Stable composition of PEGylated carfilzomib compounds

Abstract

To provide a stable modified carfilzomib formulation. [Solution] 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 vascular malignancies such as multiple myeloma. The composition can be stored in a frozen form or lyophilized in a dry solid form.

Description

[Technical Field]

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

[0002] The present invention relates to stable pharmaceutical compositions of PEGylated carfilzomib compounds, methods for preparing such compositions, and their use for treating cancers, including vascular malignancies such as multiple myeloma and solid tumors. [Background technology]

[0003] Cancer is one of the most widespread diseases and a leading cause of death worldwide. In the United States alone, cancer is the second leading cause of death, surpassed only by heart disease. Cancer is often characterized by the disruption of normal cellular processes or uncontrolled cell proliferation.

[0004] Multiple myeloma (MM) is a progressive and malignant tumor-type cancer originating from plasma cells. It is characterized by an abnormal accumulation of malignant plasma cells in the bone marrow and accounts for approximately 13% of all hematological cancers (Palumbo and Anderson, 2011). In 2015, approximately 26,850 new cases of MM were expected to be diagnosed, and approximately 11,240 deaths from the disease were expected in the United States (ACS, 2015). The incidence of MM is steadily increasing due to the rising life expectancy of the general population in the United States (Warren et al., 2013). The disease most commonly affects the elderly population, with a median age of onset of approximately 69 years (Howlander et al., 2013; ACS, 2015).

[0005] The treatment goals for managing multilamic malformation (MM) are to provide symptom relief, achieve disease control, and provide long-term remission (Kurtin, 2013). Traditionally, the combination of high-dose chemotherapy agents (melphalan, vincristine, cyclophosphamide, doxorubicin, liposomal doxorubicin, bendamamustine) followed by autologous stem cell transplantation (ASCT) has been used to treat younger, treatment-naïve, medically eligible patients (under 65 years of age) (Palumbo et al., 2011). Age, comorbidities, and geriatric assessment are key criteria for determining eligibility for ASCT following high-dose therapy (HDT) (Palumbo et al., 2014). For older patients who are not eligible for HDT and ASCT, melphalan plus prednisone has been the standard therapy for decades (Palumbo et al., 2011; Rodriguez et al., 2012). Over the past decade, the treatment algorithm for MM has undergone a paradigm shift with the introduction of new immunomodulatory agents (such as thalidomide, lenalidomide, and pomalidomide) and targeted proteasome inhibitors (bortezomib and carfilzomib) (Richardson et al., 2007; Dmoszynska, 2008; Gupta et al., 2013).

[0006] Carfilzomib is a tetrapeptide epoxyketone proteasome inhibitor that selectively and irreversibly binds to constitutive and immunoproteasomes. More specifically, an 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, its polymorphic forms, methods of preparation, formulations, uses, and other attributes of carfilzomib are described in U.S. Patent Publication No. 20050245435, No. 20140105921, PCT International Publication No. 2006017842, No. 2009045497, No. 2014169897, No. 2013169282, No. 2014011695, No. 2006063154, No. 2014015016, and No. 2010048298, each of which is incorporated herein by reference in its entirety.

[0007] Carfilzomib has demonstrated encouraging overall response rates, progression-free survival (PFS), and overall survival (OS) in patients with relapsed and refractory MM and newly diagnosed MM. Carfilzomib was first approved as monotherapy in July 2012 for the treatment of patients with relapsed and refractory MM (as Kyprolis®). More recently, Kyprolis was approved in combination with lenalidomide and dexamethasone (July 2015) and in combination with dexamethasone (January 2016) for the treatment of patients with relapsed and refractory MM who have received 1 to 3 treatments. The approved treatment regimen for carfilzomib is to administer it to the patient by infusion, either over a short 10-minute period or over a longer 30-minute period. This infusion should be administered two days a week for three consecutive weeks in a 28-day cycle. Therefore, in order to follow this treatment schedule, patients need to drive or be driven to an authorized Kyprolis® administration center, such as a physician's office, clinic, or hospital, where Kyprolis® can be administered appropriately and safely, on two consecutive days each week. This may be inconvenient or impractical, or simply burdensome for some patients. This burden may increase the likelihood of reduced or decreased compliance with a full and complete course of the prescribed Kyprolis® treatment regimen, or even complete non-compliance.

[0008] Carfilzomib is rapidly metabolized and excreted in humans. Being a small tetrapeptide compound, carfilzomib exhibits a short in vivo half-life of approximately 60 minutes or less in humans. One mechanism of carfilzomib clearance is via hepatic blood flow, which contributes to its relatively short half-life. Drug products with short half-lives or rapid clearance generally tend to exhibit a reduced target range, resulting in decreased and / or shortened biological inhibitory activity. To overcome such shortcomings, additional drugs are typically administered to provide more drug and extended efficacy at the biological site of action. Therefore, both the rapid clearance of carfilzomib and its twice-weekly dosing frequency leave room for potential improvements in efficacy, delivery, and / or patient compliance.

[0009] As currently approved (Kyprolis®), carfilzomib is a sterile, lyophilized, amorphous solid formulation containing sulfabutyl ether beta-cyclodextrin (SBECD) and sodium citrate buffer. Immediately before administration, the lyophilized material is reconstituted with sterile water and infused or injected into the patient. The SBECD excipient primarily acts as a solubilizing additive for carfilzomib, forming a complex with it and thereby improving its water solubility.

[0010] History reveals that attempts to address weaknesses in drug products and / or improve the delivery, use, or other aspects of a given drug product have led to the preparation of alternative forms of these active pharmaceutical ingredients (APIs or pharmaceutically active compounds) and / or novel formulations thereof. Some alternative compound forms include the discovery of prodrugs designed to improve the pK and / or PD properties of APIs. For example, Greenwald et al. disclose prodrugs of amine-containing compounds (J. Med. Chem., 1999, 42, 3657-3667). International Publication No. 2005063777 discloses benzyl phosphate and substituted benzyl phosphate prodrugs for the treatment of pneumonia. International Publication No. 20090152160 discloses inhaled carbaprotacyclines and prostacyclin prodrugs for the treatment of arterial hypertension. U.S. Patent Application Publication No. 20040100225 discloses an acyloxymethyl prodrug of imatinib (Gleevec®). PCT International Publication Brochure No. 2011084846 also discloses an acyloxymethyl prodrug of risperidone. These prodrug disclosures teach the concept of alkyl-acyloxymethyl linked prodrugs.

[0011] Modifications to carfilzomib have also been made to improve its properties or other attributes as an active pharmaceutical ingredient and pharmacologic agent. U.S. Patent Application Publication No. 20140105921 describes carfilzomib and other epoxy ketone proteasome inhibitor prodrugs having an acyloxymethyl linker that conjugates the inhibitor to polyethylene glycol units (PEG). However, these carfilzomib prodrug compounds have been found to release quinone methide byproducts during in vivo metabolism, which are potentially toxic and may pose a risk to human safety. It would be desirable to identify modified carfilzomib formulations and / or pharmaceutical compositions suitable for delivering modified carfilzomib to patients while maintaining or potentially improving the stability, shelf life, efficacy and / or safety of currently approved carfilzomib treatments. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] U.S. Patent Application Publication No. 2005 / 0245435 [Patent Document 2] U.S. Patent Application Publication No. 2014 / 0105921 [Patent Document 3] International Publication No. 2006 / 017842 [Patent Document 4] International Publication No. 2009 / 045497 [Patent Document 5] International Publication No. 2014 / 169897 [Patent Document 6] International Publication No. 2013 / 169282 [Patent Document 7] International Publication No. 2014 / 011695 [Patent Document 8] International Publication No. 2006 / 063154 [Patent Document 9] International Publication No. 2014 / 015016 [Patent Document 10] International Publication No. 2010 / 048298 [Patent Document 11] International Publication No. 2005 / 063777 [Patent Document 12] International Publication No. 2009 / 0152160 [Patent Document 13] U.S. Patent Application Publication No. 2004 / 0100225 [Patent Document 14] International Publication No. 2011 / 084846 [Non-patent literature]

[0013] [Non-Patent Document 1] Palumbo and Anderson, 2011 [Non-Patent Document 2] ACS, 2015 [Non-Patent Document 3] Warren et al., 2013 [Non-Patent Document 4] Howlander et al., 2013 [Non-Patent Document 5] Kurtin, 2013 [Non-Patent Document 6] Palumbo et al., 2011 [Non-Patent Document 7] Palumbo et al., 2014 [Non-Patent Document 8] Rodriguez et al., 2012 [Non-Patent Document 9] Richardson et al., 2007 [Non-Patent Document 10] Dmoszynska, 2008 [Non-Patent Document 11] Gupta et al., 2013 [Non-Patent Document 12] J.Med.Chem., 1999, 42, 3657-3667 [Overview of the project] [Means for solving the problem]

[0014] The present invention provides a novel PEGylated carfilzomib compound pharmaceutical composition, i.e., a stable PEG-carfilzomib formulation, that provides therapeutic anti-cancer benefits to patients while maintaining comparable or longer-lasting carfilzomib plasma concentrations and proteasome protein exposure. For this purpose, the formulation of the present invention provides proteasome inhibitory activity comparable to that of currently approved carfilzomib cyclodextrin IV formulations. The present invention further provides a PEGylated carfilzomib formulation that does not involve the use of cyclodextrin as a carfilzomib solubilizer.

[0015] In particular, the present invention provides stable, isotonic, cyclodextrin-free, lyophilized, and liquid PEGylated carfilzomib compound formulations. Solid lyophilized formulations can be reconstituted, for example, with sterile water and administered by parenteral methods including intravenous administration and injection, and by subcutaneous administration. These formulations are useful for treating 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 methods for preparing pharmaceutical compositions and methods for administering the compositions by infusion or injection, or parenterally such as subcutaneously, for the treatment of various forms of cancer, including multiple myeloma.

[0016] In one aspect, the present invention is (a) a PEGylated carfilzomib compound; (b) at least one excipient selected from the group consisting of sucrose, sorbital, glycerin, maltose, lactose, erythrose, dextrose, lactobiose, 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-HCl, lysine-HCl, magnesium chloride, and barium sulfate; (c) glutamate, histidine, acetate, and tris-HCl, or a combination thereof (d) a buffer selected from the group consisting of; (d) optionally a volume extender selected from the group consisting of mannitol, trehalose, PVP, cyclodextrin, glycine, dextrose, dextran, sucrose, proline, PEG33350, 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 doxert, benzalkonium chloride, Triton X100, tetrafunctional block O polymer, alcohol, SDS, protamine sulfate, and butane; or a combination of (d), (e), and (f). The present invention provides a pharmaceutical composition containing the following: The pharmaceutical composition of the present invention provides PEGylated carfilzomib compounds with preferred solubility, permeability, pharmacokinetic (pK), and / or pharmacodynamic (PD) properties compared to the corresponding approved carfilzomib product.

[0017] The pharmaceutical compositions provided by the present invention, but which are not limited, further offer potential drug product benefits, including storage stability, storage capacity, safe use after several days as a liquid formulation, frozen formulations, dried lyophilized formulations, and other conveniences for safe storage and use of carfilzomib-based drug products. The improved compositions of the present invention facilitate various modes of administration, such as intravenous administration by infusion or injection. The compositions can also be administered subcutaneously under the skin. The present invention also provides compositions comprising hyaluronidase that can facilitate subcutaneous administration. The compositions are useful in the treatment of cancers, including but not limited to multiple myeloma and solid tumors. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 is a bar graph showing the remaining API% (5K PEGylated carfilzomib - Example 34 of this specification) after storage at 25°C for 3 days and measurement by reverse-phase chromatography. [Figure 2] Figure 2 is a bar graph showing the remaining API% (20K PEGylated carfilzomib - Example 39 of this specification) after storage at 25°C for 3 days and measurement by reverse-phase chromatography. [Figure 3A] Figure 3A is a depiction of lyophilized cakes obtained from formulations G5Su2M4 (3) and H5Su2M4 (1) containing 3K PEGylated carfilzomib - Example 28 of this specification. [Figure 3B] Figure 3B is a bar graph showing the remaining API% (3K PEGylated carfilzomib - Example 28 in this specification) when measured by reverse-phase chromatography for each of the four freeze-dried cakes shown in Figure 3A. [Figure 4A] Figure 4A is a depiction of a lyophilized cake obtained from hyaluronidase containing 3K PEGylated carfilzomib—Formulation G5Su2M4+0.006% polysorbate 80+2000 units / mL, including Example 28 of this specification. [Figure 4B] Figure 4B depicts the freeze-dried cake obtained from a placebo control formulation containing only hyaluronidase. [Figure 5A] Figure 5A is a bar graph showing that the number of particles in the exemplary formulations in Table 5 before and after lyophilization exceeds 10 microns when measured under light shielding that is invisible to the naked eye; [Figure 5B] Figure 5B is a bar graph showing that the number of particles in the exemplary formulations in Table 5 before and after lyophilization exceeds 25 microns when measured under light shielding that is invisible to the naked eye; [Figure 6] Figure 6 is a graph showing the results of the in-vitro effect of an exemplary PEGylated carfilzomib compound in a cancer tumor xenograft model; [Figure 7] Figure 7 is a graph showing the results of the effects of exemplary PEGylated carfilzomib compounds described herein on cancerous tumors; [Modes for carrying out the invention]

[0019] The present invention provides novel PEGylated carfilzomib compound pharmaceutical compositions, methods for producing these formulations, and the use of these compositions for the treatment of cancer, including the treatment of hematological malignancies such as multiple myeloma, lymphoma, and leukemia, and other cancers such as solid tumors. Specifically, the formulations of the present invention are stable, resulting in improved shelf life, solution clarity, lifespan, and safety of the drug product without significant degradation of the active pharmaceutical component. The present invention provides compositions that are PEGylated carfilzomib compound cryogenic formulations and compositions that are dried cryogenic formulations. These formulations are considered to have various API pharmacokinetic (pK) and / or pharmacodynamic (PD) properties equivalent to or better than currently approved IV-administered Kyprolis® (carfilzomib).

[0020] Carfilzomib is, in particular, an epoxy ketone protease inhibitor as described in U.S. Patent Nos. 7,417,042 and 7,737,112. The present invention provides formulations comprising PEGylated carfilzomib compounds as APIs. Exemplary PEGylated carfilzomib compounds that may be included in the present invention are described in general and specifically in International Patent Application No. PCT / US2017 / 03429. This PCT application has not been published as of November 24, 2017.

[0021] A typical PEGylated carfilzomib compound that may be included in the pharmaceutical composition of the present invention is: It is as follows:

[0022] In one aspect of the present invention, the composition is formula I [ka] [In the formula, R 1 C 1~10 Alkyl or C 3~7 It is a cycloalkyl; Each R 2 Independently, C 1~6 It is alkyl, -OCH3, or halogen; o is an integer selected from 0, 1, 2, or 3; Linker, [ka] (In the formula, R 3 is either H or CH3; n is an integer selected from 1, 2, 3, or 4; p is an integer selected from 0, 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.) It is a part having the structure; PEG is a polyethylene glycol polymer moiety having a molecular weight in the range of about 500 to about 20,000 and comprises a PEGylated carfilzomib compound or a pharmaceutically acceptable salt thereof.

[0023] In aspect 1a of the present invention, the composition is of formula I

Chemical formula

Chemical formula

[0024] In aspect 2 of the present invention, the composition is of formula II

Chemical formula

[0025] In aspect 3 of the present invention, composition, R 1 C 1~10 The present invention comprises alkyl PEGylated carfilzomib compounds according to embodiments 1, 1a, and 2.

[0026] In aspect 4 of the present invention, the composition, each R 2 However, it comprises one of the PEGylated carfilzomib compounds of embodiments 1, 1a, 2, and 3, which independently is H, CH3, or a halogen.

[0027] In aspect 5 of the present invention, the composition, each R 2 However, it independently comprises one of the PEGylated carfilzomib compounds of embodiments 1, 1a, 2, 3, and 4, which is H, CH3, Cl, or F.

[0028] In aspect 5a of the present invention, the composition, each R 2 However, it comprises one of the PEGylated carfilzomib compounds of embodiments 1, 1a, 2, 3, and 4, which independently comprises H, CH3, or F.

[0029] In aspect 6 of the present invention, the composition is a linker, [ka] [In the formula, R 3 is either H or CH3; q is an integer selected from 1, 2, 3, 4, or 5; r is an integer selected from 0, 1, 2, 3, or 4. The present invention comprises a PEGylated carfilzomib compound having the structure of any one of embodiments 1, 1a, 2, 3, 4, and 5.

[0030] In aspect 6a of the present invention, the composition is a linker, [ka] [In the formula, R 3 [is H or CH3] The present invention comprises a PEGylated carfilzomib compound having the structure of any one of embodiments 1, 1a, 2, 3, 4, and 5.

[0031] In aspect 7 of the present invention, the composition and linker are [ka] [In the formula, R 3 is either H or CH3; q is 4; The present invention comprises one of the PEGylated carfilzomib compounds of embodiments 1, 1a, 2, 3, 4, 5, and 7, where r is 2.

[0032] In aspect 7a of the present invention, the composition includes a linker, [ka] [In the formula, R 3 The compound comprises one of the PEGylated carfilzomib compounds of embodiments 1, 1a, 2, 3, 4, 5, and 7, wherein [ is H or CH3].

