pH responsive block copolymer compositions, micelles, and methods of use

Abstract

Described herein are therapeutic pH-responsive compositions comprising block copolymers and a therapeutic agent that can be used to treat cancer.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 930,530, filed on November 4, 2019, which is hereby incorporated in its entirety. Background Art

[0003] Multifunctional nanoparticles have attracted attention in a wide range of applications such as biosensors, diagnostic nanoprobes, and targeted drug delivery systems. These efforts are largely due to the need to improve biological specificity and reduce side effects in diagnosis and therapy by precisely controlling the delivery of agents in various physiological systems in terms of time and space. To achieve this goal, efforts are being made to develop stimulus-responsive nanoplatforms. Environmental stimuli that have been explored for accurately determining delivery efficiency include pH, temperature, enzymatic expression, redox reactions, and light induction. Among these activation signals, pH triggers are one of the most widely studied stimuli based on two types of pH differences: (a) pathological (e.g., tumor) tissues versus normal tissues and (b) acidic intracellular compartments.

[0004] For example, due to the abnormal acidity of the extracellular microenvironment of tumor cells (pH about 6.5), it is reported that several pH-responsive nanosystems can improve the sensitivity of tumor imaging or the efficacy of therapy. However, for polymer micelle compositions that release drugs by hydrolysis in an acidic environment, the release of drugs may take several days. During the time period, the body can excrete or decompose the micelles.

[0005] In order to target the acidic endosome / lysosome compartment, nanocarriers with pH-cleavable linkers have been studied to improve the bioavailability of effective loads. In addition, several smart nanocarriers with pH-induced charge conversion have been designed to improve drug efficacy. The endocytic system comprises a series of compartments that have a unique role in the sorting, processing and degradation of internalized cargo. Due to the short residence time of nanoparticles (<several minutes) and the small pH difference in these compartments (e.g., <1 pH unit between early endosomes and lysosomes), it is particularly challenging to selectively target different endocytic compartments by pH-sensitive nanoparticles.

[0006] Immunotherapy has become a powerful strategy for cancer treatment. Immunomodulatory agents such as interleukin-2 (IL-2) can induce anti-tumor immune responses, but their clinical application is limited by unfavorable pharmacokinetic properties that may cause severe dose-limiting toxicities (e.g., broad-spectrum toxicities / side effects such as vascular leak syndrome).

[0007] What are needed are improved pH-responsive micellar compositions for therapeutic applications, particularly compositions with increased drug payload, extended blood circulation time, rapid drug delivery at the target site, and responsiveness within a specific narrow pH range (e.g., for targeting tumors or specific organelles). Summary of the invention

[0008] The block copolymers described herein are therapeutic agents that can be used to treat primary and metastatic tumor tissues, including lymph nodes. The block copolymers and micellar compositions presented herein take advantage of this ubiquitous pH difference between cancerous tissue and normal tissue and provide high sensitivity and specific response after being taken up by cells, thus allowing the deployment of therapeutic payloads to tumor tissues.

[0009] In one aspect, provided herein is a block copolymer having a structure of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0010]

[0011] in:

[0012] n1 is an integer between 10 and 200;

[0013] x1 is an integer between 40 and 300;

[0014] y1 is an integer from 0 to 6;

[0015] z1 is an integer from 0 to 10;

[0016] X 1 is halogen, -OH or -C(O)OH;

[0017] R 1 and R 2 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0018] or R 1 and R 2 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0019] Each R 3 are independently hydrogen, acyl or ICG;

[0020] L 1 is a bond or -C(O)-, or an optionally substituted C1-C 10 an alkylene linker or a PEG linker; and

[0021] Y is a therapeutic agent.

[0022] In some embodiments, each R 1 and R 2 is independently an optionally substituted C1-C6 alkyl. 1 and R 2 is independently -CH2CH3, -CH2CH2CH3 or -CH2CH2CH2CH3. In some embodiments, each R 1 and R 2 are independently -CH2CH2CH2CH3. In some embodiments, R 1 and R 2 Together with the corresponding nitrogen to which it is attached, it forms an optionally substituted 5- to 7-membered ring. 1 and R 2 Together they are -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, or -CH2(CH2)4CH2-. In some embodiments, x1 is an integer from 50-200, 60-160, or 90-140. In some embodiments, x1 is 90-140. In some embodiments, y1 is 0. In some embodiments, z1 is an integer from 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, or 1-3. In some embodiments, z1 is 0. In some embodiments, n1 is an integer from 60-150 or 100-140. In some embodiments, n1 is 100-140. In some embodiments, x1 is an integer from 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, or 1-3. In some embodiments, z1 is 0. In some embodiments, n1 is an integer from 60-150 or 100-140. In some embodiments, n1 is 100-140. 1 In some embodiments, X 1 is a bromide. In some embodiments, each R 3 are independently acyl or ICG. In some embodiments, L 1 is an optionally substituted C1-C 10 The alkylene linker, the C1-C 10 The alkylene linker is optionally substituted with a maleimide residue. In certain embodiments, the therapeutic agent is a cytokine or a fragment thereof, an engineered antibody fragment, or a small molecule having a molecular weight of less than 900 daltons. In certain embodiments, the cytokine is IL-2, IL-12, or IL-15 or a fragment thereof. In certain embodiments, the engineered antibody fragment is a bispecific T cell adaptor. In certain embodiments, the small molecule is maytansine or a derivative thereof.

[0023] In some embodiments provided herein, the block copolymer of formula (I) has a structure of formula (Ia) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0024]

[0025] in:

[0026] m1 is an integer from 10 to 200; and

[0027] A is a bond or -C(O)- optionally substituted with a maleimide residue.

[0028] In another aspect, provided herein is a block copolymer having a structure of Formula (Ib) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0029]

[0030] in:

[0031] n1 is an integer between 10 and 200;

[0032] x1 is an integer between 40 and 300;

[0033] y1 is an integer from 0 to 6;

[0034] z1 is an integer from 0 to 10;

[0035] X 1 is halogen, -OH or -C(O)OH;

[0036] R 1 and R 2 are each independently a substituted or unsubstituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0037] or R 1 and R 2 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0038] Each R 3 are independently hydrogen, acyl or ICG;

[0039] L 3 Yes key, C1-C 10 an alkylene linker or a PEG linker; and

[0040] B is maleimide,

[0041] In another aspect, provided herein is a block copolymer having a structure of formula (II) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0042]

[0043] in:

[0044] n2 is an integer from 2 to 200;

[0045] x2 is an integer between 40 and 300;

[0046] y2 is an integer from 0 to 6;

[0047] X 2 is halogen, -OH or -C(O)OH;

[0048] R 5 and R 6 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0049] or R 5 and R 6 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0050] Each R 7 are independently hydrogen, acyl or ICG;

[0051] Z 1 is -NH- or -O-;

[0052] Z 2 is -NH-, -O- or a substituted triazole;

[0053] L 2 is a bond or -C(O)-, or an optionally substituted C1-C 10 an alkylene linker or a PEG linker; and

[0054] Y is a therapeutic agent.

[0055] In some embodiments, each R 5 and R 6 is independently an optionally substituted C1-C6 alkyl. 5 and R 6 is independently -CH2CH3, -CH2CH2CH3 or -CH2CH2CH2CH3. In some embodiments, each R 5 and R 6 is -CH2CH2CH2CH3. In some embodiments, R 5 and R 6 Together with the corresponding nitrogen to which it is attached, it forms an optionally substituted 5- to 7-membered ring. 5 and R 6Together they are -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, or -CH2(CH2)4CH2-. In some embodiments, x2 is an integer from 50-200, 60-160, or 90-140. In some embodiments, x2 is 90-140. In some embodiments, y2 is an integer from 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, or 1-3. In some embodiments, y2 is 0. In some embodiments, n2 is an integer from 60-150 or 100-140. In some embodiments, n2 is 100-140. In some embodiments, x 2 In some embodiments, X 2 is -Br. In some embodiments, Z 1 is -O- or -NH-. In some embodiments, Z 2 is -O- or -NH-. In some embodiments, Z 2 is an optionally substituted triazole residue. In some embodiments, L 2 is an optionally substituted C1-C 10 The alkylene linker, the C1-C 10 The alkylene linker is optionally substituted with a maleimide residue. 2 It is an optionally substituted PEG linker, which is optionally substituted with a maleimide residue. In some embodiments, the therapeutic agent is a cytokine or a fragment thereof, an engineered antibody fragment, or a small molecule with a molecular weight of less than 900 daltons. In some embodiments, the cytokine is IL-2, IL-12 or IL-15 or a fragment thereof. In some embodiments, the engineered antibody fragment is a bispecific T cell adaptor. In some embodiments, the small molecule is maytansine or a derivative thereof.

[0056] In some embodiments, the block copolymer of formula (II) has a structure of formula (II-a) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0057]

[0058] in:

[0059] m2 is 2-200; and

[0060] A is a bond or -C(O)- optionally substituted with a maleimide residue.

[0061] In another aspect, the present invention provides a block copolymer having a structure of formula (II-b) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0062]

[0063] in:

[0064] n2 is an integer from 2 to 200;

[0065] x2 is an integer between 40 and 300;

[0066] y2 is an integer from 0 to 6;

[0067] X 2 is halogen, -OH or -C(O)OH;

[0068] R 5 and R 6 are each independently a substituted or unsubstituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0069] or R 5 and R 6 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0070] Each R 7 are independently hydrogen, acyl or ICG;

[0071] Z 1 is -NH- or -O-;

[0072] Z 2 is -NH-, -O- or a substituted triazole;

[0073] L 4 Yes key, C1-C 10 an alkylene linker or a PEG linker; and

[0074] B is maleimide,

[0075] In another aspect, there is provided a micelle comprising:

[0076] (i) a block copolymer having formula (III) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0077]

[0078] in:

[0079] n3 is an integer between 10 and 200;

[0080] x3 is an integer between 40 and 300;

[0081] y3 is an integer from 0 to 6;

[0082] z3 is an integer from 0 to 10;

[0083] X 3 is halogen, -OH or -C(O)OH;

[0084] Each R 10 independently hydrogen or ICG;

[0085] R 8 and R 9 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0086] or R 8 and R 9 together with the corresponding nitrogen to which they are attached, form an optionally substituted 5- to 7-membered ring; and (ii) a therapeutic agent encapsulated by the block copolymer.

[0087] In another aspect, there is provided a micelle comprising:

[0088] (i) a block copolymer having formula (III) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0089]

[0090] in:

[0091] n3 is an integer between 10 and 200;

[0092] x3 is an integer between 40 and 300;

[0093] y3 is an integer from 0 to 6;

[0094] z3 is an integer from 0 to 10;

[0095] X 3 is halogen, -OH or -C(O)OH;

[0096] Each R 10 independently hydrogen or ICG;

[0097] R 8 and R 9 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0098] or R 8 and R 9 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0099] (ii) a block copolymer having formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0100]

[0101] in:

[0102] n1 is an integer between 10 and 200;

[0103] x1 is an integer between 40 and 300;

[0104] y1 is an integer from 0 to 6;

[0105] z1 is an integer from 0 to 10;

[0106] X 1 is halogen, -OH or -C(O)OH;

[0107] R 1 and R 2 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0108] or R 1 and R 2 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0109] Each R 3 are independently hydrogen, acyl or ICG;

[0110] L 1 is a bond or -C(O)-, or an optionally substituted C1-C 10 Alkylene linker or PEG linker;

[0111] Y is a therapeutic agent; and / or

[0112] (iii) a block copolymer having formula (II) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0113]

[0114] in:

[0115] n2 is an integer from 2 to 200;

[0116] x2 is an integer between 40 and 300;

[0117] y2 is an integer from 0 to 6;

[0118] X 2 is halogen, -OH or -C(O)OH;

[0119] R 5 and R 6are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0120] or R 5 and R 6 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0121] Each R 7 are independently hydrogen, acyl or ICG;

[0122] Z 1 is -NH- or -O-;

[0123] Z 2 is -NH-, -O- or a substituted triazole residue;

[0124] L 2 is a bond or -C(O)-, or an optionally substituted C1-C 10 an alkylene linker or a PEG linker; and

[0125] Y is a therapeutic agent.

[0126] In some embodiments, each R 8 and R 9 is independently an optionally substituted C1-C6 alkyl. 8 and R 9 is independently -CH2CH3, -CH2CH2CH3 or -CH2CH2CH2CH3. In some embodiments, each R 8 and R 9 is -CH2CH2CH2CH3. In some embodiments, R 8 and R 9 Together with the corresponding nitrogen to which it is attached, it forms an optionally substituted 5- to 7-membered ring. 8 and R 9Together they are -CH2(CH2)2CH2-, -CH2(CH2)3CH2- or -CH2(CH2)4CH2-. In some embodiments, x3 is an integer of 50-200, 60-160 or 90-140. In some embodiments, x3 is 90-140. In some embodiments, y3 is an integer of 1-6, 1-5, 1-4 or 1-3. In some embodiments, y3 is 0. In some embodiments, z3 is an integer of 1-9, 1-8, 1-7, 1-6, 1-5, 1-4 or 1-3. In some embodiments, z3 is 0. In some embodiments, n3 is an integer of 60-150 or 100-140. In some embodiments, the therapeutic agent is a cytokine or a fragment thereof, an engineered antibody fragment or a small molecule having a molecular weight of less than 900 Daltons. In some embodiments, the cytokine or a fragment thereof is IL-12 or a fragment thereof. In some embodiments, the engineered antibody fragment is a bispecific T cell engager. In some embodiments, the small molecule is maytansine or a derivative thereof.

