US 10,159,746 B2  1  2  CYCLODEXTRIN COMPLEXATION  METHODS FOR FORMULATING PEPTIDE  PROTEASOME INHIBITORS  CROSS REFERENCE TO RELATED  APPLICATIONS  INCORPORATION BY REFERENCE OF  MATERIALS SUBMITTED ELECTRONICALLY  TECHNICAL FIELD  BACKGROUND  SUMMARY  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.  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  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):  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 