Conjugates for targeting central nervous system

Abstract

The present invention provides a conjugate comprising: (a) a CNS targeting ligand, (b) a hydrophilic polymer of polyethylene glycol (PEG), polylactic acid (PLA), polylactic acid-co-glycolic acid (PLGA), or glucan, and (c) a flavonoid. The present invention also provides a micelle nanoparticle composition comprising: (a) an outer shell comprising the conjugate, optionally (b) an inner shell comprising oligomeric (-)-epigallocatechin gallate (OEGCG), and optionally (c) a CNS disease treatment molecule encapsulated in the inner shell. The invention also provides a method of treating a CNS disease by administering to a subject an effective amount of a nanoparticle composition of the invention. The CNS targeting ligand targets a CNS tissue and delivers an active ingredient to the CNS tissue for treating a CNS pathological condition.

Description

[0001] Sequence Listing

[0002] This application contains a sequence listing that complies with ST.26, which is submitted in xml format via Patent Center and is hereby incorporated by reference in its entirety. The .xml copy created on June 1, 2023 is named SequenceListing.xml and is 15.3KB in size. Technical Field

[0003] The present invention relates to a conjugate comprising: (a) a CNS (central nervous system) targeting ligand, (b) a hydrophilic polymer of polyethylene glycol (PEG), polylactic acid (PLA), polylactic acid-co-glycolic acid (PLGA) or dextran, and (c) a flavonoid, wherein the hydrophilic polymer is covalently bound to the flavonoid and the CNS targeting ligand. The present invention relates to micellar nanoparticles comprising: (a) an outer shell comprising a CNS targeting ligand-hydrophilic polymer-(-)-epigallocatechin gallate (EGCG) conjugate, optionally (b) an inner shell comprising an oligomer (OEGCG), and optionally (c) a CNS therapeutic agent encapsulated in the inner shell. Background Art

[0004] Central nervous system (CNS) disease is a group of neurological disorders that affect the structure or function of the brain or spinal cord (which together form the CNS). The disease can be a metabolic disorder of inheritance, the result of neural damage from infection, neurodegenerative diseases, stroke, brain tumors, or other problems from unknown or multiple factors. CNS diseases include brain tumors, neurodegenerative diseases (such as amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy and prion disease), migraine, infection, addiction, arachnoid cysts, attention deficit / hyperactivity disorder (ADHD), autism, catalepsy, encephalitis, epilepsy / seizures, infection, locked-in syndrome, meningitis, migraine, myelopathy, Tourette syndrome.

[0005] CNS tumors occur when abnormal cells form within the brain and spinal cord. Brain tumors include malignant tumors and benign (noncancerous) tumors. These tumors can be further classified into primary tumors that start in the brain and secondary tumors that spread most commonly from tumors located outside the brain. Spinal tumors are tumors located in the spine or spinal cord. Brain tumors can be primary brain cancers or metastatic brain cancers.

[0006] Stroke is a medical condition in which poor blood flow to the brain causes cell death. There are two main types of stroke: ischemic stroke due to lack of blood flow and hemorrhagic stroke due to bleeding. The main risk factor for stroke is high blood pressure. Other risk factors include high blood cholesterol, smoking, obesity, diabetes, previous transient ischemic attack, end-stage renal disease and atrial fibrillation. There is no therapy to regenerate dead brain cells after a stroke. Treatment for stroke is very limited, and an FDA-approved drug for ischemic stroke is tissue plasminogen activator (tPA), which breaks down blood clots in the brain. Prevention is an important clinical strategy. Oral anticoagulants such as warfarin are the main means of preventing stroke.

[0007] Neurodegenerative diseases are a group of diseases that primarily affect neurons in the human brain. Examples of neurodegenerative diseases are Alzheimer's disease (AD) and other dementias, Parkinson's disease (PD) and Parkinson's syndrome, prion diseases, motor neuron disease (MND), Huntington's disease (HD), spinocerebellar ataxia (SCA) and spinal muscular atrophy (SMA). Some neurodegenerative disorders are caused by inherited genetic changes. Most neurodegenerative disorders are due to a combination of genetic and environmental factors. This makes it difficult to predict who will get sick. The two main neurodegenerative diseases are Alzheimer's disease and Parkinson's disease.

[0008] Alzheimer's disease (AD) is a neurodegenerative disease, which is usually slow in onset and gradually worsens. Along with the development of the disease, symptoms may include language problems, disorientation (including being easily lost), mood swings, loss of motivation, self-neglect and behavioral problems. It is believed that when an abnormal amount of amyloid beta (Aβ) is formed in the brain, Alzheimer's disease occurs, and the amyloid beta accumulates as amyloid plaques and tau protein in the extracellular space, or accumulates as neurofibrillary tangles in the cell, thereby affecting neuronal function and connection, causing progressive loss of brain function. At present, there is no treatment to stop or reverse its progress, although some treatments may temporarily improve symptoms.

[0009] Parkinson's disease (PD) is a long-term degenerative disorder of the central nervous system that primarily affects the motor system. Parkinson's disease is sometimes referred to as a class of neurodegenerative diseases called synucleinopathies due to abnormal accumulation of the protein alpha-synuclein in the brain. The most obvious early symptoms of PD are tremors, rigidity, slowed movements, and difficulty walking. In many people with PD, cognitive and behavioral problems may also occur along with depression, anxiety, and apathy. Parkinson's disease dementia becomes common in the late stages of the disease. There is currently no cure for PD; treatment aims to reduce the impact of the symptoms.

[0010] Drug delivery to the brain is the process of getting therapeutically active molecules across the blood-brain barrier to treat brain diseases. This is a complex process that must take into account the complex anatomy of the brain and the limitations imposed by the special connections of the blood-brain barrier.

[0011] The blood-brain barrier is formed by specialized tight junctions between endothelial cells that line the brain's blood vessels. The blood vessels of all tissues contain this single layer of endothelial cells, however, only brain endothelial cells have tight junctions that prevent most substances from passively diffusing into brain tissue.

[0012] The blood-brain barrier (BBB) ​​is a highly selective semi-permeable boundary of central nervous system (CNS) endothelial cells, which prevents solutes in circulating blood vessels from non-selectively crossing into the central nervous system where neurons reside. Therefore, the BBB is a barrier that hinders the accumulation of effective drugs in the brain. There are 4 approaches for BBB penetration (passive diffusion, carrier-mediated transport, receptor-mediated transcytosis, and adsorption-mediated transcytosis). Adsorption-mediated transcytosis (AMT) is the main approach. The AMT approach uses caveolae as a transport medium. Caveolae are a subgroup of lipid rafts present in BBB endothelial cells. Endocytosis mediated by caveolae is a key transport mechanism for macromolecules to be taken up from the bloodstream into the CNS.

[0013] For many therapeutic agents, only a small portion of the drug reaches the tissue to be affected, such as in chemotherapy, where about 99% of the administered drug does not reach the tumor site. Targeted drug delivery aims to concentrate the drug in the tissue of interest while reducing the relative concentration of the drug in the remaining tissues. For example, by circumventing the host's defense mechanisms and inhibiting nonspecific distribution in the liver and spleen, the system can reach the intended site of action at a higher concentration. It is believed that targeted delivery improves efficacy while reducing side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A micellar composition of the present invention is shown, wherein the drug molecule is encapsulated within the micelle, and the micelle comprises a ligand-PEG-EGCG conjugate in an outer shell and oligomeric EGCG (OEGCG) in an inner shell.

[0015] Figure 2 A micellar composition of the present invention is shown, wherein the drug molecules are encapsulated within the micelles, and the micelles contain a ligand-PEG-EGCG conjugate plus naked PEG-EGCG in the outer shell and oligomeric EGCG (OEGCG) in the inner shell.

[0016] Figure 3 Shown is a scheme for the chemical synthesis of TfR-PEG-EGCG via conjugation of the N-terminus of the TfR peptide to HOOC-PEG-EGCG.

[0017] Figure 4 Shown is a scheme for the chemical synthesis of Tetl-PEG-EGCG via conjugation of the N-terminus of the Tetl peptide to HOOC-PEG-EGCG.

[0018] Figure 5 Shown is a scheme for the chemical synthesis of TC13-PEG-EGCG via conjugation of the N-terminus of the TC13 peptide to HOOC-PEG-EGCG.

[0019] Figure 6 The successful formulation of TfR-MINC-doxorubicin (A), Tet1-MINC-doxorubicin (B), and TC13-MINC-doxorubicin (C) are shown.

[0020] Figure 7 The uptake of TfR-MINC-doxorubicin, Tet1-MINC-doxorubicin, and TC13-MINC-doxorubicin by brain endothelial cells was shown by measuring the fluorescence signal.

[0021] Figure 8 Shown is a scheme for the chemical synthesis of TfR-PEG-EGCG via conjugation of the C-terminus of the TfR peptide to HO-PEG-EGCG.

[0022] Fig. 9 Shown is a scheme for the chemical synthesis of Tet1-PEG-EGCG via conjugation of the C-terminus of the Tet1 peptide to HO-PEG-EGCG.

[0023] Fig.10 Shown is a scheme for the chemical synthesis of TCl3-PEG-EGCG via conjugation of the C-terminus of the TC13 peptide to HO-PEG-EGCG.

