Stable Composition of Pegylated Carfilzomib Compound

Abstract

The present invention provides a stable pharmaceutical composition of a pegylated carfilzomib compound, a method for preparing the composition, and the use of the composition for treating cancer, including hematological malignancies such as multiple myeloma. The composition can be stored in a frozen form or lyophilized into a dry solid form.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 587,070, filed on November 16, 2017, the specification of which is hereby incorporated by reference in its entirety. Technical Field

[0002] The present invention relates to stable pharmaceutical compositions of pegylated carfilzomib compounds, methods for preparing said compositions, and their use for the treatment of cancer, including hematological malignancies such as multiple myeloma and solid tumors. Background Art

[0003] Cancer is one of the most prevalent diseases and a leading cause of death worldwide. In the United States alone, cancer is the second leading cause of death, second only to heart disease. Cancer is generally characterized by a dysregulation of normal cellular processes or unregulated cell proliferation.

[0004] Multiple myeloma (MM) is a progressive and malignant type of cancer that originates from plasma cells. It is characterized by the abnormal accumulation of malignant plasma cells within the bone marrow and accounts for approximately 13% of all blood cancers (Palumbo and Anderson, 2011). In 2015, it was estimated that approximately 26,850 new cases of MM would be diagnosed in the United States and approximately 11,240 people would die from the disease (ACS, 2015). Due to the increasing life expectancy of the general population in the United States, the incidence of MM has been steadily increasing (Warren et al., 2013). The disease most commonly affects the elderly population, with a median age of incidence of approximately 69 years (Howlander et al., 2013; ACS, 2015).

[0005] The treatment goals in MM management are to provide symptom relief, achieve disease control, and provide prolonged remissions (Kurtin, 2013). Conventionally, a combination of high-dose chemotherapeutic agents (melphalan, vincristine, cyclophosphamide, doxorubicin, liposomal doxorubicin, bendamustine) followed by autologous stem cell transplantation (ASCT) has been used to treat young, untreated, and medically fit patients (aged less than 65 years) (Palumbo et al., 2011). Age, comorbidities, and geriatric assessment are the main criteria for determining whether a patient is eligible to tolerate high-dose therapy (HDT) followed by ASCT (Palumbo et al., 2014). For elderly patients ineligible for HDT and ASCT, melphalan plus prednisone has been the standard therapy for decades (Palumbo et al., 2011; Rodríguez et al., 2012). In the past decade, with the introduction of novel immunomodulatory agents (such as thalidomide, lenalidomide, and pomalidomide) and targeted proteasome inhibitors (bortezomib and carfilzomib), the treatment algorithm for MM has undergone a paradigm shift (Richardson et al., 2007; Dmoszyńska, 2008; Gupta et al., 2013).

[0006] Carfilzomib is a tetrapeptide epoxyketone proteasome inhibitor that selectively and irreversibly binds to constitutive proteasomes and immunoproteasomes. More precisely, the epoxyketone electrophilic warhead binds to the catalytic threonine residue of the β5 subunit of the proteasome protein. CFZ is well tolerated with an acceptable toxicity profile. Carfilzomib, polymorphic forms, methods of manufacture, formulations, its uses, and other carfilzomib properties are described in US 20050245435, US 20140105921, and PCT publications WO 2006017842, WO 2009045497, WO2014169897, WO 2013169282, WO 2014011695, WO 2006063154, WO 2014015016, and WO 2010048298, the specifications of each of these documents are hereby incorporated by reference in their entirety herein.

[0007] Carfilzomib has shown encouraging overall response rates, progression-free survival (PFS), and overall survival (OS) in patients with relapsed and refractory MM and newly diagnosed MM. In July 2012, carfilzomib was first approved (as )As a single-agent therapy for the treatment of relapsed and refractory MM patients. Recently, Kyprolis was approved in combination with lenalidomide and dexamethasone (July 2015) and in combination with dexamethasone (January 2016) for the treatment of relapsed and refractory MM patients who have received one to three prior therapies. The approved carfilzomib treatment regimen is administered to patients by infusion over either a short 10-minute period or a slower, longer 30-minute duration. In a 28-day cycle, it is infused for 2 consecutive days each week for three consecutive weeks. Thus, to comply with this treatment regimen, patients need to drive or be driven to an authorized administration center, such as a doctor's office, clinic, or hospital, where appropriate and safe administration can be performed. This can be inconvenient or impractical, or may simply be a burden for some patients. This burden increases the likelihood of reduced or decreased compliance with the full and complete course of the prescribed treatment regimen, or even complete non-compliance.

[0008] Carfilzomib is rapidly metabolized and cleared in the human body. Carfilzomib is a small tetrapeptide compound that exhibits a short in vivo half-life of approximately 60 minutes or less in the human body. One mechanism of carfilzomib clearance is via hepatic blood flow, resulting in a relatively short half-life of carfilzomib. Drug products with short half-lives or rapid clearance generally tend to exhibit reduced target coverage, leading to decreased and / or shortened biologic inhibitory activity. To overcome such deficiencies, additional drug is typically administered to provide more drug efficacy and extended efficacy at the site of biologic action. Thus, the rapid clearance and twice-weekly dosing frequency of carfilzomib leave room for potential improvement in its efficacy, delivery, and / or patient compliance.

[0009] Currently approved carfilzomib is a sterile, lyophilized, amorphous solid formulation that contains sulfobutylether β-cyclodextrin (SBECD) and sodium citrate buffer. Immediately prior to administration, the lyophilized product is reconstituted with sterile water and infused or injected into the patient. The SBECD excipient primarily acts as a solubilizing additive for carfilzomib and forms a complex with carfilzomib, thereby improving the water solubility of carfilzomib.

[0010] History has shown that attempts to address the weaknesses of a pharmaceutical product and / or to improve the delivery, use, or other aspects of a given pharmaceutical product have led to the preparation of alternative forms of these active pharmaceutical ingredients (APIs or drug compounds) and / or their new formulations. Some alternative compound forms include the discovery of prodrugs, which are designed to enhance the pK and / or PD properties of the API. For example, Greenwald et al. disclosed Prodrugs of Amine Containing Compounds (J. Med. Chem. 1999, 42, 3657-3667). WO 2005063777 disclosed benzyl phosphate and substituted benzyl phosphate prodrugs for the treatment of lung inflammation. WO 20090152160 disclosed inhaled carbaprotacyclin and prostacyclin prodrugs for the treatment of arterial hypertension. U.S. Patent Publication No. 20040100225 disclosed imatinib acyloxymethyl prodrugs. Additionally, PCT Publication WO2011084846 disclosed acyloxymethyl prodrugs of risperidone. These prodrug disclosures teach alkyl-acyloxymethyl linked prodrugs.

[0011] Carfilzomib has also been modified to improve its properties or other attributes as an active pharmaceutical ingredient and pharmaceutical product. U.S. Patent Application Publication No. US20140105921 describes carfilzomib and other epoxyketone proteasome inhibitor prodrugs having an acyloxymethyl linker connecting the inhibitor to a polyethylene glycol unit (PEG). However, it has been found that these carfilzomib prodrug compounds release quinomethide by-products during in vivo metabolism, which may have potential toxicity and may pose a human safety risk. It is desirable to identify formulations and / or pharmaceutical compositions of modified carfilzomib compounds to appropriately deliver them to a patient while maintaining or possibly improving the stability, shelf life, efficacy, and / or safety of the currently approved carfilzomib treatment. SUMMARY OF THE INVENTION

[0012] The present invention provides novel pharmaceutical compositions of pegylated carfilzomib compounds, i.e., stable formulations of pegylated carfilzomib, which provide therapeutic anti-cancer benefits to a patient while maintaining comparable or longer carfilzomib plasma concentrations and exposure to proteasomal proteins. To this end, the formulations of the present invention provide proteasome inhibitory activity comparable to that of the currently approved carfilzomib cyclodextrin IV formulation. The present invention further provides pegylated carfilzomib formulations that do not include the use of cyclodextrin as a carfilzomib solubilizer.

[0013] Specifically, the present invention provides a stable, isotonic, cyclodextrin-free, lyophilized liquid formulation of a pegylated carfilzomib compound. The solid lyophilized formulation can be reconstituted with sterile water or the like and administered by parenteral methods including intravenous administration, injection and also subcutaneous administration. These formulations can be used to treat various types of cancer, including but not limited to multiple myeloma. More specifically, the formulations provided herein maintain or exhibit suitable bioavailability. The present invention further provides a method for preparing the pharmaceutical composition, and a method for administering the composition parenterally (by infusion or injection or subcutaneously) to treat various forms of cancer, such as multiple myeloma.

[0014] In one aspect, the present invention provides a pharmaceutical composition comprising: (a) a pegylated carfilzomib compound; (b) at least one excipient selected from the group consisting of sucrose, sorbitol, glycerol, maltose, lactose, erythritol, dextrose, lactulose, cyclodextrin, proline, glycine, arginine, histidine, aspartic acid, valine, leucine, alanine, methionine, proline, glutamic acid, glutamate; and salts selected from the group consisting of sodium chloride, potassium chloride, ammonium sulfate, potassium chlorate, calcium chloride, zinc chloride, guanidine hydrochloride, ammonium chloride, potassium sulfate, ammonium aspartate, arginine hydrochloride, lysine hydrochloride, magnesium chloride and barium sulfate; (c) a buffer selected from the group consisting of glutamate, histidine, acetate and Tris-HCl, or a combination thereof; and (d) optionally a bulking agent selected from the group consisting of mannitol, trehalose, PVP, cyclodextrin, glycine, dextrose, dextran, sucrose, proline, PEG 33350 and PEG400, (e) optionally an amino acid selected from the group consisting of lysine, arginine, histidine, aspartic acid, valine, leucine, alanine, methionine, proline, glutamic acid and glutamate, (f) optionally a surfactant selected from the group consisting of polysorbate 20, polysorbate 80, pluronic F68, sodium docusate, benzaconium chloride, triton X100, tetrafunctional block O polymer, alcohol, SDS, protamine sulfate and butane, or a combination of (d), (e) and (f). When compared with the corresponding approved carfilzomib product, the pharmaceutical composition of the present invention provides suitable solubility, permeability, pharmacokinetic (pK) and / or pharmacodynamic (PD) characteristics for the pegylated carfilzomib compound.

[0015] The pharmaceutical composition provided by the present invention further provides potential pharmaceutical product benefits, including but not limited to storage stability, storage capacity, safe use after several days as a liquid formulation, a frozen formulation, a dry freeze-dried formulation, and other conveniences for the safe maintenance, storage, and use of carfilzomib-based pharmaceutical products. The improved composition of the present invention is advantageous for various modes of administration, such as intravenous administration by infusion or injection. The composition can also be administered subcutaneously under the skin. The present invention also provides a composition comprising hyaluronidase, which can facilitate subcutaneous administration. The composition can be used to treat cancer, including but not limited to multiple myeloma and solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a bar graph showing the remaining % API (5K pegylated carfilzomib - Example 34 herein) measured by reverse phase chromatography after storage at 25 °C for 3 days;

[0017] Figure 2 is a bar graph showing the remaining % API (20K pegylated carfilzomib - Example 39 herein) measured by reverse phase chromatography after storage at 25 °C for 3 days;

[0018] Figure 3-A depicts the resulting freeze-dried cakes obtained from Formulations G5Su2M4(3) and H5Su2M4(1) (with 3K pegylated carfilzomib - Example 28 herein);

[0019] Figure 3-B is a bar graph showing the remaining % API (3K pegylated carfilzomib - Example 28 herein) measured by reverse phase chromatography in each of the 4 freeze-dried cakes in Figure 3-A above;

[0020] Figure 4-A depicts the resulting freeze-dried cake of the formulation obtained from G5Su2M4 + 0.006% polysorbate 80 + 2000 units / mL hyaluronidase (with 3K pegylated carfilzomib - Example 28 herein);

[0021] Figure 4-B depicts the resulting freeze-dried cake of the placebo comparator formulation containing only hyaluronidase;

[0022] Figure 5-A is a bar graph depicting the particle count >10 microns in the exemplary pre-freeze and post-freeze formulations of Table 5 measured by subvisible light obscuration; and

[0023] Figure 5-B is a bar graph depicting the particle count >25 microns in the exemplary pre-freeze and post-freeze formulations of Table 5 measured by subvisible light obscuration;

[0024] Figure 6Graph showing the results of the in vitro action of exemplary pegylated carfilzomib compounds in a cancer tumor xenograft model; and

[0025] Figure 7 Graph showing the results of the action of exemplary pegylated carfilzomib compounds described herein on cancerous tumors. DETAILED DESCRIPTION

[0026] The present invention provides novel pharmaceutical compositions of pegylated carfilzomib compounds, methods for preparing these formulations, and the use of said compositions for the treatment of cancer, including the treatment of hematological malignancies (such as multiple myeloma, lymphoma, leukemia) and the treatment of other cancers (such as solid tumors). Specifically, the formulations of the present invention are stable, without significant degradation of the active pharmaceutical ingredient, thereby improving the shelf life, solution clarity, lifespan, and safety of the pharmaceutical product. The present invention provides compositions as frozen formulations and compositions as dry lyophilized formulations of pegylated carfilzomib compounds. It is believed that these formulations have API pharmacokinetic (pK) and / or pharmacodynamic (PD) properties comparable to or improved over those of currently approved IV-administered (carfilzomib).

[0027] Carfilzomib is an epoxyketone protease inhibitor, particularly described in U.S. Patent Nos. 7,417,042 and 7,737,112. The present invention provides formulations comprising a pegylated carfilzomib compound as the API. Exemplary pegylated carfilzomib compounds that may be included in the present invention are generally and specifically described in International Application No. PCT / US2017 / 03429. As of November 24, 2017, this PCT application has not been published.

[0028] Representative pegylated carfilzomib compounds that may be included in the pharmaceutical compositions of the present invention are as follows.

[0029] In aspect 1 of the present invention, the composition comprises a pegylated carfilzomib compound having formula I

[0030]

[0031] or a pharmaceutically acceptable salt thereof, wherein

[0032] R 1 is C 1-10 alkyl or C 3-7 cycloalkyl;

[0033] Each R 2 is independently C 1-6 alkyl, -OCH 3 or halogen;

[0034] o is an integer selected from 0, 1, 2 or 3;

[0035] The linker is a moiety having the following structure

[0036]

[0037] wherein R 3 is H or CH 3 ;

[0038] n is an integer selected from 1, 2, 3 or 4;

[0039] p is an integer selected from 0, 1, 2, 3 or 4;

[0040] q is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8 or 9;

[0041] r is an integer selected from 0, 1, 2, 3, 4 or 5; and

[0042] PEG is a polyethylene glycol polymer moiety having a molecular weight in the range of from about 500 to about 20,000.

[0043] In aspect 1a of the present invention, the composition comprises a pegylated carfilzomib compound having formula I

[0044]

[0045] or a pharmaceutically acceptable salt thereof, wherein

[0046] R 1 is C 1-10 alkyl or C 3-7 cycloalkyl;

[0047] Each R 2 is independently C 1-6 alkyl, -OCH 3 or halogen;

[0048] o is an integer selected from 0, 1, 2 or 3;

[0049] The linker is a moiety having the following structure

[0050]

[0051] wherein R 3 is H or CH 3 ; and

[0052] p is an integer selected from 0, 1, 2, 3 or 4;

[0053] n is an integer selected from 1, 2, 3 or 4; and

[0054] PEG is a polyethylene glycol polymer moiety having a molecular weight in the range of from about 500 to about 20,000.

[0055] In aspect 2 of the present invention, the composition comprises a pegylated carfilzomib compound having formula II

[0056]

[0057] wherein

[0058] R 1 is C 1-10 alkyl or C 3-7 cycloalkyl;

[0059] R 2 is C 1-6 alkyl, -OCH 3 or halogen;

[0060] The linker is a moiety having the following structure

[0061]

[0062] wherein R 3 is H or CH 3 ;

[0063] n is an integer selected from 1, 2, 3 or 4;

[0064] p is an integer selected from 0, 1, 2, 3 or 4;

[0065] q is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8 or 9;

[0066] r is an integer selected from 0, 1, 2, 3, 4 or 5;

[0067] X is a counterion salt selected from the following: chloride, bisulfate, sulfate, nitrate, phosphate, alkyl sulfonate, or aryl sulfonate; and

[0068] PEG is a polyethylene glycol polymer moiety having a molecular weight in the range of from about 2000 to about 20,000.

[0069] In aspect 3 of the present invention, the composition comprises the pegylated carfilzomib compound as described in aspects 1, 1a and 2, wherein R 1 is C 1-10 alkyl.

[0070] In aspect 4 of the present invention, the composition comprises the pegylated carfilzomib compound as described in any one of aspects 1, 1a, 2 and 3, wherein each R 2 is independently H, CH 3 or halogen.

[0071] In aspect 5 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1, 1a, 2, 3, and 4, wherein each R 2 is independently H, CH 3 , Cl, or F.

[0072] In aspect 5a of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1, 1a, 2, 3, and 4, wherein each R 2 is independently H, CH 3 or F.

[0073] In aspect 6 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1, 1a, 2, 3, 4, and 5, wherein the linker is a moiety having the following structure

[0074]

[0075] wherein R 3 is H or CH 3 ;

[0076] q is an integer selected from 1, 2, 3, 4, or 5; and

[0077] r is an integer selected from 0, 1, 2, 3, or 4.

[0078] In aspect 6a of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1, 1a, 2, 3, 4, and 5, wherein the linker is a moiety having the following structure

[0079]

[0080] wherein R 3 is H or CH 3 .

[0081] In aspect 7 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1, 1a, 2, 3, 4, 5, and 7, wherein the linker is

[0082]

[0083] wherein R 3 is H or CH 3 ;

[0084] q is 4; and

[0085] r is 2.

[0086] In aspect 7a of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1, 1a, 2, 3, 4, 5, and 7, wherein the linker is

[0087]

[0088] wherein R 3 is H or CH 3 .

[0089] In aspect 8 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1, 1a, 2, 3, 4, 6, 6a, 7, and 7a, wherein R 3 is H.

[0090] In aspect 9 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-8, wherein R 1 is methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, or heptyl.

[0091] In aspect 10 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-9, wherein R 1 is methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, or heptyl; and the linker is

[0092]

[0093] In aspect 10a of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-9, wherein R 1 is methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, or heptyl; and the linker is

[0094]

[0095] It should be noted that in aspects 1, 1a, 2, and aspects 3-10, the term "or a pharmaceutically acceptable salt thereof" may include salts that balance the quaternary nitrogen cation charge, such as those shown in formula II of aspect 2 or those shown in aspects 11-24 below. In addition, it should be noted that the term "any one of aspects 1-X" is intended to also include all sub-aspects of 1-X disclosed herein, including but not limited to sub-aspects 1a, 5a, 6a, 7a, and 10a.

[0096] In aspect 11 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-10, the compound having the following structure

[0097]

[0098] wherein R 1 is C 1-10 alkyl;

[0099] R 2 is C 1-6 alkyl, -OCH 3 or halogen;

[0100] R 3 is H or CH 3 ;

[0101] X - is a counter anion selected from chloride anions and alkyl sulfonate anions;

[0102] n is 4; and

[0103] PEG is a polyethylene glycol polymer moiety having a molecular weight in the range of from about 2000 to about 20,000.

[0104] In aspect 12 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1 - 11, wherein the compound is

[0105] wherein X is a halide, sulfonate, or alkyl sulfonate counter ion salt.

[0106] In aspect 12a of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1 - 11, wherein the compound is

[0107] wherein X is a halide, sulfonate, or alkyl sulfonate counter ion salt.

[0108] In aspect 13 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1 - 11, wherein the compound is

[0109] wherein X is a halide, sulfonate, or alkyl sulfonate counter ion salt.

[0110] In aspect 14 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1 - 11, wherein the compound is

[0111]

[0112] In aspect 15 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1 and 2, wherein R 1is methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl or heptyl;

[0113] Each R 2 independently is CH 3 or halogen;

[0114] The linker is a moiety having the following structure

[0115]

[0116] wherein R 3 is H or CH 3 ; and

[0117] PEG is a polyethylene glycol polymer moiety having a molecular weight of 2000, 3000, 5000 or 20,000.