[0033] In embodiment 8 of the present invention, the composition comprises one of the PEGylated carfilzomib compounds of embodiments 1, 1a, 2, 3, 4, 6, 6a, 7, and 7a, wherein R3 is H.

[0034] In aspect 9 of the present invention, composition, R 1 The present invention comprises one PEGylated carfilzomib compound according to any one of embodiments 1 to 8, wherein is methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl, or heptyl.

[0035] In aspect 10 of the present invention, the composition, R 1 is methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl, or heptyl; the linker is [ka] This comprises one of the PEGylated carfilzomib compounds according to any one of embodiments 1 to 9.

[0036] In aspect 10a of the present invention, the composition, R 1 is methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl, or heptyl; the linker is [ka] This comprises one of the PEGylated carfilzomib compounds according to any one of embodiments 1 to 9.

[0037] It should be noted that in aspects 1, 1a, 2, and aspects 3 to 10, the term "or a pharmaceutically acceptable salt thereof" may include counterionic salts of quaternary nitrogen cation charges, such as those shown in Formula II of aspect 2, or those shown in aspects 11 to 24 below. Furthermore, it should be noted that the term "any one of aspects 1 to X" is intended to include all sub-existences 1 to X disclosed herein, including but not limited to sub-existences 1a, 5a, 6a, 7a, and 10a.

[0038] In aspect 11 of the present invention, the composition is [ka] [In the formula, R 1 C 1~10 It is alkyl; R 2 C 1~6 It is alkyl, -OCH3, or halogen; R 3 is either H or CH3; X - This is a counter anion selected from chloride anions and alkyl-sulfonate anions; n is 4; PEG is a polyethylene glycol polymer moiety with a molecular weight in the range of approximately 2,000 to 20,000. The present invention comprises one PEGylated carfilzomib compound from any one of embodiments 1 to 10 having the structure of [the given structure].

[0039] In aspect 12 of the present invention, the composition is a compound, [ka] The present invention comprises one PEGylated carfilzomib compound according to any one of embodiments 1 to 11, wherein [X is a halide, sulfonate, or alkyl-sulfonate counterion salt].

[0040] In aspect 12a of the present invention, the composition is a compound, [ka] The present invention comprises one PEGylated carfilzomib compound according to any one of embodiments 1 to 11, wherein [X is a halide, sulfonate, or alkyl-sulfonate counterion salt].

[0041] In aspect 13 of the present invention, the composition is a compound, [ka] The present invention comprises one PEGylated carfilzomib compound according to any one of embodiments 1 to 11, wherein [X is a halide, sulfonate, or alkyl-sulfonate counterion salt].

[0042] In aspect 14 of the present invention, the composition is a compound, [ka] The present invention comprises one of the PEGylated carfilzomib compounds according to any one of embodiments 1 to 11.

[0043] In aspect 15 of the present invention, the composition, R 1 is methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl, or heptyl; Each R 2 However, independently, it is CH3 or halogen; Linker, [ka] [In the formula, R 3 [is H or CH3] It is a part having the structure; The present invention comprises one of the PEGylated carfilzomib compounds according to Embodiments 1 and 2, wherein the PEG is a polyethylene glycol polymer moiety having a molecular weight of 2,000, 3,000, 5,000, or 20,000.

[0044] In aspect 16 of the present invention, the composition, R 1 but is methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl, or heptyl; Each R 2 However, independently, it is CH3; Linker, [ka] [In the formula, R 3 [is H] It is a part having the structure; The PEGylated carfilzomib compound of embodiment 15 comprises a polyethylene glycol polymer moiety having a molecular weight of 3,000, 5,000, or 20,000.

[0045] In embodiment 17 of the present invention, the composition comprises one PEGylated carfilzomib compound from any one of embodiments 1 to 16, wherein the compound is one of the individual compounds represented in Examples 1 to 34 described in Table 2 below this specification, or a pharmaceutically acceptable salt thereof.

[0046] In aspect 18 of the present invention, the composition is a compound, [ka] [ka] [ka] The compound comprises any one of the PEGylated carfilzomib compounds of embodiments 1 to 17, or a pharmaceutically acceptable salt thereof.

[0047] In aspect 18a of the present invention, the composition is a compound, [ka] This comprises any one of the PEGylated carfilzomib compounds of embodiment 18.

[0048] In aspect 19 of the present invention, the composition is a compound, [ka] The present invention comprises one of the PEGylated carfilzomib compounds according to any one of embodiments 1 to 18.

[0049] In aspect 19a of the present invention, the composition is a compound, [ka] The present invention comprises one of the PEGylated carfilzomib compounds according to any one of embodiments 1 to 18.

[0050] In aspect 20 of the present invention, the composition is a compound, [ka] The present invention comprises one of the PEGylated carfilzomib compounds according to any one of embodiments 1 to 18.

[0051] In aspect 21 of the present invention, the composition is a compound, [ka] The present invention comprises one of the PEGylated carfilzomib compounds according to any one of embodiments 1 to 18.

[0052] In aspect 22 of the present invention, the composition is a compound, [ka] The present invention comprises one of the PEGylated carfilzomib compounds according to any one of embodiments 1 to 18.

[0053] In aspect 23 of the present invention, the composition is a compound, [ka] The present invention comprises one of the PEGylated carfilzomib compounds according to any one of embodiments 1 to 18.

[0054] In aspect 24 of the present invention, the composition is a compound, [ka] The present invention comprises one of the PEGylated carfilzomib compounds according to any one of embodiments 1 to 18.

[0055] In aspect 25 of the present invention, the composition is a compound, [ka] The present invention comprises one of the PEGylated carfilzomib compounds according to any one of embodiments 1 to 18.

[0056] In aspect 26 of the present invention, the composition is a compound, [ka] The present invention comprises one of the PEGylated carfilzomib compounds according to any one of embodiments 1 to 18.

[0057] In embodiment 27 of the present invention, the composition comprises a PEGylated carfilzomib compound from any one of embodiments 1 to 16, wherein the PEG has a weight in the range of about 2K to about 20K.

[0058] In embodiment 28 of the present invention, the composition comprises one of the PEGylated carfilzomib compounds from embodiments 1 to 16, wherein the PEG has a weight of 3K, 5K, or 20K.

[0059] In embodiment 29 of the present invention, the composition comprises one PEGylated carfilzomib compound from any one of embodiments 1 to 16, which is a pharmaceutically acceptable salt comprising a counteranion selected from a chloride anion, bisulfate anion, sulfate anion, nitrate anion, phosphate anion, alkyl-sulfonate anion, or aryl-sulfonate anion.

[0060] In embodiment 30 of the present invention, the present invention comprises the PEGylated carfilzomib compound of embodiment 29, wherein the counter anion is a chloride anion or an alkyl-sulfonate anion.

[0061] In embodiment 31 of the present invention, the composition comprises the PEGylated carfilzomib compound of embodiment 29, wherein the counter anion is a chloride anion or a methane-sulfonate anion.

[0062] In embodiment 32 of the present invention, the composition comprises one PEGylated carfilzomib compound from embodiments 1 to 26 and a pharmaceutically acceptable excipient, carrier, or diluent.

[0063] In embodiment 33 of the present invention, The present invention (a) with a PEGylated carfilzomib compound; (b) at least one excipient selected from the group consisting of sucrose, sorbital, glycerin, maltose, lactose, erythrose, dextrose, lactobiose, 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-HCl, lysine-HCl, magnesium chloride, and barium sulfate; (c) A buffer selected from the group consisting of glutamate, histidine, acetate, and tris-HCl, or combinations thereof; (d) Optionally, a volume extender selected from the group consisting of mannitol, trehalose, PVP, cyclodextrin, glycine, dextrose, dextran, sucrose, proline, PEG33350, 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 doxert, benzalkonium chloride, Triton X100, tetrafunctional block O polymer, alcohol, SDS, protamine sulfate, and butane. Or a combination of (d), (e), and (f), The present invention provides a pharmaceutical composition containing the following:

[0064] In embodiment 33a of the present invention, The present invention (a) with a PEGylated carfilzomib compound; (b) at least one excipient selected from the group consisting of sucrose, proline, glycine, and sodium chloride; (c) A buffer selected from the group consisting of glutamate, histidine, acetate, and tris-HCl, or combinations thereof; (d) Optionally, a volume extender selected from the group consisting of mannitol, trehalose, PVP, cyclodextrin, glycine, dextrose, dextran, sucrose, proline, PEG33350, 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 doxert, benzalkonium chloride, Triton X100, tetrafunctional block O polymer, alcohol, SDS, protamine sulfate, and butane. Or a combination of (d), (e), and (f), The present invention provides a pharmaceutical composition containing the following:

[0065] In embodiment 33b, the present invention provides compositions of embodiments 33 and 33a, wherein (b) at least one excipient is present in an amount ranging from 0.1 to 30% by weight / volume of the composition; (c) a buffer is present in an amount sufficient to achieve a desired pH of the formulation; (d) an optional bulking agent is present in an amount ranging from 2 to 50% by weight / volume of the composition; (e) an optional amino acid is present in an amount ranging from 0.1 to 10% by weight / volume of the composition; and (f) a surfactant is present in an amount ranging from 0.005 to 3% by weight / volume of the composition.

[0066] In embodiment 33c, the present invention is (a) with a PEGylated carfilzomib compound; (b) at least one excipient selected from the group consisting of sucrose, proline, glycine, and sodium chloride; (c) A buffer selected from the group consisting of glutamate, histidine, acetate, and tris-HCl, or combinations thereof; (d) optionally, a volume extender selected from the group consisting of mannitol, trehalose, polyvinylpyrrolidone, and dextrose, present in an amount in the range of 2 to 50% by weight. (e) Optionally, an amino acid selected from the group consisting of lysine, arginine, histidine, and proline, present in an amount ranging from 0.1 to 10% by weight. (f) Optionally, a surfactant selected from the group consisting of polysorbate 80, Pluronic F68, polysorbate 20, and Triton X100, present in an amount ranging from 0.005 to 3% by weight. Or a combination of (d), (e), and (f), The present invention provides a pharmaceutical composition containing the following:

[0067] In embodiment 33d, the present invention is (a) with a PEGylated carfilzomib compound; (b) at least one excipient selected from the group consisting of sucrose in an amount ranging from 0.1 to 30% by weight, proline or glycine in an amount ranging from 0.1 to 10% by weight, and sodium chloride in an amount at a concentration of about 300 nM; (c) A buffer selected from the group consisting of glutamate, histidine, acetate, and tris-HCl, or combinations thereof; (d) Optionally, an amount of mannitol ranging from 2 to 50% by weight, (e) Optionally, lysine or arginine in an amount ranging from 0.1 to 10% by weight, (f) Optionally, an amount of polysorbate 80 or Pluronic F68 in the range of 0.005 to 3% by weight, Or a combination of (d), (e), and (f), The present invention provides a pharmaceutical composition containing the following:

[0068] In aspect 34 of the present invention, the present invention provides pharmaceutical compositions of aspects 33 and 33a to 33d, wherein the PEGylated carfilzomib compound has the structure of formula I as described in aspects 1 and 1a of this specification, as described in aspect 7 of this specification, as described in aspect 18 of this specification, or as described in aspect 18a of this specification.

[0069] In aspect 35 of the present invention, the present invention provides one of the pharmaceutical compositions of aspects 1 to 33, 33a to 33d, and 34, wherein the composition is a frozen formulation and the pH of the formulation is in the range of 5.0 to 8.0.

[0070] In aspect 36 of the present invention, the present invention provides a pharmaceutical composition according to 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.

[0071] In embodiment 37 of the present invention, the present invention provides a pharmaceutical composition according to embodiment 36, wherein the excipient is selected from the group consisting of sucrose in an amount in the range of about 5 to 12 wt / vol%, proline in an amount in the range of about 50 to 300 mM concentration, glycine in an amount in the range of about 50 to 300 mM concentration, and sodium chloride in an amount in the range of about 30 to 160 mM concentration, or a combination thereof, and the buffer is selected from the group consisting of histidine in an amount in the range of about 10 to 30 mM concentration, acetate in an amount in the range of about 10 to 30 mM concentration, and tris-HCl in an amount in the range of about 10 to 30 mM concentration, or a combination thereof.

[0072] In embodiment 38 of the present invention, the present invention provides a pharmaceutical composition in any one of embodiments 36 and 37, wherein the excipient is selected from the group consisting of about 9 w / v% sucrose, about 220 mM L-proline, about 293 mM glycine, about 140 mM sodium chloride, and a combination of about 4.5% sucrose and about 140 mM sodium chloride; and the buffer is selected from the group consisting of about 10 mM histidine, about 10 mM acetate, and about 10 mM tris-HCl.

[0073] In aspect 39 of the present invention, the present invention relates to a pharmaceutical composition, (a) a PEGylated carfilzomib compound in an amount ranging from 150 mg to 2000 mg; (b) at least one excipient and buffer is (1) 9% sucrose and 10 mM acetate buffer at pH 5; (2) 9% sucrose and 10 mM histidine at pH 6; (3) 9% sucrose and 10 mM tris-HCl at pH 7; (4) 9% sucrose and 10 mM tris-HCl at pH 8; (5) 140 mM sodium chloride and 10 mM tris-HCl at pH 7; (6) 220 mM L-proline and 10 mM tris-HCl at pH 7; (7) 293 mM glycine and 10 mM tris-HCl at pH 7; or (8) 10 mM tris-HCl at pH 7, both with 70 mM sodium chloride and 4.5% sucrose. The present invention provides any one of the embodiments 35 to 38, which include the above.

[0074] In aspect 40 of the present invention, the present invention provides one of the pharmaceutical compositions of aspects 1 to 33, 33a to 33d, and 34, wherein the composition is a dried freeze-dried formulation.

[0075] In aspect 41 of the present invention, the present invention provides a pharmaceutical composition of claim 40, wherein at least one excipient is sucrose in an amount ranging from 0.5% by weight to 2% by weight, the bulking agent is mannitol in an amount ranging from 2% by weight to 4% by weight, the amino acid is absent or selected from lysine or arginine, the surfactant is absent or is polysorbate 80 or Pluronic F68, and the buffering agent is glutamate.

[0076] In aspect 42 of the present invention, the present invention provides a pharmaceutical composition of claim 41, wherein the excipient is sucrose in an amount in the range of about 1 to 2 wt / vol%, mannitol in an amount in the range of about 2 to 4%, an amino acid selected from lysine or arginine in an amount in the range of about 0.5 to 0.8%, the surfactant is either 0.0065 polysorbate 80 or 0.05% Pluronic F68, and the buffer is 10 mM glutamate.

[0077] In aspect 43 of the present invention, the present invention relates to a composition in which 10mg 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% Pluronic 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, PEGylated carfilzomib compound in amounts ranging from 100 mg to 3000 mg; The present invention provides one pharmaceutical composition which is essentially derived from any one of embodiments 1 to 33, 33a to 33d, 34, 41, and 42.

[0078] In aspect 44 of the present invention, the present invention provides a pharmaceutical composition of aspect 43, wherein the pH of the composition is about 5.0 when the composition is dissolved in enough water to achieve a concentration of about 10 mg / mL of a PEGylated carfilzomib compound.

[0079] In aspect 45 of the present invention, the present invention provides any one of the pharmaceutical compositions of aspects 40 to 44, wherein the pharmaceutical composition comprises a PEGylated carfilzomib compound in an amount ranging from 150 mg to 2000 mg.

[0080] Aspect 46 of the present invention provides any one of the pharmaceutical compositions of aspects 40 to 45, wherein the pharmaceutical composition contains a PEGylated carfilzomib compound in an amount ranging from 300 mg to 2000 mg.

[0081] In aspect 47 of the present invention, the present invention provides any one of the pharmaceutical compositions of aspects 40 to 46, wherein the pharmaceutical composition comprises a PEGylated carfilzomib compound in an amount ranging from 800 mg to 3000 mg.

[0082] Aspect 48 of the present invention provides a pharmaceutical composition according to any one of aspects 40 to 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.

[0083] In aspect 49 of the present invention, the present invention provides any one of the pharmaceutical compositions of aspects 1 to 48, wherein the composition further comprises hyaluronidase.

[0084] In aspect 50 of the present invention, the present invention provides the pharmaceutical composition of aspect 49, wherein hyaluronidase is present in an amount in the range of 1500 to 2500 units / mL.

[0085] In embodiment 50a of the present invention, the present invention provides the pharmaceutical compositions of embodiments 49 and 50, wherein hyaluronidase is present in an amount of 2000 units / mL.

[0086] In embodiment 51 of the present invention, the present invention provides one of the pharmaceutical compositions of embodiments 1 to 50 and 50a that does not contain cyclodextrin.

[0087] In aspect 52 of the present invention, the present invention provides one of the pharmaceutical compositions of aspects 40 to 51, wherein the lyophilized formulation, when dissolved in 1.0 mL of water at room temperature, becomes a clear solution within approximately 3 minutes.

[0088] In aspect 53 of the present invention, the present invention provides one of the pharmaceutical compositions of aspects 1 to 33, 33a to 33d, and 34 to 50, 50a, 51, and 52, which are administered parenterally by infusion, injection, or subcutaneous administration.

[0089] In aspect 54 of the present invention, the present invention provides one of the pharmaceutical compositions of aspects 1 to 52, which is administered intravenously by injection or infusion.

[0090] In aspect 55 of the present invention, the present invention provides one of the pharmaceutical compositions of aspects 1 to 33, 33a to 33d, and 34 to 50, 50a, 51, and 52, which are administered by subcutaneous injection.