[0024] Fig.11 Shown is a scheme for the chemical synthesis of adenosine-PEG-EGCG via conjugation of the primary OH group of adenosine to HOOC-PEG-EGCG.

[0025] Fig.12 Shown is a scheme for the chemical synthesis of TfR-PLA-EGCG via conjugation of the N-terminus of the TfR peptide to HOOC-PLA-EGCG.

[0026] Fig.13 Shown is a scheme for the chemical synthesis of TfR-PLGA-EGCG via conjugation of the N-terminus of the TfR peptide to HOOC-PLGA-EGCG.

[0027] Fig.14 Shown is a scheme for the chemical synthesis of TfR-dextran-EGCG via conjugation of the C-terminus of the TfR peptide to HO-dextran-EGCG. DETAILED DESCRIPTION

[0028] definition

[0029] The term "about" is defined as ±10%, preferably ±5% of the stated value.

[0030] As used herein, the term "CNS targeting ligand" refers to a molecule with a molecular weight <10000 Daltons, for example 300 Daltons to 3500 Daltons, such as a peptide or a small molecule, which binds or targets a receptor on the surface of a CNS cell or in the CNS environment.

[0031] The term "cytokine" refers to proteins (about 5 kDa to 70 kDa) that are important in cell signaling. Cytokines have been shown to participate in autocrine, paracrine, and endocrine signaling as immunomodulators. Cytokines include interferons, interleukins, lymphokines, tumor necrosis factors, and chemokines.

[0032] The term "epigallocatechin gallate" refers to the ester of epigallocatechin and gallic acid, and is used interchangeably with "epigallocatechin-3-gallate" or EGCG.

[0033] The term "oligomeric EGCG" (OEGCG) refers to 2 to 50, 3 to 50, or 3 to 20 covalently linked EGCG monomers. OEGCG preferably contains 4 to 12 EGCG monomers.

[0034] The term "nanoparticle" refers to particles having a diameter below 1 μm and between 1 nm and 999 nm.

[0035] The term "polyethylene glycol-epigallocatechin gallate conjugate" or "PEG-EGCG" refers to polyethylene glycol (PEG) conjugated to one or two EGCG molecules. The term "PEG-EGCG" refers to both PEG-mEGCG conjugate (monomeric EGCG) and PEG-dEGCG (dimeric EGCG) conjugates.

[0036] The term "MINC" (Multi-Pathway Immunomodulatory Nanocomplex Combination Therapy) is a platform technology. As used in this application, MINC utilizes the biological activity of PEG-flavonoid conjugates and oligomeric EGCG (OEGCG). MINC can encapsulate additional CNS therapeutic agents to form a MINC-agent.

[0037] As used in this application, the term "MINC-agent" is a micelle having a shell formed by a CNS targeting ligand-PEG-flavonoid conjugate and optionally an oligomeric flavonoid (such as OEGCG), and having an agent encapsulated within the shell.

[0038] Unless otherwise specified, "%" used in the present application means weight %.

[0039] Flavonoids

[0040] The flavonoids suitable for use in the present invention have the general structure of Formula I:

[0041]

[0042] in:

[0043] R1 is H or phenyl;

[0044] R2 is H, OH, gallate or phenyl; wherein the phenyl is optionally substituted with one or more (e.g., 2 to 3) hydroxyl groups;

[0045] R3 is H, OH or =O (oxo); or

[0046] R1 and R2 together form a closed loop structure; or

[0047] R2 and R3 together form a closed loop structure.

[0048] The 2-, 3-, 4-, 5-, 6-, 7- or 8-position of Formula I may be bonded to a group containing hydrocarbon, halogen, oxygen, nitrogen, sulfur, phosphorus, boron or a metal.

[0049] Examples of flavonoids of Formula I include:

[0050]

[0051] Preferred flavonoid compounds of formula I include:

[0052] EGCG(CAS#989-51-5), EC(CAS#490-46-0), EGC(CAS#970-74-1) or ECG(CAS#1257-08-5)

[0053]

[0054] Conjugate

[0055] The present invention provides a conjugate comprising: (a) a CNS targeting ligand, (b) a hydrophilic polymer of polyethylene glycol (PEG), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA) or dextran, and (c) a flavonoid of formula I, wherein PEG is covalently bound to the flavonoid and the CNS targeting ligand.

[0056] The conjugate targets the CNS via a CNS targeting ligand and delivers the active ingredient to the CNS tissue for treating CNS pathological conditions, including but not limited to inflammation, neuronal cell damage, and accumulation of pathogenic aggregates in the CNS system.

[0057] The CNS targeting ligand is covalently linked to PEG, PLA, PLGA or dextran via its -COOH group or its -NH2 group by standard chemistry known to those skilled in the art. The molecular weight of the hydrophilic polymer in the conjugate is typically 1K to 100K, preferably 3K to 80K, and more preferably 5K to 40K.

[0058] The flavonoid in the conjugate has the general formula (I), and is preferably EGCG, EC, EGC or ECG. In one embodiment, the flavonoid is epigallocatechin gallate (EGCG).

[0059] In one embodiment, PEG contains an aldehyde group conjugated to position 5, 6, 7, or 8 (preferably position 6 or 8) of the A ring of the flavonoid compound.

[0060] In another embodiment, PEG contains a thiol group conjugated to R1 or R2 of the B ring of the flavonoid (when R1 or R2 is -OH).

[0061] In one embodiment, the conjugate comprises PEG-EGCG, which is PEG connected to one or two molecules of EGCG; it can be prepared by conjugating aldehyde-terminated PEG to EGCG through the reaction of free aldehyde groups with positions 5, 6, 7 or 8 (preferably 6 or 8) of formula I. See WO2006 / 124000 and WO2009 / 054813. PEG-EGCG can also be prepared by conjugating thio-terminated PEG to EGCG through the reaction of free sulfhydryl groups with R1 or R2 (wherein R1 or R2 is phenyl) of formula I. See WO2015 / 171079.

[0062] In another embodiment, the conjugate comprises PEG-EC, PEG-EGC or PEG-ECG, and the conjugate can be prepared by conjugating the aldehyde-terminated PEG to EC, EGC or ECG via the reaction of the free aldehyde group with position 5, 6, 7 or 8 (preferably position 6 or 8) of Formula I, through the attachment of PEG.

[0063] HOOC-PEG-CHO and HO-PEG-CHO are commonly used. In one embodiment, according to WO2006 / 124000 and WO2009 / 054813, HOOC-PEG-CHO is conjugated to EGCG, EC, EGC or ECG. HOOC-PEG-flavonoids have a COOH group to react with the N-terminal of the CNS targeting peptide. Typically, the CNS targeting peptide is incubated with HOOC-PEG-flavonoids, N, N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) in DMSO. The reactants are stirred at room temperature, protected from light, and under nitrogen. The reaction mixture is dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000Da). Then, the solution is freeze-dried to obtain a lyophilized powder. In order to avoid the self-reaction of the peptide, the C-terminal of the CNS targeting peptide can be protected by, for example, a resin during the reaction. Merrifield, hydroxymethyl polystyrene, PAM and MBHA resins are commonly used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions.

[0064] In another embodiment, according to WO2006 / 124000 and WO2009 / 054813, HO-PEG-CHO is conjugated to EGCG, EC, EGC or ECG. HO-PEG-flavonoids have an OH group to react with the C-terminus of the CNS targeting peptide. Typically, the peptide is incubated with HO-PEG-EGCG and N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reactants are stirred at room temperature, protected from light, and under nitrogen. The reaction mixture is dialyzed with methanol distilled water (membrane Mw cutoff value = 2000Da). Then, the solution is freeze-dried to obtain a lyophilized powder. In order to avoid the self-reaction of the peptide, the N-terminus of the CNS targeting peptide can be protected by, for example, a resin during the reaction. Merrifield, hydroxymethyl polystyrene, PAM and MBHA resins are commonly used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions. In this reaction, the COOH group on the peptide reacts selectively with the OH group on PEG because the primary OH group on PEG is more reactive than the tertiary OH group in the aromatic ring of the flavonoid.

[0065] HOOC-PLA-CHO, HOOC-PLGA-CHO, and HO-dextran-CHO are commercially available.

[0066] In one embodiment, according to WO2006 / 124000 and WO2009 / 054813, HOOC-PLA-CHO is conjugated to EGCG, EC, EGC or ECG. HOOC-PLA-flavonoids have a COOH group to react with the N-terminus of the CNS targeting peptide. Typically, the CNS targeting peptide is incubated with HOOC-PLA-flavonoids, N, N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) in DMSO. The reactants are stirred at room temperature, protected from light, and under nitrogen. The reaction mixture is dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000Da). Then, the solution is freeze-dried to obtain a lyophilized powder. In order to avoid the self-reaction of the peptide, the C-terminus of the CNS targeting peptide can be protected by, for example, a resin during the reaction. Merrifield, hydroxymethyl polystyrene, PAM and MBHA resins are commonly used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions.

[0067] In one embodiment, according to WO2006 / 124000 and WO2009 / 054813, HOOC-PLGA-CHO is conjugated to EGCG, EC, EGC or ECG. HOOC-PLGA-flavonoids have a COOH group to react with the N-terminus of the CNS targeting peptide. Typically, the CNS targeting peptide is incubated with HOOC-PLGA-flavonoids, N, N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) in DMSO. The reactants are stirred at room temperature, protected from light, and under nitrogen. The reaction mixture is dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000Da). Then, the solution is freeze-dried to obtain a lyophilized powder. In order to avoid the self-reaction of the peptide, the C-terminus of the CNS targeting peptide can be protected by, for example, a resin during the reaction. Merrifield, hydroxymethyl polystyrene, PAM and MBHA resins are commonly used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions.