[0118] In aspect 16 of the present invention, the composition comprises a pegylated carfilzomib compound as described in aspect 15, wherein R 1 is methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl or heptyl;

[0119] Each R 2 independently is CH 3 ;

[0120] The linker is a moiety having the following structure

[0121]

[0122] wherein R 3 is H; and

[0123] PEG is a polyethylene glycol polymer moiety having a molecular weight of 3000, 5000 or 20,000.

[0124] In aspect 17 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-16, wherein the compound is a single compound represented by Examples 1-34 described in Table 2 below, or a pharmaceutically acceptable salt thereof.

[0125] In aspect 18 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-17, wherein the compound is

[0126]

[0127]

[0128] or a pharmaceutically acceptable salt thereof.

[0129] In aspect 18a of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 18, wherein the compound is

[0130]

[0131]

[0132] In aspect 19 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-18, wherein the compound is

[0133]

[0134] In aspect 19a of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-18, wherein the compound is

[0135]

[0136] In aspect 20 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-18, wherein the compound is

[0137]

[0138] In aspect 21 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-18, wherein the compound is

[0139]

[0140] In aspect 22 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-18, wherein the compound is

[0141]

[0142] In aspect 23 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-18, wherein the compound is

[0143]

[0144] In aspect 24 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-18, wherein the compound is

[0145]

[0146] In aspect 25 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-18, wherein the compound is

[0147]

[0148] In aspect 26 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-18, wherein the compound is

[0149]

[0150] In aspect 27 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-16, wherein the PEG has a weight ranging from about 2K to about 20K.

[0151] In aspect 28 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-16, wherein the PEG has a weight of 3K, 5K or 20K.

[0152] In aspect 29 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-16, and the compound is a pharmaceutically acceptable salt comprising a counter anion selected from: chloride anion, bisulfate anion, sulfate anion, nitrate anion, phosphate anion, alkylsulfonate anion, or arylsulfonate anion.

[0153] In aspect 30 of the present invention, the composition comprises the pegylated carfilzomib compound as described in aspect 29, wherein the counter anion is chloride anion or alkylsulfonate anion.

[0154] In aspect 31 of the present invention, the composition comprises the pegylated carfilzomib compound as described in aspect 29, wherein the counter anion is chloride anion or methanesulfonate anion.

[0155] In aspect 32 of the present invention, the composition comprises a pegylated carfilzomib compound as described in any one of aspects 1-26 and a pharmaceutically acceptable excipient, carrier or diluent.

[0156] In aspect 33 of the present invention, the present invention provides a pharmaceutical

[0157] composition, the pharmaceutical composition comprising:

[0158] (a) a pegylated carfilzomib compound;

[0159] (b) At least one excipient selected from the group consisting of sucrose, sorbitol, glycerol, maltose, lactose, erythritol, dextrose, lactulose, cyclodextrin, proline, glycine, arginine, histidine, aspartic acid, valine, leucine, alanine, methionine, proline, glutamic acid, glutamate; and salts selected from the group consisting of sodium chloride, potassium chloride, ammonium sulfate, potassium chlorate, calcium chloride, zinc chloride, guanidine hydrochloride, ammonium chloride, potassium sulfate, ammonium aspartate, arginine hydrochloride, lysine hydrochloride, magnesium chloride, and barium sulfate;

[0160] (c) A buffering agent selected from the group consisting of glutamate, histidine, acetate, and Tris-HCl, or a combination thereof; and

[0161] (d) Optionally, a bulking agent selected from the group consisting of mannitol, trehalose, PVP, cyclodextrin, glycine, dextrose, dextran, sucrose, proline, PEG 33350, and PEG 400,

[0162] (e) Optionally, an amino acid selected from the group consisting of lysine, arginine, histidine, aspartic acid, valine, leucine, alanine, methionine, proline, glutamic acid, and glutamate,

[0163] (f) Optionally, a surfactant selected from the group consisting of polysorbate 20, polysorbate 80, pluronic F68, sodium docusate, benzalkonium chloride, triton X100, tetrafunctionalized block O polymer, alcohol, SDS, protamine sulfate, and butane,

[0164] or a combination of (d), (e), and (f).

[0165] In aspect 33a of the present invention, the present invention provides a pharmaceutical composition comprising:

[0166] (a) A polyethylene glycolated carfilzomib compound;

[0167] (b) At least one excipient selected from the group consisting of sucrose, proline, glycine, and sodium chloride;

[0168] (c) A buffering agent selected from the group consisting of glutamate, histidine, acetate, and Tris-HCl, or a combination thereof; and

[0169] (d) Optionally, a bulking agent selected from the group consisting of mannitol, trehalose, PVP, cyclodextrin, glycine, dextrose, dextran, sucrose, proline, PEG 33350, and PEG 400,

[0170] (e) Optionally, an amino acid selected from the group consisting of lysine, arginine, histidine, aspartic acid, valine, leucine, alanine, methionine, proline, glutamic acid, and glutamate,

[0171] (f) Optionally, a surfactant selected from the group consisting of polysorbate 20, polysorbate 80, pluronic F68, sodium docusate, benzalkonium chloride, triton X100, tetrafunctionalized block O polymer, alcohol, SDS, protamine sulfate, and butane,

[0172] or a combination of (d), (e), and (f).

[0173] In aspect 33b, the present invention provides a composition as described in aspects 33 and 33a, wherein the (b) at least one excipient is present in an amount ranging from 0.1% - 30% weight / volume; the (c) buffer is present in an amount sufficient to achieve the desired pH of the formulation; the (d) optional swelling agent is present in an amount ranging from 2% - 50% weight / volume; the (e) optional amino acid is present in an amount ranging from 0.1% - 10% weight / volume; and the (f) surfactant is present in an amount ranging from 0.005% - 3% weight / volume of the composition.

[0174] In aspect 33c, the present invention provides a pharmaceutical composition comprising:

[0175] (a) A pegylated carfilzomib compound;

[0176] (b) At least one excipient selected from the group consisting of sucrose, proline, glycine, and sodium chloride;

[0177] (c) A buffer selected from the group consisting of glutamate, histidine, acetate, and Tris-HCl, or a combination thereof; and

[0178] (d) Optionally, a swelling agent selected from the group consisting of mannitol, trehalose, polyvinylpyrrolidone, and dextrose, the swelling agent being present in an amount ranging from 2% - 50% by weight,

[0179] (e) Optionally, an amino acid selected from the group consisting of lysine, arginine, histidine, and proline, the amino acid being present in an amount ranging from 0.1% - 10% by weight,

[0180] (f) Optionally, a surfactant selected from the group consisting of polysorbate 80, pluronic F68, polysorbate 20, and triton X100, the surfactant being present in an amount ranging from 0.005% - 3% by weight,

[0181] or a combination of (d), (e), and (f).

[0182] In aspect 33d, the present invention provides a pharmaceutical composition comprising:

[0183] (a) a pegylated carfilzomib compound;

[0184] (b) at least one excipient selected from the group consisting of sucrose in an amount ranging from 0.1% - 30% by weight, proline or glycine in an amount ranging from 0.1% - 10% by weight, and sodium chloride in an amount of about 300 nM concentration;

[0185] (c) a buffer selected from the group consisting of glutamate, histidine, acetate, and Tris - HCl, or a combination thereof; and

[0186] (d) optionally mannitol in an amount ranging from 2% - 50% by weight,

[0187] (e) optionally lysine or arginine in an amount ranging from 0.1% - 10% by weight;

[0188] (f) optionally polysorbate 80 or poloxamer F68 in an amount ranging from 0.005% - 3% by weight,

[0189] or a combination of (d), (e), and (f).

[0190] In aspect 34 of the present invention, the present invention provides a pharmaceutical composition as described in aspects 33 and 33a - 33d, wherein the pegylated carfilzomib compound has the structure of formula I as described in aspects 1 and 1a herein, as described in aspect 7 herein, as described in aspect 18 herein, or as described in aspect 18a herein.

[0191] In aspect 35 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 1 - 33, 33a - 33d, and 34, wherein the composition is a frozen formulation, and wherein the pH of the formulation is in the range from 5.0 to 8.0.

[0192] In aspect 36 of the present invention, the present invention provides a pharmaceutical composition as described in aspect 35, wherein the excipient is selected from the group consisting of sucrose, proline, glycine, and sodium chloride, or a combination thereof, and the buffer is selected from the group consisting of histidine, acetate, and Tris - HCl, or a combination thereof.

[0193] In aspect 37 of the present invention, the present invention provides a pharmaceutical composition as described in aspect 36, wherein the excipient is selected from the group consisting of sucrose in an amount in the range of about 5% - 12% weight / volume, proline in an amount in the concentration range of about 50 mM to 300 mM, glycine in an amount in the concentration range of about 50 mM to 300 mM, and sodium chloride in an amount in the concentration range of about 30 mM to 160 mM, or a combination thereof, and the buffer is selected from the group consisting of histidine in an amount in the concentration range of about 10 mM to 30 mM, acetate in an amount in the concentration range of about 10 mM to 30 mM, and Tris-HCl in an amount in the concentration range of about 10 mM to 30 mM, or a combination thereof.

[0194] In aspect 38 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 36 and 37, wherein the excipient is selected from the group consisting of sucrose in an amount of about 9% w / v, L-proline in an amount of about 220 mM concentration, glycine in an amount of about 293 mM concentration, sodium chloride in an amount of about 140 mM concentration, and a combination of sucrose in an amount of about 4.5% and sodium chloride in an amount of about 140 mM concentration; and the buffer is selected from the group consisting of histidine in an amount of about 10 mM concentration, acetate in an amount of about 10 mM concentration, and Tris-HCl in an amount of about 10 mM concentration.

[0195] In aspect 39 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 35 - 38, wherein the pharmaceutical composition comprises

[0196] (a) a polyethylene glycolated carfilzomib compound in an amount ranging from 150 mg to 2000 mg;

[0197] (b) the at least one excipient and buffer are (1) 9% sucrose and 10 mM acetate buffer, pH 5; (2) 9% sucrose and 10 mM histidine, pH 6; (3) 9% sucrose and 10 mM Tris-HCl, pH 7; (4) 9% sucrose and 10 mM Tris-HCl, pH 8; (5) 140 mM sodium chloride and 10 mM Tris-HCl, pH 7; (6) 220 mM L-proline and 10 mM Tris-HCl, pH 7; (7) 293 mM glycine and 10 mM Tris-HCl, pH 7; or (8) 70 mM sodium chloride and 4.5% sucrose with 10 mM Tris-HCl, pH 7.

[0198] In aspect 40 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 1 - 33, 33a - 33d and 34, wherein the composition is a dry freeze-dried formulation.

[0199] In aspect 41 of the present invention, the present invention provides a pharmaceutical composition as claimed in claim 40, wherein the at least one excipient is sucrose in an amount ranging from 0.5% to 2% weight / weight, the swelling agent is mannitol in an amount ranging from 2% to 4% weight / weight, the amino acid is absent or selected from lysine or arginine, and the surfactant is absent or is polysorbate 80 or pluronic F68, and the buffer is glutamate.

[0200] In aspect 42 of the present invention, the present invention provides a pharmaceutical composition as claimed in claim 41, wherein the excipient is sucrose in an amount in the range of from about 1% - 2% weight / volume, mannitol in an amount in the range of from about 2% to 4%, an amino acid selected from lysine or arginine in an amount ranging from about 0.5% to 0.8%, a surfactant being 0.0065% polysorbate 80 or 0.05% pluronic F68, and the buffer is 10 mM glutamate.

[0201] In aspect 43 of the present invention, the present invention provides a pharmaceutical composition as claimed in any one of aspects 1 - 33, 33a - 33d, 34, 41 and 42, wherein the composition consists essentially of:

[0202] 10 mm glutamate, 2% sucrose, and 4% mannitol, or

[0203] 10 mM glutamate, 2% sucrose, 4% mannitol, and 0.006% polysorbate 80, or

[0204] 10 mM glutamate, 2% sucrose, 4% mannitol, and 0.05% pluronic F68, or

[0205] 10 mM glutamate, 2% sucrose, 4% mannitol, 0.5% lysine, and 0.006% polysorbate 80, or

[0206] 10 mM glutamate, 2% sucrose, 4% mannitol, 0.8% lysine, and 0.006% polysorbate 80, or

[0207] 10 mM glutamate, 2% sucrose, 4% mannitol, 0.5% arginine, and 0.006% polysorbate 80, or

[0208] 10 mM glutamate, 2% sucrose, 4% mannitol, 0.8% arginine, and 0.006% polysorbate 80; and

[0209] a polyethylene glycolated carfilzomib compound in an amount ranging from 100 mg to 3000 mg.

[0210] In aspect 44 of the present invention, the present invention provides a pharmaceutical composition as described in aspect 43, wherein when the composition is dissolved in water at a concentration sufficient to achieve a concentration of about 10 mg / ml of the pegylated carfilzomib compound, the pH value of the composition is about 5.0.

[0211] In aspect 45 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 40 - 44, wherein the pharmaceutical composition comprises an amount of pegylated carfilzomib compound ranging from 150 mg to 2000 mg.

[0212] In aspect 46 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 40 - 45, wherein the pharmaceutical composition comprises an amount of pegylated carfilzomib compound ranging from 300 mg to 2000 mg.

[0213] In aspect 47 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 40 - 46, wherein the pharmaceutical composition comprises an amount of pegylated carfilzomib compound ranging from 800 mg to 3000 mg.

[0214] In aspect 48 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 40 - 45, wherein the pegylated carfilzomib compound is a 2K, 3K or 5K pegylated carfilzomib compound in an amount ranging from 200 mg to 800 mg.

[0215] In aspect 49 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 1 - 48, wherein the composition further comprises hyaluronidase.

[0216] In aspect 50 of the present invention, the present invention provides a pharmaceutical composition as described in aspect 49, wherein the hyaluronidase is present in an amount ranging from 1500 to 2500 units / mL.

[0217] In aspect 50a of the present invention, the present invention provides a pharmaceutical composition as described in aspects 49 and 50, wherein the hyaluronidase is present in an amount of 2000 units / mL.

[0218] In aspect 51 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 1 - 50 and 50a, the pharmaceutical composition being free of cyclodextrin.

[0219] In aspect 52 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 40 - 51, wherein when the lyophilized formulation is dissolved in 1.0 ml of water at room temperature, the lyophilized formulation provides a clear solution within a period of about 3 minutes.

[0220] In aspect 53 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 1-1-33, 33a-33d, and 34-50, 50a, 51, and 52, which is administered parenterally by infusion, injection, or subcutaneous administration.

[0221] In aspect 54 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 1-52, which is administered intravenously by infusion or injection.

[0222] In aspect 55 of the present invention, the present invention provides a pharmaceutical composition as described in any one of aspects 1-1-33, 33a-33d, and 34-50, 50a, 51, and 52, which is administered by subcutaneous injection.

[0223] In aspect 56 of the present invention, the present invention provides a method for treating cancer, which comprises administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition as described in any one of aspects 1-1-33, 33a-33d, and 34-50, 50a, and 51-55.

[0224] In aspect 57 of the present invention, the present invention provides the method as described in aspect 56, wherein the cancer is multiple myeloma.

[0225] In aspect 58 of the present invention, the present invention provides the method as described in aspect 57, wherein the multiple myeloma is relapsed, refractory, or relapsed and refractory multiple myeloma.

[0226] In aspect 59 of the present invention, the present invention provides the method as described in aspect 58, wherein the multiple myeloma is newly diagnosed multiple myeloma.

[0227] In aspect 60 of the present invention, the present invention provides a method for preparing a pharmaceutical composition as described in any one of aspects 1-1-33, 33a-33d, and 34-50 and 50a, the method comprising the steps of: (a) combining an amount of a pegylated carfilzomib compound effective for treating multiple myeloma with at least one excipient selected from the group consisting of sucrose, proline, glycine, and sodium chloride; and a buffer selected from the group consisting of glutamate, histidine, acetate, and Tris-HCl, or a combination thereof; and (b) mixing the combination to provide a clear solution.

[0228] Without wishing to be bound by theory, it is possible that the pharmaceutical compositions of the present invention may temporarily mask or partially mask the protease inhibitory activity of the API. This effect occurs until the polyethylene glycolylated linker moiety of the polyethylene glycolylated carfilzomib compound has been cleaved to release free carfilzomib into the systemic circulation. This delay in activity may reduce or eliminate unwanted side effects, which may be associated with various routes of administration. It should also be noted that the polyethylene glycolylated carfilzomib compounds included in the compositions of the present invention can act as prodrugs of carfilzomib. Alternatively, these compounds may themselves have very good proteasome inhibitory activity within themselves.

[0229] The beneficial properties of the compositions of the present invention may also facilitate the subcutaneous administration of the polyethylene glycolylated carfilzomib compounds. By subcutaneous administration, the present invention potentially improves dosing, patient convenience, and compliance in the treatment with the selected polyethylene glycolylated carfilzomib compounds.

[0230] In other aspects or embodiments of the present invention (which may be described later herein), these methods are characterized by treating a disease or disorder selected from the group consisting of: cancer, autoimmune diseases, graft or transplantation-related disorders, neurodegenerative diseases, fibrosis-related disorders, ischemia-related disorders, infections (viral infections, parasitic infections, or prokaryotic infections), and diseases associated with bone loss, the method comprising administering to a patient a pharmaceutical composition according to the present invention, the composition comprising a therapeutically effective amount of a polyethylene glycolylated carfilzomib compound, such as those described herein. In still further aspects, the present invention provides methods for treating cancer (e.g., multiple myeloma, e.g., relapsed and / or refractory multiple myeloma) in a patient.

[0231] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are set forth herein for this disclosure; other suitable methods and materials known in the art may also be used. These 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 herein are incorporated by reference in their entirety as if written herein. In case of conflict, the present specification, including definitions, will control. Other features and advantages of this disclosure will become apparent from the drawings, the drawings themselves, the detailed description, and the claims.

[0232] As used herein, the term "aspect" is synonymous with and used interchangeably with the term "embodiment".

[0233] Definitions

[0234] The following definitions should further assist in understanding the terms as used herein and the scope of the invention described herein.

[0235] The term "C x-y alkyl" refers to a saturated hydrocarbon group, substituted or unsubstituted, including straight-chain alkyl groups and branched alkyl groups having from x to y carbons in the chain. The term "haloalkyl" refers to an alkyl group in which at least one hydrogen atom has been replaced by a halogen (e.g., fluorine, chlorine, bromine, iodine), such as CH 2 F, CHF 2 , trifluoromethyl, and 2,2,2-trifluoroethyl.

[0236] The terms "C 2-y alkenyl" and "C 2-y alkynyl" refer to substituted or unsubstituted unsaturated aliphatic groups that are similar to the above alkyl groups in length and possible substitution, but contain at least one double bond or triple bond, respectively. In some embodiments, the divalent groups alkenylene and alkynylene include from 2 to 12 carbon atoms. In certain embodiments, alkenylene and alkynylene include from 2 to 10 carbon atoms. In certain embodiments, alkenylene and alkynylene include from 2 to 6 carbon atoms (e.g., 2, 3, 4, 5, or 6 carbon atoms).

[0237] The term "alkoxy" refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, etc. An "ether" is two hydrocarbons covalently linked by oxygen. Thus, the substituent of the alkyl group that makes it an ether is or is similar to an alkoxy.

[0238] As used herein, the term "C 3-y cycloalkyl" refers to a fully saturated, substituted or unsubstituted ring, wherein each atom of the ring is carbon, and the ring contains from 3 to γ carbon atoms in size. For example, the term C 3-7 cycloalkyl is intended to mean a carbocyclic ring containing from 3 to 7 carbon atoms in size at any position. Such rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl rings. These rings may be further substituted as specified.

