cleaves at the Gln15--Lys16 bond; in the presence of the calcium ion, the enzyme also cleaves at the Met-1--Asp1 bond, and the Asp1--Ala2 bind to release the extracellular domain of β-AP.
[0245] One embodiment, therefore, is a method of treating Alzheimer's disease, which includes administering to a patient an effective amount of the composition provided herein. This treatment includes reducing the rate of β-AP processing, reducing the rate of β-AP plaque formation, reducing the rate of β-AP generation, and reducing the clinical signs of Alzheimer's disease. .
[0246] Also provided in this context are provided methods of treatment of diseases of cachexia and loss of muscle mass. The proteasome degrades many proteins in maturing reticulocytes and growing fibroblasts. In cells lacking insulin or serum, the rate of proteolysis is practically doubled. Inhibiting the proteasome reduces proteolysis, thereby reducing both muscle protein loss and the nitrogen load on the kidneys or liver. Peptide proteasome inhibitors as provided herein are useful for treating conditions such as cancer, chronic infectious diseases, fever, muscle disuse (atrophy) and denervation, nerve damage, withdrawal, renal dysfunction associated with acidosis, and liver deficiency. See, for example, Goldberg, U.S. Pat. No. 5,340,736. Treatment methods include: reducing the rate of muscle protein breakdown in a cell; reduction in the rate of intracellular peotein degradation; reducing the rate of p53 protein degradation in a cell; and inhibition of the growth of p53-related cancers. each of these methods includes contacting a cell (in vivo or in vitro, e.g., a muscle in a patient) with an effective amount of a pharmaceutical composition disclosed herein.
[0247] Fibrosis is the excessive and persistent formation of scar tissue resulting from the hyperproliferating growth of fibroblasts and is associated with activation of the TGF-β signaling pathway. Fibrosis involves extensive extracellular matrix deposition and can occur within virtually any tissue or across many different tissues. Normally, the level of intracellular signaling protein (Smad) that activates transcription of target genes in TGF-β stimulation is regulated through proteasome activity. Nevertheless, accelerated degradation of TGF-β signaling components has been observed in cancers and other hyperproliferating conditions. Thus, in accordance with certain embodiments, a method of treating hyperproliferative conditions such as diabetic retinopathy, macular degeneration, diabetic nephropathy, glomerulosclerosis, IgA nephropathy, cirrhosis, biliary atresia, congestive heart failure, scleroderma, fibrosis is provided. radiation-induced, and pulmonary fibrosis (idiopathic pulmonary fibrosis, collagen vascular disease, sarcoidosis, interstitial lung diseases, and extrinsic lung disorders). The treatment of burn victims is often hampered by fibrosis; therefore, in accordance with some embodiments an inhibitor provided herein may be administered via topical or systemic administration to treat burns. Wound closure following surgery is often associated with disfiguring scarring, which can be prevented by inhibiting fibrosis. Thus, according to certain embodiments, a method for the prevention or reduction of scars is provided in this context.
[0248] Another protein processed through the proteasome is NF-KB, a member of the Rel protein family. The Rel family of transcriptional activator proteins can be divided into two groups. A first group requires proteolytic processing, and includes p50 (NF-kB1, 105 kDa) and p52 (NF-k2, 100 kDa). The second group does not require proteolytic processing and includes p65 (RelA, Rel (c-Rel), and RelB). Both homo- and heterodimer can be formed by members of the Rel family; NF-kB, for example, is a p50-p65 heterodimer. After phosphorylation and ubiquitination of IkB and p105, the two proteins are degraded and processed, respectively, to produce active NF-kB that translocates from the cytoplasm to the nucleus. Ubiquitinated p105 is also processed via purified proteasomes ( Palombella et al., Cell (1994) 78:773-785). Active NF-kB forms a stereospecific enhancer complex with other transcriptional activators and, for example, HMG I(Y), inducing selective expression of a particular gene.
[0249] NF-kB regulates genes involved in the immune and inflammatory response, and mitotic events. For example, NF-kB is required for expression of the immunoglobulin light chain k gene, the IL-2 receptor α chain gene, the class I major histocompatibility complex gene, and a number of cytokine genes. which encode, 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 for affecting the expression level of IL-2, MHC-I, IL-6, TNFα, IFN-β, or any of the other proteins mentioned above, with each method including administering to a patient an effective amount of a composition exposed in this context. Complexes that include p50 are rapid mediators of acute inflammatory and immune responses ( Thanos, D. and Maniatis, T., Cell (1995) 80:529-532).
[0250] NF-KB also participates in the expression of cell adhesion genes encoding selectin E, selectin P, ICAM, and VCAM-1 (Collins, T., Lab. Invest. (1993) 68:499-508 ). In accordance with some embodiments, a method of inhibiting cell adhesion (e.g., selectin E, selectin P, ICAM, or VCAM-1 mediated cell adhesion) is provided, which includes contacting a cell with (or administering to a patient) an effective amount of a pharmaceutical composition disclosed in this context.
[0251] Ischemia and reperfusion injury result in hypoxia, a condition in which there is a deficiency of oxygen reaching the tissues of the body. This condition causes an increased degradation of Ik-Bα, thereby resulting in the activation of NF-kB. It has been shown that the seriousness of the injury resulting in hypoxia can be reduced with the administration of a proteasome inhibitor. Accordingly, provided herein is a method of treating an ischemic condition or reperfusion injury which comprises administering to a patient in need of such treatment an effective amount of a compound disclosed in this context. Examples of such conditions or injuries include, but are not limited to, acute coronary syndrome (vulnerable plaques), arterial occlusive disease (cardiac, cerebral, peripheral arterial and vascular co-occlusions), atherosclerosis (coronary sclerosis, disease coronary artery), infarctions, heart failure, pancreatitis, myocardial hypertrophy, stenosis, and restenosis.
