Complex targeting the central nervous system

Abstract

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

Description

Technical Field

[0001] Sequence Listing This application includes a sequence listing compliant with ST.26, which was submitted in xml format through the Patent Center and is hereby incorporated by reference in its entirety. The ".xml" copy was created on June 1, 2023, named SequenceListing.xml, and has a size of 15.3 KB.

[0002] Field of the Invention The present invention relates to a complex comprising (a) a CNS (central nervous system)-targeting ligand; (b) a hydrophilic polymer which is polyethylene glycol (PEG), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), or dextran; and (c) a flavonoid, wherein the hydrophilic polymer is covalently bonded to the flavonoid and the CNS-targeting ligand. The present invention relates to micelle nanoparticles comprising (a) an outer shell comprising a CNS target ligand-hydrophilic polymer-(-)-epigallocatechin gallate (EGCG) complex; optionally (b) an inner shell comprising oligomers (OEGCG); and optionally (c) a CNS therapeutic agent encapsulated in the inner shell.

Background Art

[0003] Central nervous system (CNS) diseases are a group of neurological diseases that affect the structure or function of the brain or spinal cord that together form the CNS. This condition can be due to hereditary metabolic disorders, damage to nerve cells by infectious diseases, neurodegenerative conditions, stroke, brain tumors, or other problems resulting 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; prion diseases, migraine, infectious diseases, poisoning, arachnoid cysts, attention deficit / hyperactivity disorder (ADHD), autism, catatonia, encephalitis, epilepsy / spasms, infectious diseases, locked-in syndrome, meningitis, migraine, myelopathy, Tourette syndrome, and the like.

[0004] CNS tumors occur when abnormal cells form in the brain and spinal cord. Brain tumors can be malignant or benign (non-cancerous). These are further classified into primary tumors that occur within the brain and secondary tumors that most commonly spread from tumors located outside the brain. Spinal cord tumors are growths located either in the spinal column or the spinal cord. Brain tumors include primary cancer of the brain and metastatic cancer to the brain.

[0005] A stroke is a medical condition in which the deterioration of 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, a history of transient ischemic attack, end-stage renal disease, and atrial fibrillation. There is no treatment to regenerate brain cells that have died after a stroke. Treatment for stroke is very limited, but one of the FDA-approved treatments 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 mainstay of stroke prevention.

[0006] Neurodegenerative diseases are a group of diseases that mainly affect the nerve cells in the human brain. Examples of neurodegenerative diseases include 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 diseases are caused by genetic changes that are hereditary. Many neurodegenerative diseases are due to a combination of genetic and environmental factors. Therefore, it is difficult to predict who will develop the disease. The two main neurodegenerative diseases are Alzheimer's disease and Parkinson's disease.

[0007] Alzheimer's disease (AD) is a neurodegenerative disorder that usually starts slowly and worsens gradually. As the disease progresses, symptoms such as language problems, disorientation (such as getting lost easily), mood swings, loss of motivation, self-neglect, and behavioral problems appear. Alzheimer's disease is thought to be caused by the abnormal accumulation of amyloid-beta (Aβ) both extracellularly as amyloid plaques and tau protein, and intracellularly as neurofibrillary tangles, which form in the brain and affect the function and connectivity of nerve cells, leading to a progressive loss of brain function. Currently, there are treatments that can temporarily improve symptoms, but there is no treatment that can stop or reverse the progression.

[0008] Parkinson's disease (PD) is a long-term neurodegenerative disorder of the central nervous system that mainly affects the motor system. It is sometimes called a type of neurodegenerative disorder called synucleinopathy because of the abnormal accumulation of a protein called alpha-synuclein in the brain. The most obvious initial symptoms of PD are tremors, rigidity, slowness of movement, and difficulty walking. Cognitive and behavioral problems such as depression, anxiety, and apathy are also common in many PD patients. Dementia in Parkinson's disease generally occurs in the advanced stage of the disease. There is no known cure for PD, and treatment aims to reduce the impact of symptoms.

[0009] Drug delivery to the brain is the process of passing therapeutic active molecules across the blood-brain barrier for the purpose of treating brain diseases. This is a complex process that must take into account the complex anatomical structure of the brain and the constraints imposed by the special junctions of the blood-brain barrier.

[0010] The blood-brain barrier is formed by special tight junctions between endothelial cells that line the brain blood vessels. Although a single layer of these endothelial cells exists in the blood vessels of all tissues, only brain endothelial cells have tight junctions, which prevent the passive diffusion of most substances into brain tissue.

[0011] The blood-brain barrier (BBB) is a highly selective semipermeable boundary formed by endothelial cells of the central nervous system (CNS), which prevents solutes in the circulating blood vessels from non-selectively migrating into the CNS where neurons are present. Therefore, the BBB is a barrier that hinders the accumulation of effective drugs in the brain. There are four pathways for BBB penetration: passive diffusion, carrier transport, receptor-mediated transcytosis, and adsorptive-mediated transcytosis. Adsorptive-mediated transcytosis (AMT) is the main pathway. The AMT pathway utilizes caveolae as transport vehicles. Caveolae are a subgroup of lipid rafts present in the endothelial cells of the BBB. Endocytosis via caveolae is an important transport mechanism for the uptake of macromolecules from the bloodstream into the CNS.