[0127] In some embodiments presented herein, the micelle comprises: (i) a block copolymer of formula (III); and (ii) a block copolymer of formula (I). In some embodiments presented herein, the micelle comprises: (i) a block copolymer of formula (III); and (ii) a block copolymer of formula (II). In some embodiments presented herein, the micelle comprises: (i) a block copolymer of formula (III); (ii) a block copolymer of formula (I); and (iii) a block copolymer of formula (II). In some embodiments presented herein, the micelle comprises a block copolymer of (i) formula (III) and a block copolymer of (ii) formula (I) or (II) in a ratio of about 1:99 to about 99:1.

[0128] In another aspect, a pH responsive composition is provided, comprising a block copolymer or micellar composition as described herein, wherein the composition has a pH transition point and optionally an emission spectrum. In some embodiments, the pH transition point is between 4-8, 6-7.5, or 4.5-5.5. In some embodiments, the pH response of the pH responsive composition is less than 0.25 or 0.15 pH units. In some embodiments, the emission spectrum is between 700-900 nm.

[0129] In another aspect, a method for treating cancer in an individual in need thereof, the method comprising administering to the individual an effective amount of a pH-sensitive micellar composition as described herein comprising a chemotherapeutic agent. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the tumor is a cancer, wherein the cancer is breast cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, peritoneal cancer, colorectal cancer, bladder cancer, kidney cancer, esophageal cancer, head and neck cancer (HNSSC), lung cancer, brain cancer, or skin cancer (including melanoma and sarcoma).

[0130] Other objects, features and advantages of the block copolymers, micellar compositions and methods described herein will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate specific embodiments, the specific embodiments are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art based on this detailed description.

[0131] Incorporated by Reference

[0132] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Various aspects of the present disclosure are set forth with particularity in the appended claims.A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description which sets forth illustrative embodiments in which the principles of the present disclosure are utilized and the accompanying drawings set forth below.

[0134] Figure 1 Schematic diagram of an ultra-pH-sensitive nanoparticle platform capable of encapsulating and pH-dependent release of payloads (e.g., IL-2) is shown. t When the pH is <pH t , the nanoparticles disintegrate into monomers, thereby releasing the encapsulated payload.

[0135] Figure 2 The pH-dependent IL-2 release profile is shown. (Left) Quantitative measurement of acidic buffer triggering IL-2 payload release. (Right) Size changes of nanoparticles under acidic buffer conditions were measured by DLS.

[0136] Figure 3 PEG 113 -b-PDBA 90-160The micelles can be loaded with IL-2. SEC followed by dot blot of IL-2 confirmed the loading of IL-2.

[0137] Figure 4 A and Figure 4 B shows the use of pH-sensitive micelles to encapsulate bispecific antibodies. 4A shows the SEC chromatogram after bispecific antibody encapsulation, and the size distribution of micelle-encapsulated bispecific antibodies by DLS (three replicates). The smallest bispecific antibody exists in an unencapsulated free form. 4B shows quantitative analysis of bispecific antibody loading and formulation size by Western blotting and DLS.

[0138] Figure 5 pH-dependent binding of nanoparticle-encapsulated antibodies to GSU cells is shown. The nanoparticle-encapsulated bispecific antibodies show low binding affinity to cells carrying the antibody target at neutral pH. Upon acidification, the bispecific antibodies are released from the micelles. The binding of the released bispecific antibodies shows the same affinity to the target on the cells compared to the original format.

[0139] Figure 6 pH-sensitive nanoparticle non-covalently encapsulated Fab formulations (Compound 1) showing significant tumor accumulation increase and pharmacokinetic changes compared to free Fab in mice bearing orthotopic head and neck tumors from biodistribution curves are shown. Representative in vivo (A, 1 hour, 3 hours, 24 hours) and ex vivo (B, 24 hours) major organ biodistributions are shown. Quantification of in vivo tumor (C) and ex vivo organ (D) fluorescence was performed. Statistical analysis was performed by Student's t-test ( ** p<0.01), N = 3. Fabs were labeled with near-infrared fluorophores for imaging purposes.

[0140] Figure 7 Shown is a schematic for preparing covalent protein-polymer formulations in the hydrophobic / amine block.

[0141] Figure 8 pH-sensitive nanoparticles and IL-2 non-covalent formulations (Compound 2) showing significant tumor accumulation increases and pharmacokinetic changes compared to free IL-2 in mice bearing orthotopic head and neck tumors from biodistribution curves are shown. Representative in vivo (A, 1 hour, 3 hours, 24 hours) and ex vivo (B, 24 hours) major organ biodistributions are shown. Quantification of in vivo tumor (C) and ex vivo organ (D) fluorescence was performed. Statistical analysis was performed by Student's t-test ( ** p<0.01), N = 3. IL-2 was labeled with a near-infrared fluorophore for imaging purposes.

[0142] Fig. 9 pH-sensitive nanoparticles covalently conjugated to Fab formulations (Compound 3) showing significant tumor accumulation increases and pharmacokinetic changes compared to free Fab antibodies in mice bearing orthotopic head and neck tumors from biodistribution curves are shown. Representative in vivo (A, 1 hour, 3 hours, 24 hours) and ex vivo (B, 24 hours) major organ biodistributions are shown. Quantification of in vivo tumor (C) and ex vivo organ (D) fluorescence was performed. Statistical analysis was performed by Student's t-test ( ** p<0.01), N = 3. Fabs were labeled with near-infrared fluorophores for imaging purposes.

[0143] Fig.10 The method for reacting rhIL-2 with PEG is shown. 113 -PDBA 90-160 Representative scheme for the conjugation of -AMA-OPSS polymers.

[0144] Fig.11 The purification and characterization of block copolymer-IL-2 covalent conjugates are shown. (Top): PEG 113 -b-(PDBA 90 FPLC chromatogram of purification of (-160-r-OPSS4)-IL-2 covalent conjugate. (Bottom): Western blot showing FPLC fractions confirming conjugation of IL-2 by shift in electrophoretic mobility.

[0145] Fig.12 The in vitro biological activities of pH-sensitive polymer-IL-2 covalent formulations are shown. (A) PEG-PDBA-OPSS-IL-2 conjugated by SAT (PEG4) chemistry is shown. (B) PEG-PDBA-OPSS-IL-2 conjugated by Traut's reagent chemistry is shown. (C) PEG-PDBA-Mal-IL-2 conjugated by SAT (PEG4) chemistry is shown. (D) PEG-PDBA-Mal-IL-2 conjugated by Traut's reagent chemistry is shown. The parent compound used is PEG 113 -b-(PDBA 120 -r-OPSS4) or PEG 113 -b(PDBA 120 -r-Mal1).

[0146] Fig.13 A representative scheme for preparing covalent protein-block copolymer conjugates on the PEG termini is shown.

[0147] Fig.14A representative synthetic scheme of a block copolymer-small molecule (mertansine) conjugate is shown.

[0148] Figures 15A-15C Figure 15A shows the characterization of the block copolymer-small molecule (Motansin) conjugate (Compound 4). Figure 15A shows the starting material of the PDBA-AMA polymer (PEG 113 -PDBA 90-160 -AMA4) 1 H NMR spectrum. 15B shows the PDBA-AMA-SMCC-DM1 conjugate. 1 H NMR spectrum. In the case of single proton integrated peaks, the integration of the o-methoxy peak at 3.3 ppm was used to determine the drug loading of DM1 drug, and the loading of about 3.5 DM1 molecules per block copolymer chain was calculated. 15C shows the HPLC analysis of the PEG-PDBA-AMA-SMCC-DM1 modified polymer.

[0149] Fig.16 A representative synthetic scheme for the synthesis of PEG-PDBA-OPSS-DM1 is shown.

[0150] Figures 17A-17C 17A shows the characterization of PEG-PDBA-OPSS-DM1 (Compound 5). 17A shows the starting material of PEG-PDBA-OPSS using DM1 conjugate. 1 H NMR spectrum. 17B shows the PEG-PDBA-OPSS polymer material after DM1 conjugation. 1 H NMR spectrum. Integration showed that the drug loading rate of the polymer was 80%. Fig. 17C HPLC analysis of the compound 5 modified polymer is shown.

[0151] Fig.18 A representative synthesis scheme of PEG-PDBA-Mal-DM1 is shown. DETAILED DESCRIPTION

[0152] Provided herein are block copolymers conjugated to therapeutic agents.In other embodiments provided herein are micellar compositions comprising a therapeutic agent.

[0153] I. Block Copolymers

[0154] In one aspect, provided herein is a block copolymer having a structure of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0155]

[0156] in:

[0157] n1 is an integer between 10 and 200;

[0158] x1 is an integer between 40 and 300;

[0159] y1 is an integer from 0 to 6;

[0160] z1 is an integer from 0 to 10;

[0161] X 1 is halogen, -OH or -C(O)OH;

[0162] R 1 and R 2 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0163] or R 1 and R 2 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0164] Each R 3 are independently hydrogen, acyl or ICG;

[0165] L 1 is a bond or -C(O)-, or an optionally substituted C1-C 10 an alkylene linker or a PEG linker, each of which is optionally substituted with a maleimide residue; and

[0166] Y is a therapeutic agent.

[0167] In some embodiments, R 1 and R 2 In some embodiments, R 1 and R 2 Are different groups.

[0168] In some embodiments, each R 1 and R 2 R is independently an optionally substituted C1-C6 alkyl. In some embodiments, the alkyl group is a straight chain or branched chain alkyl group. In some embodiments, the alkyl group is a straight chain alkyl group. In some embodiments, each R 1 and R 2 is independently -CH2CH3, -CH2CH2CH3 or -CH2CH2CH2CH3. In some embodiments, each R 1 and R 2 It is -CH2CH2CH2CH3.

[0169] In some embodiments, each R 1 and R 2are each independently an optionally substituted C3-C 10 In some embodiments, each R 1 and R 2 R is independently optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. 1 and R 2 Independently is optionally substituted phenyl.

[0170] In some embodiments, R 1 and R 2 Together with the corresponding nitrogen to which it is attached, it forms an optionally substituted 5- to 7-membered ring. 1 and R 2 Together they are -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, or -CH2(CH2)4CH2-. In some embodiments, R 1 and R 2 Together they are -CH2(CH2)4CH2-.

[0171] In some embodiments, each R 3 is independently acyl or ICG. In some embodiments, each R 3 In some embodiments, each R 3 is independently ICG. In some embodiments, each R 3 are independently hydrogen.

[0172] In some embodiments, L 1 is an optionally substituted bifunctional linker capable of binding to the block copolymer and to the therapeutic agent. In some embodiments, L 1 is an optionally substituted C1-C 10 The alkylene linker, the C1-C 10 The alkylene linker is optionally substituted with a maleimide residue. 1 is an optionally substituted PEG linker, which is optionally substituted with a maleimide residue.

[0173] In some embodiments, L 1 yes wherein m1 is an integer from 2 to 20 or any integer therein.

[0174] In some embodiments, the block copolymer of formula (I) has a structure of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof:

[0175]

[0176] in:

[0177] m1 is an integer from 2 to 200; and

[0178] A is a bond or -C(O)- optionally substituted with a maleimide residue.

[0179] In some embodiments, m1 is an integer from 2 to 20, or any integer therein. In some embodiments, m1 is an integer from 2 to 5, 6 to 9, 10 to 14, or 15 to 20, or any integer therein.

[0180] In some embodiments, A is a bond. In some embodiments, A is -C(O)- optionally substituted with a maleimide residue.

[0181] In some embodiments, the block copolymer of formula (I) has a structure of formula (Ic) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0182]

[0183] In some embodiments of the block copolymers of formula (I), (Ia) and (Ic), the therapeutic agent is a cytokine or a fragment thereof, an engineered antibody fragment or a small molecule with a molecular weight of less than 900 Daltons. In some embodiments, the cytokine is IL-2, IL-12 or IL-15 or a fragment thereof. In some embodiments, the cytokine is IL-2 or a fragment thereof. In some embodiments, the cytokine is IL-12 or a fragment thereof. In some embodiments, the cytokine is IL-15 or a fragment thereof. In some embodiments, the cytokine is Fab or a fragment thereof. In some embodiments, the engineered antibody fragment is a bispecific T cell adaptor. In some embodiments, the small molecule is maytansine or a derivative thereof.

[0184] In another aspect, provided herein is a block copolymer having a structure of formula (II) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0185]

[0186] in:

[0187] n2 is an integer from 2 to 200;

[0188] x2 is an integer between 40 and 300;

[0189] y2 is an integer from 0 to 6;

[0190] X 2 is halogen, -OH or -C(O)OH;

[0191] R5 and R 6 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0192] or R 5 and R 6 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0193] Each R 7 are independently hydrogen, acyl or ICG;

[0194] Z 1 is -NH- or -O-;

[0195] Z 2 is -NH-, -O- or a substituted triazole;

[0196] L 2 is a bond or -C(O)-, or an optionally substituted C1-C 10 an alkylene linker or a PEG linker; and

[0197] Y is a therapeutic agent.

[0198] In some embodiments, R 5 and R 6 In some embodiments, R 5 and R 6 Are different groups.