[0239] As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological disorder in a subject that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, malignant hematological disorders or hematogenous cancers such as multiple myeloma and leukemia, and other cancers such as carcinomas, lymphomas, sarcomas, and blastomas. More specific examples of such cancers include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer. Although the term "cancer" as used herein is not limited to any particular form of the disease, it is believed that the methods of the present invention will be particularly effective against cancers in a subject that have become resistant, to some extent, to treatment with anti-cancer agents (including but not limited to chemotherapeutic agents, anti-mitotic agents, anthracyclines, etc.), and cancers that recur after treatment with such anti-cancer agents.

[0240] The term "comprising" means open-ended, including the one or more recited components but not excluding other elements.

[0241] The term or abbreviation "eg" or "eg.", as used herein, is intended to mean "for example".

[0242] The term "inhibitor" is intended to describe a compound that blocks or reduces the activity of an enzyme or enzyme system, receptor, or other pharmacological target (e.g., inhibits the proteolytic cleavage of standard fluorogenic peptide substrates such as suc-LLVY-AMC, Boc-LLR-AMC, and Z-LLE-AMC, inhibits the various catalytic activities of the 20S proteasome). The inhibitor can act with competitive, uncompetitive, or non-competitive inhibition. The inhibitor can bind reversibly or irreversibly, and thus the term includes compounds that are suicide substrates of the enzyme. The inhibitor can modify one or more sites on or near the active site of the enzyme, or it can cause a conformational change elsewhere on the enzyme. The term inhibitor is used herein more broadly than in the scientific literature so as to also encompass other classes of pharmacologically or therapeutically useful agents such as agonists, antagonists, stimulants, cofactors, etc.

[0243] As used herein, the terms "drug resistance" and "multi-drug resistance" refer to cancer cells that have developed and / or are resistant to a drug. These include cancer cells that exhibit little or no efficacy or reduced efficacy compared to the efficacy exhibited at the initial dose of the drug. The cancer cells may be resistant to one drug or to multiple drugs having different chemical structures that act on different biological targets within the cancer cells.

[0244] The term "pharmaceutically acceptable salt" includes salts commonly used to form alkali metal salts and addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts of the compounds can be prepared from inorganic acids or from organic acids. Examples of such inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Examples of organic acids include, but are not limited to, organic acids of the aliphatic, alicyclic, aromatic, arylaliphatic, heterocyclic, carboxylic, and sulfonic acid classes, examples of which are formic acid, acetic acid, adipic acid, butyric acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, mesylic (methanesulfonic) acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic (pamoic) acid, methanesulfonic acid, ethanesulfonic acid, ethanedisulfonic acid, benzenesulfonic acid, pantothenic acid, 2-hydroxyethanesulfonic acid, toluenesulfonic acid, sulfamic acid, cyclohexylaminosulfonic acid, camphoric acid, camphorsulfonic acid, digluconic acid, cyclopentanepropionic acid, dodecylsulfonic acid, glucoheptonic acid, glycerophosphoric acid, heptanoic acid, hexanoic acid, 2-hydroxy-ethanesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, palmoic acid, pectic acid, persulfuric acid, 2-phenylpropionic acid, picric acid, pivalicpropionic acid, succinic acid, tartaric acid, thiocyanic acid, methanesulfonic acid, undecanoic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, mucic acid, and galacturonic acid.

[0245] Suitable pharmaceutically acceptable base addition salts of the compounds include, but are not limited to, metal salts, such as salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc; or salts prepared from organic bases (including primary, secondary, and tertiary amines and substituted amines, including cyclic amines such as caffeine, arginine, diethylamine, N-ethylpiperidine, histidine, glucosamine, isopropylamine, lysine, morpholine, N-ethylmorpholine, piperazine, piperidine, triethylamine, and trimethylamine). All salts contemplated herein can be prepared by conventional means from the corresponding compounds by, for example, reacting the appropriate acid or base with the compound.

[0246] As used herein, the term "proteasome" is meant to include both immunoproteasome and constitutive proteasome.

[0247] As used herein, the term "refractory" is intended to mean unresponsive, resistant, or non - responsive to treatment, stimulation (therapy), or cure, including resistance to multiple therapeutic agents. When used herein in the context of characterizing a cancer or tumor, "refractory" is intended to mean a cancer or tumor that is unresponsive or resistant or has a diminished response to treatment with one or more anti - cancer agents. Over a period of time, the treatment is typically continuous, extended, and / or repeated, resulting in recurrence of the cancer or tumor, or development of resistance to the exact same treatment or becoming refractory to that treatment.

[0248] The term "subject" as used herein refers to any mammal (including humans) and animals (such as cows, horses, dogs, and cats). Thus, the present invention can be used in human patients as well as veterinary subjects and patients. In one embodiment of the invention, the compounds of the invention can be administered to human subjects.

[0249] The phrase "therapeutically effective" or "therapeutically effective amount" is intended to quantify the amount of a compound of the present invention which, when administered (to a patient, e.g., a human) as part of a desired dosage regimen, alleviates symptoms, improves the condition, or retards the onset of a disease condition, according to clinically acceptable criteria for the disorder or condition to be treated or for cosmetic purposes, e.g., with a reasonable benefit / risk ratio applicable to any medical treatment. Thus, the amount of the compounds of the present invention can treat cancer, whether multiple myeloma or other hematological malignancies or solid tumors.

[0250] The term "treat (treat, treating, and treatment)" as used herein refers to therapy, including but not limited to curative therapy, prophylactic therapy, and palliative therapy, and generally includes reversing, reducing, or preventing the symptoms, clinical signs, and underlying pathology of a disorder, thereby improving or stabilizing the condition of a patient. Prophylactic treatment generally includes completely preventing the onset of a disease in an individual or delaying the onset of the clinically apparent stage of an individual disorder. The term "prophylactic or therapeutic" treatment is well - recognized in the art and includes administering to a host one or more of the subject compositions. If administered before the clinical manifestation of an undesirable disorder (e.g., a disease or other undesirable state in a host animal) or after the subsidence of the disorder, the treatment is prophylactic (i.e., it protects the host against the development of the undesirable disorder), while if administered after the manifestation of the undesirable disorder, the treatment is therapeutic (i.e., it is intended to attenuate, improve, or stabilize the existing undesirable disorder or its side effects).

[0251] As used herein, the term PEG is intended to have its commonly understood and traditional meaning. Specifically, PEG is a moiety consisting of repeating poly(ethylene glycol) polymer units, the exact number of which determines its molecular weight. The unit of this molecular weight is Dalton. Thus, reference to the PEG molecular weight as used herein (in the specification, claims, and abstract), for example, referring to "2K", "3K", "5K", and "20K" or "2000", "3000", "5000", or "20000" relative to a given PEG is intended to mean 2000 Daltons (or 2 kilodaltons), 3000 Daltons (or 3 kilodaltons), 5000 Daltons (or 5 kilodaltons), and 20000 Daltons (or 20 kilodaltons) PEG weight, respectively. In addition, "KDa" as used herein means kilodalton.

[0252] General synthesis and representative examples of the pegylated carfilzomib compounds used in the present invention

[0253] As described, the pegylated carfilzomib compounds in Formulas I and II are cleavable polymer PEG carriers of the active pharmaceutical ingredient carfilzomib (Formulas I and II) and release free carfilzomib in vivo. The following abbreviations used throughout the general procedures and examples are intended to mean the following:

[0254] DCM Dichloromethane; methylene dichloride

[0255] DMF Dimethylformamide

[0256] DMSO Dimethyl sulfoxide

[0257] EtOAc Ethyl acetate

[0258] MeOH Methanol

[0259] mpk Milligram per kilogram; mg / kg

[0260] RT, rt Room temperature

[0261] NaCl Sodium chloride

[0262] tBuOH tert-Butanol; tert-butyl alcohol

[0263] Carfilzomib for use in preparing the pegylated carfilzomib compounds is described in PCT Publications WO 2006017842, WO 2009045497, WO 2014169897, WO2013169282, WO 2014011695, WO 2006063154, WO2014015016, WO 2010048298, and U.S. Patent Nos. 7,714,042 and 7,737,112.

[0264] Scheme 1: Cleavage of the benzyl-eliminated quaternary salt

[0265]

[0266] Enzymatic and / or chemical hydrolysis of the phenyl ester provides carboxylic acid (II) and phenolate intermediate (I), which undergoes rapid 1,6-elimination to provide free carfilzomib and quinone methide, and the quinone methide remains covalently attached to the solubilizing PEG polymer. Quinone methides are known reactive Michael acceptors and are believed to pose a risk associated with potential genotoxicity. In the present invention, the permanent attachment of the quinone methide byproduct to the polymer can attenuate toxicity by preventing cell access to serum nucleophiles and reducing reactivity towards serum nucleophiles. The most likely fate of intermediate III in vivo is to react with water to form a benzyl alcohol-polymer adduct, which is rapidly removed from the body by excretion.

[0267] Scheme 2: Cleavage of the previously described carfilzomib-polymer conjugate

[0268]

[0269] Scheme 2 shows a metabolic pathway of a carfilzomib polymer compound described in WO 2014011695. Here, as shown above, the carfilzomib-polymer conjugate interacts with esterases or undergoes chemical attack as indicated by the arrows. This attack results in the release of free quinone methide after ester hydrolysis (encapsulated above). This methide intermediate freely reacts further with cellular nucleophiles, which may lead to toxicity. As described in Scheme 1, the polyethylene glycolated carfilzomib compound of the present invention avoids this potentially toxic byproduct.

[0270] Scheme 3: Two-step PEG polymer conjugation procedure

[0271]

[0272] As shown in Scheme 3, the carfilzomib-PEG compounds provided by the present invention are prepared in a two-step procedure. First, carfilzomib is reacted with a suitably substituted p-acyloxy-substituted benzyl halide (1) to give a quaternary salt intermediate (2). The quaternary salt bromide or iodide anion can be exchanged via an ion exchange resin with a pharmaceutically acceptable anion (such as bisulfate, sulfate, nitrate, dihydrogen phosphate, or alkyl / aryl sulfonate) to give intermediate (3). This intermediate is conveniently attached with a reactive group suitable for reacting with a complementary functionalized polymer reagent (4) to obtain the desired product 5. A large number of PEG reagents are commercially available within a range of molecular weights, structures, end-group chemistries, and numbers of reactive end-groups (arms) (see Table 1). They can be directly compatible with the linker chemistries described in this disclosure or may require some additional chemical manipulations by known methods. Branched and multi-arm PEGs may have advantages over linear PEGs, such as potentially higher drug loading, improved stability, and / or lower formulation viscosities.

[0273] Scheme 4: Two-step polymer conjugation via azide / alkyne click chemistry

[0274]

[0275] Scheme 4 shows "click" chemistries, such as the Huisgen 1,3-dipolar azide / alkyne cycloaddition and the aminooxy / aldoximation, which are particularly suitable for polymer and polymeric PEG attachment due to high chemical yields, harmless by-products, large thermodynamic driving forces, and availability of starting materials.

[0276] The Huisgen 1,3-dipolar azide / alkyne cycloaddition requires that the benzyl group be capable of reacting with an azide-functionalized polymer carrier (such as PEG-azide (-N 3)) The alkyne group of the reaction (A1-(1-6)) is substituted to give a 1,2,3-triazole-linked conjugate (A4-(1-6)). The alkyne moiety can be directly linked, or linked via an alkyl spacer (A1-1), via an ether (A1-2,3), thioether, sulfoxide or sulfone (A1-4) bond, or via an amide bond (A1-5,6). Many azide-substituted PEG reagents are now commercially available in a variety of sizes and structures, but can also be readily prepared from any available PEG alcohol via mesylation or tosylation activation, followed by reaction with an azide salt. The cycloaddition reaction can be carried out using a commercially available cuprous salt catalyst, but a mixture of copper(II) (e.g., copper(II) sulfate, copper(II) methanesulfonate) and a reducing agent (e.g., sodium ascorbate) is more effective to generate Cu(I) in situ. Since Cu(I) is unstable in aqueous solution and in the presence of oxygen, a stabilizing ligand such as tris-(benzyltriazolylmethyl)amine (TBTA), tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), ethyl 2-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl] hydrogen sulfate (BTTES), or 2-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]acetic acid (BTTAA) can be optionally added. The reaction can be carried out at RT or at elevated temperature, in a variety of solvents, and in mixtures of water and a variety of miscible organic solvents including alcohols, DMSO, DMF, tBuOH and acetone. The final PEG-carfilzomib product (A4-(1-6)) can be conveniently processed by diluting the reaction mixture with water or brine, extracting with an organic solvent such as DCM, and reprecipitating from isopropanol or an ether / isopropanol mixture until a product of the desired purity is obtained. During the processing procedure, exposure of the intermediate or product to anions (such as chloride anions in brine) typically results in a mixture of anions in the final product, and a final anion exchange resin treatment may be necessary to ensure product salt uniformity.

[0277] The intermediate quaternary halide salts (bromide or iodide, (A2-(1-6)) can be converted to anions that do not precipitate with copper(I) catalysts such as methanesulfonate, hydrogen sulfate or sulfate (A3-(1-6)) to achieve high reaction yields. In addition, it may be desirable to exchange the halide anions to prevent ring opening of epoxides and the formation of possible bromohydrin or iodohydrin by-products.

[0278] Scheme 4A1-2

[0279]

[0280] Synthesis of 4-(bromomethyl)-2-(prop-2-ynyloxy)phenyl acetate (Intermediate A1-2 in Scheme 4)

[0281] Step 1: 4-Hydroxy-3-(prop-2-ynyloxy)benzaldehyde (1)

[0282] At 20 °C, 3,4-dihydroxybenzaldehyde (10 g, 72.5 mmol) in DMF (50 mL) was added to a mixture of NaOtBu in DMF (150 mL). The mixture was cooled with an ice bath and stirred while adding 3-bromoprop-1-yne (8.62 g, 72.5 mmol) portionwise, attempting to maintain the internal temperature between 15 °C and 20 °C. The reaction mixture was stirred at RT for 2 h. The mixture was diluted with water (300 mL) and extracted with EtOAc (200 mL × 3). The combined organic layers were washed with water to remove DMF, dried over anhydrous Na 2 SO 4 and concentrated to a brown solid. The residue was recrystallized from DCM / petroleum ether (30 mL / 500 mL) to give Compound 1. 1H NMR (CDCl3, 300 MHz,): δ 9.87 (s, 1H), 7.54 (d, J = 1.2 Hz, 1H), 7.49 (dd, J1 = 1.5 Hz, J2 = 8.1 Hz, 1H), 7.09 (d, J = 8.1 Hz, 1H), 4.82 (m, 2H), 2.62 (m, 1H).

[0283] Step 2: 4-Formyl-2-(prop-2-ynyloxy)phenyl acetate (2)

[0284] At 0 °C, Et 3 N (11.48 g, 113.64 mmol) was added to a solution of Compound 1 (10.00 g, 56.82 mmol) in DCM (150 mL), followed by acetyl chloride (5.35 g, 68.18 mmol). The reaction mixture was stirred at RT for 2 h. The mixture was washed with saturated 2N aqueous HCl (100 mL) and water (50 mL), dried over anhydrous MgSO 4 and concentrated to give Compound 2, which was used in the next step without further purification. 1 H NMR (CDCl 3 , 400 MHz): δ 9.96 (s, 1H), 7.63 (d, J = 1.6 Hz, 1H), 7.54 (dd, J 1 = 1.6 Hz, J 2 = 8.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 4.79 (d, J = 2.4 Hz, 2H), 2.57 (t, J = 2.4 Hz, 1H), 2.35 (s, 3H).

[0285] Step 3: 4-(Hydroxymethyl)-2-(prop-2-ynyloxy)phenyl acetate (3)

[0286] At 0 °C, NaBH 4 (3.06 g, 82.57 mmol) was added portionwise to a solution of compound 2 (12.00 g, 55.05 mmol) in DCM / MeOH (150 mL / 15 mL). The reaction mixture was stirred at RT for 30 min. The mixture was quenched with acetone (5 mL) and concentrated. The residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 2:1) to afford compound 3. 1 H NMR (CDCl 3 , 400 MHz): δ 7.16 (d, J = 1.6 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.98 (dd, J 1 = 1.6 Hz, J 2 = 8.0 Hz, 1H), 4.72 (d, J = 2.4 Hz, 2H), 4.69 (s, 2H), 2.53 (t, J = 2.4 Hz, 1H), 2.32 (s, 3H).

[0287] Step 4: 4-(Bromomethyl)-2-(prop-2-ynyloxy)phenyl acetate (4)

[0288] At 0 °C, PPh 3 (20.50 g, 78.41 mmol) and NBS (11.04 g, 62.73 mmol) were added to a solution of compound 3 (11.50 g, 52.27 mmol) in DCM (150 mL). The reaction mixture was stirred at room temperature for 0.5 h. The excess solvent was concentrated and the residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 20:1) to afford compound 4 (7.82 g, 53% yield). 1 H NMR (CDCl 3 , 400 MHz): δ 7.14 (m, 1H), 7.02 (m, 2H), 4.73 (d, J = 2.4 Hz, 2H), 4.48 (s, 2H), 2.55 (t, J = 2.4 Hz, 1H), 2.32 (s, 3H).

[0289] Scheme 5: Quaternary salt anion exchange

[0290]

[0291] Ion exchange can be accomplished by reacting the intermediate quaternary halide with a silver salt or more practically by passing it through an ion exchange resin, as shown in Scheme 5. The carfilzomib quaternary salt anion present in the intermediate or final product can be effectively converted to different strong acid anions, such as bisulfate, sulfate, dihydrogen phosphate, nitrate, or alkyl / aryl sulfonate, via an anion exchange resin. The anion exchange resin, such as Amberlyst A26(OH - form), is pretreated with the desired acid or ammonium salt and then the quaternary halide is passed through. Conjugates prepared from weak acid anions, such as acetate, formate, or lactate, are unstable due to the increased basicity of the quaternary salt and its incompatibility with the ester initiating group.

[0292] Scheme 6: Two-step polymer conjugation via aminooxy / carbonyl chemistry

[0293]

[0294]

[0295] Alternatively, the benzyl group (B1-(1-6)) can be replaced with a carbonyl (aldehyde or ketone) group that is capable of reacting with an aminooxy-functionalized polymer carrier, such as PEG-aminooxy (-ONH 2 )) to provide a stable oxime-linked conjugate (B4-(1-6)). The carbonyl moiety can be directly attached, or attached via an alkyl spacer (B1-1), via an ether (B1-2,3), thioether, sulfoxide, or sulfone (B1-4) bond, or via an amide bond (B1-5,6). Allowing carfilzomib and benzyl halide (B1-(1-6)) to react at RT or at elevated temperature in a suitable organic solvent, such as acetonitrile, to provide the quaternary salt intermediate (B2-(1-6)) as the bromide or iodide salt. It is desirable to exchange this halide anion to prevent ring opening of the epoxide and the formation of possible bromohydrin or iodohydrin by-products. The anion exchange can be accomplished by reacting the intermediate quaternary halide with a silver salt or more practically by passing it through an ion exchange resin, as described previously (Scheme 5). Then, allowing the carfilzomib quaternary salt intermediate (B3-(1-6)) and PEG-ONH 3 + Y -The polymeric reagent reacts at RT or at elevated temperatures in a suitable organic solvent (such as DCM) or a mixed aqueous organic solvent. An oximation catalyst (such as aniline, p-phenylenediamine, or 5-methoxyanthranilic acid) can be optionally added, but is usually not necessary. It should be noted that the carfilzomib quaternary salt intermediate (B3-(1-6)) and the PEG-aminooxy reagent anion salt are the same to avoid the formation of a mixed anion salt final product and the need for any additional anion manipulation. The final PEG-carfilzomib product can be conveniently processed by evaporating the reaction solvent and reprecipitating the residue from isopropanol or an ether / isopropanol mixture until a product of the desired purity is obtained.

[0296] Scheme 7: Synthesis of the PEG-aminooxy reagent

[0297]

[0298] The PEG-aminooxy reagent can be commercially available or can be readily prepared from mesyl or tosyl activated PEG-alcohol, PEG-halide (A), or PEG-amine (B) starting materials, as depicted in Scheme 7. The tert-butoxycarbonyl protected intermediate can be deprotected with a strong acid (such as hydrogen chloride, methanesulfonic acid, trifluoroacetic acid, or sulfuric acid) to give the PEG-aminooxy reagent as the chloride, trifluoroacetate, or sulfate. The PEG-aminooxy reagent anion can optionally be exchanged into a different anion via an anion exchange resin.

