How to Treat Cancer

Abstract

The present invention provides a conjugate comprising (a) a cancer targeting ligand, (b) a hydrophilic polymer, which is polyethylene glycol (PEG), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), or dextran, and (c) a flavonoid. The present invention also provides a micellar nanoparticle composition comprising (a) an outer shell comprising the conjugate, (b) an inner shell comprising oligomeric (-)-epigallocatechin gallate (OEGCG), and, optionally, (c) a cancer therapeutic molecule encapsulated in the inner shell. In one embodiment, the nanoparticle composition has at least 70% of nanoparticles having a diameter of 20-500 nm or 50-300 nm, with a single main peak in the size distribution. The present invention further provides a method of treating cancer by administering an effective amount of the nanoparticle composition to a subject. To treat cancer, the cancer targeting ligand targets and delivers an active ingredient to the tumor.

Description

[Technical field]

[0001] The present invention relates to a conjugate comprising (a) a cancer targeting ligand, (b) a hydrophilic polymer, which is polyethylene glycol (PEG), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), or dextran, and (c) a flavonoid, wherein the hydrophilic polymer is covalently attached to the flavonoid and the cancer targeting ligand.The present invention relates to a micellar nanoparticle comprising (a) an inner shell comprising oligomeric (-)-epigallocatechin gallate (OEGCG), (b) an outer shell comprising a cancer targeting ligand-hydrophilic polymer-EGCG conjugate, and optionally (c) a cancer therapeutic drug encapsulated in the inner shell. [Background technology]

[0002] Green tea catechins have health benefits in the prevention of cancer. Among tea catechins, (-)-epigallocatechin-3-gallate (EGCG) is the most abundant and plays a major role in the beneficial effects of green tea. EGCG has antioxidant, anti-inflammatory, and immunomodulatory properties. EGCG has also been shown to effectively inhibit tumor growth and metastasis by targeting multiple signaling pathways essential for cancer cell survival.

[0003] Despite these desirable activities, the clinical application of EGCG has been limited by its poor stability and low oral bioavailability. For example, EGCG is unstable in physiological environments and is easily degraded. As a result, the plasma concentrations of EGCG required to achieve the desired therapeutic effects cannot be reached after oral administration.

[0004] There are three major challenges in treating cancer, a complex disease involving multiple signaling pathways. First, cancer erupts from immune dysfunction in humans. Restoring the host immune mechanism through immunomodulation is key to long-term therapeutic solutions. Second, a single therapeutic agent can only modulate one disease pathway, resulting in limited efficacy, drug resistance, and non-responsiveness. Cancer cells can escape from single-drug treatment via alternative signaling pathways. Third, because tumor size is only a fraction of the body size, drug toxicity and ineffective administration to target tissues are common challenges in cancer treatment. Only a small fraction of the administered drug reaches the target tissue, while the majority of the drug enters non-targeted normal tissues, resulting in low efficacy to target tissues and high toxicity to normal tissues.

[0005] There is a need for pharmaceutical compositions and drug delivery systems that overcome the above challenges and that effectively penetrate target tissues without the risk of toxicity. [Brief description of the drawings]

[0006] [Figure 1] FIG. 1 shows a micelle composition of the present invention in which drug molecules are encapsulated within the micelles, which contain ligand-PEG-EGCG conjugates in the outer shell and oligomeric EGCG (OEGCG) in the inner shell. [Diagram 2] FIG. 2 shows a micelle composition of the present invention in which drug molecules are encapsulated within the micelles, the micelles containing ligand-PEG-EGCG conjugates+naked PEG-EGCG in the outer shell and oligomeric EGCG (OEGCG) in the inner shell. [Diagram 3] FIG. 3 shows a chemical synthesis scheme of RGD-PEG-EGCG via conjugating the N-terminus of RGD peptide to HOOC-PEG-EGCG. [Figure 4] FIG. 4 shows the chemical synthesis scheme of TP12-PEG-EGCG via conjugating the N-terminus of TP12 peptide to HOOC-PEG-EGCG. [Diagram 5]FIG. 5 shows the successful formulation of RGD-MINC-doxorubicin micelles (A) and TP12-MINC-doxorubicin micelles (B). [Figure 6] FIG. 6 shows the uptake of MINC-doxorubicin, RGD-doxorubicin, or TP12(TfR)-MINC-doxorubicin by tumor cells by measuring the fluorescent signal. [Figure 7] FIG. 7 shows tumor cell viability under MINC-doxorubicin treatment, RGD-MINC-doxorubicin treatment, or TP12(TfR)-MINC-doxorubicin treatment. [Figure 8] FIG. 8 shows a chemical synthesis scheme of RGD-PEG-EGCG via conjugating the C-terminus of RGD peptide to HO-PEG-EGCG. [Figure 9] FIG. 9 shows the chemical synthesis scheme of TP12-PEG-EGCG via conjugating the C-terminus of TP12 peptide to HO-PEG-EGCG. [Figure 10] FIG. 10 shows the chemical synthesis scheme of folate-PEG-EGCG via conjugating the COOH group of folic acid to HO-PEG-EGCG. [Figure 11] FIG. 11 shows a chemical synthesis scheme of RGD-PLA-EGCG via conjugating the N-terminus of RGD peptide to HOOC-PLA-EGCG. [Figure 12] FIG. 12 shows the chemical synthesis scheme of RGD-PLGA-EGCG via conjugating the N-terminus of RGD peptide to HOOC-PLGA-EGCG. [Figure 13] FIG. 13 shows a chemical synthesis scheme of RGD-dextran-EGCG via conjugating the C-terminus of the RGD peptide to HO-dextran-EGCG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] definition The term "about" is defined as ±10%, preferably ±5% of the stated value.

[0008] The term "cancer targeting ligand" as used herein refers to a molecule with a molecular weight of less than 10,000 daltons, e.g., 300-3500 daltons, such as a peptide, acid, or carbohydrate, that binds to or targets a receptor on the cancer cell surface or tumor environment.

[0009] The term "cytokine" refers to proteins (approximately 5-70 kDa) important in cell signaling. Cytokines have been shown to be involved in autocrine, paracrine, and endocrine signaling as immunomodulatory agents. Cytokines include interferons, interleukins, lymphokines, tumor necrosis factors, and chemokines.

[0010] The term "epigallocatechin gallate" refers to an ester of epigallocatechin and gallic acid and is used synonymously with "epigallocatechin-3-gallate" and EGCG.

[0011] The term "oligomeric EGCG" (OEGCG) refers to 3 to 20 covalently linked EGCG monomers. OEGCG preferably contains 4 to 12 EGCG monomers.

[0012] The term "nanoparticle" refers to a particle having a diameter of less than 1 μm, between 1 and 999 nm.

[0013] The term "polyethylene glycol-epigallocatechin gallate conjugate" or "PEG-EGCG" refers to polyethylene glycol (PEG) conjugated to one or two molecules of EGCG. The term "PEG-EGCG" refers to both PEG-mEGCG conjugates (monomeric EGCG) and PEG-dEGCG (dimeric EGCG) conjugates.

[0014] The term "MINC" (Multi-pathway Immune-modulating Nanocomplex Combination therapy) is a platform technology. As used herein, MINC leverages the bioactivity of PEG-flavonoid conjugates and oligomeric EGCG (OEGCG). MINC can be loaded with additional cancer therapeutic drugs to form MINC-drugs.

[0015] The term "MINC-drug" as used herein refers to a micelle having a shell formed by a cancer targeting ligand-PEG-flavonoid conjugate and, optionally, an oligomeric flavonoid, such as OEGCG, with the drug encapsulated within the shell.

[0016] Flavonoids Flavonoids suitable for the present invention have the general formula I: [ka] Formula I During the ceremony, R 1 is H or phenyl; R 2 is H, OH, gallic acid, or phenyl; optionally, the phenyl is substituted with one or more (e.g., 2-3) hydroxyls; R 3 is H, OH, or =O (oxo); or R 1 and R 2 taken together to form a closed ring structure; or R 2 and R 3 together form a closed ring structure.

[0017] Positions 2, 3, 4, 5, 6, 7, or 8 of formula I may be linked to a hydrocarbon, halogen, oxygen, nitrogen, sulfur, phosphorus, boron, or metal-containing group.

[0018] Examples of flavonoids of formula I include: [ka]

[0019] Preferred flavonoid compounds of formula I include: EGCG (CAS Registry Number 989-51-5), EC (CAS Registry Number 490-46-0), EGC (CAS Registry Number 970-74-1), or ECG (CAS Registry Number 1257-08-5). [ka]

[0020] Conjugates The present invention provides a conjugate comprising: (a) a cancer targeting ligand; (b) a hydrophilic polymer, which is polyethylene glycol (PEG), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), or dextran; and (c) a flavonoid of formula I, wherein the PEG is covalently attached to the flavonoid and to the cancer targeting ligand.