[0199] In some embodiments, each R 5 and R 6 R is independently an optionally substituted C1-C6 alkyl. In some embodiments, the alkyl group is a straight chain or branched chain alkyl group. In some embodiments, the alkyl group is a straight chain alkyl group. In some embodiments, each R 5 and R 6 is independently -CH2CH3, -CH2CH2CH3 or -CH2CH2CH2CH3. In some embodiments, each R 5 and R 6 It is -CH2CH2CH2CH3.

[0200] In some embodiments, each R 5 and R 6 is independently an optionally substituted C3-C 10 In some embodiments, each R 5 and R 6 R is independently optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.5 and R 6 Independently is optionally substituted phenyl.

[0201] In some embodiments, R 5 and R 6 Together with the corresponding nitrogen to which it is attached, it forms an optionally substituted 5- to 7-membered ring. 5 and R 6 Together they are -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, or -CH2(CH2)4CH2-.

[0202] In some embodiments, each R 7 is independently acyl or ICG. In some embodiments, each R 7 In some embodiments, each R 7 is independently ICG. In some embodiments, each R 7 are independently hydrogen.

[0203] In some embodiments, Z 1 is -O-. In some embodiments, Z 1 It is -NH-.

[0204] In some embodiments, Z 2 is -NH- or -O-. In some embodiments, Z 2 is -O-. In some embodiments, Z 2 is -NH-. In some embodiments, Z 2 is a substituted triazole.

[0205] In some embodiments, L 2 is an optionally substituted bifunctional linker capable of binding to the block copolymer and to the therapeutic agent. In some embodiments, L 2 is an optionally substituted C1-C 10 The alkylene linker, the C1-C 10 The alkylene linker is optionally substituted with a maleimide residue. 2 is an optionally substituted PEG linker, which is optionally substituted with a maleimide residue. In some embodiments, L 2 yes Among them, m2 is 2-200.

[0206] In some embodiments, the block copolymer of formula (II) has a structure of formula (II-a) or a pharmaceutically acceptable salt or solvate thereof:

[0207]

[0208] in:

[0209] m2 is 2-200; and

[0210] A is a bond or -C(O)- optionally substituted with a maleimide residue.

[0211] In some embodiments, m2 is an integer from 2 to 20. In some embodiments, m2 is an integer from 2-5, 6-9, 10-14, or 15-20, or any integer therein.

[0212] In some embodiments, A is a bond. In some embodiments, A is -C(O)- optionally substituted with a maleimide residue.

[0213] In some embodiments, the block copolymer of formula (II) has a structure of formula (II-c) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0214]

[0215] In some embodiments, the block copolymer of formula (II) has a structure of formula (II-a2) or a pharmaceutically acceptable salt or solvate thereof

[0216]

[0217] in:

[0218] Z 1 Yes -O-.

[0219] In some embodiments of the block copolymers of formula (II), (II-a), (II-a2) or (II-c), the therapeutic agent is a cytokine or a fragment thereof, an engineered antibody fragment or a small molecule with a molecular weight of less than 900 Daltons. In some embodiments, the cytokine is IL-2, IL-12 or IL-15 or a fragment thereof. In some embodiments, the cytokine is IL-2 or a fragment thereof. In some embodiments, the cytokine is IL-2 or a fragment thereof. In some embodiments, the cytokine is IL-15 or a fragment thereof. In some embodiments, the cytokine is Fab or a fragment thereof. In some embodiments, the engineered antibody fragment is a bispecific T cell adaptor. In some embodiments, the small molecule is maytansine or a derivative thereof.

[0220] In another embodiment, provided herein is a block copolymer having a structure of Formula (Ib) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0221]

[0222] in:

[0223] n1 is an integer between 10 and 200;

[0224] x1 is an integer between 40 and 300;

[0225] y1 is an integer from 0 to 6;

[0226] z1 is an integer from 0 to 10;

[0227] X 1 is halogen, -OH or -C(O)OH;

[0228] R 1 and R 2 are each independently a substituted or unsubstituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0229] or R 1 and R 2 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0230] Each R 3 are independently hydrogen, acyl or ICG;

[0231] L 3 Yes key, C1-C 10 an alkylene linker or a PEG linker; and

[0232] B is maleimide,

[0233] In some embodiments of the block copolymer of formula (Ib), L 3 It is C1-C 10 In some embodiments, L 3 is a PEG linker comprising 2-200 PEG units or any integer therein. 3 is the key.

[0234] In some embodiments of the block copolymer of formula (Ib), B is maleimide. In some embodiments, B is N-hydroxysuccinimide or carbonyldiimidazole.

[0235] In some embodiments, the block copolymer having the structure of formula (Ib) is:

[0236]

[0237] wherein m1 is 2-200; or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0238] In another embodiment, provided herein is a block copolymer having a structure of formula (II-b) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0239]

[0240] in:

[0241] n2 is an integer from 2 to 200;

[0242] x2 is an integer between 40 and 300;

[0243] y2 is an integer from 0 to 6;

[0244] X 2 is halogen, -OH or -C(O)OH;

[0245] R 5 and R 6 are each independently a substituted or unsubstituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0246] or R 5 and R 6 Together with the corresponding nitrogen to which it is attached, they form a substituted or unsubstituted 5- to 7-membered ring;

[0247] Each R 7 are independently hydrogen, acyl or ICG;

[0248] Z 1 is -NH- or -O-;

[0249] Z 2 is -NH-, -O- or a substituted triazole;

[0250] L 4 Yes key, C1-C 10 an alkylene linker or a PEG linker; and

[0251] B is maleimide,

[0252] In some embodiments, the block copolymer of formula (II-b) has a structure of formula (II-b2) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0253]

[0254] in:

[0255] Z 1 is -O-; and the other variables are defined in the embodiments of formula (II-b).

[0256] In some embodiments of the block copolymer of formula (II-b) or (II-b2), L 4 It is C1-C 10 In some embodiments, L 4 L is a PEG linker comprising 2-200 PEG units. 4 is the key.

[0257] In some embodiments of the block copolymer of formula (II-b) or (II-b2), B is maleimide. In some embodiments, B is N-hydroxysuccinimide or carbonyldiimidazole.

[0258] In some embodiments, the block copolymer is:

[0259]

[0260] or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0261] In some embodiments, the block copolymer is:

[0262]

[0263]

[0264] wherein m1 is 2-200, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0265] In some embodiments, the block copolymer is a diblock copolymer. In some embodiments, the block copolymer includes a hydrophilic polymer segment and a hydrophobic polymer segment. In some embodiments, the hydrophilic polymer segment includes poly (ethylene oxide) (PEO). In some embodiments, the size of the hydrophilic polymer segment is about 2kDa to about 10kDa. In some embodiments, the size of the hydrophilic polymer segment is about 2kDa to about 5kDa. In some embodiments, the size of the hydrophilic polymer segment is about 3kDa to about 8kDa. In some embodiments, the size of the hydrophilic polymer segment is about 4kDa to about 6kDa. In some embodiments, the size of the hydrophilic polymer segment is about 5kDa.

[0266] In some embodiments, each of n1, n2 and n3 is independently an integer of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-99, 100-109, 110-119, 120-129, 130-139, 140-149, 150-159, 160-169, 170-179, 180-189, 190-199, or any range derivable therein. In some embodiments, each n1, n2 and n3 is independently an integer of 60-150, 100-140 or 110-120. In some embodiments, each n1, n2 and n3 is independently 100-140.

[0267] In some embodiments, the block copolymer comprises a hydrophobic polymer segment. In some embodiments, the hydrophobic polymer segment comprises a tertiary amine. In some embodiments, the hydrophobic polymer segment is selected from the following:

[0268]

[0269] Where x is about 40-300 in total.

[0270] In some embodiments, the hydrophobic segment comprises dibutylamine. In some embodiments, the hydrophobic segment comprises

[0271]

[0272] In some embodiments, each of x1, x2 and x3 is independently an integer of 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-99, 100-109, 110-119, 120-129, 130-139, 140-149, 150-159, 160-169, 170-179, 180-189, 190-199, or any range derivable therein. In some embodiments, each x1, x2 and x3 is independently an integer of 50-200, 60-160 or 90- 140. In some embodiments, each x1, x2 and x3 is independently 90-140.

[0273] In some embodiments, each y1, y2 and y3 is independently an integer of 1-6, 1-5, 1-4 or 1-3, or any range derivable therein. In some embodiments, each y1, y2 and y3 is independently 1, 2, 3, 4, 5 or 6. In some embodiments, each y1, y2 and y3 is independently 0.

[0274] In some embodiments, each z1 and z2 is independently an integer of 1-9, 1-8, 1-7, 1-6, 1-5, 1-4 or 1-3, or any range derivable therein. In some embodiments, each z1 and z2 is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, each z1 and z2 is independently 0.

[0275] The term "r" represents the connection between different block copolymer units / segments (e.g., represented by x1, y1, and z1). In some embodiments, each r is independently a bond connecting the carbon atoms of the units / segments or an alkyl group -(CH2) n -, wherein n is 1 to 10. In some embodiments, the copolymer block segments / units (e.g., represented by x1, y1, and z1) can appear in any order, sequence, or configuration. In some embodiments, the copolymer block units appear sequentially as described in Formulas (I), (Ia), (Ib), (Ic), (II), (II-a), (II-a2), (II-b), (II-b2), (II-c), (III-c), and (III).

[0276] In some embodiments, each m1 and m2 is independently an integer from 2 to 200. In some embodiments, each m1 and m2 is independently an integer from 2 to 20.

[0277] In some embodiments, each X 1 , X 2 and X 3 is a terminal group. In some embodiments, the end-capping group is the product of an atom transfer radical polymerization (ATRP) reaction. For example, when using atom transfer radical polymerization (ATRP), the end-capping group can be a halogen, such as -Br. In some embodiments, each X 1 , X 2 and X 3 is independently Br. In some embodiments, each X 1 , X 2 and X 3 In some embodiments, each X 1 , X 2 and X 3 In some embodiments, each X 1 , X2 and X 3 is independently -C(O)OH. In some embodiments, each X 1 , X 2 and X 3 are independently H. The end groups may optionally be further modified with suitable moieties after polymerization.

[0278] In some embodiments, the linker L 1 and L 2 is a bifunctional linker having a group reactive with the block copolymer and the therapeutic agent. In some embodiments, the linker is a component used that is maleimide-PEG-NHS, NHS-carbonate (N-hydroxysuccinimide carbonate), SPDB (N-succinimidyl-4-(2-pyridyldithio)butyrate) or CDI (carbonyl diimidazole).

[0279] In some embodiments, the linker is conjugated to a therapeutic agent. In some embodiments, the linker is covalently conjugated to a therapeutic agent. Methods known in the art can be used to conjugate a therapeutic agent to, for example, a hydrophobic polymer segment.

[0280] Therapeutic agents

[0281] In some embodiments, the therapeutic agent is a cytokine or a fragment thereof, an engineered antibody fragment, or a small molecule with a molecular weight of less than 900 Daltons.

[0282] In some embodiments, the therapeutic agent is a cytokine or a fragment thereof. Cytokines are a class of extensive and loose small proteins that play an important role in cell signaling. Cytokines are peptides and cannot pass through the lipid bilayer of the cell to enter the cytoplasm. It has been shown that cytokines participate in autocrine, paracrine and endocrine signaling as immunomodulators. Interleukin-2 (IL-2) is an interleukin, i.e., a type of cytokine signaling molecule in the immune system. It is a 15.5-16kDa protein that regulates the activity of leukocytes responsible for immunity. Interleukin-15 (IL-15) is a cytokine with a similar structure to interleukin-2. Like IL-2, IL-15 binds to and signals through a complex consisting of the IL-2 / IL-15 receptor β chain and a common γ chain. IL-15 is secreted by mononuclear phagocytes after viral infection. Interleukin-21 is a cytokine that has a powerful regulatory effect on cells of the immune system, including natural killer cells and cytotoxic T cells that can destroy virally infected cells or cancer cells. Interleukin-12 (IL-12) is an interleukin naturally produced by dendritic cells, macrophages, neutrophils and human B lymphoblastoid cells (NC-37) in response to antigenic stimulation. In some embodiments, the cytokine is IL-2, IL-21, IL-12 or IL-15 or a fragment thereof. In some embodiments, the cytokine is IL-2 or IL-15 or a fragment thereof. In some embodiments, the cytokine is IL-2 or a fragment thereof. In some embodiments, the cytokine is IL-15 or a fragment thereof. In some embodiments, the therapeutic agent is a Fab or a fragment thereof.

[0283] Interferon (IFN) is a group of signaling proteins belonging to a class of proteins called cytokines, which are molecules used for communication between cells to trigger the protective defense of the immune system that helps eradicate pathogens. In some embodiments, the cytokine is interferon alpha, interferon beta or interferon gamma or a fragment thereof.

[0284] Granulocyte-macrophage colony-stimulating factor, also known as colony stimulating factor 2, is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells and fibroblasts that acts as a cytokine. In some embodiments, the cytokine is granulocyte-macrophage colony-stimulating factor GM-CSF.

[0285] In some embodiments, the therapeutic agent is an engineered antibody fragment. In some embodiments, the engineered antibody fragment is a bispecific T cell engager. Bispecific T cell engagers (BiTEs) are a class of artificial bispecific monoclonal antibodies studied as anticancer drugs. They direct the host's immune system against cancer cells, more specifically the cytotoxic activity of T cells. In some embodiments, the therapeutic agent is a bispecific T cell engager (BiTE) or a fragment thereof.