[0091] In aspect 56 of the present invention, the present invention provides a method for treating cancer, comprising administering a therapeutically effective amount of any one of the pharmaceutical compositions of aspects 1 to 33, 33a to 33d, and 34 to 50, 50a, and 51 to 55 to a patient in need of cancer treatment.

[0092] In aspect 57 of the present invention, the present invention provides the method of aspect 56, wherein the cancer is multiple myeloma.

[0093] In aspect 58 of the present invention, the present invention provides the method of aspect 57, wherein the multiple myeloma is relapsed, refractory, or relapsed and refractory multiple myeloma.

[0094] In aspect 59 of the present invention, the present invention provides the method of aspect 58, wherein the multiple myeloma is a newly diagnosed multiple myeloma.

[0095] In aspect 60 of the present invention, the present invention provides a process for producing any one of the pharmaceutical compositions of aspects 1 to 33, 33a to 33d, and 34 to 50 and 50a, comprising: (a) combining a PEGylated carfilzomib compound in an amount 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 obtain a clear solution.

[0096] While not bound by theory, the pharmaceutical compositions of the present invention may temporarily mask or partially mask the protease inhibitory activity of APIs. This effect may occur until the PEGylated linkage of the PEGylated carfilzomib compound is cleaved, thereby releasing free carfilzomib into the systemic circulation. This delay in activity may reduce or eliminate undesirable side effects, which may otherwise be related to various administration routes. It should also be noted that the PEGylated carfilzomib compounds contained in the compositions of the present invention may function as prodrugs of carfilzomib. Alternatively, these compounds may themselves possess very good active proteasome inhibitory activity.

[0097] The beneficial properties of the compositions of the present invention also facilitate subcutaneous administration of PEGylated carfilzomib compounds. Because of subcutaneous administration, the present invention potentially improves drug delivery, as well as patient convenience and compliance in treatments using selected PEGylated carfilzomib compounds.

[0098] In other aspects or embodiments of the present invention that may be described later herein, the method is characterized by treating a disease or condition selected from the group consisting of cancer, autoimmune diseases, graft or transplant-related conditions, neurodegenerative diseases, fibrosis-related conditions, ischemia-related conditions, infections (viral, parasitic or prokaryotic) and bone loss-related diseases, the method comprising administering a pharmaceutical composition according to the present invention to a patient, comprising administering to the patient a therapeutically effective amount of a PEGylated carfilzomib compound, such as those described herein. In further embodiments, the present invention provides a method for treating cancer in a patient (e.g., multiple myeloma, e.g., relapsed and / or refractory multiple myeloma).

[0099] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs. Methods and materials are described herein for use in this disclosure, and other suitable methods and materials known in the art may also be used. Materials, methods and examples are illustrative and not intended to limit. All publications, patent applications, patents, sequences, database entries and other references referred herein are incorporated herein by reference in their entirety as if they were described herein. In case of any conflict, this specification, including definitions, shall prevail. Other features and advantages of this disclosure will become apparent from the brief descriptions of the figures, the figures themselves, the detailed descriptions and the claims.

[0100] As used herein, the term "aspect" is synonymous with and interchangeable with the term "embodiment."

[0101] definition The following definitions will further aid in understanding the terms used herein and the scope of the invention as described herein.

[0102] The term “C x~y The term "alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including linear alkyl groups and branched alkyl groups, that contain x to y carbon atoms in the chain. The term "haloalkyl" refers to alkyl groups in which at least one hydrogen atom is substituted with a halo (e.g., fluoro, chloro, bromo, iodo), such as CH2F, CHF2, trifluoromethyl, and 2,2,2-trifluoroethyl.

[0103] The term “C 2~y "Alkenil" and "C 2~y"Alkynyl" refers to a substituted or unsubstituted unsaturated aliphatic group similar to the alkyl group above in length and possible substitutions, each containing at least one double or triple bond. In some embodiments, the divalent alkenylenes and alkynylenes contain 2 to 12 carbon atoms. In certain embodiments, the alkylenes and alkynylenes contain 2 to 10 carbon atoms. In certain embodiments, the alkylenes and alkynylenes contain 2 to 6 carbon atoms (e.g., 2, 3, 4, 5, or 6 carbon atoms).

[0104] The term "alkoxyl" refers to an alkyl group that has an oxygen atom bonded to it. Representative alkoxyl groups include methoxy, ethoxy, propoxy, and tert-butoxy. An "ether" is formed when two hydrocarbons are covalently bonded by an oxygen atom. Therefore, the substituents on the alkyl group that convert it to an ether are either alkoxy or similar.

[0105] The term "C" as used in this specification 3~y A "cycloalkyl" refers to a fully saturated, substituted, or unsubstituted ring in which each atom of the ring is carbon, and the ring contains 3 to γ ​​carbon atoms in size. For example, C 3~7 The term cycloalkyl is intended to mean a carbocyclic ring containing 3 to 7 carbon atoms in size at some point. Examples of such rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl rings. These rings may be further substituted as specified.

[0106] The terms “cancer” and “cancerous” refer to or describe a physiological condition in an object typically characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, hematological malignancies or blood-borne 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. As used herein, the term “cancer” is not limited to any one specific form of disease, however, the methods of the present invention are considered particularly effective for cancer in patients who have developed some resistance to treatment with anticancer agents, including but not limited to chemotherapeutic agents, antimitotic agents, and anthracyclines, and for cancer that has relapsed and has received subsequent treatment with such anticancer agents.

[0107] The term "includes" means that it is open-ended, including the listed components but not excluding other elements.

[0108] As used herein, the terms or abbreviations "eg" or "eg." are intended to mean "example."

[0109] The term "inhibitor" refers to a compound that blocks or reduces the activity of an enzyme or the system of an enzyme, receptor, or other pharmacological target (e.g., inhibition of proteolytic cleavage of standard fluorescent peptide substrates such as suc-LLVY-AMC, Box-LLR-AMC, and Z-LLE-AMC; inhibition of various catalytic activities of the 20S proteasome). Inhibitors may act by competitive, non-competitive, or non-competitive inhibition. Inhibitors may bind reversibly or irreversibly, and therefore the term includes compounds that are suicide substrates of enzymes. Inhibitors may modify one or more sites on or near the active site of an enzyme, or inhibitors may cause structural changes at other sites on the enzyme. The term "inhibitor" is used herein more broadly than in the scientific literature to also encompass other categories of pharmacologically or therapeutically useful agents, such as agonists, antagonists, stimulants, and cofactors.

[0110] As used herein, the terms “drug resistance” and “multidrug resistance” refer to cancer cells that have developed and / or are resistant to drugs. These include cancer cells that have little or no efficacy, or whose efficacy shown at the initial dose of a drug has decreased. Cancer cells may be resistant to one drug, or to multiple drugs with different chemical structures directed to act on different biological targets within the cancer cell.

[0111] The term “pharmaceutically acceptable salt” encompasses salts commonly used to form alkali metal salts and addition salts of free acids or free bases. The properties of the salt are not important as long as it is pharmaceutically acceptable. Suitable acid addition salts of pharmaceutically acceptable compounds can be prepared from inorganic or 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, though not limited to them, include aliphatic, alicyclic, aromatic, aromaticaliphatic, heterocyclic, carbocyclic, and sulfonic acids. Examples include 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 acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, ethanedisulfonic acid, benzenesulfonic acid, pantothenic acid, and 2-hydroxyethanolic acid. These include sulfonic acid, toluenesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, camphor acid, camphor sulfonic acid, digluconic acid, cyclopentanepropionic acid, dodecylsulfonic acid, glucoheptanoic acid, glycerophosphate, heptanoic acid, hexanoic acid, 2-hydroxyethanesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, palmoic acid, pectinic acid, persulfate, 2-phenylpropionic acid, picric acid, pivalic acid, propionic acid, succinic acid, tartaric acid, thiocyanic acid, mesylic acid, undecanoic acid, stearic acid, alginic acid, β-hydroxybutyric acid, salicylic acid, galactaric acid, and galacturonic acid.

[0112] 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 made from organic bases including cyclic amines, such as caffeine, arginine, diethylamine, N-ethylpiperidine, histidine, glucamine, isopropylamine, lysine, morpholine, N-ethylmorpholine, piperazine, piperidine, triethylamine, and trimethylamine, as well as primary, secondary, tertiary amines and substituted amines. All salts intended herein can be prepared by conventional means from the corresponding compounds, for example, by reacting the compound with a suitable acid or base.

[0113] As used herein, the term "proteasome" includes immune and constituent proteasomes.

[0114] As used herein, the term “refractory” is intended to mean unresponsive, resistant to, or unresponsive to treatment, stimulation (therapy), or cure, including resistance to multiple therapeutic agents. As used herein, “refractory” is intended to mean, in the context of characterizing cancer or tumors, cancer or tumor that is unresponsive to, resistant to, or has a diminished response to, treatment with one or more anticancer agents. The treatment is typically continuous, persistent, and / or repeated over a period of time that leads to relapse, resistance, or unresponsiveness to the very same treatment.

[0115] As used herein, the term “subject” refers to humans and any mammals, including animals such as cattle, horses, dogs, and cats. Therefore, the present invention may be used in human patients and veterinary subjects and patients. In one embodiment of the present invention, the compounds of the present invention may be administered to human subjects.

[0116] The terms “therapeutic effectiveness” or “therapeutic effective dose” are intended to quantify the amount of the compound of the present invention that, when administered to a patient (e.g., a human) as part of a desired drug regimen, alleviates symptoms, improves a condition, or delays the onset of a disease in accordance with clinically acceptable standards for the disorder or condition being treated or for cosmetic purposes, for example, in a reasonable benefit / risk ratio applicable to any medical treatment. Therefore, that is the amount of the compound of the present invention that can treat cancer, whether it be multiple myeloma or other hematological malignancies or solid tumors.

[0117] As used herein, the terms “to treat,” “to treat,” and “treatment” include, but are not limited to, curative, prophylactic, and preventive therapies, and generally include overturning, reducing, or preventing symptoms, clinical signs, and underlying pathological conditions of a patient’s condition by means of improving or stabilizing the patient’s condition. Prophylactic treatment generally consists of either completely preventing the onset of a disorder or delaying the onset of a preclinically apparent stage of the disorder in an individual. The terms “prophylactic or therapeutic” treatment are recognized in the art and include the administration of one or more of the composition in question to a host. If administered before the clinical signs of an undesirable condition (e.g., disease or other undesirable condition in a host animal) or after the condition has subsided, the subsequent treatment is prophylactic (i.e., protects the host from developing the undesirable condition), but if administered after the signs of an undesirable condition, the treatment is therapeutic (i.e., intended to reduce, improve, or stabilize an existing undesirable condition or its side effects).

[0118] The term PEG, as used herein, is intended to have its commonly understood traditional meaning. In particular, PEG is a part composed of repeating poly(ethylene glycol) polymer units, the exact number of which determines its molecular weight. The unit of this molecular weight is the dalton. Therefore, references to the molecular weight of PEG as used herein (in the specification, claims, and abstract), for example, "2K," "3K," "5K," and "20K," or "2000," "3000," "5000," or "20000" for a given PEG, are intended to mean 2000 daltons (or 2 kilodaltons), 3000 daltons (or 3 kilodaltons), 5000 daltons (or 5 kilodaltons), and 20000 daltons (or 20 kilodaltons), respectively, in weight of PEG. Furthermore, as used herein, "KDa" means kilodalton.

[0119] General synthesis and representative examples of PEGylated carfilzomib compounds used in the present invention As described, the PEGylated carfilzomib compounds in Formulas I and II are cleavable polymer PEG carriers for the active pharmaceutical ingredient, releasing carfilzomib (Formulas I and II) and free carfilzomib in vivo. The following abbreviations used throughout both the general scheme and the examples are intended to mean the following: DCM Dichloromethane; Methylenedichloride DMF Dimethylformamide DMSO (Dimethyl Sulfoxide) HCl ethyl acetate MeOH methanol mpk: milligrams per kilogram; mg / kg RT, rt room temperature NaCl (Sodium Chloride) tBuOH t-butanol; t-butyl alcohol

[0120] Carfilzomib is used to prepare PEGylated carfilzomib compounds, as described in International Publication Nos. 2006017842, 2009045497, 2014169897, 2013169282, 2014011695, 2006063154, 2014015016, 2010048298, and U.S. Patent Nos. 7,714,042 and 7,737,112.

[0121] Scheme 1: Cleavage of benzyl elimination quaternary salt [ka]

[0122] Enzymatic and / or chemical hydrolysis of the phenyl ester yields a carboxylic acid (II) and a phenolate intermediate (I), which undergoes rapid 1,6-elimination to provide free carfilzomib and quinone methide, with the quinone methide remaining covalently bound to the solubilized PEG polymer. Quinone methide is known to be a reactive Michael receptor and is thought to pose a risk associated with potential genotoxicity. In this invention, permanent binding of the quinone methide linker byproduct to the polymer may reduce toxicity by hindering cellular access and reducing reactivity to serum nucleophiles. The most likely in vivo outcome of intermediate III is its reaction with water, forming a benzyl alcohol-polymer adduct that is rapidly removed from the body by excretion.

[0123] Scheme 2: Cleavage of the carfilzomib-polymer complex described above. [ka]

[0124] Scheme 2 shows the metabolic pathway for the carfilzomib polymer compound described in International Publication No. 2014011695. Here, as shown above, the carfilzomib-polymer complex interacts with esterase enzymes or is subjected to chemical attack as indicated by the arrows. This attack results in the release of a free quinone methide (boxed above) during ester hydrolysis. This methide intermediate can further react freely with cellular nucleophiles, potentially leading to toxicity. The PEGylated carfilzomib compound of the present invention avoids this potentially toxic byproduct, as described in Scheme 1.

[0125] Scheme 3: Two-step PEG polymer composite formation procedure [ka]

[0126] The carfilzomib-PEG compounds provided by the present invention are prepared in a two-step procedure as shown in Scheme 3. Carfilzomib is first reacted with a suitably substituted para-alkanoyloxy-substituted benzyl halide (1) to obtain a quaternary salt intermediate (2). The quaternary salt bromide or iodide anion is exchanged via an ion exchange resin for a pharmaceutically acceptable anion such as a bisulfate, sulfate, nitrate, dihydrogen phosphate, or alkyl / aryl sulfonate to obtain intermediate (3). This intermediate is conveniently modified with reactive groups suitable for reaction with a complementaryly functionalized polymer reagent (4) to obtain the desired product 5. Numerous PEG reagents are commercially available in a range of molecular weights, structures, chemical properties of end groups, and the number of reactive end groups (arms) (see Table 1). They may be directly compatible with the chemical properties of the linker described herein, or they may require some further chemical manipulation by known methods. Branched and multi-arm PEGs may offer advantages over linear PEGs, such as higher drug load, improved stability, and / or potential lower formulation viscosity.

[0127] Scheme 4: Two-step Polymer Complexation via Azide / Alkyne Click Chemistry

Chem.

[0128] Scheme 4 shows "click" chemistries such as Huisgen 1,3-dipolar azide / alkyne cycloaddition and aminooxy / aldehyde oxime formation, which are particularly well-suited for polymers and polymer PEG conjugates due to high chemical yields, harmless by-products, large thermodynamic driving forces, and availability of starting materials.

[0129] The Hysgen 1,3-bipolar azide / alkyne cyclization requires the substitution of a benzyl group with an alkyne group (A1-(1-6)) that can react with an azide-functionalized polymer support such as PEG-azide (-N3) to obtain a 1,2,3-triazole-linked complex (A4-(1-6)). The alkyne moiety may be directly linked, linked via an alkyl spacer (A1-1), linked via an ether (A1-2,3), thioether, sulfoxide or sulfone (A1-4) linkage, or linked via an amide linkage (A1-5,6). Many azide-substituted PEG reagents are currently commercially available in a wide variety of sizes and structures, and can also be readily prepared from any available PEG-alcohol via reaction with an azide salt following activation by mesylation or tosylation. The cycloaddition reaction can be carried out using commercially available cuprous salt catalysts, but it proceeds more efficiently using a mixture of copper(II) (e.g., copper(II) sulfate, copper(II) methanesulfonate) and a reducing agent (e.g., sodium ascorbate) to produce Cu(I) in situ. Since copper(I) is unstable in aqueous solution and in the presence of oxygen, stabilizing ligands, such as tris-(benzyltriazolylmethyl)amine (TBTA), tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 2-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]ethyl 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), may be optionally added. The reaction may be carried out at room temperature or at high temperatures in a variety of solvents, and in mixtures of water and various miscible organic solvents, including alcohols, DMSO, DMF, tBuOH, and acetone.The final PEG-calfilzomib product (A4-(1-6)) can be conveniently post-treated 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. Exposure of the intermediate or product to anions during the post-treatment procedure, such as chloride anions in brine, typically results in a mixture of anions in the final product and may require a final anion exchange resin treatment to ensure product salt homogeneity.

[0130] The intermediate quaternary halide salt (bromide or iodide, (A2-(1-6)) is converted to an anion that does not precipitate with a copper(I) catalyst such as methanesulfonate, bisulfate or sulfate to (A3-(1-6)), achieving a high reaction yield. Also, it may be desirable to exchange the halide anion to prevent the cleavage of the epoxide and the possibility of forming bromohydrin or iodohydrin by-products.

