Patent Application
20240115514
Provided herein are nanoparticles useful for binding therapeutic agents (e.g., anticancer agents). Also provided are methods of using the nanoparticles to treat cancer.
Chemotherapy, targeted therapy, radiation therapy, and hormonal therapy are commonly used methods in the prevention, diagnosis, and treatment of cancer (see, e.g., A. Gadducci, S. Cosio, A. R. Genazzani, Crit. Rev. Oncol. Hematol. 2006, 58, 242-256). Immune cells play roles in regulating tumor growth, and as such, can be harnessed for anticancer therapy. For example, immunotherapies targeting T-cell immune functions have improved survival rates of cancer patients. The tumor microenvironment (TME) includes a diverse set of host cell types that can be therapeutically targeted, including tumor-associated macrophages (TAMs), TAMs are abundant immune cells in the tumor stroma in a broad range of cancers, and a high abundance of these cells in tumors can be associated with a poor clinical outcome.
Provided herein is a nanoparticle, comprising at least two host macrocycles, wherein the at least two host macrocycles are covalently crosslinked by a linker, wherein the linker comprises a moiety of Formula (I):
wherein:
Q is selected from a bond or methylene;
X is selected from O, S, and NR1;
each Y is independently selected from C1-10 alkylene optionally substituted with one or more R2;
Z is selected from A-B, wherein A is selected from a bond and C1-10 alkylene, and B is selected from C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, and C3-10 cycloalkyl;
wherein A is optionally substituted with one or more R3, and B is optionally substituted with one or more R4;
R1 is selected from H and C1-3 alkyl;
each R2 is independently selected from C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, C3-10 cycloalkyl, hydroxy, halo, CN, oxo, C1-C6 alkyl, C1-C6 alkoxy, NH2, COOC1-C6 alkyl, CONH2, CONHC1-C6 alkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, OCOC1-C6 alkyl, OCOC6-C10 aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), and NHCOC2-C6 alkynyl;
each R3 is independently selected from C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, C3-10 cycloalkyl, hydroxy, halo, CN, oxo, C1-C6 alkyl, C1-C6 alkoxy, NH2, COOC1-C6 alkyl, CONH2, CONHC1-C6 alkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, OCOC1-C6 alkyl, OCOC6-C10 aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), and NHCOC2-C6 alkynyl;
each R4 is independently selected from C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, C3-10 cycloalkyl, hydroxy, halo, CN, C1-C6 alkyl, C1-C6 alkoxy, NH2, COOC1-C6 alkyl, CONH2, CONHC1-C6 alkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, OCOC1-C6 alkyl, OCOC6-C10 aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), and NHCOC2-C6 alkynyl; and
R5 is selected from H, C1-C6 alkyl, CO2H, C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, C3-10 cycloalkyl, hydroxy, halo, CN, C1-C6 alkoxy, NH2, COOC1-C6 alkyl, CONH2, CONHC1-C6 alkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, OCOC1-C6 alkyl, OCOC6-C10 aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), and NHCOC2-C6 alkynyl.
In some embodiments, R5 is CO2H. In some embodiments, Q is a bond. In some embodiments, each Y is ethylene. In some embodiments, X is NH. In some embodiments, Z is n-butylene.
In some embodiments, the at least two host macrocycles comprise less than 1×109 host macrocycles. In some embodiments, the at least two host macrocycles comprise less than 5×106 host macrocycles. In some embodiments, the at least two host macrocycles comprise less than 5000 host macrocycles.
In some embodiments, at least one of the at least two host macrocycles is selected from the group consisting of: cyclodextrin, pillar[n]arenes, calix[n]arenes, and cucurbit[n]urils. In some embodiments, at least two of the at least two host macrocycles are selected from the group consisting of: cyclodextrin, pilllar[n]arenes, calix[n]arenes, and cucurbit[n]urils. In some embodiments, the at least two host macrocycles comprise at least two cyclodextrins.
In some embodiments, each cyclodextrin comprises α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β- cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, a cyclodextrin sulfobutylether, a cyclodextrin thioether, a cyanoethylated cyclodextrin, a succinyl-cyclodextrin, or an aminated cyclodextrin, In some embodiments, each cyclodextrin comprises β-cyclodextrin.
In some embodiments, the nanoparticle comprises at least one linear or branched polymer. In some embodiments, the at least one polymer is selected from the group consisting of: a dextran derivative, a hyaluronic acid derivative, a chitosan derivative, a fucoidan derivative, an alginate derivative, a cellulose derivative, a collagen derivative, a poly(ethylene glycol) derivative, a poly(hydroxyethyl acrylate) derivative, a poly(hydroxyethyl methacrylate) derivative, a poly(N-isopropylacrylamide) derivative, a poly(glycolic acid), a poly(lactic acid) derivative, a poly(lactic acid-glycolic acid) derivative, a oligo(poly(ethylene glycol)fumarate) derivative, a poly(vinyl alcohol) derivative, and a poly(vinyl acid) derivative.
In some embodiments, the nanoparticle comprises at least one therapeutic agent. In some embodiments, the therapeutic agent forms a host-guest complex with at least one of the host macrocycles. In some embodiments, the at least one therapeutic agent comprises an anticancer or immunomodulating agent. In some embodiments, the at least one therapeutic agent comprises an anticancer agent. In some embodiments, the anticancer agent is a toll-like receptor (TLR) agonist. In some embodiments, the anticancer agent is a TLR3, TLR4, TLR 7/8, or TLR9 agonist.
In some embodiments, one or more of the at least one therapeutic agents is selected from the group consisting of: GW2580, CEP32496, BLZ945, OS930, PLX3397, dasatinib, sunitinib, ABT869, imatinib, foretinib, XL228, gefitinib, PD0325901, trametinib, bentamapimod, dabrafenib, vemurafinib, crizotinib, UNC2025, indoximod, celecoxib, rapamycin, NIK12192, trichostatin A, MET151, TMP195, BYL719, GDC0941, BKM120, imiquimod, gardiquimod, resiquimod (R848), motolimod, and GS9620. In some embodiments, one or more of the at least one therapeutic agents is a compound selected from the group consisting of: imiquimod, indoximod, gardiquimod, motolimod, and resiquimod (R848). In some embodiments, the anticancer agent is resiquimod (R848).
In some embodiments, the nanoparticle comprises two or more therapeutic agents, wherein one of the two or more therapeutic agents improves the efficacy of one or more of the other therapeutic agents.
In some embodiments, the nanoparticle further comprises an imaging agent. In sonic embodiments, the imaging agent comprises a magnetic resonance imaging (MRI) agent, a positron emission tomography (PET) agent, a single-photon emission computed tomography (SPECT) agent, or a near-infrared fluorophore. In some embodiments, the near-infrared fluorophore is selected from the group consisting of Vivo Tag 680-XL, ZW800-1C, ZW800-1, ZW800-3C, ZW700-1, indocyanine green (ICG), Cy5, Cy 5.5, Cy7, Cy7.5, IRDye800-CW (CW800), BODIPY 630, and ZWCC.
In some embodiments, the at least one therapeutic agent is conjugated with a fluorescent dye.
In some embodiments, the at least one therapeutic agent is conjugated with adamantane.
In some embodiments, the stoichiometric ratio of the cyclodextrin to the therapeutic agent is from about 100:1 to about 1:100. In some embodiments, the stoichiometric ratio of the cyclodextrin to the therapeutic agent is about 1:1. In some embodiments, the stoichiometric ratio of the cyclodextrin to the therapeutic agent is about 1.1:1.
In some embodiments, the equilibrium binding constant (KD) of the at least one therapeutic agent to the host macrocycle is from about 1×10−12 M to about 0.1 M. In some embodiments, the equilibrium binding constant (KD) of the at least one therapeutic agent to the cyclodextrin is from about 5.5 mM to about 7.2 mM. In some embodiments, the equilibrium binding constant (KD) of the at least one therapeutic agent to the cyclodextrin is about 6.3 mM.
In some embodiments, the half-life of the therapeutic agent in vivo after release from the nanoparticle is from about 45 minutes to about 90 minutes. In some embodiments, the half-life of the therapeutic agent in vivo after release from the nanoparticle is about 62 minutes.
In some embodiments, the nanoparticle has an overall negative charge. In some embodiments, the nanoparticle has a zeta potential of from about −5 mV to about −50 mV. In some embodiments, the nanoparticle has a zeta potential of about −10 mV
In some embodiments, the average molecular weight of the nanoparticle is from about 1,500 g/mol to about 5×1011 g/mol. In some embodiments, the average molecular weight of the nanoparticle is from about 15×103 g/mol to about 20×106 g/mol. In some embodiments, the average molecular weight of the nanoparticle is about 20×106 g/mol.
In some embodiments, the nanoparticie comprises an average of from about 10 to about 10,000 cyclodextrins. In some embodiments, the nanoparticle comprises an average of from about 100 to about 2,000 cyclodextrins. In some embodiments, the nanoparticle comprises an average of about 1,000 cyclodextrins.