[0286] In some embodiments, the therapeutic agent is a small molecule. In some embodiments, the therapeutic agent is a small molecule with a molecular weight of less than 900 daltons. In some embodiments, the small molecule is maytansine, paclitaxel, doxorubicin, temozolomide, sunitinib, dacarbazine, gemcitabine, melphalan, fenretinide or its derivatives, or EGFR-TKI (tyrosine kinase inhibitor). In some embodiments, the small molecule is maytansine, temozolomide, sunitinib, dacarbazine, gemcitabine, melphalan, fenretinide or its derivatives, or EGFR-TKI (tyrosine kinase inhibitor). In some embodiments, the small molecule is not doxorubicin or paclitaxel. In some embodiments, the small molecule is maytansine or its derivatives. Maitansine or maytansine are cytotoxic agents. It inhibits the assembly of microtubules by binding to tubulin at the root mycin binding site. It is a macrolide of ansamycin type and can be isolated from Maytenus plants. Derivatives are called maytansinoids. Maytansine and its analogs (maytansinoid DM1 and DM4) are potent microtubule targeting compounds that inhibit cell proliferation during mitosis. It inhibits the assembly of microtubules by binding to tubulin at the root mycin binding site. In certain embodiments, small molecules are maytansinoid DM1 (Motansen) or its derivatives; or maytansinoid DM4 or its derivatives. In certain embodiments, maytansine has any structure in the following structures:

[0287]

[0288] In certain embodiments, the block copolymer comprises a fluorescent dye conjugated to the block copolymer via an amine. In some embodiments, the fluorescent dye is conjugated to the hydrophobic block of the block copolymer via an amine on the block copolymer. In some embodiments, the fluorescent dye is a cyanine dye or a derivative thereof. In some embodiments, the fluorescent dye is indocyanine green (ICG) or a derivative thereof. Indocyanine green (ICG) is used in medical diagnosis. In some embodiments, the structure of the ICG derivative is:

[0289]

[0290] In one aspect, the compounds described herein are in the form of pharmaceutically acceptable salts. Likewise, active metabolites of these compounds having the same type of activity are also included within the scope of the present disclosure. In addition, the compounds described herein may exist in unsolvated forms as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. The solvated forms of the compounds presented herein are also considered to be disclosed herein.

[0291] II. Micelles and Compositions

[0292] One or more block copolymers described herein can be used to form pH-sensitive micellar compositions. In some embodiments, the composition comprises a single type of micelle. In some embodiments, two or more different types of micelles can be combined to form a mixed micellar composition. In some embodiments, the micelle comprises a block copolymer covalently conjugated to a therapeutic agent. In some embodiments, the micelle comprises one or more block copolymers that non-covalently encapsulate a therapeutic agent.

[0293] In some embodiments, the block copolymer of formula (I), (Ia), (Ib) or (Ic) or a pharmaceutically acceptable salt, solvate or hydrate thereof is in the form of micelles. In some embodiments, the block copolymer of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof is in the form of micelles. In some embodiments, the block copolymer of formula (Ic) or a pharmaceutically acceptable salt, solvate or hydrate thereof is in the form of micelles.

[0294] In some embodiments, the block copolymer of formula (II), (II-a), (II-b) or (II-c) or a pharmaceutically acceptable salt, solvate or hydrate thereof is in the form of micelles. In some embodiments, the block copolymer of formula (II) or a pharmaceutically acceptable salt, solvate or hydrate thereof is in the form of micelles. In some embodiments, the block copolymer of formula (II-c) or a pharmaceutically acceptable salt, solvate or hydrate thereof is in the form of micelles.

[0295] In another aspect, presented herein is a micelle comprising:

[0296] (i) a block copolymer having a structure of formula (III) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0297]

[0298] in:

[0299] n3 is an integer between 10 and 200;

[0300] x3 is an integer between 40 and 300;

[0301] y3 is an integer from 0 to 6;

[0302] z3 is an integer from 0 to 10;

[0303] X 3 is halogen, -OH or -C(O)OH;

[0304] Each R 10 independently hydrogen or ICG;

[0305] R 8 and R 9 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0306] or R 8 and R 9 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring; and

[0307] (ii) a therapeutic agent encapsulated by the block copolymer.

[0308] In some embodiments, the encapsulation is non-covalent encapsulation, wherein the therapeutic agent is physically located within the micelle. In some embodiments, the therapeutic agent is non-covalently encapsulated.

[0309] Therapeutic agents can be incorporated into micelles using methods known in the art. In some embodiments, the therapeutic agent is a cytokine or a fragment thereof, an engineered antibody fragment, or a small molecule with a molecular weight of less than 900 Daltons. In some embodiments, the cytokine is IL-2, IL-21, IL-12, or IL-15, or a fragment thereof. In some embodiments, the cytokine is IL-2 or IL-15, or a fragment thereof. In some embodiments, the cytokine is IL-2 or a fragment thereof. In some embodiments, the cytokine is IL-15, or a fragment thereof. In some embodiments, the cytokine is interferon alpha, interferon beta, or interferon gamma, or a fragment thereof. In some embodiments, the cytokine is a Fab, or a fragment thereof. In some embodiments, the engineered antibody fragment is a bispecific T cell adaptor (BiTE), or a fragment thereof. In some embodiments, the small molecule is maytansine, paclitaxel, doxorubicin, temozolomide, sunitinib, dacarbazine, gemcitabine, melphalan, fenretinide or its derivatives, or EGFR-TKI (tyrosine kinase inhibitor). In some embodiments, the small molecule is maytansine or its derivatives.

[0310] In some embodiments, when y3 and z3 are both 0, the block copolymer of formula (III) does not non-covalently encapsulate paclitaxel or doxorubicin.

[0311] In some embodiments of the micelle, the block copolymer of formula (III) has a structure of formula (III-c) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0312]

[0313] In some embodiments, the micelle comprises (i) a block copolymer of formula (III-c) and (ii) a therapeutic agent non-covalently encapsulated by the block copolymer. In some embodiments, the therapeutic agent is a cytokine or a fragment thereof, or an engineered antibody fragment or a small molecule with a molecular weight of less than 900 Daltons. In some embodiments, the therapeutic agent is a cytokine or a fragment thereof. In some embodiments, the cytokine is IL-2 or a fragment thereof. In some embodiments, the engineered antibody fragment is a bispecific T-cell adapter (BiTE) or a fragment thereof.

[0314] In another aspect, presented herein is a micelle comprising:

[0315] (i) a block copolymer having a structure of formula (III) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0316]

[0317] in:

[0318] n3 is an integer between 10 and 200;

[0319] x3 is an integer between 40 and 300;

[0320] y3 is an integer from 0 to 6;

[0321] z3 is an integer from 0 to 10;

[0322] X 3 is halogen, -OH or -C(O)OH;

[0323] Each R 10 independently hydrogen or ICG;

[0324] R 8 and R 9 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0325] or R 8 and R 9 together with the corresponding nitrogen to which they are attached, form an optionally substituted 5- to 7-membered ring; and

[0326] (ii) a block copolymer having a structure of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0327]

[0328] in:

[0329] n1 is an integer between 10 and 200;

[0330] x1 is an integer between 40 and 300;

[0331] y1 is an integer from 0 to 6;

[0332] z1 is an integer from 0 to 10;

[0333] X 1 is halogen, -OH or -C(O)OH;

[0334] R 1 and R 2 are each independently an optionally substituted C1-C6 alkyl, C3-C 10Cycloalkyl or aryl;

[0335] or R 1 and R 2 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0336] Each R 3 are independently hydrogen, acyl or ICG;

[0337] L 1 is a bond or -C(O)-, or an optionally substituted C1-C 10 Alkylene linker or PEG linker;

[0338] Y is a therapeutic agent; or

[0339] (ii) a block copolymer having a structure of formula (II) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0340]

[0341] in:

[0342] n2 is an integer from 2 to 200;

[0343] x2 is an integer between 40 and 300;

[0344] y2 is an integer from 0 to 6;

[0345] X 2 is halogen, -OH or -C(O)OH;

[0346] R 5 and R 6 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0347] or R 5 and R 6 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0348] Each R 7 are independently hydrogen, acyl or ICG;

[0349] Z 1 is -NH- or -O-;

[0350] Z 2 is -NH-, -O- or a substituted triazole;

[0351] L 2is a bond or -C(O)-, or an optionally substituted C1-C 10 an alkylene linker or a PEG linker; and

[0352] Y is a therapeutic agent.

[0353] In another aspect is a micelle comprising:

[0354] (i) a block copolymer having a structure of formula (III) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0355]

[0356] in:

[0357] n3 is an integer between 10 and 200;

[0358] x3 is an integer between 40 and 300;

[0359] y3 is an integer from 0 to 6;

[0360] z3 is an integer from 0 to 10;

[0361] X 3 is halogen, -OH or -C(O)OH;

[0362] Each R 10 independently hydrogen or ICG;

[0363] R 8 and R 9 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 cycloalkyl or aryl; and

[0364] or R 8 and R 9 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0365] (ii) a block copolymer having a structure of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0366]

[0367] in:

[0368] n1 is an integer between 10 and 200;

[0369] x1 is an integer between 40 and 300;

[0370] y1 is an integer from 0 to 6;

[0371] z1 is an integer from 0 to 10;

[0372] X 1 is halogen, -OH or -C(O)OH;

[0373] R 1 and R 2 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0374] or R 1 and R 2 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0375] Each R 3 are independently hydrogen, acyl or ICG;

[0376] L 1 is a bond or -C(O)-, or an optionally substituted C1-C 10 an alkylene linker or a PEG linker; and

[0377] Y is a therapeutic agent; and

[0378] (iii) a block copolymer having a structure of formula (II) or a pharmaceutically acceptable salt, solvate or hydrate thereof:

[0379]

[0380] in:

[0381] n2 is an integer from 2 to 200;

[0382] x2 is an integer between 40 and 300;

[0383] y2 is an integer from 0 to 6;

[0384] X 2 is halogen, -OH or -C(O)OH;

[0385] R 5 and R 6 are each independently an optionally substituted C1-C6 alkyl, C3-C 10 Cycloalkyl or aryl;

[0386] or R 5 and R 6 together with the corresponding nitrogen to which it is attached, form an optionally substituted 5- to 7-membered ring;

[0387] Each R 7are independently hydrogen, acyl or ICG;

[0388] Z 1 is -NH- or -O-;

[0389] Z 2 is -NH-, -O- or a substituted triazole residue;

[0390] L 2 is a bond or -C(O)-, or an optionally substituted C1-C 10 an alkylene linker or a PEG linker; and

[0391] Y is a therapeutic agent.

[0392] In some embodiments of Formula (III) or (III-c), R 8 and R 9 In some embodiments, R 8 and R 9 Are different groups.

[0393] In some embodiments of Formula (III) or (III-c), each R 8 and R 9 R is independently an optionally substituted C1-C6 alkyl. In some embodiments, the alkyl group is a straight chain or branched chain alkyl group. In some embodiments, the alkyl group is a straight chain alkyl group. In some embodiments, each R 8 and R 9 is independently -CH2CH3, -CH2CH2CH3 or -CH2CH2CH2CH3. In some embodiments, each R 8 and R 9 is -CH2CH2CH2CH3. In some embodiments, each R 8 and R 9 is independently an optionally substituted C3-C 10 In some embodiments, each R 8 and R 9 R is independently optionally substituted cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. 8 and R 9 Independently is optionally substituted phenyl.

[0394] In some embodiments of Formula (III) or (III-c), R 8 and R 8 Together with the corresponding nitrogen to which it is attached, it forms an optionally substituted 5- to 7-membered ring. 8 and R 9Together they are -CH2(CH2)2CH2-, -CH2(CH2)3CH2-, or -CH2(CH2)4CH2-. In some embodiments, R 8 and R 9 Together they are -CH2(CH2)4CH2-.

[0395] In some embodiments, micelles include one or more different types of block copolymer components from various monomers. In some embodiments, micelles include (i) block copolymers of formula (III) and (ii) block copolymers of formula (I) or formula (II). In some embodiments, micelles include components (i) to (ii) in a ratio of 1:99 to 99:1; or any ratio therein. In some embodiments, micelles include components (i) and (ii) in a ratio of 1:99, 10:90, 20:80, 30:70, 40:50, or 50:50. In some embodiments, micelles include components (i) and (ii) in a ratio of 1:1.

[0396] In some embodiments, the micelle comprises a block copolymer of formula (III) and a block copolymer of formula (I) at a ratio of 1:99. In some embodiments, the micelle comprises a block copolymer of formula (III) and a block copolymer of formula (I) at a ratio of 99:1. In some embodiments, the micelle comprises a block copolymer of formula (III) and a block copolymer of formula (II) at a ratio of 1:99. In some embodiments, the micelle comprises a block copolymer of formula (III) and a block copolymer of formula (II) at a ratio of 99:1.

[0397] In some embodiments, the micelle comprises (i) a block copolymer of formula (III); (ii) a block copolymer of formula (I); and (iii) a block copolymer of formula (II). In some embodiments, the micelle comprises equal portions of components (i), (ii), and (iii). In some embodiments, the micelle comprises unequal portions of components (i), (ii), and (iii).

[0398] In some embodiments, each different type of block copolymer is conjugated to a different therapeutic agent. In some embodiments, each different type of block copolymer is conjugated to the same therapeutic agent.