[0131] Scheme 4A1-2 [Chemical formula]

[0132] Synthesis of 4-(bromomethyl)-2-(prop-2-ynyloxy)phenyl acetate (intermediate A1-2 in Scheme 4) Step 1: 4-Hydroxy-3-(prop-2-ynyloxy)benzaldehyde (1) To a mixture of NaOtBu in DMF (150 mL), 3,4-dihydroxybenzaldehyde (10 g, 72.5 mmol) from DMF (50 mL) was added at 20°C. The mixture was cooled in an ice bath, and 3-bromoprop-1-yin (8.62 g, 72.5 mmol) was added gradually while stirring, attempting to maintain the internal temperature at 15-20°C. The reaction mixture was stirred at room temperature for 2 hours. The mixture was diluted with water (300 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic layer was washed with water to remove the DMF, dried on anhydrous Na2SO4, and concentrated to a brown solid. Compound 1 was obtained by repeated crystallization of the residue from DCM / petroleum ether (30 mL / 500 mL). 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).

[0133] Step 2: 4-Formyl-2-(prop-2-inyloxy)phenylacetate (2) To a solution of compound 1 (10.00 g, 56.82 mmol) in DCM (150 mL), Et3N (11.48 g, 113.64 mmol), followed by acetyl chloride (5.35 g, 68.18 mmol), was added at 0°C. The reaction mixture was stirred at room temperature for 2 hours. The mixture was washed with 2N saturated aqueous HCl (100 mL) and water (50 mL), dried on anhydrous MgSO4, and concentrated to obtain compound 2, which was used in the next step without further purification. 1 H NMR (CDCl3, 400 MHz): δ 9.96 (s, 1H), 7.63 (d, J = 1.6 Hz, 1H), 7.54 (dd, J1= 1.6Hz, J2= 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).

[0134] Step 3: 4-(hydroxymethyl)-2-(prop-2-inyloxy)phenylacetate (3) To a solution of compound 2 (12.00 g, 55.05 mmol) in DCM / MeOH (150 mL / 15 mL), NaBH4 (3.06 g, 82.57 mmol) was gradually added at 0°C. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was quenched with acetone (5 mL) and concentrated. The residue was purified on silica gel by flash column chromatography (petroleum ether / alkyl = 2:1) to obtain compound 3. 1 H NMR (CDCl3, 400 MHz): δ 7.16 (d, J = 1.6 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.98 (dd, J1= 1.6 Hz, J2= 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).

[0135] Step 4: 4-(bromomethyl)-2-(prop-2-inyloxy)phenylacetate (4) To a solution of compound 3 (11.50 g, 52.27 mmol) in DCM (150 mL), PPh3 (20.50 g, 78.41 mmol) and NBS (11.04 g, 62.73 mmol) were added at 0°C. The reaction mixture was stirred at room temperature for 0.5 hours. The excess solvent was concentrated, and the residue was purified by flash column chromatography (petroleum ether / alkyl = 20:1) on silica gel to obtain compound 4 (7.82 g, 53% yield). 1 H NMR (CDCl3, 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).

[0136] Scheme 5: Quaternary salt anion exchange [ka]

[0137] Ion exchange can be achieved by the reaction of the intermediate quaternary halide with the silver salt, as shown in Scheme 5, or more practically, by passing through an ion exchange resin. The carfilzomib quaternary salt anion present in the intermediate or final product can be efficiently converted via the anion exchange resin to different strong acid anions such as bisulfate, sulfate, dihydrogen phosphate, nitrate, or alkyl / aryl sulfonate. AmberlystA26(OH - Anion exchange resins such as (type) are pretreated with the desired acid or ammonium salt, and then passed through a quaternary halide salt. Composites prepared from weak acid anions such as acetate, formate, or lactate are unstable due to the increased basicity of the quaternary salt and incompatibility with the ester trigger group.

[0138] Scheme 6: Two-step polymer composite formation via aminooxy / carbonyl chemistry [ka]

[0139] Alternatively, the benzyl group (B1-(1-6)) may be substituted with a carbonyl (aldehyde or ketone) group that can react with an aminooxy-functionalized polymer support such as PEG-aminooxy(-ONH2) to provide a stable oxime-bonded complex (B4-(1-6)). The carbonyl moiety may be directly bonded, bonded via an alkyl spacer (B1-1), bonded via an ether (B1-2,3), thioether, sulfoxide, or sulfone (B1-4) bond, or bonded via an amide bond (B1-5,6). Carfilzomib and the benzyl halide (B1-(1-6)) are reacted in a suitable organic solvent such as acetonitrile at room temperature or high temperature to provide a quaternary intermediate (B2-(1-6)) as a bromide or iodide salt. It is desirable to replace this halide anion to prevent epoxide cleavage and the formation of bromohydrin or iodohydrin byproducts. Anion exchange can be achieved, as described above (Scheme 5), by the reaction of the intermediate quaternary halide with the silver salt, or more practically, by passing it through an ion exchange resin. Carfilzomib quaternary salt intermediate (B3-(1-6)) and PEG-ONH3 + The Y-polymer reagent is then reacted at room temperature or high temperature in a suitable organic substance such as DCM or a mixed aqueous organic solvent. Oximetion catalysts such as aniline, p-phenylenediamine, or 5-methoxyanthranilic acid may be optionally added, but are usually not necessary. It should be noted that the carfilzomib quaternary salt intermediate (B3-(1-6)) and the PEG-aminooxy reagent anionic salt are identical in order to eliminate the formation of a mixed anionic salt final product and the need for any further anionic operations. The final PEG-carfilzomib product can be conveniently post-treated by evaporation of the reaction solvent and reprecipitation of the residue from isopropanol or an ether / isopropanol mixture until a product of the desired purity is obtained.

[0140] Scheme 7: Synthesis of PEG-aminooxy reagents [ka]

[0141] The PEG-aminooxy reagent is either commercially available or can be readily prepared from mesyl or tosyl activated PEG-alcohol, PEG-halide (A) or PEG-amine (B) starting materials as illustrated in Scheme 7. The tert-butyloxycarbonyl protected intermediate is deprotected with a strong acid such as hydrogen chloride, methanesulfonic acid, trifluoroacetic acid, or sulfuric acid to afford the PEG-aminooxy reagent as a chloride, trifluoroacetate or sulfate salt. The PEG-aminooxy reagent anion can be optionally exchanged with a different anion via an anion exchange resin.

[0142] Scheme 8: Oxime Isomers

Chem.

[0143] As illustrated in Scheme 8, it will be readily appreciated by those skilled in the art that oximes can exist as two geometric isomers: the syn (Z)-isomer and the anti (E)-isomer. Many of the examples in the present disclosure are aromatic aldoximes and exist only as the (E)-isomer. Non-aromatic aldoximes and ketoximes are usually fully separated and can be obtained as the (Z)-isomer and the (E)-isomer. The PEGylated non-aromatic aldoximes and ketoximes described in the present invention can exist as separated (Z) and (E)-isomers or as a mixture of (Z) and (E)-isomers.

[0144] Scheme 9: Direct Polymer Complexation to Form Quaternary Salts

Chem.

[0145] Alternatively, the carfilzomib-polymer complex described in the present invention may be prepared by a one-step reaction of carfilzomib with a para-alkanoyloxy-substituted benzyl halide to which the desired polymer chain has been pre-attached, as shown in Scheme 9. The polymer chain may be attached via a wide variety of known chemistry or the aforementioned alkyne / azide or carbonyl / aminooxy chemistry. This route may be less desirable because it is difficult to separate the PEG-containing product from the unreacted PEGylated starting material.

[0146] Examples of representative compounds for use in the present invention The following PEGylated carfilzomib compounds are representative examples of PEGylated carfilzomib compounds that may be used in the present invention and are not intended to be construed as limiting the scope of the invention. The PEGylated carfilzomib compounds were prepared using the following two common PEG conjugation methods (A and B).

[0147] PEG-triazole-linker method A: [ka]

[0148] Carfilzomib quaternary salt 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 obtain a cream-colored suspension. The mixture was vigorously stirred for 5 minutes, and a solution of copper(II) sulfate pentahydrate (0.3 equivalents) in water (10 mL / mmol PEG-azide) was rapidly added dropwise. The reaction immediately darkened to a yellowish-brown color, and the suspension became clear within 5 minutes. After 1 hour, the second part of the ascorbic acid (0.75 equivalents) was added, and the reaction mixture was stirred for 60 minutes. The third part of the ascorbic acid (0.38 equivalents) was added, and the reaction mixture was stirred overnight at room temperature. 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 (3 × 35 mL / mmol PEG-azide). The extract was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum at 40°C. The residue was dissolved in isopropanol (125 mL / mmol PEG-azide) at 40°C. After the solid was 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 filter cake was washed twice each with 2-propanol and diethyl ether. The filter cake was dissolved in DCM and concentrated under vacuum. The residue was dissolved in warm (40°C) isopropanol (200 mL / mmol PEG-azide) and then cooled in an ice bath. The resulting solid was filtered, and the filter cake was washed twice each with 2-propanol and diethyl ether and then dried under vacuum.

[0149] PEG oxime-linker method B [ka]

[0150] Carfilzomib quaternary salt intermediate B3-(1-6) (1 equivalent) in DCM (15 mL / mmol B3-(1-6)), PEG-ONH3 + MsO -(0.8 equivalents) and 5-methoxyanthranilic acid (oxime catalyst, 0.3 equivalents) were stirred at room temperature 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 room temperature, 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 formed solid was collected by filtration. Recrystallization from isopropanol / ether was repeated 1-2 more times until all unreacted carfilzomib quaternary salt intermediate B3-(1-6) was detected and removed by HPLC. The final solid was dried under vacuum at 30°C. Typical yield: 60-80%; Typical reaction time: 10-30 minutes for intermediates containing aldehyde functional groups, and 24 hours for intermediates containing ketone functional groups.

[0151] Examples of prepared PEG-carfilzomib compounds, PEG structures, and PEG linker means are listed in Table 1. Table 1 further includes the size and weight (Daltons) of the PEG adducts, as well as the methods used to add the PEG portion to the carfilzomib skeleton.

[0152] [Table 1]

[0153] [Table 2]

[0154] [Table 3]

[0155] [Table 4]

[0156] [Table 5]

[0157] [Table 6]

[0158] Example 2: 4-(4-acetoxy-2-((1-PEG 5K -1H-1,2,3-triazole-4-yl)methoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxiran-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholine-4-ium methanesulfonate (9) [ka]

[0159] 2-Hydroxy-4-(methoxymethoxy)benzaldehyde(1) To a solution of compound 2,4-dihydroxybenzaldehyde (5.04 g, 36.24 mmol) in THF (100 mL), DIPEA (6.52 g, 54.35 mmol) and chloro(methoxy)methane (3.21 g, 39.86 mmol) were added. The reaction mixture was stirred overnight at room temperature. The excess solvent was concentrated, and the residue was purified by flash column chromatography (petroleum ether / siRNA = 15:1) on silica gel to obtain compound 1 (3.96 g, 60% yield). 1 H NMR (300 MHz, CDCl3): 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).

[0160] 4-(methoxymethoxy)-2-(prop-2-inyloxy)benzaldehyde(2) Compound 1 (2.0 g, 10.63 mmol) in DMSO (50 mL) was added at 20°C to a mixture of NaH (900 mg, 21.252 mmol) in DMSO (100 mL). The mixture was stirred at the same temperature for 30 minutes, and then 3-bromoprop-1-in (1.90 g, 15.94 mmol) was added dropwise. The reaction mixture was stirred at the same temperature for 4 hours, and then poured into ice water (100 mL). The resulting solution was adjusted to pH 2-3, and ELISA (100 mL) was added. The two phases were separated, and the aqueous phase was extracted with ELISA (100 mL x 3). The combined organic phase was dried and concentrated. The residue was purified on silica gel by flash column chromatography (petroleum ether / ELISA = 3:1) to obtain compound 2 (1.89 g, 80% yield). 1 H NMR (300 MHz, CDCl3): 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).

[0161] 4-Hydroxy-2-(prop-2-inyloxy)benzaldehyde(3) To a solution of compound 2 (5.1 g, 23.18 mmol) in propan-2-ol (100 mL), CBr4 (760 mg, 2.32 mmol) was added. The reaction mixture was refluxed overnight. The excess solvent was concentrated, and the residue was purified by flash column chromatography (petroleum ether / siRNA = 3:1) on silica gel to obtain compound 3 (2.44 g, 60% yield). 1H 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).

[0162] 4-(hydroxymethyl)-3-(prop-2-inyloxy)phenol(4) To a solution of compound 3 (2.45 g, 13.92 mmol) in MeOH (40 mL), NaBH4 (618 mg, 16.698 mmol) was gradually added at 0°C. The reaction mixture was stirred at the same temperature for 1 hour, and then quenched with water (1.5 mL). The excess solvent was concentrated, and the residue was redissolved in SiO2 (100 mL). The resulting solution was dried and concentrated to obtain 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).

[0163] 4-(hydroxymethyl)-3-(prop-2-inyloxy)phenylacetate (5) To a solution of compound 4 (1.20 g, 6.74 mmol) in DCM (30 mL), TEA (1.70 g, 16.85 mmol), followed by acetyl chloride (634 mg, 8 mmol), was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 30 minutes. The excess solvent was concentrated, and the residue was purified by flash column chromatography (petroleum ether / alkyl = 5:1) on silica gel to obtain compound 5 (360 mg, 30% yield). 1H 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).

[0164] 4-(bromomethyl)-3-(prop-2-inyloxy)phenylacetate(6) To a solution of compound 5 (360 mg, 1.64 mmol) in DCM (15 mL), PPh3 (515 mg, 1.96 mmol), followed by NBS (318 mg, 1.80 mmol), was added gradually at 0°C. The reaction mixture was stirred at the same temperature for 30 minutes. The excess solvent was concentrated, and the residue was purified by flash column chromatography (petroleum ether / dimethyl = 50:1) on silica gel to obtain compound 6 (190 mg, 41% yield). 1 H 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 = 2.0Hz, 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).

[0165] 4-(4-acetoxy-2-(prop-2-in-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-tetraazahexadecyl)morpholine-4-ium methanesulfonate (8) To a solution of compound 6 (190 mg, 0.67 mmol) in MeCN (10 mL), (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropane-2-yl)-2-((S)-2-(2-morpholinoacetamide)-4-phenylbutanamide)pentanamide (480 mg, 0.67 mmol) was added. The reaction mixture was stirred overnight at 45°C. The excess solvent was concentrated, and the residue was purified by flash column chromatography (MeOH / siRNA = 1:50) on silica gel to obtain the desired compound 7, which was converted to the corresponding mesylate (340 mg, 74% yield) by treatment with an ion exchange resin; 1 H NMR (400 MHz, CDCl3): 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).

[0166] The compound of Example 2 was PEGylated with compound 8 and PEG according to the general PEGylation procedure A. 5K Prepared from N3

[0167] Example 13: 4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxiran-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-((1-(PEG20K-4-arm)-1H-1,2,3-triazole-4-yl)methoxy)-4-(pivaloyloxy)benzyl)morpholine-4-iumformate(7) [ka]

[0168] 4-Hydroxy-3-(prop-2-inyloxy)benzaldehyde(1) To a mixture of NaH in DMSO (300 mL), 3,4-dihydroxybenzaldehyde (30 g, 217.39 mmol) in DMSO (50 mL) was added at 20°C. The mixture was stirred for 30 minutes, and 3-bromoprop-1-yin (25.87 g, 217.39 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour, then poured into ice water. The resulting solution was adjusted to pH=2 and then extracted with ELISA (500 mL x 3). The combined organic phase was dried over anhydrous MgSO4 and concentrated. The residue was repeatedly crystallized from DCM / petroleum ether (30 mL / 500 mL) to obtain compound 1 (30 g, 78% yield). 1 H NMR (CDCl3, 300 MHz,): δ 9.89 (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).

[0169] 4-Formyl-2-(prop-2-inyloxy)phenylpivalate(2) To a solution of compound 1 (3.0 g, 17 mmol) in DCM (120 mL), Et3N (3.45 g, 34 mmol), followed by pivaloyl chloride (2.34 g, 20.4 mmol), was added at 0°C. The reaction mixture was stirred at room temperature for 2 hours. The mixture was washed with saturated NaHCO3 (20 mL) and water (20 mL), dried on anhydrous MgSO4, and concentrated. The residue was purified on silica gel by flash column chromatography (petroleum ether / siRNA = 50:1) to obtain compound 2 (2.10 g, 47% yield) as a white solid; 1 H NMR (CDCl3, 300 MHz): δ 9.99 (s, 1H), 7.61 (d, J = 1.8 Hz, 1H), 7.55 (dd, J1= 1.8 Hz, J2= 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).

[0170] 4-(hydroxymethyl)-2-(prop-2-inyloxy)phenylpivalate(3) To a solution of compound 2 (1.8 g, 6.9 mmol) in DCM / MeOH (100 mL / 10 mL), NaBH4 (0.37 g, 10.4 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 30 minutes. The mixture was quenched with acetone (3 mL), and the solvent was concentrated. The residue was purified on silica gel by flash column chromatography (petroleum ether / alkyl = 3:1) to obtain compound 3 (1.50 g, 83% yield). 1 H NMR (CDCl3, 300 MHz): δ 7.11 (m, 1H), 7.00 (m, 2H), 4.68 (m, 4H), 2.53 (m, 1H), 1.41 (s, 9H).