[0252] NF-kB also specifically binds to the enhancer/promoter of HIV. When compared to Nef from mac239, the HIV regulatory protein, Nef from pbj14, differs by two amino acids in the region that controls protein kinase binding. The protein kinase is believed to signal phosphorylation of IKB, triggering IKB degradation through the ubiquitin-proteasome pathway. After degradation, NF-KB is released into the nucleus, thereby enhancing HIV transcription (Cohen, J., Science, (1995) 267:960). Provided herein is a method of inhibiting or reducing HIV infection in a patient, and a method of increasing the level of expression of viral genes, with each method including administering to the patient an effective amount of a composition as set out in this context. .
[0253] Viral infections contribute to the pathology of many illnesses. Cardiac conditions such as ongoing myocarditis and dilated cardiomyopathy have been linked to coxsackievirus B3. In a comparative whole-genome microarray analysis of infected mouse hearts, specific proteasome subunits were evenly regulated in the hearts of mice that developed chronic myocarditis (Szalay et al, Am J Pathol 168:1542-52, 2006). Some viruses use the ubiquitin-proteasome system in the viral entry step where the virus is released from the endosome into the cytosol. Mouse hepatitis virus (MHV) belongs to the Coronaviridae family, which also includes 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 a decrease in viral replication, correlating with reduced viral titer compared to that of untreated cells. Human hepatitis B virus (HBV), a member of the Hepadnaviridae virus family, similarly requires virally encoded envelope proteins to propagate. Inhibition of the proteasome degradation pathway causes a significant reduction in the amount of secreted envelope proteins (Simsek et al, J Virol 79:12914-12920, 2005). In addition to HBV, other hepatitis viruses (A, C, D and E) can also use the ubiquitin-proteasome degradation pathway for segregation, morphogenesis and pathogenesis. Therefore, in accordance with certain embodiments, there is provided a method of treating viral infection, such as SARS or hepatitis A, B, C, D and E, which comprises contacting a cell with (or administering to a patient) an effective amount of the compound disclosed in this context.
[0254] The overproduction of polysaccharide (LPS)-induced cytokines such as TNFα is considered to be central to the processes associated with septic shock. Furthermore, it is generally accepted that the first step in cell activation through 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 signal transduction may be an important therapeutic target in the treatment or prevention of sepsis (Qureshi, N. et al. , J. Immun. (2003) 171: 1515-1525). Therefore, in accordance with certain embodiments, compositions as provided herein can be used for the inhibition of TNFα to prevent and/or treat septic shock.
[0255] Intracellular proteolysis generates small peptides for presentation to T lymphocytes to induce MHC class I-mediated immune responses. The immune system screens out autologous cells that are virally infected or have undergone oncogenic transformation. One embodiment is comprised of a method of inhibiting antigen presentation in a cell, which includes exposing the cell to a composition described in this context. Another embodiment is comprised of a method of suppressing a patient's immune system (e.g., inhibiting transplant rejection, allergy, asthma), which includes administering to the patient an effective amount of a composition described in this context. Compositions provided herein can also be used to treat autoimmune disorders such as lupus, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disorders such as ulcerative colitis and Crohn's disease.
[0256] Another embodiment is comprised of a method to alter the repertoire of antigenic peptides produced by the proteasome or other Ntn with multicatalytic activity. 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 cell surfaces different from what would be produced and presented either without any enzyme inhibition, or with, for example, selective inhibition of chymotrypsin-like activity of the proteasome.
[0257] Certain proteasome inhibitors block both the degradation and processing of NF-KBubiquitinated in vitro and in vivo. Proteasome inhibitors also block IKB-α degradation and NF-kB activation ( Palombella, et al. Cell (1994) 78:773-785; and Traenckner, et al., EMBO J. (1994) 13:5433 -5441). According to some embodiments, a method of inhibiting IkB-α degradation is provided, which includes contacting the cell with a composition described in this context. Another embodiment is comprised of a method for reducing the cellular content of NF-kB in a cell, muscle, organ, or patient, which includes contacting the cell, muscle, organ or patient with a composition described in this context.
[0258] Other eukaryotic transcription factors that require proteolytic processing include general transcription factor TFIIA, herpes simplex virus accessory protein VP16 (host cell factor), virus-inducible IFN regulatory factor 2 protein, and protein 1 binding membrane binding sterol regulatory element.
[0259] Further provided in this context are methods for affecting cyclin-dependent eukaryotic cell cycles, which include exposing a cell (in vitro or in vivo) to a composition as set out in this context. Cyclins are proteins involved in controlling cell cycles. The proteasome participates in the degradation of cyclins. Examples of cyclins include mitotic cyclins, G1 cyclins, and B cyclins. Degradation of cyclins allows a cell to move from one cell cycle stage (eg, mitosis) to another (eg, division). All cyclins are believed to be associated with the p34cdc2 protein kinase or related kinases. The proteolysis targeting signal is localized to amino acids 42-RAALGNISEN-50 (destruction box). There is evidence that cyclin is converted to a form vulnerable to a ubiquitin ligase or that a specific cyclin ligase is activated during mitosis (Ciechanover, A., Cell, (1994) 79:13-21). Inhibition of the proteasome inhibits cyclin degradation, and therefore inhibits cell proliferation, for example, in cyclin-related cancers (Kumatori et al., Proc. Natl. Acad. Sci. USA (1990) 87:7071-7075 ). A method of treating a proliferating disorder in a patient (e.g., cancer, psoriasis or restenosis) which includes administering to the patient an effective amount of a composition disclosed in this context is provided in this context. Also provided herein is a method of treating cyclin-related inflammation in a patient, which includes administering to a patient a therapeutically effective amount of a composition described in this context.