In some embodiments, the average hydrodynamic diameter of the nanoparticle is from about 10 nm to about 1000 nm. In some embodiments, the average hydrodynamic diameter of the nanoparticle is from about 10 nm to about 70 nm. In some embodiments, the average hydrodynamic diameter of the nanoparticle is from about 20 nm to about 60 nm. In some embodiments, the average hydrodynamic diameter of the nanoparticle is about 50 nm, In some embodiments, the average hydrodynamic diameter of the nanoparticle is about 30 nm.
Also provided herein is a nanoparticle, comprising:
at least two cyclodextrins, wherein the at least two cyclodextrins are covalently crosslinked by a linker, and wherein the linker comprises a moiety of Formula (I):
wherein:
Q is selected from a bond or methylene;
X is selected from O, S, and NR1;
each Y is independently selected from C1-10 alkylene optionally substituted with one or more R2;
Z is selected from A-B, wherein A is selected from a bond and C1-10 alkylene, and B is selected from C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, and C3-10 cycloalkyl;
wherein A is optionally substituted with one or more R3, and B is optionally substituted with one or more R4;
R1 is selected from H and C1-3 alkyl;
each R1 is independently selected from C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, C3-10 cycloalkyl, hydroxy, halo, CN, oxo, C1-C6 alkyl, C1-C6 alkoxy, NH2, COOC1-C6 alkyl, CONH2, CONHC1-C6 alkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, OCOC1-C6 alkyl, OCOC6-C10 aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), and NHCOC2-C6 alkynyl;
each R3 is independently selected from C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, C3-10 cycloalkyl, hydroxy, halo, CN, oxo, C1-C6 alkyl, C1-C6 alkoxy, NH2, COOC1-C6 alkyl, CONH2, CONHC1-C6 alkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, OCOC1-C6 alkyl, OCOC6-C10 aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocydoalkyl), and NHCOC2-C6 alkynyl;
each R4 is independently selected from C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, C3-10 cycloalkyl, hydroxy, halo, CN, C1-C6 alkyl, C1-C6 alkoxy, NH2, COOC1-C6 alkyl, CONH2, CONHC1-C6 alkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, OCOC1-C6 alkyl, OCOC6-C10 aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), and NHCOC2-C6 alkynyl; and
R3 is selected from H, C1-C6 alkyl, CO2H, C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, C3-10 cycloalkyl, hydroxy, halo, CN, C1-C6 alkoxy, NH2, COOC1-C6 alkyl, CONH2, CONHC1-C6 alkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, OCOC1-C6 alkyl, OCOC6-C10 aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), and NHCOC2-C6 alkynyl; and a therapeutic agent.
In some embodiments, R5 is CO2H. In some embodiments, Q is a bond. In some embodiments, each Y is ethylene. In some embodiments, X is NH. In some embodiments, Z is n-butylene.
In some embodiments, the at least two host macrocycles comprise less than 1×109 host macrocycles. In some embodiments, the at least two host macrocycles comprise less than 5×106 host macrocycles. In some embodiments, the at least two host macrocycles comprise less than 5000 host macrocycles.
In some embodiments, each cyclodextrin comprises α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, a cyclodextrin sulfobutylether, a cyclodextrin thioether, a cyanoethylated cyclodextrin, a succinyl-cyclodextrin, or an aminated cyclodextrin. In some embodiments, each cyclodextrin comprises β-cyclodextrin.
In some embodiments, the linker comprises L-lysine.
In some embodiments, the nanoparticle comprises at least one linear or branched polymer. In some embodiments, the at least one polymer is selected from the group consisting of: a dextran derivative, a hyaluronic acid derivative, a chitosan derivative, a fucoidan derivative, an alginate derivative, a cellulose derivative, a collagen derivative, a poly(ethylene glycol) derivative, a poly(hydroxyethyl acrylate) derivative, a poly(hydroxyethyl methacrylate) derivative, a poly(N-isopropylacrylamide) derivative, a poly(glycolic acid), a poly(lactic acid) derivative, a poly(lactic acid-glycolic acid) derivative; a oligo(poly(ethylene glyco)fumarate) derivative, a poly(vinyl alcohol) derivative, and a poly(vinyl acid) derivative.
In some embodiments, the therapeutic agent forms a host-guest complex with at least one of the cyclodextrins.
In some embodiments, the at least one therapeutic agent comprises an anticancer agent. In some embodiments, the anticancer agent is a toll-like receptor (TLR) agonist. In some embodiments, the anticancer agent is a TLR 7/8 agonist.
In some embodiments, one or more of the at least one therapeutic agents is selected from the group consisting of: GW2580, CEP32496, BLZ945, OSI930, PLX3397, dasatinib, sunitinib, ABT869, imatinib, foretinib, XL228, gefitinib, PD0325901, trametinib, bentamapimod, dabrafenib, vemurafinib, critzotinib, UNC2025, indoximod, celecoxib, rapamycin, NIK12192, trichostatin A, IBET151, TMP195, BYL719, GDC0941, BKM120, resiquimod (R848), motolimod, GS9620, and a compound comprising an imidazoquinoline. In some embodiments, one or more of the at least one therapeutic agents is a compound selected from the group consisting of: imiquimod, indoximod, gardiquimod, motolimod, or resiquimod (R848), In sonic embodiments, one or more of the at least one therapeutic agents is resiquimod (R848).
In sonic embodiments, the nanoparticle comprises two or more therapeutic agents, wherein one of the two or more therapeutic agents improves the efficacy of one or more of the other therapeutic agents.
In some embodiments, the nanoparticle further comprises an imaging agent.
In some embodiments, the imaging agent comprises a magnetic resonance imaging (MRI) agent, a positron emission tomography (PET) agent, a single-photon emission computed tomography (SPECT) agent, or a near-infrared fluorophore. In some embodiments, the near-infrared fluorophore is selected from the group consisting of Vivi Tag 680-XL, ZW800-1C, ZW800-1, ZW800-3C, ZW700-1, indocyanine green (ICG), Cy5, Cy5.5, Cy7, Cy7.5, IRDye800-CW (CW800), BODIPY 630, and ZWCC.
In some embodiments, the at least one therapeutic agent is conjugated with a fluorescent dye.
In some embodiments, the at least one therapeutic agent is conjugated with adamantane.
In some embodiments, the stoichiometric ratio of the cyclodextrin to the therapeutic agent is from about 100:1 to about 1:100. In some embodiments, the stoichiometric ratio of the cyclodextrin to the therapeutic agent is about 1:1. In some embodiments, the stoichiometric ratio of the cyclodextrin to the therapeutic agent is about 1.1:1.
In some embodiments, the equilibrium binding constant (KD) of the at least one therapeutic agent to the cyclodextrin is from about 1×10−12 M to about 0.1 M. In some embodiments, the equilibrium binding constant (KD) of the at least one therapeutic agent to the cyclodextrin is from about 5.5 mM to about 7.2 mM. In some embodiments, the equilibrium binding constant (KD) of the at least one therapeutic agent to the cyclodextrin is about 6.3 mM.
In some embodiments, the half-life of the therapeutic agent in vivo after release from the nanoparticle is from about 45 minutes to about 90 minutes. In some embodiments, the half-life of the therapeutic agent in vivo after release from the nanoparticle is about 62 minutes.
In sonic embodiments, the nanoparticle has an overall negative charge. In some embodiments, the nanoparticle has a zeta potential of from about −5 mV to about −15 mV. In some embodiments, the nanoparticle has a zeta potential of about −10 mV.
In some embodiments, the average molecular weight of the nanoparticle is from about 1,500 g/mol to about 5×1011 g/mol. In some embodiments, the average molecular weight of the nanoparticle is from about 15×103 g/mol to about 20×106 g/mol. In some embodiments, the average molecular weight of the nanoparticle is about 20×106 g/mol.
In some embodiments, the nanoparticle comprises an average of from about 10 to about 10,000 cyclodextrins. In some embodiments, the nanoparticle comprises an average of from about 100 to about 2,000 cyclodextrins, In some embodiments, the nanoparticle comprises an average of about 1,000 cyclodextrins.
In some embodiments, the average hydrodynamic diameter of the nanoparticle is from about 10 nm to about 1000 nm. In some embodiments, the average hydrodynamic diameter of the nanoparticle is from about 10 nm to about 70 nm. In some embodiments, the average hydrodynamic diameter of the nanoparticle is from about 20 nm to about 60 nm. In some embodiments, the average hydrodynamic diameter of the nanoparticle is about 50 nm. In some embodiments, the average hydrodynamic diameter of the nanoparticle is about 30 nm.
Also provided herein is a pharmaceutical composition comprising any of the foregoing nanoparticles that comprise a therapeutic agent, and a pharmaceutically, acceptable excipient.
Also provided herein is a method of treating cancer in a patient, the method comprising administering a therapeutically effective amount of any of the foregoing nanoparticles that comprise a therapeutic agent, or the foregoing pharmaceutical composition, to the patient.