[0299] Scheme 8: Oxime isomers

[0300]

[0301] One of ordinary skill in the art can readily understand that an oxime can exist as two geometric isomers: the cis (Z)-isomer and the trans (E)-isomer, as depicted in Scheme 8. Many of the examples in this disclosure are aromatic aldehyde oximes and exist only as the (E)-isomer. Non-aromatic aldehyde oximes and ketoximes can generally be fully separated and obtained as the (Z)-isomer and the (E)-isomer. The polyethylene glycolated non-aromatic aldehyde oximes and ketoximes described in the present invention can exist as the individual (Z) and (E)-isomers or as a mixture of the (Z) and (E)-isomers.

[0302] Scheme 9: Direct polymer conjugation to form the quaternary salt

[0303]

[0304] Alternatively, the carfilzomib-polymer conjugate described in the present invention can be prepared in a one-step reaction of carfilzomib and an alkoxycarbonyl-substituted benzyl halide pre-attached with the desired polymer chain, as shown in Scheme 9. The polymer chain can be attached via a variety of known chemistries or the previously described alkyne / azide or carbonyl / aminooxy chemistries. This route may not be ideal due to the difficulty in separating the PEG-containing product from the unreacted PEGylated starting material.

[0305] Representative compound examples for use in the present invention

[0306] The following PEGylated carfilzomib compounds are representative examples of PEGylated carfilzomib compounds useful in the present invention and should not be construed as limiting the scope of the present invention. The PEGylated carfilzomib compounds are prepared using the following two general PEG-linking methods (A and B).

[0307] PEG triazole-linker method A:

[0308]

[0309] The carfilzomib mesylate intermediate A3-(1-6) (1.5 equivalents), PEG-azide (1 equivalent), and (L)-ascorbic acid (0.75 equivalents) were mixed in DMF (50 mL / mmol PEG-azide) to give a cream-colored suspension. The mixture was stirred vigorously for 5 minutes, and a solution of copper(II) sulfate pentahydrate (0.3 equivalents) in water (10 mL / mmol PEG-azide) was added dropwise rapidly. The reaction immediately darkened to light yellow-brown, and the suspension became clear within 5 minutes. After 1 hour, a second portion of ascorbic acid (0.75 equivalents) was added, and the reaction mixture was stirred for 60 minutes. A third portion of ascorbic acid (0.38 equivalents) was added, and the reaction mixture was stirred overnight at RT. Water (100 mL / mmol PEG-azide) and NaCl (15 g / mmol PEG-azide) were added, and the mixture was stirred until the NaCl dissolved. The product was extracted with DCM (3X 35 mL / mmol PEG-azide). The extract was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo at 40 °C. The residue was dissolved in isopropanol (125 mL / mmol PEG-azide) at 40 °C. Once the solid had completely dissolved, diethyl ether (90 mL / mmol PEG-azide) was added, and the solution was cooled in an ice bath. The resulting solid was filtered, and the cake was washed twice with 2-propanol and twice with diethyl ether. The cake was dissolved in DCM and concentrated in vacuo. The residue was dissolved in warm (40 °C) isopropanol (200 mL / mmol PEG-azide), and then allowed to cool in an ice bath. The resulting solid was filtered, and the cake was washed twice with 2-propanol and twice with diethyl ether, and then dried in vacuo.

[0310] PEG oxime-linker method B

[0311]

[0312] Carfilzomib mesylate intermediate B3-(1-6) (1 equivalent) in DCM (15 mL / mmol B3-(1-6)) was stirred at RT, PEG-ONH 3 + MsO -(0.8 eq), and 5-methoxyanthranilic acid (oximation catalyst, 0.3 eq) until complete consumption of the PEG reagent was observed by HPLC (ELS detector). The reaction mixture was evaporated to dryness and the residue was dissolved in isopropanol (15 mL / mmol B3-(1-6)) at 40 °C. The clear solution was cooled to RT and ether (5 mL / mmol B3-(1-6)) was added to induce crystallization. The mixture was cooled in an ice bath for 5 - 10 minutes and the solid formed was collected by filtration. Recrystallization from isopropanol / ether was repeated one or more times until all unreacted carfilzomib quaternary salt intermediate B3-(1-6) was removed as detected by HPLC. The final solid was dried under vacuum at 30 °C. Typical yield: 60% - 80%; typical reaction time: 10 - 30 min for intermediates with aldehyde functional groups, 24 h for intermediates with ketone functional groups.

[0313] Examples of the prepared PEG-carfilzomib compounds, PEG structures, and PEG linker methodologies are listed in Table 1. Table 1 further includes the size and weight (in daltons) of the PEG adducts and the methods used to attach the PEG moiety to the carfilzomib backbone.

[0314] Table 1

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321] Example 2: 4-(4-acetoxy-2-((1-PEG 5K -1H-1,2,3-triazol-4-yl)methoxy)benzyl)-4- ((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5, 8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazacyclohexadecyl)morpholin-4-ium methanesulfonate (9).

[0322]

[0323] 2-Hydroxy-4-(methoxymethoxy)benzaldehyde (1)

[0324] To a solution of compound 2,4-dihydroxybenzaldehyde (5.04 g, 36.24 mmol) in THF (100 mL) was added DIPEA (6.52 g, 54.35 mmol) and chloro(methoxy)methane (3.21 g, 39.86 mmol). The reaction mixture was stirred overnight at RT. The excess solvent was concentrated and the residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 15:1) to afford compound 1 (3.96 g, 60% yield); 1 H NMR (300 MHz, CDCl 3 ): δ 11.41 (s, 1H), 9.76 (s, 1H), 7.48 (dd, J1 = 2.7 Hz, J2 = 8.4 Hz, 1H), 6.67 (dd, J1 = 2.4 Hz, J2 = 8.7 Hz, 1H), 6.62 (d, J = 2.1 Hz, 1H), 5.25 (d, J = 2.7 Hz, 2H), 3.51 (d, J = 3.0 Hz, 3H).

[0325] 4-(Methoxymethoxy)-2-(prop-2-ynyloxy)benzaldehyde (2)

[0326] At 20 °C, to a mixture of NaH (900 mg, 21.252 mmol) in DMSO (100 mL) was added compound 1 (2.0 g, 10.63 mmol) in DMSO (50 mL). The mixture was stirred at the same temperature for 30 min and then 3-bromoprop-1-yne (1.90 g, 15.94 mmol) was added dropwise. The reaction mixture was stirred at the same temperature for 4 h and then poured into ice water (100 mL). The resulting solution was adjusted to pH = 2 - 3 and EtOAc (100 mL) was added. The two phases were separated and the aqueous phase was extracted with EtOAc (100 mL × 3). The combined organic phases were dried and concentrated. The residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 3:1) to afford compound 2 (1.89 g, 80% yield); 1 H NMR (300 MHz, CDCl 3 ): δ 10.34 (s, 1H), 7.85 (d, J = 9.3 Hz, 1H), 6.76 (m, 2H), 5.26 (s, 2H), 4.83 (d, J = 2.4 Hz, 2H), 3.52 (s, 3H), 2.60 (q, J = 2.4 Hz, 1H).

[0327] 4-Hydroxy-2-(prop-2-ynyloxy)benzaldehyde (3)

[0328] To a solution of compound 2 (5.1 g, 23.18 mmol) in propan-2-ol (100 mL) was added CBr 4(760 mg, 2.32 mmol). The reaction mixture was refluxed overnight. The excess solvent was concentrated and the residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 3:1) to afford compound 3 (2.44 g, 60% yield); 1 H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1H), 10.11 (s, 1H), 7.60 (d, J = 8.8 Hz, 1H), 6.59 (d, J = 2.0 Hz, 1H), 6.52 (dd, J1 = 2.0 Hz, J2 = 8.8 Hz, 1H), 4.92 (d, J = 2.4 Hz, 2H), 3.70 (q, J = 2.4 Hz, 1H).

[0329] 4-(Hydroxymethyl)-3-(prop-2-ynyloxy)phenol (4)

[0330] At 0 °C, NaBH 4 (618 mg, 16.698 mmol) was added portionwise to a solution of compound 3 (2.45 g, 13.92 mmol) in MeOH (40 mL). The reaction mixture was stirred at the same temperature for 1 h and then quenched with water (1.5 mL). The excess solvent was concentrated and the residue was redissolved in EtOAc (100 mL). The resulting solution was dried and concentrated to afford compound 4 (1.80 g, 74% yield), which was used in the next step without further purification; 1 H NMR (400 MHz, DMSO-d6): δ 6.98 (d, J = 8.0 Hz, 1H), 6.32 (s, 1H), 6.26 (d, J = 8.0 Hz, 1H), 4.65 (d, J = 2.0 Hz, 2H), 4.32 (s, 2H), 3.54 (m, 1H).

[0331] 4-(Hydroxymethyl)-3-(prop-2-ynyloxy)phenyl acetate (5)

[0332] At 0 °C, TEA (1.70 g, 16.85 mmol) was added dropwise to a solution of compound 4 (1.20 g, 6.74 mmol) in DCM (30 mL), followed by acetyl chloride (634 mg, 8 mmol). The reaction mixture was stirred at RT for 30 min. The excess solvent was concentrated and the residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 5:1) to afford compound 5 (360 mg, 30% yield); 11H NMR (400 MHz, DMSO-d6): δ 7.38 (d, J = 8.0 Hz, 1H), 6.79 (d, J = 2.0 Hz, 1H), 6.75 (dd, J1 = 2.0 Hz, J2 = 8.0 Hz, 1H), 5.09 (m, J = 5.6 Hz, 1H), 4.82 (d, J = 2.4 Hz, 1H), 4.46 (d, J = 5.6 HZ, 2H), 3.60 (q, J = 2.4 Hz, 1H), 2.26 (s, 3H).

[0333] 4-(Bromomethyl)-3-(prop-2-ynyloxy)phenyl acetate (6)

[0334] At 0 °C, PPh 3 (515 mg, 1.96 mmol) was added portionwise to a solution of compound 5 (360 mg, 1.64 mmol) in DCM (15 mL), followed by NBS (318 mg, 1.80 mmol). The reaction mixture was stirred at the same temperature for 30 min. The excess solvent was concentrated and the residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 50:1) to afford compound 6 (190 mg, 41% yield); 1 1H NMR (400 MHz, CDCl3): δ 7.36 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 2.0 Hz, 1H), 6.73 (dd, J1 = 2.0 Hz, J2 = 8.4 Hz, 1H), 4.77 (d, J = 2.4 Hz, 2H), 4.54 (s, 2H), 2.56 (q, J = 2.4 Hz, 1H), 2.31 (s, 3H).

[0335] 4-(4-Acetoxy-2-(prop-2-yn-1-yloxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7- isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9, 12-tetraazacyclohexadecyl)morpholin-4-ium methanesulfonate (8)

[0336] (S)-4-Methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxirane-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide (480 mg, 0.67 mmol) was added to a solution of compound 6 (190 mg, 0.67 mmol) in MeCN (10 mL). The reaction mixture was stirred at 45 °C overnight. The excess solvent was concentrated and the residue was purified by flash column chromatography on silica gel (MeOH / EtOAc = 1:50) to afford the desired compound 7, which was converted to the corresponding mesylate (340 mg, 74% yield) by treatment with an ion exchange resin; 1 1H NMR (400 MHz, CDCl 3): δ 9.68 (m, 1H), 7.88 (m, 1H), 7.63 (m, 1H), 7.33 - 7.16 (m, 10H), 6.89 (m, 3H), 6.50 (m, 1H), 5.16 (m, 1H), 5.05 (m, 1H), 4.87 (m, 1H), 4.75 (m, 2H), 4.47 (m, 2H), 4.45 - 4.12 (m, 8H), 4.02 (m, 3H), 3.72 (m, 1H), 3.54 (m, 1H), 3.38 (m, 1H), 3.20 (m, 1H), 3.06 (m, 2H), 2.80 (s, 3H), 2.74 (m, 2H), 2.63 (m, 2H), 2.40 - 2.08 (m, 5H), 1.64 (m, 2H), 1.47 (s, 3H), 0.85 (m, 12H).

[0337] The compound of Example 2 was prepared from Compound 8 and PEG according to the general PEGylation procedure A 5K N 3 Prepare

[0338] Example 13: 4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methylcyclo oxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazacyclohexadecyl)-4-(3-((1- (PEG 20K -4-arm)-1H-1,2,3-triazol-4-yl)methoxy)-4-(pivaloyloxy)benzyl)morpholin-4-ium formate (7)

[0339]

[0340] 4-Hydroxy-3-(prop-2-ynyloxy)benzaldehyde (1)

[0341] At 20 °C, 3,4-dihydroxybenzaldehyde (30 g, 217.39 mmol) in DMSO (50 mL) was added to a mixture of NaH in DMSO (300 mL). The mixture was stirred for 30 min and 3-bromoprop-1-yne (25.87 g, 217.39 mmol) was added. The reaction mixture was stirred at RT for one hour and then poured into ice water. The resulting solution was adjusted to pH = 2 and then extracted with EtOAc (500 mL × 3). The combined organic phases were dried over anhydrous MgSO 4 and concentrated. The residue was recrystallized from DCM / petroleum ether (30 mL / 500 mL) to give Compound 1 (30 g, 78% yield); 1 H NMR (CDCl 3 , 300 MHz,): δ 9.89 (s, 1H), 7.54 (d, J = 1.2 Hz, 1H), 7.49 (dd, J 1 = 1.5 Hz, J 2 = 8.1 Hz, 1H), 7.09 (d, J = 8.1 Hz, 1H), 4.82 (m, 2H), 2.62 (m, 1H).

[0342] 4-Formyl-2-(prop-2-ynyloxy)pivalic acid phenyl ester (2)

[0343] At 0 °C, Et 3 N (3.45 g, 34 mmol) was added to a solution of Compound 1 (3.0 g, 17 mmol) in DCM (120 mL), followed by pivaloyl chloride (2.34 g, 20.4 mmol). The reaction mixture was stirred at RT for 2 h. The mixture was washed with saturated NaHCO 3 (20 mL) and water (20 mL), dried over anhydrous MgSO 4 and concentrated. The residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 50:1) to afford Compound 2 (2.10 g, 47% yield) as a white solid; 1 1H NMR (CDCl 3 , 300 MHz): δ 9.99 (s, 1H), 7.61 (d, J = 1.8 Hz, 1H), 7.55 (dd, J 1 = 1.8 Hz, J 2 = 8.1 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 4.77 (d, J = 2.4 Hz, 2H), 2.58 (t, J = 2.4 Hz, 1H), 1.42 (s, 9H).

[0344] 4-(Hydroxymethyl)-2-(prop-2-ynyloxy)pivalic acid phenyl ester (3)

[0345] At 0 °C, NaBH 4 (0.37 g, 10.4 mmol) was added to a solution of Compound 2 (1.8 g, 6.9 mmol) in DCM / MeOH (100 mL / 10 mL). The reaction mixture was stirred at RT for 30 min. The mixture was quenched with acetone (3 mL) and the solvent was concentrated. The residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 3:1) to afford Compound 3 (1.50 g, 83% yield); 1 1H NMR (CDCl 3 , 300 MHz): δ 7.11 (m, 1H), 7.00 (m, 2H), 4.68 (m, 4H), 2.53 (m, 1H), 1.41 (s, 9H).

[0346] Phenyl 4-(bromomethyl)-2-(prop-2-ynyloxy)pivalate (4)

[0347] At 0 °C, PPh 3(1.80 g, 6.8 mmol) and NBS (1.11 g, 6.3 mmol). The reaction mixture was stirred at RT for 0.5 h. The excess solvent was concentrated and the residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 50:1) to afford compound 4 (1.34 g, 81% yield); 1 H NMR(CDCl 3 , 300 MHz): δ 7.12 (d, J = 1.5 Hz, 1H), 7.03 (m, 2H), 4.70 (d, J = 2.4 Hz, 2H), 4.51 (d, J = 3.9 Hz, 2H), 2.56 (t, J = 2.4 Hz, 1H), 1.40 (s, 9H).

[0348] 4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2- carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazacyclohexadecyl)-4-(4-(pivaloyloxy)-3- (prop-2-yn-1-yloxy)benzyl)morpholin-4-ium methanesulfonate (6)

[0349] To a solution of compound 4 (2.38 g, 7.3 mmol) in MeCN (30 ml) was added (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxirane-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide (2.64 g, 3.7 mmol). The reaction mixture was stirred at 45 °C overnight. The excess solvent was concentrated and the residue was purified by flash column chromatography on silica gel (EtOAc / MeOH = 100:6) to afford the desired compound 5, which was converted to the corresponding mesylate (1.23 g, 25% yield) by treatment with an ion exchange resin; 1 H NMR(CDCl 3 , 300 MHz): δ 9.83 (m, 1H), 7.92 (m, 1H), 7.50 - 7.11 (m, 13H), 7.03 (m, 1H), 6.62 (m, 1H), 5.25 (m, 1H), 5.15 - 4.90 (m, 2H), 4.88 - 4.75 (m, 2H), 4.70 - 4.20 (m, 7H), 4.20 - 3.90 (m, 3H), 3.70 - 3.40 (m, 4H), 3.26 (m, 1H), 3.15 (m, 2H), 2.90 (s, 3H), 2.85 (m, 2H), 2.40 - 2.10 (m, 2H), 1.87 - 1.63 (m, 5H), 1.55 (m, 3H), 1.41 (s, 9H), 1.38 (m, 2H), 0.89 - 1.05 (m, 12H).

[0350] Compound Example 13 was prepared from Compound 6 and PEG according to General PEGylation Procedure A. In the various figures shown herein, Compound Example 13 is also designated as OP-59381. 20K (N 3 ) 4 1 H NMR (500 MHz, relaxation time = 10 sec, DMSO-d 6 ) δ 8.47 (s, 4H), 8.42 (d, J = 8.5 Hz, 4H), 8.29 (d, J = 7.5 Hz, 4H), 8.11 (s, 4H), 8.07 (d, J = 8 Hz, 4H), 7.53 (s, 4H), 7.26 - 7.29 (m, 4H), 7.11 - 7.19 (m, 32H), 7.05 - 7.06 (m, 4H), 5.21 (s, 8H), 4.95 (dd, J = 12.5 Hz and 39.0 Hz, 8H), 4.52 - 4.54 (m, 12H), 4.28 - 4.38 (m, 16H), 4.17 - 4.20 (m, 4H), 4.06 (m, 20H), 3.78 (t, J = 5.5 Hz, 8H), 3.61 - 3.65 (m, 8H), 3.50 (s, 2133H), 3.35 - 3.37 (m, 8H), 3.10 (d, J = 5 Hz, 4H), 2.94 - 2.98 (m, 12H), 2.73 - 2.78 (m, 4H), 2.50 - 2.65 (m, 8H), 1.90 - 1.98 (m, 4H), 1.78 - 1.88 (m, 4H), 1.51 - 1.68 (m, 8H), 1.39 (s, 12H), 1.25 - 1.38 (m, 16H), 1.18 (s, 36H), 0.833 - 0.881 (m, 24H), 0.782 - 0.815 (m, 24H); Loading: 86%.

[0351] Example 18: 4-(3-acetoxy-4-((PEG 5K -imino)methyl)benzyl)-4-((4S,7S,10S,13S)- 10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4- phenethyl-3,6,9,12-tetraazacyclohexadecyl)morpholin-4-ium methanesulfonate (8)

[0352]

[0353] 2-Hydroxy-5-(hydroxymethyl)benzaldehyde (1)

[0354] 2-Hydroxybenzaldehyde (10.3 g, 84.4 mmol) and concentrated HCl (42 mL) were added to an aqueous formaldehyde solution (37%, 17 mL). The reaction mixture was heated under reflux overnight. The mixture was cooled to RT and then extracted with EtOAc (200 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 3:1) to afford Compound 1 (1.97 g, 15% yield); 1 H NMR (DMSO-d 6 , 300 MHz): δ 10.61 (s, 1H), 10.26 (s, 1H), 7.60 (d, J = 2.1 Hz, 1H), 7.46 (dd, J = 2.4, 8.7 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 5.18 (m, 1H), 4.42 (d, J = 3.3 Hz, 2H).