[0260] Additional embodiments include methods for affecting the proteasome-dependent regulation of oncoproteins and methods of treating or inhibiting cancer growth, with each method including exposing a cell (in vivo, for example, in a patient, or in vitro) to a composition as set out in this context. HPV-16 and HPV-18-derived E6 proteins stimulate ubiquitin-dependent ATP-e conjugation and p53 degradation in crude reticulocyte lysates. The recessive oncogene p53 has been shown to accumulate under non-permissive temperature in a cell line with a mutant heat labile E1. Elevated levels of p53 can lead to apoptosis. Examples of proto-oncoproteins degraded via the ubiquitin system include c-Mos, c-Fos, and c-Jun. One embodiment is comprised of a method of treating p53-related apoptosis, which includes administering to a patient an effective amount of the composition set forth in this context.
[0261] According to another embodiment, the disclosed compositions are useful for the treatment of a parasitic infection, such as infections caused by protozoan parasites. The proteasome of these parasites is considered to be primarily involved in cell differentiation and replication activities (Paugam et al., Trends Parasitol. 2003, 19(2): 55-59). Furthermore, Entamoeba species have been shown to lose the ability to encyst when exposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res. 1997, 28, Spec No: 139-140). According to some of these embodiments, compositions are disclosed which are useful for the treatment of parasitic infections in humans caused by means of a protozoan parasite selected from Plasmodium sps. (which includes P. falciparum, P. vivax, P. malariae, and P. ovale, which cause malaria), Trypanosoma sps. (which includes T. cruzi, which causes Chagas disease, and T. brucei, which causes African sleeping sickness), Leishmania sps. (which includes L. amazonesis, L. donovani, L. infantum, L. mexicana, and the like), Pneumocystis carinii (a protozoan known to cause pneumonia in AIDS and other immunosuppressed patients), Toxoplasma gondii, Entamoeba histolytica, Entamoeba invadens, and Giardia lamblia. According to certain embodiments, the disclosed compositions are useful for the treatment of parasitic infections in animals and livestock caused by a protozoan parasite selected from Plasmodium hermani, Cryptosporidium sps., Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona, and Neurospora gross. 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.
[0262] According to certain embodiments, the exposed compositions inhibit the proteasome activity irreversibly in a parasite. This irreversible inhibition has been shown to induce closure in enzyme activity without recovery in red blood cells and white blood cells. According to some of these embodiments, the long half-life of blood cells may provide prolonged protection with regard to therapy against relapsed exposures to parasites. According to certain embodiments, the long half-life of blood cells can provide prolonged protection with respect to chemoprophylaxis against future infection.
[0263] Prokaryotes are endowed with what is equivalent to the eukaryote 20S proteasome particle. While the subunit composition of the 20S particle of prokaryote is simpler than that of eukaryotes, it has the ability to hydrolyze peptide bonds in a similar manner. For example, nucleophilic attack of the peptide bond occurs through the threonine residue at the N-terminus of the β subunits. In accordance with some embodiments, a method of treating prokaryotic infections is provided, which comprises administering to a patient an effective amount of the proteasome inhibitory composition disclosed herein. Prokaryotic infections can include illnesses caused by either mycobacteria (such as tuberculosis, leprosy or Buruli Ulcer) or archaebacteria.
[0264] Inhibitors that bind to the 20S proteasome have also been shown to stimulate bone formation in bone organ cultures. Furthermore, when these inhibitors were administered to mice systemically, certain proteasome inhibitors increased bone volume and bone formation rates by more than 70% (Garrett, IR et al., J. Clin. Invest. (2003) 111 : 1771-1782), thereby suggesting that the ubiquitin-proteasome mechanism regulates osteoblast differentiation and bone formation. For that reason, the disclosed compositions may be useful for treating and/or preventing diseases associated with bone loss, such as osteoporosis.
[0265] Provided in this context is a method for the treatment of a disease or condition selected from cancer, autoimmune disease, graft or transplant-related condition, neurodegenerative disease, associated fibrotic condition, conditions related to ischemia, infection ( viral, parasitic or prokaryotic) and disorders associated with bone loss, which comprises administering a proteasome inhibitor as provided herein. For example a compound of formula (5).
[0266] Bone tissue is an excellent source for factors that have the ability to stimulate bone cells. Thus, extracts from bovine bone tissue contain not only structural proteins that are responsible for maintaining the structural integrity of bone, but also biologically active bone growth factors that can stimulate bone cell proliferation. Among these latter factors are a recently described family of proteins called bone morphogenetic proteins (BMPs). All of these growth factors have effects on other cell types as well as on bone cells, including Hardy, MH, et al., Trans Genet (1992) 8:55-61 describes evidence that bone morphogenetic proteins ( BMPs) are differentially expressed in hair follicles during development. Harris, S.E., et al., J Bone Miner Res (1994) 9:855-863 describes the effects of TGF-β on the expression of BMP-2 and other substances in bone cells. Expression of BMP-2 in mature follicles also occurs during maturation and after the period of cell proliferation (Hardy, et al. (1992, supra) Therefore, the compounds provided in this context may also be useful for stimulating cancer. hair follicle growth.
[0267] Finally, the exposed compositions are also useful as diagnostic agents (eg in diagnostic kits or for use in clinical laboratories) for sieving proteins (eg enzymes, transcription factors) processed by hydrolases of Ntn, including the proteasome. The disclosed compositions are also useful as screening reagents to specifically bind the X/MB1 subunit or α chain and inhibit proteolytic activities associated with it. For example, the activity of (and specific inhibitors of) other subunits of the proteasome can be determined.