[0399] In another aspect, presented herein are micelles comprising: (i) a block copolymer of formula (III); (ii) a block copolymer of formula (I) and / or a block copolymer of formula (II); and (iii) a therapeutic agent encapsulated by the block copolymer. In some embodiments, the therapeutic agent is non-covalently encapsulated within the micelle.

[0400] The use of micelles in cancer therapy can enhance anti-tumor efficacy and reduce toxicity to healthy tissues, in part due to the size of the micelles. Although small molecules such as certain chemotherapeutic agents can enter both normal tissues and tumor tissues, non-targeted micellar nanoparticles can preferentially pass through leaky tumor vessels. The size of the micelle will generally be nanoscale (i.e., a diameter between about 1nm and 1 μm). In some embodiments, the size of the micelle is about 10 to about 200nm. In some embodiments, the size of the micelle is about 20 to about 100nm. In some embodiments, the size of the micelle is about 30 to about 50nm. In some embodiments, the diameter of the micelle is less than about 1 μm. In some embodiments, the diameter of the micelle is less than about 100nm. In some embodiments, the diameter of the micelle is less than about 50nm.

[0401] pH responsive compositions

[0402] In another aspect, presented herein are pH responsive compositions. The pH responsive compositions disclosed herein include one or more pH responsive micelles and / or nanoparticles comprising a block copolymer and a therapeutic agent. Each block copolymer includes a hydrophilic polymer segment and a hydrophobic polymer segment, wherein the hydrophobic polymer segment includes an ionizable amine group to provide pH sensitivity. This pH sensitivity is utilized to provide a composition suitable as a drug / therapeutic conjugate therapeutic agent.

[0403] Micelles can have different pH transition values ​​within the physiological range to target specific cells or microenvironments. In some embodiments, the pH transition value of the micelle is about 5 to about 8, or any value therein. In some embodiments, the pH transition value of the micelle is about 5 to about 6. In some embodiments, the pH transition value of the micelle is about 6 to about 7. In some embodiments, the pH transition value of the micelle is about 7 to about 8. In some embodiments, the pH transition value of the micelle is about 6.3 to about 6.9. In some embodiments, the pH transition value of the micelle is about 5.0 to about 6.2. In some embodiments, the pH transition value of the micelle is about 5.9 to about 6.2. In some embodiments, the pH transition value of the micelle is about 5.0 to about 5.5. In some embodiments, the pH transition point is 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5. In some embodiments, the pH transition point is about 4.8. In some embodiments, the pH transition point is about 4.9. In some embodiments, the pH transition point is about 5.0. In some embodiments, the pH transition point is about 5.1. In some embodiments, the pH transition point is about 5.2. In some embodiments, the pH transition point is about 5.3. In some embodiments, the pH transition point is about 5.4. In some embodiments, the pH transition point is about 5.5.

[0404] Compared with other pH-sensitive compositions with very wide pH responses (i.e., 2 pH units), the pH-sensitive micellar compositions of the present disclosure can advantageously have a narrow pH transition range. In some embodiments, the pH transition range of the micelle is less than about 1 pH unit. In various embodiments, the pH transition range of the micelle is less than about 0.9, less than about 0.8, less than about 0.7, less than about 0.6, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1 pH units. In some embodiments, the pH transition range of the micelle is less than about 0.5 pH units. In some embodiments, the pH transition range of the micelle is less than about 0.25 pH units. The narrow pH transition range advantageously provides a more dramatic pH response, wherein the micelle can be opened to release cargo at a specific location (e.g., in a tumor or a specific organelle).

[0405] In some embodiments, the pH responsive composition has an emission spectrum. In some embodiments, the emission spectrum is 600-800 nm. In some embodiments, the emission spectrum is 700-800 nm.

[0406] III. How to use

[0407] Aerobic glycolysis, known as the Warburg effect, in which cancer cells preferentially take up glucose and convert it to lactate or other acids, occurs in all solid cancers. Lactic acid or other acids preferentially accumulate in the extracellular space due to monocarboxylate transporters or other transporters. The resulting acidification of the extracellular space promotes remodeling of the extracellular matrix for further tumor invasion and metastasis.

[0408] Some embodiments provided herein describe compounds that form micelles at physiological pH (7.35-7.45). In some embodiments, the compounds described herein are covalently or non-covalently conjugated to a therapeutic agent. In some embodiments, the molecular weight of the micelle is greater than 2×10 7 Daltons. In some embodiments, the molecular weight of the micelle is about 2.7×10 7 Daltons. In some embodiments, the therapeutic agent is sequestered within the micelle core at physiological pH (7.35-7.45) (e.g., during blood circulation). In some embodiments, when the micelle encounters an acidic environment (e.g., tumor tissue), the micelle dissociates into individual compounds, such as those with an average molecular weight of about 3.7×10 4 Dalton diblock copolymer monomers, thereby releasing the therapeutic agent. In some embodiments, the micelles dissociate at a pH below the pH transition point (eg, the acidic state of the tumor microenvironment).

[0409] In some embodiments, the therapeutic agent can be incorporated into the interior of the micelle. Specific pH conditions (e.g., the acidic pH present in tumors and endocytic compartments) may cause the micelles to rapidly protonate and dissociate into monomers, thereby releasing the therapeutic agent (e.g., drug). In some embodiments, the micelles provide stable drug encapsulation at physiological pH (pH 7.4), but can rapidly release the drug in an acidic environment.

[0410] In some cases, the pH sensitive micellar compositions described herein have a narrow pH transition range. In some embodiments, the pH transition range (ΔpH 10-90％ ) is less than 1 pH unit. In various embodiments, the pH transition range of the micelle is less than about 0.9, less than about 0.8, less than about 0.7, less than about 0.6, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1 pH unit. In some embodiments, the pH transition range of the micelle is less than about 0.5 pH unit. In some embodiments, the pH transition range is less than 0.25 pH unit. In some embodiments, the pH transition range is less than 0.15 pH unit. This sharp transition point allows the micelle to dissociate from the acidic pH of the tumor microenvironment.

[0411] Micelles described herein can be used as drug delivery agents. Micelles comprising drugs can be used to treat, for example, cancer or other diseases, where due to local pH differences (e.g., pH different from physiological pH (7.4)), the drug can be delivered to the appropriate location. In some embodiments, the condition treated is cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the tumor is a secondary tumor from a primary tumor metastasis. In some embodiments, drug delivery can reach lymph nodes or reach the peritoneal surface or pleural surface.

[0412] In some embodiments is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the block copolymers, micelles, or compositions disclosed herein.

[0413] In some embodiments, the cancer is carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma.

[0414] In some embodiments, the tumor is from cancer. In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, bladder cancer, urethral cancer, kidney cancer, esophageal cancer, colorectal cancer, peritoneal metastasis, brain cancer or skin cancer (including melanoma and sarcoma). In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), esophageal cancer, kidney cancer or colorectal cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma (NHSCC). In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is colorectal cancer.

[0415] In some embodiments, the cancer is a solid tumor.

[0416] In some embodiments, the tumor is reduced by about 5%, about 10%, about 15%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In some embodiments, the tumor is reduced by about 50%. In some embodiments, the tumor is reduced by about 60%. In some embodiments, the tumor is reduced by about 70%. In some embodiments, the tumor is reduced by about 75%. In some embodiments, the tumor is reduced by about 80%. In some embodiments, the tumor is reduced by about 85%. In some embodiments, the tumor is reduced by about 90%. In some embodiments, the tumor is reduced by about 95%. In some embodiments, the tumor is reduced by about 99%.

[0417] In some embodiments, the cancer is not a solid tumor.

[0418] Dosage and treatment regimen

[0419] The pharmaceutical compositions disclosed herein may be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are set forth herein. In some embodiments, the pharmaceutical compositions disclosed herein are in the form of administration or administration by oral, intravenous (IV), intramuscular, subcutaneous, intradermal injection, or intratumoral injection. In some embodiments, the pharmaceutical compositions are formulated for oral administration, intramuscular administration, subcutaneous administration, or intravenous administration. In some embodiments, the pharmaceutical compositions are formulated for intravenous administration. In some embodiments, the pharmaceutical compositions are formulated for aqueous solutions or suspensions for intravenous (IV) administration. In some embodiments, the pharmaceutical compositions are formulated for single-dose administration. In some embodiments, the pharmaceutical compositions disclosed herein are formulated for administration by IV bolus injection. In some embodiments, the pharmaceutical compositions disclosed herein are formulated for administration by injection into a tumor.

[0420] In some embodiments, compositions containing compounds disclosed herein are administered for preventive and / or therapeutic treatments. In certain therapeutic applications, compositions are administered to patients who already have a disease or condition in an amount sufficient to cure or at least partially prevent at least one of the symptoms of the disease or condition. The amount effective for this use depends on the severity and course of the disease or condition, previous therapy, the patient's health, body weight, and response to the drug, and the judgment of the attending physician. The therapeutically effective amount is optionally determined by methods including, but not limited to, a dose escalation clinical trial.

[0421] Typical dosage ranges are from about 0.001 mg / kg to about 100 mg / kg per dose. In some embodiments, the dosage range is from about 0.01 mg / kg to about 50 mg / kg. In some embodiments, additional dosage ranges are from about 0.05 mg / kg to about 10 mg / kg per dose. In some embodiments, the dosage is about 50 mg / kg. In some embodiments, the dosage is about 100 mg / kg. The exact dosage will depend on the frequency of administration and mode of administration, the sex, age, weight and general health of the subject being treated, the nature and severity of the condition being treated, and any concomitant diseases to be treated, as well as other factors apparent to those skilled in the art.

[0422] In some embodiments, the dose of the composition being administered may be temporarily reduced or temporarily suspended for a certain length of time (ie, a "drug holiday").

[0423] In some embodiments, the method comprises administering the composition once. In some embodiments, the method comprises administering the composition two or more times. In some embodiments, the composition is administered once daily.

[0424] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0425] Combination therapy

[0426] In another aspect, the compositions disclosed herein are administered with one or more additional therapies. In some embodiments, the method further comprises a second anticancer therapy. In some embodiments, the second anticancer therapy is surgery, chemotherapy, radiotherapy, gene therapy, or immunotherapy. In some embodiments, the second anticancer therapy is immunotherapy. In some embodiments, immunotherapy is checkpoint therapy. In some embodiments, the second anticancer therapy is radiotherapy. In some embodiments, the second therapy is surgery.

[0427] definition

[0428] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one of ordinary skill in the art will appreciate that the present invention can be practiced without these details. In other instances, well-known structures are not shown or described in detail to avoid unnecessary confusion in the description of the embodiments. Unless the context otherwise requires, throughout the specification and the appended claims, the words "comprise" and variations thereof, such as "comprises" and "comprising", will be interpreted in an open and inclusive sense, i.e., interpreted as "including but not limited to". Further, the titles provided herein are merely for convenience and do not explain the scope or meaning of the claimed invention.

[0429] As used in this specification and the appended claims, the singular forms "a / an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in a sense including "and / or" unless the content clearly dictates otherwise.

[0430] Unless otherwise specified, the following terms used herein have the following meanings:

[0431] "Oxo" refers to a =0 substituent.

[0432] "Thioxo" refers to a =S substituent.

[0433] "Alkyl" refers to a straight or branched hydrocarbon chain radical having from one to twenty carbon atoms and which is attached to the rest of the molecule by a single bond. Alkyl groups comprising up to 10 carbon atoms are referred to as C1-C 10 Alkyl groups, for example, include up to 6 carbon atoms is C1-C6 alkyl. Alkyl groups (and other moieties defined herein) including other numbers of carbon atoms are represented in a similar manner. Alkyl groups include but are not limited to C1-C6. 10Alkyl, C1-C9 alkyl, C1-C8 alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C3-C8 alkyl and C4-C8 alkyl.Representative alkyl includes but is not limited to methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, isobutyl, sec-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, 1-ethyl-propyl etc.In certain embodiments, alkyl is methyl, ethyl, sec-butyl or 1-ethyl-propyl.Unless otherwise specifically stated in the specification, alkyl can be optionally substituted as described below." alkylene" or "alkylene chain" refers to the straight or branched divalent hydrocarbon chain connecting the rest of the molecule to a free radical.In certain embodiments, alkylene is saturated. In some embodiments, the alkylene group is -CH2-, -CH2CH2-, or -CH2CH2CH2-. In some embodiments, the alkylene group is -CH2-. In some embodiments, the alkylene group is -CH2CH2-. In some embodiments, the alkylene group is -CH2CH2-.

[0434] "Alkoxy" refers to a radical of the formula -OR, where R a is an alkyl group as defined. Unless otherwise specifically stated in the specification, an alkoxy group may be optionally substituted as described below. Representative alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, and pentoxy. In some embodiments, an alkoxy group is a methoxy group. In some embodiments, an alkoxy group is an ethoxy group.

[0435] "Assorted alkylene" refers to an alkyl group as described above, wherein one or more carbon atoms in the alkyl group are replaced by O, N or S atoms. "Assorted alkylene" or "assorted alkylene chain" refers to a straight or branched divalent heteroalkyl chain that connects the remainder of the molecule to a free radical. Unless otherwise specifically stated in the specification, assorted alkyl or assorted alkylene can be optionally substituted as described below. Representative assorted alkylene includes, but is not limited to, -OCHOMe, -OCHCHOMe or -OCHCHOCHCHNH. Representative assorted alkylene includes, but is not limited to, -OCHCHO-, -OCHCHOCHCHO- or -OCHCHOCHCHOCHCHO-.