[0021] The conjugate treats cancer by targeting the cancer with a cancer-targeting ligand and delivering the active ingredient to the cancer tissue.

[0022] The cancer targeting ligand is a PEG, PLA, PLGA, or dextran that is attached to its -COOH group or its -NH 2 The hydrophilic polymer of the conjugate is usually covalently linked via a group by standard chemistry known to those skilled in the art. The molecular weight of the hydrophilic polymer of the conjugate is usually 1K to 100K, preferably 3K to 80K, more preferably 5K to 40K.

[0023] The flavonoid of the conjugate has the general formula (I) and is preferably EGCG, EC, EGC, or ECG. In one embodiment, the flavonoid is epigallocatechin gallate (EGCG).

[0024] In one embodiment, the PEG contains an aldehyde group conjugated to the 5-, 6-, 7-, or 8-position (preferably the 6- or 8-position) of the A ring of the flavonoid compound.

[0025] In another embodiment, PEG is present at the R 1 or R 2 (R 1 or R 2 is -OH).

[0026] In one embodiment, the conjugate comprises PEG-EGCG, in which PEG is linked to one or two molecules of EGCG; it can be prepared by conjugating an aldehyde-terminated PEG to EGCG by attachment of PEG via reaction of a free aldehyde group with the 5-, 6-, 7-, or 8-position (preferably 6- or 8-position) of formula I. See WO 2006 / 124000 and WO 2009 / 054813. PEG-EGCG can also be prepared by conjugating an aldehyde-terminated PEG to EGCG by attachment of PEG via reaction of a free aldehyde group with the 5-, 6-, 7-, or 8-position (preferably 6- or 8-position) of formula I. 1 or R 2 R of formula I is a phenyl group 1 or R 2 It can also be prepared by conjugating thio-terminated PEG to EGCG by attachment of PEG via reaction with, see WO 2015 / 171079.

[0027] In another embodiment, the conjugate comprises PEG-EC, PEG-EGC, or PEG-ECG, which may be prepared by conjugating an aldehyde-terminated PEG to EC, EGC, or ECG by attachment of PEG via reaction of a free aldehyde group with the 5-, 6-, 7-, or 8-position (preferably 6- or 8-position) of Formula I.

[0028] HOOC-PEG-CHO and HO-PEG-CHO are publicly available. In one embodiment, HOOC-PEG-CHO is conjugated to EGCG, EC, EGC, or ECG according to WO 2006 / 124000 and WO 2009 / 054813. HOOC-PEG-flavonoid has a COOH group for reacting with the N-terminus of a cancer targeting peptide. Typically, a cancer targeting peptide is incubated with HOOC-PEG-flavonoid, N,N'-dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide (NHS) in DMSO. The reaction is stirred at room temperature, protected from light, and placed under nitrogen. The reaction mixture is dialyzed against methanol and distilled water (membrane molecular weight cutoff=2000 Da). The solution is then lyophilized to obtain a lyophilized powder. To avoid peptide self-reaction, the C-terminus of cancer targeting peptide may be protected with, for example, resin during reaction.Commonly, Merrifield resin, hydroxymethyl polystyrene resin, PAM resin, and MBHA resin are used to prevent undesired peptide conjugation.After reaction, the resin can be removed under acidic conditions.

[0029] In another embodiment, HO-PEG-CHO is conjugated to EGCG, EC, EGC, or ECG according to WO 2006 / 124000 and WO 2009 / 054813. HO-PEG-flavonoid has an OH group to react with the C-terminus of the cancer targeting peptide. Typically, the peptide is incubated with HO-PEG-EGCG and N,N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction is stirred at room temperature, protected from light, and placed under nitrogen. The reaction mixture is dialyzed against methanol distilled water (membrane molecular weight cutoff=2000 Da). The solution is then lyophilized to obtain a lyophilized powder. To avoid self-reaction of the peptide, the N-terminus of the cancer targeting peptide may be protected during the reaction, for example with a resin. Typically, Merrifield resin, hydroxymethyl polystyrene resin, PAM resin, and MBHA resin are used to prevent undesired peptide conjugation. After the reaction, the resin can be removed under acidic conditions, where the COOH groups on the peptide react selectively with the OH groups on the PEG because the OH groups attached to the primary carbons on the PEG are more reactive than the OH groups attached to the tertiary carbons of the aromatic rings of the flavonoids.

[0030] HOOC-PLA-CHO, HOOC-PLGA-CHO, and HO-Dextran-CHO are commercially available.

[0031] In one embodiment, HOOC-PLA-CHO is conjugated to EGCG, EC, EGC, or ECG according to WO 2006 / 124000 and WO 2009 / 054813. HOOC-PLA-flavonoid has a COOH group for reacting with the N-terminus of the cancer targeting peptide. Typically, the cancer targeting peptide is incubated with HOOC-PLA-flavonoid, N,N'-dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide (NHS) in DMSO. The reaction is stirred at room temperature, protected from light, and placed under nitrogen. The reaction mixture is dialyzed against methanol and distilled water (membrane molecular weight cutoff=2000 Da). The solution is then lyophilized to obtain a lyophilized powder. To avoid self-reaction of the peptide, the C-terminus of the cancer targeting peptide may be protected during the reaction, for example with a resin. Commonly, Merrifield resin, hydroxymethyl polystyrene resin, PAM resin, and MBHA resin are used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions.

[0032] In one embodiment, HOOC-PLGA-CHO is conjugated to EGCG, EC, EGC, or ECG according to WO 2006 / 124000 and WO 2009 / 054813. HOOC-PLGA-flavonoid has a COOH group for reacting with the N-terminus of the cancer targeting peptide. Typically, the cancer targeting peptide is incubated with HOOC-PLGA-flavonoid, N,N'-dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide (NHS) in DMSO. The reaction is stirred at room temperature, protected from light, and placed under nitrogen. The reaction mixture is dialyzed against methanol and distilled water (membrane molecular weight cutoff=2000 Da). The solution is then lyophilized to obtain a lyophilized powder. To avoid self-reaction of the peptide, the C-terminus of the cancer targeting peptide may be protected during the reaction, for example with a resin. Commonly, Merrifield resin, hydroxymethyl polystyrene resin, PAM resin, and MBHA resin are used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions.

[0033] In one embodiment, HO-dextran-CHO is conjugated to EGCG, EC, EGC, or ECG according to WO 2006 / 124000 and WO 2009 / 054813. HO-dextran-flavonoid has an OH group to react with the C-terminus of the cancer targeting peptide. Typically, the peptide is incubated with HO-dextran-EGCG and N,N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction is stirred at room temperature, protected from light, and placed under nitrogen. The reaction mixture is dialyzed against methanol distilled water (membrane molecular weight cutoff=2000 Da). The solution is then lyophilized to obtain a lyophilized powder. To avoid self-reaction of the peptide, the N-terminus of the cancer targeting peptide may be protected during the reaction, for example with a resin. Commonly, Merrifield resin, hydroxymethyl polystyrene resin, PAM resin, and MBHA resin are used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions. In this reaction, the COOH groups on the peptide are converted to CH groups on the dextran. 2 It reacts selectively with the OH at the OH terminal because this OH is the only OH group bonded to a primary carbon in dextran and this OH group bonded to a primary carbon is more reactive than the other OH groups bonded to secondary carbons in dextran and tertiary carbons in the aromatic rings of flavonoids.