In some embodiments, the cancer comprises a tumor-associated macrophage, and wherein the phenotype of the macrophage is M2. In some embodiments, the treating further comprises converting the phenotype of the macrophage from M2 to M1.
In some embodiments, the cancer is selected from the group consisting of Ewing sarcoma, osteosarcoma, glioblastoma, meningioma, oligodendrial cancer, melanoma metastasis, melanoma primary, breast cancer, gastric cancer, germ cell tumors, astrocytoma, ovarian cancer, lung large cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, colon cancer, head and neck cancer, bladder cancer, thyroid cancer, liver cancer, pancreas cancer, kidney cancer, cervical cancer, testicular cancer, prostate cancer, and bone cancer.
In some embodiments, the cancer is metastatic.
In some embodiments, the uptake of the nanoparticle is higher into tumor associated macrophages than into any other organ or tissue type in the subject after administration.
In sonic embodiments, less than 20 mol % of the therapeutic agent is released prior to uptake of the nanoparticle into tumor macrophage cells. In some embodiments, less than 10 mol % of the nanoparticle is released prior to uptake of the nanoparticle into tumor macrophage cells. In some embodiments, less than 5 mol % of the nanoparticle is released prior to uptake of the nanoparticle into tumor macrophage cells. In some embodiments, less than 1 mol % of the nanoparticle is released prior o uptake of the nanoparticle into cancer cells.
In some embodiments, the nanoparticle or composition is administered intravenously, intraarterially, intratumorally, subcutaneously, or intraperitoneally.
In some embodiments, the method further comprises administering an additional therapeutic agent that improves the efficacy of the nanoparticle. In some embodiments, the additional therapeutic agent is a PD-1 antibody, a CTLA-4 antibody, a PD-L1 antibody, an IDO inhibitor, a CSF-1R inhibitor, kinase inhibitor, an HDAC inhibitor, a PI3K inhibitor, a MerTK inhibitor, or an Ax1 inhibitor. In some embodiments, the additional therapeutic agent is a PD-1 antibody. In some embodiments, the PD-1 antibody is selected from the group consisting of: nivolumab, pembrolizumab, pidilizumab, BMS-936559, atezolizumab, and avelumab.
In some embodiments, further comprising treating the patient with radiation, chemotherapy, antibody checkpoint therapy, immunotherapy, or any combination thereof.
In some embodiments, the treating comprises slowing the formation of cancer cells. In some embodiments, the treating comprises preventing the formation of cancer cells. In some embodiments, the treating comprises killing cancer cells.
In some embodiments, the patient is a human.
Also provided herein is a method of altering the phenotype of a tumor-associated macrophage in a cancer cell, comprising contacting the anticancer agent of the any of the foregoing nanoparticles that comprise an anticancer agent, with the cancer cell.
In some embodiments, the altering comprises converting an M2 phenotype to an M1 phenotype.
Also provided herein is a method of reducing the toxicity, side effects, or both of a chemotherapeutic agent in a patient, comprising administering a therapeutically effective amount of any of the foregoing nanoparticles that comprise a therapeutic agent, or the foregoing pharmaceutical composition, to the patient.
In some embodiments, the chemotherapeutic agent is administered systemically, and comprises a TLR7/8 inhibitor. In some embodiments, the TLR7/8 inhibitor comprises resiquimod (R848).
As used herein, the terms “about” and “approximately” are used interchangeably, and when used to refer to modify a numerical value, encompass a range of uncertainty of the numerical value of from 0% to 10% of the numerical value.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein, the term “derived from” refers to a compound or moiety that is structurally identical in most respects to the compound to which it refers. In some embodiments, the compound that the moiety is derived from was used as a reagent or intermediate in the synthesis of the compound that is substituted with the moiety. In some embodiments, the moiety , only differs structurally from the compound it is derived from at the portion of the moiety that links to the remainder of the molecule that the moiety substitutes. As used herein, a “derivative” of a particular compound or moiety encompasses compounds and moieties that are derived from the particular compound. For example, 2-hydroxypropyl-α-cyclodextrin is derived from α-cyclodextrin.
As used herein, the term “host macrocycle” refers to any compound or chemical group that is a cyclic group comprising a minimum of 12 ring members (e.g., 12 or more contiguous atoms that form a ring), wherein the cyclic group is capable of binding a compound (e.g., a therapeutic agent, e.g., an anticancer agent) by means of intermolecular forces that, under certain conditions, last greater than 1 second (e.g., greater than 2 seconds, 4 seconds, 10 seconds, 60 seconds, 1 minute, 2 minutes, 5 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, 2 months, 6 months, 1 year, 2 years, 5 years, or 10 years). Example classes of host macrocycles include cyclodextrins, pillar[n]arenes, calix[n]arenes, and cucurbit[n]urils. Included in each of the foregoing classes are host macrocycles derived from any members of that class through, for example, chemical derivatization. For example, “cyclodextrin” encompasses α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, as well as any chemically derivatized versions of the same including, but not limited to, 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, a cyclodextrin sulfobutylether, a cyclodextrin thioether, a cyanoethylated cyclodextrin, a succinyl-cyclodextrin, or an aminated cyclodextrin.
As used herein, the term “patient,” refers to any animal, including mammals (e.g. domesticated mammals). Example patients include, but are not limited to, mice, rats, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
Tumor-associated macrophages (TAMs) play roles in tumor metastasis and resistance to therapeutic drugs. TAMs can assume opposing phenotypes that can be either tumorigenic (e.g., M2-like cells) or tumoricidal (e.g., M1-like cells). In some tumors, the tumorigenic M2 phenotype prevails. TAMs having the M2 phenotype can accelerate the progression of untreated tumors and adversely influence the effectiveness and/or efficacy of anticancer drugs. Small molecules that inhibit receptors, tyrosine kinases, and/or other transduction pathways in TAMs, and that convert (i.e., re-educate) M2 TAMs into M1 TAMs, have been developed. Such drugs are administered systemically and as such are not delivered to the tumor selectively, leading to side effects. Disclosed herein are nanoparticles that, in some embodiments, include a therapeutic agent (e.g., an anticancer agent, e.g., an anticancer agent that converts M2 TAMs to M1 TAMs, e.g., resiquimod (R848)). In some embodiments, when administered to a patient, the therapeutic nanoparticles provided herein can release the therapeutic agent into the tumor microenvironment (
The nanoparticles disclosed herein comprise at least two host macrocycles, wherein the at least two host macrocycles are covalently crosslinked by a linker.
In some embodiments, at least one of the at least two host macrocycles (e.g., at least two of the at least two host macrocycles) is selected from a cyclodextrin (CD), a pillar[n]arene, a calix[n]arene, or a cucurbit[n]uril. In some embodiments, at least one of the host macrocycles is a cyclodextrin. Examples of cyclodextrins include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, a cyclodextrin sulfobutylether, a cyclodextrin thioether, a cyanoethylated cyclodextrin, a succinyl-cyclodextrin, or an aminated cyclodextrin. In some embodiments, the cyclodextrin is β-cyclodextrin.
In some embodiments, the linker is formed by means of, for example, metal-catalyzed cross-coupling reactions, condensation reactions, addition reactions, or free radical polymerizations. In some embodiments, the linker crosslinks two host macrocycles through a reactive group (e.g., a hydroxyl, amino, amino, sulfoxyl, sulfhydryl, haloacyl, or carboxyl group) on each host macrocycle.
In some embodiments, the linker comprises chemical groups derived from natural and/or unnatural amino acids (e.g., lysine (e.g., L-lysine or D-lysine), arginine, histidine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, tyrosine, tryptophan), succinimides (e.g., N-hydroxysuccinimide), alkylene diamines, epoxides, or epichlorohydrin.
In some embodiments, the linker comprises a moiety of Formula (I):
wherein:
Q is selected from a bond or methylene;
X is selected from O, S, and NR1;
each Y is independently selected from C1-10 alkylene optionally substituted with one or more R2;
Z is selected from A-B, wherein A is selected from a bond and C1-10 alkylene, and B is selected from C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, and C3-10 cycloalkyl;
wherein A is optionally substituted with one or more R3, and B is optionally substituted with one or more R4;
R1 is selected from H and C1-3 alkyl;
each R2 is independently selected from C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, C3-10 cycloalkyl, hydroxy, halo, CN, oxo, C1-C6 alkyl, C1-C6 alkoxy, NH2, COOC1-C6 alkyl, CONH2, CONHC1-C6 alkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, OCOC1-C6 alkyl, OCOC6-C10 aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), and NHCOC2-C6 alkynyl;
each R3 is independently selected from C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, C3-10 cycloalkyl, hydroxy, halo, CN, oxo, C1-C6 alkyl, C1-C6 alkoxy, NH2, COOC1-C6 alkyl, CONH2, CONHC1-C6 alkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, OCOC1-C6 alkyl, OCOC6-C10 aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), and NHCOC2-C6 alkynyl;
each R4 is independently selected from C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, C3-10 cycloalkyl, hydroxy, halo, CN, C1-C6 alkyl, C1-C6 alkoxy, NH2, COOC1-C6 alkyl, CONH2, CONHC1-C6 alkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, OCOC1-C6 alkyl, OCOC6-C10 aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), and NHCOC2-C6 alkynyl; and
R5 is selected from H, C1-C6 alkyl, CO2H, C1-10 arylene, 3-10 membered heteroarylene, 3-10 membered heterocycloalkyl, C3-10 cycloalkyl, hydroxy, halo, CN, C1-C6 alkoxy, NH2, COOC1-C6 alkyl, CONH2, CONHC1-C6 alkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, OCOC1-C6 alkyl, OCOC6-C10 aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), and NHCOC2-C6 alkynyl ; and
wherein each through a bond indicates a point of attachment to a host macrocycle or an additional moiety that attaches to the host macrocycle.