[0436] "Alkylamino" refers to a radical of the formula -NHR or -NRR, wherein each R is independently an alkyl group as defined above. Unless stated otherwise specifically in the specification, an alkylamino group may be optionally substituted as described below.

[0437] The term "aromatic" refers to a planar ring having a delocalized π-electron system containing 4n+2π electrons, where n is an integer. Aromatics may be optionally substituted. The term "aromatic" includes both aryl (e.g., phenyl, naphthyl) and heteroaryl (e.g., pyridyl, quinolinyl).

[0438] "Aryl" refers to an aromatic ring in which each of the atoms forming the ring is a carbon atom. Aryl can be optionally substituted. Examples of aryl include, but are not limited to, phenyl and naphthyl. In some embodiments, aryl is phenyl. Depending on the structure, aryl can be a monoradical or a diradical (i.e., arylene). Unless otherwise specifically stated in the specification, the term "aryl" or the prefix "ar-" (as in "aralkyl") means an aryl group that is optionally substituted.

[0439] "Carboxyl" refers to -CO2H. In some embodiments, the carboxyl moiety can be replaced with a "carboxylic acid bioisostere," which refers to a functional group or moiety that exhibits physical and / or chemical properties similar to a carboxylic acid moiety. The biological properties of a carboxylic acid bioisostere are similar to those of a carboxylic acid group. A compound having a carboxylic acid moiety can have a carboxylic acid moiety exchanged with a carboxylic acid bioisostere and have similar physical and / or biological properties when compared to a compound containing a carboxylic acid. For example, in one embodiment, a carboxylic acid bioisostere will ionize at physiological pH to about the same extent as a carboxylic acid group. Examples of bioisosteres of carboxylic acids include, but are not limited to:

[0440]

[0441] "Cycloalkyl" refers to a monocyclic or polycyclic non-aromatic radical, in which each atom in the atoms (i.e., backbone atoms) forming the ring is a carbon atom. Cycloalkyl can be saturated or partially unsaturated. Cycloalkyl can be fused with an aromatic ring (in this case, the cycloalkyl is bonded by a non-aromatic ring carbon atom). Cycloalkyl includes groups with 3 to 10 ring atoms. In some embodiments, cycloalkyl is a C3-C6 cycloalkyl. In some embodiments, cycloalkyl is a 3- to 6-membered cycloalkyl. Representative cycloalkyls include, but are not limited to, cycloalkyls with three to ten carbon atoms, three to eight carbon atoms, three to six carbon atoms, or three to five carbon atoms. Monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, monocyclic cycloalkyls are cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decahydronaphthyl, and 3,4-dihydronaphthalene-1 (2H)-one. Unless stated otherwise specifically in the specification, a cycloalkyl group may be optionally substituted.

[0442] "Fused" refers to any ring structure described herein that is fused to an existing ring structure. When the fused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atom on the existing ring structure that becomes part of the fused heterocyclyl ring or the fused heteroaryl ring may be replaced by a nitrogen atom.

[0443] "Halo" or "halogen" refers to bromo, chloro, fluoro or iodo.

[0444] "Haloalkyl" refers to an alkyl group as defined above substituted with one or more halo groups as defined above, for example, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, etc. Unless specifically stated otherwise in the specification, a haloalkyl group may be optionally substituted.

[0445] "Haloalkyl" refers to an alkoxy group as defined above substituted with one or more halo groups as defined above, for example, trifluoromethoxy, difluoromethoxy, fluoromethoxy, trichloromethoxy, 2,2,2-trifluoroethoxy, 1,2-difluoroethoxy, 3-bromo-2-fluoropropoxy, 1,2-dibromoethoxy, etc. Unless specifically stated otherwise in the specification, a haloalkoxy group may be optionally substituted.

[0446] "Heterocycloalkyl" or "heterocyclyl" or "heterocycle" refers to a stable 3- to 14-membered non-aromatic ring radical comprising 2 to 13 carbon atoms and one to 6 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. In some embodiments, heterocycloalkyl is C2-C7 heterocycloalkyl. In some embodiments, heterocycloalkyl is C2-C6 heterocycloalkyl. In some embodiments, heterocycloalkyl is C2-C5 heterocycloalkyl. In some embodiments, heterocycloalkyl is 3- to 8-membered heterocycloalkyl. In some embodiments, heterocycloalkyl is 3- to 7-membered heterocycloalkyl. In some embodiments, heterocycloalkyl is 3- to 6-membered heterocycloalkyl. In some embodiments, heterocycloalkyl is 3- to 5-membered heterocycloalkyl. Unless otherwise specifically stated in the specification, heterocycloalkyl can be a monocyclic or bicyclic system, which can contain a fused ring system (when fused to an aryl ring or a heteroaryl ring, the heterocycloalkyl is bonded by a non-aromatic ring atom) or a bridged ring system. The nitrogen atom, carbon atom or sulfur atom in the heterocyclic group may be optionally oxidized. The nitrogen atom may be optionally quaternized. The heterocycloalkyl group is partially or completely saturated. Examples of such heterocycloalkyl groups include, but are not limited to, dioxolanyl, thienyl [1,3] dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidone, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithienyl, tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes all ring forms of carbohydrates, including but not limited to monosaccharides, disaccharides and oligosaccharides. Unless otherwise specified, heterocycloalkyl has 2 to 10 carbons in the ring. In certain embodiments, heterocycloalkyl has 2 to 8 carbons in the ring. In certain embodiments, heterocycloalkyl has 2 to 8 carbons and 1 or 2 N atoms in the ring. It should be understood that when referring to the number of carbon atoms in heterocycloalkyl, the number of carbon atoms in heterocycloalkyl is different from the total number (including heteroatoms) of the atoms (that is, the skeleton atoms of heterocycloalkyl ring) constituting heterocycloalkyl. Unless otherwise specified in the specification, heterocycloalkyl can be optionally substituted.

[0447] "Heteroaryl" refers to an aryl group containing one or more ring heteroatoms selected from nitrogen, oxygen and sulfur. Heteroaryl is monocyclic or bicyclic. In some embodiments, heteroaryl is a 5-membered or 6-membered heteroaryl. In some embodiments, heteroaryl is a 5-membered heteroaryl. In some embodiments, heteroaryl is a 6-membered heteroaryl. Illustrative examples of monocyclic heteroaryl groups include pyridyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, furazolyl, indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine and pteridine. Illustrative examples of monocyclic heteroaryls include pyridyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl and furazolyl. Illustrative examples of bicyclic heteroaryls include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine and pteridine. In certain embodiments, heteroaryl is pyridyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl or furanyl. In certain embodiments, heteroaryl contains 0-4 N atoms in the ring. In certain embodiments, heteroaryl contains 1-4 N atoms in the ring. In some embodiments, the heteroaryl group contains 0-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl group contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring.

[0448] The term "optionally substituted" or "substituted" means that the group mentioned can be substituted by one or more additional groups individually and independently selected from the following: alkyl, haloalkyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, -OH, alkoxy, aryloxy, alkylthio, arylthio, alkyl sulfoxide, aryl sulfoxide, alkyl sulfone, aryl sulfone, -CN, alkyne, C1-C6 alkylalkyne, halogen, acyl, acyloxy, -CO2H, -CO2alkyl, nitro and amino, including mono- and di-substituted amino (e.g., -NH2, -NHR, -N(R)2) and protected derivatives thereof. In some embodiments, the optional substituents are independently selected from alkyl, alkoxy, haloalkyl, cycloalkyl, halogen, -CN, -NH2, -NH(CH3), -N(CH3)2, -OH, -CO2H and -CO2alkyl. In some embodiments, the optional substituents are independently selected from fluorine, chlorine, bromine, iodine, -CH3, -CH2CH3, -CF3, -OCH3 and -OCF3. In some embodiments, the optional substituents are independently selected from fluorine, chlorine, -CH3, -CF3, -OCH3 and -OCF3. In some embodiments, the substituted groups are substituted by one or two of the aforementioned groups. In some embodiments, the optional substituents on aliphatic carbon atoms (acyclic or cyclic, saturated or unsaturated carbon atoms, excluding aromatic carbon atoms) include oxo (=O).

[0449] "Maleimide residue" refers to a compound structure resulting from the reaction of a maleimide group with a thiol sulfur atom of, for example, a protein.

[0450] "Tautomer" refers to a proton transfer from one atom of a molecule to another atom of the same molecule. The compounds presented herein may exist in the form of tautomers. Tautomers are compounds that can be interconverted by the migration of a hydrogen atom, accompanied by the switching of a single bond and an adjacent double bond. In bonding arrangements where tautomerism may occur, a chemical equilibrium of tautomers will exist. All tautomeric forms of the compounds disclosed herein are contemplated. The exact ratio of tautomers depends on several factors, including temperature, solvent, and pH. Some examples of tautomeric interconversions include:

[0451]

[0452] As used herein, the terms "co-administered," "co-administered," and the like are intended to encompass administration of selected therapeutic agents to a single patient, and are intended to encompass treatment regimens in which the agents are administered by the same or different routes of administration, or at the same or different times.

[0453] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of an agent or compound administered that will alleviate to some extent one or more of the symptoms of the disease or condition being treated. The result may be a reduction and / or alleviation of the signs, symptoms, or causes of the disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a compound disclosed herein required to clinically significantly alleviate the symptoms of the disease. Techniques such as dose escalation studies can be used to determine the appropriate "effective" amount in any individual case.

[0454] Unless otherwise indicated, the following terms used in this application have the definitions given below. The use of the term "including" and other forms such as "include", "includes" and "included" is not limiting. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0455] As used herein, "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the block copolymer and is relatively nontoxic, i.e., the material can be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any component of the composition contained therein.

[0456] The term "pharmaceutically acceptable salt" refers to a form of a therapeutically active agent that consists of a cationic form of the therapeutically active agent in combination with a suitable anion, or in an alternative embodiment, consists of an anionic form of the therapeutically active agent in combination with a suitable cation. Handbook of Pharmaceutical Salts: Properties, Selection and Use. International Union of Pure and Applied Chemistry, Wiley - VCH 2002. SM Berge, LD Bradley, DC Monkhouse, J. Pharm. Sci. Sci. 1977, 66, 1-19. PH Stahl and CG Wermuth, eds., Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zürich: Wiley-VCH / VHCA, 2002. Pharmaceutical salts are generally more soluble and more rapidly soluble in gastric and intestinal fluids than nonionic species and can therefore be used in solid dosage forms. Furthermore, since their solubility is generally a function of pH, selective dissolution in one part of the digestive tract or another is possible and this ability can be manipulated as an aspect of delayed and sustained release behavior. Likewise, since salt-forming molecules can equilibrate with the neutral form, passage through biological membranes can be regulated.

[0457] In some embodiments, a pharmaceutically acceptable salt is obtained by reacting the block copolymer with an acid. In some embodiments, the block copolymer disclosed herein (i.e., free base form) is alkaline and reacts with an organic acid or an inorganic acid. Inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and metaphosphoric acid. Organic acids include, but are not limited to, 1-hydroxy-2-naphthoic acid; 2,2-dichloroacetic acid; 2-hydroxyethanesulfonic acid; 2-oxoglutaric acid; 4-acetamidobenzoic acid; 4-aminosalicylic acid; acetic acid; adipic acid; ascorbic acid (L); aspartic acid (L); benzenesulfonic acid; benzoic acid; camphoric acid (+); camphor-10-sulfonic acid (+); caprylic acid (decanoic acid); caprylic acid (caproic acid); caprylic acid (octanoic acid); carbonic acid; cinnamic acid; citric acid; cyclohexylaminosulfonic acid; dodecyl sulfuric acid; ethane-1,2-disulfonic acid; ethanesulfonic acid; formic acid; fumaric acid; mucic acid; gentian acid; glucoheptanoic acid (D); gluconic acid (D); glucuronic acid (D); glutamic acid; glutaric acid; glycerophosphoric acid; glycolic acid; hippuric acid; isobutyric acid; lactic acid (DL); lactobionic acid; lauric acid; maleic acid; malic acid (-L); malonic acid; mandelic acid (DL); methanesulfonic acid; naphthalene-1,5-disulfonic acid; naphthalene-2-sulfonic acid; nicotinic acid; oleic acid; oxalic acid; palmitic acid; pamoic acid; phosphoric acid; propionic acid; pyroglutamic acid (-L); salicylic acid; sebacic acid; stearic acid; succinic acid; sulfuric acid; tartaric acid (+L); thiocyanic acid; toluenesulfonic acid (p); and undecylenic acid.

[0458] In some embodiments, the block copolymers disclosed herein are prepared as chloride salts, sulfate salts, bromide salts, methanesulfonate salts, maleate salts, citrate salts, or phosphate salts.

[0459] In some embodiments, pharmaceutically acceptable salts are obtained by reacting the block copolymer disclosed herein with a base. In some embodiments, the block copolymer disclosed herein is acidic and reacts with a base. In such cases, the acidic protons of the block copolymer disclosed herein are replaced by metal ions, such as lithium ions, sodium ions, potassium ions, magnesium ions, calcium ions or aluminum ions. In some cases, the block copolymers described herein can be coordinated with organic bases, such as but not limited to ethanolamine, diethanolamine, triethanolamine, tromethamine, meglumine, N-methylglucamine, dicyclohexylamine, tris(hydroxymethyl)methylamine. In other cases, the block copolymers described herein form salts with amino acids, such as but not limited to arginine, lysine, etc. The acceptable inorganic bases used to form salts with the block copolymers containing acidic protons include but are not limited to aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium hydroxide, lithium hydroxide, etc. In some embodiments, the block copolymers provided herein are prepared into sodium salts, calcium salts, potassium salts, magnesium salts, melamine salts, N-methylglucamine salts or ammonium salts.

