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    • 专利摘要:
    MICELAR NANOCOMPLEX, CONJUGATEFLAVONOID POLYMER AND METHODS FOR ITS FORMATIONS. presentinvention relates to micellar nanocomplexes and a method for theirformation. The micellar nanocomplex comprises a micelle and an agentencapsulated within said micelle, wherein the micelle comprises aflavonoid polymer conjugate, wherein said polymer is linked to thering B of said flavonoid. The micellar nanocomplex may haveUseful applications as a drug delivery system. presentinvention also relates to a flavonoid polymer conjugate whichcomprises a polymer linked to the B ring of a flavonoid and a methodfor your training.
    公开号:BR112016026260A2
    申请号:R112016026260-3
    申请日:2015-05-08
    公开日:2021-08-03
    发明作者:Motoichi Kurisawa；Yongvongsoontorn Nunnarpas；Jackie Y Ying；Joo Eun Chung；Ki Hyun Bae；Min-Han Tan；Esther Lee
    申请人:Agency For Science, Technology And Research；
    IPC主号:
    专利说明:

    [001] [001] The present invention generally refers to micellar nanocomplexes for drug delivery and method for its formation. The present invention also relates to a polymer-flavonoid conjugate comprising a polymer linked to the B ring of a flavonoid and a method for its formation. HISTORY OF ART
    [002] [002] Chemotherapy, which is one of the most common treatments for cancer, uses cytotoxic drugs administered perorally and parenterally. The main challenge with the administration of conventional anticancer drugs is their specific distribution in the body, leading to toxicity with serious side effects. Furthermore, the therapeutic effect of oral medications is limited by their low bioavailability because the medications must pass through the digestive ducts. In recent decades, researchers have focused on drug delivery systems to overcome the limitations of conventional drug delivery by improving drug pharmacokinetics and biodistribution.
    [003] [003] In recent years, green tea catechins have been extensively studied for their health benefits, including the prevention of cardiovascular disease and cancer. Among the tea catechins, epigalallocatechin-3-gallate (EGCG) is the most abundant and has been considered to play a major role in the beneficial effects of green tea. Several studies have shown that EGCG has antioxidant, antidiabetic, antibacterial, anti-inflammatory and hypocholesterolemic effects. Furthermore, it has been shown to effectively inhibit tumor growth and metastasis by targeting multiple signal transduction pathways essential for cancer cell survival.
    [004] [004] Despite these desirable activities, the clinical applications of EGCG were limited by its unsatisfactory stability and low oral bioavailability. For example, EGCG is unstable and easily decomposed in a physiological environment. EGCG was reported to have a short half-life of less than 30 minutes at 0.05 M of
    [005] [005] Therefore, there is a need to provide a drug delivery system that overcomes, or at least ameliorates, one or more of the disadvantages described above. There is also a need to provide a method of training such a drug delivery system. SUMMARY OF THE INVENTION
    [006] [006] According to a first aspect, a micellar nanocomplex is provided which comprises a micelle and an agent encapsulated within said micelle, wherein said micelle comprises a polymer-flavonoid conjugate, wherein said polymer is linked to ring B of said flavonoid.
    [007] [007] Advantageously, micellar nanocomplexes can be used as drug delivery systems. Micellar nanocomplexes are small in size and have high drug-carrying capacity favorable for delivery of drugs destined for the tumor. Also advantageously, sustained release of the agent can be achieved using micellar nanocomplexes under physiological conditions. Even more advantageously, nanocomplexes may be promising delivery vehicles for a variety of water-insoluble anti-cancer agents. Even more advantageously, the micellar nanocomplex can significantly suppress tumor growth, with reduced toxicity associated with administration of the agent. Even more advantageously, micelle nanocomplexes can represent a unique and effective drug delivery system with therapeutic synergistic effects from the drug delivery system or micelle vehicle and agent.
    [008] [008] The agent may be doxorubicin. Advantageously, micellar nanocomplexes that encapsulate doxorubicin can present a sustained release of the drug. The sustained release of the drug may be due to the strong
    [009] [009] The agent may be Sunitinib (SU) and the flavonoid may be epigalallocatechin-3-gallate (EGCG). Advantageously, micellar nanocomplexes can show a sustained release of SU. Advantageously, in some configurations, hardly any burst release was observed, suggesting that SU molecules were stably encapsulated in micellar nanocomplexes.
    [010] [010] In one setting, the flavonoid can be a monomeric flavonoid. In another configuration, the flavonoid can be a dimeric flavonoid. Advantageously, the micellar nanocomplex comprising the monomeric flavonoid may exhibit greater and faster SU release compared to the micellar nanocomplexes comprising the dimeric flavonoid. Advantageously, there may be a stronger interaction between SU and the dimeric flavonoid.
    [011] [011] Advantageously, micellar nanocomplexes can minimize the adverse side effects of agents such as SU by stably encapsulating the agent in its interior, and delivering them to the target site. The micellar nanocomplex may therefore offer beneficial synergistic effects between SU and EGCG.
    [012] [012] Still advantageously, the micellar nanocomplex comprising SU may have enhanced tumor effects in vivo compared to free SU. More advantageously, the micellar nanocomplex comprising SU may have fewer adverse effects on the tumor in vivo compared to free SU. Advantageously, less dosage of the micellar nanocomplex comprising SU may be required compared to free SU to obtain the same effects. still way
    [013] [013] Still advantageously, the micellar nanocomplex comprising SU can lead to a reduction in plasma concentrations of free SU, resulting in fewer adverse effects of SU. Still advantageously, this reduction in plasma concentration may be due to the interaction between the flavonoid and the SU, as well as the effect of improving permeability and retention (EPR) offered by micellar nanoparticles.
    [014] [014] According to a second aspect, a method of formation of a micellar nanocomplex comprising a micelle and an agent encapsulated within said micelle is provided, the method comprising the steps of: (a) adding said agent to a solvent suitable for a polymer-flavonoid conjugate, wherein said polymer is attached to the B ring of said flavonoid; and (b) allowing self-assembly of a micelle comprising said polymer-flavonoid conjugate and encapsulating said agent within said micelle to thereby form said micellar nanocomplex.
    [015] [015] Advantageously, the nanocomplex is self-assembled in the presence of the polymer-flavonoid conjugate and the agent. Still advantageously, the formation of the nanocomplex was obtained using the property of binding the flavonoid with the agents.
    [016] [016] According to a third aspect, a polymer-flavonoid conjugate is provided that comprises a polymer linked to the B ring of a flavonoid.
    [017] [017] Advantageously, a flavonoid is conjugated to a polymer. In one configuration, the polymer can be polyethylene glycol (PEG). Advantageously, polymer-based nanoparticles avoid both renal elimination and becoming trapped by the reticuloendothelial system (RES), allowing subsequent accumulation within tumor tissues due to the EPR effect. More advantageously, PEG-stabilized micelles have a longer extended plasma half-life than unmodified micelles, as PEG surface chains impede RES recognition and clearance in the body. Advantageously, PEG
    [018] [018] According to a fourth aspect, a method is provided for forming the polymer-flavonoid conjugate as defined above, which comprises the step of conjugating said flavonoid with said polymer by means of nucleophilic addition under basic conditions, in which the said polymer has a free nucleophilic group.
    [019] [019] Advantageously, polymer-flavonoid conjugates can be synthesized by nucleophilic addition at basic pH. Advantageously, the conjugation can be carried out by nucleophilic addition of a nucleophilic group such as a thiol group of the polymer such as a PEG at the C2' position of the B ring of the flavonoid under conditions of controlled pH.
    [020] [020] In one configuration, the polymer is polyethylene glycol (PEG) and the free nucleophilic group is thiol. Advantageously, the flavonoid electron deficient orthochitone like EGCG can react with a nucleophilic group like thiol groups. Thiol groups are present in a diverse range of biomolecules, including cysteine, glutathione and proteins. EGCG can covalently bind to cysteine residues in human erythrocyte membrane proteins and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, covalent adducts of EGCG can form when oxidized in the presence of cysteine and glutathione. Still advantageously, cysteine conjugates resulting from EGCG may have higher pro-oxidant activities than EGCG, while retaining their growth-inhibitory and anti-inflammatory activities. Most advantageously, the EGCG N-acetylcysteine conjugate may enhance the growth-inhibitory and apoptosis-inducing effects of EGCG against human and murine lung cancer cells.
    [021] [021] According to a fifth aspect, the use of a micelle nanocomplex comprising a micelle and an agent encapsulated within said micelle as a drug delivery vehicle is envisaged, wherein said micelle comprises a polymer-flavonoid conjugate, and wherein said polymer is attached to the B ring of said flavonoid.
    [022] [022] According to a sixth aspect, a method for treating a tumor is envisaged which comprises the step of administering the micellar nanocomplex as defined above to a cancer agent.
    [023] [023] Advantageously, the micellar nanocomplex may have a greater anticancer effect compared to a free agent. More advantageously, micellar nanocomplexes can minimize the adverse side effects of agents such as Suhitinib (SU) by stably encapsulating the agent within them, and delivering them to the target site. These delivery systems also provide for beneficial synergistic effects.
    [024] [024] According to a seventh aspect, the micellar nanocomplex as defined above is envisaged to treat a tumor.
    [025] [025] According to an eighth aspect, the use of micellar nanocomplex as defined above is envisaged in the manufacture of a drug for the treatment of a tumor. DEFINITIONS
    [026] [026] The following words and terms used in this instrument shall have the indicated meaning:
    [027] [027] The "B ring" of a flavonoid refers to an optionally substituted phenyl that is attached to a bicyclic structure (the bicyclic structure composed of a benzene ring (A) condensed with a six-membered ring (C)). The optionally substituted phenyl is attached to the 2-position of the C ring. For purposes of the present disclosure, the rings are labeled as follows: [Chem. 1]
    [028] [028] The term “epigallocatechin gallate” refers to an ester of epigallocatechin and gallic acid and can be used interchangeably with “epigallocatechin-3 gallate” or EGCG.