In some embodiments, R5 is CO2H. In some embodiments, Q is a bond. In some embodiments, each Y is ethylene. In some embodiments, X is NH. In some embodiments, Z is n-butylene.
In some embodiments, the at least two host macrocycles comprise less than 1×109 host macrocycles (e.g., less than 1×108, less than 1×107, less than 5×106, less than 1×106, less than 1×105, less than 1×104, less than 5000, less than 2500, less than 1000, less than 500, or less than 100). For example, the nanoparticles comprise from 2 to 1×109 host macrocycles, from 2 to 1×108 host macrocycles, from 2 to 1×107 host macrocycles, from 2 to 5×106 host macrocycles, from 2 to 1×106 host macrocycles, from 2 to 1×105 host macrocycles, from 2 to 1×104 host macrocycles, from 2 to 5000 host macrocycles, from 2 to 2500 host macrocycles, from 2 to 1000 host macrocycles, from 2 to 500 host macrocycles, or from 2 to 100 host macrocycles.
In some embodiments, the nanoparticle comprises at least one polymer. In some embodiments, the polymer is linear. In some embodiments, the polymer is branched. Example polymers include a dextran derivative, a hyaluronic acid derivative, a chitosan derivative, a fucoidan derivative, an alginate derivative, a cellulose derivative, a collagen derivative, a poly(ethylene glycol) derivative, a poly(hydroxyethyl acrylate) derivative, a poly(hydroxyethyl methacrylate) derivative, a poly(N-isopropylacrylamide) derivative, a poly(glycolic acid), a poly(lactic acid) derivative, a poly(lactic acid-glycolic acid) derivative, a oligo(poly(ethylene glyco)fumarate) derivative, a poly(vinyl alcohol) derivative, and a poly(vinyl acid) derivative.
In some embodiments, the nanoparticle comprises at least one therapeutic agent. In some embodiments, the at least one therapeutic agent forms a host-guest complex with at least one of the host macrocycles. In some embodiments, the at least one therapeutic agent is covalently bonded with at least one of the host macrocycles. In some embodiments, the at least one therapeutic agent comprises an anticancer or immunomodulating agent. In some embodiments, the at least one therapeutic agent comprises an anticancer agent. In some embodiments, the anticancer agent is a toll-like receptor (TLR) agonist. In some embodiments, the anticancer agent is a TLR3, TLR4, TLR 7/8, or TLR9 agonist. For example, the anticancer agent is a TLR 7/8 agonist. In some embodiments, the TLR 7/8 agonist is imiquimod, gardiquimod, resiquimod (R848), motolimod, or GS9620. For example the TLR 7/8 agonsit is resiquimod (R848), in some embodiments, one or more of the at least one therapeutic agents is selected from the group consisting of: GW2580, CEP32496, BL1945, OSI930, PLX3397, dasatinib, sunitinib, ABT869, imatinib, foretinib, XL228, gefitinib, PD0325901, trametinib, bentamapimod, dabrafenib, vemurafinib, crizotinib, UNC2025, indoximod, celecoxib, rapamycin, NIK12192, trichostatin A, IBET151, TMP195, BYL719, GDC0941, BKM120, imiquimod, gardiquimod, resiquimod (R848), motolimod, and GS9620. In some embodiments, one or more of the at least one therapeutic agents is a compound selected from the group consisting of: imiquimod, indoximod, gardiquimod, motolimod, or resiquimod (R848). For example, one or more of the at least one therapeutic agents is resiquimod (R848). In some embodiments, one or more of the at least one therapeutic agents comprises a 1H-imidazo[4,5-c]quinoline. In some embodiments, one or more of the at least one therapeutic agents comprises a 4-amino-1H-imidazo[4,5-c]quinoline. In some embodiments, the therapeutic agent is a TKi, CSF1R, or HDAC inhibitor.
In some embodiments, the nanoparticle comprises two or more therapeutic agents. In some embodiments, one of the two or more therapeutic agents improves the efficacy of one or more of the other therapeutic agents (e.g., is synergistic with one or more of the other therapeutic agents).
In some embodiments, nanoparticle further comprises an imaging agent. In some embodiments, the imaging agent comprises a magnetic resonance imaging (MRI) agent, a positron emission tomography (PET) agent, a single-photon emission computed tomography (SPECT) agent, or a near-infrared fluorophore. In some embodiments, the imaging agent comprises a near-infrared fluorophore. In some embodiments, the near-infrared fluorophore is selected from the group consisting of Vivo Tag 680-XL, ZW800-1C, ZW800-1, ZW800-3C, ZW700-1, indocyanine green (ICG), Cy5, Cy5.5, Cy7, Cy7.5, IRDye800-CW (CW800), BODIPY 630, and ZWCC.
In some embodiments, the one or more therapeutic agents are conjugated with a fluorescent dye. In certain instances, conjugating a fluorescent dye to the therapeutic agent enables tracking (e.g., imaging) of the therapeutic agent in vivo. In some embodiments, the fluorescent dye includes a xanthene derivative (e,g., fluorescein, rhodamine, Oregon green, eosin, or Texas red), cyanine derivative (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, or merocyanine), squaraine derivative or ring-substituted squaraine (e.g., seta, setau, and square dyes), naphthalene derivative (e.g., dansyl or prodan derivatives), coumarin derivative, oxadiazole derivative (e.g., pyridyloxazole, nitrobenzoxadiazole, or benzoxadiazole), anthracene derivative (e.g., anthraquinones, including DRAQ5, DRAQ7, or CyTRAK orange), pyrene derivative (e.g., cascade blue), oxazine derivative (e.g., nile red, nile blue, cresyl violet, or oxazine 170), acridine derivative (e.g., proflavin, acridine orange, or acridine yellow), arylmethine derivative (e.g., auramine, crystal violet, or malachite green), tetrapyrrole derivative (e.g., porphin, phthalocyanine, or bilirubin), ZW800 (e.g., ZW800-1C, ZW800-1, or ZW800-3C), ZW700-1, indocyanine green (ICG), Cy5, Cy5.5, Cy7, Cy7.5, IRDye800-CW (CW800), or ZWCC. In some embodiments, the fluorescent dye is a xanthene derivative (e.g., fluorescein, rhodamine, Oregon green, eosin, or Texas red), cyanine derivative cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, or merocyanine), squaraine derivative or ring-substituted squaraine (e.g., seta, setae, and square dyes), naphthalene derivative (e.g., dansyl or prolan derivatives), coumarin derivative, oxadiazole derivative (e.g., pyridyloxazole, nitrobenzoxadiazole, or benzoxadiazole), anthracene derivative (e.g., anthraquinones, including DRAQ5, DRAQ7, or CyTRAK, orange), pyrene derivative (e.g., cascade blue), oxazine derivative (e.g., nile red, nile blue, cresyl violet, or oxazine 170), acridine derivative (e.g., proflavin, acridine orange, or acridine yellow), arylmethine derivative (e.g., auramine, crystal violet, or malachite green), tetrapyrrole derivative (e.g., porphin, phthalocyanine, or bilirubin), ZW800 (e.g., ZW800-1C, ZW800-1, or ZW800-3C), ZW700-1, indocyanine green (ICG), Cy5, Cy7, Cy7.5, IRDye800-CW (CW800), or ZWCC. In some embodiments, the at least one therapeutic agent is conjugated with adamantane.
In some embodiments, the stoichiometric ratio of the cyclodextrin to the therapeutic agent is from about 100:1 to about 1:100 (e.g., from about 100:1 to about 1:100, from about 100:1 to about 1:1, from about 50:1 to about 1:1, from about 20:1 to about 1:1, from about 10:1 to about 1:1, from about 5:1 to about 1:1, from about 2:1 to about 1:1, from about 1:1 to about 100:1, from about 1:1 to about 50:1, from about 1:1 to about 20:1, from about 1:1 to about 10:1, from about 1:1 to about 5:1, from about 1:1 to about 2:1, from about 50:1 to about 1:50, from about 20:1 to about 1:20, from about 10:1 to about 1:10, from about 2:1 to about 1:2, about 1.1:1, or about 1:1. In some embodiments, the stoichiometric ratio of the cyclodextrin to the therapeutic agent is about 1.1:1.