[0460] It should be understood that reference to pharmaceutically acceptable salts includes solvent addition forms. In some embodiments, solvates contain a stoichiometric or non-stoichiometric amount of solvent and are formed during the process of crystallization with a pharmaceutically acceptable solvent such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of the compounds described herein are conveniently prepared or formed during the processes described herein. In addition, the compounds provided herein are optionally present in unsolvated forms as well as solvated forms.

[0461] The methods and formulations described herein include the use of N-oxides (if appropriate) or pharmaceutically acceptable salts of block copolymers having a structure of any of Formula (I), (Ia), (Ib), (I-b2), (Ic), (II), (II-a), (II-b), (II-b2), (III) or (III-c), as well as active metabolites of these compounds having the same type of activity.

[0462] In another embodiment, the compounds described herein are isotopically labeled (eg, with a radioisotope) or labeled by other means, including but not limited to the use of a chromophore or a fluorescent moiety, a bioluminescent label, or a chemiluminescent label.

[0463] The compounds described herein include isotopically labeled compounds which are identical to those set forth in the various formulas and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine, chlorine, iodine, phosphorus, such as 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 35 S. 18 F. 36 Cl, 123 I. 124 I. 125 I. 131 I. 32 P and 33 In one aspect, an isotopically labeled compound described herein, for example, is incorporated into 3 H and 14Compounds containing radioactive isotopes such as C are useful in drug and / or substrate tissue distribution assays. In one aspect, substitution with isotopes such as deuterium may result in greater metabolic stability, thereby conferring certain therapeutic advantages, such as increased in vivo half-life or reduced dosage requirements.

[0464] As used herein, "pH responsive system", "pH responsive composition", "micelle", "pH responsive micelle", "pH sensitive micelle", "pH activatable micelle" and "pH activatable micelle (pHAM) nanoparticle" are used interchangeably herein to indicate micelles comprising one or more compounds that dissociate according to pH (e.g., above or below a certain pH). As a non-limiting example, at a certain pH, the block copolymer of formula (II) is substantially in micellar form. As the pH changes (e.g., decreases), the micelles begin to dissociate, and as the pH further changes (e.g., further decreases), the block copolymer of formula (II) exists substantially in a dissociated (non-micelle) form.

[0465] As used herein, "pH transition range" indicates the pH range over which micelles dissociate.

[0466] As used herein, the "pH transition value" (pH) indicates the pH at which half of the micelles are dissociated.

[0467] "Nanoprobe" is used herein to refer to a pH-sensitive micelle that includes an imaging labeling moiety. In some embodiments, the labeling moiety is a fluorescent dye. In some embodiments, the fluorescent dye is an indocyanine green dye.

[0468] As used herein, the term "administer / administering / administration" and the like refers to methods that can be used to enable a compound or composition to be delivered to a desired biological site of action. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injections (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular or infusion), topical and rectal administration. Those skilled in the art are familiar with administration techniques that can be used with the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally. In some embodiments, the compositions described herein are administered intravenously.

[0469] As used herein, the terms "co-administered," "co-administered," and the like are intended to encompass administration of selected therapeutic agents to a single patient, and are intended to encompass treatment regimens in which the agents are administered by the same or different routes of administration, or at the same or different times.

[0470] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of an agent or compound administered that will alleviate to some extent one or more of the symptoms of the disease or condition being treated. Results include alleviation and / or relief of signs, symptoms, or causes of the disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a compound disclosed herein required to clinically significantly alleviate the symptoms of the disease. Techniques such as dose escalation studies are optionally used to determine an appropriate "effective" amount in any individual case.

[0471] As used herein, the term "enhance" or "enhancing" means to increase or prolong the potency or duration of a desired effect. Thus, with respect to enhancing the effect of a therapeutic agent, the term "enhance" refers to the ability to increase or prolong, in potency or duration, the effect of another therapeutic agent on a system. As used herein, an "enhancing-effective amount" refers to an amount sufficient to enhance the effect of another therapeutic agent in a desired system.

[0472] The term "subject" or "patient" encompasses mammals. Examples of mammals include, but are not limited to, any member of the mammalian species: humans, non-human primates, such as chimpanzees and other ape species and monkey species; farm animals, such as cattle, horses, sheep, goats, pigs; livestock, such as rabbits, dogs and cats; laboratory animals, including rodents, such as rats, mice and guinea pigs, etc. In one aspect, the mammal is a human.

[0473] As used herein, the terms "treat", "treating" or "treatment" include alleviating, reducing or ameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting a disease or condition, such as arresting the development of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating symptoms caused by a disease or condition, or prophylactically and / or therapeutically arresting the symptoms of a disease or condition.

[0474] The term "or" is used in the claims to mean "and / or" unless explicitly indicated to refer to only alternatives or the alternatives are mutually exclusive, but the present disclosure supports definitions referring to only alternatives as well as to "and / or". Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method used to determine the value. Under long-standing patent law, "a" and "an" when used with the word "including" in a claim or specification means one or more unless otherwise noted.

[0475] Examples

[0476] Example 1: Synthesis of block copolymers

[0477] General synthetic method

[0478] The block copolymers and micelles described herein are synthesized using standard synthetic techniques or using methods known in the art.

[0479] Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are employed. Block copolymers are prepared using standard organic chemistry techniques, such as those described in, for example, March's Advanced Organic Chemistry, 6th Edition, John Wiley and Sons, Inc.

[0480] Some abbreviations used in this article are as follows:

[0481] DCM: Dichloromethane

[0482] DMAP: 4-dimethylaminopyridine

[0483] DMF: Dimethylformamide

[0484] DMF-DMA: N,N-dimethylformamide dimethyl acetal

[0485] EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0486] EtOAc: Ethyl acetate

[0487] EtOH: Ethanol

[0488] FPLC fast protein liquid chromatography

[0489] ICG-OSu: Indocyanine green succinamide ester

[0490] MeOH: Methanol

[0491] PMDETA: N,N,N',N",N"-pentamethyldiethylenetriamine

[0492] CDI Carbonyldiimidazole

[0493] NHS-Carbonate N-Hydroxysuccinimide Carbonate

[0494] SPDB N-succinimidyl-4-(2-pyridyldithio)butyrate

[0495] TEA: Triethylamine

[0496] Hr Hours

[0497] Reanalysis of samples generated by ISR

[0498] IV

[0499] kg kilogram

[0500] mg milligram

[0501] mL milliliters

[0502] μg microgram

[0503] NC Not calculated

[0504] NR Not reported

[0505] Suitable PEG polymers can be purchased (e.g., purchased from Sigma Aldrich) or can be synthesized according to methods known in the art. In some embodiments, hydrophilic polymers can be used as initiators for polymerizing hydrophobic monomers to form block copolymers. For example, MPC polymers (e.g., narrowly distributed MPC polymers) can be prepared by atom transfer radical polymerization (ATRP) with commercially available small molecule initiators such as ethyl 2-bromo-2-methylpropionate (Sigma Aldrich). These resulting MPC polymers can be used as macromolecular ATRP initiators to further copolymerize with other monomers to form block polymers, which can be synthesized using atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT) methods.

[0506] In some embodiments, suitable block copolymers and micelles may be synthesized using standard synthetic techniques or using methods known in the art in conjunction with the methods described in Patent Publication Nos. WO 2012039741 and WO 2015188157, which are herein incorporated by reference in their entirety.

[0507] Example 2: Micelle Formation

[0508] General Methods

[0509] Methanol was added to the block copolymer in a glass round bottom flask and dissolved with the aid of an ultrasonic bath. After dissolution, the resulting solution was quantitatively transferred to a HDPE bottle containing a stirring bar and cooled to 0°C with an ice bath. While stirring, water was added dropwise to the methanol polymer solution in the HDPE bottle using a peristaltic pump. The HDPE bottle containing the polymer solution was maintained in an ice bath to form micelles. The micelles were then stirred for 100 min at 4 °C. 2 Micro-Ultrafiltration Modules Methanol was removed from the micellar solution using 5 cycles of tangential flow filtration (TFF).

[0510] PEG-PDBA-IL-2 formulation prepared by simple mixing

[0511] The polymer micelle aqueous solution was diluted with water for injection (WFI). A phosphate buffer containing 10% (w / w) of IL-2 (which accounts for % of the polymer) was added to prepare a solution of 1 mg / mL micelles and 0.1 mg / mL IL-2 by pipetting. The solution was incubated at room temperature for 10 minutes. The sample was then centrifuged at high speed (Eppendorf, 21,130xg, 10 minutes) in a microcentrifuge at ambient temperature. The solution was purified by membrane ultrafiltration (Amicon, 0.5mL, MWCO 100kDa) to remove any unencapsulated IL-2. Then 0.5mL of the formulation was added to an Amicon ultracentrifugation device and centrifuged at 5,000rcf for 2-3 minutes. The permeate was discarded, and the retentate containing the micellar IL-2 formulation was diluted to 0.5mL in water for injection. This process was repeated 10 times. The IL-2 concentration in the formulation was determined by protein blotting or dot blotting relative to a standard curve.

[0512] Purification of PDBA-IL-2 formulations by FPLC

[0513] PEG-PDBA-IL-2 non-covalent formulations or conjugates are prepared by one of the methods (e.g., simple mixing, acid-base titration, etc.). The crude PDBA-IL-2 formulation is purified by FPLC using an Akta Pure 25M (GE) system equipped with a Superdex 200Increase 10 / 300GL column (GE). Balance at a rate of 0.75 ml / min in 1X PBS. Sample injection is performed using a sample loop or super loop of appropriate size. Isocratic elution is performed at a flow rate of 0.5 ml / min in 1X PBS while monitoring the absorbance at multiple wavelengths (e.g., 214nm, 280nm, 700nm). Fractions (0.5mL) are collected in 1.5mL tubes. The fractions containing formulations and free proteins indicated by the chromatogram are analyzed by SDS-PAGE, Western blot or dot blot. The fractions containing IL-2 in the formulation are combined.

[0514] Double emulsion solvent evaporation (DESE) of PEG-PDBA-IL-2 formulation

[0515] A solution of 1.0 mg / mL of polymer in dichloromethane (DCM) and a phosphate buffer containing 1.0 mg / mL of IL-2 were cooled in an ice-water bath for 5 minutes. In an ice-water bath, under ultrasonic treatment conditions, the IL-2 solution was added dropwise to the polymer solution with a total amount of 10% (w / w, IL-2 / polymer) to form a first emulsion solution. In ice water, under ultrasonic treatment conditions, the first emulsion was added dropwise to a frozen PVA / THL solution to form a second emulsion solution. The second emulsion solution was stirred at room temperature overnight. The solution was purified by membrane ultrafiltration (Amicon, 0.5 mL, MWCO 100 kDa) to remove unencapsulated IL-2. Then 0.5 mL of the formulation was added to an Amicon ultracentrifugation device and centrifuged at 5,000 rcf for 2-3 minutes. The permeate was discarded, and the retentate containing the micellar IL-2 formulation was diluted to 0.5 mL in water for injection. This process was repeated 10 times. IL-2 concentrations in formulations were determined by Western blotting or dot blotting relative to a standard curve.

[0516] Preparation of PEG-PDBA-IL-2 formulations by acid-base titration

[0517] Phosphate buffer containing 10% (w / w) IL-2 was added to the polymer solution in pH 4.47 phosphate buffer and vortexed at room temperature. 1M NaOH solution was added to the solution under ultrasonic treatment conditions. The solution was diluted with a final concentration of 1.0 mg / mL polymer and 0.1 mg / mL IL-2 by WFI. The solution was purified by membrane ultrafiltration (Amicon, 0.5 mL, MWCO 100 kDa) to remove unencapsulated IL-2. Next, 0.5 mL of the formulation was added to an Amicon ultracentrifugation device and centrifuged at 5,000 rcf for 2-3 minutes. The permeate was discarded, and the retentate containing the micellar IL-2 formulation was diluted to 0.5 mL in water for injection. This process was repeated 10 times. The IL-2 concentration in the formulation was determined by protein blotting or dot blotting relative to a standard curve.

[0518] Quantification of IL-2 and micelles in formulations by dot blot

[0519] The IL-2 content and micelle content of the formulations were determined by dot blot. The dot blot apparatus was assembled with a 0.2 μm nitrocellulose membrane. Each well was washed with 200 μL 1x PBS under vacuum and then rehydrated with 100 μL PBS. Samples and standards (10-100 μL) were added and vacuum was applied to the membrane. The membrane was washed 2 times with PBS.

[0520] IL-2 immunoblotting was performed by probing and blocking with PBS-T supplemented with 2% BSA (PBS containing 0.05% Tween-20), probing with anti-IL-2 rabbit monoclonal antibody (Invitrogen, 2H20L7, 1:1000 dilution in PBS-T, 1 hour), washing 4 times with PBS-T, and then staining with labeled Donkey anti-rabbit IgG of 680RD (LI-COR, diluted 1:5000 in PBS-T) was used for detection. Detection was performed by using ChemiDoc MP (Bio-Rad), and images were quantified by densitometry analysis using ImageLab (Bio-Rad). IL-2 content was determined by fitting the standard curve.