    [029] [029] For purposes of this application, the term "PEG-EGCG conjugates" refers to
    [030] [030] The term "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. Where necessary, the term "substantially" may be omitted from the definition of the invention.
    [031] [031] Unless otherwise specified, the terms "comprising" and "comprises" and their grammatical variants are intended to represent an "open" or "inclusive" language in order to include recited elements, but also allow the inclusion of additional elements, not recited.
    [032] [032] As used herein, the term "over", in the context of concentrations of formulations components, typically means +/‐ 5% of the declared value, more typically +/‐ 4% of the declared value, more typically +/ ‐ 3% of declared value, more typically +/‐ 2% of declared value, even more typically +/‐ 1% of declared value, and even more typically +/‐ 0.5% of declared value.
    [033] [033] Throughout this disclosure, certain configurations may be disclosed in band format. It should be understood that the description in band format is for convenience and brevity purposes only and is not to be construed as an inflexible limitation on the scope of the bands posted. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual numerical values within that range. For example, describing a range as 1 to 6 should be considered to have specifically disclosed sub-ranges as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, of 3 to 6, etc., as well as individual numbers within that range, for example 1, 2, 3, 4, 5, and 6. This applies regardless of the range's amplitude.
    [034] [034] Certain settings may also be described comprehensively and generically in this instrument. Each of the subgeneric groupings and the narrower species falling within the generic disclosure are also part of the disclosure. This includes the generic description of the
    [035] [035] The accompanying drawings illustrate a disclosed configuration and serve to explain the principles of the disclosed configuration. It should be understood, however, that the drawings are intended for illustrative purposes only, and not as a definition of the limits of the invention: Figure 1 is a synthetic scheme conjugated PEG-mEGCG (108). Thiol-functionalized PEG (PEG-SH) (102) was conjugated to EGCG (104) in a 1:3 (v/v) mixture of DMSO and water at basic pH pH (106); Figure 2 is a UV-Vis spectrum of PEG-EGCG (202) and PEG (204) conjugate dissolved in de-ionized water at a concentration of 0.5 mg mL-1; Figure 3 refers to the HPLC chromatograms of EGCG (302) and PEG-mEGCG conjugate (304). Arrows indicate peaks of samples monitored at 280 nm; Figure 4 is the degree of conjugation of PEG-mEGCG conjugates as a function of reaction time; Figure 5 is an H NMR spectrum of the PEG-mEGCG conjugate dissolved in D20. Figure 6 is a schematic showing the formation of doxorubicin/PEG-mEGCG micellar nanocomplexes; Figure 7 refers to the graphs showing (A) Size and (B) the zeta potential of doxorubicin/PEG-mEGCG micellar nanocomplexes prepared in different PEG-mEGCG:doxorubicin ratios by weight. The size and zeta potential of as-prepared nanocomplexes (black bars, 702) were compared to those of reconstituted nanocomplexes (cross bars, 704); Figure 8 refers to the graphs showing (A) drug loading efficiency and (B) loading content of doxorubicin/PEG-mEGCG micellar nanocomplexes prepared with different PEG-mEGCG:doxorubicin ratios by weight;
    [036] [036] Exemplary, non-limiting configurations of a micellar nanocomplex will be disclosed hereafter.
    [037] [037] A micellar nanocomplex may comprise a micelle and an agent encapsulated within said micelle, wherein said micelle comprises a polymer-flavonoid conjugate, wherein said polymer is attached to the B ring of said flavonoid.
    [038] [038] At least one flavonoid can be attached to said polymer. At least two flavonoids can be attached to said polymer.
    [039] [039] The polymer can be linked to said flavonoid through a binder. The linker can be any chemical group that can link the polymer and the flavonoid. The linker can be selected from the group consisting of a thioether, imine, amine, azo and 1,2,3-triazole group. The linker can be an alkane group. The binder can be present between any part of the polymer and any part of the flavonoid. The binder can be present between a terminus of the polymer and any part of the flavonoid.
    [040] [040] The flavonoid can be selected from the group consisting of a monomeric flavonoid or a dimeric flavonoid. A monomeric flavonoid can comprise a flavonoid molecule. A dimeric flavonoid can comprise two flavonoid molecules linked by a linker. One of the flavonoid molecules of the dimeric flavonoid can be bound by the polymer. Both flavonoid molecules of the dimeric flavonoid can be independently linked by the polymer. When a flavonoid is present in said conjugate, the flavonoid is attached to said polymer via the B ring. When a flavonoid is present in said conjugate, the flavonoid is attached to said polymer via the D ring.
    [041] [041] When more than one flavonoid is present in said conjugate, at least one flavonoid is attached to said polymer through ring B. The other of said at least one flavonoid is attached to said polymer through ring A.
    [042] [042] When more than one flavonoid is present in said conjugate, at least one flavonoid is attached to said polymer through the D ring. The other of said at least one flavonoid is attached to said polymer through the A ring. of a flavonoid is present in said conjugate, at least one flavonoid is attached to said polymer via ring D. The other of said at least one flavonoid is attached to said polymer via ring B. When more than one flavonoid is present in said conjugate, at least one flavonoid is attached to said polymer via the D ring. The other of said at least one flavonoid is attached to said polymer via the D ring.
    [043] [043] The polymer may be a hydrophilic polymer. The hydrophilic polymer may comprise monomers selected from the group consisting of acrylamides, alkys, oxazolines, alkenyls, imines, acrylic acids, methacrylates, diols, oxiranes, alcohols, amines, anhydrides, esters, lactones, carbonates, carboxylic acids, acrylates, hydroxys, phosphates, terephthalate, amides and ethers.
    [044] [044] The hydrophilic polymer can be selected from the group consisting of polyacrylamide, poly(N-isopropylacrylamide), poly(oxazoline), polyethyleneimine, poly(acrylic acid), polymethacrylate, poly(ethylene glycol), poly(oxygen oxide). ethylene), poly(vinyl alcohol), poly(vinylpyrrolidinone), polyethers, poly(allylamine), polyanhydrides, poly(β-amino ester), poly(butylene succinate), polycaprolactone, polycarbonate, polydioxanone, poly(glycerol), polyglycolic acid , poly(3-hydroxypropionic acid), poly(2-hydroxyethyl methacrylate), poly(N-(2-hydroxypropyl)methacrylamide), polylactic acid, poly(lactic-co-glycolic acid), poly(ortho esters), poly( 2-oxazoline), poly(sebacic acid), poly(terephthalate-co-phosphate) and their copolymers.
    [045] [045] The hydrophilic polymer can be a polysaccharide. The polymer can be a
    [046] [046] The hydrophilic polymer can be polyethylene glycol (PEG). PEG is a synthetic polymer that has been used in biomedical applications due to its hydrophilic, flexible and biocompatible nature. Specifically, PEG was used to modify the surface of nanoparticles and polymeric micelles to produce antifouling surfaces.
    [047] [047] Advantageously, polyethylene glycol (PEG) was selected as the polymer to be conjugated to the flavonoid. Conjugation was performed by nucleophilic addition of a thiol group of PEG at the C2' position of the flavonoid B ring under controlled pH conditions.
    [048] [048] The flavonoid can be selected from the group consisting of flavones, isoflavones, flavan, proanthocyanidins and anthocyanidins.
    [049] [049] Flavones can be selected from the group consisting of apigenin, luteolin, tangeritin, chrysin, 6-hydroxyflavone, baicalein, scutellarein, wogonin, diosmin, flavoxate and 7,8-dihydroxyflavone.
    [050] [050] Isoflavones can be selected from the group consisting of genistein, daidzein, glycitein, genistin, daidzin, glycitin, acetyl-genistin, acetyl-daidzin, acetyl-glycitin, malonyl genistin, malonyl-daidzin and malonyl-glycitin.
    [051] [051] Flavans can be selected from the group consisting of (‐)‐epicatechin, (+)‐epicatechin, (‐)‐catechin, (+)‐catechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, physetinidol, gallocatechin , Gallocatechin gallate, Mesquitol and Robinetinidol, ellagitanin, galotanin, olongthean, phlorotanin, tannin, theacitrin, theadibenzotropolone, theaflavin, theanadthoquinone, thearubigines, teasinensinae and mixtures thereof.
    [052] [052] Anthocyanidins can be selected from the group consisting of aurantinidine, capensinidin, cyanidin, delphinidin, europinidin, hirsutinidin, malvidin, pelargondin, peonidin, petunidin, pulquelidin, and rosinidin.
    [053] [053] The agent can be a therapeutic agent. The therapeutic agent can be a
    [054] [054] The chemotherapeutic agent can be selected from the group consisting of Actinomycin, Afatinib, All-Trans Retinoic Acid, Axitinib, Azacitidine, Azathioprine, Bevacizumab, Bleomycin, Bosutinib, Bortezomib, Carboplatin, Capecitabine, Cetuximabamide, Cisplatin, Crizoftinibucci , Cytarabine, Dasatinib, Daunorubicin, Docetaxel, Doxyfluridine, Doxorabicin, Epirubicin, Epothilone A (C26H39NO6S), Epothilone B (C27H41NO6S), Epothilone C (C26H39NO5S), Epothilone D (C27H39NOH5S), Epothilone D (C27H41NO5S), Epothilone D (C27H41NO5S), , Erlotinib, Etoposide, Fluorouracil, Fostamatinib, Gefitinib, Gemcitabine, Hydroxyurea, Idarabicin, Imatinib, Irinotecan, Lapatinib, Lenvatinib, Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Nilotinib, Pebbanib, Oxalipa, Oxalipatin , Ruxolitinib, Sorafenib, Sunitinib, Trastuzumab, Teniposide, Thioguanine, Tofacitinib, Topotecan, Valrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorrelbine.