[0068] In one embodiment, according to WO2006 / 124000 and WO2009 / 054813, HO-glucan-CHO is conjugated to EGCG, EC, EGC or ECG. HO-glucan-flavonoids have an OH group to react with the C-terminus of the CNS targeting peptide. Typically, the peptide is incubated with HO-glucan-EGCG and N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reactants are stirred at room temperature, protected from light, and under nitrogen. The reaction mixture is dialyzed with methanol distilled water (membrane Mw cutoff value = 2000Da). Then, the solution is freeze-dried to obtain a lyophilized powder. In order to avoid the self-reaction of the peptide, the N-terminus of the CNS targeting peptide can be protected by, for example, a resin during the reaction. Merrifield, hydroxymethyl polystyrene, PAM and MBHA resins are commonly used to prevent unwanted peptide conjugation. After the reaction, the resin can be removed under acidic conditions. In this reaction, the COOH group on the peptide reacts selectively with the OH in the CH2OH terminus of dextran because this is the only primary OH group in dextran, which is more reactive than other secondary OH in dextran and the tertiary OH in the aromatic ring of flavonoids.

[0069] The CNS targeting ligands in the present invention are ligands selected for targeting receptors on the surface of CNS cells (neuronal cells) or the CNS environment. For example, the CNS targeting ligands of the present invention target the following receptors on neuronal cells or the CNS environment:

[0070] (i) Receptors on neuronal cells: This group includes, but is not limited to, glutamatergic neuronal receptors, GABAergic neuronal receptors, dopaminergic neuronal receptors, serotonergic neuronal receptors, cholinergic neuronal receptors, nicotinic receptors, muscarinic receptors, dopamine receptors, adenosine receptors, glutamate receptors, GABA receptors, AMPA receptors, NMDA receptors, TrkB receptors, CB1 receptors, scavenger receptors, synaptophysin, PSD95, VGLUT1, VGLUT2, NMDAR1, NMDAR28, GAT1, DAT, SERT, Pet1, and VAChT.

[0071] (ii) Receptors present in the CNS environment: This group includes, but is not limited to, oligodendrocyte receptor, oligodendrocyte precursor cell receptor, intermediate progenitor cell receptor, neuroepithelial cell receptor, Schwann cell receptor, radial glial cell receptor, astrocyte receptor, microglia receptor, pericyte receptor, B cell receptor, T cell receptor, RAGE receptor, Fc receptor, toll-like receptor, TfR, IR, LDLR, Dhh, P75NTR, NCAM, E-cadherin, N-cadherin, PDGFRA, NG2, MOG, TN-C-glycoprotein, α(2-3)-sialoglycoprotein receptor, Notch, E-cadherin, S100, MBP, MPZ, EAAT1, TMEM119, CD11b, CD45, CX3CR1, F4 / 80, CD68 and CD40.

[0072] In one embodiment, the CNS targeting ligand is a TfR peptide having the amino acid sequence of THRPPMWSPVWP (SEQ ID NO: 1), which targets the transferrin receptor on neuronal cells or the CNS microenvironment.

[0073] In one embodiment, the CNS targeting ligand is a Tet1 peptide having an amino acid sequence of HLNILSTLWKYRC (SEQ ID NO: 2), which targets the GT1b receptor on neuronal cells or the CNS microenvironment.

[0074] In one embodiment, the CNS targeting ligand is a TC13 (TGN) peptide having an amino acid sequence of TGNYKALHPHNGC (SEQ ID NO: 3), which is targeted to neuronal cells or the CNS microenvironment.

[0075] In one embodiment, the CNS targeting ligand is apamin having an amino acid sequence of CNCKAPETALCARRCQQH (SEQ ID NO: 4), which targets apamin receptors on neuronal cells or the CNS microenvironment.

[0076] In one embodiment, the CNS targeting ligand is a Regulon polypeptide having the amino acid sequence of PTVIHGKREVTLHL (SEQ ID NO: 5), which targets the low density lipoprotein (LDL) receptor on neuronal cells or the CNS microenvironment.

[0077] In one embodiment, the CNS targeting ligand is a RAP peptide having the amino acid sequence of ELKHFEAKIEKHNHYQKQLE (SEQ ID NO: 6), which targets the LDL receptor on neuronal cells or the CNS microenvironment.

[0078] In one embodiment, the CNS targeting ligand is an Angiopep-2 peptide having the amino acid sequence of TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 7), which targets the LDL receptor on neuronal cells or the CNS microenvironment.

[0079] In one embodiment, the CNS targeting ligand is a TAT peptide having an amino acid sequence of GGGGYGRKKRRQRRR (SEQ ID NO: 8), which is targeted to neuronal cells or the CNS microenvironment.

[0080] In one embodiment, the CNS targeting ligand is a SynB1 peptide having the amino acid sequence of RGGRLSYSRRRFSTSTGR (SEQ ID NO: 9), which is targeted to neuronal cells or the CNS microenvironment.

[0081] In one embodiment, the CNS targeting ligand is a Leptin 30 peptide having an amino acid sequence of YQQVLTSLPSQNVLQIANDLENLRDLLHLLC (SEQ ID NO: 10), which targets a leptin receptor on a neuronal cell or the CNS microenvironment.

[0082] In one embodiment, the CNS targeting ligand is a LNP peptide having an amino acid sequence of KKRTLRKNDRKKRC (SEQ ID NO: 11), which targets caveolae-mediated endocytosis and macropinocytosis on neuronal cells or the CNS microenvironment.

[0083] In one embodiment, the CNS targeting ligand is an ApoB peptide having the amino acid sequence of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS (SEQ ID NO: 12), which targets the LRP2 receptor on neuronal cells or the CNS microenvironment.

[0084] In one embodiment, the CNS targeting ligand is a RVG-29 peptide having an amino acid sequence of YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 13), which targets nAChR receptors on neuronal cells or the CNS microenvironment.

[0085] In one embodiment, the CNS targeting ligand is a T7 peptide having the amino acid sequence of HAIYPRH (SEQ ID NO: 14), which targets the transferrin receptor on neuronal cells or the CNS microenvironment.

[0086] In one embodiment, the CNS targeting ligand is a GSH (glutathione) peptide having the amino acid sequence of ECG, which is targeted to neuronal cells or the CNS microenvironment.

[0087] In one embodiment, the CNS targeting ligand is a CRT peptide having the amino acid sequence of CRTIGPSVC (SEQ ID NO: 15), which targets the transferrin receptor on neuronal cells or the CNS microenvironment.

[0088] In one embodiment, the CNS targeting ligand is a CAQK peptide having the amino acid sequence of CAQK (SEQ ID NO: 16), which targets proteoglycan complexes on neuronal cells or the CNS microenvironment.

[0089] In one embodiment, the CNS targeting ligand is a TACL05 peptide having an amino acid sequence of SACPSHLTKMCGGG (SEQ ID NO: 17), which is targeted to neuronal cells or the CNS microenvironment.

[0090] In one embodiment, the CNS targeting ligand is adenosine, which targets the adenosine A1 receptor.

[0091] In one embodiment, the CNS targeting ligand is 5'-N-ethylcarboxamidoadenosine (NECA), which targets the adenosine A2A receptor.

[0092] In one embodiment, the CNS targeting ligand is glutamate, which targets a glutamate receptor.

[0093] In one embodiment, the CNS targeting ligand is gamma-aminobutyric acid (GABA), which targets the 5-HT4 receptor.

[0094] Nanoparticle composition

[0095] The term "MINC" (Multiple Pathway Immunomodulatory Nanocomplex Combination Therapy) is a platform technology. The present invention provides a nanoparticle micelle (MINC) composition having an outer shell comprising one or more CNS targeting conjugates of the present invention, an optional inner shell comprising one or more flavonoid oligomers, and optional drugs encapsulated within these shells.

[0096] In one embodiment, the micellar composition comprises both an outer shell and an inner shell as described above; the composition optionally has a drug encapsulated with these shells.

[0097] In one embodiment, the micellar composition comprises an outer shell as described above and has no inner shell; the composition optionally has a drug encapsulated with the shell.

[0098] In one embodiment, the micellar composition comprises a CNS targeting ligand-polymer-flavonoid conjugate in an outer shell and an oligomeric flavonoid in an inner shell, wherein the flavonoid in the outer shell and the flavonoid in the inner shell are independently EGCG, EC, EGC or ECG, and the polymer is PEG, PLA, PLGA or dextran. The preferred polymer is PEG. The preferred flavonoid is EGCG. Figure 1 Preferred micellar compositions are shown.CNS targeting ligands allow the nanoparticle compositions to be specifically targeted to CNS tissues.

[0099] In one embodiment, the micelle shell further comprises a naked PEG-flavonoid conjugate, such as PEG-EGCG, which has no CNS targeting ligand attached to the PEG-flavonoid. Figure 2 In such micellar shells, the ratio of ligand-PEG-EGCG to ligand-PEG-EGCG plus PEG-EGCG is typically greater than 10%, or greater than 20%, or greater than 30%, or greater than 50%, and up to 100%. In one embodiment, the ratio of ligand-PEG-EGCG to ligand-PEG-EGCG plus PEG-EGCG is 10%-90%, or 20%-80%, or 40%-60%.