In some embodiments, the equilibrium binding constant (KD) of the at least one therapeutic agent to the host macrocycle is from about 1×10−12 M to about 0.1 M (e.g., from about 1×10−11 M to about 0.1 M, from about 1×10−10 M to about 0.1 M, from about 1×10−9 M to about 0.1 M, from about 1×10−8 M to about 0.1 M, from about 1×10−7 M to about 0.1 M, from about 1×10−6 M to about 0.1 M, from about 1×10−5 M to about 0.1 M, from about 1×10−4 M to about 0.1 M, from about 1×10−3 M to about 0.1 M, from about 1×10−2 M to about 0.1 M, from about 1 mM to about 10 mM, from about 2 mM to about 8 mM, from about 5 mM to about 8 mM, from about 5.5 mM to about 7.2 mM, or about 0.1 M). In some embodiments, the equilibrium binding constant (KD) of the at least one therapeutic agent to the host macrocycle is from about 5.5 mM to about 7.2 mM (e.g., about 5.5 mM, about 6 mM, about 6.3 mM, about 7 mM, or about 7.2
In some embodiments, the half-life of the therapeutic agent in vivo after release from the nanoparticle is from about 30 to about 120 minutes (e.g., from about 30 to about 90 minutes, from about 40 to about 90 minutes, from about 90 to about 120 minutes, from about 90 to about 100 minutes, from about 45 to about 90 minutes, about 55 minutes, about 60 minutes, about 62 minutes, or about 65 minutes). In some embodiments, the half-life of the therapeutic agent in vivo after release from the nanoparticle is about 62 minutes.
In some embodiments, the nanoparticle has an overall negative charge. In some embodiments, the nanoparticle has a zeta potential of from about −5 mV to about −50 mV (e.g., from about −10 mV to about −50 mV, from about −15 mV to about −50 mV, from about −20 mV to about −50 mV, from about −30 mV to about −50 mV, from about −40 mV to about −50 mV, from about −5 mV to about −40 mV, from about −5 mV to about −30 mV, from about −-5 mV to about −20 mV, from about −5 mV to about −10 mV, or about −10 mV). In some embodiments, the nanoparticle has a zeta potential of about −10 mV.
In some embodiments, the average molecular weight of the nanoparticle is from about 1,500 g/mol to about 5×1011 g/mol (e.g., from about 1,500 g/mol to about 5×1010 g/mol, from about 1,500 g/mol to about 5×109 g/mol, from about 1,500 g/mol to about 5×108 g/mol, from about 1,500 g/mol to about 5×107 g/mol, from about 1,500 g/mol to about 5×106 g/mol, from about 1,500 g/mol to about 5×105 g/mol, from about 1,500 g/mol to about 5×104 g/mol, from about 1,500 g/mol to about 5×103 g/mol, from about 5×103 g/mol to about 5×1011 g/mol, 5×104 g/mol to about 5×1011 g/mol, 5×105 g/mol to about 5×1011 g/mol, 5×106 g/mol to about 5×1011 g/mol, 5×107 g/mol to about 5×1011 g/mol, 5×108 g/mol to about 5×1011 g/mol, 5×109 g/mol to about 5×1011 g/mol, 15×103 g/mol to about 20×106 g/mol, or about 20×106 g/mol). In some embodiments, the average molecular weight of the nanoparticle is from about 15×103 g/mol to about 20×106 g/mol. In some embodiments, the average molecular weight of the nanoparticle is about 20×106 g/mol.
In some embodiments, the nanoparticle comprises an average of from about 10 to about 10,000 cyclodextrins (e.g., from about 10 to about 10,000 cyclodextrins, from about 100 to about 10,000 cyclodextrins, from about 1000 to about 10,000 cyclodextrins, from about 2000 to about 10,000 cyclodextrins, from about 5000 to about 10,000 cyclodextrins, from about 8000 to about 10,000 cyclodextrins, from about 100 to about 8,000 cyclodextrins, from about 100 to about 5,000 cyclodextrins, from about 100 to about 2,000 cyclodextrins, from about 100 to about 1,000 cyclodextrins, about 500 cyclodextrins, about 1,000 cyclodextrins, or about 2,000 cyclodextrins. In some embodiments, the nanoparticle comprises an average of from about 100 to about 2,000 cyclodextrins, In some embodiments, the nanoparticle comprises an average of about 1,000 cyclodextrins.
In some embodiments, the average hydrodynamic diameter of the nanoparticle is from about 10 nm to about 1000 nm (e.g, from about 10 nm to about 500 nm, from about 10 nm to about 300 nm, from about 10 nm to about 200 nm, from about 10 nm to about 100 nm, from about 100 nm to about 1000 nm, from about 200 nm to about 1000 nm, from about 500 nm to about 1000 nm, from about 700 nm to about 1000 nm, from about 10 nm to about 70 nm, from about 20 to about 60 nm, about 50 nm, or about 30 nm). In some embodiments, the average hydrodynamic diameter of the nanoparticle is from about 10 nm to about 70 nm. In some embodiments, the average hydrodynamic diameter of the nanoparticle is from about 20 to about 60 nm. In some embodiments, the average hydrodynamic diameter of the nanoparticle is about 50 nm. In some embodiments, the average hydrodynamic diameter of the nanoparticle is about 30 nm.
The present application further provides methods of treating a disease or disorder in a patient (e.g., cancer), including administering a therapeutically effective amount of the nanoparticle (or a composition (e.g., a pharmaceutical composition) comprising the nanoparticle) provided herein to the patient. In some embodiments, the nanoparticle comprises one or more therapeutic agents. In such embodiments, for example, a therapeutically effective amount of the nanoparticle can be determined based upon the amount of therapeutic agent to be administered to the patient by the nanoparticle.
In some embodiments, the cancer comprises a tumor-associated macrophage (TAM). In some embodiments, the phenotype of the tumor-associated macrophage is M2. It is understood that, in some embodiments, the M2 tumor-associated macrophage encourages tissue repair and/or deactivates immune system activation in tumors (by, for example, metabolizing arginine to the ornithine, which facilitates the repair or by producing anti-inflammatory cytokines such as IL-10). In some embodiments, the treating further comprises converting (i.e., re-educating) the phenotype of the macrophage from M2 to M1. In some embodiments, the M1 tumor-associated macrophage encourages inflammation and tissue disrepair (by, for example, secreting high levels of IL-12 and low levels of IL-10, and/or by metabolizing arginine to nitric oxide). Not wishing to be bound by theory, it is understood that the phenotype conversion of tumor-associated macrophages from M2 to M1 exerts an anticancer effect by, for example, slowing cancer growth (e.g., reducing the rate of cancer growth, e.g., reducing the rate of formation of cancer cells.), stopping cancer growth, or killing cancer cells.
In some embodiments, the cancer is selected from the group consisting of Ewing sarcoma, osteosarcoma, glioblastoma, meningioma, oligodendrial cancer, melanoma metastasis, melanoma primary, breast cancer, gastric cancer, germ cell tumors, astrocytoma, ovarian cancer, lung large cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, colon cancer, head and neck cancer, bladder cancer, thyroid cancer, liver cancer, pancreas cancer, kidney cancer, cervical cancer, testicular cancer, prostate cancer, and bone cancer. In some embodiments, the cancer is metastatic.
In some embodiments, the nanoparticle comprising the anticancer agent kills the cancer faster than the anticancer agent alone. In some embodiments, the nanoparticle comprising the anticancer agent kills more cancer cells than the anticancer agent alone after 6 hours, 12 hours, 1 day, 2 days, 4 days, 6 days. 8 days, 2 weeks, 1 month, 2 months, 4 months, 6 months, or 1 year following administration of one or more doses of the anticancer agent.
In some embodiments, the uptake of the nanoparticle is higher into the tumor and/or into tumor associated macrophages than into any other organ or tissue type in the subject after administration (e.g., muscle, heart, or liver). In some embodiments, less than 50 mol % of the nanoparticle is released prior to uptake of the nanoparticle into tumor macrophage cells (e.g., less than 40 mol %, less than 30 mol %, less than 20 mol %, less than 10 mol %, less than 7 mol %, less than 5 mol %, less than 2 mol %, or less than 1 mol % of the nanoparticle is released prior to uptake of the nanoparticle into tumor macrophage cells). In some embodiments, less than 10 mol % of the nanoparticle is released prior to uptake of the nanoparticle into tumor macrophage cells. In some embodiments, less than 5 mol % of the nanoparticle is released prior to uptake of the nanoparticle into tumor macrophage cells. In some embodiments, less than 1 mol % of the nanoparticle is released prior to uptake of the nanoparticle into tumor macrophage cells.
