WO2006063154A1 - Composition for proteasome inhibition - Google Patents

Abstract

Compositions comprising one or more practically insoluble proteasome inhibitors and a cyclodextrin, particularly a substituted cyclodextrin, substantially increase the solubility of these proteasome inhibitors and facilitate their administration. Such compositions optionally comprise a buffer. Methods of treatment using such compositions are also disclosed

Description

COMPOSITION FOR PROTEASOME INHIBITION

Cross-Reference to Related Applications

This application claims the benefit of U.S. Provisional Application No. 60/634,366, filed December 7, 2004, and U.S. Provisional Application No. 60/655,930 filed February 23, 2005, the specifications of which are hereby incorporated by reference in their entirety.

Background of the Invention

The proteasome has been validated as a therapeutic target, as demonstrated by the recent FDA approval of bortezomib, a boronic acid proteasome inhibitor, for the treatment of multiple myeloma. However, other more highly proteasome-specific inhibitors that could have fewer toxic side effects have recently been described. These compounds include peptide epoxy ketones such as epoxomicin and peptide (b), described in U.S. Patent No. 6,831,099, the contents of which are hereby incorporated by reference, and peptide (a), described in U.S. Provisional Application No. 60/569,096, filed May 7, 2004, the contents of which are hereby incorporated by reference. However, the low aqueous solubility of some of these compounds makes it difficult to formulate compositions with optimal bioavailability. Thus, additional methods of formulating peptide epoxy ketones are needed.

Summary of the Invention

It has now been found that the solubility of proteasome inhibitors, such as the peptide epoxy ketones peptide (a) and peptide (b) (structures or definitions for these peptides are provided in Group 3 and Group 1, respectively), is significantly enhanced when formulated with a cyclodextrin. In one embodiment, the present invention is a pharmaceutical composition that includes a practically insoluble proteasome inhibitor, a cyclodextrin and optionally a buffer. Such pharmaceutical compositions typically include a pharmaceutically effective amount of the proteasome inhibitor, e.g., which ameliorates the effects of neurodegenerative disease (such as Alzheimer's disease), immunological conditions, muscle-wasting diseases, cancer, chronic infectious diseases, fever, muscle disease, denervation, nerve injury, and/or fasting, among others, when administered to a patient.

In another aspect, the invention provides anti-inflammatory compositions that include a therapeutically effect amount of a proteasome inhibitor, a cyclodextrin and optionally a buffer. In another aspect, the invention provides methods that involve administering to or contacting a subject, a cell, a tissue, an organ or an organism with an effective amount of a composition comprising one or more proteasome inhibitors disclosed herein. These methods include, but are not limited to, inhibiting or reducing HIV infection in a subject; affecting the level of viral gene expression in a subject; altering the variety of antigenic peptides produced by the proteasome in an organism; determining whether a cellular, developmental, or physiological process or output in an organism is regulated by the proteolytic activity of a particular Ntn hydrolase; treating Alzheimer's disease in a subject; reducing the rate of muscle protein degradation in a cell; reducing the rate of intracellular protein degradation in a cell; reducing the rate of p53 protein degradation in a cell; inhibiting the growth of p53-related cancers in a subject; inhibiting antigen presentation in a cell; suppressing the immune system of a subject (e.g., conditions such as septic shock, psoriasis, graft rejection, and rheumatoid arthritis); inhibiting I/cB-α degradation in an organism; reducing the content of NF-κB in a cell, muscle, organ or subject; affecting cyclin-dependent eukaryotic cell cycles; treating proliferative disease in a subject; affecting proteasome-dependent regulation of oncoproteins in a cell; treating cancer growth in a subject; treating p53-related apoptosis in a subject; and screening proteins processed by N- terminal nucleophile hydrolases in a cell.

The invention, in another aspect, is directed to medical devices that include a pharmaceutical composition disclosed herein.

Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

Brief Description of the Drawings Figure 1 shows the solubility of peptide (a) at various pH values in aqueous 10%

(w/v) sulfobutyl ether beta-cyclodextrin (SBECD )/ 10 mM sodium citrate solutions. Figure 2 shows the percentage of peptide (a) remaining in aqueous 10% (w/v) SBECD/10 mM sodium citrate solutions over time at various pH values.