Summary of the Invention

[0012] For many therapeutic agents, only a small proportion of the drug reaches the tissue where it is intended to act. For example, in chemotherapy, approximately 99% of the administered drug does not reach the tumor site. Targeted drug delivery aims to concentrate the drug in the target tissue while reducing the relative concentration of the drug in other tissues. For example, by avoiding the host's defense mechanisms and suppressing non-specific distribution in the liver and spleen, the system can reach the target site of action at a higher concentration. It is believed that targeted delivery improves efficacy while suppressing side effects.

Brief Description of the Drawings

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[0014] **Definitions** The term "about" is defined as ±10%, preferably ±5% of the stated value.

[0015] As used herein, the term "CNS-targeting ligand" refers to a molecule, such as a peptide or small molecule, having a molecular weight of less than 10,000 daltons, for example 300 - 3500 daltons, that binds to or targets receptors on the CNS cell surface or in the CNS environment.

[0016] The term "cytokine" refers to proteins (about 5 - 70 kDa) that are important for cell signaling. Cytokines have been shown to be involved in autocrine, paracrine, and endocrine signaling as immunomodulatory substances. Examples of cytokines include interferons, interleukins, lymphokines, tumor necrosis factors, and chemokines.

[0017] The term "epigallocatechin gallate" refers to an ester of epigallocatechin and gallic acid and is used interchangeably with "epigallocatechin-3-gallate" or "EGCG".

[0018] The term "oligomeric EGCG" (OEGCG) refers to a molecule in which 2 - 50, 3 - 50, or 3 - 20 monomers of EGCG are covalently bonded. OEGCG preferably contains 4 - 12 EGCG monomers.

[0019] The term "nanoparticle" refers to particles having a diameter of less than 1 μm and between 1 and 999 nm.

[0020] The term "polyethylene glycol-epigallocatechin gallate complex" or "PEG-EGCG" refers to a conjugate in which one or two molecules of EGCG are linked to polyethylene glycol (PEG). The term "PEG-EGCG" refers to both the PEG-mEGCG complex (monomeric EGCG) and the PEG-dEGCG (dimeric EGCG) complex.

[0021] The term "MINC" (Multi-pathway Immune-modulating Nanocomplex Combination therapy) is one of the platform technologies. The MINC used in this application utilizes the biological activities of the PEG-flavonoid complex and oligomeric EGCG (OEGCG). An additional CNS therapeutic agent can be encapsulated in MINC to form MINC-drug.

[0022] The term "MINC-drug" used in this application is a micelle having a shell formed by a CNS-targeting ligand-PEG-flavonoid complex and optionally an oligomeric flavonoid such as OEGCG, and having a drug encapsulated therein.

[0023] Unless otherwise specified, "%" used in this application means weight %.

[0024] Flavonoid A flavonoid suitable for the present invention has the general structure of Formula I:

Chemical formula

[0025] The 2nd, 3rd, 4th, 5th, 6th, 7th, or 8th position of Formula I can be linked to a group containing hydrocarbon, halogen, oxygen, nitrogen, sulfur, phosphorus, boron, or metal.

[0026] Examples of flavonoids of Formula I include the following:

Chemical formula

[0027] Preferred flavonoid compounds of Formula I include the following: EGCG (CAS# 989-51-5), EC (CAS# 490-46-0), EGC (CAS# 970-74-1), or ECG (CAS# 1257-08-5)

Chemical formula

[0028] Complex The present invention provides a complex comprising (a) a CNS-targeting ligand; (b) a hydrophilic polymer that is polyethylene glycol (PEG), polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), or dextran; and (c) a flavonoid of Formula I, wherein the PEG is covalently bonded to the flavonoid and the CNS-targeting ligand.

[0029] This complex is targeted to the CNS by the CNS-targeting ligand and delivers the active ingredient to the CNS tissue for treating CNS pathologies including, but not limited to, inflammation, nerve cell damage, accumulation of pathogenic aggregates in the CNS system, etc.

[0030] The CNS target ligand is covalently bonded to PEG, PLA, PLGA, or dextran via its -COOH group or its -NH2 group by standard chemical reactions known to those skilled in the art. The molecular weight of the hydrophilic polymer in the complex is generally 1K - 100K, preferably 3K - 80K, more preferably 5K - 40K.

[0031] The flavonoid in the complex has the general formula (I) and is preferably EGCG, EC, EGC, or ECG. In one embodiment, the flavonoid is epigallocatechin gallate (EGCG).

[0032] In one embodiment, PEG contains an aldehyde group linked to the 5th, 6th, 7th, or 8th position (preferably the 6th or 8th position) of the A ring of the flavonoid compound.

[0033] In another embodiment, PEG contains a thiol group linked to R1 or R2 (when R1 or R2 is -OH) of the B ring of the flavonoid.