[0268] Most cellular proteins undergo proteolytic processing during maturation or activation. Enzyme inhibitors set forth in this context can be used to determine whether a cellular, developmental, or physiological process or yield is regulated by the proteolytic activity of a particular Ntn hydrolase. Such a method includes obtaining an organism, preparing an intact cell, or cell extract, exposing the organism, cell preparation, or cell extract to a signal, and monitoring the process or yield. The high selectivity of compounds exposed in this context allows for the rapid and accurate elimination or implication of Ntn (eg, the 20S proteasome) in a particular cellular, developmental, or physiological process. Management
[0269] The compositions prepared as described in this context can be administered in various forms, depending on the disorder to be treated and the age, condition and body weight of the patient, as is known in the art. For example, where the compositions are to be administered orally, they can be formulated as tablets, capsules, granules, powders, or syrups; or for parenteral administration, they may be formulated as injections (intravenous, intramuscular, or subcutaneous), infusion preparations, drops, or suppositories. For application via the ophthalmic mucous membrane, they can be formulated as eye drops or ointments. These formulations may be prepared by conventional means in conjunction with the methods described herein, and, if desired, the active ingredient may be mixed with any conventional additive or excipient, such as a binder, a disintegrating agent, a lubricant, a corrective. , a solubilizing agent, a suspending aid, an emulsifying agent, or a coating agent in addition to a cyclodextrin and a buffer. Although the dosage may vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration and the form of the drug, in general, a daily dosage varies from 0.01 to 2000 mg of the compound is recommended for an adult human patient, and it 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 will generally be that amount of the compound that produces a therapeutic effect. In general, compositions intended for parenteral use (eg, intravenous injection, subcutaneously) include a substituted cyclodextrin. Compositions administered by other routes, particularly the oral route, include a substituted or unsubstituted cyclodextrin.
[0270] The precise time of administration and/or amount of the composition that will provide the most effective results in terms of treatment efficacy in a given patient will be dependent on the activity, pharmacokinetics, and bioavailability of a particular compound, physiological condition of the patient ( including age, sex, type of illness and stage, general physical condition, responsiveness to a certain dosage, and type of medication), route of administration, and others. Nevertheless, the guidelines set out above can be used as the basis for fine-tuning treatment, for example, determining the optimal time and/or amount of administration, which will require no more than routine experimentation consisting of patient monitoring and adjusting the dosage and/or timing.
[0271] The phrase "pharmaceutically acceptable" is used in this context to refer to those binders, materials, compositions, and/or dosage forms that are, within the scope of healthy medical judgment, suitable for use in contact with the tissues from humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, measured with a reasonable benefit/risk ratio.
[0272] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition, or carrier, such as a solid or liquid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and saccharin; (2) starches, such as corn starch, potato starch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) pulverized tragacanth; (5) bad; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations. According to certain embodiments, the pharmaceutical compositions provided in this context are non-pyrogenic, that is, they do not induce significant temperature elevations when administered to a patient.
[0273] The term "pharmaceutically acceptable salt" refers to the relatively non-toxic, inorganic and organic acid addition salts of the inhibitor(s). These salts can be prepared in situ during the final isolation and purification of the inhibitor(s), or by reacting separately a purified peptide proteasome inhibitor in its free base form with a suitable organic or inorganic acid, and isolation of the salt that is formed in this way. The salts that are representative include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate salts , naphthylate, mesylate, glycoeptonate, lactobionate, laurylsulfonate, and amino acid salts, and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19.)
[0274] According to some embodiments, the peptide proteasome inhibitors provided herein may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these cases refers to the relatively non-toxic inorganic and organic base addition salts of an inhibitor(s). Similarly, these salts can be prepared in situ during the final isolation and purification of the inhibitor(s), or by reacting separately from the purified inhibitor(s) in their free acid form. with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable primary, secondary, or tertiary organic amine. Representative alkaline or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines of utility for forming the base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et al., supra).
[0275] Wetting agents, emulsifiers, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavorings, and perfuming agents, preservatives and antioxidants they can also be present in the compositions.
[0276] 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, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
[0277] Formulations that are suitable for oral administration may be in the form of capsules, wafers, pills, tablets, lozenges (using a flavored base, usually saccharin and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or in the form of an elixir or syrup, or in the form of tablets (using an inert matrix such as gelatine and glycerin, or saccharin and acacia) and/or in the form of mouthwashes, and the like, each containing a predetermined amount of an inhibitor(s) as an active ingredient. The composition may also be administered in the form of a bolus, electuary or paste.
[0278] In solid dosage forms for oral administration (capsules, tablets, pills, pills, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or sodium phosphate dicalcium, and/or any of the following: (1) fillers or extenders, such as starches, cyclodextrins, lactose, saccharin, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, saccharin, 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 delaying agents such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols, and the like.
[0279] A tablet can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binder (eg, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), dispersing agent or active surface. Molded tablets may be prepared by molding in a suitable machine the mixture of the powdered inhibitor(s) moistened with an inert liquid diluent.
[0280] Tablets and other solid dosage forms, such as tablets, capsules, pills, and granules, can optionally be labeled or prepared with coatings and shells, such as enteric coatings and other coatings widely known in the pharmaceutical formulation art. They can also be formulated to provide slow or controlled release of the active ingredient provided therein, using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes, and/or microspheres . They can be sterilized, for example, by filtering through a bacteria-retaining filter, or by including sterilizing agents in sterile solid form as compositions 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 capable of releasing the active ingredient(s) only, or preferentially, in a certain part of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the excipients described above.