Detailed Description

Pharmaceutical compositions of the invention include a practically insoluble proteasome inhibitor, a cyclodextrin and optionally a buffer. The amount of proteasome inhibitor that can be solubilized is dependent on several parameters. One such parameter is pH. As shown in Figure 1, higher pH results in poorer solubility of a basic compound, and lower pH would be expected to decrease solubility of an acidic compound, as is well known in the art. However, a pH should be selected to provide suitable stability of the proteasome inhibitor. For example, lower pH results in decreased chemical stability of one such compound, as demonstrated in Figure 2. The effects of pH on a compound's stability and solubility can be readily determined using methods widely known in the art and disclosed herein. For formulations to be administered to a mammal, the pH is preferably from pH 2.5 to pH 9. In many compositions of the invention, the primary source of pH control is the buffer. Typically, the buffer is present as an acid or a base and its conjugate base or acid, respectively. In one embodiment, the range of buffering salt is 1-100 niM, preferably between 5-50 mM, most preferentially about 10 mM (in solid formulations, the amount of buffer is selected to produce this concentration after reconstirution/dilution). The concentration of buffer and the pH of the solution are advantageously chosen to give optimal balance of solubility and stability.

Examples of suitable buffers include mixtures of weak acids and alkali metal salts (e.g., sodium, potassium) of the conjugate base of weak acids such as sodium tartrate and sodium citrate. The preferred buffer is sodium citrate/citric acid. Cyclodextrins of the invention include alpha-, beta- and gamma-cyclodextrin. In one embodiment, the cyclodextrin is either a substituted or non-substituted /3-cyclodextrin, present, for example, at from 5-20% (w/v). In a certain embodiment, the preferred amount of a cyclodextrin is about 10% (w/v). In another preferred embodiment, the cyclodextrin is a substituted /?-cyclodextrin. Substituted cyclodextrins increase the solubility of the cyclodextrin and mitigate toxic effects associated with unsubstituted cyclodextrins. Examples of substituted /3-cyclodextrins include those substituted with one or more hydrophilic groups, such as monosaccharide (e.g., glucosyl, maltosyl), carboxyalkyl (e.g., carboxylmethyl, carboxy ethyl), hydroxyalkyl-substituted (e.g., hydroxyethyl, 2- hydroxypropyl) and sulfoalkylether-substituted beta-cyclodextrin. Particularly suitable beta-cyclodextrins include hydroxypropyl beta-cyclodextrin (HPBCD) and sulfobutylether beta-cyclodextrin (SBECD), preferentially SBECD. However, it is understood that typically any substitution to the cyclodextrin, including substitution by hydrophobic groups such as alkyls, will improve its aqueous solubility by disrupting the hydrogen-bonding network within the crystal lattice of the solid cyclodextrin, thereby lowering the lattice energy of the solid. The degree of substitution is not believed to be critical; however, the degree of substitution is advantageously at least 1% and typically 2% to 10%, such as 3% to 6%.

One particular embodiment of this invention is a pharmaceutical formulation comprising 1 to 5 mg/ml of a proteasome inhibitor, 5% to 25% (w/v) of a cyclodextrin such as HPBCD or SBECD and 5 mM to 20 mM of a buffer producing a pH of about pH 3 to about pH 6, e.g., a solution of 2 mg/ml of a proteasome inhibitor (peptide (a)), 10% (w/v) SBECD, 10 mM sodium citrate, pH 3.5.

Proteasome Inhibitors

Suitable proteasome inhibitors, particularly those with epoxide and aziridine moieties, are described in U.S. Patent 6,831,099 and U.S. Provisional Application Nos.

60/562,340, filed April 15, 2004, 60/569,096, filed May 7, 2004, 60/596,885, filed May 10,

2004, 60/572,072, filed May 17, 2004, 60/599,401, August 6, 2004, 60/610,001, filed September 14, 2004, 60/610,002, filed September 14, 2004, 60/610,040, September 14,

2004, 60/610,159, filed September 14, 2004, 60/620,573, filed October 20, 2004, and

60/634,366, filed December 7, 2004, the contents of which are incorporated herein by reference.