    [055] [055] The chemotherapeutic agent may be doxorabicin.
    [056] [056] The chemotherapeutic agent may be Sunitinib (SU). SU is a multi-target tyrosine kinase inhibitor and a first-line therapy to eliminate renal cell carcinoma (ccRCC). Specifically, SU targets vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors, which play a role in tumor angiogenesis and proliferation, leading to reduced tumor vascularization, as well as death of cancer cells. It has been approved for use in advanced RCC, gastrointestinal stromal tumors (GIST), and pancreatic neuroendocrine tumors (pNET). It has also been shown to have the potential to cure metastatic breast cancer, breast cancer.
    [057] [057] The micellar nanocomplex can have a size in the range from 30 to 300 nm, from 50 to 300 nm, from 100 to 300 nm, from 30 to 50 nm, from 30 to 100 nm, from 30 to 150 nm, from 150 at 300 nm, from 200 to 300 nm, from 250 to 300 nm, from 100 to 150 nm, from 100 to 200 nm, from 100 to 250 nm, from 130 to 180 nm, or from 130 to 250 nm.
    [058] [058] The micellar nanocomplex may have a loading efficiency of said agent present within said micelle greater than 30%, greater than 35%, greater than 40%, greater than 45%, d greater than 50% , greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, or 80%.
    [059] [059] The micellar nanocomplex may have a loading content of said agent present within said micelle in the range of 1 to 10 w/w%, from 5 to 25 w/w%, from 20 to 45 w/w%, of 30 to 50 w/w%, from 35 to 50 w/w%, from 40 to 50 w/w%, from 45 to 50 w/w%, from 30 to 35 w/w%, from 30 to 40 w/w/ p% or from 30 to 45 w/w%.
    [060] [060] A method of forming a micellar nanocomplex can comprise a micelle and an agent encapsulated within said micelle, the method comprising the steps of: a. adding said agent in a suitable solvent to a polymer-flavonoid conjugate, wherein said polymer is attached to ring B of said flavonoid; and b. allowing self-assembly of a micelle comprising said polymer-flavonoid conjugate and encapsulating said agent within said micelle to thereby form said micellar nanocomplex.
    [061] [061] Step (a) may further comprise the steps of: a. removing said solvent to form a dry film of said agent and said polymer-flavonoid conjugate; and b. hydrate said dry film with an aqueous solvent.
    [062] [062] The method may further comprise the step of isolating the micellar nanocomplex
    [063] [063] A polymer-flavonoid conjugate can comprise a polymer attached to the B ring of a flavonoid.
    [064] [064] The polymer-flavonoid conjugate polymer can be selected from the group consisting of a polysaccharide, polyacrylamide, poly(N-isopropylacrylamide), poly(oxazoline), polyethyleneimine, poly(acrylic acid), polymethacrylate, poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidinone), polyethers, poly(allylamine), polyanhydrides, poly(β-amino ester), poly(butylene succinate), polycaprolactone, polycarbonate, polydioxanone, poly(glycerol), polyglycolic acid, poly(3-hydroxypropionic acid), poly(2-hydroxyethyl methacrylate), poly(N-(2-hydroxypropyl)methacrylamide), polylactic acid, poly(lactic-co-glycolic acid), poly (ortho esters), poly(2-oxazoline), poly(sebacic acid), poly(terephthalate-co-phosphate) and their copolymers.
    [065] [065] The flavonoid of the polymer-flavonoid conjugate can be selected from the group consisting of (‐)‐epicatechin, (+)‐epicatechin, (‐)‐catechin, (+)‐catechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, Fisetinidol, Gallocatechin, Gallocatechin gallate, Mesquitol and Robinetinidol, ellagitanin, galotanin, olongthean, phlorotanin, tanin, theacitrin, theadibenzotropolone, theaflavin, theanadthoquinone, thearubigines, teasinensinae and mixtures thereof.
    [066] [066] The polymer can be conjugated to a flavonoid in the polymer-flavonoid conjugate through a linker selected from the group consisting of a group of thioether, imine, amine, azo and 1,2,3-triazole. The linker can be an alkane group. The binder can be present between any part of the polymer and any part of the flavonoid. The binder can be present between a terminus of the polymer and any part of the flavonoid.
    [067] [067] The polymer of the polymer-flavonoid conjugate may be poly(ethylene glycol), the said flavonoid of the polymer-flavonoid conjugate may be epigallocatechin-3-gallate and the said polymer-flavonoid conjugate ligand may be thioether.
    [068] [068] The flavonoid polymer can have the following formula [Chem. two]
    [069] [069] Where n is in the range from 20 to 910.
    [070] [070] A method for forming the polymer-flavonoid conjugate may comprise the step of conjugating said flavonoid with said polymer by means of nucleophilic addition under basic conditions, wherein said polymer has a free nucleophilic group.
    [071] [071] The nucleophilic group can be selected from the group consisting of a sulfhydryl amine, carbonyl, carboxylic acid, azide, halogen, alkali and alkene. The nucleophilic group can be selected from the group consisting of a thiol, an amine, a diazoalkane and an azide.
    [072] [072] The nucleophilic group can be a thiol. EGCG can undergo oxidation in the presence of oxygen to form an orf/zo-quinone via a pathway that involves semiquinone radicals and reactive oxygen species. The electron-deficient EGCG orf/zo-quinone may react with a nucleophilic thiol group present in several biomolecules, including cysteine, glutathione, and proteins. EGCG can covalently bind to cysteine residues in human erythrocyte membrane proteins and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Covalent adducts of EGCG can form when oxidized in the presence of cysteine and glutathione. Cysteine conjugates resulting from EGCG may have higher pro-oxidant activities than EGCG while retaining their growth-inhibitory and anti-inflammatory activities. In addition, EGCG N-acetylcysteine conjugate may enhance the growth-inhibitory and apoptosis-inducing effects of EGCG against human and murine lung cancer cells.
    [073] [073] The conjugation step can be carried out in a reaction time from approximately 1 hour to 24 hours, from approximately 1 hour to 2 hours, from
    [074] [074] The method may further comprise the step of conducting the conjugation step in a solvent that substantially prevents the aggregation of said flavonoid.
    [075] [075] The method may further comprise the step of adding a scavenging agent to prevent the H 2 O 2 mediated oxidation of said nucleophilic group to thereby increase the efficiency of said conjugation step.
    [076] [076] Basic conditions can be in the pH range of more than 7 to 10, more than 8 to 10, more than 9 to 10, more than 7 to 11, more than 8 to 11, more than 9 to 11, more 10 to 11, more than 7, more than 8, more than 9, more than 10 or more than 11.
    [077] [077] The use of a micellar nanocomplex may comprise a micelle and an agent encapsulated within said micelle as a drug delivery vehicle, wherein said micelle comprises a polymer-flavonoid conjugate, and wherein said polymer is linked to the ring B of said flavonoid.
    [078] [078] The micellar nanocomplex can deliver the encapsulated agent to a target tumor site in vivo.
    [079] [079] A cancer treatment method may comprise the step of administering the micellar nanocomplex to a cancer patient. A tumor treatment method may comprise the step of administering the micellar nanocomplex to a cancer patient.
    [080] [080] The micellar nanocomplex can be administered parenterally, by administration of inhalation spray, topical, rectal, nasal, buccal, vaginal, through an implanted reservoir, by injection, subdermal, intraperitoneal, transmucosal, oral or in an ophthalmic preparation .
    [081] [081] Parenteral administration can include subcutaneous routes,
    [082] [082] The agent present in said micellar nanocomplex can be administered at a dose of approximately 1 to approximately 80 mg/kg per day, from approximately 1 to approximately 2 mg/kg per day, from approximately 1 to approximately 5 mg/kg per day, from approximately 1 to approximately 10 mg/kg per day, from approximately 1 to approximately 20 mg/kg per day, from approximately 1 to approximately 50 mg/kg per day, from approximately 2 to approximately 5 mg/kg per day, from approximately 2 to approximately 10 mg/kg per day, from approximately 2 to approximately 20 mg/kg per day, from approximately 2 to approximately 50 mg/kg per day, from approximately 2 to approximately 80 mg/kg per day, from approximately 5 to approximately 10 mg/kg per day, from approximately 5 to approximately 20 mg/kg per day, from approximately 5 to approximately 50 mg/kg per day, from approximately 5 to approximately 80 mg/kg per day, from approximately 10 to approximately 20 mg/kg per day, from approximately 10 to approximately 50 mg/kg per day, from approximately 10 to approximately 80 mg/kg per day, from approximately 20 to approximately 50 mg/kg per day, from approximately 20 to approximately 80 mg/kg per day or from approximately 50 to approximately 80 mg/kg per day.
    [083] [083] The cancer patient may be suffering from a cancer selected from the group consisting of adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendix cancer, grade I astrocytoma (anaplastic), grade II astricutin, grade astricutin III, grade IV astricutin, atypical central nervous system teratoid/rhabdoid tumor, basal cell carcinoma, bladder cancer, bronchial cancer, bronchioalveolar carcinoma, Burkitt's lymphoma, cervical cancer, colon cancer, colorectal cancer, craniopharyngioma, cell lymphoma Skin T, endometrial cancer, uterine endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, fibrous histiocytoma, gallbladder cancer biliary, gastric cancer, carcinoid tumor
    [084] [084] The tumor patient may be suffering from a cancer selected from the group consisting of adrenocortical carcinoma, anal cancer, appendix cancer, grade I astrocytoma (anaplastic), grade II astricutin, grade III astricutin, grade astricutin IV, atypical teratoid/rhabdoid tumor of the central nervous system, basal cell carcinoma, bladder cancer, bronchial cancer, bronchioalveolar carcinoma, cervical cancer, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, uterine endometrial cancer, ependymoblastoma, ependymoma,
    [085] [085] The micellar nanocomplex may be for the treatment of cancer. The micellar nanocomplex can be used to treat a tumor.