[0034] The cancer targeting ligand of the present invention is a ligand selected to target a receptor on the cancer cell surface or tumor environment. The cancer targeting ligand of the present invention is, for example, the following integrin receptors (α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, αLβ2, αMβ2, ICAM-1, αIIbβ3, αVβ1, αVβ3, αVβ3αVβ5αVβ5) αVβ3, αVβ5, αVβ5, αVβ6, αVβ8, α6β4), transferrin receptor, FGFR1, FGFR2, FGFR3, FGFR4, EGFR1 (HER1), EGFR2 (HER2), EGFR3 (HER3), EGFR4 (HER4), TNFR1, TNFR2, c-MET (HGFR), NOTCH1, NOTCH2, NOTCH3, NOTCH4, IR, AR, ER, PRPTK7 receptor, TrkA, TrkB, TrkC, GPCR, Eph receptor, AXL Targeting receptors on cancer including, but not limited to, receptors, Frizzled receptors, RET, ROS, folate receptors, IL-2R, IL-2RG, IL-2RB, IL-4R, IL-7R, IL-9R, CD133 receptor, LHRHR, LRP5, LRP6, CD44 receptor, CD47 receptor, CD20 receptor, Fas receptor, DR4, DR5, LEP-R, MUC1 receptor, adiponectin receptor, alpha-adrenergic receptor, nucleolin receptor, PD-L1, ASGPR, lectin receptors, annexin receptors, glycyrrhetinic acid receptor cadherin, and EpCAM. The cancer targeting ligands of the invention interact with targets within the tumor microenvironment, for example, α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, αLβ2, αMβ2, ICAM-1, αIIbβ3, αVβ1, αVβ3, αVβ3αVβ5αVβ5αVβ3, αVβ5, αVβ5, αVβ6, αVβ8, α6β4MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP 18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, CCL2, CCL5, CXCL12, ICAM-1, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, CCR2, CXCR4, CSF-1R, VEGFR1, VEGFR2, VEGFR3, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FG F12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WN T5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 , IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-21, IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, I Examples of target proteins include, but are not limited to, IL-11R, IL-12R, IL-13R, IL-15R, IL-17R, IL-21R, PDGF, PDGFR-α, PDGFR-β, IGF1, IGF2, TGF-β, EGF, HB-EGF, TNF-α, TNFβ, TNFγ, CD19, CD103, IDO, PD-1, CTLA-4, collagen, elastin, glycosaminoglycans, proteoglycans, cancer-associated fibroblasts, tumor-associated macrophages, T cells, B cells, and dendritic cells.

[0035] In one embodiment, the cancer targeting ligand is RGD, which targets integrin αvβ3.

[0036] In one embodiment, the cancer targeting ligand is TP12, having the amino acid sequence THRPPMWSPVWP (SEQ ID NO:1), which targets the transferrin receptor in a variety of cancers.

[0037] In one embodiment, the cancer targeting ligand is MC11, having the amino acid sequence MQLPLATGGGC (SEQ ID NO:2), which targets FGFR in a variety of cancers.

[0038] In one embodiment, the cancer targeting ligand is FV12, having the amino acid sequence FCDGFYACYMDV (SEQ ID NO:3), which targets HER2 in a variety of cancers.

[0039] In one embodiment, the cancer targeting ligand is YI12 having the amino acid sequence YHWYGYTPQNVI (SEQ ID NO: 4), which targets EGFR. In one embodiment, the cancer targeting ligand is CTT having the amino acid sequence CTTHWGFTLC (SEQ ID NO: 5), which targets MMP2 / MMP9 in the tumor microenvironment.

[0040] In one embodiment, the cancer targeting ligand is the H2009.1 peptide having the amino acid sequence RGDLATLRQLAQEDGVVGVR (SEQ ID NO: 6), which targets the integrin αvβ6 receptor.

[0041] In one embodiment, the cancer targeting ligand is an IL-13 peptide having the amino acid sequence GSETWKTIITKN (SEQ ID NO:7), which targets the IL-13Rα2 receptor.

[0042] In one embodiment, the cancer targeting ligand is an AP-1 peptide having the amino acid sequence RKRLDRN (SEQ ID NO:8), which targets the IL-4 receptor.

[0043] In one embodiment, the cancer targeting ligand is CVKTPAQSC (SEQ ID NO: 9), which targets the CD133+ receptor.

[0044] In one embodiment, the cancer targeting ligand is a CC9 peptide having the amino acid sequence CDCRGDCFC (SEQ ID NO: 10), which targets integrins in the cancer and tumor environment, including αvβ3, αvβ5, αvβ6, αvβ8, αIIbβ3, α8β1, and α5β1.

[0045] In one embodiment, the cancer targeting ligand is the RGDS peptide having the amino acid sequence RGDS (SEQ ID NO:11), which targets the integrin αvβ3 receptor.

[0046] In one embodiment, the cancer targeting ligand is the NR7 peptide having the amino acid sequence NSVRGSR (SEQ ID NO: 12), which targets .

[0047] In one embodiment, the cancer targeting ligand is an LHRH peptide having the amino acid sequence EHWSYGLRPG (SEQ ID NO: 13), which targets the LHRHR.

[0048] In one embodiment, the cancer targeting ligand is angiopep-2 peptide having the amino acid sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 14), which targets the LRP.

[0049] In one embodiment, the cancer targeting ligand is a TbFGF peptide having the amino acid sequence KRTGQYKLC (SEQ ID NO: 15), which targets EGFR.

[0050] In one embodiment, the cancer targeting ligand is an EGF peptide having the amino acid sequence YHWYGYTPQNVI (SEQ ID NO: 16), which targets EGFR.

[0051] In one embodiment, the cancer targeting ligand is folic acid or folate (C 19 H 19 N 7 O 6 ), which targets the folate receptor in cancer.

[0052] In one embodiment, the cancer targeting ligand is hyaluronic acid, which targets the CD44 receptor.

[0053] In one embodiment, the cancer targeting ligand is lactose or galactosamine, which targets the ASGPR.

[0054] In one embodiment, the cancer targeting ligand is galactose, which targets ASGPR and lectin receptors.

[0055] In one embodiment, the cancer targeting ligand is glycyrrhetinic acid, which targets the glycyrrhetinic acid receptor.

[0056] Nanoparticle Composition The term "MINC" (Multi-pathway Immune-modulating Nanocomplex Combination therapy) is a platform technology. The present invention provides a nanoparticle micelle (MINC) composition. The micelle contains a cancer targeting ligand-PEG-flavonoid conjugate in the outer shell and an oligomeric EGCG (OEGCG) in the inner shell (see Figure 1). The cancer targeting ligand enables the nanoparticle composition to specifically target cancer tissue.

[0057] In one embodiment, the nanoparticle micelle composition has a well-defined and narrow size distribution in that at least 70% of the nanoparticles have a diameter between 20 and 500 nm or between 50 and 300 nm, and the size distribution of the nanoparticles has only one major peak that contains more than 90% of the total nanoparticles.

[0058] The micelles optionally contain a cancer therapeutic molecule (drug) encapsulated within the micelle (MINC-drug).

[0059] The MINC-drug composition contains three active ingredients that are functionally complementary to each other, addressing both immune response and signaling pathways through the MINC-drug backbone components (PEG-flavonoid / OEGCG) and additional signaling pathways through selected drug molecules for treating cancer. Each nanoparticle is a fixed-dose combination containing the three active ingredients in a fixed molar ratio.

[0060] The present invention delivers MINC-drugs to targeted cancer tissues by active delivery of micelles via cancer targeting ligands to tumors with specific receptors. Additionally, the present invention delivers MINC-drugs to tumors by passive delivery based on nanoparticle size.

[0061] The size of the formulation determines how much of the drug will go preferentially to the tumor compared to other unintended tissues. Normal, unintended healthy tissues usually have blood vessel openings less than 10 nm. Nanoparticles with a single size distribution around 100 nm can be taken up more efficiently by targeted cells. Nanoparticles with a single peak and limited size around 100 nm have been shown to be able to deliver more therapeutic agents to targeted cells. The nanoparticle micelle composition of the present invention is dominated by particles with sizes of 20-500 nm or 50-300 nm. If the particle size is smaller than 50 nm, there is a high risk that the particles will not be sufficiently taken up by the cells. If the particle size is larger than 300 nm, it may cause excessive uptake by the reticuloendothelial (RE) system, resulting in side effects. The composition contains more than 70% of the particles in the size range of 20-500 nm, allowing the therapeutic agent to enter the lesion preferentially over normal tissues and the RE system. Furthermore, when the nanoparticle size is limited to 20-500 nm, selective uptake by targeted cells becomes possible, increasing the efficacy of drugs (agents).

[0062] The nanoparticle micelle compositions of the present invention have a narrow particle size distribution in that they have only one main peak that contains more than 90% of the total nanoparticles. It is important that the therapeutic composition have only one particle size distribution peak, rather than several or many peaks. If the therapeutic composition has more than one molecular size, serious variations in therapeutic efficacy and patient response rates can occur.

[0063] The nanocomplex of the present invention contains two first active ingredients, OEGCG and PEG-flavonoid such as PEG-EGCG, in the backbone of a micelle composition. These active ingredients are derivatives of EGCG, which are potent immunomodulators and control a wide range of disease signal pathways. EGCG controls both innate and adaptive immunity. However, the bioavailability of EGCG is low and EGCG is not stable. The nanocomplex composition overcomes the problem of EGCG bioavailability by forming a nanocarrier to carry EGCG to target sites for treatment, and overcomes the problem of EGCG stability by forming a complex between OEGCG and PEG-EGCG, which effectively enables EGCG to be a highly effective therapeutic agent.