[0281] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents, and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, acetate of ethyl, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (with particularity, cottonseed oils, peanuts, corn, germ, olive, castor, and sesame), glycerol, alcohol tetrahydrofuryl, polyethylene glycols, and sorbitan fatty acid esters, and mixtures thereof.
[0282] In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring, coloring, perfuming and preservatives.
[0283] Suspensions, in addition to active inhibitors may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, as well like their blends.
[0284] Formulations for rectal or vaginal administration may be presented in the form of a suppository, which may be prepared by mixing one or more inhibitors with one or more suitable non-irritating excipients or carriers comprising, for example, buttermilk. cocoa, polyethylene glycol, a suppository wax or salicylate, which is solid at room temperature but liquid at body temperature and will therefore melt in the rectum or vaginal cavity and release the active agent.
[0285] Formulations that 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.
[0286] Dosage forms for topical or transdermal administration of an inhibitor include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active component can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
[0287] Ointments, pastes, creams, and gels may contain, in addition to the inhibitor(s), excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.
[0288] The powders and sprays can contain, in addition to one or more inhibitors, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain conventional propellants such as chlorofluorohydrocarbons and unsubstituted volatile hydrocarbons such as butane and propane.
[0289] A peptide proteasome inhibitor can be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation, or solid particles containing the composition. A non-aqueous suspension (eg fluorocarbon propellant) may be used. According to some embodiments, sonic nebulizers are preferred because they minimize the agent's exposure to shear, which can result in compound degradation.
[0290] Typically, an aqueous aerosol is prepared by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary with the requirements of the particular composition, but typically include nonionic surfactants (Tweens, Pluronics, sorbitan esters, lecithin, Cremophors), pharmaceutically acceptable co-solvents such as polyethylene glycol, innocuous albumin-like proteins of serum, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars, or sugar alcohols. Aerosols are generally prepared from isotonic solutions.
[0291] Transdermal patches have the added advantage of providing controlled delivery of an inhibitor(s) to the body. Such dosage forms can be prepared by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the inhibitor(s) through the skin. The rate of this flux can be controlled either by providing a rate-controlling membrane or by dispersing the inhibitor(s) in a polymer matrix or gel.
[0292] Pharmaceutical compositions that are 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 which may be reconstituted into injectable solutions or dispersions just before use, which may contain antioxidants, buffers, bacteriostats, solutes that make the formulation isotonic with the intended recipient's blood, or suspending or thickening agents.
[0293] Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions provided herein include water for injection (e.g. sterile water for injection), ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol , and the like), buffer (such as citrate buffer), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. proper fluidity can be maintained, for example, through the use of coating materials such as lecithin, through maintenance of the required particle size in the case of dispersions, and through the use of surface-active agents.
[0294] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used in this context, the term "pharmaceutically acceptable carrier" includes buffer, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, absorption delaying and isotonic agents, and the like, compatible with pharmaceutical administration . In accordance with some embodiments, a pharmaceutically acceptable carrier is a buffer (e.g., citrate buffer). In accordance with some embodiments, a pharmaceutically acceptable carrier is comprised of sterile water for injection. According to some embodiments, a pharmaceutically acceptable carrier comprises citric acid.
[0295] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can also be ensured through the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include tonicity adjusting agents such as sugars and the like within the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
[0296] In some cases, in order to prolong the effect of a drug, it is desirable to delay drug absorption from a subcutaneous or intramuscular injection. For example, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle.
[0297] Injectable depot forms are prepared by forming microencapsule matrices of inhibitor(s) in biodegradable polymers such as polylactide-polyglycolide. Depending on the drug to polymer ratio, and the nature of the particular polymer employed, the drug release rate can be controlled. 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 tissue.
[0298] Agent preparations can be administered orally, parenterally, topically, or rectally. They are, of course, administered in ways suitable for each route of administration. For example, they are administered in the form of tablets or capsules, by injection, inhalation, eye lotion, ointment, suppository, infusion; topically through lotion and rectally through suppositories. In some embodiments, administration is oral.
[0299] The phrases "parenteral administration" and "parenterally administered" as used in this context mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous injection , intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal, and infusion.
[0300] The phrases "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered" as used in this context mean the administration of a binder, drug or other material that is not directly within of the central nervous system, in such a way that it enters the patient's system and thus undergoes metabolism or other similar processes, eg, subcutaneous administration.
[0301] The peptide proteasome inhibitors described in this context can be administered to humans and other animals for therapy by any suitable routes of administration, including orally, nasally, such as, for example, by means of a spray, rectally, intravaginally, parenterally, intracysteically, and topically, such as by means of powders, ointments or drops, including buccally and sublingually.
[0302] Regardless of the selected route of administration, a peptide proteasome inhibitor, which can be used in a suitable hydrated form, and/or the pharmaceutical compositions provided herein, is formulated in a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.
[0303] The actual dosage levels of the active ingredients in the pharmaceutical compositions provided herein can be varied in order to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration , without being toxic to the patient.
[0304] The concentration of a compound exposed 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(s) used, and the route of administration. In general, compositions provided herein may be provided in an aqueous solution containing about 0.1-10% w/v of a compound disclosed herein, among other substances, for parenteral administration. Typical dosage ranges range from about 0.01 to about 50 mg/kg of body weight per day, administered in 1-4 divided doses. Each divided dose can contain the same or different compounds. The dosage will be an effective amount dependent on several factors including the general health of a patient, and the formulation and route of administration of the selected compound(s).
[0305] According to another embodiment, the pharmaceutical composition is an oral solution or a parenteral solution. Another embodiment is comprised of a lyophilized preparation that can be reconstituted prior to administration. In the form of a solid, this formulation can also include tablets, capsules or powders.