    [086] [086] The use of micellar nanocomplex can be in the manufacture of a drug for the treatment of cancer. The use of micellar nanocomplex can take place in
    [087] [087] Non-limiting examples of the invention and a comparative example will be described in more detail by referring to specific Examples, which should not be construed to limit the scope of the invention. EXAMPLE 1: MATERIALS AND CELL CULTURE MATERIALS
    [088] [088] Methoxy-polyethylene glycol with a thiol end terminus (PEG-SH, Mw = 5000 Da) was obtained from JenKem Technology (China). Methoxy-polyethylene glycol with a terminal aldehyde terminal end (PEG-CHO, Mw = 5000 Da) was obtained from NOF Co., Japan. (-)-Epigallocatechin-3-gallate (EGCG, >95% purity) was obtained from Kurita Water Industries (Tokyo, Japan). Sodium pyruvate solution (100 mM) was purchased from Invitrogen (Singapore). PBS saline without Ca2+ and Mg2+ (150 mM, pH 7.3) was provided by the medium preparation facility in Biopolis, Singapore. DMSO and triethylamine (TEA) were purchased from Sigma-Aldrich (Singapore). Doxorubicin hydrochloride (DOX-HCl) was purchased from Boryung Pharm. Inc. (Korea). SU (free base form) was purchased from BioVision (USA). All other chemicals were of analytical grade. CELL CULTURE
    [089] [089] A498 human renal cell carcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, 1% penicillin. streptomycin, 2 mM glutamine and 0.1 mM non-essential amino acids. The stable A498 cell clone expressing the luciferase gene (A498-luc) was generated as described. In summary, A498 cells were seeded on a six-position slide at a density of 5x105 cells/position and transfected with d plasmid pRC-CMV2-luc using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 1 day, transfected cells were transferred to a 100 mm cell culture dish, q 1 mg ml-1 geneticin was added to the medium
    [090] [090] In the study, two types of PEG-EGCG conjugates were used to form micellar nanocomplexes, PEG-mEGCG and PEG-dEGCG, which have one and two EGCG media at one end of the PEG, respectively. SYNTHESIS OF THE PEG-mEGCG CONJUGATE
    [091] [091] The PEG-mEGCG conjugate was synthesized by conjugating EGCG to PEG with a thiol end terminus. Typically, EGCG (18.3 mg, 40 pmol) was dissolved in 20 ml of a 1:1 (v/v) mixture of PBS and DMSO. PEG-SH (Mw= 5000 Da, JenKem Technology, China) (100 mg, 20 pmol) was dissolved separately in 20 mL PBS. The PEG-SH solution was added dropwise to a stirred EGCG solution. As a control experiment, the unmodified PEG solution was added to an EGCG solution at the same concentration. The resulting mixture has a pH of 8.4. The mixture was stirred for 7 hours at 25 °C. To this solution, 1.6 ml of 10% acetic acid was added to adjust the pH to 4 to stop the reaction. The resulting solution was transferred to dialysis tubes with molecular weight cut-off (MWCO) of 1000 Da. The tubes were dialyzed against deionized water. The purified solution was lyophilized to obtain the PEG-mEGCG conjugate. The structure of the PEG-mEGCG conjugate was confirmed by 1H NMR spectroscopy. The PEG-mEGCG conjugate was dissolved in D20 at a concentration of 20 mg mL-1 and then analyzed with a Bruker AV-400 NMR spectrometer operating at 400 MHz. Yield: 89%. 1H NMR (D20): δ 2.9 (t, PEG H-α), 3.4 (s, PEG H-γ), 3.5-3.8 (m, PEG protons), 5, 5 (s, H-2 of ring C), 5.85 (s, H-3 of ring C), 6.15 (d, H-6 and H-8 of ring A), 6.9 (s, H-6' from ring B), 7.05 (s, H-2” and H-6” from ring D).
    [092] [092] Fig. 1 illustrates the synthetic scheme of PEG-mEGCG conjugates (108). Thiol-functionalized PEG (PEG-SH) (102) was incubated with a 2-fold molar excess of EGCG (PEG-SH: EGCG = 1:2) (104) in a 1:3 (v/v) mixture of DMSO and water at basic pH (106). It has been reported that pH critically influences the autooxidation process of EGCG. In the basic pH range of 7–9.5, the gallyl medium in ring B was more susceptible to autooxidation than the gallal medium in ring D. As a result, only the galyl medium in ring B forms an orf/zo-quinone . Under a strong alkaline condition (pH > 10), the gallate medium in the D ring can also be autooxidized to form an orf/zo-quinone. In the present study, the reaction was conducted at pH 8.4 to allow the formation of an orf/zo-quinone only in the B-ring of EGCG. Subsequent nucleophilic addition of PEG-SH to orf/zo-quinone produced PEG-mEGCG conjugates linked by a covalent thioether bond.
    [093] [093] It should be noted that the conjugation reaction proceeded in the presence of dimethyl sulfoxide (DMSO). As EGCG would aggregate upon contact with PEG in aqueous solution, aggregation should be avoided during conjugation of EGCG to PEG-SH. It was found that DMSO effectively prevented aggregation. Based on this finding, the conjugation reaction was carried out in a mixture of DMSO and water. In addition, sodium pyruvate was used as a scavenger for H2O2 generated during EGCG autooxidation. As sodium pyruvate protects the free thiol groups from H2O2 mediated oxidation, it may increase the number of PEG-SH molecules available for an EGCG conjugation reaction. The PEG-mEGCG conjugate obtained was purified by dialysis under a nitrogen atmosphere and then lyophilized to obtain a white powder. UV-Vis CHARACTERIZATION OF THE PEG-mEGCG CONJUGATE
    [094] [094] PEG-mEGCG conjugates were characterized using visible ultraviolet (UV-Vis) spectroscopy (Fig. 2).
    [095] [095] The UV-Vis spectra of the PEG-mEGCG conjugates were measured on a Hitachi U-2810 (Japan) spectrophotometer. For UV-Vis spectroscopy, dry PEG-mEGCG conjugate and PEG were dissolved in deionized water at a concentration of 0.5 mg mL-1. Unlike unmodified PEG (204), PEG-mEGCG conjugates
    [096] [096] The PEG-mEGCG conjugate has also been evaluated by reversed-phase high-performance liquid chromatography (HPLC). Reversed phase HPLC was performed using the Waters 2695 Separations Module equipped with a Spirit™ C18 organic column (5 pm, 4.6 x 250 mm i.d., AAPPTec). EGCG, PEG/EGCG mixture and PEG-mEGCG conjugates were dissolved in deionized water at a concentration of 1 mg mL-1. Samples were eluted with a solvent mixture of 1% acetic acid in acetonitrile and 1% acetic acid in water at a flow rate of 1 ml/minute at 25°C. For the mobile phase, the acetonitrile:water volume ratio gradually increased from 3:7 at 0 minutes to 4:6 at 10 minutes. The eluted samples were monitored at 280 nm. The degree of EGCG conjugation was determined by comparing the integrated peak area with those obtained from a series of EGCG standard solutions at various concentrations. As shown in Fig. 3, EGCG (302) eluted at a retention time of 4.8 min, whereas the PEG-mEGCG conjugate (304) eluted at 8 min. This dramatic change in retention time could be explained by the attachment of a hydrophilic PEG chain to EGCG. Furthermore, no EGCG peak was observed in the HPLC chromatogram of the PEG-mEGCG conjugates, suggesting that unreacted EGCG molecules were completely removed by dialysis. The degree of conjugation of EGCG increased from ~63 to 98% as the reaction time increased from 6 to 7 hours (Fig. 4). However, when the reaction time was 8 h, the degree of conjugation was slightly reduced, presumably because EGCG dimers and other oxidative products started to form. Therefore, the ideal reaction time was 7 h. 1H NMR CHARACTERIZATION OF PEG-MEGCG CONJUGATE
    [097] [097] The structure of PEG-mEGCG conjugates was determined by 1H nuclear magnetic resonance (NMR) spectroscopy. As shown in Fig. 5, the
    [098] [098] PEG-dEGCG conjugates were synthesized by conjugating EGCG to PEG with an aldehyde end group (PEG-CHO). PEG-CHO (Mw= 5000 Da, NOF Co., Japan) (1.75 g) and EGCG (3.25 g, 7.09 mmol) were dissolved separately in a mixture of acetic acid, water and DMSO. The reaction was started with the addition of drops of PEG-CHO solution, and was carried out at 20 °C for 72 h. The resulting solution was dialyzed (MWCO = 3500 Da) against deionized water. The purified solution was lyophilized to obtain PEG-dEGCG conjugates. EXAMPLE 3: THE DOXORUBICIN/PEG-mEGCG CONJUGATE
    [099] [099] For cancer therapy applications, PEG-mEGCG conjugates were designed to form micellar nanocomplexes capable of carrying a large number of anti-cancer drugs in their interior. In the study, PEG-mEGCG micellar nanocomplexes were used as a delivery vehicle for doxorubicin. Doxorubicin is one of the most widely used chemotherapeutic agents and has strong cytotoxic activity against several types of cancers, such as leukemia, breast, ovarian and lung. However, it can cause severe cardiotoxicity and increase the risk of congestive heart failure, cardiac arrhythmias,
    [100] [100] Doxorubicin/PEG-mEGCG micellar nanocomplexes were prepared using a dialysis method. In summary, 5 mg of DOX-HCl was dissolved in 4.5 ml of dimethylformamide. To this solution, TEA was added at a molar ratio of TEA:DOX-HCl of 5:1. This mixture was mixed for 30 minutes to form deprotonated doxorubicin (DOX). The resulting DOX solution was mixed with PEG-mEGCG conjugates dissolved in 0.5 mL of dimethylformamide in varying weight ratios of PEG-mEGCG/DOX. This mixture was mixed for 90 minutes and then transferred to dialysis tubes with MWCO of 2000 Da. The tubes were dialyzed against deionized water for 24 hours to obtain the micellar nanocomplexes doxorubicin/PEG-mEGCG. CHARACTERIZATION OF OXORUBICINE/PEG-mEGCG MECLAR NANOCOMPLEXES
    [101] [101] The hydrodynamic diameters, polydispersion indices, and zero potentials of micellar doxorubicin/PEG-mEGCG nanocomplexes were evaluated by dynamic light scattering (Zetasizer Nano ZS, Malvern, UK). The measurement was carried out in triplicate in water at 25°C. To measure the amount of loading of doxorubicin, 20 µl of the nanocomplexes dispersed in water was mixed with 980 µl of dimethylformamide to extract the doxorubicin. The absorbance of doxorubicin at 480 nm was measured using a Hitachi U-2810 spectrophotometer (Japan). The drug loading efficiency and loading content were determined by comparing the absorbance values with those obtained from a series of doxorubicin standard solutions with varying concentrations.