[0100] The micelles optionally contain a CNS therapeutic molecule (drug or agent) encapsulated within the micelle (MINC-agent).

[0101] In one embodiment, the MINC-pharmaceutical composition comprises three active ingredients that are functionally complementary to address both immune responses and signaling pathways through its backbone components (PEG-flavonoids / OEGCG), as well as additional signaling pathways through selected drug molecules for the treatment of CNS diseases. Each nanoparticle is a fixed-dose combination drug with a fixed molar ratio of the three active ingredients.

[0102] The present invention delivers MINC-agents to targeted CNS tissues by actively delivering micelles to the brain with specific receptors via CNS targeting ligands.

[0103] The nanocomposite of the present invention contains the first two active ingredients, i.e., flavonoids such as (OEGCG) and PEG-flavonoids (such as PEG-EGCG), in the skeleton of the micellar composition. They are derivatives of EGCG, which is a strong immunomodulator and regulates a variety of disease signal transduction pathways. EGCG regulates both innate immunity and adaptive immunity. However, the bioavailability of EGCG is low and EGCG is unstable. The nanocomposite composition of the present invention overcomes the bioavailability problem of EGCG by forming a nanocarrier that carries EGCG to the target position for treatment, and overcomes the stability problem of EGCG by forming an OEGCG and PEG-EGCG complex, which effectively enables EGCG to become an efficient therapeutic agent.

[0104] The nanocomplex of the present invention optionally contains a third active ingredient, which is a drug molecule encapsulated in nanoparticles for the treatment of CNS diseases. In one embodiment, the CNS disease is Alzheimer's disease, and the drug is anti-CD3, anti-CD33, anti-CD36, anti-CD39, anti-CD73, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA4, anti-GZM-A, anti-GZM-B, anti-TAM, anti-FcγRI, anti-RAGE, anti-APOE, anti-CR1, anti-NLRP3, anti-β-amyloid protein, anti-tau, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, GDNF, NRTN, PDGF-BB, CDNF or BDNF.

[0105] In one embodiment, the CNS disease is Parkinson's disease and the drug is anti-CD3, anti-CD33, anti-CD36, anti-CD39, anti-CD73, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA4, anti-GZM-A, anti-GZM-B, anti-TAM, anti-FcγRI, anti-RAGE, anti-APOE, anti-CR1, anti-NLRP3, anti-α-synuclein, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, GDNF, NRTN, PDGF-BB, CDNF or BDNF.

[0106] In one embodiment, the CNS disease is dementia with Lewy bodies and the drug is anti-CD3, anti-CD33, anti-CD36, anti-CD39, anti-CD73, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA4, anti-GZM-A, anti-GZM-B, anti-TAM, anti-FcγRI, anti-RAGE, anti-APOE, anti-CR1, anti-NLRP3, anti-β-amyloid or anti-α-synuclein, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, GDNF, NRTN, PDGF-BB, CDNF, or BDNF.

[0107] In one embodiment, the CNS disease is a brain tumor and the drug is doxorubicin, disulfiram, celecoxib, temsirolimus, everolimus, vorinostat, cabozantinib, marizomib, fimepinostat, acetazolamide, metformin, vinblastine, cyclophosphamide, anti-HER2, anti-EGFR, anti-PD-1, anti-PD-L1, anti-PDGFRA, anti-VEGF, anti-VEGFR2, IL-2, IL-4, IL-12, IFN-α, IFN-β, IFN-γ, or TNF-α.

[0108] In one embodiment, the CNS disease is stroke and the drug is a MMP inhibitor, an eNOS inhibitor, anti-TLR4, anti-HSP, anti-IL6, anti-IL-12, S100β, fibronectin, MCP-1, MMP9, UCH-L1, BDNF, GDNF, NRTN, PDGF-BB, or CDNF.

[0109] In one embodiment, the CNS disease is Huntington's disease and the drug is anti-CD3, anti-mHtt, anti-α-synuclein, anti-SEMA4D, anti-TNFα, Tetrabenazine, deutetrabenazine, valbenazine, bevantolol, pridopidine, branaplam, nilotinib, mitoconix, or azathioprine.

[0110] In one embodiment, the CNS disease is multiple sclerosis and the drug is anti-CD3, anti-CD4, anti-IL-17, anti-CD19, anti-CD20, anti-CD25, anti-CD52, anti-RGMA, anti-IL-12, anti-IL-23, anti-α4 integrin, anti-IL-2R, LINGO-1, or anti-NOGO-A.

[0111] In one embodiment, the CNS disease is amyotrophic lateral sclerosis (ALS) and the drug is anti-NOGO-A, a PKC inhibitor, IGF-1, NOGO-A, GDNF, VEGF, anti-SOD1, S1R, GLT-1, anti-Ataxin2, anti-TDP43, anti-hnRNP, CK-1 inhibitor, anti-FET or HDAC inhibitor, EPO or IL-2.

[0112] In one embodiment, the CNS disease is acute spinal cord injury and the drug is Nogo receptor extracellular domain, 5-HT1A receptor, FGF, GSK-3bβ inhibitor, anti-IN-1, TNF-α, IL-12, SDF-1α, SOD1, NEC-1, anti-P-selectin or anti-CD11d.

[0113] In one embodiment, the CNS disease is encephalitis and the drug is anti-FcRn, anti-IL-6, anti-CD20, anti-CD19, anti-CD38, anti-C5, or IL-2.

[0114] In one embodiment, the CNS disease is epilepsy or seizures and the drug is an mTOR inhibitor, a P13K inhibitor, a GABA inhibitor, an anti-Glu3B peptide antibody, an anti-NR1 antibody, anti-CASPR2, or anti-LGI-1.

[0115] In one embodiment, the CNS disease is meningitis and the drug is a C1 inhibitor, anti-C5, anti-MASP-2, anti-PD-L1, anti-CTLA-4, or anti-PD-1.

[0116] In one embodiment, the CNS disease is motor neuron disease (MND) and the drug is anti-SOD1, anti-TDP-43, anti-C9ORF72, anti-Nogo-A, anti-MuSK, anti-IL-6R, anti-NRP-1, anti-myostatin, anti-CD40L, anti-DR-6, anti-IFN-g, anti-GD1a, anti-CTGF, or anti-HMGB1.

[0117] Nanoparticles are stable in hydrophilic environments, such as blood circulation, but dissociate in hydrophobic environments, such as CNS tissues.

[0118] Methods for preparing nanoparticle compositions

[0119] The nanoparticle composition of the present invention can be prepared by a method comprising the following steps: (a) mixing the drug molecule with a flavonoid oligomer (e.g., OEGCG) and a CNS targeting ligand conjugate of the present invention in an aqueous solution; and (b) filtering the mixture through a membrane with a molecular weight cutoff of 8,000 Daltons to 300,000 Daltons to remove small molecular weight molecules and retain large molecular weight molecules.

[0120] In a preferred embodiment, the method further comprises step (c), filtering the large molecular weight molecules through a 0.2 μm to 0.3 μm membrane and collecting the filtrate.

[0121] In step (a), the drug molecule is dissolved in an aqueous solvent such as phosphate buffered saline, saline, water, bicarbonate buffer, oxygenated hemoglobin buffer, bis-tris paraffin, Tris-HCl, HEPES, histidine buffer, NP-40, RIPA (radioimmunoprecipitation assay 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 generally 0.01 mg / ml to 50 mg / ml, preferably 0.05 mg / ml to 10 mg / ml, more preferably 0.1 mg / ml to 5 mg / ml.

[0122] The flavonoid oligomer and CNS targeting ligand conjugate are dissolved in ketones, acetonitrile, alcohols, aldehydes, ethers, acetates, sulfoxides, benzene, organic acids, amides, aqueous buffers, and any combination thereof. Preferred solvents are alcohols, acetonitrile, sulfoxides, amides, and any combination thereof. For example, the concentrations of OEGCG / EGCG and PEG-EGCG are typically independently 0.001 mg / ml to 10 mg / ml, preferably 0.005 mg / ml to 1 mg / ml or 0.1 mg / ml to 5 mg / ml.

[0123] It is important that OEGCG is in molar excess relative to the drug agent. Generally speaking, the molar ratio of EGCG to drug molecules in OEGCG is between 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 by the number of moles of monomeric EGCG in OEGCG to the number of moles of drug molecules. The molar excess of EGCG ensures that most or all of the drug agent is encapsulated by the OEGCG molecules. In the method of the present invention, unencapsulated drug agents are avoided by controlling the molar ratio of OEGCG to protein, which will not be selectively distributed to the target tissue and will lead to lower efficacy and safety issues.

[0124] In step (b), the mixture is filtered through a membrane having a molecular weight cut-off 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 small molecular weight molecules and retain large molecular weight molecules. The ultrafiltration membrane material is selected from the group consisting of: cellulose (and derivatives thereof), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, polyvinylidene fluoride or polyvinylidene fluoride (PVDF) and polypropylene (PP); preferably cellulose (and derivatives thereof), PTFE and PVDF.

[0125] Prior to ultrafiltration, the mixture is optionally diluted in an aqueous solvent, such as those described above in step (a).