[0355] 5-(((tert-Butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde (2)

[0356] Imidazole (1.43 g, 21 mmol) was added to a solution of Compound 1 (2.01 g, 13.2 mmol) in DCM (60 mL). The solution was cooled to 0 °C and tert-butylchlorodimethylsilane (2.57 g, 17.1 mmol) was added. The reaction mixture was stirred at RT for 3 h and then poured into water (50 mL). The two phases were separated and the organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 50:1) to afford Compound 2 (3.2 g, 91% yield); 1 H NMR (CDCl 3 , 400 MHz): δ 10.85 (br, s, 1H), 9.78 (s, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.35 (dd, J = 2.0, 8.4 Hz, 1H), 6.85 (d, J = 8.4 Hz, 1H), 4.59 (s, 2H), 0.82 (s, 9H), 0.00 (s, 6H).

[0357] 4-((tert-Butyldimethylsilyloxy)methyl)-2-formylphenyl acetate (3)

[0358] To a solution of compound 2 (25 g, 94 mmol) in DCM (500 mL) was added TEA (19.0 g, 188 mmol). The mixture was cooled to 0 °C and acetyl chloride (11.1 g, 141 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The mixture was washed with water (500 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 100:1) to give compound 3 (19.7 g, 68% yield); 1 H NMR(CDCl 3 , 400 MHz): δ 9.98 (s, 1H), 7.70 (d, J = 2.0 Hz, 1H), 7.49 (dd, J = 2.4, 8.4 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H), 4.66 (s, 2H), 2.28 (s, 3H), 0.83 (s, 9H), 0.00 (s, 6H).

[0359] Phenyl 2-formyl-4-(hydroxymethyl)acetate (4)

[0360] Compound 3 (3.6 g, 11.7 mmol) was dissolved in AcOH / THF / H 2 O (50 mL / 25 mL / 25 mL). The reaction mixture was stirred at 30 °C for 3 h. Excess THF was removed and the resulting solution was adjusted to pH = 7 - 8 and then extracted with EtOAc (50 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated. The residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 3:1) to give compound 4 (2.04 g, 90% yield); 1 H NMR(DMSO-d 6 , 400 MHz): δ 10.08 (s, 1H), 7.85 (d, J = 2.0 Hz, 1H), 7.67 (dd, J = 2.4, 8.4 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 4.57 (s, 2H), 2.35 (s, 3H).

[0361] Phenyl 4-(bromomethyl)-2-formylacetate (5a)

[0362] At 0 °C, PBr 3 (2.79 g, 10.3 mmol) was added to a solution of compound 4 (2.03 g, 10.3 mmol) in DCM (80 mL). The reaction mixture was stirred at RT for 4 h. The reaction was quenched by the addition of water (20 mL) and the resulting mixture was washed with saturated NaHCO 3The aqueous solution was adjusted to pH = 7. The organic phase was separated, dried over anhydrous sodium sulfate and concentrated. The residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 3:1) to give compound 5a (300 mg, 11% yield); 1 H NMR(CDCl 3 , 300 MHz): δ 10.12 (s, 1H), 7.92 (d, J = 2.1 Hz, 1H), 7.68 (dd, J = 2.4, 8.4 Hz, 1H), 7.22 (d, J = 8.1 Hz, 1H), 4.54 (s, 2H), 2.42 (s, 3H).

[0363] Phenyl 4-(iodomethyl)-2-formylacetate (5b)

[0364] At 0 °C, SOCl 2 (6.13 g, 51.55 mmol) was added to a solution of compound 4 (5.0 g, 27.55 mmol) in DCM (300 mL). The reaction mixture was heated at reflux overnight. The mixture was concentrated and the residue was purified by flash column chromatography on silica gel (petroleum ether / EtOAc = 10:1) to give the corresponding benzyl chloride (2.4 g, 44% yield); 1 HNMR(CDCl 3 , 300 MHz): δ 10.12 (s, 1H), 7.92 (d, J = 2.4 Hz, 1H), 7.68 (dd, J = 2.4, 8.4 Hz, 1H), 7.22 (d, J = 2.4 Hz, 1H), 4.64 (s, 2H), 2.42 (s, 3H).

[0365] To a solution of benzyl chloride (2.4 g, 11.29 mmol) in acetone (160 mL) was added NaI (16.94 g, 112.94 mmol). The reaction mixture was stirred at 30 °C overnight. The mixture was concentrated and the residue was dissolved in DCM (100 mL). The resulting solution was washed with saturated Na 2 S 2 O 3 aqueous solution (50 mL × 3) and water (50 mL), dried over anhydrous sodium sulfate and concentrated to give compound 5b (2.1 g, 61% yield), which was used in the next step without further purification. 1 HNMR(CDCl 3 , 300 MHz): δ 10.10 (s, 1H), 7.90 (d, J = 2.4 Hz, 1H), 7.66 (dd, J = 2.1, 8.4 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H), 4.49 (s, 2H), 2.41 (s, 3H).

[0366] 4-(4-Acetoxy-3-formylbenzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-meth yl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazacyclohexadecyl)morpholin-4-ium methanesulfonate (7) 4-(4-Acetoxy-3-formylbenzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-meth

[0367] To a solution of compound 5b (380 mg, 1.48 mmol) in MeCN (5 mL) was added (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxirane-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide (532 mg, 0.74 mmol). The reaction mixture was stirred at 45 °C overnight. The solvent was removed under reduced pressure. The residue was purified by flash column chromatography on silica gel (DCM / MeOH = 10:1) to afford the desired compound ( 6 ), which was then converted to the corresponding mesylate (280 mg, 39% yield) by treatment with an ion-exchange resin; 1 H NMR (CDCl 3 , 400 MHz): δ 10.15 (s, 1H), 9.53 (br s, 1H), 8.03 (d, J = 2.0 Hz, 1H), 7.85 (m, 1H), 7.68 (br s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.26 - 7.13 (m, 10H), 6.84 (br s, 1H), 6.52 (br s, 1H), 5.20 (m, 2H), 4.97 (m, 1H), 4.50 - 3.96 (m, 7H), 3.46 - 3.28 (m, 2H), 3.16 (m, 1H), 3.06 - 2.92 (m, 3H), 2.85 - 2.61 (m, 7H), 2.44 (s, 3H), 2.14 (m, 2H), 1.69 - 1.17 (m, 11H), 0.89 - 0.83 (m, 12H). Compound 5a can also be used in this reaction.

[0368] Example 18 was prepared from compound 7 and PEG 5K ONH 3 + .MsO- according to the general PEGylation procedure A.

[0369] yl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazacyclohexadecyl)morpholin-4-ium methanesulfonate (7) 3K Example 23: 4-(3-Acetoxy-4-((PEG -imino)methyl)benzyl)-4-((4S,7S,10S,13S)- 10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-

[0370]

[0371] phenethyl-3,6,9,12-tetraazacyclohexadecyl)morpholin-4-ium methanesulfonate (8) 4-(4-Acetoxy-3-formylbenzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazacyclohexadecyl)morpholin-4-ium methanesulfonate (7)

[0372] (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxirane-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide (532 mg, 0.74 mmol) was added to a solution of compound 5b (380 mg, 1.48 mmol) in MeCN (5 mL). The reaction mixture was stirred overnight at 45 °C. The solvent was removed under reduced pressure. The residue was purified by flash column chromatography on silica gel (DCM / MeOH = 10:1) to afford the desired compound ( 6 ), which was then converted to the corresponding mesylate (280 mg, 39% yield) by treatment with an ion exchange resin; 1 H NMR (CDCl 3 , 400 MHz): δ 10.15 (s, 1H), 9.53 (br s, 1H), 8.03 (d, J = 2.0 Hz, 1H), 7.85 (m, 1H), 7.68 (br s, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.26 - 7.13 (m, 10H), 6.84 (br s, 1H), 6.52 (br s, 1H), 5.20 (m, 2H), 4.97 (m, 1H), 4.50 - 3.96 (m, 7H), 3.46 - 3.28 (m, 2H), 3.16 (m, 1H), 3.06 - 2.92 (m, 3H), 2.85 - 2.61 (m, 7H), 2.44 (s, 3H), 2.14 (m, 2H), 1.69 - 1.17 (m, 11H), 0.89 - 0.83 (m, 12H).

[0373] Example 23 was prepared by a method similar to that described in Example 16, wherein the intermediate was prepared in a similar manner (using acetyl chloride to generate the putative intermediate 1 shown in Example 16 and compound 7 in International Application No. PCT / US2017 / 03429) and PEG 3K ONH 3 + .MsO - was prepared according to General PEGylation Procedure A.

[0374] Example 26: 4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2- carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazacyclohexadecyl)-4-(4-(isobutyryl (Benzyloxy)-3-((PEG 5K -imino)methyl)benzyl)morpholin-4-ium methanesulfonate (6)

[0375]

[0376] 4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2- -2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazacyclohexadecyl)-4-(3-formyl-4-(isobutyryl yloxy)benzyl)morpholin-4-ium methanesulfonate (5)

[0377] (S)-4-Methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxirane-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide (393 mg, 0.547 mmol) was added to a solution of compound 3 (550 mg, 1.657 mmol) in MeCN (8 mL). The reaction mixture was stirred overnight at 40 °C. The excess solvent was concentrated and the residue was recrystallized from (EtOAc / Et 2 O = 1:5) to afford the desired compound 4, which was converted to the corresponding mesylate 5 (115 mg, 7.5% yield) by treatment with an ion-exchange resin; 1 H NMR (400 MHz, CDCl 3 ): δ 10.18 (s, 1H), 9.68 (m, 1H), 8.04 (m, 1H), 7.89 (m, 1H), 7.81 (s, 1H), 7.35 (m, 1H), 7.30 (m, 1H), 7.11 - 7.29 (m, 9H), 6.79 (s, 1H), 6.44 (m, 1H), 5.18 (m, 2H), 4.99 (m, 1H), 4.41 (m, 3H), 4.20 (m, 3H), 3.99 (m, 3H), 3.40 (m, 1H), 3.30 (m, 1H), 3.20 (m, 1H), 2.95 (m, 2H), 2.92 (m, 1H), 2.79 (m, 3H), 2.75 (m, 2H), 2.21 (m, 1H), 2.09 (m, 1H), 1.83 (m, 4H), 1.62 (m, 2H), 1.49 (m, 4H), 1.38 (m, 6H), 1.24 (m, 2H), 0.88 (m, 12H).

[0378] Example 26 was prepared by a method similar to that described in Example 16, wherein the intermediate was prepared in a similar manner (using isopropyl chloride to generate the inferential intermediate 1 and compound 5 shown in Example 16 (in International Application No. PCT / US2017 / 03429)) and PEG 5K ONH 3 + .MsO - was prepared according to General PEGylation Procedure B.

[0379] Example 32: 4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methylcyclo ethane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazacyclohexadecyl)-4-(4-(isobutyryl (Benzyloxy)-3-((4-(PEG 5K -imino)methyl)benzyloxy)benzyl)morpholin-4-ium methanesulfonate (8)

[0380]

[0381] 4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methylcycloethane-2- carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazacyclohexadecyl)-4-(3-(4-formylbenzyloxy yl)-4-(isobutyryloxy)benzyl)morpholin-4-ium methanesulfonate (7)

[0382] (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxirane-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide (286 mg, 0.30 mmol) was added to a solution of compound 5 (310.4 mg, 0.80 mmol) in MeCN (2 mL). The reaction mixture was stirred at 45 °C for 48 h. The excess solvent was evaporated and the residue was recrystallized from MeCN / Et 2 O (1 / 5, v / v) to afford the desired product 6, which was then converted to the corresponding mesylate compound 7 (280 mg, 83% yield) by treatment with an ion exchange resin.

[0383] Compound 6 (280 mg, 0.25 mmol), 2-amino-5-methoxybenzoic acid (14.0 mg, 0.026 mmol), and PEG-O-NH 2 (mesylate, 1.16 g, 0.227 mmol) in DCM (3 mL) was stirred at r.t. for 2 h. The reaction mixture was then concentrated and the residue was dissolved in i-PrOH at 40 °C. The solution was cooled to room temperature and Et 2 O was added to induce crystallization. The mixture was kept in an ice bath for 10 min and the solid formed was collected by filtration. Crystallization from i-PrOH / Et 2 O (5:2) was repeated twice until all 7 was removed to afford 8 (1.0 g, 72% yield).

[0384] Example 34: 4-(4-Acetoxy-3-((1-PEG 3K -1H-1,2,3-triazol-4-yl)methoxy)benzyl)-4- ((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methylcycloethane-2-carbonyl)-2,5, 8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazacyclohexadecyl)morpholin-4-ium chloride

[0385]

[0386] Example 34 was prepared using a method similar to that taught in Examples 5-11 of International Application No. PCT / US2017 / 03429 and Method A, but using a chloride salt intermediate having a chloride anion as the counter ion.

[0387] Example 35: 4-(4-acetoxy-3-((1-PEG 3K -1H-1,2,3-triazol-4-yl)methoxy)benzyl)-4- ((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methylcycloethane-2-carbonyl)-2,5, 8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazacyclohexadecyl)morpholin-4-ium methanesulfonate

[0388]

[0389] Example 35 was prepared using a method similar to that taught in Examples 5-11 of International Application No. PCT / US2017 / 03429 and Method A using PEG 3K N 3 by Method A. 1 1H NMR (DMSO-d6, 400 MHz): δ 9.19 (m, 1H), 8.24 (m, 2H), 8.12 (m, 1H), 7.90 (m, 1H), 7.62 (m, 1H), 7.22 (m, 13H), 7.0 (m, 1H), 5.26 (m, 2H), 4.88 (m, 2H), 4.53 (m, 3H), 4.37 (br s, 4H), 4.05 (m, 5H), 3.81 (m, 2H), 3.68 (m, 4H), 3.52 (br s, 339H), 3.30 (m, 4H), 3.24 (s, 4H), 2.94 (m, 2H), 2.75 (m, 1H), 2.63 (m, 2H), 2.24 (s, 3H), 1.87 (m, 2H), 1.59 (m, 2H), 1.40 (m, 7H), 0.84 (m, 12H)

[0390] The present invention provides pharmaceutical compositions comprising a pegylated carfilzomib compound having Formula I or II and a range of excipients and buffers to be selected therefrom. As described herein, the present invention provides stable, isotonic, freeze-dried lyophilized formulations. These pharmaceutical compositions can be used to deliver bioactive pegylated carfilzomib compounds for the treatment of cancer. These compositions (also referred to herein as formulations) include, but are not limited to, stable formulations that can be administered to a patient in need of treatment by parenteral administration routes, including intravenous and subcutaneous administration. The compositions are stable enough as liquids for use in both clinical and commercial cancer settings.

[0391] The letters and numbers used in the formulations as described herein are defined as follows:

[0392] "A" represents an acetate buffer system at the noted concentration;

[0393] "G" represents a glutamate buffer system at the indicated concentration;

[0394] "H" represents histidine;

[0395] "M" represents mannitol;

[0396] "T" represents Tris-HCl;

[0397] "Na" represents sodium chloride;

[0398] "Pro" represents proline;

[0399] "Gly" represents glycine;

[0400] The numbers 5, 6, 7, and 8 represent the pH of the formulation;

[0401] "Su" represents sucrose, and the following number represents the % sucrose contained in the formulation;

[0402] Accordingly, a formulation designated as "A5Su" herein means that in addition to the amount of active carfilzomib present in the polyethylene glycolated carfilzomib API compound (the amount is expressed in mg or the concentration is expressed in mg / mL), the formulation also contains 10 mM acetate, 9.0% sucrose and has a pH of 5.0. The solutions made and tested herein were prepared by adding half of the required water to a batch container and then measuring out the amounts of each component (i.e., acetate, sucrose, and excipients such as PS80) to achieve the desired (or specified) concentration of each component. The exact concentration / amount was calculated using the molar mass of each component / chemical. The calculated amounts were added to the batch container; the resulting solution was stirred well until all components were mixed and dissolved. The initial pH of the solution was measured and the solution was titrated to the appropriate pH using 10N NaOH or 37% HCl stock solution according to the desired / stipulated pH. The remaining volume of water required to reach the final concentration was added to the batch container, and the final pH and temperature of the solution were obtained. The solution was sterile filtered through a 0.22 micron cellulose acetate filter into a container of appropriate size. The starting reagents were commercially available and were purchased from Sigma-Aldrich.

[0403] Some of the tests conducted herein involved measuring the osmolality of representative formulations or solutions. Freezing point depression osmolality measurements were collected using an Advanced Instruments 3250 single sample osmometer. All samples were sterile filtered through a 0.2 micron PES filter prior to measurement to ensure no particles. Three measurements were averaged for each sample point. The instrument operation was verified with 100, 200, and 290 mOsm standards prior to collecting sample data.

[0404] Carfilzomib (CFZ) is sold under the trade name Commercially available proteasome inhibitors and are indicated for the treatment of relapsed or refractory multiple myeloma. The current administration route of Kyprolis is IV (intravenous). From the perspective of patient convenience, it is highly desirable that a subcutaneous formulation convert the 30 - 90 - minute intravenous administration into a subcutaneous injection of 5 minutes or less. A major challenge in preparing a subcutaneous formulation is the solubility of carfilzomib. The currently approved formulation is a dry lyophilized formulation which, when reconstituted with an appropriate amount of sterile water according to the instructions on the approved label, produces a clear administrable liquid solution which has carfilzomib at a concentration of about 2 mg / mL in In order to improve the solubility of carfilzomib in water, pegylated carfilzomib compounds with different lengths of PEG attached thereto have been discovered and prepared. The PEG groups are attached to carfilzomib via a covalently - linked linker which is designed to be cleaved once the PEG - CFZ construct is parenterally administered into the human body.

[0405] The following pegylated carfilzomib compounds are used in the experiments described herein to demonstrate the stability, shelf - life and clarity of the formulations of the present invention. Example 39 (OP - 0059381) is carfilzomib with 20kPEG attached; Example 26 (OP - 0214575) has 5kPEG attached thereto; and Example 34 (OP - 0214576 - 1) has 3kPEG attached thereto.

[0406] To identify stable formulations of PEG - CFZ, various formulations were prepared in which the desired pegylated carfilzomib compound (API) was dissolved in experimental compositions of excipients, and the stability and quantity of intact, non - degraded API were monitored and measured in the frozen state (-20 °C or below). Formulations of the API were also prepared and tested to determine the ability of each exemplary formulation to provide sufficient stability in the liquid state for suitable clinical and commercial administration. Here, these formulations were stored at room temperature for at least 8 hours and at temperatures in the range of 2 °C - 8 °C for 2 days. All formulations tested contained the desired API compound at a concentration of 1 mg / mL.

[0407] For each formulation, the stability of the API was tested using three observable and / or measurable characteristics, as follows:

[0408] 1. Integrity of the molecule as evaluated by reverse - phase assay;

[0409] 2. Clarity of the solution as evaluated visually; and

[0410] 3. Concentration of API material in the solution as evaluated by material recovery after centrifugation

[0411] Results

[0412] Integrity of the API: The first research criterion focused on reducing or minimizing the hydrolysis of the carfilzomib epoxide ring, as hydrolysis produces inactive degradation by-products and impurities. Various exemplary formulations in which the API (example 26 of a pegylated carfilzomib compound) is dissolved in a solution as described in Table 2.

[0413] Table 2

[0414] Formulation Name pH Buffer System One or More Excipients Included A5Su 5 10 mM Acetate 9% Sucrose H6Su 6 10 mM Histidine 9% Sucrose T7Su 7 10 mM Tris-HCl 9% Sucrose T8Su 8 10 mM Tris-HCl 9% Sucrose T7Na 7 10 mM Tris-HCl 140 mM Sodium Chloride T7Pro 7 10 mM Tris-HCl 220 mM L-Proline T7Gly 7 10 mM Tris-HCl 293 mM Glycine T7NaSu 7 10 mM Tris-HCl 70 mM Sodium Chloride, 4.5% Sucrose

[0415] The pH range tested in the appropriate buffer was 5 - 8. The excipient sucrose remained constant from pH 5 to 8 to more directly test the effects of different pHs (from slightly acidic to slightly basic pH) at an isotonic level of 9%.