    [102] [102] Fig. 6 illustrates the formation of doxorubicin/PEG-mEGCG micellar nanocomplexes. PEG-mEGCG (602) and doxorubicin (604) conjugates were dissolved in
    [103] [103] The size and surface charge of micellar doxorubicin/PEG-mEGCG nanocomplexes were characterized by dynamic light scattering (DLS) analysis. Fig. 7 refers to graphs showing (A) size and (B) zeta potential of the micellar nanocomplexes doxorubicin/PEG-mEGCG.
    [104] [104] Fig. 7A shows the hydrodynamic diameter of micellar nanocomplexes prepared in different weight ratios different from PEG-mEGCG to doxorubicin. Notably, nanocomplexes were produced with a size range of 130–180 nm. This small size is favorable in achieving prolonged circulation in the bloodstream and targeting the tumor through the enhanced permeability and retention effect (EPR).
    [105] [105] Micellar nanocomplexes formed at a PEG-mEGCG:doxorubicin weight ratio of 0.5:1 are larger in diameter than those formed at 1:1. Trapping higher amounts of doxorubicin was responsible for the formation of larger micellar nanocomplexes. The nanocomplexes were highly monodisperse, as is evident from the value of the small polydispersion index (PDI) being within the range of 0.1–0.2.
    [106] [106] As shown in Fig. 7B, micellar nanocomplexes had positive zeta potentials in the range of +15–25 mV. This cationic surface charge has been attributed to the encapsulation of positively charged doxorubicin molecules within nanocomplexes. We also evaluated whether micellar nanocomplexes maintained their structural integrity during the freeze-drying process. Freeze drying is one of the most popular techniques used for the long-term storage of colloidal nanoparticles. The nanocomplexes were lyophilized and then redispersed in de-ionized water at the same concentration. Reconstituted nanocomplexes were found to retain the original particle size and surface charge even without any lyoprotectants. This high colloidal stability would be advantageous in the clinical translation and commercialization of micellar nanocomplexes.
    [107] [107] Fig. 8 shows the drug loading efficiency and loading content of doxorubicin/PEG-mEGCG micellar nanocomplexes. The drug loading efficiency was greater than 75%, indicating that doxorubicin was efficiently incorporated into PEG-mEGCG nanocomplexes. As the weight ratio of PEG-mEGCG:doxorubicin was reduced, both drug loading efficiency and loading content increased. The observed loading content (35–50 w/w%) was significantly higher than those obtained with other polymeric micellar systems. π-π stacking and/or hydrophobic interactions between EGCG and doxorubicin may have played an important role in the high drug-carrying capacity of PEG-mEGCG micellar nanocomplexes. DOXORUBICIN RELEASE STUDY
    [108] [108] For release experiments, 0.5 mL of nanocomplexes loaded with doxorubicin (2 mg ml−1) were placed in dialysis tubes with 2000 Da MWCO. Tubes were immersed in 25 ml of PBS in a shaking incubator at 37°C. At one point, 1 ml of the release medium was collected and then replaced with an equivalent volume of fresh PBS. The amount of doxorubicin released into the medium was determined by measuring the absorbance of doxorubicin at 480 nm
    [109] [109] The drug release profile of doxorubicin/PEG-mEGCG micellar nanocomplexes has also been investigated at physiological temperature and pH. As shown in Fig. 9, micellar nanocomplexes showed a sustained release of doxorubicin in PBS. Approximately 11% of the loaded doxorubicin was released within 7 days. The observed release rate is considerably lower than that of other previously reported doxorubicin delivery systems. This sustained drug release was likely caused by the strong interaction between EGCG and doxorubicin within micellar nanocomplexes. Furthermore, only a marginal burst release was observed in the initial stage, suggesting that doxorubicin molecules were stably encapsulated in micellar nanocomplexes. This low drug leakage would be essential to ensure maximum therapeutic efficacy with minimal side effects, as drug molecules encapsulated in nanocomplexes would not prematurely leak during circulation in the blood stream. Taken together, these results demonstrated that PEG-mEGCG micellar nanocomplexes could be applied for systemic administration of doxorubicin for cancer treatment. EXAMPLE 4: SU/PEG-EGCG CONJUGATES FORMATION OF SU/PEG-EGCG MICELLAR NANOCOMPLEXES
    [110] [110] Fig. 10 illustrates the formation of SU/PEG-EGCG conjugates using the solid dispersion method. Briefly, 2 mg of SU (1006) was dissolved in 1 ml of chloroform. Then SU solution was added to PEG-EGCG conjugates (either PEG-mEGCG (1002) or PEG-dEGCG (1004) in glass vials in varying weight ratios of PEG-EGCG: SU (1008) and mixed. the chloroform from the solution was evaporated under reduced pressure (1010). The resulting thin film of the mixture of PEG-EGCG and SU (1012) was hydrated by adding 2 ml of water to the surface (1014), and incubated at room temperature for 24 h. As the resulting solid film was hydrated, the PEG-EGCG self-assembled to form micellar nanocomplexes by isolation of SU and EGCG media from the hydrated PEG chains.
    [111] [111] It should be noted that PEG-EGCG conjugates refer to both PEG-mEGCG and PEG-dEGCG unless otherwise specified.
    [112] [112] As EGCG has a polyphenol structure capable of interacting with SU through hydrophobic interaction and π-π stacking, it was predicted that EGCG enriched in the core of micellar nanocomplexes would provide a favorable environment for trapping SU. In addition, surface exposed PEG chains would form a highly hydrated shield around micellar nanocomplexes to prevent RES clearance, thus allowing prolonged circulation in the bloodstream and reducing side effects. CHARACTERIZATION OF SU/PEG-mEGCG MICELLAR NANOCOMPLEXES
    [113] [113] The hydrodynamic diameters, size distribution, and surface charge of SU/PEG-mEGCG micellar nanocomplexes were evaluated by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern, UK). Measurements were conducted in triplicate in water at 25°C. Fig. 11A shows the hydrodynamic diameter of micellar nanocomplexes prepared in different weight ratios of PEG-EGCG:SU. Notably, micellar nanocomplexes were produced in the 130–250 nm size range. The size of the nanometer would be favorable in prolonging circulation and targeting the tumor through the EPR effect. The characteristics of micellar nanocomplexes were controlled by varying the weight ratios of PEG-EGCG: SU. Fig. 11B demonstrates that micellar nanocomplexes formed at PEG-EGCG:SU weight ratios of 8 and 16 were highly monodosperse. The micellar nanocomplexes reduced in positive charge as the weight ratio of PEG-EGCG: SU increased (Fig. 11C). Its slightly positive surface charge has been attributed to encapsulation of positively charged SU molecules.
    [114] [114] To measure drug loading efficiency and quantity, 10 pL of micellar nanocomplexes in water were dissolved in 990 pL of DMF, and the absorbance of SU was measured at 431 nm using a Hitachi U-2810 ultraviolet visible spectrophotometer (UV-Vis) (Japan). The calibration curve obtained with the SU standard solutions was used to determine the efficiency and quantity of loading.
    [115] [115] Fig. 12 shows the drug loading efficiency and loading content of SU/PEG-EGCG micellar nanocomplexes. As the PEG-EGCG:SU weight ratio increased from 1 to 16, the drug loading efficiency increased from ~30% to ~80%. The loading efficiency of SU/PEG-dEGCG micellar nanocomplexes was higher than SU/PEG-mEGCG micellar nanocomplexes, indicating greater interaction of SU with PEG-dEGCG compared to PEG-mEGCG. It was also found that the loading efficiency of micellar nanocomplexes was related to the amount of uncharged SU precipitate before filtration. When the PEG-EGCG:SU weight ratio was increased to 8, no SU precipitate was found. As expected, the loading content of the micellar nanocomplexes was reduced as the weight ratio of PEG-EGCG:SU increased due to the higher content of PEG-EGCG in the micellar nanocomplexes. SU RELEASE STUDY
    [116] [116] The drug release profile of SU/PEG-EGCG micellar nanocomplexes was further investigated under physiological condition (phosphate-buffered saline (PBS), pH 7.3 at 37 °C). For SU release experiments, 0.5 mL of micellar nanocomplexes loaded with SU/PEG-EGCG (0.4 mg ml-1) were placed in dialysis tubes (MWCO = 2000 Da). Tubes were immersed in 25 ml of PBS in a shaking incubator at 37 °C. At one point, 1 ml of the release medium was collected and then replaced with an equivalent volume of fresh PBS. The amount of SU released into the medium was determined by measuring the absorbance at 431 nm using a Hitachi U-2810 spectrophotometer.