[0306] Also provided in this context is a conjoint therapy wherein one or more other therapeutic agents are administered with a peptide proteasome inhibitor or pharmaceutical composition comprising a peptide proteasome inhibitor. This conjoint treatment can be achieved through simultaneous, successive, or separate dosing of the individual components of the treatment.
[0307] According to certain embodiments, the composition provided herein is administered in conjunction with one or more other proteasome inhibitor(s).
[0308] According to certain embodiments, the composition provided in this context is administered in conjunction with a chemotherapeutic. Chemotherapeutic agents that are suitable may include, natural products, such as vinca alkaloids (i.e., vinblastine, vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (i.e., etoposide, teniposide), antibiotics (dactinomycin (actinomycin D), daunorubicin, doxorubicin and idarubicin), anthracyclines, mitoxantrone, bleomycins, plicamycin (mitramycin) and mitomycin, enzymes (L-asparaginase that systemically metabolizes L-asparagine and removes cells that do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferating/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and the like, melphalan, chlorambucil), ethyleneimines and methylmelamines (hexamethyl melamine and thiotepa), alkyl sulfonates (busulfan), nitrosoureas (carmustine (BCNU) and zocins ), trazenes-dacarbazinine (DTIC); antiproliferating/antimitotic antimetabolites such as folic acid analogues (methotrexate), pyrimidine analogues (fluorouracil, floxuridine, and cytarabine), purine analogues and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine); aromatase inhibitors (anastrozole, exemestane, and letrozole); and platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; histone deacetylase (HDAC) inhibitors (tricostatin, sodium butyrate, apicidan, suberoyl anilide hydroamic acid); hormones (ie estrogen) and hormonal agonists such as leutinizing hormone releasing hormone (LHRH) agonists (goserelin, leuprolide and triptorelin). Other chemotherapeutic agents may include mechlorethamine, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine, or any analog or variant derived from the foregoing.
[0309] According to certain embodiments, the pharmaceutical composition as provided herein is administered in conjunction with a cytokine. Cytokines include, but are not limited to, Interferon-Y, -α, and -β, Interleukins 1-8, 10, and 12, Granulocyte Monocyte Colony Stimulating Factor (GM-CSF), TNF-α, and -β , and TGF-β.
[0310] According to certain embodiments, the pharmaceutical composition provided herein is administered in conjunction with a steroid. Steroids that are suitable may include, but are not limited to, 21-acetoxypregnenolone, alclomethasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chlorprednisone, clobetasol, clocortolone, cloprednol, corticosterone, deflasterone, cortisone , deoxymethasone, dexamethasone, diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort, fluchloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, butyl fluocortin, fluocortolone, fluocortonacetate, fluodrenoprelone forone, flucortonone, fluoropredolone acetate, flucornione acetate, flucornioneforone forone , halcinonide, halobetasol propionate, halomethasone, hydrocortisone, loteprednol etabonate, mazipredone, medrisone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone 25-prednisolone , thixocorto l, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, and salts and/or derivatives thereof.
[0311] According to some embodiments, the pharmaceutical composition provided in this context is administered in conjunction with an immunotherapeutic agent. Immunotherapeutic agents that are suitable may include, but are not limited to, MDR modulators (verapamil, valspordar, biricodar, tariquidar, laniquidar), cyclosporin, thalidomide, and monoclonal antibodies. Monoclonal antibodies can be either naked or conjugated, such as rituximab, tositumomab, alemtuzumab, epratuzumab, ibritumomab tiuxetan, gemtuzumab ozogamicin, bevacizumab, cetuximab, erlotinib and trastuzumab. EXAMPLES Example 1. Preparation of a suspension of carfilzomib-active pharmaceutical ingredient (CFZ-API) in sulfobutyl ether beta-cyclodextrin (SBECD)
[0312] This Example describes the preparation of a suspension of CFZ-API in SBECD under batch sizes of 400 L. Smaller batch sizes were performed at equivalent proportions of the constituents, such as under batch sizes of 290 L, 90 L, and 1-3 L.
[0313] In a refrigerated tank lined with a 525 L stainless steel jacket controlled to 2°C - 8°C, a suspension of 2.0 kg of carfilzomib-API (CFZ-API), 246 kg of water for injection (WFI), and 100 kg of sulfobutyl ether beta-cyclodextrin (SBECD). Specifically, in the 525 L stainless steel jacket lined refrigerated tank controlled to 2°C - 8°C, 100 kg of SBECD was dissolved in 246 kg of WFI. Carfilzomib suspension was then prepared using 2.0 kg of CFZ-API. Mixing was carried out using an impeller mixer to keep the CFZ-API solids suspended and dissolve the SBECD. A probe-style rotor-stator high-shear mixer (homogenizer) was used in the same vessel as well as the low-shear impeller. The high shear mixer was operated for approximately 1 hour providing uniform suspension and particle size reduction for any larger primary particles or agglomerated API. After a suspension was obtained, 1.96 kg of citric acid was added as a 16% aqueous solution. The pH of the solution was then lowered inducing partial solubilization of CFZ-API followed by complexation due to the presence of SBECD. Mixing was continued for a further 24 hours with the impeller and high shear mixer and a dissolved concentration of CFZ-API greater than 5.1 mg/ml was achieved. The suspension containing more than 5.1 mg/ml of dissolved complexed CFZ-API was filtered through a 0.45 micrometer clarification filter, then precisely diluted to a dissolved concentration of 5.0 mg/ml and pH adjusted with 1 N sodium hydroxide solution to achieve pH 3.5. The solution was sterile filtered, with two sequential 0.22 micrometer sterilization filters, then poured into vials of 12.36 ml each, containing 61.8 mg per vial of CFZ-API. The vials were partially capped and loaded into a lyophilizer and freeze dried for 103 hours using a freezing temperature of -45°C, main drying temperature of -15°C, and secondary drying of +30°C. Lyophilized vials were fully capped, capped, then stored at the product stability temperature of 2°C - 8°C for up to two years prior to use. When in use, the vial has been reconstituted with sterile water for injection to produce 2 mg/ml of a reconstituted solution for injection, which has a pH 3.5 and acceptable tonicity for direct injection into patients. Alternatively, the reconstituted solution was further diluted in an intravenous bag for further dilution and infusion without inducing precipitation.