[0416] Another aspect of the study was to test the role of the excipients at the same pH. For this purpose, the following excipients were tested in a constant composition of 10 mM Tris-HCl at pH 7: sucrose (a polyol), sodium chloride (a salt), proline (a representative amino acid), glycine (a representative amino acid), and a combination of sodium chloride and sucrose.

[0417] Figure 1 Results obtained after incubating each formulation at 25 °C for 3 days are shown for the exemplary pegylated carfilzomib compound 26 (5kPEG-CFZ construct). This figure depicts two outputs for each formulation in the study: (a) the percentage of the main peak at time 0 (T 0 ) and at time 3 days @25 °C (T 3 ), measured on the left y-axis by reverse-phase assay, and (b) the percentage of API material recovered at T 3 . The main peak in the reverse-phase assay represents the intact API, while chemical modifications in the compound appear before or after the main peak, thus reducing the percentage of the main peak. Figure 1 Indicates that after 3 days at 25 °C, the formulations with the highest levels of intact API peaks are A5Su, T7NaSu, and T7Na. Underperforming formulations include H6Su and T8Su. The material recovery for A5Su, T7NaSu, and T7Na is also high.

[0418] After 3 days at 25 °C, all formulations shown in Figure 1 were visually inspected, and the results are provided in Table 3 below.

[0419] Table 3

[0420] Formulation Visual Observation A5Su Clear H6Su Turbid T7Su Turbid T8Su Turbid T7Na Turbid T7Pro Turbid T7Gly Turbid T7NaSu Turbid

[0421] Turbidity means that the solution is cloudy, which indicates that the carfilzomib API has not fully entered the solution. Turbidity indicates that the API requires more time to completely dissolve. However, turbidity also indicates that its solubility limit has been reached. Turbidity is generally undesirable. After 3 days at 25 °C, the only formulation that showed clear clarity was A5Su. All other formulations showed turbidity, which indicates that some impurities have formed that are not completely soluble in the formulation. Such impurities may be undesirable and indicate the degradation of the API or other excipients of the composition and a reduction in API efficacy after that time period under those specific storage conditions.

[0422] Figure 2 Results of similar measurements obtained with the same formulation prepared with exemplary pegylated carfilzomib compound number 39 instead of example 26 are shown. The API material recovery is similarly shown in red and shows the percentage of the main API peak at T o and T 3 at 3 days of storage at 25 °C as measured by the same reverse phase assay. Solutions for temperature and time storage conditions were generally prepared as follows - by dissolving the CFZ API in the formulation buffer by stirring for a period of 5 minutes at room temperature until completely dissolved. The sample was then sterile filtered through a 0.2 micron PES filter into 3 cc vials (using 1 ml fill) under sterile conditions. During the stability study, the samples were capped and crimped and placed in the desired or specified storage conditions, such as in an incubator at 4 °C, 25 °C or 37 °C. At each time point, the samples were removed from the incubator and aliquots were taken for analysis.

[0423] Measure the percent recovery of exemplary pegylated carfilzomib compounds as a function of a reverse phase assay of one or more peaks representative of the active molecule / compound. As described below, the percent is determined based on the area under the curve. The reverse phase assay is performed as follows - CFZ samples are analyzed by reverse phase (using a Phenomenex Gemini C18, 50 x 4.6 mm, 3 micron particle size column, using a 47 minute HPLC method). During sample run, the column is held at 28 C and the autosampler is held at 5 C. The CFZ samples are diluted to 0.4 mg / mL in mobile phase A for HPLC injection. The samples are eluted over 38 minutes using a gradient method of 100% mobile phase A (0.1 M sodium perchlorate buffer, pH 3.1 / acetonitrile, 60 / 40, v / v) to 100% mobile phase B (0.1 M sodium perchlorate buffer, pH 3.1 / acetonitrile, 10 / 90, v / v). A wash step of 100% B for 5 minutes follows this gradient. A 5 minute re-equilibration step with 100% mobile phase A ensures bringing the column back to the initial sample loading conditions. CFZ elutes at approximately 20 - 25 minutes. The percent of the main peak is determined by taking the integral of the area under the curve. To calculate the recovery % of the samples, a standard curve is generated using a CFZ standard (1 mg / mL CFZ in ACN diluted to 0.4 mg / mL CFZ in mobile phase A). The total integrated area for each point on the curve is plotted relative to the injection load and fit to a trend line. By inserting the total integrated area, the equation of the trend line generated from this calibration curve is used to calculate the concentration of each unknown sample.

[0424] Figure 2 The results in indicate that the formulations that were stable (i.e., lowest loss of the main API peak) after 3 days at 25 C were A5Su, T8Su, T7NaSu, T7Na, and T7Pro. However, the material recovery curve (red dots) for formulation A5Su was the highest.

[0425] Figure 1 and Figure 2 The results of indicate that for each of the 5kPEG-CFZ and 20kPEG-CFZ compounds, formulation A5Su exhibited the highest stability among the formulations prepared and tested.

[0426] The increased stability may be at least partially attributed to the reduced pH. To help determine if pH has this effect, a second study was conducted to compare pH 3 and pH 4 formulations to the A5Su formulation at pH 5. The study design is shown in Table 4 below.

[0427] Table 4

[0428] Formulation Name pH Buffer Excipient C3Su 3 10 mM Citrate 9% Sucrose C4Su 4 10 mM Citrate 9% Sucrose A5Su 5 10 mM Acetate 9% Sucrose

[0429] Then these three compositions were tested using all three representative PEG-CFZ constructs, namely 3kPEG-CFZ (Example 34), 5kPEG-CFZ (Example 26), and 20kPEG-CFZ (Example 39). The major API peak loss for Example 34 (shown as Compound 576 in Table 5) is shown in Table 5.

[0430] Table 5

[0431] 25C 4C -70C Loss / 8 h Loss / 1 week Loss / 2 weeks 576 C3Su 0.0 7.7 5.3 576 C4Su 0.0 8.0 5.1 576 A5Su 0.0 3.9 1.2

[0432] As shown in Table 5, no loss was detected (expressed as a percentage; within experimental variability) for all 3 formulations after 8 hours at 25 °C. This indicates that these exemplary liquid formulations of the pegylated carfilzomib compounds are clinically or potentially commercially viable for administration to patients after storage at room temperature for 8 hours. For the exemplary formulations of Example 39 stored at each of 4 °C and -70 °C after a 2-week incubation period, both representative compositions showed the lowest loss of the major API peak in the A5Su formulation. In addition, all three representative formulations of 3kPEG-CFZ (Example 34) in Table 5 were clear, with no visibly detectable particles or particulate matter.

[0433] Table 6

[0434] 25C 4C -70C Loss / 8 h Loss / 1 week Loss / 2 weeks C3Su 1.1 1.6 0.9 C4Su 1.5 2.6 4.1 A5Su 0.0 0.0 0.0

[0435] Table 6 shows the major API peak loss rates (expressed as a percentage) for the indicated formulations at pH 3, 4, and 5, at 25 °C, 4 °C, and -70 °C, for the representative 5kPEG-CFZ construct (Compound Example 26). Similar to that observed for the 3kPEG-CFZ construct (Example 34) in Table 5 above, there was minimal major API peak loss at each of the storage conditions of 25 °C (8 h), 4 °C (1 week), and -70 °C (2 weeks).

[0436] Table 7

[0437] 25C 4C -70C Loss / 8 h Loss / 1 week Loss / 2 weeks C3Su 20.3 33.5 25.7 C4Su 9.5 31.1 16.1 A5Su 7.0 28.1 6.4

[0438] Table 7 shows the primary API peak loss rates of the 20kPEG-CFZ construct (Compound Example 39 herein) at 25 °C, 4 °C, and -70 °C, respectively, for formulations at pH 3, 4, and 5. Similar to what was observed in the 3kPEG-CFZ construct, minimal primary API peak loss was seen for the representative A5Su compositions at 25 °C (8 h), 4 °C (1 week), or -70 °C (2 weeks). Of note for these results, on an absolute scale, the 20kPEG-CFZ compound in the A5Su formulation was observed to be less stable than the 3kPEG-CFZ and 5kPEG-CFZ constructs in the same A5Su composition. Even more notably, the 20K pegylated carfilzomib compounds were generally less stable over time to storage conditions than the corresponding 3K and 5K pegylated carfilzomib compounds in the same composition.

[0439] Table 8 shows the results of a stability investigation of each of the 3kPEG-CFZ, 5kPEG-CFZ, and 20kPEG-CFZ representative pegylated carfilzomib compounds in A5Su at a concentration of 20 mg / mL. This study showed that the higher the API concentration in a given formulation, the better the stability of the construct under the test conditions. It was also found that at lower concentrations, the carfilzomib API with a larger PEG size (20kPEG-CFZ construct) was less stable in the A5Su formulation compared to the 3kPEG-CFZ and 5kPEG-CFZ representative compounds.

[0440] Table 8

[0441] 25℃ 4℃ -70℃ Loss / 8 h Loss / 1 week Loss / 2 weeks 3K PEG-CFZ 0.0 0.0 0.4 5K PEG-CFZ 0.0 0.0 0.0 20K PEG-CFZ 1.8 1.4 0.0

[0442] Each of the 3 PEG-CFZ construct examples was prepared as an A5Su formulation for animal administration. These formulations were evaluated by visual assessment. Visual observation indicated that there were no visible particles in any of the 3 prepared samples. Endotoxin testing showed that all 3 samples contained endotoxin at a level of <1.0 EU / mL. The measured osmolality values for all 3 formulations were in the range of 299 - 307 mOsm. Reverse phase chromatography was used to monitor the primary API peak of all constructs before and after administration. No significant loss of the primary API peak of any construct was detected in any of the administration studies. No significant loss of the area under the curve was detected between the pre-administration samples and the post-administration samples. This indicates that the active biocompounds in these formulations will be efficacious in terms of the expected biological activity.

[0443] Stable, isotonic lyophilized formulations for intravenous and subcutaneous administration

[0444] The present invention also provides a stable, isotonic, dry freeze-dried pharmaceutically acceptable composition of a pegylated carfilzomib compound. Representative exemplary stable, dry freeze-dried formulations include, but are not limited to, G5Su2M4, which is a composition of 10 mM glutamate (buffer), 2.0% sucrose, 4% mannitol, 0.006% polysorbate 80, and API (pH 5.0). In addition, the formulations of the present invention may further include hyaluronidase. It is believed that hyaluronidase facilitates the subcutaneous delivery of the API and potentially alleviates, reduces, or prevents the aggregation of the API and / or formulation excipients at the injection site. It is also believed that hyaluronidase reduces the degree of local skin irritation at the injection site.

[0445] To identify a stable freeze-dried formulation of a representative 3K-pegylated carfilzomib compound (Example 28 in Table 2), the compound API was dissolved in representative freeze-drying compatible solutions, and the stability of each exemplary solution was compared. The comparison was made between the formulations immediately after dissolution and after completion of the freeze-drying cycle. In addition, after reconstituting the freeze-dried batches (lyophilizates), the stability of each reconstituted liquid formulation was tested prior to drug administration (clinical or commercial administration) to determine the safe and acceptable or allowable handling time. It was found that the representative reconstituted formulations could be safely administered for at least 8 hours at room temperature and for 2 days at 2°C - 8°C. All initial screenings of the constructs were carried out at an API concentration of 5 - 40 mg / mL.

[0446] For each exemplary formulation, the stability of the API was tested or measured by the following six different criteria or methods:

[0447] 1. Integrity of the molecule as evaluated by reverse-phase assay;

[0448] 2. Clarity of the solution as determined by visual evaluation;

[0449] 3. Concentration of API material in the solution as evaluated by API material recovery after centrifugation;

[0450] 4. pH of the formulation;

[0451] 5. Osmolality of the exemplary solution; and

[0452] 6. Subvisible particle count by light obscuration.

[0453] Table 9 below describes and provides the freeze-drying cycle parameters used in the experimental preparation, namely temperature, ramp, and hold time. The hold step indicates that throughout the step, the conditions for holding the sample at a given temperature and pressure remain unchanged. The ramp step means that the sample is brought to the desired temperature by gradually increasing or decreasing until the specified temperature is reached.

[0454] Table 9

[0455] Temperature (°C) Time (min) Hold / Ramp step 5 60 H -45 100 R -45 120 H -12 65 R -12 200 H -45 65 R -45 150 H -25 60 R -25 1800 H 25 550 R 25 780 H

[0456] The freeze-drying process employed utilizes a vacuum pressure of 150 mTorr.

[0457] Result

[0458] Preliminary freeze-drying studies were conducted on an exemplary formulation of API Compound Example 28 (3KPEG-CFZ) herein. The formulation consisted of the desired amount or concentration of API (amount in mg or concentration in mg / mL), 10 mM glutamate, containing 2.0% sucrose, 4% mannitol, 0.006% polysorbate 80, and had a solution pH of 5.0. The following test formulations had different solution concentrations of API Example 28: 5 mg / mL, 20 mg / mL, and 40 mg / mL. Additionally, 10 mM histidine was examined at 20 mg / mL of the exemplary polyethylene glycolated carfilzomib Compound Example 8 (same 3K-PEG CFZ), pH 5.0, 2.0% sucrose, 4% mannitol, 0.006% polysorbate 80 formulation.

[0459] Figure 3-A shows an image of the resulting dry solid lyophilized cake obtained from the formulations prepared above. As shown, lyophilized cakes of Compound Example 28 were formulated at 3 different concentrations (40 mg / mL; 20 mg / mL and 5 mg / mL) in G5Su2M4 solution (10 mM glutamate, pH 5.0, 2.0% sucrose, 4% mannitol, 0.006% polysorbate 80). Each prepared composition solution was frozen and lyophilized in a standard manner. Figure 3-A also shows the resulting dry solid lyophilized cake of the formulation solution from H5Su2M4 (10 mM histidine, pH 5.0, 2.0% sucrose, 4% mannitol, 0.006% polysorbate 80 formulation) at 40 mg / mL concentration of Compound Example 28.

[0460] Figure 3-B depicts a bar graph of the percentage of the major Compound Example 28 peak in a reversed-phase intact API measurement assay. In Figure 3-B, the black bars represent the pre-lyophilization assay measurements, while the red bars represent the post-lyophilization API peak measurements. The green bars represent the measurements taken after reconstituting the lyophilized cake to form a clear solution and storing it at 25 °C for 8 hours. Finally, the yellow bars represent the assay measurements taken after reconstituting the corresponding lyophilized cake to form a solution and storing it at 2 °C - 8 °C for 24 hours. Figure 3-B highlights that there is no loss of the major API peak during the lyophilization process and no loss of the major API peak after incubation of the reconstituted solution. The concentration of the material in the solution was obtained from the area under the reversed-phase curve and did not change after lyophilization and subsequent incubation of the solution.

[0461] Reconstitution of these dry solid lyophilized cakes with sterile water produces a clear, particulate-free solution. Reconstitution times are within one minute for all formulations except the 40 mg / mL formulation. For the 40 mg / mL formulation, a clear solution is formed only after a slightly longer time (i.e., within two minutes) upon reconstitution. The measured osmolality values for all formulations are in the range of 300 - 307 mOsm. The pH readings are all in the range of 5.0 - 5.1. As previously described, the percentage of remaining compound of API reverse phase integrity is determined immediately before and after completion of the lyophilization cycle. In addition, a temperature and time period of 8 hours at 25°C and 24 hours between 2°C - 8°C are selected, under which the reconstituted solution is allowed to stand to simulate a representative clinical and commercial setting for the administration and application of FDA-approved carfilzomib in the real world.

[0462] The present invention further provides a pharmaceutically acceptable composition for subcutaneous administration. Hyaluronidase has been used in the pharmaceutical industry as an additive in formulations with certain drug products for the purpose of converting an IV-administered formulation into a subcutaneous-administered formulation. However, hyaluronidase has not successfully converted every tested drug into a subcutaneous-administered formulation. It is not possible to predict whether the addition of hyaluronidase will meet the requirements for subcutaneous administration of any given drug product, let alone the pegylated carfilzomib compound. As described herein, the present invention contemplates and provides compositions and formulations that further comprise hyaluronidase. To this end, the tests performed using Compound Example 28 are described below and referenced Figures 4-A and 4-B to, in which this compound is co-formulated with 2000 units / mL of hyaluronidase (designated as PH 20 and commercially available from different sources). A formulation solution is prepared consisting of 10 mM glutamate, pH 5.0, 2.0% sucrose, 4% mannitol, 0.006% polysorbate 80, and Compound Example 28 at a concentration of 20 mg / mL. This formulation (the first vial) also includes 2000 units / mL of hyaluronidase. It is lyophilized, and the resulting dry solid lyophilized cake (see Figure 4-A) is assayed as described above (see Figure 4-B) to determine the percentage of the remaining major API peak. The first vial in Figure 4-A is an image of the resulting lyophilized cake obtained from the above-described G5Su2M4 formulation. The second vial depicted in Figure 4-A is the resulting placebo lyophilized cake from a solution formulation containing only hyaluronidase. This vial is prepared and lyophilized only as a control. As described above with respect to Figure 3-B, the percentage of the API main peak of both vials is measured by reverse phase assay. As shown in Figure 4-B, the black bars represent the API main peak of the formulation solution before lyophilization, while the red bars represent the API main peak of the lyophilized cake after reconstitution with sterile water. The green bars represent the main API peak readings after reconstituting each vial to form a solution that is stored at 25°C for 8 hours and at 2°C - 8°C for 24 hours (yellow bars).

[0463] Reconstitution of two vials (the first vial and the second vial in Figure 4-A) with sterile water produced a clear, particulate-free solution. The reconstitution time for both vials was within one minute. The measured osmolarity values of the two reconstituted formulations were in the range of 300 - 302 mOsm. The pH readings of the two reconstituted formulations were found to be in the range of 5.0 - 5.1. The percentage of the main peak of pegylated carfilzomib Compound Example 28 was measured by reverse phase assay immediately before lyophilization and immediately after completion of the lyophilization cycle. In addition, the two reconstituted solutions were stored at 25 °C for 8 hours and at 2 °C - 8 °C for 24 hours to measure the stability of the API under these test conditions or storage over these time periods. As shown in Figure 4-B, the reconstituted API formulation and the placebo hyaluronidase formulation showed no loss of the main peak during the lyophilization process. More importantly, after incubation and storage of the reconstituted solution in the specified storage environment for the specified time, neither of the two formulations showed any loss of the main API peak. Additionally, the concentration of Compound Example 28 in solution, as measured by the area under the reverse phase curve, did not change after lyophilization and subsequent storage of the reconstituted solution. This supports and demonstrates that under the test conditions and similar conditions, the formulations of the present invention provide a suitable stable and intact solution that can be stored for a long time and can be safely and reliably administered to patients in a research, clinical, or commercial setting. The formulations of the present invention reduce the degree of degradation of the pegylated carfilzomib Compound API such that the formulations can be conveniently stored without forming impurities, and the solution remains relatively clear of particulate matter, impurities and contains a significant proportion of, or even as much active pharmaceutical ingredient as when the drug product was initially manufactured.

[0464] Hyaluronidase was sourced from Calbiochem (ovine testes, 38594 - 100 KU, Calbiochem) and its buffer was exchanged into the above formulation before adding sufficient 3K PEG-CFZ API to achieve a concentration of 20 mg / mL.

[0465] The stability and safety of the formulations of the present invention were also tested or measured by particle count and particle size (expressed as a percentage of the solution volume) in the reconstituted solution. Figures 5-A and 5-BShows the results of these particle counts, where the measured particle size is a diameter > 10 μm or > 25 μm. The solutions used in the tests included a formulation solution consisting essentially of G5Su2M4 and 0.006% polysorbate 80, with a concentration of 20 mg / mL of pegylated compound example 28 (as the API). Table 10 below shows the various conditions for measuring particulate matter formation in each test formulation. Each test formulation listed in Table 10 below was prepared in a mixed manner and freeze-dried in 3 mL vials with a 1 mL fill volume. Then, they were screened for their particle formation content after freeze-drying as a measure of stability.