[0171] 4-(bromomethyl)-2-(prop-2-inyloxy)phenylpivalate(4) To a solution of compound 3 (1.50 g, 5.7 mmol) in DCM (60 mL), PPh3 (1.80 g, 6.8 mmol) and NBS (1.11 g, 6.3 mmol) were added at 0°C. The reaction mixture was stirred at room temperature for 0.5 hours. The excess solvent was concentrated, and the residue was purified by flash column chromatography (petroleum ether / SiO7 = 50:1) on silica gel to obtain compound 4 (1.34 g, 81% yield). 1 H NMR (CDCl3, 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).

[0172] 4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxiran-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(pivaloyloxy)-3-(prop-2-in-1-yloxy)benzyl)morpholine-4-ium methanesulfonate(6) To a solution of compound 4 (2.38 g, 7.3 mmol) in MeCN (30 ml), (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropane-2-yl)-2-((S)-2-(2-morpholinoacetamide)-4-phenylbutanamide)pentanamide (2.64 g, 3.7 mmol) was added. The reaction mixture was stirred overnight at 45°C. The excess solvent was concentrated, and the residue was purified by flash column chromatography (siRNA / MeOH = 100:6) on silica gel to obtain the desired compound 5, which was converted to the corresponding mesylate (1.23 g, 25% yield) by treatment with an ion exchange resin; 1H NMR (CDCl3, 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).

[0173] Compound Example 13 was prepared by following the general PEGylation procedure A, using compound 6 and PEGylation. 20K It was prepared from (N3)4. Compound Example 13 is also designated as OP-59381 in the various figures illustrated herein. 1H NMR (500 MHz, relaxation time = 10 sec, DMSO-d6) 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%.

[0174] 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-tetraazahexadecyl)morpholine-4-ium methanesulfonate(8) [ka]

[0175] 2-Hydroxy-5-(hydroxymethyl)benzaldehyde(1) To an aqueous solution of formaldehyde (37%, 17 mL), 2-hydroxybenzaldehyde (10.3 g, 84.4 mmol) and concentrated HCl (42 mL) were added. The reaction mixture was heated overnight under reflux. The mixture was cooled to room temperature and then extracted with HCl (200 mL). The organic phase was dried on anhydrous sodium sulfate and concentrated. The residue was purified on silica gel by flash column chromatography (petroleum ether / HCl = 3:1) to obtain compound 1 (1.97 g, 15% yield). 1 H NMR (DMSO-d6, 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).

[0176] 5-(((tert-butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde(2) To a solution of compound 1 (2.01 g, 13.2 mmol) in DCM (60 mL), imidazole (1.43 g, 21 mmol) was added. The solution was cooled to 0°C, and tert-butylchlorodimethylsilane (2.57 g, 17.1 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours, 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 on silica gel by flash column chromatography (petroleum ether / siRNA = 50:1) to obtain compound 2 (3.2 g, 91% yield). 1H NMR (CDCl3, 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).

[0177] 4-((tert-butyldimethylsilyloxy)methyl)-2-formylphenylacetate(3) To a solution of compound 2 (25 g, 94 mmol) in DCM (500 mL), TEA (19.0 g, 188 mmol) was added. 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 hours. The mixture was washed with water (500 mL). The organic phase was dried on anhydrous sodium sulfate and concentrated. The residue was purified on silica gel by flash column chromatography (petroleum ether / alkyl = 100:1) to obtain compound 3 (19.7 g, 68% yield). 1 H NMR (CDCl3, 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).

[0178] 2-Formyl-4-(hydroxymethyl)phenylacetate(4) Compound 3 (3.6 g, 11.7 mmol) was dissolved in AcOH / THF / H2O (50 mL / 25 mL / 25 mL). The reaction mixture was stirred at 30°C for 3 hours. Excess THF was removed, and the resulting solution was adjusted to pH 7-8, then extracted with HCl (50 mL x 3). The combined organic phase was dried on anhydrous sodium sulfate and concentrated. The residue was purified on silica gel by flash column chromatography (petroleum ether / HCl = 3:1) to obtain compound 4 (2.04 g, 90% yield). 1 H NMR (DMSO-d6, 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).

[0179] 4-(bromomethyl)-2-formylphenylacetate (5a) To a solution of compound 4 (2.03 g, 10.3 mmol) in DCM (80 mL), PBr3 (2.79 g, 10.3 mmol) was added at 0°C. The reaction mixture was stirred at room temperature for 4 hours. The reaction was quenched by adding water (20 mL), and the resulting mixture was adjusted to pH=7 with saturated NaHCO3 aqueous solution. The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated. The residue was purified on silica gel by flash column chromatography (petroleum ether / alkyl = 3:1) to obtain compound 5a (300 mg, 11% yield). 1 H NMR (CDCl3, 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).

[0180] 4-(iodomethyl)-2-formylphenylacetate (5b) To a solution of compound 4 (5.0 g, 27.55 mmol) in DCM (300 mL), SOCl2 (6.13 g, 51.55 mmol) was added at 0°C. The reaction mixture was heated overnight under reflux. The mixture was concentrated, and the residue was purified by flash column chromatography (petroleum ether / alkyl = 10:1) on silica gel to obtain the corresponding benzyl chloride (2.4 g, 44% yield). 1 H NMR (CDCl3, 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).

[0181] To a solution of benzyl chloride (2.4 g, 11.29 mmol) in acetone (160 mL), NaI (16.94 g, 112.94 mmol) was added. The reaction mixture was stirred overnight at 30°C. The mixture was concentrated, and the residue was dissolved in DCM (100 mL). The resulting solution was washed with saturated Na2S2O3 aqueous solution (50 mL x 3) and water (50 mL), dried on anhydrous sodium sulfate, and concentrated to obtain compound 5b (2.1 g, 61% yield), which was used in the next step without further purification. 1 H NMR (CDCl3, 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).

[0182] 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-tetraazahexadecyl)morpholine-4-ium methanesulfonate (7) To a solution of compound 5b (380 mg, 1.48 mmol) in MeCN (5 mL), (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropane-2-yl)-2-((S)-2-(2-morpholinoacetamide)-4-phenylbutanamide)pentanamide (532 mg, 0.74 mmol) was added. The reaction mixture was stirred overnight at 45°C. The solvent was removed under reduced pressure. The residue was purified on silica gel by flash column chromatography (DCM / MeOH = 10:1) to obtain 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 (CDCl3, 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 could also have been used for this reaction.

[0183] Example 18 involves compound 7 and PEG according to the general PEGylation procedure A. 5K ONH3 + .MsO - It was prepared from.

[0184] Example 23: 4-(3-acetoxy-4-((PEG 3K-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-tetraazahexadecyl)morpholine-4-ium methanesulfonate(8) [ka]

[0185] 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-tetraazahexadecyl)morpholine-4-ium methanesulfonate (7) To a solution of compound 5b (380 mg, 1.48 mmol) in MeCN (5 mL), (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropane-2-yl)-2-((S)-2-(2-morpholinoacetamide)-4-phenylbutanamide)pentanamide (532 mg, 0.74 mmol) was added. The reaction mixture was stirred overnight at 45°C. The solvent was removed under reduced pressure. The residue was purified on silica gel by flash column chromatography (DCM / MeOH = 10:1) to obtain the desired compound (6), which was then converted to the corresponding mesylate (280 mg, 39% yield) by treatment with an ion exchange resin; 1H NMR (CDCl3, 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).

[0186] Example 23 was prepared by a method similar to that described in Example 16, where the intermediate was prepared by a similar method (using acetyl chloride to produce intermediate 1 of the result shown in Example 16 and compound 7 of International Patent Application No. PCT / US2017 / 03429), and PEG was prepared according to the general PEGylation procedure A. 3K ONH3 + .MsO - .

[0187] 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-tetraazahexadecyl)-4-(4-(isobutyryloxy)-3-((PEG5K-imino)methyl)benzyl)morpholine-4-ium methanesulfonate (6) [ka]

[0188] 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-tetraazahexadecyl)-4-(3-formyl-4-(isobutyryloxy)benzyl)morpholine-4-ium methanesulfonate(5) To a solution of compound 3 (550 mg, 1.657 mmol) in MeCN (8 mL), (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropane-2-yl)-2-((S)-2-(2-morpholinoacetamide)-4-phenylbutanamide)pentanamide (393 mg, 0.547 mmol) was added. The reaction mixture was stirred overnight at 40°C. The excess solvent was concentrated, and the residue was repeatedly crystallized from (siRNA / Et2O=1:5) to obtain 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, CDCl3): 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).

[0189] Example 26 was prepared by a method similar to that described in Example 16, where the intermediates were prepared in a similar manner (intermediate 1 of the product shown in Example 16 using isopropanoyl chloride and compound 5 (of International Patent Application No. PCT / US2017 / 03429) were generated), and according to the general PEGylation procedure B, PEG 5K ONH3 + .MsO - .

[0190] Example 32: 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-tetraazadecyl)-4-(4-(isobutyryloxy)-3-((4-(PEG 5K -imino)methyl)benzyloxy)benzyl)morpholin-4-ium methanesulfonate (8) [Chemical formula]

[0191] 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-tetraazadecyl)-4-(3-(4-formylbenzyloxy)-4-(isobutyryloxy)benzyl)morpholin-4-ium methanesulfonate (7) To a solution of compound 5 (310.4 mg, 0.80 mmol) in MeCN (2 mL), (S)-4-methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropane-2-yl)-2-((S)-2-(2-morpholinoacetamide)-4-phenylbutanamide)pentanamide (286 mg, 0.30 mmol) was added. The reaction mixture was stirred at 45°C for 48 hours. The excess solvent was evaporated, and the residue was repeatedly crystallized from MeCN / Et2O (1 / 5, v / v) to obtain 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.

[0192] Compound 6 (280 mg, 0.25 mmol), 2-amino-5-methoxybenzoic acid (14.0 mg, 0.026 mmol), and PEG-O-NH2 (mesylate salt, 1.16 g, 0.227 mmol) in DCM (3 mL) were stirred at room temperature for 2 hours. 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 Et2O was added to induce crystallization. The mixture was kept in an ice bath for 10 minutes, and the formed solid was collected by filtration. Crystallization from i-PrOH / Et2O (5:2) was repeated twice until all of compound 7 was removed to obtain compound 8 (1.0 g, 72% yield).

[0193] Example 34: 4-(4-acetoxy-3-((1-PEG 3K -1H-1,2,3-triazole-4-yl)methoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxiran-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholine-4-ium chloride [ka]

[0194] Example 34 was prepared using a method similar to that taught in Examples 5-11 and Method A of International Patent Application No. PCT / US2017 / 03429, except that a chloride salt intermediate having a chloride anion as a counterion was used.

[0195] Example 35: 4-(4-acetoxy-3-((1-PEG 3K -1H-1,2,3-triazole-4-yl)methoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxiran-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholine-4-ium mesylate [ka]

[0196] Example 35 is PEG 3K N3 was used to prepare the material using a method similar to that taught in Examples 5-11 and Method A of International Patent Application No. PCT / US2017 / 03429. 1 H 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, 13 H), 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)

[0197] The present invention provides pharmaceutical compositions comprising a PEGylated carfilzomib compound of formula I or II and a number of various excipients and buffers to be selected. The present invention provides stable, isotonic, lyophilized and freeze-dried formulations as described herein. These pharmaceutical compositions are useful for delivering PEGylated carfilzomib compounds that are biologically active 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 by parenteral routes, including intravenous and subcutaneous administration, to patients in need of treatment. The compositions are sufficiently stable as liquids for use in both clinical and commercial cancer settings.

[0198] The letters and numbers used in the formulations described herein are defined as follows: "A" refers to the acetate buffer system at the stated concentration; "G" refers to the glutamate buffer system at the indicated concentration; "H" stands for histidine; "M" stands for mannitol; "T" refers to tris-HCl; "Na" refers to sodium chloride; "Pro" refers to proline; "Gly" refers to glycine; The numbers 5, 6, 7, and 8 represent the pH of the formulation; "Su" stands for sucrose, followed by a number indicating the percentage of sucrose contained in the preparation.

[0199] Therefore, the formulation referred to herein as "A5Su" means a formulation containing 10 mM acetate, 9.0% sucrose, and having a pH of 5.0, in addition to the amount of active carfilzomib present in the PEGylated carfilzomib API compound in mg or mg / mL. The solutions prepared and tested herein were prepared by adding half the desired amount of water to a batch container, followed by measuring the amount of each component (i.e., acetate, sucrose, and excipients such as PS80) to reach 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, depending on the desired / described pH. The remaining volume of water required to achieve the final concentration was added to the batch container, and the final pH and temperature of the solution were measured. The solution was aseptically filtered through a 0.22 micron cellulose acetate filter and placed in a container of appropriate size. The starting reagents were commercially available and purchased from Sigma-Aldrich.

[0200] Some of the tests performed herein involve measuring the osmotic pressure of representative formulations or solutions. Freezing point depression osmotic pressure measurements were collected using an Advanced Instruments 3250 single-sample osmometer. All samples were sterile filtered through a 0.2-micron PES filter before measurement to ensure the absence of particulate matter. For each sample point, the average of three measurements was performed. Instrument operation was validated with 100, 200, and 290 mOsm standards before collecting sample data.

[0201] Carfilzomib (CFZ), marketed under the trade name Kyprolis®, is a proteasome inhibitor necessary for the treatment of relapsed and refractory multiple myeloma. The current route of administration for Kyprolis is intravenous (IV). From a patient convenience standpoint, a subcutaneous formulation would be highly desirable, converting the 30-90 minute intravenous administration to a subcutaneous injection of 5 minutes or less. One significant challenge in manufacturing a subcutaneous formulation is the solubility of carfilzomib. The currently approved formulation is a dry, lyophilized formulation, which, when reconstituted with the appropriate amount of sterile water as indicated on the approved label, yields a clear, administerable liquid solution with a concentration of approximately 2 mg / mL of carfilzomib in Captisol®. To increase the water solubility of carfilzomib, PEGylated carfilzomib compounds have been discovered and are manufactured by attaching PEGs of various lengths. The PEG group is attached to carfilzomib by a covalent linker designed to cleave when the PEG-CFZ construct is parenterally administered to the human body.

[0202] In the experiments described herein, the following PEGylated carfilzomib compounds were used to demonstrate the stability, lifespan, and clarity of the formulations of the present invention: Example 39 (OP-0059381) is carfilzomib conjugated with 20 kPEG; Example 26 (OP-0214575) has carfilzomib conjugated with 5 kPEG; and Example 34 (OP-0214576-1) has carfilzomib conjugated with 3 kPEG.

[0203] To identify stable formulations of PEG-CFZ, various formulations were prepared by dissolving the desired PEGylated carfilzomib compound (API) in experimental compositions of excipients, monitoring the stability and quantity of intact, undegraded API, and measuring them under frozen conditions (below -20°C). API formulations were also prepared and tested to determine the ability of each exemplary formulation to provide adequate stability in liquid state for clinical and commercial administration. These formulations were then stored at room temperature for at least 8 hours and at temperatures ranging from 2 to 8°C for 2 days. All formulations tested contained the desired API compound at a concentration of 1 mg / mL.

[0204] For each formulation, the stability of the API was tested using three observable and / or measurable properties as follows: 1. Molecular integrity assessed by reverse-phase assay; 2. Visual evaluation of the transparency of the solution; and 3. The concentration of the API material in the solution, as evaluated by the material recovery rate after centrifugation.

[0205] result API integrity: Since hydrolysis yields inert degradation by-products and impurities, the primary research criterion focused on reducing or minimizing the hydrolysis of the carfilzomib epoxide ring. Various exemplary formulations were prepared by dissolving the API (PEGylated carfilzomib compound Example 26) in the solutions listed in Table 2.

[0206] [Table 7]

[0207] The tested pH range was 5–8 in appropriate buffer. The excipient sucrose was kept constant at pH 5–8, and the effect of pH changes from slightly acidic to slightly basic at a 9% isotonic level was tested more directly.

[0208] Another aspect of this test was to examine the effect of 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 (polyol), sodium chloride (salt), proline (representative amino acid), glycine (representative amino acid), and combinations of sodium chloride and sucrose.

[0209] Figure 1 shows the results obtained after incubating each formulation of exemplary PEGylated carfilzomib compound 26 (5kPEG-CFZ construct) at 25°C for 3 days. The graph shows two outputs for each formulation in this test: (a) the percentage of the main peak at time 0 (T0) and time 3 days (T3) at 25°C, measured from left to right along the y-axis, and (b) the percentage of API material recovered at T3. The main peak in the reversed-phase assay represents the intact API, but chemical modifications of the compound are shown as pre-main peak or post-main peak, thus reducing the percentage of the main peak. From Figure 1, it can be seen that the highest levels of intact API peaks after 3 days at 25°C were A5Su, T7NaSu, and T7Na. Formulations that did not function well contained H6Su and T8Su. Material recovery rates were also high for A5Su, T7NaSu, and T7Na.

[0210] Visual inspection was performed on all formulations shown in Figure 1 after 3 days at 25°C, and the results are provided in Table 3 below.