    [117] [117] As shown in Fig. 13, micellar nanocomplexes showed a sustained release of SU in PBS, which could be attributed to the strong interaction
    [118] [118] To study toxicity and therapeutic effect, in vivo studies were conducted on micellar nanocomplexes. A model of subcutaneous renal cell carcinoma was established. Athymic Balb/c adult female nude mice, immunoincompetent (mean weight = 19 g, age = 6–8 weeks) were used.
    [119] [119] To study the therapeutic effect of SU/PEG-EGCG micellar nanocomplexes by intravenous injection, a xenograft tumor model was established by inoculating 6x106 A498-luc cells subcutaneously into the root of the mouse's left thigh. On day 6 after tumor inoculation, the animals were divided into four groups for tail vein injection of various solutions (n = 8 per group) twice a week for 5 weeks, while one group received daily SU, with gavage at 60 mg/kg. For tail vein injection, a volume of 200 µl of the sample solution was used.
    [120] [120] To monitor bioluminescent signals from A498-luc cells, isoflurane gas-anesthetized animals were injected intraperitoneally with 200 µl of D-luciferin (5 mg ml-1, Promega) in PBS, and placed in a warm stage (30°C). ) inside the camera housing of the IVIS imaging system (Xenogen, Alameda, CA, USA) with a CCD camera. Luminescent images were taken 20 minutes after
    [121] [121] All data were represented as mean + standard error of the mean (SEM). Statistical significance of differences between mean values was determined by Student's t test. Multiple comparisons were evaluated by ANOVA with Bonferroni multiple comparison tests using SigmaStat 3.5. A P value of < 0.05 was considered statistically significant.
    [122] [122] SU/PEG-mECGC micellar nanocomplexes (with PEG-EGCG:SU weight ratios of 8 and 16) and SU/PEG-dEGCG micellar nanocomplexes (with PEG-EGCG:SU weight ratios of 8) were selected for in vivo studies based on micellar nanocomplex size, size distribution and surface charge. SU/PEG-mEGCG micellar nanocomplexes were injected intravenously twice a week for 5 weeks and one group received daily SU gavage at 60 mg/kg. The oral drug dose of 60 mg/kg per day was selected based on previous reports that demonstrated the optimal preclinical dose of SU for antitumor efficacy in mice as 40–80 mg/kg per day. For our studies, a dose of 60 mg/kg per day represented an effective antitumor dose, as other studies indicated that a dose of < 40 mg/kg per day would be sub-effective and a dose of 120 mg/kg per day would test the effects of even higher drug administration.
    [123] [123] Fig. 14 shows significant weight loss in the group that received free oral SU one week after starting treatment. This was not observed in the other groups that received the SU/PEG-EGCG micellar nanocomplex treatment. THE
    [124] [124] To investigate the therapeutic effect of SU/PEG-mEGCG MNC via oral administration, a xenograft tumor model was established by inoculating g 4x10 6 ACHN cells suspended in 100 µl PBS and 100 µl Matrigel (BD Bioscience ) subcutaneously at the root of the mouse's right thigh. Once the tumors reached a volume of 200 mm3, the animals were divided into four groups for oral gavage of various solutions (n = 8 per group) daily for 5 weeks: control (citrate buffer at pH5), SU/PEG- mEGCG 8:1 (SU at 15 mg/kg), SU at 15 and 40 mg/kg. Tumors were measured twice weekly with a digital caliper, and tumor volumes (mm3) were calculated from the formula: volume = (length x width 2/2) (Figs. 16 and 17).
    [125] [125] As the oral SU dose of 60 mg/kg per day has been shown to be too toxic, the oral SU dose has been reduced to 40 mg/kg per day in the present disclosure. This oral dose of 40 mg/kg per day is the optimal preclinical dose for antitumor efficacy in mice (40–80 mg/kg per day) based on previous reports. Fig. 16 shows significant weight loss in the group receiving 40 mg/kg SU during treatment. This was not observed in the other groups that received treatment with SU/PEG-mEGCG MNC 8:1 and SU at 15 mg/kg. At the same dose of SU at 15 mg/kg, SU/PEG-mEGCG MNC had a significantly higher therapeutic effect compared to SU alone (Fig. 17). This antitumor effect with the MNC of SU/PEG-mEGCG was obtained at less than half the concentration of the optimal oral dose of SU at 40 mg/kg. The MNC inhibitory effect of SU/PEG-EGCG was maintained by a
    [126] [126] The EPR effect considers the anatomical-physiological nature of tumor blood vessels to facilitate the transport of >40 kDa macromolecules that selectively leak from tumor vessels and accumulate in tumor tissue. Most solid tumors have blood vessels with defective architecture, which usually results in large amounts of vascular permeability. This does not occur in normal tissues. The present invention discloses the use of SU/PEG-mEGCG micellar nanocomplexes via intravenous and oral administrations as a possible therapy for ccRCC for the first time. EGCG was observed to interact with SU and pharmacokinetic studies in rats demonstrated that administration of EGCG markedly reduced plasma concentrations of SU. The reported interaction of green tea with SU and the EPR effect of micellar nanoparticles on various tumors suggested the possibility of using PEG-EGCG as a nanoparticle vehicle for SU delivery. In glioblastoma, a highly angiogenic tumor, anti-angiogenic therapy has demonstrated high but transient efficacy. This tumor stimulates the formation of new blood vessels by processes driven primarily by VEGF, but the resulting vessels are structurally and functionally abnormal. The use of SU/PEG-EGCG micellar nanocomplexes could potentially improve the anti-angiogenic activity in these cases. INDUSTRIAL APPLICABILITY
    [127] [127] Micellar nanocomplexes can be applied for use in systemic administration of doxorubicin for the treatment of cancer.
    [128] [128] Micellar nanocomplexes can be used as a nanoparticle vehicle for SU delivery. In glioblastoma, a highly angiogenic tumor, anti-angiogenic therapy has demonstrated high but transient efficacy. This tumor stimulates the formation of new blood vessels by processes driven primarily by VEGF, but the resulting vessels are structurally and functionally abnormal. The use of micellar nanocomplexes comprising SU can potentially improve the anti-angiogency activity in these cases.
    [129] [129] Micellar nanocomplexes can be used for sustained release of
    [130] [130] Micellar nanocomplexes can be used to encapsulate a variety of therapeutic agents for different types of cancer treatment.
    [131] [131] Micellar nanocomplexes can also be used to encapsulate a variety of therapeutic agents for non-cancerous treatment. This can include small molecules for antibiotics and other medical applications.
    [132] [132] It will be apparent that various other modifications and adaptations of the invention will be apparent to a person skilled in the art after reading the above disclosure without departing from the spirit and scope of the invention, and all such modifications and adaptations are intended to be within the scope of the appended claims.