[0466] Table 10

[0467]

[0468] Before and after freeze-drying, each formulation vial was tested for particle counts by subvisible light obscuration methodology. This methodology involves using a liquid particle counting system (HIAC / Royco 9703 or 9703+). In the case of the freeze-dried samples, they were reconstituted before measurement and allowed to equilibrate for two hours before analysis. Before measurement, all samples were degassed using a vacuum chamber for one hour. After the samples were degassed, the water control and particle standards were measured by the instrument. Before the standard particle control and sample measurements, a water control sample measurement reading of zero must be obtained. Briefly, the samples were gently rotated by hand to avoid generating any bubbles, and then measured by the instrument using a 0.2 mL pipette / syringe. A total of 4 pipettes were taken for each sample, and the last three pipettes were averaged. No sample dilution was performed on any of the samples tested.

[0469] As Figures 5-A and 5-B shown, a significant increase in particle counts can be seen in formulations 1 - 3. The addition of the surfactants polysorbate 80 and pluronic F68 only slightly reduced or decreased the tendency to form particulate matter in the formulation (10 mM glutamate + 2% sucrose + 4% mannitol, pH 5.0). It was found that the addition of a small amount of amino acids (such as lysine or arginine) in combination with a surfactant (such as polysorbate 80) resulted in a decrease in particle counts in two test formulations (count size > 10 μm and count size > 25 microns).

[0470] As a summary, representative formulations prepared and tested by freeze-drying are shown in Table 11 below.

[0471] Table 11

[0472]

[0473] Administration of the pharmaceutical composition of the present invention

[0474] The pharmaceutical compositions of the present invention can be administered parenterally. For example, compositions for parenteral administration can be formulated as injections (intravenous, intramuscular or subcutaneous), drip infusion preparations, or suppositories. These formulations can be prepared by conventional means in combination with the methods set forth herein, and if desired, the active ingredient can be mixed with any conventional additives or excipients (such as binders, disintegrants, lubricants, flavoring agents, solubilizers, suspending aids, emulsifying agents or coating agents). Compositions that can be administered parenterally (suitable for infusion, injection or subcutaneous administration) generally include sterile aqueous solutions (in the case of water solubility) or dispersions for infusion or injection and / or sterile powders for the extemporaneous preparation of sterile solutions or dispersions. As noted above, for intravenous administration, suitable carriers include sterile water for injection, sterile buffers. In all cases, the compositions (especially those for human use, treatment and consumption) must be sterile and should be in a liquid form that is readily admissible to or fillable into a syringe or infusion bag. The compositions should be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Thus, in some aspects, the present invention provides compositions that can include antibacterial or antifungal agents. In some aspects of the present invention, the compositions provided herein can include isotonic agents (such as sugars), polyols (such as mannitol, sorbitol) and sodium chloride in the composition, as exemplified and tested above. Prolonged absorption of injectable compositions can be achieved by incorporating agents that delay absorption (such as aluminum monostearate and gelatin) into the composition.

[0475] Sterile injectable solutions can be prepared by incorporating the required amount of the active PEG carfilzomib compound into a suitable solvent having one or a combination of the ingredients listed above, if needed, followed by filtration sterilization. Generally, dispersions are prepared by incorporating carfilzomib into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders (such as the lyophilized cakes prepared and described herein) for the preparation of sterile injectable solutions, a suitable method of preparation is freeze-drying (lyophilization), which provides the active polyethylene glycolated carfilzomib compound and any additional desired ingredients from its previously sterile filtered solution in powder form.

[0476] The dosage and precise administration time of the pegylated carfilzomib compound used in the pharmaceutical composition of the present invention described herein depend on the nature and type of the cancer to be treated, as well as the age, condition, and weight of the patient. In terms of the therapeutic efficacy for a given patient, the composition that produces the most effective result will also depend on the activity, pharmacokinetics, and bioavailability of the specific PEG-carfilzomib compound, the physiological conditions of the patient as described above (including age, gender, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), the route of administration, etc. Although the dosage will vary depending on the symptoms and severity of the disorder to be treated or prevented, the route of administration of the drug, and the drug form, a daily dosage of the compound of from 0.01 mg to 2000 mg is generally recommended for adult human patients, and this daily dosage can be administered as a single dose or in divided doses. More information regarding the dosage of the compounds of the present invention is provided below. Generally, compositions intended for parenteral use (e.g., intravenous injection, subcutaneous injection) include solubilizing agents. The solubilizing agent can be a substituted cyclodextrin.

[0477] The actual dosage of the PEG-carfilzomib compound utilized in the pharmaceutical composition provided by the present invention can be an amount clinically proven effective and / or commercially approved as effective to achieve the desired therapeutic response for cancer patients (including, but not limited to, for patients with multiple myeloma). In some aspects, the present invention provides a pharmaceutical composition in an aqueous solution containing from about 0.1% - 20% w / v of the compounds disclosed herein and other substances for parenteral administration. The typical dosage range of the PEG-carfilzomib compound is from about 0.01 to about 50 mg / kg body weight / day, administered in 1 - 4 divided doses per day. Each divided dose will contain one or more of the compounds provided by the present invention. The specific dosage of the compound required should be an amount sufficient to provide a therapeutically effective dose of free-acting carfilzomib in the patient's plasma, said effective dose being based on the regulatory-approved use for the regulatory-approved indication. Such effective amount may vary from patient to patient and generally depends on several factors, including the overall health of the patient, as well as the specific formulation composition and route of administration of the one or more compounds selected. In some embodiments, the PEG-carfilzomib compounds useful in the present invention are described in U.S. Patent No. 9309283.

[0478] The currently approved dosage of carfilzomib, which, within a 28-day cycle, for 3 consecutive weeks, is once daily for 2 consecutive days each week, in an amount sufficient to provide a range from 20 mg / m 2 to 56 mg / m 2Provided is the amount of the patient plasma concentration. Therefore, the higher molecular weight PEG carfilzomib compounds of the present invention should be administered in an amount sufficient to provide approximately the same amount pharmacokinetically as the approved dose range. For example, the 2K PEG compound of the present invention is approximately 24% of free carfilzomib by weight. Therefore, using an average male with a body surface area of 1.9 m2, to achieve an equivalent dose of about 27 mg / m 2 , approximately 215 mg of the 2kPEG CFZ compound must be administered. Similarly, about 1100 mg of the 20K PEG CFZ compound can be administered to deliver the same amount of carfilzomib as the 70 mg / m 2 dose of the currently approved carfilzomib formulation.

[0479] The pharmaceutical compositions of the present invention include a variety of excipients as described herein. For example, at least one excipient may include a sugar additive such as sucrose, sorbitol, glycerol, maltose, lactose, erythritol, dextrose, lactulose or cyclodextrin, or a charged amino acid such as proline, glycine, arginine, histidine, aspartic acid, glutamic acid or glutamate, or a neutral hydrophobic amino acid such as valine, leucine, alanine, methionine. The excipient may be a salt selected from the group consisting of sodium chloride, potassium chloride, ammonium sulfate, potassium chlorate, calcium chloride, zinc chloride, guanidine hydrochloride, ammonium chloride, potassium sulfate, ammonium aspartate, arginine hydrochloride, lysine hydrochloride, magnesium chloride and barium sulfate. If the excipient is a sugar, it is usually included in an amount in the range of from about 0.1% - 30% by total weight of the composition or % weight / volume of the solution formulation, or if the excipient is an amino acid, it is usually included in an amount in the range of from about 0.1% - 10% by total weight of the composition.

[0480] The pharmaceutical compositions of the present invention may further include other pharmaceutically acceptable excipients. As used herein with respect to excipients, carriers and / or diluents in the compositions of the present invention, the term "pharmaceutically acceptable" refers to those ligands, materials, compositions and / or dosage forms that are suitable for contact with the tissues of humans and animals within the scope of reasonable medical judgment, without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio. As used herein, "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0481] Additional excipients include the use of surfactants, which include but are not limited to PS20, PS80, PLF68 (and other Pluronics in the F and L series), triblock surfactant polymers, sodium docusate, benzalkonium chloride, Triton X 100, and tetrafunctionalized block O polymers. Surfactants that may be included are typically surfactants that reduce surface tension (such as alcohols), SDS, protamine sulfate, or butane. If a surfactant is included in the composition of the present invention, it should be included in an amount ranging from 0.005% to 3% (by weight).

[0482] For the dry lyophilized compositions provided by the present invention, sugars such as sucrose, sorbitol, glycerol, maltose, lactose, erythritol, dextrose, lactulose, cyclodextrin, sugar derivatives, and adducts may be included. Sugars or sugar derivatives are typically included in an amount ranging from 0.1% - 30% (by weight). In cases where the excipient can be an amino acid, it can be any suitable amino acid, including proline, glycine, lysine, arginine, histidine, aspartic acid, valine, leucine, alanine, methionine, proline, glutamic acid, glutamate. Amino acids are typically present in an amount ranging from about 0.1% - 10% (by weight). The compositions of the present invention may further include salts. The salts can act as buffers or provide other advantages to the composition. The salts can be, for example, NaCl, KCl, ammonium sulfate, potassium chlorate, calcium chloride, ZnCl, guanidine hydrochloride, ammonium chloride, potassium sulfate, amino acid salts (including but not limited to ammonium aspartate, arginine hydrochloride, and lysine hydrochloride), magnesium chloride, and barium sulfate. When salts are included, they are typically included in an amount ranging from about 30 - 300 mM by volume of the solution composition.

[0483] Additional examples of materials that can typically be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch, potato starch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) diols, such as propylene glycol; (11) polyols, such as glycerol, 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) ethanol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical formulations. In certain embodiments, the pharmaceutical compositions provided herein are pyrogen-free, i.e., they do not induce a significant temperature increase upon administration to a patient.

[0484] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the phrase "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., that are compatible with the administration of a drug. In some embodiments, the pharmaceutically acceptable carrier is an acid-base buffer system, such as a citrate buffer, to maintain a stable pH of the resulting solution. In some embodiments, the pharmaceutically acceptable carrier is sterile water for injection. In some embodiments, the pharmaceutically acceptable carrier contains citric acid.

[0485] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action can be ensured by incorporating various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be necessary to incorporate tonicity regulators, such as sugars, etc., into the composition. Additionally, the absorption of injectable pharmaceutical forms can be prolonged by incorporating agents that delay absorption (e.g., aluminum monostearate and gelatin). In some cases, in order to prolong the action of a drug, it is desirable to slow the absorption of the drug from a subcutaneous or intramuscular injection. For example, delayed absorption of parenterally administered pharmaceutical forms is accomplished by dissolving or suspending the drug in an oily vehicle.

[0486] As used herein, the phrases "parenteral administration" and "administered parenterally" mean a mode of administration other than enteral and topical administration, usually effected by injection, and include (but are not limited to) intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intrasternal, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0487] The pharmaceutical compositions of the invention described herein can be administered to humans and other animals (including mammals) by any suitable route of administration for therapy.

[0488] Method of use

[0489] The biological effects of proteasome inhibition are useful and desired. Proteasome inhibition has been proposed for the prevention and / or treatment of a variety of diseases, including but not limited to proliferative diseases, neurotoxic / degenerative diseases, Alzheimer's disease, ischemic conditions, inflammation, autoimmune diseases, HIV, cancer, organ transplant rejection, septic shock, antigen presentation inhibition, reduction of viral gene expression, parasitic infections, acidosis-related conditions, macular degeneration, pulmonary conditions, muscle atrophy diseases, fibrotic diseases, bone and hair growth diseases. Accordingly, a pharmaceutical formulation comprising a therapeutically effective dose of a PEG carfilzomib compound of the invention provides a means of administering the drug to a patient and treating these conditions.

[0490] Accumulation of polyubiquitinated proteins, alterations in cell morphology, and apoptosis have been reported at the cellular level following treatment of cells with various proteasome inhibitors. Proteasome inhibition has also been disclosed and has been demonstrated clinically and commercially to be a useful anti-tumor treatment strategy. For this reason, the compounds of the invention and compositions comprising said compounds can be used to treat cancer, including but not limited to newly diagnosed multiple myeloma and / or relapsed and refractory multiple myeloma.

[0491] Both in vitro and in vivo models have shown that malignant cells are generally vulnerable to proteasome inhibition. In fact, proteasome inhibition has been demonstrated to be a therapeutic strategy for the treatment of multiple myeloma. This may be due in part to the high proliferative malignant cells' dependence on the proteasome system for rapid protein removal (Rolfe et al., J. Mol. Med. (1997) 75:5-17; Adams, Nature (2004) 4:349-360). Methods of treating cancer are provided herein, said methods comprising administering to a patient in need of such treatment a therapeutically effective amount of a polyethylene glycolated carfilzomib compound of Formula I and II, or any specifically exemplified PEG carfilzomib compound, as provided or described herein.

[0492] As used herein, the term "cancer" includes, but is not limited to, hematogenous cancers and solid tumors. Cancers can damage components of the blood, bones, organs, skin tissues, and vascular system, including but not limited to bladder cancer, blood cancer, bone cancer, brain cancer, breast cancer, cervical cancer, chest cancer, colon cancer, endometrial cancer, esophageal cancer, eye cancer, head cancer, kidney cancer, liver cancer, lung cancer, lymph node cancer, oral cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, stomach cancer, testicular cancer, laryngeal cancer, and uterine cancer. Specific cancers include, but are not limited to, leukemia (acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia), mature B-cell neoplasms (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (such as Waldenström macroglobulinemia ( macroglobulinemia), splenic marginal zone lymphoma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, extranodal marginal zone B-cell lymphoma (MALT lymphoma), nodal marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, and Burkitt lymphoma / leukemia), mature T-cell and natural killer (NK) cell neoplasms (T-cell prolymphocytic leukemia, T-cell large granular lymphocyte leukemia, aggressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides (Sezary syndrome), primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, peripheral T-cell lymphoma not otherwise specified, and anaplastic large cell lymphoma), Hodgkin lymphoma (nodular sclerosis Hodgkin lymphoma, mixed cellularity Hodgkin lymphoma, lymphocyte-rich Hodgkin lymphoma, lymphocyte depletion or undifferentiated Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma), non-Hodgkin lymphoma, myeloma (multiple myeloma, indolent myeloma, smoldering myeloma), chronic myeloproliferative disorders, myelodysplastic / myeloproliferative diseases, myelodysplastic syndromes, immunodeficiency-related lymphoproliferative disorders, histiocytic and dendritic cell neoplasms, mastocytosis, chondrosarcoma, Ewing sarcoma, fibrosarcoma, malignant giant cell tumor, myeloma bone disease, osteosarcoma, breast cancer (hormone-dependent breast cancer, non-hormone-dependent breast cancer), gynecologic cancers (cervical cancer, endometrial cancer, fallopian tube cancer, gestational trophoblastic disease, ovarian cancer, peritoneal cancer, uterine cancer, vaginal cancer, and vulvar cancer), basal cell carcinoma (BCC), squamous cell carcinoma (SCC), malignant melanoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, Kaposi'ssarcoma), astrocytoma, pilocytic astrocytoma, dysembryoplastic neuroepithelial tumor, oligodendroglioma, ependymoma, glioblastoma multiforme, mixed glioma, oligoastrocytoma, medulloblastoma, retinoblastoma, neuroblastoma, germ cell tumor, teratoma, malignant mesothelioma (peritoneal mesothelioma, pericardial mesothelioma, pleural mesothelioma), gastroenteropancreatic or gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid, pancreatic endocrine tumor (PET), colorectal adenocarcinoma, colorectal cancer, invasive neuroendocrine tumor, leiomyosarcoma, mucinous adenocarcinoma, signet ring cell adenocarcinoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, hemangioma, hepatic adenoma, focal nodular hyperplasia (nodular regenerative hyperplasia, hamartoma), non-small cell lung cancer (NSCLC) (lung squamous cell carcinoma, adenocarcinoma, large cell lung cancer), small cell lung cancer, thyroid cancer, prostate cancer (hormone refractory prostate cancer, non-androgen dependent prostate cancer, androgen dependent prostate cancer, hormone insensitive prostate cancer) and soft tissue sarcoma (fibrosarcoma, malignant fibrous histiocytoma, dermatofibrosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, hemangioendothelioma, synovial sarcoma, malignant peripheral nerve sheath tumor / neurofibrosarcoma, extraskeletal osteosarcoma).

[0493] In one aspect, the present invention provides a pharmaceutically acceptable composition comprising a pegylated carfilzomib compound or a pharmaceutically acceptable salt thereof, the composition being administered to a patient for the treatment of multiple myeloma. Additionally, and in another aspect of the present invention, multiple myeloma can include one or both of newly diagnosed multiple myeloma or relapsed and / or refractory multiple myeloma.

[0494] Figure 6 and Figure 7 Demonstrated the efficacy of representative pegylated carfilzomib compound Example 13 in a murine xenograft model of human colorectal adenocarcinoma cancer cells. Tumors in the vehicle group grew linearly during the study. Intravenous administration of compound Example 13 (200 mpk, or escalating from 150 mpk to 250 mpk after 3 weeks) or CFZ-captisol (5 mpk) once a week for 19 days within the first dose administration provided a significant attenuation of tumor growth (compared to vehicle control). Additionally, intravenous administration of both formulations was associated with a significant attenuation of body weight gain.

[0495] Additional embodiments include methods of affecting the proteasome-dependent regulation of oncoproteins and methods of treating or inhibiting cancer growth, each method comprising exposing a cell (in vivo, such as in a patient, or in vitro) to a composition disclosed herein. The HPV-16 and HPV-18-derived E6 proteins stimulate the ATP-dependent and ubiquitin-dependent conjugation and degradation of p53 in crude reticulocyte lysates. The recessive oncogene p53 has been shown to accumulate at non-permissive temperatures in cell lines with a mutant heat-labile E1. Elevated p53 levels can lead to apoptosis. Examples of proto-oncoproteins degraded by the ubiquitin system include c-Mos, c-Fos, and c-Jun. One embodiment is a method for treating p53-related apoptosis, the method comprising administering to a patient an effective amount of a composition disclosed herein.

[0496] It has also been demonstrated that inhibitors that bind to the 20S proteasome stimulate bone formation in bone organ cultures. In addition, when such inhibitors have been administered systemically to mice, certain proteasome inhibitors increase bone volume and bone formation rate by more than 70% (Garrett, I.R. et al., J. Clin. Invest. [Journal of Clinical Investigation] (2003) 111:1771-1782), which thus indicates that the ubiquitin-proteasome mechanism regulates osteoblast differentiation and bone formation. Accordingly, the disclosed compositions can be used to treat and / or prevent diseases associated with bone loss, such as osteoporosis.

[0497] In addition, the present invention provides compositions that can also be used as diagnostic reagents (e.g., in a diagnostic kit or for use in a clinical laboratory) for screening proteins (e.g., enzymes, transcription factors) processed by Ntn hydrolases, including the proteasome. The disclosed compositions can also be used as research reagents for specifically binding to the X / MB1 subunit or the α-chain and inhibiting the associated proteolytic activity. For example, the activities of other subunits of the proteasome (as well as the activities of specific inhibitors) can be determined.