[0126] Ultrafiltration step (b) removes unwanted small molecular weight impurities, such as unreacted OEGCG or EGCG, or reaction byproducts. These impurities can reduce drug efficacy and safety. Excessive unreacted OEGCG or EGCG can also cause single nanoparticles to aggregate into particles of about 1000nm size, which will reduce efficacy and cause potential toxicity.

[0127] In step (c), the retained large molecular weight molecules are filtered through a membrane having a pore size of about 0.2 μm to 0.3 μm (such as 0.22 μm), and the filtrate is collected. This is to remove unwanted large molecular impurities, such as giant aggregates. These aggregates can be discharged from the tissues into which they enter due to their huge size. These aggregates reduce the overall efficacy / safety and have a higher chance of inducing immunogenicity in patients. Large size nanoparticles are also more likely to be taken up by the RES in the liver, lungs, and more undesirable organs.

[0128] The membrane material of step (c) is selected from the group consisting of cellulose (and its derivatives), PES, PTFE, nylon, PVDF and PP; preferably cellulose (and its derivatives), PES and PP.

[0129] In one embodiment, steps (b) and (c) are repeated at least once, for example 1, 2, 3 or 4 times, before step (d) to effectively remove unwanted small molecule impurities and large aggregates.

[0130] After step (c), the filtrate is stored at 2°C to 8°C and is stable for at least 100 days.

[0131] The method of the present invention optionally further comprises a freeze-drying step (d) after step (c) to provide long-term stability of the nanoparticle composition.

[0132] Pharmaceutical composition

[0133] The present invention provides a pharmaceutical composition comprising a nanoparticle composition of the present invention and optionally one or more pharmaceutically acceptable carriers. For tablets, powders or parenteral preparations, the nanoparticle composition in the pharmaceutical composition is generally about 1% to 90%, preferably 20% to 90% or 30% to 80%. For capsule preparations, the nanoparticle composition in the pharmaceutical composition is generally 1% to 100%, preferably 20% to 100%, 50% to 100% or 70% to 100%. For liquid suspension preparations, the nanoparticle composition in the pharmaceutical composition is generally 1% to 50%, 5% to 50% or 10% to 40%.

[0134] In one embodiment, the pharmaceutical composition may be in the form of tablets, capsules, granules, fine granules, powders, suspensions, patches, parenteral preparations, injectable preparations, etc. The pharmaceutical composition may be prepared by conventional methods.

[0135] Pharmaceutically acceptable carriers are inactive ingredients that can be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers can contain a variety of ingredients, including but not limited to 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 salts of hydroxides, phosphates, citrates, acetates, borates and triethanolamine; antioxidants such as salts, acids and / or bases of bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol and ascorbyl palmitate; surfactants such as lecithin and phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine and phosphatidylcholine. alcohols; poloxamers and poloxamines; polysorbates such as polysorbate 80, polysorbate 60 and polysorbate 20; polyethers such as polyethylene glycol and polypropylene glycol; polyethylenes such as polyvinyl alcohol and polyvinyl pyrrolidone (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, soybean oil; mono-, di- and triglycerides; polysaccharides such as dextran; and glycosaminoglycans such as sodium hyaluronate. Such pharmaceutically acceptable carriers may be preserved to prevent bacterial contamination with 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 non-preserved preparations for single or multiple use.

[0136] For example, the tablet, capsule or parenteral preparation of the active compound may contain other excipients that are not biologically active and do not react with the active compound. The excipients of tablets or capsules may include fillers, binders, lubricants and glidants, disintegrants, wetting agents and release rate regulators. Examples of excipients of tablets or capsules include but are not limited to carboxymethyl cellulose, cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, karaya gum, starch, tragacanth gum, gelatin, magnesium stearate, titanium dioxide, poly (acrylic acid) and polyvinyl pyrrolidone.

[0137] 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. Powder oral formulations may contain inactive ingredients such as silica gel, sodium benzoate, sodium citrate, sucrose, and xanthan gum.

[0138] Treatment

[0139] The present invention relates to a method for treating CNS diseases, comprising the step of administering an effective amount of the nanoparticle composition of the present invention to a subject in need thereof. CNS diseases suitable for treatment by the present invention include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, migraine, multiple sclerosis, autism, cerebral palsy, epilepsy (seizures), amyotrophic lateral sclerosis, spinal cord injury.

[0140] As used in this application, an "effective amount" is an amount effective for treating a disease by ameliorating the pathological condition or reducing the symptoms of the disease.

[0141] The ligand-polymer-flavonoid (eg, ligand-polymer-EGGC) for injection is typically administered at 0.01 mg / kg to 100 mg / kg (total weight of polymer-flavonoid / body weight of subject) or 0.001 mg / kg to 1000 mg / kg.

[0142] In one embodiment, the method comprises the step of administering to a subject in need thereof an effective amount of a MINC having a shell formed from one or more ligand-hydrophilic polymer-flavonoid conjugates (and optionally together with a naked polymer-flavonoid conjugate), with or without a flavonoid oligomer, optionally with a pharmaceutical agent encapsulated within the shell.

[0143] In one embodiment, the shell is formed from Ligand-PEG-EGCG, optionally together with PEG-EGCG.

[0144] In one embodiment, the shell is formed from the ligands - PEG-EGCG and OEGCG, and optionally PEG-EGCG.

[0145] The ligand-polymer-flavonoid conjugates of the present invention can pass through the blood-brain barrier (BBB) ​​from the circulatory blood vessels to reach the brain. The ligand-polymer-flavonoid conjugates of the present invention can target CNS lesions after BBB penetration. The ligand-polymer-flavonoid conjugates of the present invention additionally have neuronal cell repair or regeneration activity, and have immune and disease regulatory functions for the treatment of CNS disorders.

[0146] The flavonoid oligomers used in the present method are able to cross the BBB from the circulatory blood vessels to the brain and have immune and disease regulatory functions for treating CNS disorders. The flavonoid oligomers of the present invention additionally have neuronal cell repair or regeneration activity for treating CNS disorders.

[0147] The dosing of the MINC-agent is based on the known doses of the agent used to treat the specific disease and the condition of the subject. The dose can be a dose approved by the Food and Drug Administration (FDA) or a dose used in a clinical trial.

[0148] In the MINC-agent, the total weight of the ligand-PEG-EGCG and PEG-EGCG (if present) is close to the encapsulated pharmaceutical agent. The weight of OEGCG (if present) is variable. Typically, the dosage of the ligand-PEG-EGCG and PEG-EGCG (if present) combined with OEGCG is between 0.01 mg / kg and 1000 mg / kg.

[0149] The concentration of encapsulated drug agent can be as low as 0.01 mg / kg (eg, for a cytokine drug, rhBDNF) and as high as 100 mg / kg (for an antibody drug, eg, anti-alpha synuclein antibody is at this level).

[0150] For the treatment of Alzheimer's disease, anti-beta amyloid (Aducanumab, Solanezumab, Crenezumab, Gantenerumab, Donanemab, or Lecanemab) is administered IV at 0.01 mg / kg to 100 mg / kg or 0.01 mg / kg to 1000 mg / kg every one to four weeks. An effective dose of MINC-anti-beta amyloid in the same dosage range can be used to treat Alzheimer's disease.

[0151] For the treatment of Parkinson's disease, anti-alpha synuclein (Prasinezumab or Cinpanemab) is given IV at 0.01 mg / kg to 100 mg / kg or 0.01 mg / kg to 1000 mg / kg every one to four weeks. An effective dose of MINC-anti-alpha-synuclein within the same dosage range can be used to treat Parkinson's disease.

[0152] For treatment of Huntington's disease, anti-mHtt (C6 / 17) is administered IV at 0.01 mg / kg to 100 mg / kg or 0.01 mg / kg to 1000 mg / kg every one to four weeks. Effective doses of MINC-anti-mHtt within the same dose range can be used to treat Huntington's disease.

[0153] For the treatment of migraine, anti-CGRP (Eptinezumab, Fremanezumab, Galcanezumab, Erenumab) is given IV at 0.01 mg / kg to 100 mg / kg or 0.01 mg / kg to 1000 mg / kg every one to four weeks. Effective doses of MINC-anti-CGRP in the same dose range can be used to treat migraine.

[0154] For the treatment of multiple sclerosis, anti-CD20 (Ocrelizumab, Rituximab, Ofatumumab, or Ublituximab) is given IV at 0.01 mg / kg to 100 mg / kg or 0.01 mg / kg to 1000 mg / kg one to four times per year. Effective doses of MINC-anti-CD20 within the same dose range can be used to treat multiple sclerosis.

[0155] The present invention can be used to treat humans and non-human animals. For example, the present invention can be used to treat mammalian subjects, such as humans, horses, pigs, cats and dogs.

[0156] The following examples further illustrate the present invention. These examples are intended only to illustrate the present invention and should not be construed as limiting.

[0157] Example

[0158] Example 1: Conjugation of TfR peptide with HOOC-PEG-EGCG

[0159] Material

[0160] TfR peptide was purchased from Hangzhou Xinbosi Biomedical Co., Ltd.

[0161] HOOC-PEG-CHO was purchased from NBC chemical.

[0162] method

[0163] According to WO2006 / 124000 and WO2009 / 054813, HOOC-PEG-CHO was conjugated to EGCG; TfR peptide was conjugated to HOOC-PEG-EGCG via conjugation between the COOH group on PEG and the NH2 group on TfR to form TfR-PEG-EGCG (N′ linked) ( Figure 3 ).