[0314] As shown in Figure 1, the slurry-based complexation process results in increased solubilization of CFZ-API over time (greater than 5 milligrams per milliliter, which is substantially higher than the intrinsic aqueous solubility of CFZ-API, which is less than 10 micrograms per milliliter). Furthermore, the process is less dependent on CFZ-API physicochemical properties (eg, particle size, surface area, degree of agglomeration, polymorphic shape, and others). Unlike most pharmaceutical production or testing, the dissolution rate (or solubilization rate) in this process is effectively independent of the API particle size (see, for example, Figure 2) as the process delivers an equivalent extent of solubilization over the 24-hour time period for complexation to occur regardless of whether the API initially had a large or small API particle size (21.1 micrometers, and 7.5 micrometers respectively). It was further determined that in the process described above, higher concentrations of SBECD increased the solubility of CFZ-API (see Figure 3). Finally, it was observed that the complexed solubility of CFZ/SBECD was effectively independent of processing or storage temperature (see, for example, Figure 4 where the solubilized extent is illustrated as a function of the concentration of SBECD at pH 3.5 for two temperatures 5oC and 25oC not showing any apparent difference). For this reason lower processing temperatures (2oC - 8oC) are preferred to minimize the potential for any thermally induced degradation reactions that may occur. In other processes, higher temperatures are more commonly needed to increase the solubility, however in this process, the highest solubility is achieved by increasing the cyclodextrin concentration and/or pH rather than by increasing the temperature and this allows that thermal degradations of the product are reduced to a minimum in this process. Example 2. Effect of Chloride Ion on Carfilzomib Stability
[0315] A multivariate statistical design of experiments was conducted to evaluate factors that control the level of chlorohydrin degradation product as a function of processing parameters and storage time for six months. Complexation was carried out in the proportion and parameters given in Example 1, with the following modifications: (i) the complexation process was carried out under a batch size of 2 L; (ii) the final pH of the solution prior to pouring into the vials was subjected to variations for experimental purposes from 3.0 to 4.0; (iii) sodium chloride was enriched in SBECD in some experiments to create a high sodium chloride condition; (iv) the water content of the final freeze-dried product in the stoppered vials was produced under both high and low sodium chloride conditions through early termination and capping of the vials to create a higher residual water content condition. Materials. Table 2. Materials

Methods. Complexation Process:
[0316] Complexed Carfilzomib solution for pre-lyophilization of bulk solution for injection included 5 mg/ml aqueous carfilzomib, 250 mg/ml Captisol® (SBECD) and 4.86 mg/ml citric acid, pH adjusted with aqueous sodium hydroxide. The composition of the bulk solutions for lyophilization followed the procedure detailed in Example 1 with the following manipulations to create solutions with different specific attributes: 1. pH was adjusted to 3.0 and 4.0 2. Sodium chloride was enriched in Captisol® to create a "High Chloride Content" condition
[0317] Captisol® manufactured by Cydex, a subsidiary of Ligand, has a standard product analysis range for sodium chloride from 0.05% to 0.2% (w/v). A batch of Captisol® was made available for experimentation that had a low chloride content of only 0.05% (w/v) as sodium chloride. 400 g of this Captisol® per batch was required for the process to be carried out under 2 L scale batches of complexation processing (in the same proportions and general parameters used for Example 1). To create the "high chloride content" condition, 0.6 g of NaCl was added to 399.4 g of Captisol® which, in this way, mimicked what a batch containing 0.2% of Captisol® chloride would be comprised of. . Freeze drying:
[0318] In order to generate two (2) conditions of moisture content in the final lyophilized vials, two (2) sets of samples of 61.8 mg/vial (of CFZ-API) were lyophilized. The first cycle generated the “dry” sample set of vials that contained approximately 0.6% residual water for the lyophilization parameters of Example 1. For the second set of samples, lyophilization was completed and the vials stoppered earlier in the Secondary drying phase to generate the jars in the “wet” condition, with residual moisture of approximately 2.4% water for each jar initially.
[0319] One (1) batch of placebo was prepared as a control that contained 250 mg/ml Captisol® and 4.86 mg/ml citric acid, adjusted to pH 3.5 with NaOH. Analytical Test:
[0320] Most of the complexed carfilzomib solution was analyzed during manufacturing by means of High Performance Liquid Chromatography (HPLC) to accurately quantify the concentration of the dissolved and complexed drug substance Carfilzomib. Subsequently, additional water was added to carefully dilute the complexed solution in bulk. After this dilution step, HPLC was used again to ensure that a target concentration of 5.0 mg/ml was obtained. Samples from the final three (3) bulk solutions were analyzed for potency and purity confirmed by means of the HPLC test. The stability of the samples was analyzed after six months of storage at 5oC and 25oC by means of HPLC for potency and purity. The Karl Fischer Coulometry method was used to determine the water content in the lyophilized drug. Data Processing:
[0321] Stat-Ease DX7 was used to analyze the results. Results.