[0498] In Example 71 of the present invention, a method for treating cancer in a subject in need of treatment is provided, the method comprising administering to the subject an effective dose of a pharmaceutical composition comprising an effective amount of a PEG carfilzomib compound of Formula I. In Example 72, the present invention provides the method as described in Example 71, wherein the cancer is multiple myeloma. In Example 73, the present invention provides the method as described in any one of Examples 71-72, wherein the effective dose of PEG carfilzomib is in the range from about 100 mg to about 2000 mg. In Example 74, the present invention provides the method as described in any one of Examples 71-73, wherein the effective dose is in the range from about 150 mg to about 1000 mg / day. In Example 75, the present invention provides the method as described in any one of Examples 71-74, wherein the effective dose of the PEG carfilzomib compound administered is in the range from about 200 mg to about 500 mg / day. In Example 76, the present invention provides the method as described in any one of Examples 71-73, wherein the effective dose of the 2K PEG carfilzomib compound administered is in the range from about 150 mg to about 600 mg / day. In Example 77, the present invention provides the method as described in any one of Examples 71-73, wherein the effective dose of the 3K PEG carfilzomib compound administered is in the range from about 300 mg to about 2000 mg / day. In Example 78, the present invention provides the method as described in any one of Examples 71-73, wherein the effective dose of the 5K PEG carfilzomib compound administered is in the range from about 800 mg to about 3000 mg / day. In Example 79, the present invention provides the method as described in any one of Examples 71-73, wherein the effective dose of the 20K PEG carfilzomib compound administered is in the range from about 800 mg to about 3000 mg / day. In Example 80, the present invention provides the method as described in any one of Examples 71-73, wherein the effective dose of the PEG carfilzomib compound administered is in the range from about 200 mg to about 1500 mg / day. In Example 81, the present invention provides the method as described in any one of Examples 71-73, wherein the effective dose of the PEG carfilzomib compound administered is in the range from about 5 mg / kg to about 50 mg / kg / day based on the body weight of the subject. In Example 82, the present invention provides the method as described in any one of Examples 71-73, wherein the effective dose of the 2K, 3K or 5K PEG carfilzomib compound administered is in the range from about 200 mg to about 800 mg / day. In Example 83, the present invention provides the method as described in any one of Examples 71-73, wherein the effective dose of the 2K or 3K PEG carfilzomib compound administered is in the range from about 200 mg to about 500 mg / day.In Example 84, the present invention provides a method as described in any one of Examples 71 - 73, wherein the effective dose of the 5K or 20K PEG carfilzomib compound administered is in the range from about 400 mg to about 1000 mg / day. In Example 85, the present invention provides a method as described in any one of Examples 71 - 84, wherein the method further comprises administering a steroid. In Example 86, the present invention provides a method as described in Example 85, wherein the steroid is selected from the group consisting of dexamethasone and prednisone. In Example 87, the present invention provides a method as described in any one of Examples 85 - 86, wherein the steroid is dexamethasone. In Example 88, the present invention provides a method as described in any one of Examples 85 - 86, wherein the steroid is prednisone. In Example 89, the present invention provides a method as described in any one of Examples 71 - 88, wherein the method further comprises administering an immunomodulator selected from the group consisting of thalidomide, lenalidomide, and pomalidomide. In Example 90, the present invention provides a method as described in Example 89, wherein the immunomodulator is lenalidomide or pomalidomide. In Example 91, the present invention provides a method as described in any one of Examples 89 - 90, wherein the immunomodulator is lenalidomide. In Example 92, the present invention provides a method as described in any one of Examples 89 - 90, wherein the immunomodulator is pomalidomide. In Example 93, the present invention provides a method as described in any one of Examples 71 - 88, wherein the method further comprises administering a CD - 38 inhibitor. In Example 94, the present invention provides a method as described in Example 93, wherein the CD - 38 inhibitor is daratumumab. In Example 95, the present invention provides a method as described in any one of Examples 71 - 94, wherein the cancer is relapsed or refractory multiple myeloma. In Example 96, the present invention provides a method as described in any one of Examples 71 - 94, wherein the cancer is newly diagnosed multiple myeloma. In Example 97, the present invention provides a method as described in Example 96, wherein the cancer is newly diagnosed multiple myeloma, and wherein the patient is a stem cell transplantation eligible patient as determined by a qualified and authorized practicing physician. In Example 98, the present invention provides a method as described in Example 96, wherein the cancer is newly diagnosed multiple myeloma, and wherein the patient is not a stem cell transplantation eligible patient as determined by a qualified and authorized practicing physician. In Example 99, the present invention provides a method as described in any one of Examples 71 - 98, wherein the method comprises administering to the subject a pharmaceutical composition comprising a PEG carfilzomib compound having Formula I.In Example 101, the present invention provides the method according to any one of Examples 99-100, wherein the pharmaceutical composition is a lyophilized formulation that can be reconstituted before administration.

[0499] Combination

[0500] Although the PEG-carfilzomib compounds of the present invention can be administered or applied as the sole active agent, it can also be used in combination with one or more agents (such as a second anti-cancer agent). When administered in combination, the PEG-carfilzomib active ingredient and other agent formulation components can be formulated as separate compositions, which are administered simultaneously or sequentially at different times, or the two active agents can be administered as a single composition.

[0501] In defining the use of the PEG-carfilzomib compounds of the present invention and another anti-cancer agent, the phrase "co-therapy" (or "combination therapy") is intended to include administering each agent in a sequential manner in a regimen that provides a beneficial drug combination effect, and is also intended to include co-administering these agents in a substantially simultaneous manner, such as in a single-dose formulation having a fixed ratio of these active agents or in multiple separate-dose formulations of each active agent. Accordingly, the present invention is not limited by the order of administration, i.e., one or more PEG-carfilzomib compounds can be administered before, simultaneously with, or after the administration of other agents.

[0502] In certain embodiments, the PEG-carfilzomib compounds described herein are administered in combination with one or more other proteasome inhibitors. Another proteasome inhibitor can include, for example, bortezomib, oprozomib, or ixazomib. In another embodiment, the PEG-carfilzomib compounds described herein are administered in combination with immunomodulatory compounds (including thalidomide, lenalidomide, and pomalidomide). In an embodiment from the immediately preceding embodiments, PEG-carfilzomib is administered in combination with an immunomodulator selected from lenalidomide and pomalidomide. In another embodiment, the present invention provides a method of treating cancer in a subject by administering to the subject a combination therapy comprising a PEG-carfilzomib compound having Formula I or II and an immunomodulator. In another embodiment, the cancer is multiple myeloma.

[0503] In certain embodiments, the PEG carfilzomib compounds described herein are administered in combination with one or more chemotherapeutic agents. Suitable chemotherapeutic agents can include natural products such as vinca alkaloids (i.e., vinblastine, vincristine, and vinorelbine), taxanes (e.g., docetaxel, paclitaxel, such as docetaxel), epipodophyllotoxins (i.e., etoposide, teniposide), antibiotics (actinomycin D, daunorubicin, doxorubicin, and idarubicin; e.g., doxorubicin), anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin, enzymes (L-asparaginase, which metabolizes L-asparagine systemically and depletes cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents such as nitrogen mustards (dichloromethyldiethylamine, ifosfamide, cyclophosphamide, and analogs, melphalan, chlorambucil, such as melphalan), ethyleneimines and methylmelamines (hexaamethylmelaamine and thiotepa), alkyl sulfonates (busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin), triazenes-dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate), pyrimidine analogs (fluorouracil, floxuridine, and cytarabine), purine analogs and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine), aromatase inhibitors (anastrozole, exemestane, and letrozole); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; DNA-binding / cytotoxic agents (e.g., Zalypsis); histone deacetylase (HDAC) inhibitors (e.g., trichostatin, sodium butyrate, apicidan, suberoylanilide hydroxamic acid (“SAHA” (vorinostat)), trichostatin A, depsipeptide, apicidan, A-161906, scriptaid, PXD-101, CHAP, butyric acid, depudecin, oxaliplatin, phenylbutyrate, valproic acid, MS275 (N-(2-aminophenyl)-4-[N-(pyridin-3-ylmethoxy-carbonyl)aminomethyl]benzamide), LAQ824 / LBH589, CI994, MGCD0103, ACY-1215, panobinostat; hormones (i.e., estrogen) and hormone agonists such as luteinizing hormone-releasing hormone (LHRH) agonists (goserelin, leuprolide, and triptorelin). Other chemotherapeutic agents can include dichloromethyldiethylamine, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine, or any analogs or derivative variants of the foregoing items.

[0504] In certain embodiments, the PEG carfilzomib compounds described herein are administered in combination with cytokines. Cytokines include, but are not limited to, interferon gamma, interferon alpha and interferon beta, interleukins 1-8, 10 and 12, granulocyte macrophage colony stimulating factor (GM-CSF), TNF-alpha and -beta, and TGF-beta.

[0505] In certain embodiments, the PEG carfilzomib compounds described herein are administered in combination with a steroid. Suitable steroids can include, but are not limited to, 21 - acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoxymethasone, dexamethasone, diflorasone, diflucortolone, difuprednate, glycyrrhetinic acid, flazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinolone acetonide acetate, fluocortin butyl, flucortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, hydrocortisone, loteprednol etabonate, maprednone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25 - diethylaminoacetate, prednisolone sodium phosphate, prednisone, prednisolone valerate, prednylidene, rimcortolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, and salts and / or derivatives thereof (e.g., hydrocortisone, dexamethasone, methylprednisolone, and prednisolone; e.g., dexamethasone).In certain embodiments, the PEG carfilzomib compounds described herein are administered in combination with dexamethasone. In certain embodiments, the combination therapy includes the dosing regimens provided on the KYPROLIS (carfilzomib) label as approved by the US FDA and EMA.

[0506] In some embodiments, the PEG carfilzomib compounds described herein are administered in combination with an immunotherapeutic agent. Suitable immunotherapeutic agents can include, but are not limited to, MDR modulators (verapamil, valspodar, biricodar, tariquidar, lanquidar), cyclosporine, thalidomide, and monoclonal antibodies. Monoclonal antibodies can be naked or conjugated, such as rituximab, tositumomab, alemtuzumab, epratuzumab, tectum-eretumomab, ozogamicin, bevacizumab, cetuximab, erlotinib, and trastuzumab.

[0507] In certain embodiments, the PEG carfilzomib compounds described herein are administered in combination with one or more histone deacetylase (HDAC) inhibitors (e.g., trichostatin, sodium butyrate, apicitadine, suberoylanilide hydroxamic acid (“SAHA” (vorinostat)), trichostatin A, depudecin, apicitadine, A-161906, scriptaid, PXD-101, CHAP, butyric acid, depudecin, oxaliplatin, phenylbutyrate, valproic acid, MS275 (N-(2-aminophenyl)-4-[N-(pyridin-3-ylmethoxy-carbonyl)aminomethyl]benzamide), LAQ824 / LBH589, CI994, MGCD0103, ACY-1215, panobinostat; e.g., SAHA, ACY-1215, panobinostat).

[0508] In certain embodiments, the PEG carfilzomib compounds described herein are administered in combination with one or more nitrogen mustards (dichloromethyldiethylamine, ifosfamide, cyclophosphamide and analogs, melphalan, chlorambucil, e.g., melphalan).

[0509] In certain embodiments, the PEG carfilzomib compounds described herein are administered in combination with one or more DNA-binding agents / cytotoxic agents (e.g., zaliprost).

[0510] In certain embodiments, the PEG carfilzomib compounds described herein are administered in combination with one or more taxanes (e.g., docetaxel, paclitaxel, e.g., docetaxel).

[0511] In certain embodiments, the PEG carfilzomib compounds described herein are administered in combination with one or more antibiotics (actinomycin D, daunorubicin, doxorubicin, and idarubicin; e.g., doxorubicin).

[0512] The foregoing is only illustrative of the present invention and is not intended to limit the present invention to the disclosed uses. Changes and modifications that are routine to those skilled in the art are intended to fall within the scope and nature of the present invention as defined by the appended claims.

Claims

1. A pharmaceutical composition comprising (a) a pegylated carfilzomib compound having the structure of Formula I: or a pharmaceutically acceptable salt thereof, wherein R 1 is C 1-10 alkyl or C 3-7 cycloalkyl; Each R 2 is independently C 1-6 alkyl, -OCH 3 or halogen; o is an integer selected from 0, 1, 2 or 3; the linker is a moiety having the following structure wherein n is an integer selected from 1, 2, 3 or 4; q is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8 or 9; r is an integer selected from 0, 1, 2, 3, 4 or 5 ； and PEG is a polyethylene glycol polymer moiety having a molecular weight in the range of from 500 to 20,000; (b) at least one excipient selected from the group consisting of sucrose, sorbitol, glycerol, maltose, lactose, erythritol, dextrose, lactulose, cyclodextrin, proline, glycine, arginine, histidine, aspartic acid, valine, leucine, alanine, methionine, glutamic acid, glutamate; and salts selected from the group consisting of sodium chloride, potassium chloride, ammonium sulfate, potassium chlorate, calcium chloride, zinc chloride, guanidine hydrochloride, ammonium chloride, potassium sulfate, ammonium aspartate, arginine hydrochloride, lysine hydrochloride, magnesium chloride and barium sulfate; (c) a buffer selected from the group consisting of glutamate, histidine, acetate and Tris-HCl, or a combination thereof; and (d) a bulking agent selected from the group consisting of mannitol, trehalose, PVP, cyclodextrin, glycine, dextrose, dextran, sucrose, proline, PEG 33350 and PEG400, (e) an amino acid selected from the group consisting of lysine, arginine, histidine, aspartic acid, valine, leucine, alanine, methionine, proline, glutamic acid and glutamate, (f) a surfactant selected from the group consisting of polysorbate 20, polysorbate 80, pluronic F68, sodium docusate, benzalkonium chloride, triton X100, tetrafunctional block O polymer, alcohol, SDS, protamine sulfate and butane, or a combination of (d), (e) and (f).

2. The composition according to claim 1, wherein the linker is a moiety having the following structure wherein q is 4; and r is 2.

3. The composition according to any one of claims 1-2, wherein the pegylated carfilzomib compound has the following structure 4. The composition according to any one of claims 1-2, wherein the pegylated carfilzomib compound has the following structure 5. The pharmaceutical composition according to any one of claims 1-2, wherein the composition is a frozen formulation, wherein the pH of the formulation is in the range of from 5.0 to 8.

0.

6. The pharmaceutical composition according to claim 5, wherein the excipient is selected from the group consisting of sucrose, proline, glycine, and sodium chloride, or a combination thereof, and the buffer is selected from the group consisting of histidine, acetate, and Tris-HCl, or a combination thereof.

7. The pharmaceutical composition according to claim 6, wherein the excipient is selected from the group consisting of: sucrose in an amount in the range of 5% - 12% weight / volume, proline in an amount in the concentration range of 50 mM to 300 mM, glycine in an amount in the concentration range of 50 mM to 300 mM, and sodium chloride in an amount in the concentration range of 30 mM to 160 mM, or a combination thereof; and the buffer is selected from the group consisting of: histidine in an amount in the concentration range of 10 mM to 30 mM, acetate in an amount in the concentration range of 10 mM to 30 mM, and Tris-HCl in an amount in the concentration range of 10 mM to 30 mM, or a combination thereof.

8. The pharmaceutical composition according to any one of claims 6 and 7, wherein the excipient is selected from the group consisting of: sucrose in an amount of 9% w / v, L-proline in an amount of 220 mM concentration, glycine in an amount of 293 mM concentration, sodium chloride in an amount of 140 mM concentration, and a combination of sucrose in an amount of 4.5% and sodium chloride in an amount of 140 mM concentration; and the buffer is selected from the group consisting of: histidine in an amount of 10 mM concentration, acetate in an amount of 10 mM concentration, and Tris-HCl in an amount of 10 mM concentration.

9. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition comprises (a) a polyethylene glycolated carfilzomib compound in an amount ranging from 150 mg to 2000 mg; (b) the at least one excipient and buffer are (1) 9% sucrose and 10 mM acetate buffer, pH 5; (2) 9% sucrose and 10 mM histidine, pH 6; (3) 9% sucrose and 10 mM Tris-HCl, pH 7; (4) 9% sucrose and 10 mM Tris-HCl, pH 8; (5) 140 mM sodium chloride and 10 mM Tris-HCl, pH 7; (6) 220 mM L-proline and 10 mM Tris-HCl, pH 7; (7) 293 mM glycine and 10 mM Tris-HCl, pH 7; or (8) 70 mM sodium chloride and 4.5% sucrose with 10 mM Tris-HCl, pH 7.

10. The pharmaceutical composition according to any one of claims 1 - 2, wherein the pharmaceutical composition is a dry freeze-dried formulation.

11. The pharmaceutical composition according to claim 10, wherein the at least one excipient is sucrose in an amount ranging from 0.5% to 2% w / w, the bulking agent is mannitol in an amount ranging from 2% to 4% w / w, the amino acid is absent or selected from lysine or arginine, and the surfactant is absent or is polysorbate 80 or pluronic F68, and the buffer is glutamate.

12. The pharmaceutical composition according to claim 11, wherein the excipient is sucrose in an amount in the range of 1% - 2% weight / volume, mannitol in an amount in the range of 2% to 4% weight / weight, an amino acid selected from lysine or arginine in an amount in the range of 0.5% to 0.8%, a surfactant being 0.006% polysorbate 80 or 0.05% poloxamer F68, and the buffer is 10 mM glutamate.

13. The pharmaceutical composition according to any one of claims 1, 11, and 12, wherein the composition consists of the following components: 10 mm glutamate, 2% sucrose, and 4% mannitol, or 10 mM glutamate, 2% sucrose, 4% mannitol, and 0.006% polysorbate 80, or 10 mM glutamate, 2% sucrose, 4% mannitol, and 0.05% poloxamer F68, or 10 mM glutamate, 2% sucrose, 4% mannitol, 0.5% lysine, and 0.006% polysorbate 80, or 10 mM glutamate, 2% sucrose, 4% mannitol, 0.8% lysine, and 0.006% polysorbate 80, or 10 mM glutamate, 2% sucrose, 4% mannitol, 0.5% arginine, and 0.006% polysorbate 80, or 10 mM glutamate, 2% sucrose, 4% mannitol, 0.8% arginine, and 0.006% polysorbate 80; and a polyethylene glycolylated carfilzomib compound in an amount ranging from 100 mg to 3000 mg.

14. The pharmaceutical composition according to claim 13, wherein when the composition is dissolved in a certain amount of water to achieve 10 mg / mL of the polyethylene glycolylated carfilzomib compound, the pH of the composition is 5.

0.

15. The pharmaceutical composition according to claim 10, wherein the pharmaceutical composition contains the polyethylene glycolylated carfilzomib compound in an amount ranging from 150 mg to 2000 mg.

16. The pharmaceutical composition according to any one of claims 1 - 2, 6 - 7, 9, 11 - 12, and 15, wherein the composition further comprises hyaluronidase.

17. The pharmaceutical composition according to claim 16, wherein the hyaluronidase is present in an amount of 2000 units / mL.

18. The pharmaceutical composition according to any one of claims 1 - 2, 6 - 7, 9, 11 - 12, 15, and 17, which does not contain cyclodextrin.

19. The pharmaceutical composition according to claim 10, wherein when the lyophilized formulation is dissolved in 1.0 ml of water at room temperature, the lyophilized formulation provides a clear solution within a period of 3 minutes.

20. The pharmaceutical composition according to any one of claims 1 - 2, 6 - 7, 9, 11 - 12, 15, 17, and 19, which is administered parenterally by infusion or injection.

21. The pharmaceutical composition according to any one of claims 1 - 2, 6 - 7, 9, 11 - 12, 15, 17, and 19, which is administered intravenously by infusion or injection.

22. The pharmaceutical composition according to any one of claims 1 - 19, which is administered by subcutaneous injection.

23. Use of a therapeutically effective amount of a pharmaceutical composition as described in any one of claims 1-22 in the preparation of a medicament for the treatment of cancer, wherein the cancer is multiple myeloma.

24. The use according to claim 23, wherein the multiple myeloma is relapsed, refractory, or relapsed and refractory multiple myeloma.

25. The use according to claim 23, wherein the multiple myeloma is newly diagnosed multiple myeloma.

26. A method for preparing a pharmaceutical composition as described in any one of claims 1-18 for the treatment of multiple myeloma, the method comprising the steps of: (a) combining an amount of a pegylated carfilzomib compound effective for treating multiple myeloma with a combination of at least one excipient selected from the group consisting of sucrose, proline, glycine, and sodium chloride; and a buffer selected from the group consisting of glutamate, histidine, acetate, and Tris-HCl, or a combination thereof; and (b) mixing the combination to provide a clear solution.

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