[0064] The nanocomplexes of the present invention optionally contain a third active ingredient, which is a drug molecule encapsulated in the nanoparticles for treating cancer.

[0065] Nanoparticles with sizes between 20 and 500 nm preferentially reach the intended treatment site. Large nanoparticles (500-999 nm) or micron-sized particles (1000-5000 nm) due to aggregation of smaller nanoparticles may result in toxicity because larger nanoparticles are often efficiently taken up by the reticuloendothelial system (RES), also known as the mononuclear phagocyte system (MPS), located in the liver, lungs, and bone marrow.

[0066] The inventors have discovered nanoparticle micelle compositions of the invention for targeted delivery to target tissues, wherein at least 70% of the nanoparticles have a diameter between 20-500 nm or 50-300 nm, and the size distribution of the nanoparticles has only one major peak containing more than 90% of all particles. The inventors have also discovered methods for preparing such nanoparticle compositions.

[0067] The present invention relates to a nanoparticle composition comprising nanoparticles having (a) an inner core comprising oligomeric (-)-epigallocatechin gallate (OEGCG), (b) an outer core comprising a cancer targeting ligand-PEG-flavonoid conjugate, and (c) a drug molecule encapsulated in the inner shell. In one embodiment, at least 70% of the nanoparticles have a diameter of 20-500 nm or 50-300 nm, and the size distribution of the nanoparticles has only one major peak that includes more than 90% of all particles. The flavonoid in the conjugate is preferably EGCG, EC, EGC, or ECG, with EGCG being more preferred. The structure of the nanoparticles of the present invention is shown in FIG. 1.

[0068] In one embodiment, the shell of the micelle further comprises a naked PEG-flavonoid conjugate, such as PEG-EGCG, which does not have a cancer targeting ligand linked to the PEG-flavonoid. In such a shell of the micelle, the ratio of ligand-PEG-EGCG to ligand-PEG-EGCG+PEG-EGCG is usually more than 10%, or more than 20%, or more than 30%, or more than 50%, and is up to 100%. See FIG. 2.

[0069] In one embodiment, at least 80%, or at least 85%, or at least 90%, or at least 95% of the nanoparticles have a diameter of 20 to 500 nm, or 50 to 300 nm.

[0070] In one embodiment, the median diameter of nanoparticles in the nanoparticle composition is 50 to 250 nm, 50 to 200 nm, 80 to 200 nm, 100 to 200 nm, or 50 to 150 nm.

[0071] In one embodiment, the size distribution of the nanoparticles exhibits only one major narrow peak comprising more than 80%, more than 85%, more than 90%, more than 95%, or more than 98% of all particles.

[0072] In one embodiment, the cancer therapeutic drug molecule of the MINC-agent is a cytokine, an antibody, a chemotherapeutic drug, or a small molecule compound inhibitor.

[0073] Cytokines suitable for the present invention for treating cancer include, but are not limited to, IL-2, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, TARIL, IGF1, GLP-1, IFN-α, IFN-β, IFN-γ, CCL5, CXCL9, CXCL10, CXCL11, CX3CL1, and recombinant cytokine products.

[0074] Antibodies suitable for the present invention for cancer treatment include, but are not limited to, monoclonal antibodies, polyclonal antibodies, antibody-drug conjugates, and bispecific antibodies. Preferred antibodies for the present invention are monoclonal antibodies. Antibodies suitable for the present invention include anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-LAG3 antibodies, anti-TIGIT antibodies, anti-TIM3 antibodies, anti-HER2 antibodies, anti-HER3 antibodies, anti-HGFR antibodies, anti-EGFR antibodies, anti-EpCAM, anti-FOLR1 antibodies, anti-c-Met antibodies, anti-GD2 ganglioside antibodies, anti-GD3 ganglioside antibodies, anti-VEGFR1 antibodies, anti-VEGF antibodies, anti-TGF-β antibodies, anti-TNF-α antibodies, anti-IGF-1R antibodies, anti-IL-4 antibodies, anti-IL-10 antibodies, anti-IL-13 antibodies, anti-C-cell antibodies, anti-IL-2 antibodies, anti-IL-3 antibodies, anti-IL-4 antibodies, anti-IL-5 antibodies, anti-IL-6 antibodies, anti-IL-7 antibodies, anti-IL-8 antibodies, anti-IL-9 antibodies, anti-IL-10 antibodies, anti-IL-13 antibodies, anti-IL-14 antibodies, anti-IL-15 antibodies, anti-IL-16 antibodies, anti-IL-17 antibodies, anti-IL-18 antibodies, anti-IL-19 antibodies, anti-IL-20 antibodies, anti-IL-21 antibodies, anti-IL-22 antibodies, anti-IL-23 antibodies, anti-IL-24 antibodies, anti-IL-25 antibodies, anti-IL-26 antibodies, anti-IL-27 antibodies, anti-IL-28 antibodies, anti-IL-29 antibodies, anti-IL-30 antibodies, anti-IL-31 antibodies, anti-IL-32 antibodies, anti-IL-33 antibodies, anti-IL-34 antibodies, anti-IL-35 antibodies, anti-IL-36 antibodies, anti-IL-37 antibodies, anti-IL-38 D3 antibody, anti-CD4 antibody, anti-CD40 antibody, anti-CD40L antibody, anti-CD43 antibody, anti-CD19 antibody, anti-CD27 antibody, anti-CD70 antibody, anti-CD71 antibody, anti-CD28 antibody, anti-CD38 antibody, anti-CD20 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, Anti-DR5 antibody, anti-MUC1 antibody, anti-Tau antibody, anti-β amyloid antibody, avagovomab, abituzumab, adalimumab, aducanumab, alemtuzumab, amatuximab, amivantamab, anif Lorumab, atezolizumab, avelumab, bapineuzumab, basiliximab, belimumab, benralizumab, besilesomab, bevacizumab, bezlotoxumab, blinatumomab, brazikumab, brontixumab, cabiralizumab, camrelizumab, carlumab, carotuximab, catumaxomab, cedelizumab, cetrelimab, cetuximab, civisatamab, crenezumab, cusatuzumab, daclizumab, daclizumab, darotuzumab Zumab, daratumumab, detumomab, dinutuximab, drozitumab, durigotuzumab, dupilumab, durvalumab, ecromeximab, emibetuzumab, epcolitamab, epratuzumab, eptinezumab, erenumab, ertumaxomab, etaracizumab, etesevimab, farletuzumab, fezakinumab, ficlatuzumab, figitumumab, fretikumab, foralumab,fresolimumab, futuximab, ganitumab, gantenerumab, gatipotuzumab, gevokizumab, golimumab, guselkumab, icrucumab, igovomab, imalumab, imgatuzumab, inebilizumab, infliximab, intetumumab, ipilimumab , istiratumab, ixekizumab, letolizumab, lexatumumab, lintuzumab, mapatuzumab, matuzumab, mavrilimumab, mepolizumab, mogamulizumab, monalizumab, mosunetuzumab, natalizumab, naxitumab, necitumumab, nimotuzumab, nivolumab, ocralizumab, ocrelizumab, ofatumumab, olaratumab, olaratumab opisinumab, panitumumab, pembrolizumab, pertuzumab, ponezumab, ramucirumab, ranibizumab, rituximab, samalizumab, sarilumab, secukinumab, sintilimab, solanezumab, teprotumumab, tigatuzumab, tildrakizumab, timigutuzumab, tocilizumab, tomuzotuximab, trastuzumab Mab, ustekinumab, vanucizumab, varisacumab, varlilumab, vedolizumab, vepalimomab, besencumab, visilizumab, bonlerolizumab, zanolimumab, zatuximab, zenoctuzumab, zolbetuximab, ado-trastuzumab etansine, anetumab ravtansine ravtansine, brentuximab vedotin, cantuzumab mertansine, certolizumab pegol, coltuximab ravtansine, depatuximab mafodotin, enapotamab vedotin, gemtuzumab ozogamicin, grembatumumab vedotin, iradatuzumab vedotin, inatuzumab vedotin, indatuximab ravtansine, indusatumab vedotin, rifastuzumab vedotin,These include rilotumab satetrazole, lorvetuximab mertansine, rosatuximab vedotin, lulizumab pegol, mirvetuximab soravtansine, naratuximab emtansine, inotuzumab ozogamicin, polatuzumab vedotin-piiq, rovalpituzumab tesirin, sacituzumab govitecan, samrotamab vedotin, terisotuzumab vedotin, trastuzumab deruxtecan, and tucotuzumab celmoleukin. Antibodies also include antibody fragments capable of binding to the corresponding antigen, such as Fab, (Fab)2, or single chain antibodies.