[0164] Specifically, by incubating with 1mg to 1000mg HOOC-PEG-EGCG, 1mg to 1000mg N, N'-dicyclohexylcarbodiimide (DCC) and 1mg to 1000mg N-hydroxysuccinimide (NHS) in DMSO, 1mg to 1000mg TfR is PEGylated. The reaction mixture is stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture is dialyzed with methanol and distilled water (membrane molecular weight cutoff = 2000Da) for 3 days. Then, the solution is freeze-dried to obtain a lyophilized powder.

[0165] result

[0166] HPLC was used to confirm the preparation of TfR-PEG-EGCG. HPLC was performed under the following conditions: column: C18, 4.6 mm×150 mm, 4 μm; eluent: A=0.1% TFA / H2O, B=0.1% TFA / ACN; oven temperature: 40°C; flow rate: 1 ml / min; autosampler temperature: 15°C; measurement: UV280.

[0167] The retention time of HOOC-PEG-EGCG was 6.04 min, and after TfR conjugation, there was a new peak with a retention time of 6.46 min. The HPLC results indicated the successful conjugation of TfR-PEG-EGCG.

[0168] Example 2: Conjugation of Tet1 peptide with HOOC-PEG-EGCG

[0169] Material

[0170] Tet1 peptide was purchased from Hangzhou Xinbaisi Biopharmaceutical Co., Ltd.

[0171] HOOC-PEG-CHO was purchased from NBC Chemicals

[0172] method

[0173] According to WO2006 / 124000 and WO2009 / 054813, HOOC-PEG-CHO was conjugated to EGCG; Tet1 peptide was conjugated to HOOC-PEG-EGCG via conjugation between the COOH group on PEG and the NH2 group on Tet1 to form Tet1-PEG-EGCG (N′-linked) ( Figure 4 ).

[0174] Specifically, 1 mg to 1000 mg of Tet1 was PEGylated by incubation with 1 mg to 1000 mg of HOOC-PEG-EGCG, 1 mg to 1000 mg of N, N′-dicyclohexylcarbodiimide (DCC) and 1 mg to 1000 mg of N-hydroxysuccinimide (NHS) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane molecular weight cutoff = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0175] result

[0176] HPLC was used to confirm the preparation of Tet1-PEG-EGCG. HPLC was performed under the following conditions: column: C18, 4.6 mm×150 mm, 4 μm; eluent: A=0.1% TFA / H2O, B=0.1% TFA / ACN; oven temperature: 40°C; flow rate: 1 ml / min; autosampler temperature: 15°C; measurement: UV280.

[0177] The retention time of HOOC-PEG-EGCG was 6.04 min, and after Tet1 conjugation, a new peak with a retention time of 7.54 min was present. The HPLC results indicated the successful conjugation of Tet1-PEG-EGCG.

[0178] Example 3: Conjugation of TC13 peptide with HOOC-PEG-EGCG

[0179] Material

[0180] TC13 peptide was purchased from Hangzhou Xinbaisi Biopharmaceutical Co., Ltd.

[0181] HOOC-PEG-CHO was purchased from NBC Chemicals

[0182] method

[0183] According to WO2006 / 124000 and WO2009 / 054813, HOOC-PEG-CHO was conjugated to EGCG; TC13 peptide was conjugated to HOOC-PEG-EGCG via conjugation between the COOH group on PEG and the NH2 group on TC13 to form TC13-PEG-EGCG (N′ linked) ( Figure 5 ).

[0184] Specifically, 1mg to 1000mg TC13 is PEGylated by incubating with 1mg to 1000mg HOOC-PEG-EGCG, 1mg to 1000mg N, N'-dicyclohexylcarbodiimide (DCC) and 1mg to 1000mg N-hydroxysuccinimide (NHS) in DMSO. The reaction mixture is stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture is dialyzed with methanol and distilled water (membrane molecular weight cutoff value = 2000Da) for 3 days. Then, the solution is freeze-dried to obtain a lyophilized powder.

[0185] result

[0186] HPLC was used to confirm the preparation of TC13-PEG-EGCG. HPLC was performed under the following conditions: column: C18, 4.6 mm×150 mm, 4 μm; eluent: A=0.1% TFA / H2O, B=0.1% TFA / ACN; oven temperature: 40°C; flow rate: 1 ml / min; autosampler temperature: 15°C; measurement: UV280.

[0187] The retention time of HOOC-PEG-EGCG was 6.04 min, and after TC13 conjugation, a new peak with a retention time of 6.54 min was present. The HPLC results indicated the successful conjugation of TC13-PEG-EGCG.

[0188] Example 4: Preparation of TfR-MINC-doxorubicin, Tet1-MINC-doxorubicin and TC13-MINC-doxorubicin

[0189] Material

[0190] TffR-PEG-EGCG was prepared according to Example 1.

[0191] Tet1-PEG-EGCG was prepared according to Example 2.

[0192] TC13-PEG-EGCG was prepared according to Example 3.

[0193] Doxorubicin was purchased from Sigma-Aldrich.

[0194] method

[0195] TfR-MINC-doxorubicin nanoparticles, Tet1-MINC-doxorubicin nanoparticles, and TC13-MINC-doxorubicin nanoparticles were prepared according to the following protocol:

[0196] 1. Incubate 5 μg to 500 μg of doxorubicin in 1 mL of DMSO for 15 minutes to 1 hour.

[0197] 2. Add 1 μg to 100 μg of OEGCG and 1 μg to 10,000 μg of TfR-PEG-EGCG, Tet1-PEG-EGCG or TC13-PEG-EGCG. Incubate the mixture at 25°C for 3 hours.

[0198] 3. Filter out the liquid using a 10KMWCO filter unit. Wash the filter 3 times with 0.9% NaCl.

[0199] 4. Freeze-dried into dry powder.

[0200] result

[0201] Nanoparticle size was measured by DLS (Anton Paar, Litesizer 500). Figure 6 The successful formulation of TfR-MINC-doxorubicin (A), Tetl-MINC-doxorubicin (B), and TC13-MINC-doxorubicin (C) are shown.

[0202] Example 5: Different ligands for drug delivery into brain endothelial cells - MINC-doxorubicin

[0203] Material

[0204] TfR-MINC-doxorubicin, Tet1-MINC-doxorubicin, TC13-MINC-doxorubicin or MINC-doxorubicin were formulated according to Example 4.

[0205] method

[0206] The brain endothelial cell line bEnd.3 was seeded in 96-well plates at 8×103 cells / well and incubated overnight. On the second day, the cells were treated with 2.5 μM of MINC-doxorubicin, TfR-MINC-doxorubicin, Tet1-MINC-doxorubicin or TC13-MINC-doxorubicin for 2 hours (n=2). After treatment, the fluorescence intensity was measured by a Molecular Devices Gemini XPS fluorescence microplate reader to assess the delivery efficiency. The data are shown as mean ± SD and statistically analyzed by GraphPad Prism7. Statistical significance was calculated by one-way ANOVA, and the difference was considered significant when *: p<0.05, **: p<0.01; ***: p<0.001.

[0207] result

[0208] Doxorubicin is a red fluorescent compound, and its delivery into cells was visualized using fluorescence microscopy. Higher fluorescence intensity means more doxorubicin was delivered into the cells. Figure 7 In the , the fluorescence signals of TfR-MINC-doxorubicin and Tetl-MINC-doxorubicin were significantly stronger in bEnd.3 cells compared with MINC-doxorubicin. We also observed a trend of higher fluorescence signals in TC13-MINC-doxorubicin treated cells. These results demonstrate that CNS targeting peptides increase specific drug delivery to brain endothelial cells.

[0209] Example 6: Conjugation of TfR peptide with HO-PEG-EGCG ( Prophetic Embodiment)

[0210] Purpose

[0211] This experiment was designed to demonstrate the conjugation of TfR peptide to HO-PEG-EGCG. HPLC was used to detect the formation of a new product (TfR-PEG-EGCG) with a different retention time than HO-PEG-EGCG. NMR was used to confirm the structure.

[0212] Material

[0213] TfR peptide was purchased from Hangzhou Xinbaisi Biopharmaceutical Co., Ltd.

[0214] HO-PEG-CHO was purchased from Huanteng Pharma

[0215] method

[0216] According to WO2006 / 124000 and WO2009 / 054813, HO-PEG-CHO was conjugated to EGCG to prepare HO-PEG-EGCG.

[0217] The TfR peptide was conjugated to HO-PEG-EGCG via conjugation between the OH group on PEG and the COOH group on TfR to form TfR-PEG-EGCG (C' linked). Figure 8 .

[0218] Specifically, 1 mg to 1000 mg TfR was PEGylated by incubation with 1 mg to 1000 mg HO-PEG-EGCG and 1 mg to 1000 mg N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0219] Example 7: Conjugation of Tet1 peptide with HO-PEG-EGCG (prophetic example)

[0220] Purpose

[0221] This experiment aims to demonstrate the conjugation of Tet1 peptide to HO-PEG-EGCG. HPLC is used to detect the formation of a new product (Tet1-PEG-EGCG) with a different retention time from HO-PEG-EGCG. NMR can be used to confirm the structure.