In some embodiments, the method further comprises administering an additional therapeutic agent in combination with a nanoparticle that comprises a therapeutic agent that improves the efficacy of the therapeutic agent (e.g., is synergistic with the therapeutic agent). In some embodiments, the additional therapeutic agent, in combination with the nanoparticle comprising the therapeutic agent, kills the cancer faster than the nanoparticle comprising the therapeutic agent alone (i.e., without an additional therapeutic agent). In some embodiments, the additional therapeutic agent is a PD-1 antibody, a CTLA-4 antibody, a PD-L1 antibody, an inhibitor, a CSF-1R inhibitor, kinase inhibitor, an HDAC inhibitor, a PI3K inhibitor, a MerTK inhibitor, or an Ax1 inhibitor. In some embodiments, the additional therapeutic agent is a PD-1 antibody. In some embodiments, the PD-1 antibody is selected from the group consisting of: nivolumab, pembrolizumab, pidilizumab, BMS-936559, atezolizumab, and avelumab. In some embodiments, the additional therapeutic agent is selected from afatinib, AG 879, alectinib (Alecensa), altiratinib, apatinib (Tykerb), ARQ-087, ARRY-112, ARRY-523, ARRY-651, AUY-922, AXD7451, AZ-23, AZ623, AZ64, AZD4547, AZD6918, AZD7451, BGJ398, binimetinib, BLU6864, BLU9931, brivatinib, cabozantinib, CEP-751 and CEP-701 (lestaurtinib), cetuximab (Erbitux), CH5183284, crizotinib (Xalkori), CT327, dabrafenib (Tafinlar), danusertib, DCC-2036 (rebastinib), DCC-2157, dovitinib, DS-6051, encorafenib, erdafitinib, erlotinib, EWMD-2076, gefitinib (Iressa), GNF-4256, GNF-5837, Gö 6976, GTx-186, GW441756, imatinib (Gleevec), K252a, Iapatinib, lenvatinib (Lenvima), Loxo-101, Loxo-195 (ARRY-656), lucitanib, LY2874455, MGCD516 (sitravatinib), motesanib, nilotinib (Tasigna), nintedanib, NVP-AST487, ONO-5390556, orantinib (TSU-68, panitumumab (Vectibix), pazopanib (Votrient), PD089828, PD166866, PD173074, pertuzumab (Perjeta), PF-477736, PHA-739358 (danusertib), PHA-848125AC (Milciclib), PLX7486, ponatinib (AP-24534), PZ-1, quercetin, regorafenib (Stivarga), RPI-1, ruxolitinib, RXDX101 (Entrectinib), RXDX105, semaxanib (SU5416), sorafenib, SPP86, SSR128129E, SU4984, SU5402, SU6668, SUN11602, Sunitinib, TAS120, TG101209, TPX-0005, trastuzumab, TSR-011, vandetanib (Caprelsa), vatalanib, VSR-902A, and XL-184 (cabozantinib).
In some embodiments, the method further comprises treating the patient with radiation, chemotherapy, antibody checkpoint therapy, immunotherapy, or any combination thereof.
The present application further provides methods of imaging a tissue in a subject, including administering the nanoparticle provided herein to the patient. In some embodiments, the tissue includes cancer cells. In some embodiments, the tissue includes kidney tissue, bladder tissue, or both.
In some embodiments, the patient is a mammal (e.g., a human or a domesticated mammal).
The present application further provides methods of altering the phenotype of a tumor-associated macrophage in a cancer cell, comprising contacting a therapeutic agent (e.g., an anticancer agent) of a nanoparticle disclosed herein with the cancer cell. In some embodiments, the altering comprises converting an M2 phenotype to an M1 phenotype.
The present application further provides methods of reducing the toxicity, side effects, or both of a chemotherapeutic agent in a patient, comprising administering a therapeutically effective amount of a nanoparticle comprising the chemotherapeutic agent as disclosed herein to the patient. In some embodiments, the nanoparticle comprising the chemotherapeutic agent is administered systemically (e.g., intraperitoneally, intravenously, intraarterially), and comprises a TLR7/8 inhibitor (e.g., resiquimod).
The nanoparticles disclosed herein (e.g., nanoparticles comprising cyclodextrins) may be formed by, for example, reacting a 6′-hydroxyl group of a cyclodextrin with N-hydroxysuccinimide (NHS) to form an ester bond to result in a succinyl-β-cyclodextrin. Subsequent amide bond formation between a free carboxyl group on a succinyl-β-cyclodextrin and a free carboxyl group on another succinyl-β-cyclodextrin with two amino groups of L-lysine results in a crosslinked nanoparticle (Scheme 1). In some embodiments, a molar ratio of 1:2 lysine to succinyl moieties is used in the crosslinking step. In some embodiments, a solution comprising from about 0.5% to about 109 wt/vol is used in the crosslinking step (e.g., from about 0.5% to about 8% wt/vol, from about 0.5% to about 5% wt/vol, from about 0.5% to about 3% wt/vol, from about 0.5% to about 2% wt/vol, from about 2% to about 10% wt/vol, from about 3% to about 10% wt/vol, from about 5% to about 10% wt/vol, about 1.25% wt/vol, about 2.5% wt/vol, about 3.3% wt/vol, or about 5% wt/vol). In some embodiments, a solution comprising about 3.3% wt/vol is used in the crosslinking step.
When employed as pharmaceuticals, the nanoparticles (e.g., nanoparticles comprising one or more therapeutic agents) provided herein can be administered via various routes (e.g., intravenous, intraarterial, intratumoral, intranasal, subcutaneous, intradermal, intraperitoneal, or oral administration) in the form of pharmaceutical compositions. These compositions can be prepared as described herein or elsewhere, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. In some embodiments, the administration is parenteral. Parenteral administration includes, for example, intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular or injection or infusion; or intracranial administration, (e.g., intrathecal or intraventricular, administration). Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. In some embodiments, the compounds, salts, and pharmaceutical compositions provided herein are suitable for parenteral administration. In some embodiments, the nanoparticles provided herein are suitable for intravenous administration. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Also provided are pharmaceutical compositions which contain, as the active ingredient, a nanoparticle provided herein (e.g., a nanoparticle comprising a therapeutic agent), in combination with one or more pharmaceutically acceptable carriers (e.g., excipients). In making the compositions provided herein, the active ingredient is typically, mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, tablet, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
Some examples of suitable excipients include, without limitation, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include, without limitation, lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; flavoring agents, or combinations thereof.
The nanoparticles (e.g., nanoparticles comprising one or more therapeutic agents) can be effective over a wide dosage range and are generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the nanoparticle (e.g., nanoparticles comprising one or more therapeutic agents) actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the like.
The following examples are offered for illustrative purposes, and are not intended to limit the invention.
Unless otherwise indicated, solvents and reagents were purchased from Sigma-Aldrich and used without further purification. Water used for all experiments was purified using a MilliQ filtration system (Waters). All pharmacological drugs were purchased from commercial suppliers (Selleckchem, MedchemExpress, InvivoGen, or LC Laboratories). The Rat IgG2a kappa anti-mouse PD1 29F.1A12 clone was provided by Gordon Freeman (DFCI). Ferumoxytol (AMAG Pharmaceuticals) and amino-dextran (500kDa, Thermo Fisher Scientific) were used for intravital imaging, and were fluorescently labeled by Pacific Blue (label concentration: 40.1±2.6 nM m−1 Dextran, 1.79 mg injected).
Cells were maintained in the indicated medium at 37° C. and 5% CO2 and screened regularly for mycoplasma. RAW 264.7 cells were sourced from ATCC and maintained in Dutbecco's Modified Eagles Medium supplemented with 10% heat inactivated fetal calf serum (Atlanta Biologicals), 100 IU penicillin (Invitrogen), and 100 μg mL−1 streptomycin (Invitrogen), and 200 mM L-glutamine. The MC38 mouse colon adenocarcinoma cell lines were provided by Mark Smyth (QIMR Berghofer Medical Research Institute) with stable transfection of the H2B-Apple reporter to yield a MC38-H2B-mApple cell line employed in intravital microscopy studies. Murine bone marrow-derived macrophages (BMDMs) were isolated and derived by adaptation of published procedures known to those of skill in the art. Briefly, bone marrow was extracted from the surgically resected femur and tibia of naive C57BL/6 mice, dissociated and passed through a 40 μm strainer, and red blood cells lysed by ammonium chloride (StemCell Tech). Resultant bone marrow cells were plated in either 24-well (Corning 3527, for PCR analysis) or optical-bottom 384-well plates (Thermo Fisher 142761, for image analysis) at 1×106 cells mL−1 in Iscove's Modified Dulbecco's Medium supplemented with 10% heat inactivated fetal calf serum, 100 IU penicillin, 100 μg ml−1 streptomycin (Invitrogen) and 10 ng mL−1 recombinant murine M-CSF (PeproTech, 315-02); media was replenished every two days. Human macrophages were derived from peripheral blood mononuclear cells isolated using Ficoll-Paque PLUS (GE Healthcare) gradient separation and derived in the presence of 50 ng mL−1 recombinant human M-CSF (PeproTech, 300-25). Cell proliferation was assessed by PrestoBlue (Fisher) following manufacturer's protocols.