    权利要求:
    Claims (1)
    [1]
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    1) "MICELAR NANOCOMPLEX, POLYMER-FLAVONOID CONJUGATE AND METHODS FOR THEIR FORMATIONS", characterized in that it comprises a micelle and an agent encapsulated within said micelle, wherein said micelle comprises a polymer-flavonoid conjugate, wherein said polymer is attached to the B-ring of said flavonoid. 2) "MICELAR NANOCOMPLEX", according to claim 1, characterized in that at least one flavonoid is linked to said polymer. 3) "MICELAR NANOCOMPLEX", according to claims 1 and 2 characterized in that said polymer is linked to said flavonoid through a binder. 4) "MICELAR NANOCOMPLEX", according to claim 3, characterized in that said ligand is selected from the group consisting of athioether, imine, amine, azo and 1,2,3-triazole group. 5) "MICELAR NANOCOMPLEX", according to claim 3 characterized in that said flavonoid is a monomeric flavonoid or a dimeric flavonoid. 6) "MICELAR NANOCOMPLEX", according to claims 2 to 5, characterized in that more than one flavonoid is present in said conjugate, at least one flavonoid is linked to said polymer through ring B. 7) "MICELAR NANOCOMPLEX", of according to claim 6, characterized in that another of said at least one flavonoid is linked to said polymer through ring A. 8) "MICELAR NANOCOMPLEX", according to the preceding claims, characterized in that said polymer is a hydrophilic polymer. 9) "MICELAR NANOCOMPLEX", according to claim 8 characterized in that said hydrophilic polymer comprises monomers selected from the group consisting of acrylamides, alkys, oxazolines, alkenis, imines, acrylic acids, methacrylates, diols, oxiranes, alcohols, amines , anhydrides, esters, lactones, carbonates, carboxylic acids, acrylates, hydroxys, phosphates, terephthalate, amides and ethers. 10) "MICELAR NANOCOMPLEX", according to claim 8 characterized
    2 / 9 that said hydrophilic polymer is selected from the group consisting of polyacrylamide, poly(N-isopropylacrylamide), poly(oxazoline), polyethyleneimine, poly(acrylic acid), polymethacrylate, poly(ethylene glycol), poly(oxide ethylene), poly(vinyl alcohol), poly(vinylpyrrolidinone), polyethers, poly(allylamine), polyanhydrides, poly(β-amino ester), poly(butylene succinate), polycaprolactone, polycarbonate, polydioxanone, poly(glycerol), polyglycolic acid , poly(3-hydroxypropionic acid), poly(2-hydroxyethyl methacrylate), poly(N-(2-hydroxypropyl)methacrylamide), polylactic acid, poly(lactic-co-glycolic acid), poly(ortho esters), poly( 2-oxazoline), poly(sebacic acid), poly(terephthalate-co-phosphate) and their copolymers. 11) "MICELAR NANOCOMPLEX", according to claim 8 characterized in that said hydrophilic polymer is a polysaccharide. 12) "MICELAR NANOCOMPLEX", according to claim 8 characterized in that said hydrophilic polymer is a polysaccharide selected from the group consisting of hyaluronic acid, dextran, pullulan, chitosan, cellulose, amylose, starch, gelatin, carrageenan, cyclodextrin, dextran sulfate, Fucoll, gellan, guar gum, pectin, polysaccharose, pullulan, scleroglucan, xanthan, xyloglucan and alginate. 13) "MICELAR NANOCOMPLEX", according to the preceding claims characterized in that said flavonoid is selected from the group consisting of flavans, isoflavones, flavans, proanthocyanidins and anthocyanidins. 14) "MICELAR NANOCOMPLEX", according to claim 13 characterized in that said flavans are selected from the group consisting of (‐)‐ epicatechin, (+)‐ epicatechin, (‐)‐ catechin, (+)‐catechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, physetinidol, gallocatechin, gallocatechin gallate, mesquitol and robinetinidol, ellagitanin, galotanin, olongthean, phlorotanin, tanin, theacithrin, theadibenzotropolone, theaflavin, theanadtoquinone, and its teasubiginine. 15) "MICELAR NANOCOMPLEX", according to any one of the preceding claims, characterized in that said agent is a therapeutic agent. 16) "MICELAR NANOCOMPLEX", according to any of the claims
    3 / 9 above characterized in that said therapeutic agent is a chemotherapeutic agent selected from the group consisting of alkylating agents, anthracyclines, di-skeletal disruptors, epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, monoclonal antibodies , antibody-drug conjugates, nucleotide analogues, precursor analogues, peptide antibiotics, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids, cytokines, antimetabolites and derivatives of vinca alkaloids and other cytotoxics. 17) "MICELAR NANOCOMPLEX", according to claim 16 characterized in that said chemotherapeutic agent is selected from the group consisting of Actinomycin, Afatinib, fully trans retinoic acid, Axitinib, Azacitidine, Azathioprine, Bevacizumab, Bleomycin, Bosutinib, Bortezomib, Carboplatin, Capecitabine, Cetuximab, Cisplatin, Chlorambucil, Crizotinib, Cyclophosphamide, Cytarabine, Dasatinib, Daunorubicin, Docetaxel, Doxyfluridine, Doxorabicin, Epirubicin, Epothilone A (C26H39NO6S), Epothilone B (C27H41NOH6S), Epothilone B (C27H41NOH6S), Epothilone E (C26H39NO7S), Epothilone F (C27H41NO7S), Erlotinib, Etoposide, Fluorouracil, Phostamatinib, Gefitinib, Gemcitabine, Hydroxyurea, Idarabicin, Imatinib, Irinotecan, Lapatinib, Lenvatinib, Meritinylchlorine, Methorethamine Panitumumab, Pazopanib, Pegaptanib, Pemetrexed, Ranibizumab, Regorafenib, Ruxolitinib, Sorafenib, Sunitinib, Trastuzumab, Teniposide, Thioguanine, Tofacitinib, Topotecan, Valrrubicin, Vemurafenib, Vinblastine, Vincristine, Vindesine, Vinorrelbine. 18) "MICELAR NANOCOMPLEX", according to any one of the preceding claims characterized in that said micellar nanocomplex has a size in the range from 30 to 300 nm, from 50 to 300 nm, from 100 to 300 nm, from 30 to 50 nm, of 30 to 100 nm, from 30 to 150 nm, from 150 to 300 nm, from 200 to 300 nm, from 250 to 300 nm, from 100 to 150 nm, from 100 to 200 nm, from 100 to 250 nm, from 130 to 180 nm, or from 130 to 250 nm. 19) "MICELAR NANOCOMPLEX", according to any one of the preceding claims characterized by loading efficiency of said agent present
    4 / 9 within said micelle is greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, or 80%. 20) "MICELAR NANOCOMPLEX", according to any one of the preceding claims characterized in that the loading content of said agent present within said micelle is in the range from 1 to 10 w/w%, from 5 to 25 w/w%, of 20 to 45 w/w%, from 30 to 50 w/w%, from 35 to 50 w/w%, from 40 to 50 w/w%, from 45 to 50 w/w%, from 30 to 35 w/w/ w%, from 30 to 40 w/w% or from 30 to 45 w/w%. 21) "METHOD FOR THE FORMATION OF A MICELAR NANOCOMPLEX", characterized in that it comprises a micelle and an agent encapsulated within said micelle, the method comprising the steps of: a. adding said agent in a suitable solvent to a polymer-flavonoid conjugate, wherein said polymer is attached to ring B of said flavonoid; and b. allowing self-assembly of a micelle comprising said polymer-flavonoid conjugate and encapsulating said agent within said micelle to thereby form said micellar nanocomplex. 22) "FORMATION METHOD OF A MICELAR NANOCOMPLEX", according to claim 21, characterized in that step (a) further comprises the steps of: a. removing said solvent to form a dry film of said agent and said polymer-flavonoid conjugate; and b. hydrate said dry film with an aqueous solvent. 23) "METHOD FOR FORMATION OF A MICELAR NANOCOMPLEX", according to claim 21 or 22, characterized in that it further comprises the step of isolating the micellar nanocomplex formed by filtration. 24) "METHOD FOR FORMATION OF A MICELAR NANOCOMPLEX", according to claim 21, characterized in that step (a) further comprises the step of dialysis of the agent in a suitable solvent. 25) "POLYMER-FLAVONOID CONJUGATE", characterized in that it comprises a polymer linked to the B ring of a flavonoid. 26) "POLYMER-FLAVONOID CONJUGATE", according to claim 25
    5 / 9 characterized in that said polymer is selected from the group consisting of a polysaccharide, polyacrylamide, poly(N-isopropylacrylamide), poly(oxazoline), polyethyleneimine, poly(acrylic acid), polymethacrylate, poly(ethylene glycol), poly (ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidinone), polyethers, poly(allylamine), polyanhydrides, poly(β-amino ester), poly(butylene succinate), polycaprolactone, polycarbonate, polydioxanone, poly(glycerol) , polyglycolic acid, poly(3-hydroxypropionic acid), poly(2-hydroxyethyl methacrylate), poly(N-(2-hydroxypropyl)methacrylamide), polylactic acid, poly(lactic-co-glycolic acid), poly(ortho esters) , poly(2-oxazoline), poly(sebacic acid), poly(terephthalate-co-phosphate) and their copolymers. 27) "POLYMER-FLAVONOID CONJUGATE", according to claims 25 to 26, characterized in that said flavonoid is selected from the group consisting of (‐)‐epicatechin, (+)‐epicatechin, (‐)‐ catechin, (+ )‐catechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, physetinidol, gallocatechin, gallocatechin gallate, Mesquitol and Robinetinidol, ellagitanin, gallotanin, olongthean, phlorotanin, tannin, theacithrin, theadibenzotropolone, theaflavin, teanabiquinone, teanabiquinone. 28) "POLYMER-FLAVONOID CONJUGATE", according to any one of claims 25 to 27 characterized in that said polymer is conjugated to a flavonoid in the polymer-flavonoid conjugate through a ligand selected from the group consisting of a thioether group, imine , amine, azo and 1,2,3-triazole. 29) "POLYMER-FLAVONOID CONJUGATE", according to any one of claims 25 to 28, characterized in that said polymer is poly(ethylene glycol), said flavonoid being epigallocatechin-3-gallate and said being thioether binder. 30) "POLYMER-FLAVONOID CONJUGATE", according to claim 29, characterized in that said conjugate has the following formula [Chem. two]
    6 / 9
    - Where n is in the range from 20 to 910. 31) "METHOD FOR FORMING THE POLYMER-FLAVONOID CONJUGATE", according to any one of claims 25 to 30, characterized in that it comprises the step of conjugating said flavonoid with said polymer by means of nucleophilic addition under basic conditions, wherein said polymer has a free nucleophilic group. 32) "METHOD FOR FORMING THE POLYMER-FLAVONOID CONJUGATE", according to claim 31, characterized in that said conjugation step is carried out in a reaction time between 1 and 24 hours. 33) METHOD FOR FORMING THE POLYMER-FLAVONOID CONJUGATE", according to claims 31 and 32, characterized in that it further comprises the step of conducting the conjugation step in a solvent that substantially prevents the aggregation of said flavonoid. 34) METHOD FOR FORMING THE POLYMER-FLAVONOID CONJUGATE, according to claim 31 to 33, characterized in that it further comprises the step of adding a cleaning agent to prevent the H2O2 mediated oxidation of said nucleophilic group to thus increase the efficiency of said conjugation step. 35) METHOD FOR FORMING THE POLYMER-FLAVONOID CONJUGATE", according to claim 31 to 34, characterized in that said basic conditions are in the pH range of more than 7 to 10. 36) METHOD FOR FORMING THE POLYMER-FLAVONOID CONJUGATE", of according to claims 28 to 31 characterized in that said nucleophilic group is selected from the group consisting of a thiol, an amine, a diazoalkane and an azide. 37) "USE OF A MICELAR NANOCOMPLEX", characterized in that it comprises a micelle and an agent encapsulated within said micelle as a drug delivery vehicle, wherein said micelle comprises a polymer-flavonoid conjugate, and wherein said polymer is connected to ring B of the above
    7 / 9 flavonoid. 38) "USE OF A MICELAR NANOCOMPLEX", according to claim 37 characterized in that micellar nanocomplex delivers the encapsulated agent to a target tumor site in vivo. 39) "METHOD FOR TREATMENT OF A TUMOR", characterized in that it comprises the step of administering the micellar nanocomplex of any one of claims 1 to 24 to a patient with cancer. 40) "METHOD FOR TREATMENT OF A TUMOR", according to claim 39, characterized in that said micellar nanocomplex is administered parenterally, by administration of an inhalation spray, topical, rectal, nasal, buccal, vaginal, through an implanted reservoir, by injection, subdermal, intraperitoneal, transmucosal, oral or in an ophthalmic preparation. 41) "METHOD FOR TREATMENT OF A TUMOR", according to claim 40, characterized in that said parenteral administration comprises subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional and injection routes. intracranial or infusion techniques. 42) "METHOD FOR TREATMENT OF A TUMOR", according to claims 39 to 41, characterized in that the agent present in said micellar nanocomplex is administered at a dose of 1 to 80 mg/kg per day. 43) "METHOD FOR TREATMENT OF A TUMOR", according to claims 39 to 41 characterized in that said cancer patient suffers from a cancer selected from the group consisting of adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendix cancer , grade I astrocytoma (anaplastic), grade II astricutin, grade III astricutin, grade IV astricutin, atypical central nervous system teratoid/rhabdoid tumor, basal cell carcinoma, bladder cancer, bronchial cancer, bronchial alveolar carcinoma, lymphoma of Burkitt, cervical cancer, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, endometrial cancer, uterine endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, germ cell tumor
    8/9 extracranial, extragonadal germ cell tumor, extrahepatic bile duct cancer, fibrous histiocytoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic tumor, gestational trophoblastic tumor, glioma, cancer of head and neck, heart cancer, hepatocellular cancer, Hilar's cholangiocarcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, Langerhans cell histocytosis, laryngeal cancer, labral cancer, lymphoma, macroglobulinemia , malignant fibrous histiocytoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, endocrine neoplasia, multiple myeloma, fungoid myeloma, myelodysplasia, myelodysplastic/myeloproliferative neoplasms, myelopliferative disorders, lymphoma, nasal cavity cancer of Hodgkin, oral cancer, oropharyngeal cancer, the steosarcoma, ovarian cell carcinoma and elimination, ovarian epithelial cancer, ovarian germ cell tumor, papillomatosis, paranasal sinus cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pineal parenchymal tumor, pineoblastoma, pituitary tumor, cell neoplasm plasma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, respiratory tract cancer with chromosome 15 alterations, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sezary syndrome, bowel cancer thin, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, 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, Waldenst's macroglobulinemia rom and Wilms' tumor. 44) "MICELAR NANOCOMPLEX", according to any one of claims 1 to 24, characterized in that it treats a tumor. 45) "USE OF MYCELLAR NANOCOMPLEX", according to any one of claims 1 to 24 characterized by the manufacture of a drug for the treatment of a tumor.