[0222] Material

[0223] Tet1 peptide was purchased from Hangzhou Xinbaisi Biopharmaceutical Co., Ltd.

[0224] HO-PEG-CHO was purchased from Huanteng Pharmaceuticals

[0225] method

[0226] According to WO2006 / 124000 and WO2009 / 054813, HO-PEG-CHO was conjugated to EGCG to prepare HO-PEG-EGCG.

[0227] Tetl peptide was conjugated to HO-PEG-EGCG via conjugation between the OH group on PEG and the COOH group on Tetl to form Tetl-PEG-EGCG (C' linked). Fig. 9 .

[0228] Specifically, 1 mg to 1000 mg of Tet1 was PEGylated by incubation with 1 mg to 1000 mg of HO-PEG-EGCG and 1 mg to 1000 mg of N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0229] Example 8: Conjugation of TC13 peptide with HO-PEG-EGCG (Pre Exemplary Embodiment)

[0230] Purpose

[0231] This experiment was designed to demonstrate the conjugation of TC13 peptide to HO-PEG-EGCG. HPLC was used to detect the formation of a new product (TC13-PEG-EGCG) with a different retention time than HO-PEG-EGCG. NMR was used to confirm the structure.

[0232] Material

[0233] TC13 peptide was purchased from Hangzhou Xinbaisi Biopharmaceutical Co., Ltd.

[0234] HO-PEG-CHO was purchased from Huanteng Pharmaceuticals

[0235] method

[0236] According to WO2006 / 124000 and WO2009 / 054813, HO-PEG-CHO was conjugated to EGCG to prepare HO-PEG-EGCG.

[0237] The TC13 peptide was conjugated to HO-PEG-EGCG via conjugation between the OH group on PEG and the COOH group on TC13 to form TC13-PEG-EGCG (C′ linked). Fig.10 .

[0238] Specifically, 1 mg to 1000 mg of TC13 was PEGylated by incubation with 1 mg to 1000 mg of HO-PEG-EGCG and 1 mg to 1000 mg of N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0239] Example 9: Conjugation of adenosine to HOOC-PEG-EGCG (Prophetic Example)

[0240] Purpose

[0241] This experiment was designed to demonstrate the conjugation of adenosine to HOOC-PEG-EGCG. HPLC was used to detect the formation of a new product (adenosine-PEG-EGCG) with a different retention time than HO-PEG-EGCG. NMR was used to confirm the structure.

[0242] Material

[0243] Adenosine was purchased from Sigma-Aldrich

[0244] HOOC-PEG-CHO was purchased from NBC Chemicals

[0245] method

[0246] According to WO2006 / 124000 and WO2009 / 054813, HOOC-PEG-CHO was conjugated to EGCG.

[0247] Adenosine was conjugated to HOOC-PEG-EGCG via conjugation between the COOH group on PEG and the COOH group on adenosine to form adenosine-PEG-EGCG. Fig.11 .

[0248] Specifically, 1 mg to 1000 mg of adenosine was PEGylated by incubation with 1 mg to 1000 mg of HOOC-PEG-EGCG and 1 mg to 1000 mg of N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0249] Example 10: Conjugation of TfR peptide to HOOC-PLA-EGCG (prophetic example)

[0250] Purpose

[0251] This experiment was designed to demonstrate the conjugation of TfR peptide to HOOC-PLA-EGCG. HPLC was used to detect the formation of a new product (TfR-PLA-EGCG) with a different retention time than HOOC-PLA-EGCG. NMR was used to confirm the structure.

[0252] Material

[0253] TfR peptide was purchased from Hangzhou Xinbaisi Biopharmaceutical Co., Ltd.

[0254] HOOC-PLA-CHO was purchased from Merck (Sigma-Aldrich).

[0255] method

[0256] According to WO2006 / 124000 and WO2009 / 054813, HOOC-PLA-CHO was conjugated to EGCG; TfR peptide was conjugated to HOOC-PLA-EGCG via conjugation between the COOH group on PLA and the NH2 group on TfR to form TfR-PLA-EGCG (N′ linked). Fig.12 .

[0257] Specifically, 1 mg to 1000 mg of TfR was incubated with 1 mg to 1000 mg of HOOC-PLA-EGCG, 1 mg to 1000 mg of N, N'-dicyclohexylcarbodiimide (DCC) and 1 mg to 1000 mg of N-hydroxysuccinimide (NHS) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane molecular weight cutoff = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0258] Example 11: Conjugation of TfR peptide to HOOC-PLGA-EGCG (prophetic example)

[0259] Purpose

[0260] This experiment was designed to demonstrate the conjugation of TfR peptide to HOOC-PLGA-EGCG. HPLC was used to detect the formation of a new product (TfR-PLGA-EGCG) with a different retention time from HOOC-PLGA-EGCG. NMR was used to confirm the structure.

[0261] Material

[0262] TfR peptide was purchased from Hangzhou Xinbaisi Biopharmaceutical Co., Ltd.

[0263] HOOC-PLGA-CHO was purchased from Merck (Sigma-Aldrich).

[0264] method

[0265] According to WO2006 / 124000 and WO2009 / 054813, HOOC-PLGA-CHO was conjugated to EGCG; TfR peptide was conjugated to HOOC-PLGA-EGCG via conjugation between COOH groups on PLGA and NH2 groups on TfR to form TfR-PLGA-EGCG (N′ linked). Fig.13.

[0266] Specifically, 1 mg to 1000 mg of TfR was incubated with 1 mg to 1000 mg of HOOC-PLGA-EGCG, 1 mg to 1000 mg of N, N'-dicyclohexylcarbodiimide (DCC) and 1 mg to 1000 mg of N-hydroxysuccinimide (NHS) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light and nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane molecular weight cutoff = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0267] Example 12: Conjugation of TfR peptide with HO-dextran-EGCG (prophetic example)

[0268] Purpose

[0269] This experiment was designed to demonstrate the conjugation of TfR to HO-Glucan-EGCG. HPLC was used to detect the formation of a new product (TfR-Glucan-EGCG) with a different retention time from HO-Glucan-EGCG. NMR was used to confirm the structure.

[0270] Material

[0271] TfR peptide was purchased from Hangzhou Xinbaisi Biopharmaceutical Co., Ltd.

[0272] HO-dextran-CHO was purchased from Merck (Sigma-Aldrich).

[0273] method

[0274] According to WO2006 / 124000 and WO2009 / 054813, HO-dextran-CHO was conjugated to EGCG to prepare HO-dextran-EGCG.

[0275] TfR was conjugated to HO-dextran-EGCG via conjugation between the OH group on dextran and the COOH group on TfR to form TfR-dextran-EGCG (C′ linked). Fig.14 .

[0276] Specifically, 1 mg to 1000 mg of TfR was incubated with 1 mg to 1000 mg of HO-glucan-EGCG and 1 mg to 1000 mg of N, N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction mixture was stirred for 24 hours at room temperature, protected from light, and under nitrogen. The reaction mixture was dialyzed with methanol and distilled water (membrane Mw cutoff value = 2000 Da) for 3 days. Then, the solution was freeze-dried to obtain a lyophilized powder.

[0277] Example 13: Formulation of Adenosine-MINC-Doxorubicin (Prophetic Example)

[0278] Purpose

[0279] This experiment was designed to demonstrate the formulation of adenosine-MINC-doxorubicin. DLS was used to measure the size of the nanoparticles.

[0280] Material

[0281] Adenosine-PEG-EGCG was formulated according to Example 9.

[0282] Doxorubicin was purchased from Sigma-Aldrich or other suppliers.

[0283] method

[0284] Adenosine-MINC-doxorubicin nanoparticles were prepared according to the following protocol:

[0285] 1. Incubate 5 μg to 500 μg of doxorubicin in 1 mL of DMSO for 15 minutes to 1 hour.

[0286] 2. Add 1 μg to 100 μg OEGCG and 1 μg to 10,000 μg adenosine-PEG-EGCG. Incubate the mixture at 25°C for 3 hours.

[0287] 3. Filter out the liquid using a 10KMWCO filter unit. Wash the filter 3 times with 0.9% NaCl.

[0288] 4. Freeze-dried into dry powder.

[0289] Example 14: TfR-MINC-anti-CD3, Tetl-MINC-anti-CD3, TC13-MINC-anti-CD3 and adenosine-MINC-anti Efficacy study of CD3 in a mouse model of Alzheimer's disease (prophetic example).

[0290] Purpose

[0291] This experiment aims to demonstrate the therapeutic efficacy of TfR, Tet1, TC13 peptide and adenosine conjugation. Histological anti-Aβ staining is used to detect brain Aβ content. Behavioral studies are used to evaluate spatial working memory and exploratory activities.

[0292] Material

[0293] TfR-MINC-anti-CD3 nanoparticles, Tetl-MINC-anti-CD3 nanoparticles, TCl3-MINC-anti-CD3 nanoparticles, adenosine-MINC-anti-CD3 and MINC-anti-CD3 nanoparticles were prepared using the method taught in Example 4 by replacing doxorubicin with anti-CD3.

[0294] Anti-CD3 antibody was purchased from Biolegend.