Animal research was conducted in compliance with the Institutional Animal Care and Use Committees at Massachusetts General Hospital (MGH). Unless otherwise stated, experiments were performed using female C57BL/6 that were 6- to 8-week old at the start of the experiment, and animals were sourced from The Jackson Laboratory.
For morphological analysis, media was replenished with M-CSF-tree media on day 7 followed by drug dosing (Table 1).
After 48 hours, cells were fixed with formaldehyde (30 min, 37° C.) and stained for actin (5.0 μg mL−1 DyLight 554 Phalloidin, Cell Signaling Technology), cell membrane (5.0 μg mL−1 Alexa Fluor 647 wheat germ agglutinin, Thermo Fisher) and nuclei (DAPI, Invitrogen) for 25 min at room temperature. Plates were washed by PBS prior to imaging on a custom Olympus-based automated high-content screening microscope. Four images were acquired per well in a 2×2 grid, imported into CellProfiler (Broad Institute) for pre-processing and segmentation (Table 2).
Computational cell classification was performed in CellProfiler Analyst (Broad Institute) by random forest assignment. Training data (examples provided, Supplementary Figure S1) consisted of approximately 50 healthy cells each representing undifferentiated (M0), M1-like, or M2-like phenotypes. The fast gentle algorithm was trained on the selected cells for unsupervised determination of weights and thresholds for cell shape features. The resulting set of parameters was used to score all other images. The enrichment score for M1 cells was output back into the database and imported into KNIME to generate per-well and per-treatment averages.
For transcriptional analysis, derived murine macrophages were treated with 10 ng mL−1 recombinant mouse IL-4 (PeproTech 214-14) for 24 hours to induce an M2-like polarization state and subsequently dosed with fresh media supplemented by pharmacologic drugs at the prescribed concentrations. Macrophages treated only with IL-4 (10 ng mL−1) or LPS (100 ng mL−1) and IFNg (50 ng mL−1) served as internal controls for M2-like and M1-like transcription profiles, respectively. After 24 hours, RNA was isolated by standard protocols (QIAGEN 74106) and subject to reverse transcription (Thermo Fisher 4368814) and qPCR (Thermo Fisher 44-445-57) for analysis of hrpt (Mm01545399_m1), arg1 (Mm00475988 m1), mrc1(Mm01329362_1), cd80 (Mm00711660_m1), il12b (Mm01288989_m1), and nos2 (Mm00440502_ m1). For analysis of human macrophages, cells were similarly treated and processed prior to analysis for expression of β-actin (Hs01060665_g1) and il12b (Hs01011518_m1). Data are presented as the gene expression (fold change relative to hprt or/β-actin, as indicated) or M1-likeness, calculated as described in Example 2,
Polyglucose (succinyl-β-cyclodextrin (CD) or 10 kDa carboxymethyl dextran (5% carboxylated, TdB), 1.0 eq. carboxylate), N-(3-Dimethylaminopropyl)-N′-ethlycarbodiimide hydrochloride (Sigma; 10.0 eq. to carboxylate), and N-hydroxysuccinimide (Sigma, 5.0 eq. to carboxylate) were combined and dissolved in MES buffer (50 mM, pH 6.0) at the desired glucose concentration (1.25 to 20.0 % wt/v). The reaction was stirred for 30 min at room temperature prior to the addition of L-lysine (0.5 eq. to carboxylate, unless otherwise noted) and overnight crosslinking. The product was recovered by addition of brine (0.05 volumetric equivalents) and precipitation from a 10-fold excess of iced ethanol. Upon re-dissolution in water, the product was concentrated by centrifugal filtration (10 kDa MWCO, Amicon), washed repeatedly by water, passed through a 0.22. μM spin filter (Costar, Spin-x), and lyophilized. The final products were re-dissolved at a concentration of 20 mg mL−1 prior to use. Particle size was calculated by dynamic light scattering (Malvern, Zetasizer APS) at a typical concentration of 5 mg mL −1 in 100 mM PBS. Zeta potential was determined at 100 μg mL−1 in 10 mM PBS (Malvern, Zetasizer ZS) following calibration measurements on manufacturer standards. For scanning electron microscopy, samples were prepared at 1.0 μg mL−1 in water, spotted on silica wafers, freeze-dried and sputter coated prior to imaging. Analysis of R848 affinity for CD was performed by a standard colorimetric competitive binding assay. Briefly, phenolphthalein (200 mM) was freshly prepared in 125 mM carbonate buffer (pH 10.5). Decrease in absorbance at 550 nm due to nanoparticle-phenolphthalein complexati on and absorbance recovery due to R848 competitive binding were measured (Tecan, Spark), and results are presented as absorbance relative to nanoparticle-free controls. The dissociation constant, KD, was determined by treatment of β-cyclodextrin by increasing concentrations of R848 and fit to a one-site competitive inhibition model in GraphPad Prism 6 (GraphPad Software, Inc.). Drug loading in CDNP-R848 was analytically determined as a function of the molar ratio of R848 to CD in the nanoparticle, assuming the appropriate reaction equilibrium for one-to-one association: KD=[R848]*[CD]/[CD-R848], where [R848] is the concentration of unbound R848, [CD] is the concentration of unbound cyclodextrin in the nanoparticle, and [CD-R848] is the concentration of R848 bound by the nanoparticle. A molar ratio of guest-to-host ranging from 0.01 to 100 was examined, and drug loading (% wt/wt) was defined as 100*(MR848/(MR848+MCDNP)), where MCDNP is the mass of nanoparticle, and MR848 is the mass of R848 bound by cyclodextrin.
For intravital imaging and assessment of biodistribution, cyclodextrin nanoparticles were fluorescently labeled. The CDNP nanoparticle was dissolved at 20 mg mL−1 in carbonate buffer (0.1 M, pH 8.5) prior to addition of VivoTag 680 XL (PerkinElmer, 1.0 mg mL−1 in anhydrous DMSO) at a final concentration of 50 μM. The reaction was allowed to proceed for 3 hours at room temperature prior to product recovery by centrifugal filtration (10 kDa MWCO, Amicon), repeated washing by water to remove unbound dye, and lyophilization. Resultant CDNP-VT680 was re-dissolved at a concentration of 10 mg mL−1. Absorption at 668 nm (Nanodrop) was used to determine the label concentration (1.79±0.03 nM mg−1) by the Beer-Lambert equation, (A=εbc, where A is the absorbance, ε is the molar absorptivity 210,000 M−1cm−1, and c is the concentration).
The blood half-lite of CDNP-VT680 was determined by time-lapse confocal fluorescence microscopy of vessels in the ear during and immediately following tail vein injection of Pacific Blue Dextran and CDNP-VT680 (0.5 mg). Time-lapse images were acquired continually over the first 3 hours after CDNP-VT680 injection, after which the mice were allowed to recover before subsequent imaging at 24 hours. Across three separate C57BL/6 mice, multiple fields of view were analyzed by identification of regions of interest within the labeled vasculature. Mean fluorescence intensity was determined as a function of time, background subtracted, and normalized to the to peak fluorescence intensity. Resulting data was fit to a mono-exponential decay in GraphPad Prism 6,
At 24 hours following injection, examination of CDNP biodistribution was performed. Surgically resected tissues of interest were thoroughly washed in PBS, weighed, and placed in an OV110 (Olympus) for brightfield imaging to identify regions of interest and fluorescence reflectance imaging (1000 ms exposure time; λex=620-650 nm, λem=680-710 nm). Integrated fluorescence density was determined for ROIs representing each tissue (ImageJ, NIH). Values were background-subtracted for tissue autofluorescence by imaging of corresponding tissues from a vehicle treated control. Percentage of injected dose was determined relative to standards of CDNP-VT680 prepared in 1.0% intralipid (McKesson, 988248), to account for optical scattering of tissue, and values are presented following normalization to tissue mass.
Intravital examination CDNP-VT680 distribution into macrophages and tumor cells was examined using dorsal skinfold window chambers installed on recently developed MerTK-GFP mice inoculated with MC38-H2B-mApple. Mice received CDNP-VT680 i.v. (0.5 mg) 24 hours prior to imaging. Intravital examination of IL12 expression was similarly performed using p40-IRES-eYEP IL12 reporter mice (#015864, Jackson). Prior to imaging, mice received intravenous administration of R848 (2.0 mg kg−1), CDNP-VT680 (16.5 mg kg−1 CDNP), or CDNP-VT680+R848 (16.5 mg kg−1 CDNP-VT680+2.0 mg kg−1 R848; 1/1.1 R848/CD molar ratio). IL12 expression was examined at 24 hours following treatment. In both cases, macrophages and vasculature were labeled by Pacific Blue-ferumoxytol and Pacific Blue-dextran, respectively. Images were acquired on a FV1000MPE confocal imaging system (Olympus). Pacific Blue, GFP/YFP, mApple, and VT680 were imaged sequentially using 405-, 473-, 559-, and 633-nm light sources and BA430-455, BA490-540, BA575-620, and BA575-675 emission filters with DM473, SDM560, and SDM640 beam splitters.