[0211] [Table 8]

[0212] Turbidity means the solution is cloudy and indicates that the carfilzomib API has not completely dissolved. Turbidity indicates that more time is needed for the API to completely dissolve. However, turbidity can also indicate that the solubility limit has been reached. Turbidity is generally undesirable. Only the A5Su formulation appeared visually clear after 3 days at 25°C. All other formulations appeared turbid, indicating the formation of several impurities that did not completely dissolve in the formulation. Such impurities would be undesirable and, under their specific storage conditions, indicate the degradation of the API or other excipients in the composition after that period and the effectiveness of the API.

[0213] Figure 2 shows the results of similar measurements performed with a similar formulation prepared with exemplary PEGylated carfilzomib Compound Number 39 instead of Example 26. The recovery of the API material was measured in the same reverse phase assay after storage at 25°C for 3 days and is shown as T o and T3, again in red and in percent main API peak. Solutions for temperature and time storage conditions were generally prepared as follows - the CFZ API was dissolved in the formulation buffer by stirring at room temperature for 5 minutes until completely dissolved. The sample was then sterile filtered using a 0.2 micron PES filter into 3 cc vials using 1 mL fills under aseptic conditions. During the stability testing period, the samples were capped and shrink wrapped and placed in the desired or specified storage conditions such as 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.

[0214] The recovery percentage of exemplary PEGylated carfilzomib compounds was measured as a function of the reversed-phase assay of the peak representing the active molecule / compound. As described below, the percentage was determined based on the area of ​​the r curve. The reversed-phase assay was performed as follows: CFZ samples were analyzed by reversed-phase HPLC using a Phenomenex Gemini C18, 50 × 4.6 mm, 3 micron particle size column for 47 minutes. During sample analysis, the column was maintained at 28C and the autosampler at 5C. For HPLC injection, the CFZ samples were diluted to 0.4 mg / mL in mobile phase A. The samples were eluted over 38 minutes using a gradient method from 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). Following the gradient, a 5-minute washing step with 100% B was performed. A 5-minute re-equilibrium step with 100% mobile phase A ensured the column returned to its initial sample-loaded state. CFZ was eluted in approximately 20–25 minutes. The main peak percentage was determined using the area under the curve integral. A standard curve was created using the CFZ standard (1 mg / mL CFZ in ACN diluted to 0.4 mg / mL CFZ in mobile phase A) to calculate the sample recovery %. The total integrated area of ​​each point on the curve was plotted against the injection load and aligned with a trend line. Each unknown sample concentration was calculated using the equation of the trend line generated from this calibration curve by connecting them in the total integrated area.

[0215] The results in Figure 2 show that the formulations that appeared stable after 3 days at 25°C, i.e., those with the lowest loss of the main API peak, were A5Su, T8Su, T7NaSu, T7Na, and T7Pro. However, the material recovery profile (red points) was highest for formulation A5Su.

[0216] The results in Figures 1 and 2 show that the A5Su formulation exhibits the highest stability among the 5kPEG-CFZ and 20kPEG-CFZ compounds prepared and tested, respectively.

[0217] The improved stability may be at least partially due to the decrease in pH. To confirm whether pH has this effect, a second test was conducted comparing formulations at pH 3 and pH 4 with the A5Su formulation at pH 5. The test design is shown in Table 4 below.

[0218] [Table 9]

[0219] These three compositions were then tested using three representative PEG-CFZ constructs: 3kPEG-CFZ (Example 34), 5kPEG-CFZ (Example 26), and 20kPEG-CFZ (Example 39). The main API peak loss for Example 34 (shown as compound 576 in Table 5) is shown in Table 5.

[0220] [Table 10]

[0221] As shown in Table 5, no loss was detected (as a percentage; within experimental variability) for all three formulations after 8 hours at 25°C. This indicates that these exemplary liquid formulations of the PEGylated carfilzomib compound are viable for clinical or possibly commercial administration to patients after 8 hours of storage at room temperature. Regarding the storage of the exemplary formulations of Example 39 after incubation for 2 weeks at 4°C and -70°C, both representative compositions showed the lowest loss at the main API peak of the A5Su formulation. Furthermore, all three representative formulations of 3kPEG-CFZ (Example 34) in Table 5 appeared clear, and no visible particles or particulate matter were detected.

[0222] [Table 11]

[0223] Table 6 shows the percentage loss of the main API peak for a representative 5kPEG-CFZ construct (Compound Example 26) for specified formulations at pH 3, 4, and 5 at 25°C, 4°C, and -70°C. Similar to what was observed in Table 5 above for the 3kPEG-CF construct (Example 34), the minimum loss of the main API peak was observed under each of the storage conditions of 25°C (8 hours), 4°C (1 week), and -70°C (2 weeks).

[0224] [Table 12]

[0225] Table 7 shows the main API peak loss rates for the 20kPEG-CFZ construct (compound example 39 as it is specified) for formulations at pH 3, 4, and 5 at 25°C, 4°C, and -70°C, respectively. Similar to what was observed for the 3kPEG-CFZ construct, there was minimal main API peak loss for representative A5Su compositions at 25°C (8 hours), 4°C (1 week), or -70°C (2 weeks). Of note in these results is that, on an absolute scale, the 20kPEG-CFZ compound was observed to be less stable in A5Su formulations than the 3kPEG-CFZ and 5kPEG-CFZ constructs in the same A5Su formulation composition. More noteworthy is that the 20K PEGylated carfilzomib compound was generally less stable over time with respect to storage conditions than the corresponding 3K and 5K PEGylated carfilzomib compounds in the same composition composition.

[0226] Table 8 shows the results of stability studies in A5Su, performed at a concentration of 20 mg / mL for each of the representative PEGylated carfilzomib compounds: 3kPEG-CFZ, 5kPEG-CFZ, and 20kPEG-CFZ. This study indicates that higher API concentrations in a given formulation resulted in greater stability of the construct under the tested conditions. It was also found that at lower concentrations, carfilzomib APIs with larger PEG sizes (20kPEG-CFZ constructs) were more unstable in A5Su compared to those of the representative compounds in 3kPEG-CFZ and 5kPEG-CFZ.

[0227] [Table 13]

[0228] Each of the three PEG-CFZ constructs was prepared as an A5Su formulation for animal administration. The three formulations were evaluated visually. Visual observation revealed no particle formation in any of the three prepared samples. Endotoxin testing showed that all three samples contained endotoxin at concentrations less than 1.0 EU / mL. Osmotic pressure measurements for all three formulations ranged from 299 to 307 mOsm. Reverse-phase chromatography was used to monitor the main API peak of all constructs before and after administration. No significant loss of the main API peak was detected for any of the constructs in any of the administration tests. No significant loss of the area under the curve was detected between pre- and post-administration of the samples. This indicates that the active biological compounds in these formulations possess the potency in their intended biological activity.

[0229] A stable, isotonic, lyophilized formulation for intravenous and subcutaneous administration. The present invention also provides stable, isotonic, dry, lyophilized, and pharmaceutically acceptable compositions of PEGylated carfilzomib compounds. A typical and exemplary stable, dry, lyophilized formulation includes, but is not limited to, G5Su2M4, which is a composition of the API in 10 mM glutamate (buffer), 2.0% sucrose, 4% mannitol, 0.006% polysorbate 80, and pH 5.0. Furthermore, the formulations of the present invention may further contain hyaluronidase. Hyaluronidase is thought to assist in the subcutaneous delivery of the API and potentially reduce, decrease, or prevent aggregation of the API and / or formulation excipients at the injection site. Hyaluronidase is also thought to reduce the degree of local skin irritation at the injection site.

[0230] To identify stable lyophilized formulations of representative 3K-PEGylated carfilzomib compounds (Example 28 in Table 2), the compound API was dissolved in representative lyophilization-compatible solutions, and the stability of each representative solution was compared. Comparisons were made between formulations immediately after dissolution and after completion of the lyophilization cycle. In addition, after reconstitution of the lyophilized batches (lyophilized products), the safety of each reconstituted liquid formulation was tested to determine safe, acceptable, or permitted handling times before drug administration, whether clinical or commercial. Representative reconstituted formulations were found to be safe for administration after at least 8 hours at room temperature and 2 days at 2–8°C. All initial screening of constructs was performed at API concentrations of 5–40 mg / mL.

[0231] For each exemplary formulation, the stability of the API was tested or measured using the following six different criteria or methods: 1. Molecular integrity assessed by reverse-phase assay; 2. Transparency of the solution as measured by visual inspection; 3. The concentration of the API material in the solution, as evaluated by the recovery rate of the API material after centrifugation; 4. pH of the formulation; 5. Osmotic pressure of exemplary solutions; and 6. The number of particles invisible to the naked eye due to light shielding.

[0232] The freeze-drying cycle parameters used in the experimental preparations, namely temperature, gradient, and holding time, are listed and provided in Table 9 below. The holding step means that the sample remains unchanged at a given temperature and pressure throughout the process. The gradient step means that the sample is gradually increased or decreased until it reaches the specified temperature.

[0233] [Table 14]

[0234] The freeze-drying process used involved a reduced pressure of 150 mTorr.

[0235] result Initial lyophilization tests were performed with the exemplary formulation of API compound Example 28 (3KPEG-CFZ) as specified herein. The formulation consisted of 10 mM glutamate with the desired mg amount or mg / mL concentration of API, 2.0% sucrose, 4% mannitol, and 0.006% polysorbate 80, and had a solution pH of 5.0. The following test formulations had various solution concentrations of API Example 28 at 5, 20, and 40 mg / mL. Furthermore, formulations of 10 mM histidine, pH 5.0, 2.0% sucrose, 4% mannitol, and 0.006% polysorbate 80 were tested with an exemplary PEGylated carfilzomib compound Example 8 (3K-PEG-CFZ) at 20 mg / mL.

[0236] Figure 3-A shows an image of the dried solid lyophilized cake obtained from the formulations prepared above. As shown, the lyophilized cake of Compound Example 28 was compounded into a G5Su2M4 solution (10 mM glutamate, pH 5.0, 2.0% sucrose, 4% mannitol, 0.006% polysorbate 80) at three different concentrations: 40 mg / mL; 20 mg / mL and 5 mg / mL. Each prepared composition solution was frozen and lyophilized using a standard method. Figure 3-A also shows the dried solid lyophilized cake obtained from a formulation solution of Compound Example 28 at a concentration of 40 mg / mL (H5Su2M4 formulation: 10 mM histidine, pH 5.0, 2.0% sucrose, 4% mannitol, 0.006% polysorbate 80).

[0237] Figure 3-B shows a bar graph of the main peak % for compound example 28 in a reverse-phase intact API measurement assay. In Figure 3-B, black bars represent assay measurements before lyophilization, and red bars represent API peak measurements after lyophilization. Green bars represent measurements taken after reconstituting the lyophilized cake to form a clear solution and storing it at 25°C for 8 hours. Finally, yellow bars represent assay measurements taken after reconstituting the corresponding lyophilized cake to form a solution and storing it at 2–8°C for 24 hours. Figure 3-B highlights that there was no loss of the main API peak during lyophilization and no loss of the main API peak after incubation of the reconstituted solution. The concentration of the material in the solution was obtained from the reverse-phase region below the curve and did not change during lyophilization and subsequent solution incubation.

[0238] Reconstitution of these dried solid lyophilized cakes with sterile water yielded clear, particulate-free solutions. Reconstitution times were all less than 1 minute, except for the 40 mg / mL formulation. The 40 mg / mL cake formed a clear solution only after a slightly longer time, i.e., less than 2 minutes, upon reconstitution. Osmotic pressure measurements for all formulations ranged from 300 to 307 mOsm. pH readings were all in the range of 5.0 to 5.1. As described, the percentage of intact residual API reversed-phase compounds was analyzed before and immediately after the completion of the lyophilization cycle. In addition, the temperatures and times—8 hours at 25°C and 24 hours at 2–8°C—for which the reconstituted solutions were left to stand were chosen to mimic the actual, globally representative clinical and commercial environments for the administration and administration of FDA-approved carfilzomib.

[0239] The present invention further provides pharmaceutical compositions that can be administered subcutaneously. Hyaluronidase has been used in the pharmaceutical industry with certain drug products as an additive to formulations aimed at converting intravenous formulations into subcutaneous formulations. However, hyaluronidase has not been successful in converting all drugs tested to subcutaneous administration. It is unpredictable whether the addition of hyaluronidase will satisfy the subcutaneous administration requirements of any given drug product, not to mention the case of PEGylated carfilzomib compounds. The present invention intends to provide compositions and formulations further comprising hyaluronidase as described herein. For this purpose, a test using Compound Example 28 co-formulated with 2000 units / mL of hyaluronidase (shown as PH20 and commercially available from various suppliers) is described below, with reference to Figures 4-A and 4-B. A formulation solution was 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 (first vial) also contained 2000 units / mL of hyaluronidase. As described above herein, it was lyophilized, and the resulting dried solid lyophilized cake (see Figure 4-A) was analyzed (see Figure 4-B) to determine the percentage of the remaining main API peak. The first vial in Figure 4-A is an image of the lyophilized cake obtained from the G5Su2M4 formulation described above. The second vial shown in Figure 4-A is a placebo lyophilized cake obtained from a solution formulation containing only hyaluronidase. This vial was prepared as a control and simply lyophilized. As described above, referring to Figure 3-B, the percentage main API peaks of both vials were measured by reverse-phase assay. As shown in Figure 4-B, the black bars represent the main API peaks of the formulation solution before lyophilization, and the red bars represent the main API peaks of the lyophilized cake after reconstitution with sterile water. The green bars represent the main API peak readings after each vial was reconstituted to form a solution and stored at 25°C for 8 hours, and after storage at 2-8°C for 24 hours (yellow bars).

[0240] Reconstitution of both cakes (the first and second vials in Figure 4-A) with sterile water yielded clear, particle-free solutions. The dissolution time for both vials was less than 1 minute. Osmotic measurements for both reconstituted formulations ranged from 300 to 302 mOsm. pH readings for both reconstituted formulations were found to be in the range of 5.0 to 5.1. Percent main peaks for PEGylated carfilzomib compound Example 28 were measured by reverse-phase assay both before and immediately after the completion of the lyophilization cycle. In addition, both reconstituted solutions were stored at 25°C for 8 hours and at 2–8°C for 24 hours to measure the stability of the API over these periods under these tested conditions or storage. As shown in Figure 4-B, the reconstituted API formulations and placebo hyaluronidase formulations showed no loss of main peaks during lyophilization. More importantly, both of these formulations showed no loss of main API peaks after incubation and storage of the reconstituted solutions for the specified time in the specified storage environment. Furthermore, the concentration of compound example 28 in the solution remained unchanged during lyophilization and subsequent storage of the reconstituted solution, as measured by the reversed-phase region below the curve. This supports and suggests that, under test and similar conditions, the formulations of the present invention provide a suitable, stable, intact solution that can be stored for extended periods and confidently and safely administered to patients in research, clinical, or commercial settings. The formulations of the present invention reduce the degree of degradation of the PEGylated carfilzomib compound API, provide a formulation that can be conveniently stored without forming impurities, and result in a solution containing a significant portion, or even more, of the active pharmaceutical ingredient as the drug product would be if it were originally manufactured, while keeping particulate matter and impurities relatively clear.

[0241] Hyaluronidase was supplied from Calbiome (sheep testes, 38594-100KU, Calbiome) and buffered with the above formulation before adding enough 3K PEG-CFZ API to achieve a concentration of 20 mg / mL.

[0242] The stability and safety of the formulations of the present invention were also tested and measured by the number and particle size of particles in the reconstituted solution as a percentage of the solution volume. Figures 5-A and 5-B show the results for such particle counts, where particle size is either >10 μm or >25 μm in diameter. The solutions used in the tests contained compound example 28 PEGylated as an API at a concentration of 20 mg / mL in a formulation solution essentially consisting of a composition of G5Su2M4 and 0.006% polysorbate 80. The various conditions under which particulate matter formation was measured in each test formulation are shown in Table 10 below. Each test formulation listed in Table 10 below was prepared according to the formulation method and lyophilized in a 3 mL vial with a filling volume of 1 mL. These were then screened after lyophilization for particle formation content as a measure of stability.

[0243] [Table 15]

[0244] Each formulation vial was tested for particulate count before and after lyophilization using a light-shielding methodology that prevented detection by the naked eye. This methodology included the use of a liquid particle counting system (HIAC / Royco 9703 or 9703+). For lyophilized samples, reconstruction was performed before measurement, and the samples were equilibrated for 2 hours before analysis. Before measurement, all samples were degassed for 1 hour using a vacuum chamber. After degassing, water controls and particle standards were measured instrumentally. Before measuring the standard particle controls and samples, the water control sample measurement readings had to be zero. In summary, samples were slowly rotated by hand to avoid generating bubbles, and then measured instrumentally using 0.2 mL sip per measurement. A total of four sips were performed per sample, and the last three sips were averaged. None of the tested samples were diluted.

[0245] As shown in Figures 5-A and 5-B, a significant increase in particle count was observed in formulations 1-3. The addition of the surfactants polysorbate 80 and Pluronic F68 only slightly reduced or decreased the tendency for particulate matter to form in the formulation (10 mM glutamate + 2% sucrose + 4% mannitol, pH 5.0). The addition of small amounts of amino acids such as lysine or arginine in combination with surfactants such as polysorbate 80 resulted in a reduction in particle count in both formulations tested, with sizes exceeding 10 μm and 25 microns.

[0246] To summarize, representative formulations manufactured and tested by freeze-drying are shown in Table 11 below.