[0075] Chemotherapeutic agents for the treatment of cancer suitable for the present invention include, but are not limited to, altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, trabectedin, 5-fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, clofarabine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate, trifluridine, vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan, topotecan, daunorubicin, doxorubicin, epirubicin, idarubicin, and valrubicin.

[0076] Suitable small molecule inhibitors for cancer therapy according to the present invention include, but are not limited to, imatinib, gefitinib, erlotinib, sunitinib, lapatinib, nilotinib, sorafenib, temsirolimus, everolimus, pazopanib, crizotinib, ruxolitinib, vandetanib, axitinib, bosutinib, cabozantinib, ponatinib, regorafenib, ibrutinib, trametinib, or perifosine for targeting tyrosine kinases and serine / threonine kinases.

[0077] Small molecule compound inhibitors for cancer therapy suitable for the present invention include, but are not limited to, bortezomib, carfilzomib, or marizomib for targeting the proteasome.

[0078] Suitable small molecule inhibitors for cancer treatment according to the present invention include, but are not limited to, batimastat, neovastat, prinomastat, levimastat, marimastat, ganetespib, or NVP-AUY922 for targeting MMPs and HSPs.

[0079] Suitable small molecule compound inhibitors for treating cancer according to the present invention include, but are not limited to, obatoclax or navitoclax to induce apoptosis.

[0080] The nanoparticle composition of the present invention has a diameter of 20-500 nm or 50-300 nm, and OEGCG, PEG-EGCG, and drug molecules are held together by hydrophobic interactions. The nanoparticle composition of the present invention is stable in a hydrophilic environment such as blood circulation, and dissociates in a hydrophobic environment such as tumor tissue. The nanoparticle composition of the present invention can selectively diffuse from blood vessels to surrounding tissues that have leaky blood vessels due to inflammation and other hyperactivity such as rapid and uncontrolled tumor growth. Due to its size, the nanoparticle composition of the present invention is limited in its entry into normal tissues that have less leaky blood vessels. Upon entering the hydrophobic tissue, the nanoparticle complex dissociates and releases its active ingredients, OEGCG, PEG-flavonoid such as PEG-EGCG, and drug molecules in the nanocomplex. The released active ingredients regain their biological activity in retarding cancer. The active ingredients in the nanoparticles have a longer circulation half-life and act as a sustained release mechanism, which further reduces the required dose of the drug. As a result, any adverse effects on normal tissues are further attenuated.

[0081] The MINC-pharmaceutical composition further has the following advantages: MINCs are stable in the circulation for more than 15 days. During circulation, the encapsulated drug molecules are protected within the shell of the MINC nanoparticles. MINC nanoparticles utilize the enhanced permeability and retention (EPR) effect to deliver the majority of drug molecules to target cells. ·MINC nanoparticles do not aggregate during freeze-thaw cycles. ·MINC drugs retain their original biological activity after lyophilization. ·MINC drugs are stable at temperatures between 2 and 8°C.

[0082] Methods for preparing nanoparticle compositions The present invention provides a method for preparing a fixed dose combination nanoparticle composition, which is optimized to produce only nanometer sized particles, with at least 70% of the particles having a diameter between 20-500 nm or 50-300 nm and having one main peak.

[0083] The method includes the steps of: (a) mixing a drug molecule with OEGCG and a cancer targeting ligand-PEG-EGCG conjugate in an aqueous solution; and (b) filtering the mixture through a membrane having a molecular weight cut-off of 8,000 to 300,000 daltons to remove low molecular weight molecules and retain high molecular weight molecules.

[0084] In one preferred embodiment, the above method further comprises the step (c) of filtering high molecular weight molecules through a 0.2 to 0.3 μm membrane and recovering the filtrate.

[0085] The method optionally further comprises a freeze-drying step (d) after step (c): freeze-drying the filtrate by stepwise freezing at (i) about 0 to 5°C, (ii) about -20 to -30°C, and (iii) about -60 to -100°C, followed by drying.

[0086] In step (a), the drug molecule is dissolved in an aqueous solvent such as phosphate buffered saline, saline, water, bicarbonate buffer, oxyhemoglobin buffer, bis-trisalkane, tris-HCl, HEPES, histidine buffer, NP-40, RIPA (radioimmunoprecipitation buffer), tricine, TES, TAPS, TAPSO, bicine, MOPS, PIPES, cacodylate, or MES. Preferred solvents are phosphate buffered saline, saline, or water. The protein drug concentration is usually 0.01-50 mg / ml, preferably 0.05-10 mg / ml, and more preferably 0.1-5 mg / ml.

[0087] OEGCG, PEG-EGCG, and optionally EGCG are dissolved in a ketone, acetonitrile, alcohol, aldehyde, ether, acetate, sulfoxide, benzene, organic acid, amide, aqueous buffer, and any combination thereof. Preferred solvents are alcohol, acetonitrile, sulfoxide, amide, and any combination thereof. The OEGCG / EGCG concentration and the PEG-EGCG concentration are usually, independently of each other, 0.001-10 mg / ml, preferably 0.005-1 mg / ml, or 0.1-5 mg / ml.

[0088] It is important that OEGCG is in excess of the drug in molar ratio. Usually, the molar ratio of EGCG to drug molecules in OEGCG is 1-500:1, 2-500:1, 3-500:1, or 5-500:1, preferably 3-100:1, 5-100:1, or 10-50:1. The molar ratio is calculated as the number of moles of monomeric EGCG to the number of moles of drug molecules in OEGCG. By using an excess molar ratio of EGCG, most or all of the drug is encapsulated by the OEGCG molecules. Unencapsulated drug, which would not be selectively distributed to the target tissue and would cause problems of low efficacy and safety, is avoided by adjusting the molar ratio of OEGCG to protein in this method.

[0089] The drug, OEGCG, and PEG-EGCG are mixed for 1 minute to 2 days, preferably 1 minute to 12 hours, at a temperature of about 0°C to 60°C, preferably 0°C to 45°C, or 0°C to 37°C.

[0090] In step (b), the mixture is filtered through a membrane having a molecular weight cutoff of 8,000 to 300,000 daltons, preferably 8,000 to 200,000 daltons, 8,000 to 150,000 daltons, or 8,000 to 12,000 daltons, to remove low molecular weight molecules and retain high molecular weight molecules. The ultrafiltration material is selected from the group consisting of cellulose (and its derivatives), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, polyvinylidene fluoride or polyvinylidene difluoride (PVDF), and polypropylene (PP); preferably, it is selected from the group consisting of cellulose (and its derivatives), PTFE, and PVDF.

[0091] The mixture is optionally diluted with an aqueous solvent, such as those described above, in step (a) prior to ultrafiltration.

[0092] Ultrafiltration step (b) removes undesired low molecular weight impurities such as unreacted OEGCG or EGCG or reaction by-products. These impurities may reduce efficacy and safety. Excess unreacted OEGCG or EGCG may cause individual nanoparticles to aggregate, resulting in particles of about 1000 nm size, which may reduce efficacy and cause potential toxicity.

[0093] In step (c), the retained high molecular weight molecules are filtered through a membrane with a pore size of about 0.2-0.3 μm (e.g., 0.22 μm) and the filtrate is collected. This removes unwanted high molecular size impurities such as mega-aggregates. These aggregates may be excluded from entering tissues due to their mega size. These aggregates reduce overall efficacy / safety and are more likely to induce immunogenicity in patients. Large size nanoparticles are also more easily taken up by the liver, lungs, and RES of less desirable organs.

[0094] The membrane material in step (c) is selected from the group consisting of cellulose (and its derivatives), PES, PTFE, nylon, PVDF, and PP; preferably, it is selected from the group consisting of cellulose (and its derivatives), PES, and PP.

[0095] In one embodiment, steps (b) and (c) are repeated at least once, e.g., 1, 2, 3, or 4 times, prior to step (d) to effectively remove unwanted small molecule impurities and large aggregates.

[0096] After step (c), the filtrate is stored at 2-8°C and is stable for at least 100 days.

[0097] The method optionally further comprises a freeze-drying step (d) after step (c) to allow for long-term stability of the nanoparticle composition.

[0098] In step (d), the filtrate recovered after filtration through a 0.2-0.3 μm membrane is freeze-dried by first stepwise freezing the filtrate (i) at about 0-5° C., e.g., for about 1-3 hours, (ii) at about −25° C. to −30° C., e.g., for about 1-3 hours, and then (iii) freezing at −60° C. to −100° C. or −70° C. to −100° C., e.g., for at least 8 hours.

[0099] After freezing, the material is lyophilized for 1 to 7 days.