[0521] Polymer content was determined by immunoblotting for polyethylene glycol relative to a polymer standard curve. Immunoblotting was performed by blocking the membrane with PBS supplemented with 2% BSA and staining with THE TM Anti-PEG IGM mAb (Genscript, diluted 1:1000 in PBS) was used for detection, and the cells were washed 4 times with PBS and labeled with The samples were probed with goat anti-mouse IgM (μ chain specific) of 680RD (LI-COR, diluted 1:5000 in PBS). Detection was performed using ChemiDoc MP (Bio-Rad), and images were quantified by densitometry analysis using ImageLab (Bio-Rad). The polymer content was determined by fitting the PEG-PDBA standard curve.

[0522] Example 3: Block copolymers covalently conjugated with IL-2 and Fab

[0523] Conjugation of PEG-PDBA to IL-2 in the amine block

[0524] 13.5ul SAT (PEG) 4 (Thermo, 25mM in DMSO) is added to the solution (Genscript Z00368-1) of 1mg / ml rhIL-2 in pH 7.5PBS buffer of 500ul. After 30 minutes, the reaction is quenched with 1M Tris-HCl, and the solution is stirred at room temperature for 15 minutes. The solution is transferred to 2mL desalting column (Thermo Zeba, 7kDa MWCO), and then 100 μL 1x PBS is added at the top of the column to purify the intermediate. 167 μL of deacetylation solution (0.5M hydroxylamine, 25mM EDTA in 1x PBS solution) is added to the collected solution, and the reaction solution is kept at room temperature for 2 hours. Then the solution is transferred to 2ml desalting column, and then 100 μL 1x PBS buffer is added at the top of the column to purify the protein precursor to be conjugated with polymer in the next step.

[0525] Add 5.6 mg of PEG-PDBA to the solution 100 -AMA4-OPSS polymer, followed by addition of 5mL pH 4.5PBS buffer. The mixture is passed through ultrasonic treatment to prepare a clear solution. The polymer solution (1mL) is diluted with 1.35mL pH 4.5 buffer solution and 1.35mL1x PBS solution. Modified rhIL-2 solution is then added. The reaction is kept overnight at room temperature. The solution is then transferred to a 5mL desalting column to purify the conjugate. The conjugate is concentrated to 0.4mg / mL (based on rhIL-2 as API). Sodium acetate buffer is used, pH 4.5 is used as mobile phase, the conjugate is purified by FPLC, and IL-2 content is determined by Western blotting. The micellization of PEG-PDBA-IL-2 conjugate is carried out by blending with PEG-PDBA and forming micelles by acid-base titration.

[0526] Example 4: Block copolymers covalently conjugated with the small molecule Mortensen

[0527] PEG-PDBA-OPSS

[0528] Mortensen (DM1) (13.35 mg, 0.018 mmol, 4.1 eq.) was added to a solution of PEG-PDBA-OPSS (150 mg, 0.00441 mmol, 1.0 eq.) in 2.5 ml of anhydrous THF / DMF (4 / 1 v:v). (The parent compound used was PEG 113 -b-(PDBA 120-r-OPSS4)). The reaction mixture was stirred at 37°C for 20 hours. The crude reaction mixture was purified by diluting it to 30 ml with a methanol / water solution (1:1). The solution was transferred to an Amicon ultracentrifugal membrane device (10kMWCO). The solution was concentrated to about 1 mL by centrifuge (2,500 rpm, 40-60 minutes), and the process was repeated 5-7 times. The supernatant from each cycle was analyzed by HPLC to monitor and confirm the complete removal of unconjugated DM1. Once purified, the polymer-DM1 conjugate was removed to a vial and the solvent MeOH / water was removed under a stream of nitrogen, followed by lyophilization. The final product was purified by 1 H NMR characterization to determine the drug loading.

[0529] PEG-PDBA-AMA-DM1

[0530] NHS-ester conjugated Mortensen (SMCC-DM1) (13.79 mg, 0.0128 mmol, 3.0 equiv) was added to a solution of PEG-PDBA-AMA (150 mg, 0.00428 mmol, 1.0 equiv) in 3 ml of anhydrous MeOH. The reaction mixture was stirred at 37 ° C for 20 hours. Purification was performed by adding water (3 mL) to the crude reaction mixture and then diluting it to 15 ml with a methanol / water solution (1: 1). The solution was transferred to an Amicon ultracentrifugal membrane device (10k MWCO). The solution was concentrated to about 1 mL by centrifuge (2,500 rpm, 40-60 minutes), and the process was repeated 5-7 times. The supernatant from each cycle was analyzed by HPLC to monitor and confirm the complete removal of unconjugated DM1. Once purified, the polymer-DM1 conjugate was removed to a vial and the solvent MeOH / water was removed under a stream of nitrogen and then lyophilized. The final product was purified by RP-HPLC and 1 H NMR characterization. 1 NMR was used to determine drug loading by comparing the integration of the o-methoxy singlet from DM1 (δ 3.4 ppm, 3H) with the aryl CH (δ 6.75 ppm, 1H) and vinyl CH (δ 4.7 ppm, 1H).

[0531] Example 5: General Procedures for In Vivo Tumor Mouse Models

[0532] Female NOD scid mice (strain NOD.CB17-Prkdc) aged approximately 6-8 weeks were used for scid / J ) with 1.5×10 6HN5 tumor cells were inoculated in the submandibular triangle and the tumors were allowed to grow for approximately 1 week. PEG-PDBA-IL-2 formulations or PEG-PDBA-Fab formulations were prepared with rhIL-2, which was 800CW (LiCOR) was used for fluorescence labeling and the 800CW fluorescence (λ Ex 760 nm, λ Em 780 nm) to normalize the dose. Unencapsulated fluorescently labeled proteins were used as controls. Micellar IL-2 formulations or proteins were administered by tail vein injection. Animals were anesthetized with isoflurane and in vivo small animal imaging was performed using Pearl Trilogy (LI-COR) in white light and 800nm ​​channels 1 hour, 3 hours, and 24 hours after the test article was administered. After the final in vivo imaging time point, animals were killed by CO2 asphyxiation and cervical dislocation, and ex vivo imaging of major organs was performed. Fluorescence was quantified by ROI analysis using ImageStudio software (LI-COR).

[0533] Example 6: General Procedure for In Vitro IL-2 Bioactivity Assay

[0534] According to the manual, thaw and use IL-2 bioassay (Promega) to measure the IL-2 bioactivity in the formulation. In the acid release or encapsulation state, the micelles conjugated to IL-2 or IL-2 were evaluated in the dose response assay. By mixing 20 μL of the formulation with 20 μL of the combined human serum, 40 μL of acidic sodium acetate buffer (0.1M sodium acetate, 0.9% saline, pH about 4.5) were incubated at room temperature for 15 minutes, and then 40 μL of 20X PBS were added for acid release. For the encapsulated samples, the acidic acetate buffer was replaced with a neutral acetate buffer (0.1M sodium acetate, 0.9% saline, pH 7-7.6), and a similar process was used for mixing. Three times serial dilutions of the released or encapsulated formulation were prepared in assay buffer (90% RPMI 1640 / 10% fetal bovine serum). According to the manufacturer's recommendations, the formulation diluent (25 μL) was added to the wells containing IL-2 bioassay cells, which were pre-seeded in white opaque 96-well microplates or half-well microplates (Corning). The assay buffer alone and untreated cells were used as negative controls, while IL-2 alone was used as a positive control. The plate was covered and incubated for 6 hours in a humidified incubator (37° C., 5% CO2). After incubation, 75 μL Bio-Glo reagent (Promega) was added, incubated for 10 minutes, and the bioluminescence was read using a microplate reader (Tecan M200 Pro). The data were plotted in Prism (GraphPad) and the ED50 was calculated by nonlinear fitting.

[0535] Example 7: General Procedure for SDS-PAGE Analysis of Formulations

[0536] Micellar IL-2 formulations were evaluated by SDS-PAGE to confirm IL-2 loading into micelles and IL-2 integrity. Samples were prepared to target 100-200 ng of protein loaded on each lane. For characterization of formulations loaded with IL-2 purified by FPLC, the loading sample constituted the crude formulation without any purification, the spin loading sample constituted the formulation after purification by high-speed centrifugation to remove aggregates and large particles, the micellar pool was prepared by combining the fractions containing micelles, and the free IL-2 sample contained the fraction containing unencapsulated protein. Depending on the reduction requirement, the formulation samples were diluted in 4X Laemmli buffer (Bio-Rad) with or without β-mercaptoethanol and denatured at 65°C for 5 minutes. The samples were loaded onto Any kD by stacking at 50V for 30 minutes, followed by separation at 100V for 90 minutes. TMOr 4-20% SDS-PAGE gradient Mini-Protean gel (Bio-Rad). IL-2 detection is performed by Simply Blue Stain (Invitrogen). After transfer to 0.2 μm nitrocellulose membrane, IL-2 is also determined by protein blotting, i.e., by detecting with anti-IL-2Ab clone (Cell Signaling Technology, clone D7A5, 1:4000 dilution), followed by detection with HRP-conjugated anti-rabbit secondary (LI-COR, 1:2000 dilution), and detected by ECL reagent (Pierce), and chemiluminescence is captured with ChemiDoc MP imager (Bio-Rad). Image processing and density determination analysis are performed using ImageLab (Bio-Rad). If necessary, IL-2 is quantified by fitting the IL-2 standard curve.

[0537] Example 8: Treatment Method

[0538] A therapeutically effective amount of a therapeutic agent encapsulated by a block copolymer as disclosed herein (e.g., in the form of micelles) is administered to a human subject having cancer (e.g., a solid tumor cancer) by injection, e.g., by intravenous injection or in the range of 1 mg / kg to 100 mg / kg, e.g., 10 mg / kg to 50 mg / kg.

[0539] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art will now expect many variations, changes and substitutions. It should be understood that each alternative of the embodiments of the present invention described herein can be used to practice the present invention. The appended claims are intended to define the scope of the present invention, and thus cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A micelle, comprising: (i) a block copolymer having formula (III) or a pharmaceutically acceptable salt thereof: in: n3 is an integer between 10 and 200; x3 is an integer between 40 and 300; y3 is an integer from 0 to 6; z3 is an integer from 1 to 10; X 3 is halogen, -OH or -C(O)OH; R 8 and R 9 are each independently an optionally substituted C1-C6 alkyl; and Each R 10 are independently hydrogen or ICG; and (ii) a block copolymer having a structure of formula (I) or a pharmaceutically acceptable salt thereof: in: n1 is an integer between 10 and 200; x1 is an integer between 40 and 300; y1 is an integer from 0 to 6; z1 is an integer from 1 to 10; X 1 is halogen, -OH or -C(O)OH; R 1 and R 2 Each is independently an optionally substituted C1-C6 alkyl; Each R 3 are independently hydrogen, acyl or ICG; Each r is independently a bond connecting the carbon atoms of the unit / segment or an alkyl group -(CH2) n -, where n is 1 to 10; L 1 yes and Y is a therapeutic agent, wherein the therapeutic agent is IL-2, Wherein "optionally substituted" means that the group is replaced by one or more C1-C 10 Alkyl substitution.

2. The micelle according to claim 1, wherein the ratio of the block copolymer of formula (III) to the block copolymer of formula (I) is 1:99 to 99:1 or any ratio therein.

3. A pH responsive composition comprising the micelle according to claim 1 or 2, wherein the composition has a pH transition point and optionally an emission spectrum.

4. The pH responsive composition of claim 3, wherein the pH transition point is between 4-8, 6-7.5 or 4.5-5.

5.

5. The pH responsive composition of claim 3, wherein the pH response of the composition is less than 0.25 or 0.15 pH units.

6. The pH responsive composition of claim 3, wherein the emission spectrum is between 700-900 nm.

7. The micelle according to claim 1, wherein R 1 and R 2 Each is independently -CH2CH3, -CH2CH2CH3 or -CH2CH2CH2CH3.

8. The micelle according to any one of claims 1, 2 and 7, wherein R 1 and R 2 Each is -CH2CH2CH2CH3.

9. The micelle according to claim 1, wherein x1 is an integer of 50-200, 60-160 or 90-140.

10. The micelle according to claim 9, wherein x1 is 90-140.

11. The micelle according to any one of claims 1, 2, 7, 9 and 10, wherein y1 is an integer of 1-6, 1-5, 1-4 or 1-3.

12. The micelle according to any one of claims 1, 2, 7, 9 and 10, wherein y1 is 0.

13. The micelle of any one of claims 1, 2, 7, 9 and 10, wherein z1 is an integer of 1-9, 1-8, 1-7, 1-6, 1-5, 1-4 or 1-3.

14. The micelle according to any one of claims 1, 2, 7, 9 and 10, wherein z1 is 1.

15. The micelle according to any one of claims 1, 2, 7, 9 and 10, wherein n1 is an integer of 60-150 or 100-140.

16. The micelle according to any one of claims 1, 2, 7, 9 and 10, wherein n1 is 100-140.

17. The micelle according to any one of claims 1, 2, 7, 9 and 10, wherein X 1 It's a halogen.

18. The micelle according to claim 17, wherein X 1 Yes -Br.

19. The micelle according to any one of claims 1, 2, 7, 9 and 10, wherein each R 3 are independently acyl or ICG.

20. The micelle according to any one of claims 1, 2, 7, 9 and 10, wherein each R 3 are independently hydrogen.

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