[0247] [Table 16]

[0248] Administration of the pharmaceutical composition of the present invention The pharmaceutical compositions of the present invention may be administered parenterally. For example, compositions administered parenterally may be formulated as injections (intravenous, intramuscular, or subcutaneous), intravenous infusions, or suppositories. These formulations can be prepared by conventional means in conjunction with the methods described herein, and, if desired, the active ingredient may be mixed with any conventional additives or excipients, such as binders, disintegrants, lubricants, correctors, solubilizers, suspension aids, emulsifiers, or coatings. Suitable parenterally administered compositions for infusion, injection, or subcutaneous administration generally include sterile aqueous solutions (if water-soluble) or dispersions, and / or sterile powders for the immediate preparation of sterile solutions or dispersions for either infusion or injection. For intravenous administration, suitable carriers include sterile water for injection and sterile buffer solutions, as described above. In all cases, compositions particularly for human use, treatment, and ingestion must be sterile and fluid enough to be easily added to or drawn from syringes or infusion bags. The composition must be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coating materials such as lecithin, maintaining the particle size required in the case of dispersions, and the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Accordingly, in some embodiments, the present invention provides compositions that may contain antimicrobial or antifungal agents. In some embodiments of the present invention, the compositions provided herein may contain isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride, as exemplified and tested herein above. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0249] Sterile injectable solutions can be prepared by incorporating the active PEG carfilzomib compound, along with one or a combination of the above components as needed, in the required amount in a suitable solvent, followed by filtration sterilization. Generally, dispersion systems are prepared by incorporating carfilzomib into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders, such as lyophilized cakes, prepared for the preparation of sterile injectable solutions, a preferred preparation method is freeze-drying, which yields a powder form of the active PEGylated carfilzomib compound plus any further desired components from their pre-sterilized filtered solutions.

[0250] The dosage and precise administration time of the PEGylated carfilzomib compound used in the pharmaceutical compositions of the present invention described herein depend on the type of cancer being treated, the patient's age, condition, and body weight. The composition that yields the most effective results in terms of therapeutic efficacy in a given patient will also depend on the activity, pharmacokinetics, and bioavailability of the particular PEGylated carfilzomib compound, the patient's physiological condition as described above (age, sex, type and stage of disease, overall health, response to a given dosage and type of drug), the route of administration, etc. The dosage will vary depending on the symptoms and severity of the disorder being treated or prevented, but the route of administration and drug form are generally such that a daily dose of 0.01 to 2000 mg of the compound is recommended for adult human patients, which may be administered as a single dose or in divided doses. Further information on dosages for the compounds of the present invention is provided below herein. Generally, compositions intended for parenteral use (e.g., intravenous, subcutaneous injection) include a solubilizer. The solubilizer may be a substituted cyclodextrin.

[0251] The actual dosage of the PEG-carfilzomib compound used in the pharmaceutical compositions provided by the present invention may be an amount that has been clinically proven and / or commercially approved as effective in achieving a desired therapeutic response for cancer patients, including but not limited to patients with multiple myeloma. In some embodiments, the present invention provides pharmaceutical compositions as aqueous solutions containing about 0.1 to 20% w / v of the compounds disclosed herein, in particular substances for parenteral administration. A typical dose range for the PEG-carfilzomib compound is about 0.01 to about 50 mg / kg body weight per day, given in 1 to 4 divided doses each day. Each divided dose contains one or more of the compounds provided by the present invention. The desired, specific compound dosage must be sufficient to provide a therapeutically effective dosage of free active carfilzomib in the patient's plasma and, for regulatory approval indicators, is the effective dosage based on regulatory approval use. This effective dosage varies from patient to patient and generally depends on a variety of factors, including the overall patient assessment, as well as the specific formulation composition and the route of administration of the selected compound. In some embodiments, the PEG-carfilzomib compounds that may be used in the present invention are described in U.S. Patent No. 9,309,283.

[0252] Carfilzomib is currently administered in 28-day cycles, once daily for the first two consecutive days of each week, followed by three consecutive weeks, at a dose of 20 mg / m². 2 ~56 mg / m² 2 It is approved in amounts sufficient to provide patient plasma concentrations within the approved range. Therefore, the high molecular weight PEG carfilzomib compound of the present invention must be administered in amounts sufficient to provide pharmacokinetically an amount approximately equivalent to the approved dosage range. For example, the 2K PEG compound of the present invention is approximately 24% by weight of free carfilzomib. Therefore, using an average male with an average body surface area of ​​1.9 m2, approximately 27 mg / m2 2 To achieve an equivalent dose, approximately 215 mg of the 2k PEG CFZ compound must be administered. Similarly, the currently approved formulation of carfilzomib is 70 mg / m². 2To deliver the same amount of carfilzomib as the dose, approximately 1100 mg of the 20K PEG CFZ compound may be administered.

[0253] The pharmaceutical compositions of the present invention include various excipients as described herein. For example, at least one excipient may include sugar additives such as sucrose, sorbital, glycerin, maltose, lactose, erythrose, dextrose, lactobiose, or cyclodextrin, or amino acids such as proline, glycine, arginine, histidine, aspartic acid, glutamic acid, or glutamate, or neutral, hydrophobic amino acids such as valine, leucine, alanine, or methionine. The excipient may also 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-HCl, lysine-HCl, magnesium chloride, and barium sulfate. When the excipient is a sugar, it is generally present in an amount ranging from approximately 0.1% to 30% of the total weight of the composition or in a weight percentage per volume of the solution formulation; or when the excipient is an amino acid, it is generally present in an amount ranging from approximately 0.1% to 10% of the total weight of the composition.

[0254] The pharmaceutical compositions of the present invention may also further contain other pharmaceutically acceptable excipients. With respect to excipients, carriers and / or diluents in the compositions of the present invention, the term “pharmaceutically acceptable” as used herein means ligands, materials, compositions and / or dosage forms that, within the bounds of sound medical judgment, are suitable for use in contact with human and animal tissues, without excessive toxicity, irritation, allergic reactions or other problems or complications, and are balanced by 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 that it is compatible with the other components of the formulation and is not harmful to the patient.

[0255] Further excipients include, but are not limited to, the use of surfactants such as PS20, PS80, PL F68 (and other Pluronic F and L series) triblock surfactant polymers, sodium doxert, benzalkonium chloride, Triton X100, and tetrafunctional block O polymers. Surfactants that may be included are generally those with lower surface tension (such as alcohols), SDS, protamine sulfate, or butane. If surfactants are included in the compositions of the present invention, they must be present in an amount ranging from 0.005% to 3% by weight.

[0256] The dried freeze-dried compositions provided by the present invention may contain sugars, such as sucrose, sorbitol, glycerin, maltose, lactose, erythrose, dextrose, lactobiose, cyclodextrin, sugar derivatives, and adducts. The sugars or sugar derivatives are generally included in an amount ranging from about 0.1 to 30% by weight. If the excipient can be an amino acid, it can be any suitable amino acid, including prolinglysine, lysine, arginine, histidine, aspartic acid, valine, leucine, alanine, methionine, proline, glutamic acid, and glutamate. The amino acid should generally be present in an amount ranging from about 0.1 to 10% by weight. The composition of the present invention may further contain a salt. The salt may function as a buffer or provide other advantages to the composition. The salt may be, for example, an amino acid salt, but is not limited to NaCl, KCl, ammonium sulfate, potassium chlorate, calcium Cl, ZnCl, guanidine hydrochloride, ammonium chloride, potassium sulfate, ammonium aspartate, arginine-HCl, and lysine-HCl, magnesium chloride, and barium sulfate. If a salt is present, it is generally present in an amount ranging from about 30 to 300 mM by volume of the solution composition.

[0257] Typically, further examples of substances that can serve as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch, potato starch, and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, e.g., cocoa butter and suppository waxes; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, etc. Examples include (10) leaf oil, corn oil and soybean oil, (11) glycols, such as propylene glycol, (12) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol, (13) esters, such as ethyl oleate and ethyl laurate, (14) agar, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffer solution, and (21) other non-toxic and suitable substances used in pharmaceutical formulations. In certain embodiments, the pharmaceutical compositions provided herein are non-pyrogenic, i.e., they do not induce a significant increase in body temperature when administered to a patient.

[0258] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retarders, etc., that are compatible with the administration of the drug. In some embodiments, the pharmaceutically acceptable carrier is an acid-base buffer system, such as a citrate buffer, to maintain a stable pH for 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.

[0259] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial activity can be ensured by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include tonic modifiers such as sugars in the composition. In addition, sustained absorption of injectable pharmaceutical forms can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin. In some cases, it is desirable to slowly absorb the drug from subcutaneous or intramuscular injection to prolong the effect of the drug. For example, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0260] The terms "parenteral administration" and "administered parenterally," as used herein, usually mean methods of administration other than enteral and topical administration by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, subarachnoid, intracapsular, intraorbital, intracardiac, intracutaneous, intraperitoneal, percutaneous, subtracheal, subcutaneous, intra-articular, subcapsular, subarachnoid, intrathecal, and intrastemmal injections and infusions.

[0261] The pharmaceutical compositions of the present invention described herein may be administered to humans and other animals, including mammals, for therapeutic purposes via any preferred route of administration.

[0262] How to use The biological effects of proteasome inhibition are useful and desirable. Proteasome inhibition has been proposed as a preventive and / or therapeutic agent for numerous diseases, including but not limited to proliferative disorders, neurotoxic / degenerative diseases, Alzheimer's disease, ischemic conditions, inflammation, autoimmune diseases, HIV, cancer, organ graft rejection, septic shock, antigen presentation inhibition, decreased viral gene expression, parasitic infections, acidosis-related conditions, macular degeneration, lung diseases, muscle wasting diseases, fibrous diseases, and bone and hair growth disorders. Therefore, the pharmaceutical formulations of the present invention, containing the PEG-carfilzomib compound in a therapeutically effective dosage, provide a means of administering the drug to patients and treating these conditions.

[0263] At the cellular level, accumulation of polyubiquitinated proteins, changes in cell morphology, and apoptosis have been reported upon treatment of cells with various proteasome inhibitors. Proteasome inhibition has also been disclosed and has been clinically and commercially proven as a useful antitumor therapeutic strategy. For this purpose, the compounds of the present invention and compositions comprising these compounds are useful for treating cancers, including newly diagnosed and / or relapsed and refractory multiple myeloma, but are not limited to these.

[0264] In both in vitro and in vivo models, malignant cells have been shown to be generally susceptible to proteasome inhibition. In fact, proteasome inhibition has already been demonstrated as a therapeutic strategy for the treatment of multiple myeloma. This may be partly due to the reliance of more proliferative malignant cells on the proteasome system for rapid protein removal (Rolfe et al., J.Mol.Med. (1997) 75:5-17; Adams, Nature (2004) 4:349-360). This specification provides a method for treating cancer, comprising administering to a patient in need of such treatment a therapeutically effective dose of a PEGylated carfilzomib compound of formulas I and II provided or described herein, or any specifically exemplified PEG-carfilzomib compound.

[0265] As used herein, the term “cancer” includes, but is not limited to, blood-derived cancers and solid tumors. Cancers can affect components of the blood, bones, organs, skin tissues, and vascular system, including, but not limited to, cancers of the bladder, blood, bones, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidneys, liver, lungs, lymph nodes, mouth, neck, ovaries, pancreas, prostate, rectum, kidneys, skin, stomach, testes, throat, and uterus. Specific cancers include leukemia (acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia, mature B-cell neoplasm (small lymphocytic leukemia), B-cell prelymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenström's macroglobulinemia), perisplenic zone lymphoma, plasmacytoma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, extranodal perisplenic zone B Cellular lymphoma (MALT lymphoma), peripheral zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse B-cell lymphoma, large mediastinal (thymic) B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma and Burkitt lymphoma / leukemia), mature T-cell and natural killer (NK) cell neoplasms (pre-lymphocytic T-cell leukemia, macrogranulocytic T-cell lymphocytic leukemia, progressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK-cell leukemia) T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides (Sézary syndrome), primary cutaneous regressive large cell lymphoma, lymphomatoid papular disease, angioimmunoblastic T-cell lymphoma, unspecified peripheral T-cell lymphoma and regressive large cell lymphoma), Hodgkin lymphoma (tubular sclerosis, mixed cell solid, lymphocyte-rich, lymphocyte-depleted or non-depleted type, nodular lymphocyte-dominant type), non-Hodgkin lymphoma, myeloma (multiple myeloma, painless bone marrow Myeloma, smoldering myeloma), chronic myeloproliferative disorders, myelodysplastic / myeloproliferative disorders, myelodysplastic syndromes, immunodeficiency-associated lymphoproliferative disorders, histiocytic and dendritic cell neoplasms, mastocytosis, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant giant cell tumor, myeloma bone disease, osteosarcoma, breast cancer (hormone-dependent, hormone-independent), gynecological cancers (cervical, endometrial, fallopian tube, gestational trophoblastic disease, ovarian, abdominal, uterine, vaginal and vulvar), basal cell carcinoma (BCC), squamous cell carcinoma (SCC),Malignant melanoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, Kaposi's sarcoma, astrocytoma, pilocytic astrocytoma, embryonic teratogenic neuroepidermal tumor, oligodendroglioma, ependymal cell tumor, pleomorphic gliablastoma, mixed glioma, oligoastrocytoma, medulloblastoma, retinoblastoma, neuroblastoma, germ cell tumor, teratoma, malignant mesothelioma (intraperitoneal mesothelioma, pericardial mesothelioma, pleural mesothelioma), neuroendocrine tumors of the gastrointestinal tract-pancreas or gastrointestinal pancreas (GEP-NET), carcinoid tumors, pancreatic endocrine tumors (PET), colorectal adenocarcinoma, colorectal carcinoma, progressive neuroendocrine tumors, leiomyosarcoma, glial adenocarcinoma, signet ring cell This includes, but is not limited to, adenocarcinoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, hemangioma, hepatic adenoma, focal nodular hyperplasia (nodular regenerative hyperplasia, hamartoma), non-small cell lung cancer (NSCLC) (squamous cell lung cancer, adenocarcinoma, large cell lung cancer), small cell lung cancer, thyroid cancer, prostate cancer (hormone-refractory, androgen-independent, androgen-dependent, hormone-insensitive), and soft tissue sarcomas (fibrosarcoma, malignant fibrous histiocytoma, dermatofibrosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, hemangioendothelioma, synovial sarcoma, malignant peripheral nerve sheath tumor / neurofibrosarcoma, skeletal exosarcoma).

[0266] In one embodiment, the present invention provides a pharmaceutically acceptable composition comprising a PEGylated carfilzomib compound or a pharmaceutically acceptable salt thereof, which can be administered to treat multiple myeloma in a patient. Furthermore, and in another embodiment of the present invention, multiple myeloma may include newly diagnosed or relapsed and / or refractory multiple myeloma, or both.

[0267] Figures 6 and 7 illustrate the efficacy of representative PEGylated carfilzomib compound Example 13 in a mouse xenograft model of human colon adenocarcinoma cells. Tumors in the vehicle group grew linearly during the study period. Intravenous administration of compound Example 13 (200 mpk, or 150 increasing to 250 mpk after 3 weeks) or CFZ-captisol (5 mpk) once weekly significantly inhibited tumor growth (compared to the vehicle control) within 19 days of the initial dose. In addition, intravenous administration of both formulations was associated with a significant reduction in weight gain.

[0268] Further embodiments include methods for influencing the proteasome-dependent regulation of oncoplastic proteins and methods for treating or inhibiting cancer growth, each method comprising exposing cells (in vivo, e.g., in a patient, or in vitro) to the compositions disclosed herein. HPV-16 and HPV-18-induced E6 proteins stimulate ATP and ubiquitin-dependent complexation, as well as the degradation of p53 in reticulocyte lysates. The recessive oncogene p53 has been shown to accumulate at non-permissible temperatures in cell lines with mutant thermal instability E1. Elevated levels of p53 can lead to apoptosis. Examples of proto-oncoplastic proteins degraded by the ubiquitin system include c-Mos, c-Fos, and c-Jun. One embodiment is a method for treating p53-related apoptosis, comprising administering an effective amount of the compositions disclosed herein to a patient.

[0269] Furthermore, inhibitors that bind to the 20S proteasome have been shown to stimulate osteogenicity in bone tissue culture. Moreover, when such inhibitors were systemically administered to mice, certain proteasome inhibitors increased bone volume and osteogenicity by more than 70% (Garrett, IR et al., J. Clin. Invest. (2003) 111:1771-1782), suggesting that the ubiquitin-proteasome mechanism regulates osteoblast differentiation and osteogenicity. Therefore, the disclosed compositions may be useful in the treatment and / or prevention of bone loss-related diseases such as osteoporosis.

[0270] Furthermore, the present invention provides compositions useful as diagnostic reagents (e.g., in diagnostic kits or for use in clinical laboratories) for screening proteins (e.g., enzymes, transcription factors) treated with Ntn hydrolases containing proteasomes. The disclosed compositions are also useful as research reagents for specifically binding to the X / MB1 subunit or α chain and inhibiting associated proteolytic activity. For example, the activity of other subunits of the proteasome (and their specific inhibitors) can be determined.

