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## CYCLODEXTRIN COMPLEXATION METHODS FOR FORMULATING PEPTIDE PROTEASOME INHIBITORS 

## CROSS REFERENCE TO RELATED APPLICATIONS 

This application claims the benefit of U.S. Provisional Application No. 61/644,122, filed on May 8, 2012 and U.S. Provisional Application No. 61/777,475, filed on Mar. 12, 2013, each of which is incorporated herein by reference in its entirety. 

## INCORPORATION BY REFERENCE OF MATERIALS SUBMITTED ELECTRONICALLY 

This application contains, as a separate part of disclosure, a Sequence Listing in computer-readable form (filename: 40073_SeqListing.txt; 989 byte-ASCII text file; created Dec. 16, 2015) which is incorporated by reference in its entirety. 

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 cyclo› dextrin(s). Such methods substantially increase the solubil› ity and stability of these proteasome inhibitors and facilitate both their manufacture and administration. 

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. 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, described in U.S. Pat. No. 6,831,099, the contents of which are hereby incorporated by reference, and those described in U.S. Pat. No. 7,232,818, 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 at sufficiently high concentration to enable practical administration with desired antineoplastic or other pharmacological effects. Thus, addi› tional methods of formulating peptide epoxy ketones are needed. 

Provided herein are cyclodextrin complexation methods of formulating a peptide proteasome inhibitor ( e.g., a com› pound of formula (1)-(5) or a pharmaceutically acceptable salt thereof) with one or more cyclodextrins. Many peptide proteasome inhibitors have been shown to have low solu› bility in water. This low solubility can be overcome through complexation of the compound with one or more cyclodex› trins using the methods provided herein. For example, homogenous solutions of a compound of formula (5) (carfil- zomib) can be obtained at a pharmaceutically useful pH (e.g., about 3.5) and at higher concentrations (e.g., about 5 mg/mL) than could be obtained without one or more cyclo› dextrins and the processes of complexation between the compound and one or more cyclodextrins provided herein. In addition to increasing the solubility of a peptide protea› some inhibitor in solution, the formulations prepared by the 

## TECHNICAL FIELD 

## BACKGROUND 

## SUMMARY 

methods provided herein result in pharmaceutical solutions having surprising stability. The stability of a complexed inhibitor is reflected in the lack of precipitation from the homogeneous complexed inhibitor solution over extended periods of time and thermal stresses. For example, the complexed inhibitor can remain soluble for periods of time and under thermal stresses exceeding those typical for practical use of aseptically manufactured injectable pharma› ceutical products. Although the high concentrations achieved by the processing methods provided herein may not be expected to be thermodynamically stable, the physi› cal stability of the solutions have been shown to be unaf› fected by storage temperature ( e.g., the solutions can be stable from -20° C. to 25° C.), freeze thaw cycling, and lyophilization and reconstitution. The stability of the super- saturated solutions of complexed peptide proteasome inhibi› tor and one or more cyclodextrins is sufficient to tolerate adjustments to pH following complexation without precipi› tation. For example, performing complexation in the pH range 2.5-3, then titrating the pH with sodium hydroxide solution to pH 3 .5. This solution physical stability allows for use of the complexed material in a pH range acceptable for injection and other pharmaceutical purposes, as well exhib› iting stability in a pH range where suitable chemical stability and shelf life is obtained. Accordingly, the pharmaceutical compositions prepared by the methods provided herein can be supersaturated solutions that do not precipitate or decrease in concentration to a significant extent during their use in any number of medical applications (e.g., a bulk solution during sterile product manufacture may not pre- cipitate for several days post sterile filtration while being held in a vial filling sterile hold tank. Likewise, final reconstituted pharmaceutical compositions may be stable for a range of hours to days facilitating their use as medicinal agents). 

In addition to producing stable, highly concentrated solu- tions of a peptide proteasome inhibitor, the formulations prepared by the complexation methods provided herein can be achieved without the chemical degradation and stability limitations of other methods of formulation. For example, the methods provided herein avoid the use of strong acids (e.g., HCl) to lower the pH during complexation. Although decreasing the pH of the formulation to a value less than 2 can facilitate the dissolution of the peptide proteasome inhibitor and produce a homogenous solution prior to com› plexation, the acidity of the solution can result in degrada- tion of the peptide proteasome inhibitor. For example, in the case of the peptide proteasome inhibitor carfilzomib, 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 formation of a chlorohydrin adduct as a degradant (CDP): 

<img src='content_image/929382.jpg'>

Based on its structure, this degradant is classified as an alkylator, which is a class of compound considered by the FDA to be a potentially genotoxic impurity. Importantly, from a regulated product safety standpoint, using the meth› ods provided herein avoids such strong acids and therefore degradation reactions of the peptide proteasome inhibitor to 