[0100] Freezing and lyophilization often cause nanoparticles to form complexes and aggregates. These large particles may be too large to penetrate the blood vessels and enter the tissue environment. As a result, efficacy and safety may be reduced, and immunogenicity may increase. To avoid these possibilities caused by lyophilization, the present method preserves nanoparticle size during lyophilization by using a stepwise freezing method instead of a continuous freezing method (gradually lowering the temperature during freezing).

[0101] Pharmaceutical Compositions The present invention provides a pharmaceutical composition comprising the nanoparticle composition of the present invention and, optionally, one or more pharma- ceutically acceptable carriers. In the case of a tablet, powder, or parenteral formulation, the nanoparticle composition in the pharmaceutical composition is usually about 1-90%, preferably 20-90%, or 30-80%. In the case of a capsule formulation, the nanoparticle composition in the pharmaceutical composition is usually 1-100%, preferably 20-100%, 50-100%, or 70-100%. In the case of a suspension formulation, the nanoparticle composition in the pharmaceutical composition is usually 1-50%, 5-50%, or 10-40%.

[0102] In one embodiment, the pharmaceutical composition may be in the form of tablets, capsules, granules, microgranules, powders, suspensions, patches, parenterals, injections, or the like. The pharmaceutical compositions described above may be prepared by conventional methods.

[0103] Pharmaceutically acceptable carriers are inert ingredients and can be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers include saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents, such as sodium chloride, potassium chloride, glycerol, and glucose; pH adjusters and buffers, such as hydroxide salts, phosphate salts, citrate salts, acetate salts, borates, and salts of trolamine; antioxidants, such as bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, and salts of ascorbyl palmitate, acids, and / or bases; surfactants, such as lecithin and phospholipids, including, but not limited to, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol; poloxamers and poloxamines; polysorbate 80, ... It may contain ingredients including, but not limited to, polysorbates such as resorbate 60 and polysorbate 20; polyethers such as polyethylene glycol and polypropylene glycol; polyvinyls such as polyvinyl alcohol and polyvinylpyrrolidone (PVP, povidone); cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose, and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, and soybean oil; monoglycerides, diglycerides, and triglycerides; polysaccharides such as dextran; and glycosaminoglycans such as sodium hyaluronate. Such pharma- ceutically acceptable carriers may be preserved against microbial contamination through the use of well-known preservatives, including, but not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated as unpreserved preparations for single or multiple use.

[0104] For example, tablets, capsules, or parenteral formulations of an active compound may contain other excipients that are not biologically active and do not react with the active compound. Tablet or capsule excipients may include fillers, binders, lubricants and glidants, disintegrants, wetting agents, and release rate modifiers. Examples of tablet or capsule excipients include, but are not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, poly(acrylic acid), and polyvinylpyrrolidone. For example, tablet formulations may contain inactive ingredients such as colloidal silicon dioxide, crospovidone, hypromellose, magnesium stearate, microcrystalline cellulose, polyethylene glycol, sodium starch glycolate, and titanium dioxide. Capsule formulations may contain inactive ingredients such as gelatin, magnesium stearate, and titanium dioxide. Powdered oral formulations may contain inactive ingredients such as silica gel, sodium benzoate, sodium citrate, sucrose, and xanthan gum.

[0105] Treatment methods The present invention relates to a method of treating cancer comprising administering to a subject in need thereof an effective amount of a nanoparticle composition of the present invention.

[0106] An "effective amount," as used herein, is an amount effective to treat a disease by ameliorating a pathological condition or reducing the symptoms of the disease.

[0107] Cancers suitable for treatment by the present invention include adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendix cancer, grade I (anaplastic) astrocytoma, grade II astrocytoma, grade III astrocytoma, grade IV astrocytoma, atypical teratoid / rhabdoid tumor of the central nervous system, basal cell carcinoma, bladder cancer, breast cancer, bronchial carcinoma, bronchioloalveolar carcinoma, Burkitt's lymphoma, cervical cancer, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endometrial cancer, endometrial carcinoma, and uterine cancer. cancer), ependymomas, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, fibrous histiocytoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic tumors, gestational trophoblastic tumors, gliomas, head and neck cancer, cardiac cancer, hepatocellular carcinoma, hilar cholangiocarcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, Kaposi's sarcoma, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, endocrine neoplasia neoplasia), multiple myeloma, mycosis fungoides, myelodysplasia, myelodysplastic / myeloproliferative neoplasms, myeloproliferative disorders, nasal cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian clear cell carcinoma, ovarian epithelial cancer, ovarian germ cell tumor, papilloma, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pineal parenchymal tumor, pineoblastoma, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm, pleuropulmonary blastoma, primary central nervous system lymphoma, These include prostate cancer, rectal cancer, renal cell carcinoma, airway cancer with chromosome 15 alterations, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sezary syndrome, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical carcinoma, supratentorial primitive neuroectodermal tumor, supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymic carcinoma, thymoma, thyroid cancer, renal pelvis cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's hypergammaglobulinemia, and Wilms' tumor.

[0108] The dosage of the ligand-PEG-EGGC conjugate for injection is usually 0.01 to 1200 mg / kg (total weight of the conjugate / body weight of the subject), or 0.1 to 1000 mg / kg.

[0109] Dosing of the MINC-drug is based on known doses of the drug to treat a particular disease and the condition of the subject. The doses may be Food and Drug Administration (FDA) approved doses or doses used in clinical trials.

[0110] In MINC-drugs, the weight of the ligand-PEG-EGCG is approximated to the amount of the encapsulated drug. Typically, the dose of PEG-EGCG combined with OEGCG is 10ug / kg to 100mg / kg.

[0111] Dosing of the nanoparticle composition is based on known doses of protein drugs to treat a particular disease and the condition of the subject. For example, to treat breast cancer in adults, trastuzumab is administered at 4-8 mg / kg by IV infusion once a week for 52 weeks. Effective amounts of Ligand-MINC-Trastuzumab are within the same dose range, but administered less frequently, once every 12-16 weeks for 52 weeks.

[0112] For example, in the case of melanoma treatment, interferon-α induction is 20 million IU / m 2 as an IV infusion for 5 consecutive days / week for 4 weeks. Effective doses of Ligand-MINC-Interferon-α are within the same dose range and can be given 1 day / week for 2 weeks to achieve the same efficacy and reduce toxicity.

[0113] For example, in the treatment of renal cancer, 600,000 International Units / kg (0.037 mg / kg) of IL-12 is administered three times a day for up to 14 doses. After a nine-day rest period, the above schedule is repeated for an additional 14 doses, as tolerated. An effective dose of Ligand-MINC-IL-12 within the same dose range is administered once a day for three days for a total of nine doses.

[0114] The invention is useful in treating humans and non-human animals. For example, the invention is useful in treating mammalian subjects such as humans, horses, pigs, cats, and dogs.

[0115] The following examples further illustrate the present invention. These examples are intended merely to illustrate the invention and should not be construed as limiting. EXAMPLES

[0116] Example 1: Conjugation of RGD peptide to HOOC-PEG-EGCG material RGD peptide was purchased from Biotools. HOOC-PEG-CHO was purchased from NBC Chemical Co.

[0117] method HOOC-PEG-CHO was conjugated to EGCG according to WO 2006 / 124000 and WO 2009 / 054813; the COOH group on PEG and the NH group on RGD were 2 The RGD peptide was conjugated to HOOC-PEG-EGCG via conjugation between the HOOC group to form RGD-PEG-EGCG (N'-linked) (Figure 3).

[0118] Specifically, 1-1000 mg of RGD was PEGylated by incubating with 1-1000 mg of HOOC-PEG-EGCG, 1-1000 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 1-1000 mg of N-hydroxysuccinimide (NHS) in DMSO. The reaction was kept under nitrogen for 24 hours with stirring at room temperature, protected from light. The reaction mixture was dialyzed against methanol and distilled water for 3 days (membrane molecular weight cutoff = 2000 Da). The solution was then lyophilized to obtain a lyophilized powder.

[0119] result The formulation of RGD-PEG-EGCG was confirmed using HPLC. HPLC was performed under the following conditions: Column: C18, 4.6×150 mm, 4 μm; Elution: A=0.1% TFA / H 2 O, B = 0.1% TFA / ACN; oven temperature: 40°C; flow rate: 1 ml / min; autosampler temperature: 15°C; measurement: UV280.

[0120] HOOC-PEG-EGCG had a retention time of 5.95 min, but after RGD conjugation, a new peak appeared with a retention time of 6.38 min. HPLC results indicate the successful conjugation of RGD-PEG-EGCG.

[0121] Example 2: Conjugation of transferrin peptide (TP12) to HOOC-PEG-EGCG material TP12 peptide was purchased from Biotools. HOOC-PEG-CHO was purchased from NBC Chemical Company.