[0322] The results for the formation of a chlorohydrin degradation product (CDP) in 6 months at 5°C and 25°C are summarized in Table 3 below. Table 3. Results for CDP formation after 6 months at 5°C and 25°C

[0323] The ANOVA analyzes below (Table 4 and 5) for CDP show that chloride content is the main factor in the formation of CDP. The higher chloride content leads to higher levels of CDP. Even at the low level of chloride content (0.05% (w/v)), the formation of chlorohydrin is still observed, but at an acceptably low concentration compared to 0.2% chloride. Furthermore, medicine containing low levels of chloride ion showed unacceptable formation of chlorohydrin under 25°C after 6 months of storage. Figure 5 illustrates the relationship between CDP and the interaction of two factors of water and chloride content. The top line is a high chloride content and the bottom line is a low chloride content. The x-axis represents water content, with 0.7% on the left and 2% on the right. At higher chloride levels, levels of CDP production increase. This increase is even more evident under conditions of higher water content, as can be seen from the slope of the top curve. Under low chloride levels, there is little difference observed between low or high water content conditions. Table 4. ANOVA Analysis - CDP (RRT 0.86) in 6 Months, 5°C
Table 5. ANOVA Analysis - CDP (RRT 0.86) in 6 Months, 25°C
example 3. Effect of hydrochloric and citric acids on the chlorohydrin degradation product
[0324] A study was conducted to determine the impact of using hydrochloric acid in the complexation process by comparing CDP degradation product impurity levels on storage time for batch produced without HCl, and stored for the same period of time. During production, the pH of all batches was adjusted at the end of the process to 3.5 using sodium hydroxide.
[0325] As shown in Table 6, batches produced with the addition of HCl (2, 3, and 4) showed a clear formation of the chlorohydrin degradation product (CDP) over storage time, while under temperature recommended storage of 5°C, CDP was mostly below the reported HPLC limit (0.1%) or not detected (ND) in lots 1 and 5 (where no HCl was used). Clearly, higher chloride content from HCl as the ad to initiate complexation resulted in higher formation (and unacceptable levels of formation of) of CDP. For this reason, the use of weaker citric acid alone to initiate complexation in SBECD reduced to a minimum the formation of CDP. Table 6. Results for CDP formation (% Area) at 5°C and 25°C
Example 4.
[0326] The solubility of Carfilzomib as a function of SBECD cyclodextrin concentration was determined in aqueous solutions containing citric acid (30 mM), under pH 1.5 and pH 3.5, and under temperatures that included 5°C and 25 °C. the solubility profile is illustrated in Figure 6. No significant differences in solubility were observed between the low and high temperatures that were tested. The experiments were carried out under acidic conditions below the target pH values and titrated to pH 1.5 or 3.5 using aqueous sodium hydroxide solution. Measurements of solubilized concentration were those from samples analyzed after 24 hours of time to equilibrate. Other Achievements
[0327] It should be understood that although the exposition is carried out in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the claims set out below.

权利要求:
Claims (16)
[0001]
1. Method for preparing a pharmaceutical composition with low chloride content, the method CHARACTERIZED by: (i) providing a first combination comprising: (a) a compound:
[0002]
2. Method according to claim 1, CHARACTERIZED by the fact that the second combination comprises a complex of the compound and SBECD with a low chloride content.
[0003]
3. Method according to claim 1, CHARACTERIZED by the fact that providing a first combination (step (i)) comprises adding the compound to a solution of SBECD with low chloride and water content.
[0004]
4. Method according to claim 1, CHARACTERIZED by the fact that it comprises: (i) providing a first combination comprising: (a) a compound:
[0005]
5. Method according to claim 1 or 4, CHARACTERIZED by the fact that the first combination contains less than 0.5% w/w or w/v of organic solvent.
[0006]
6. Method according to claim 1 or 4, CHARACTERIZED by the fact that the first combination contains less than 0.5% w/w or w/v buffer.
[0007]
7. Method according to claim 4, CHARACTERIZED by the fact that the second combination comprises a complex of the compound and SBECD with a low chloride content.
[0008]
8. Method according to claim 1 or 4, CHARACTERIZED by the fact that the molar ratio of chloride ion to the compound in the first combination is less than 1.5.
[0009]
9. Method according to claim 8, CHARACTERIZED by the fact that the molar ratio of chloride ion to compound in the first combination is up to 0.32.
[0010]
10. Method according to claim 4, CHARACTERIZED by the fact that providing a first combination (step (i)) comprises adding the compound to a solution of SBECD with low chloride and water content.
[0011]
11. Method according to claim 3 or 10, CHARACTERIZED by the fact that the compound is a crystalline solid.
[0012]
12. The method of claim 11, CHARACTERIZED by the fact that the crystalline form of the compound has an X-ray powder diffraction pattern comprising characteristic peaks expressed in degrees 2θ at 6.10, 9.32, 10 .10, 12.14, 13.94, 18.44, 20.38 and 23.30.
[0013]
13. Method according to claim 1 or 4, CHARACTERIZED in that the method further comprises mixing the first combination before contacting the first combination with acid.
[0014]
14. Method according to claim 1 or 4, CHARACTERIZED by the fact that it further comprises mixing the second combination to achieve a third homogeneous combination.
[0015]
15. Method according to claim 14, CHARACTERIZED by the fact that the dissolved and complexed concentration of the compound in the third combination is 4 to 8 mg/mL.
[0016]
16. Method according to claim 14, CHARACTERIZED by the fact that the pH of the third combination is from 2 to 4.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-08-20| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2020-06-30| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-12-22| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2021-05-04| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-07-13| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 13/09/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261644122P| true| 2012-05-08|2012-05-08|

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US61/644,122|2012-05-08|

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PCT/US2012/055127|WO2013169282A1|2012-05-08|2012-09-13|Cylodextrin complexation methods for formulating peptide proteasome inhibitors|

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