[0295] method

[0296] APPsw (strain 2576) mice, APP / PS1 mice or Wistar rats were used. Mice or rats were treated once a week with vehicle (PBS or saline as untreated control), 5 μg to 125 μg of anti-CD3 / mouse MINC-anti-CD3, TfR-MINC-anti-CD3 nanoparticles, Tet1-MINC-anti-CD3 nanoparticles, TC13-MINC-anti-CD3 or adenosine-MINC-anti-CD3 via iv injection for 4 to 8 weeks. These mice or rats were sacrificed at 6 to 24 months of age to analyze Aβ levels and Aβ load in the brain. Quantitative Aβ image analysis was performed using anti-β-amyloid protein (clone 4G8).

[0297] To measure spatial working memory and exploratory activity, animals were placed individually in one arm of a radially symmetrical Y-maze made of opaque grey acrylic or other appropriate maze for behavioral testing. RAWM test or appropriate test for behavioral assessment in mice or rats was performed.

[0298] Example 15: TfR-MINC-trastuzumab, Tet1-MINC-trastuzumab, TC13-MINC-trastuzumab Efficacy study of anti- and adenosine-MINC-trastuzumab in a glioma mouse model (prophetic example).

[0299] Purpose

[0300] This experiment aims to demonstrate the therapeutic efficacy of TfR, Tet1, TC13 peptide and adenosine conjugation in the treatment of glioma. In vivo imaging system (IVIS) was used to measure tumor volume.

[0301] Material

[0302] TfR-MINC-trastuzumab nanoparticles, Tet1-MINC-trastuzumab nanoparticles, TC13-MINC-trastuzumab nanoparticles, adenosine-MINC-trastuzumab and MINC-trastuzumab nanoparticles were prepared using the method taught in Example 4.

[0303] Trastuzumab was purchased from EirGenix.

[0304] A172 glioma cell line was from ATCC (CRL1620).

[0305] D-Luciferin was purchased from Sigma-Aldrich.

[0306] method

[0307] The efficacy of TfR-MINC-trastuzumab nanoparticles, Tetl-MINC-trastuzumab nanoparticles, TC13-MINC-trastuzumab nanoparticles, adenosine-MINC-trastuzumab, and MINC-trastuzumab nanoparticles in treating glioma was evaluated using an orthotopic glioma mouse model. To image tumor size, the A172 cell line was engineered with the luciferase gene (A172-Luc). To generate the mouse model, a skull burr hole was created in the right forebrain region and 1×10 6 A172-Luc cells (suspended in 3 μl DMEM) were slowly injected into the mouse brain. Two weeks after tumor implantation, TfR-MINC-trastuzumab, Tet1-MINC-trastuzumab, TC13-MINC-trastuzumab, adenosine-MINC-trastuzumab and MINC-trastuzumab were injected iv twice a week at 0.1 mg / kg to 10 mg / kg for 5 to 10 weeks. Tumor size was checked every two weeks using an in vivo imaging system (IVIS). After anesthetizing the mice, the luciferase substrate solution was injected intraperitoneally into the mice and then transferred to the IVIS chamber for image acquisition.

[0308] List of abbreviations

[0309] ADO adenosine

[0310] HENECA N-ethylcarboxamidoadenosine

[0311] GDNF Glial cell line-derived neurotrophic factor

[0312] PDGF Platelet-derived growth factor

[0313] CDNF Brain dopamine neurotrophic factor

[0314] TfR transferrin receptor

[0315] IR Insulin receptor

[0316] LDLR low-density lipoprotein receptor

[0317] Dhh Desert Hedgehog

[0318] P75NTR p75 neurotrophin receptor

[0319] NCAM neural cell adhesion molecule

[0320] PDGFRA Platelet-derived growth factor receptor alpha

[0321] NG2 Neuronal-glial antigen 2

[0322] MOG Myelin oligodendrocyte glycoprotein

[0323] MBP Myelin basic protein

[0324] TMEM119 Transmembrane protein 119

[0325] CX3CR1 CX3C chemokine receptor 1

[0326] CD45 Cluster of differentiation 45

Claims

1. A conjugate comprising: (a) a CNS targeting ligand, (b) a hydrophilic polymer of polyethylene glycol (PEG), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA) or dextran, and (c) a flavonoid of EGCG, EC, EGC or ECG, as shown in the following structures: wherein the hydrophilic polymer is covalently bound to the flavonoid and the CNS targeting ligand, wherein the CNS targeting ligand is selected from the group consisting of: a TfR peptide having an amino acid sequence of THRPPMWSPVWP (SEQ ID NO: 1), a Tet1 peptide having an amino acid sequence of HLNILSTLWKYRC (SEQ ID NO: 2), a TC13 peptide having an amino acid sequence of TGNYKALHPHNGC (SEQ ID NO: 3), apamin having an amino acid sequence of CNCKAPETALCARRCQQH (SEQ ID NO: 4), A regulator polypeptide having an amino acid sequence of PTVIHGKREVTLHL (SEQ ID NO: 5), a RAP peptide having an amino acid sequence of ELKHFEAKIEKHNHYQKQLE (SEQ ID NO: 6), Angiopep-2 peptide having an amino acid sequence of TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 7), a TAT peptide having an amino acid sequence of GGGGYGRKKRRQRRR (SEQ ID NO: 8), a SynB1 peptide having an amino acid sequence of RGGRLSYSRRRFSTSTGR (SEQ ID NO: 9), a leptin 30 peptide having an amino acid sequence of YQQVLTSLPSQNVLQIANDLENLRDLLHLLC (SEQ ID NO: 10), A LNP peptide having an amino acid sequence of KKRTLRKNDRKKRC (SEQ ID NO: 11), an ApoB peptide having the amino acid sequence of SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS (SEQ ID NO: 12), an RVG-29 peptide having an amino acid sequence of YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 13), a T7 peptide having the amino acid sequence of HAIYPRH (SEQ ID NO: 14), A GSH peptide having an amino acid sequence of ECG, a CRT peptide having an amino acid sequence of CRTIGPSVC (SEQ ID NO: 15), a CAQK peptide having the amino acid sequence of CAQK (SEQ ID NO: 16), a TACL05 peptide having an amino acid sequence of SACPSHLTKMCGGG (SEQ ID NO: 17), Adenosine, '-N-ethylcarboxamidoadenosine, glutamic acid or glutamate Glutamate and gamma-aminobutyric acid (GABA).

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

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

4. A nanoparticle composition comprising nanoparticles having: (a) an outer shell comprising the conjugate according to any one of claims 1 to 3, optionally (b) an inner shell comprising one or more flavonoid oligomers, and (c) a drug encapsulated in the shell, wherein the flavonoid is EGCG, EC, EGC or ECG, and the drug is effective in treating a CNS disease.

5. The nanoparticle composition of claim 4, wherein the encapsulated drug is anti-CD3, anti-CD4, anti-IL-17, anti-CD19, anti-CD20, anti-CD38, anti-β-amyloid, anti-tau, anti-α-synuclein, anti-mHtt, anti-IL6R, anti-IL-1β, anti-TREM2, BDNF, CDNF, GDNF, NRTN, PDGF-BB, anti-HER2, anti-EGFR, anti-PD-1, anti-PD-L1, anti-PDGFRA, anti-VEGF, anti-VEGFR2, IL-2, IL-4, IL-12, IFN-α, IFN-β, IFN-γ or TNF-α.

6. The nanoparticle composition of claim 4 or 5, wherein the shell further comprises a naked hydrophilic polymer-flavonoid conjugate to which the CNS targeting ligand is not covalently bound.

7. A method for treating a CNS disease, comprising the step of administering an effective amount of the nanoparticle composition according to claim 4 or 5 to a subject in need thereof.

8. The method of claim 7, wherein the CNS disease is Alzheimer's disease and the drug is anti-CD3, anti-CD33, anti-CD36, anti-CD39, anti-CD73, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA4, anti-GZM-A, anti-GZM-B, anti-TAM, anti-FcγRI, anti-RAGE, anti-APOE, anti-CR1, anti-NLRP3, anti-β-amyloid, anti-tau, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, GDNF, NRTN, PDGF-BB, CDNF or BDNF.

9. The method of claim 7, wherein the CNS disease is Parkinson's disease and the drug is anti-CD3, anti-CD33, anti-CD36, anti-CD39, anti-CD73, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA4, anti-GZM-A, anti-GZM-B, anti-TAM, anti-FcγRI, anti-RAGE, anti-APOE, anti-CR1, anti-NLRP3, anti-α-synuclein, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, GDNF, NRTN, PDGF-BB, CDNF or BDNF.

10. The method of claim 7, wherein the CNS disease is Lewy body dementia and the drug is anti-CD3, anti-CD33, anti-CD36, anti-CD39, anti-CD73, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA4, anti-GZM-A, anti-GZM-B, anti-TAM, anti-FcγRI, anti-RAGE, anti-APOE, anti-CR1, anti-NLRP3, anti-β-amyloid or anti-α-synuclein, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, GDNF, NRTN, PDGF-BB, CDNF or BDNF.

11. The method of claim 7, wherein the CNS disease is a brain tumor and the drug is doxorubicin, disulfiram, celecoxib, temsirolimus, everolimus, vorinostat, cabozantinib, marizomib, feminostat, acetazolamide, metformin, vinblastine, cyclophosphamide, anti-HER2, anti-EGFR, anti-PD-1, anti-PD-L1, anti-PDGFRA, anti-VEGF, anti-VEGFR2, IL-2, IL-4, IL-12, IFN-α, IFN-β, IFN-γ or TNF-α.

Images