[0122] method For the TP12-PEG-EGCG formulation, HOOC-PEG-CHO was conjugated to EGCG according to WO 2006 / 124000 and WO 2009 / 054813; the TP12 peptide was conjugated to HOOC-PEG-EGCG via conjugation between the COOH group on PEG and the NH2 group on TP12 to form TP12-PEG-EGCG (N'-linked) (Figure 4).

[0123] Specifically, 1-1000 mg of TP12 was PEGylated by incubating with 1-1000 mg of HOOC-PEG-EGCG, 1-1000 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 1-1000 mg of N-hydroxysuccinimide (NHS) in DMSO. The reaction was kept under nitrogen for 24 hours with stirring at room temperature, protected from light. The reaction mixture was dialyzed against methanol and distilled water for 3 days (membrane molecular weight cutoff = 2000 Da). The solution was then lyophilized to obtain a lyophilized powder.

[0124] result The formulation of TP12-PEG-EGCG was confirmed using HPLC. HPLC was performed under the following conditions: Column: C18, 4.6×150 mm, 4 μm; Elution: A=0.1% TFA / HO, B=0.1% TFA / ACN; Oven temperature: 40° C.; Flow rate: 1 ml / min; Autosampler temperature: 15° C.; Measurement: ELSD.

[0125] HOOC-PEG-EGCG had a retention time of 6.04 min. After conjugation of TP12, a new peak appeared with a retention time of 6.28 min. These results indicate the successful conjugation of TP12-PEG-EGCG.

[0126] Example 3: Preparation of RGD-MINC-doxorubicin and TP12-MINC-doxorubicin material RGD-PEG-EGCG was prepared according to Example 1. TP12-PEG-EGCG was prepared according to Example 2. Doxorubicin was purchased from Sigma-Aldrich or other suppliers.

[0127] method RGD-MINC-doxorubicin nanoparticles and TP12-MINC-doxorubicin nanoparticles were prepared according to the following protocol: 1. Incubate 5-500 μg of doxorubicin in 1 mL of DMSO for 15 min to 1 h. 2. Add 1-100 μg of OEGCG and 1-10,000 μg of RGD-PEG-EGCG or TP12-PEG-EGCG. Incubate the mixture at 25° C. for 3 hours. 3. Filter the liquid through a 10K MWCO filter unit and wash the filter three times with 0.9% NaCl. 4. Freeze-dry to a dry powder.

[0128] result Nanoparticle size was measured by DLS (Anton Paar, Litesizer 500). Figure 5 shows the successful formulation of RGD-MINC-doxorubicin (A) and TP12-MINC-doxorubicin (B).

[0129] Example 4: Differences between Ligand-MINC-doxorubicin in drug delivery to cancer cells material RGD-MINC-doxorubicin, TfR-MINC-doxorubicin (TP12-MINC-doxorubicin), or MINC-doxorubicin were formulated according to Example 3.

[0130] method Breast cancer cell line MDA-MB-231 was cultured at 1 × 10 5 Cells / well were seeded in 12-well plates with cover slips and incubated overnight. On the second day, cells were treated with 2.5 μM MINC-doxorubicin, RGD-MINC-doxorubicin, or TfR-MINC-doxorubicin for 2 and 24 hours. After treatment, cells were fixed with ice-cold methanol. Fluorescent images were taken with a fluorescent microscope to evaluate the delivery efficiency. Fluorescence intensity was measured by Image J using the parameter fixed region: 10.309. The stronger the fluorescent signal, the more doxorubicin was taken up by the cells.

[0131] result Doxorubicin is a red fluorescent compound, and the delivery of doxorubicin to cells was observed using a fluorescence microscope. The stronger the fluorescence intensity, the more doxorubicin was delivered to the cells. In Figure 6, the fluorescent signals of RGD-MINC-doxorubicin or TfR-MINC-doxorubicin were stronger in MDA-MB-231 cancer cells compared to MINC-doxorubicin. These results indicate that the tumor targeting peptides increased the specific drug delivery to cancer cells.

[0132] Example 5: Efficacy study of different ligands -MINC-doxorubicin against cancer cells material RGD-MINC-doxorubicin, TfR-MINC-doxorubicin (TP12-MINC-doxorubicin), or MINC-doxorubicin were formulated according to Example 3. An acid phosphatase (ACP) assay kit was purchased from LSBio. The MDA-MB-231 cell line was purchased from ATCC (HTB26).

[0133] method Breast cancer cell line MDA-MB-231 was cultured at 1 × 10 4 Cells / well were seeded in a 96-well plate and incubated overnight. On the second day, cells were treated with different concentrations of MINC-doxorubicin, RGD-MINC-doxorubicin, or TfR-MINC-doxorubicin for 72 h. After treatment, cell viability was assessed with an ACP kit according to the manufacturer's instructions. Optical density (OD) values ​​were measured with a spectrophotometer. Acid phosphatase is an enzyme present in cancer cells. This enzyme activity is positively correlated with cell viability and can be measured by OD values. Cell viability (%) was calculated as [(OD value of treated group - blank) / (OD value of untreated group - blank)] x 100%.

[0134] result In Figure 7, the RGD-MINC-doxorubicin and TfR-MINC-doxorubicin treatment groups had lower cell viability compared to naked MINC-doxorubicin at the same concentration of doxorubicin. The calculated median inhibitory concentration (IC50) was much lower in the RGD-MINC-doxorubicin and TfR-MINC-doxorubicin treatment groups than in the MINC-doxorubicin treatment group. These results indicate that the tumor targeting ligand improved the anticancer efficacy of MINC-doxorubicin.

[0135] Example 6: Conjugation of RGD peptide to HO-PEG-EGCG (hypothetical example) the purpose This experiment is intended to demonstrate that RGD is conjugated to HO-PEG-EGCG. HPLC is used to detect the formation of a new product (RGD-PEG-EGCG) that has a different retention time than HO-PEG-EGCG. NMR can be used to confirm the structure.

[0136] material RGD peptide is purchased from Biotools. HO-PEG-CHO is purchased from Hunan Huateng Pharmaceutical Co., Ltd.

[0137] method HO-PEG-CHO is conjugated to EGCG to prepare HO-PEG-EGCG according to WO 2006 / 124000 and WO 2009 / 054813.

[0138] RGD is conjugated to HO-PEG-EGCG via conjugation between the OH group on PEG and the COOH group on RGD to form RGD-PEG-EGCG (C'-linked). See Figure 8.

[0139] Specifically, 1-1000 mg of RGD is PEGylated by incubating with 1-1000 mg of HO-PEG-EGCG and 1-1000 mg of N,N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction is kept under nitrogen for 24 hours with stirring at room temperature, protected from light. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cutoff = 2000 Da). The solution is then lyophilized to obtain a lyophilized powder.

[0140] Example 7: Conjugation of transferrin peptide (TP12) to HO-PEG-EGCG (hypothetical example) the purpose This experiment is intended to demonstrate that TP12 is conjugated to HO-PEG-EGCG. HPLC is used to detect the formation of a new product (TP12-PEG-EGCG) that has a different retention time than HO-PEG-EGCG. NMR can be used to confirm the structure.

[0141] material TP12 peptide is purchased from Biotools. HO-PEG-CHO is purchased from Hunan Huateng Pharmaceutical Co., Ltd.

[0142] method For the formulation of TP12-PEG-EGCG, HO-PEG-CHO is conjugated to EGCG according to WO 2006 / 124000 and WO 2009 / 054813.

[0143] The TP12 peptide is conjugated to HO-PEG-EGCG via conjugation between the OH group on PEG and the COOH group on TP12 to form TP12-PEG-EGCG (C'-linked). See Figure 9.

[0144] Specifically, 1-1000 mg of TP12 is PEGylated by incubating with 1-1000 mg of HO-PEG-EGCG and 1-1000 mg of N,N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction is kept under nitrogen for 24 hours, protected from light, with stirring at room temperature. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cutoff = 2000 Da). The solution is then lyophilized to obtain a lyophilized powder.

[0145] Example 8: Conjugation of Folate to HO-PEG-EGCG (hypothetical example) the purpose This experiment is intended to demonstrate that folate is conjugated to HO-PEG-EGCG. HPLC is used to detect the formation of a new product (folate-PEG-EGCG) that has a different retention time than HO-PEG-EGCG. NMR can be used to confirm the structure.

[0146] material Folate is purchased from TCI Chemicals. HO-PEG-CHO is purchased from Hunan Huateng Pharmaceutical Co., Ltd.

[0147] method HO-PEG-CHO is conjugated to EGCG according to WO 2006 / 124000 and WO 2009 / 054813.