In each of the following groups, the values for various moieties (e.g., for R¹, etc.) are understood to be consistent within a group, but values for one group (e.g., Group 1) do not apply to another group (Group 9).

Group 1

In one embodiment, the proteasome inhibitor is an epoxide- or aziridine-containing compound, which preferably contains groups proximate to the heteroatom-containing, three-membered rings, such that a ring-opening reaction of the heteroatom-containing three- membered ring is facilitated. Such groups include electron-withdrawing groups (E.W.G.) adjacent to (for example, at a carbon vicinal to a carbon atom of the three-membered, heteroatom-containing ring), or in electronic communication with (for example, via a carbon atom, or an alkenyl or alkynyl linkage), epoxide or aziridine functionalities. The E.W.G. can be bonded to one of the carbon atoms of the heteroatom-containing, three- membered ring. E.W.G. include, for example, cyano, isocyano, nitro, amide, sulfonyl, β- carboxy vinyl, sulfinyl, /3,/3-dicyano vinyl, formyl, carboxyl, alkyloxy- and aryloxy- carbonyl, 1 -tetrazolyl, carbamoyl, sulfamoyl, carbonyl, sulfoxide groups, and halogenated or dihalogenated carbon atoms such as -CHX-, -CXX'-, and -CRX- (where X and X' are independently selected halogens, and R is a carbon-containing substituent such as alkyl, aryl alkenyl, alkynyl and the like). In some preferred embodiments, E.W.G. is a carbonyl group. In some embodiments, it may be desirable to utilize E.W.G. that are of size, charge, and polarity sufficient to interact electronically with particular polar or charged moieties within an Ntn hydrolase. For example, an ionized aspartate or glutamate side chain can be present in the Ntn, and interact with, and stabilize, an electron-withdrawing group present in a peptide epoxide. Such groups act as an "anion hole," with which E.W.G. can interact when enzyme inhibitors are bound to Ntn, resulting in increased electrophilicity of E.W.G.

Some peptide epoxide or peptide aziridine compounds have a ketone functionality as the electron-withdrawing group, along with epoxide or aziridine functional groups. Particular examples are peptide α',j8'-epoxy ketones or peptide α',/J'-aziridine ketones, in which the carbon atoms forming two of the three members of the epoxide or aziridine ring are one (α') and two (β') carbons from the ketone, and the ketone carbon is bonded to one of the carbon atoms of the heteroatom-containing, three-membered ring. Further groups can be bonded to α' or /3' carbons such as hydrogen and Q^alkyl groups, including methyl, ethyl, propyl and butyl groups. Groups bonded to α' or /3' carbons can be further substituted with hydroxy, halogen, amino, carboxy, carbonyl, thio, sulfide, ester, amide or ether functionality.

For example, a carboxylic acid group can be bonded directly to the a' carbon, or via a linker. The linker can be CM alkylene, C_2-5 alkenylene, C_2-5 alkynylene, aryl, oxygen, sulfur, or amine. This carboxylic acid can be part of a peptide moiety extending from the cC carbon of the heteroatom-containing, three-membered ring. In this way, peptides containing side chains can be constructed. Such side chains can be labeled as Pl ', P2', and so forth, with Pl ' being the first side chain adjacent to the ά" carbon, P2' being the second, and so forth. Optimization of side chains for Pl ', P2' and other positions can result in enzyme inhibitors with desirable specificity, or desirable inhibition rates. Side chains for Pl ', P2' and so forth can be any of those side chains discussed herein. In embodiments including such groups bonded to a.' carbons, the stereochemistry of the α' -carbon (that carbon forming a part of the epoxide or aziridine ring) can be (R) or (S). The invention is based, in part, on the structure-function information disclosed herein, which suggests the following preferred stereochemical relationships. Note that a preferred compound may have a number of stereocenters having the indicated up-down (or β-a, where β as drawn herein is above the plane of the page) or (R)-(S) relationship (that is, it is not required that every stereocenter in the compo