[0271] Embodiment 71 of the present invention provides a method for treating cancer in a subject requiring treatment, the method comprising administering an effective dose of a pharmaceutical composition containing an effective amount of the PEG-carfilzomib compound of formula I to the subject. Embodiment 72 provides the method of Embodiment 71, wherein the cancer is multiple myeloma. Embodiment 73 provides one of the methods of Embodiments 71 to 72, wherein the effective dose of PEG-carfilzomib is in the range of about 100 mg to about 2000 mg. Embodiment 74 provides one of the methods of Embodiments 71 to 73, wherein the effective dose is in the range of about 150 mg to about 1000 mg per day. Embodiment 75 provides one of the methods of Embodiments 71 to 74, wherein the effective dose of the PEG-carfilzomib compound administered is in the range of about 200 mg to about 500 mg per day. Embodiment 76 provides one of the methods from Embodiments 71 to 73, wherein the effective dose of the 2K PEG-carfilzomib compound administered is in the range of about 150 mg to about 600 mg per day. Embodiment 77 provides one of the methods from Embodiments 71 to 73, wherein the effective dose of the 3K PEG-carfilzomib compound administered is in the range of about 300 mg to about 2000 mg per day. Embodiment 78 provides one of the methods from Embodiments 71 to 73, wherein the effective dose of the 5K PEG-carfilzomib compound administered is in the range of about 800 mg to about 3000 mg per day. Embodiment 79 provides one of the methods from Embodiments 71 to 73, wherein the effective dose of the 20K PEG-carfilzomib compound administered is in the range of about 800 mg to about 3000 mg per day. Embodiment 80 provides one of the methods from Embodiments 71 to 73, wherein the effective dose of the PEG-carfilzomib compound administered is in the range of about 200 mg to about 1500 mg per day. Embodiment 81 provides one of the methods from Embodiments 71 to 73, wherein the effective dose of the PEG-carfilzomib compound administered is in the range of about 5 mg / kg to about 50 mg / kg of the subject's body weight per day.Embodiment 82 provides one of the methods of Embodiments 71 to 73, wherein the effective dose of the 2K, 3K, or 5K PEG-carfilzomib compound administered is in the range of about 200 mg to about 800 mg per day. Embodiment 83 provides one of the methods of Embodiments 71 to 73, wherein the effective dose of the 2K or 3K PEG-carfilzomib compound administered is in the range of about 200 mg to about 500 mg per day. Embodiment 84 provides one of the methods of Embodiments 71 to 73, wherein the effective dose of the 5K or 20K PEG-carfilzomib compound administered is in the range of about 400 mg to about 1000 mg per day. Embodiment 85 provides one of the methods of Embodiments 71 to 84, wherein the method further comprises the administration of a steroid. Embodiment 86 provides the method of Embodiment 85, wherein the steroid is selected from the group consisting of dexamethasone and prednisone. Embodiment 87 provides one of the methods of Embodiments 85 to 86, wherein the steroid is dexamethasone. Embodiment 88 provides one of the methods of Embodiments 85 to 86, wherein the steroid is prednisone. Embodiment 89 provides one of the methods of Embodiments 71 to 88, further comprising the administration of an immunomodulator selected from the group consisting of thalidomide, lenalidomide, and pomalidomide. Embodiment 90 provides the method of Embodiment 89, wherein the immunomodulator is lenalidomide or pomalidomide. Embodiment 91 provides one of the methods of Embodiments 89 to 90, wherein the immunomodulator is lenalidomide. Embodiment 92 provides one of the methods of Embodiments 89 to 90, wherein the immunomodulator is pomalidomide. Embodiment 93 provides one of the methods of Embodiments 71 to 88, wherein the method further comprises the administration of a CD-38 inhibitor. Embodiment 94 provides the method of Embodiment 93, wherein the CD-38 inhibitor is daratumumab. Embodiment 95 provides any one of the methods of Embodiments 71 to 94, wherein the cancer is recurrent or refractory multiple myeloma.Embodiment 96 provides a method according to any one of Embodiments 71 to 94, wherein the cancer is newly diagnosed multiple myeloma. Embodiment 97 provides a method according to Embodiment 96, wherein the cancer is newly diagnosed multiple myeloma and the patient is eligible for stem cell transplantation as determined by a licensed and accredited physician. Embodiment 98 provides a method according to Embodiment 96, wherein the cancer is newly diagnosed multiple myeloma and the patient is not eligible for stem cell transplantation as determined by a licensed and accredited physician. Embodiment 99 provides a method according to any one of Embodiments 71 to 98, wherein the method comprises administering a pharmaceutical composition comprising a PEG-carfilzomib compound of formula I to a subject. Embodiment 101 provides a method according to any one of Embodiments 99 to 100, wherein the pharmaceutical composition is a freeze-dried preparation that can be reconstituted before administration.

[0272] combination The PEG-carfilzomib compound of the present invention may be administered or given as a sole active pharmaceutical agent, or it may be used in combination with one or more agents, such as a second anticancer agent. When administered in combination, the PEG-carfilzomib active ingredient and the other agents may be formulated as separate compositions to be administered simultaneously or sequentially at different times, or both activators may be given as a single composition.

[0273] The term “co-therapy” (or “combination therapy”) used in defining the use of the PEG-carfilzomib compound and another anticancer agent in this invention is intended to utilize the sequential administration of each agent in a regimen that provides a beneficial effect of the drug combination, and also to utilize the co-administration of these agents in a substantially simultaneous manner, such as a single-dose formulation having these active agents in fixed ratios, or multiple separate dosing formulations for each active agent. Therefore, the invention is not limited to the order of administration. That is, the PEG-carfilzomib compound may be administered before, simultaneously with, or after the administration of other agents.

[0274] In certain embodiments, the PEG-carfilzomib compound described herein is administered in combination with one or more other proteasome inhibitors. Other proteasome inhibitors may include, for example, bortezomib, optrozomib, or ixazomib. In another embodiment, the PEG-carfilzomib compound described herein is administered in combination with an immunomodulatory compound comprising thalidomide, lenalidomide, and pomalidomide. In one embodiment from the preceding embodiment, PEG-carfilzomib is administered in combination with an immunomodulator selected from lenalidomide and pomalidomide. In further embodiments, the present invention provides a method for treating cancer in a subject by administering a combination therapy comprising a PEG-carfilzomib compound of formula I or II and an immunomodulator to the subject. In further embodiments, the cancer is multiple myeloma.

[0275] In certain embodiments, the PEG-carfilzomib compounds described herein are administered in combination with one or more chemotherapeutic agents. Suitable chemotherapeutic agents include natural products, e.g., vinca alkaloids (i.e., vinblastine, vincristine, and violerbin), taxanes (e.g., docetaxel, paclitaxel, e.g., docetaxel), epidipodophilotoxins (i.e., etoposide, teniposide), antibiotics (dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin; e.g., doxorubicin), anthracyclines, mitoxantrone, bleomycin, plicamycin (mitramycin), and mitomycin, enzymes (L-asparaginase, which systemically metabolizes L-asparagine and depletes cells that lack the ability to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents, e.g., nitrogen mustard (mechloretamine, ifosfamide, cyclophosphamide, and analogs, melf Phalanes, chlorambucil (e.g., melphalan), ethyleneimine and methylmelamine (hexamethylmelamine and thiotepa), alkyl sulfonates (busulfan), nitrosourea (carmustine (BCNU) and analogs, streptozocin), trazene-decarbazinine (DTIC); antiproliferative / antimitotic antimetabolites, e.g., folic acid analogs (methotrexate), pyrimidine analogs (fluorouracil, phloxuridine, and cytarabine), purine analogs and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine); aromatase inhibitors (anastrozole, exemestane, and letrozole); platinum-coordinate complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; DNA-binding / cytotoxic agents (e.g., zalipsis);Histone deacetylase (HDAC) inhibitors (e.g., trichostatin, sodium butyrate, apicidan, subeloylanilide hydroxamate (SAHA (vorinostat)), trichostatin A, depsideptide, apicidine, A-161906, scriptide, PXD-101, CHAP, butyrate, depdesin, oxamfratin, phenylbutyrate, valproic acid, MS275 (N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide), LAQ824 / LBH589, CI994, MGCD0103, ACY-1215, panobinostat); hormones (i.e., estrogens) and hormonal agonists, such as luteinizing hormone-releasing hormone (LHRH) agonists (goserelin, leuprolide, and triptorelin). Other chemotherapeutic agents may include mechloretamine, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine, or any of the above analogs or derivative variants.

[0276] In certain embodiments, the PEG-carfilzomib compounds described herein are administered in combination with cytokines. Examples of cytokines include, but are not limited to, interferon-γ, -α, and -β, interleukin 1-8, 10, and 12, granulocyte-monocyte colony-stimulating factor (GM-CSF), TNF-α and -β, and TGF-β.

[0277] In certain embodiments, the PEG-carfilzomib compounds described herein are administered in combination with a steroid. Suitable steroids include 21-acetoxypregnolone, alclomethasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, crocortol, cloprednol, corticosterone, cortisone, cortivazole, deflazacort, desonide, desoxymethasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, fluchloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, flupredniden acetate, fluprednisolone, flulandrenolide, fluticasone propionate, formocortal, and hal. Examples of synonides, halobetazole propionate, halomethasone, hydrocortisone, loteprednol etabonate, mazipredone, medrisone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, thixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, and salts and / or derivatives thereof (e.g., hydrocortisone, dexamethasone, methylprednisolone, and prednisolone; e.g., dexamethasone) may be found, but are not limited to these. In certain embodiments, the PEG-carfilzomib compounds described herein are administered in combination with dexamethasone. In certain embodiments, combination therapies include drug regimens provided under the KYPROLIS (carfilzomib) label, which are approved by the U.S. FDA and EMA.

[0278] In some embodiments, the PEG-carfilzomib compounds described herein are administered in combination with an immunotherapy agent. Suitable immunotherapy agents include, but are not limited to, MDR modifiers (verapamil, valspordal, viricodal, tariquidal, lanicidal), cyclosporine, thalidomide, and monoclonal antibodies. The monoclonal antibody may be either naked or complexed, and may be, for example, rituximab, tocitumomab, alemtuzumab, epratuzumab, ibritumomab tiuxetan, gemtuzumab ozogamicin, bevacizumab, cetuximab, erlotinib, and trastuzumab.

[0279] 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, apicidan, subeloylanilide hydroxamate ("SAHA" (vorinostat)), trichostatin A, depsipeptide, apicidine, A-161906, scriptide, PXD-101, CHAP, butyrate, depdesin, oxamfratin, phenylbutyrate, valproic acid, MS275 (N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide), LAQ824 / LBH589, CI994, MGCD0103, ACY-1215, panobinostat; e.g., SAHA, ACY-1215, panobinostat).

[0280] In certain embodiments, the PEG-carfilzomib compounds described herein are administered in combination with one or more nitrogen mustards (mechloretamine, ifosfamide, cyclophosphamide and its analogues, melphalan, chlorambucil, e.g., melphalan). In certain embodiments, the PEG-carfilzomib compounds described herein are administered in combination with one or more DNA-binding / cytotoxic agents (e.g., Zalypsis).

[0281] 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).

[0282] In certain embodiments, the PEG-carfilzomib compounds described herein are administered in combination with one or more antibiotics (dactinomycin (actinomycin D), daunorubicin, doxorubicin, and idarubicin; for example, doxorubicin).

[0283] The above description is merely illustrative of the present invention and is not intended to limit the invention to the disclosed uses. Modifications and alterations that are routine to those skilled in the art are intended to be within the scope and nature of the invention as defined in the appended claims.

Claims

1. (a) PEG-modified carfilzomib compound, (b) At least one excipient selected from the group consisting of sucrose, sorbital, glycerin, maltose, lactose, erythrose, dextrose, lactobiose, 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-HCl, lysine-HCl, magnesium chloride, and barium sulfate, (c) A buffer selected from the group consisting of glutamate, histidine, acetate, and tris-HCl, or combinations thereof, (d) Optionally, a volume extender selected from the group consisting of mannitol, trehalose, PVP, cyclodextrin, glycine, dextrose, dextran, sucrose, proline, PEG-33350, and PEG-400. (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 doxert, benzalkonium chloride, Triton X100, tetrafunctional block O polymer, alcohol, SDS, protamine sulfate, and butane. Or a combination of (d), (e), and (f), A pharmaceutical composition containing the following:

2. The PEG-modified carfilzomib compound, Formula I 【Chemistry 1】 [In the formula, R １ C １～１０ Alkyl or C ３～７ It is a cycloalkyl, Each R ２ Independently, C １～６ Alkyl, -OCH ３ , or halogen, o is an integer selected from 0, 1, 2, or 3. Linker, 【Chemistry 2】 (In the formula, R ３ is H or CH ３ And, n is an integer selected from 1, 2, 3, or 4. p is an integer selected from 0, 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.) It is a part having the structure, PEG is a polyethylene glycol polymer portion having a molecular weight in the range of approximately 500 to 20,000. A pharmaceutical composition according to claim 1, having the structure of or a pharmaceutically acceptable salt thereof.

3. The aforementioned linker, 【Transformation 3】 [Wherein, R ３ is H or CH ３ and q is 4, The composition according to claim 2, wherein r is a part having the structure of r = 2.

4. The PEG-modified carfilzomib compound 【Chemistry 4】 【Transformation 5】 A composition according to any one of claims 1 to 3, having the structure of [the specified structure].

5. The PEG-modified carfilzomib compound 【Transformation 6】 A composition according to any one of claims 1 to 4, having the structure of [the specified structure].

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the composition is a cryogenic preparation and the pH of the preparation is in the range of 5.0 to 8.

0.

7. The pharmaceutical composition according to claim 6, 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.

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

9. The pharmaceutical composition according to claim 7 or 8, 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, glycine in an amount of about 293 mM, sodium chloride in an amount of about 140 mM, and a combination of sucrose in an amount of about 4.5% and sodium chloride in an amount of about 140 mM, and the buffer is selected from the group consisting of histidine in an amount of about 10 mM, acetate in an amount of about 10 mM, and tris-HCl in an amount of about 10 mM.

10. The aforementioned pharmaceutical composition (a) an amount of the PEG-modified carfilzomib compound in the range of 150 mg to 2000 mg, (b) The at least one excipient and buffer is (1) 9% sucrose and 10 mM acetate buffer at pH 5, (2) 9% sucrose and 10 mM histidine at pH 6, (3) 9% sucrose and 10 mM tris-HCl at pH 7, (4) 9% sucrose and 10 mM tris-HCl at pH 8, (5) 140 mM sodium chloride and 10 mM Tris-HCl at pH 7, (6) 220 mM L-proline and 10 mM Tris-HCl at pH 7, (7) 293 mM glycine and 10 mM Tris-HCl at pH 7, or (8) 70 mM sodium chloride, 4.5% sucrose, and 10 mM Tris-HCl at pH 7. A pharmaceutical composition according to any one of claims 6 to 9, comprising:

11. The pharmaceutical composition according to any one of claims 1 to 5, wherein the composition is a dried freeze-dried formulation.

12. The pharmaceutical composition according to claim 11, wherein the at least one excipient is sucrose in an amount ranging from 0.5 w / w% 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, the surfactant is absent or is polysorbate 80 or Pluronic F68, and the buffer is glutamate.

13. The pharmaceutical composition according to claim 12, wherein the excipient is a surfactant which is sucrose in an amount in the range of about 1 to 2 wt / vol%, mannitol in an amount in the range of about 2 to 4 wt / vol%, an amino acid selected from lysine or arginine in an amount in the range of about 0.5% to 0.8%, 0.0065 polysorbate 80, or 0.05% Pluronic F68, and the buffer is 10 mM glutamate.

14. The composition is 10 mg 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% Pluronic 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, PEG-modified carfilzomib compound in amounts ranging from 100 mg to 3000 mg A pharmaceutical composition according to any one of claims 1, 12, and 13, comprising essentially the above.

15. The pharmaceutical composition according to claim 14, wherein the pH of the composition is about 5.0 when the composition is dissolved in an amount of water that achieves about 10 mg / mL of the PEGylated carfilzomib compound.

16. The pharmaceutical composition according to any one of claims 11 to 15, wherein the pharmaceutical composition comprises the PEGylated carfilzomib compound in an amount ranging from 150 mg to 2000 mg.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the composition further comprises hyaluronidase.

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

19. A pharmaceutical composition according to any one of claims 1 to 18, which does not contain cyclodextrin.

20. The pharmaceutical composition according to any one of claims 11 to 19, wherein the lyophilized preparation becomes a clear solution within approximately 3 minutes when dissolved in 1.0 mL of water at room temperature.

21. A pharmaceutical composition according to any one of claims 1 to 20, which is administered parenterally by injection or infusion.

22. A pharmaceutical composition according to any one of claims 1 to 20, which is administered intravenously by injection or injection.

23. A pharmaceutical composition according to any one of claims 1 to 20, administered by subcutaneous injection.

24. A method for treating cancer, comprising administering a therapeutically effective amount of the pharmaceutical composition described in any one of claims 1 to 23 to a patient in need of cancer treatment.

25. The method according to claim 24, wherein the cancer is multiple myeloma.

26. The method according to claim 25, wherein the multiple myeloma is relapsed, refractory, or relapsed and refractory multiple myeloma.

27. The method according to claim 25, wherein the multiple myeloma is a newly diagnosed multiple myeloma.

28. A process for manufacturing a pharmaceutical composition according to any one of claims 1 to 19 for the treatment of multiple myeloma, comprising: (a) combining a PEGylated carfilzomib compound in an amount 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; (b) A step of mixing the above combination to obtain a transparent solution, A process that includes this.

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