[0148] Folate is conjugated to HO-PEG-EGCG via conjugation between the OH group on PEG and the COOH or COOR group on folate to form folate-PEG-EGCG, see FIG.

[0149] Specifically, 1-1000 mg of folate is PEGylated by incubating with 1-1000 mg of HO-PEG-EGCG and 1-1000 mg of N,N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction is kept under nitrogen for 24 hours with stirring at room temperature, protected from light. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cutoff = 2000 Da). The solution is then lyophilized to obtain a lyophilized powder.

[0150] Example 9: Conjugation of RGD peptide to HOOC-PLA-EGCG (hypothetical example) the purpose This experiment is intended to demonstrate the conjugation of RGD peptide to HOOC-PLA-EGCG. HPLC is used to detect the formation of a new product (RGD-PLA-EGCG) with a different retention time than HOOC-PLA-EGCG. NMR can be used to confirm the structure.

[0151] material RGD peptide is purchased from Biotools. HOOC-PLA-CHO was purchased from Merck (Sigma-Aldrich).

[0152] method HOOC-PLA-CHO was conjugated to EGCG according to WO 2006 / 124000 and WO 2009 / 054813; the COOH groups on PLA and the NH groups on RGD were 2 The RGD peptide is conjugated to the HOOC-PLA-EGCG via conjugation between the N' groups to form RGD-PLA-EGCG (N'-linked). See FIG. 11.

[0153] Specifically, 1-1000 mg of RGD is incubated with 1-1000 mg of HOOC-PLA-EGCG, 1-1000 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 1-1000 mg of N-hydroxysuccinimide (NHS) in DMSO. The reaction is kept under nitrogen for 24 hours with stirring at room temperature, protected from light. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cutoff = 2000 Da). The solution is then lyophilized to obtain a lyophilized powder.

[0154] Example 10: Conjugation of RGD peptide to HOOC-PLGA-EGCG (hypothetical example) the purpose This experiment is intended to demonstrate the conjugation of RGD peptide to HOOC-PLGA-EGCG. Using HPLC, the formation of a new product (RGD-PLGA-EGCG) with a different retention time from HOOC-PLGA-EGCG is detected. Using NMR, the structure can be confirmed.

[0155] material RGD peptide is purchased from Biotools. HOOC-PLGA-CHO was purchased from Merck (Sigma-Aldrich).

[0156] method HOOC-PLGA-CHO is conjugated to EGCG according to WO 2006 / 124000 and WO 2009 / 054813; RGD peptide is conjugated to HOOC-PLGA-EGCG via conjugation between the COOH group on PLGA and the NH2 group on RGD to form RGD-PLGA-EGCG (N'-linked). See Figure 12.

[0157] Specifically, 1-1000 mg of RGD is incubated with 1-1000 mg of HOOC-PLGA-EGCG, 1-1000 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 1-1000 mg of N-hydroxysuccinimide (NHS) in DMSO. The reaction is kept under nitrogen for 24 hours with stirring at room temperature, protected from light. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cutoff = 2000 Da). The solution is then lyophilized to obtain a lyophilized powder.

[0158] Example 11: Conjugation of RGD peptide to HO-dextran-EGCG (hypothetical example) the purpose This experiment is intended to demonstrate that RGD is conjugated to HO-Dextran-EGCG. HPLC is used to detect the formation of a new product (RGD-Dextran-EGCG) that has a different retention time than HO-Dextran-EGCG. NMR can be used to confirm the structure.

[0159] material RGD peptide is purchased from Biotools. HO-dextran-CHO was purchased from Merck (Sigma-Aldrich).

[0160] method HO-dextran-CHO is conjugated to EGCG to prepare HO-dextran-EGCG according to WO 2006 / 124000 and WO 2009 / 054813.

[0161] RGD is conjugated to HO-dextran-EGCG via conjugation between the OH group on PEG and the COOH group on dextran to form RGD-dextran-EGCG (C'-linked). See Figure 13.

[0162] Specifically, 1-1000 mg of RGD is incubated with 1-1000 mg of HO-dextran-EGCG and 1-1000 mg of N,N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction is kept under nitrogen for 24 hours with stirring at room temperature, protected from light. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cutoff = 2000 Da). The solution is then lyophilized to obtain a lyophilized powder.

[0163] Example 12: Folate-MINC-doxorubicin Combination (Hypothetical Example) the purpose This experiment is designed to demonstrate the Folate-MINC-Doxorubicin formulation. DLS is used to measure the size of the nanoparticles.

[0164] material Folate-PEG-EGCG is formulated according to Example 8. Doxorubicin is purchased from Sigma-Aldrich or other suppliers.

[0165] method Folate-MINC-doxorubicin nanoparticles are prepared according to the following protocol: Incubate 1.5 to 500 µg of doxorubicin in 1 mL of DMSO for 15 min to 1 h. 2. Add 1-100 μg of OEGCG and 1-10,000 μg of folate-PEG-EGCG. Incubate the mixture at 25° C. for 3 hours. 3. Filter the liquid through a 10K MWCO filter unit. Wash the filter 3 times with 0.9% NaCl. 4. Freeze-dry to a dry powder.

[0166] [Table 1]

Claims

1. (a) Cancer targeting ligands, (b) A hydrophilic polymer which is polyethylene glycol (PEG), polylactic acid (PLA), glycolic acid lactate copolymer (PLGA), or dextran, (c) Flavonoids that are EGCG, EC, EGC, or ECG, A conjugate that includes the following structure: 【Chemistry 1】 The hydrophilic polymer is covalently bonded to the flavonoid and the cancer targeting ligand. The aforementioned cancer targeting ligand is RGD, TP12 having the amino acid sequence THRPPMWSPWPP (SEQ ID NO: 1), MC11 having the amino acid sequence MQLPLATGGGC (SEQ ID NO: 2), FV12 having the amino acid sequence FCDGFYACYMDV (SEQ ID NO: 3), YI12 having the amino acid sequence YHWYGYTPQNVI (SEQ ID NO: 4), CTT having the amino acid sequence CTTHWGFTLC (SEQ ID NO: 5), H2009.1 peptide having the amino acid sequence RGDLATLRQLAQEDGVVGVR (SEQ ID NO: 6), IL-13 peptide having amino acid sequence GSETWKTIITKN (SEQ ID NO: 7), AP-1 having the amino acid sequence RKRLLDRN (SEQ ID NO: 8), CVKTPAQSC (Sequence ID 9), CC9 peptide having amino acid sequence CDCRGDCFC (SEQ ID NO: 10), RGDS peptide having the amino acid sequence RGDS (SEQ ID NO: 11), NR7 peptide having amino acid sequence NSVRGSR (SEQ ID NO: 12), LHRH peptide having amino acid sequence EHWSYGLRPG (SEQ ID NO: 13), Angiopep-2 peptide having the amino acid sequence TFFYGGSRRGKRNNFKTEEY (SEQ ID NO: 14), TbFGF peptide having amino acid sequence KRTGQYKLC (SEQ ID NO: 15), EGF peptide having amino acid sequence YHWYGYTPQNVI (SEQ ID NO: 16), Folic acid or folate, hyaluronic acid, lactose, galactose, galactosamine, and glycyrrhetinic acid, A conjugate selected from the group consisting of the following.

2. The conjugate according to claim 1, wherein the flavonoid is EGCG.

3. The conjugate according to claim 1, wherein the hydrophilic polymer is PEG.

4. (a) an outer shell including the conjugate described in claim 1, (b) An inner shell containing oligomer (-)-epigallocatechin gallate (OEGCG), (c) A drug molecule for cancer treatment, which is a cytokine, antibody, or chemotherapeutic agent, enclosed in the inner shell, Contains nanoparticles having; At least 70% of the aforementioned nanoparticles have a diameter of 20 to 500 nm. The size distribution of the aforementioned nanoparticles has only one main peak containing more than 90% of all the particles. Nanoparticle composition.

5. The nanoparticle composition according to claim 4, further comprising a bare hydrophilic polymer-flavonoid conjugate whose outer shell is not covalently bonded to the cancer targeting ligand.

6. The nanoparticle composition according to claim 4, wherein at least 80% of the nanoparticles have a diameter of 50 to 300 nm.

7. The nanoparticle composition according to claim 4, wherein at least 90% of the nanoparticles have a diameter of 50 to 300 nm.

8. The nanoparticle composition according to claim 4, wherein the median diameter of the nanoparticles is about 50 to 250 nm.

9. The nanoparticle composition according to claim 4, wherein the size distribution of the nanoparticles has only one main peak containing more than 95% of all the particles.

10. A pharmaceutical composition for use in a method for treating cancer, comprising the nanoparticle composition according to any one of claims 4 to 9.

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