Images were pseudo-colored and processed in FIJI (ImageJ, NIH) by adjusting brightness/contrast, creating z-projections of image stacks, and performing a rolling ball background subtraction. For quantification of IL12 expression, the sum of YFP, Pacific Blue, and VT680 channels were segmented by automated thresholding using the RenyEntropy method to generate a mask and corresponding ROIs for individual macrophages. The fluorescence intensity was determined for YFP within each ROI, and data are presented following normalization to the average intensity for CDNP control treatment.
Flow Cytometry For examination of CDNP-VT680 biodistribution in MerTK-GFP mice, MC38 tumors and tissues of interest were excised 10 days after tumor implantation, 24 hours after intravenous injection of CDNP-VT680 (0.5 mg). For examination of IL12 expression, MC38 tumors were harvested 9 days after intradermal implantation into IL12-eYFP mice, 24 hours following intravenous administration of R848 (2.0 mg kg−1), CDNP-VT680 (16.5 mg kg−1 CDNP), or CDNP-VT680±R848 (16.5 mg kg−1 CDNP-VT680±2.0 mg kg−1 R848; 1/1.1 R848/CD molar ratio) in saline. Tissues were minced, incubated in RPMI containing 0.2 mg mL−1 collagenase I (Worthington Biochemical) for 30 min at 37° C. and then passed through a 40 μm filter. Red blood cells were lysed using ACK lysis buffer (Thermo Fisher Scientific) prior to pre-treatment with low affinity Fe receptor blocking reagent (Tru Stain FcX anti-CD16/32 clone 93, BioLegend) and staining in phosphate buffered saline containing 0.5% BSA and 2 mM EDTA with fluorochrome labeled antibodies against CD45 (30-F11, eBioscience), CD11c (N418, BioLegend), Ly6G (1A8, Biolegend), F4/80 (BM8, BioLegend), and 7-AAD. Samples were run on a LSR II flow cytometer (BD) and analyzed in FlowJo v.8.8.7 (Tree Star, Inc.) to identify macrophages (CD45+MerTK+Ly6G-F4/80+) in IL12-eYFP mice as well as macrophages (CD45+MerTK+Ly6G−), neutrophils (CD45+MerTK-Ly6G+), and other immune cells (CD45+) in MerTK-GFP mice. Identically treated tissue from MC38 tumors grown in wild type C57BL/6 mice served as a control for thresholding cutoffs for IL12+ and CDNP-VT680+ cells in analysis of IL12-eYFP induction.
Tumor growth studies were initiated by intradermal injection of 2×106 MC38 cells suspended in 50 μL of PBS. Tumors were allowed to grow to 25 mm2 (8 days) at which time treatment cohorts were assigned such that tumor size and body weight were normalized across groups at baseline. For repeated dosing experiments, animals were treated 3 times weekly by i.v. administration of R848 (2.0 mg kg−1), CDNP (16.5 mg kg−1), or CDNP (16.5 mg kg−1) with R848 (2.0 mg kg−1) in saline. For single dosing experiments, animals were treated by i.v. administration of R848 (3.0 mg kg−1), CDNP (24.6 mg kg−1), or equivalent dosing of CDNP (24.6 mg kg−1) with R848 (3.0 mg kg−1) in saline. For aPD-1 treatment, the 29F.1.A12 aPD-1 clone was administered at a dose of 200 μg by intraperitoneal injection. At set time points, tumor growth was assessed by caliper measurement (A=length×width) and values are reported following normalization to area at the time treatment was initiated.
Data are presented as mean±standard error unless otherwise indicated. Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc.). Statistical significance was determined by analysis of variance, using repeated measures where appropriate, in conjunction with post hoc Tukeys HSD. For in vivo studies of tumor growth, temporal comparisons were made by Friedmans test and comparison at set time points were performed by Kruskal-Wallis, each using post hoc Dunn's test for multiple comparisons. Survival analysis was performed by log-rank test. Significance was determined at P=0.05.
Cell morphology was used as an integrated biomarker of cell function, including as an indicator for age and immunosuppressive capacity. The method that was used uses high-content image analysis via computational automated segmentation to extract features of single cells such as cellular radius, axis lengths, compactness, and eccentricity which are associated with the polarization state.
Having established a HTS for examination of cell state, drugs capable of macrophage re-educating were then identified. A panel of 38 drugs was curated from the literature, representing specific drugs or drug classes which have been implicated in macrophage polarization.
A subset of drugs having a range of M1 enrichment activities were further scrutinized by qPCR analysis of representative M1-like (nos2, il12, and cd80) and M2-like (mrc1, arg1) transcripts.
It is understood that certain dextran nanoparticles have native macrophage avidity which results in rapid, preferential distribution to TAMs relative to other cells present in the TME. β-cyclodextrin (CD) shares similar chemical composition with linear dextran, suggesting potential for macrophage avidity. Moreover, host-guest inclusion by macrocycles, such as CD, is an established mechanism for drug solubilization and nanoparticle-mediated drug delivery which forgoes chemical modification of established drug compounds (see Zhang & Ma, Nature Protocols, 2016, 11:1757); and Rodell et al, Bioconjug Chem. 2015, 26:2279-2289). The interaction of CD with R848 was therefore used to enable formation of drug-loaded nanoparticles.
To examine the pharmacokinetics and biodistribution of the newly developed CDNP, a fluorescent derivative (CDNP-VT680) was developed, where the covalently bound fluorochrome readily allows for examination in vivo by fluorescence microscopy. Systemic circulation and biodistribution were examined in an immunocompetent mouse model of colorectal cancer (MC38) in C57BL/6 mice. First, time-lapse confocal fluorescence microscopy was performed of vessels within the ear for assessment of systemic circulation, demonstrating a vascular half-life (t1/2) of 62.5±4.75 min.
To further interrogate the intratumoral kinetics and cellular distribution, a dorsal window chamber setup was employed for intravital imaging. Tumors were generated by inoculation with 1×106 MC38-H2B-mApple cells, allowing identification of tumor cells. To enable identification of TAMs, a CRISPR-CAS reporter mouse was used, wherein TAMs are detectable through MerTK-GFP expression. The distribution of CDNP-VT680 was examined by confocal fluorescence microscopy in a MerTKGFP/+ mouse bearing an MC38-H2B-mApple tumor in a dorsal window chamber model 60 min following administration (
At 24 hours following administration of CDNP-VT680, relevant tissues were harvested from a MerTKGFP/+mouse bearing an MC38-H2B-mApple tumor, and flow cytometry was performed to identify the distribution (
R848 delivery to TAMs was also examined in an orthotopic lung adenocarcinoma model (eGFP-expressing KrasG12D p53−/− mutant (KP) lung adenocarcinoma) by imaging of CDNP-VT680 and a newly developed fluorescent drug conjugate, R848-BODIPY TMR.
To examine macrophage re-education, both murine and human macrophages were polarized to an M2-state (IL-4 induction, 24 hrs) prior to drug treatment in the absence of IFNγ. For all experiments, dosing was matched (100 nM R848, 24 hr).
The pharmacodynamics of M1 induction in vivo were further explored by employing an IL12-YFP reporter mouse in which TAMs co-express YFP with the prototypical M1 marker IL12-p40.
Observation of tumor recession for CDNP-R848 treatment was also observed. Tumor size was monitored following single-dose administration in IL12-YFP mice on day 0 (8 days following tumor inoculation). Treatment with single-dose CDNP-R848 (16.5 mg kg−1 CDNP with 2.0 mg kg−1 R848) resulted in rapid tumor recession, observed by confocal fluorescence microscopy of MC38-H2B-mApple tumors, outlined (
The tumor growth experiments were repeated using single dosage of free or nano-encapsulated R848, and it was found that CDNP assisted delivery of R848 to significantly improve therapeutic response (
To examine the role of adaptive immune involvement, antibody depletion of CD8+T-cells was examined in treatment groups, relative to CDNP controls in C57BL/6 mice bearing MC38 tumours.
Given the productive diversion of TAMS from immune-suppressive to immune-supportive phenotypes and the demonstrated involvement of adaptive immunity, it is understood that CDNP-R848 monotherapy could potentiate checkpoint blockade. Combination of CDNP-R848 with anti-PD-1 was synergistic and resulted in tumor shrinkage, stabilization and homogenization of response, as shown in
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims,
This application is a continuation of U.S. patent application Ser. No. 16/757,119, filed Apr. 17, 2020, which is a § 371 National Stage Application of PCT/US2018/056780, filed Oct. 19, 2018, which claims the benefit of U.S. Provisional Application Ser. No. 62/574,942, filed Oct. 20, 2017, the disclosure of which is incorporated herein by reference in its entirety.
This invention was made with Government support under Grant Nos. T32CA079443; R01CA206890; U01CA206997; and R01HL131495, awarded by the National Institute of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62574942 | Oct 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16757119 | Apr 2020 | US |
| Child | 18215309 | US |