    FIG. 1 104 106 102 108 1/14
    1.6
    1.4 202 FIG. 2 1.2 204 1
    0.8 Petition 870160066193, of 11/09/2016, p. 142/164
    0.6 Absorbance
    0.4
    0.2 0 200 250 300 350 400 450 500 Wavelength [nm]
    FIG. 3
    302 2/14
    Absorbance [a.u.]
    Petition 870160066193, of 11/09/2016, p. 143/164 304
    0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Retention time [min]
    Reaction time [h] 7 6 100
    80
    60
    40
    20
    0
    Degree of Conjugation [%] FIG. 4
    Petition 870160066193, of 11/09/2016, p. 145/164 FIG. 5
    1.00
    7.0
    0.50
    6.5
    0.84
    6.0
    0.51
    0.47
    5.5
    5.0
    4.5
    4.0
    2.71
    1.59
    549.07
    48.24
    5.73
    3.5
    3.34
    4.32
    0.23
    0.23
    3.0
    2.05
    0.78 ppm 4/14
    FIG. 6
    FIG. 7A FIG. 7B
    200 30
    25 150 20 6/14
    100 15
    10
    Size [nm] 50
    Zeta potential [mV] 5
    0 0 1:1 0.5:1 1:1 0.5:1
    Petition 870160066193, of 11/09/2016, p. 147/164 PEG-EGCG: Doxorubicin (w/w) PEG-EGCG: Doxorubicin (w/w)
    702 704
    FIG. 8A FIG. 8B Loading efficiency [%]
    Loading content [%] 100 60
    80 50 40 60 30 40 20 20 10 0 0 1:1 0.5:1 1:1 0.5:1 PEG-EGCG: Doxorubicin (w/w) PEG-EGCG: Doxorubicin (w/w)
    FIG. 9
    14 Cumulative release [%]
    12 10 8 6 4 2 0 0 1 2 3 4 5 6 7 8
    Time [d]
    FIG. 10
    FIG. 11A FIG. 11B 300 0.6 250 0.5 200 0.4 150 0.3
    PDI 100 0.2 Size [nm] 50 0.1 0 0 9/14 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 PEG-EGCG:SU (w/w) PEG-EGCG:SU (w/w p) FIG. 11C 10 8 SU/PEG-mEGCG MNCs 6 SU/PEG-dEGCG MNCs Petition 870160066193, dated 11/09/2016, p. 150/164 4 2 Zeta potential [mV] 0 0 2 4 6 8 10 12 14 16 PEG-EGCG:SU (w/w)
    FIG. 12A
    100 Loading efficiency [%]
    80
    60
    40
    20
    0 0 2 4 6 8 10 12 14 16
    PEG-EGCG:SU (w/w)
    FIG. 12B 60 Loading content [%]
    50
    40
    30
    20
    10
    0 0 2 4 6 8 10 12 14 16 PEG-EGCG:SU (w/w)
    SU/PEG-mEGCG MNCs SU/PEG-dEGCG MNCs
    FIG. 13A FIG. 13B
    120 120 SU release [%]
    SU release [%] 100 100
    80 80 PEG-EGCG:SU 1 60 60 2 4 40 40 8 16 20 20
    0 0 2 4 6 0 2 4 6 Time [h] Time [h]
    FIG. 14 body weight
    22 SU/PEG-mEGCG 16:1 SU/PEG-mEGCG 8:1 SU/PEG-dEGCG 8:1 SU isolated (oral) 20 Untreated Body Weight [g]
    18
    16 End of treatment
    0 1 2 3 4 5 6 7 8 9 10 11
    Weeks after Treatment
    FIG. 15A Tumor Size
    1e+10 SU/PEG-mEGCG 16:1 SU/PEG-mEGCG 8:1 SU/PEG-dEGCG 8:1 1e+9 SU isolated (oral) No treatment ** 1e+8 * 12/14
    1e+7
    1e+6
    Luminescent Signal [photons/s] End of treatment
    Petition 870160066193, of 11/09/2016, p. 153/164 1e+5 0 1 2 3 4 5 6 7 8 9 10 11
    Weeks after Treatment
    *P<0.01; **P < 0.005
    SU/PEG- SU/PEG- SU/PEG- mEGCG mEGCG dEGCG 16:1 8:1 8:1 Oral No treatment
    FIG. 15B Day 14
    High Day 28 5
    4 13/14
    Day 42 3 6 x 10 2 Day 56 1
    Petition 870160066193, of 11/09/2016, p. 154/164 Low Day 70
    day 84
    Body weight
    FIG. 16 22
    21
    20 Body Weight [g]
    19
    18 SU/PEG-mEGCG 8:1 (SU 15mg/kg) 17 SU 15mg/kg SU 40mg/kg 16 Control End of treatment 15 0 10 20 30 40 50 60 70 Days after Treatment
    FIG. 17 Tumor Size
    1600 SU/PEG-mEGCG 8:1 (SU 15mg/kg) 1400 SU 15mg/kg SU 40mg/kg Tumor volume [mm³]
    1200 Control
    1000
    800
    600
    400
    200 End of treatment
    0 0 10 20 30 40 50 60 70 Days after Treatment
    *P<0.05; **P < 0.001
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    同族专利:
    公开号 | 公开日
    US10463646B2|2019-11-05|
    US10052307B2|2018-08-21|
    AU2015256667A1|2016-11-17|
    CA2948460C|2020-10-13|
    EP3139915A4|2017-12-20|
    EA201692213A1|2017-06-30|
    AU2015256667C1|2018-07-05|
    US20190008830A1|2019-01-10|
    MX2016014606A|2017-08-24|
    JP2017518280A|2017-07-06|
    IL248632D0|2017-01-31|
    EA035820B1|2020-08-17|
    EP3139915B1|2020-11-04|
    CU24520B1|2021-06-08|
    EP3139915A1|2017-03-15|
    ZA201608424B|2019-12-18|
    WO2015171079A1|2015-11-12|
    KR20170007780A|2017-01-20|
    KR20190095510A|2019-08-14|
    SA516380256B1|2021-02-25|
    JP6307631B2|2018-04-04|
    US20170258926A1|2017-09-14|
    IL248632A|2021-03-25|
    AU2015256667B2|2018-04-19|
    ES2847602T3|2021-08-03|
    SG11201609333YA|2016-12-29|
    CA2948460A1|2015-11-12|
    CU20160164A7|2017-05-10|
    KR102102093B1|2020-04-17|
    UA118479C2|2019-01-25|
    CN106659708A|2017-05-10|
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    法律状态:
    2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A61K 31/353 (2006.01), A61K 47/00 (2006.01), C08G |
    2019-10-01| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |
    2021-08-17| B350| Update of information on the portal [chapter 15.35 patent gazette]|
    2021-09-08| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|Free format text: NOTIFICACAO DE DEVOLUCAO DO PEDIDO EM FUNCAO DA REVOGACAO DO ART. 229-C DA LEI NO 9.279, DE 1996, POR FORCA DA LEI NO 14.195, DE 2021 |
    2021-09-21| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    优先权:
    申请号 | 申请日 | 专利标题
    SG10201402244S|2014-05-09|
    SG10201402244S|2014-05-09|
    PCT/SG2015/050104|WO2015171079A1|2014-05-09|2015-05-08|A micellar nanocomplex|
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