[0034] In one embodiment, the complex contains PEG-EGCG in which PEG is linked to 1 or 2 molecules of EGCG, which can be prepared by linking aldehyde-terminated PEG and EGCG, and the linking is by the reaction of the free aldehyde group with the 5th, 6th, 7th, or 8th position (preferably the 6th or 8th position) of formula I for the binding of PEG. See WO 2006 / 124000 pamphlet and WO 2009 / 054813 pamphlet. PEG-EGCG can also be prepared by linking thio-terminated PEG and EGCG, and the linking is by the reaction of the free thiol group with R1 or R2 of formula I (wherein R1 or R2 is a phenyl group) for the binding of PEG. See WO 2015 / 171079 pamphlet.

[0035] In another embodiment, the conjugate comprises PEG-EC, PEG-EGC, or PEG-ECG, and the conjugate can be prepared by linking aldehyde-terminated PEG to EC, EGC, or ECG, and the linking is by the reaction of the free aldehyde group with the 5-, 6-, 7-, or 8-position (preferably the 6- or 8-position) of Formula I to effect the attachment of PEG.

[0036] HOOC-PEG-CHO and HO-PEG-CHO are generally available. In one embodiment, HOOC-PEG-CHO is linked to EGCG, EC, EGC, or ECG according to WO 2006 / 124000 pamphlet and WO 2009 / 054813 pamphlet. HOOC-PEG-flavonoid has a COOH group and reacts with the N-terminus of the CNS-targeting peptide. Generally, the CNS-targeting peptide is incubated in DMSO with HOOC-PEG-flavonoid, N,N'-dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide (NHS). The reaction is carried out with stirring at room temperature under light-shielding and nitrogen atmosphere. The reaction mixture is dialyzed against methanol and distilled water (membrane molecular weight cut-off = 2000 Da). Then, the solution is lyophilized to obtain a lyophilized powder. To avoid self-reaction of the peptide, the C-terminus of the CNS-targeting peptide may be protected, for example, by a resin during the reaction. Merrifield, hydroxymethylpolystyrene, PAM, and MBHA resins are generally used to prevent the linking of unwanted peptides. After the reaction, the resin can be removed under acidic conditions.

[0037] In another embodiment, HO-PEG-CHO is linked to EGCG, EC, EGC, or ECG according to WO 2006 / 124000 pamphlet and WO 2009 / 054813 pamphlet. The HO-PEG-flavonoid has an OH group and reacts with the C-terminus of the CNS-targeting peptide. Generally, the peptide is incubated with HO-PEG-EGCG and N,N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction is carried out with stirring at room temperature under light-shielding and nitrogen atmosphere. The reaction mixture is dialyzed against methanol-distilled water (membrane molecular weight cut-off = 2000 Da). Next, the solution is lyophilized to obtain a lyophilized powder. To avoid self-reaction of the peptide, the N-terminus of the CNS-targeting peptide may be protected by, for example, a resin during the reaction. Merrifield, hydroxymethylpolystyrene, PAM, and MBHA resins are generally used to prevent the ligation of unwanted peptides. After the reaction, the resin can be removed under acidic conditions. In this reaction, the COOH group on the peptide selectively reacts with the OH group on PEG. This is because the primary OH group on PEG is more reactive than the tertiary OH of the aromatic ring of the flavonoid.

[0038] HOOC-PLA-CHO, HOOC-PLGA-CHO, and HO-dextran-CHO are commercially available.

[0039] In one embodiment, HOOC-PLA-CHO is linked to EGCG, EC, EGC, or ECG according to WO 2006 / 124000 pamphlet and WO 2009 / 054813 pamphlet. HOOC-PLA-flavonoid has a COOH group and reacts with the N-terminus of the CNS-targeting peptide. Generally, the CNS-targeting peptide is incubated in DMSO with HOOC-PLA-flavonoid, N,N'-dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide (NHS). The reaction is carried out with stirring at room temperature under light shielding and nitrogen atmosphere. The reaction mixture is dialyzed against methanol and distilled water (membrane fractional molecular weight = 2000 Da). Next, the solution is lyophilized to obtain a lyophilized powder. To avoid self-reaction of the peptide, the C-terminus of the CNS-targeting peptide may be protected, for example, by a resin during the reaction. Merrifield, hydroxymethylpolystyrene, PAM, and MBHA resins are generally used to prevent the ligation of unwanted peptides. After the reaction, the resin can be removed under acidic conditions.

[0040] In one embodiment, HOOC-PLGA-CHO is linked to EGCG, EC, EGC, or ECG according to WO 2006 / 124000 pamphlet and WO 2009 / 054813 pamphlet. HOOC-PLGA-flavonoid has a COOH group and reacts with the N-terminus of the CNS-targeting peptide. Generally, the CNS-targeting peptide is incubated in DMSO with HOOC-PLGA-flavonoid, N,N'-dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide (NHS). The reaction is carried out with stirring at room temperature under light shielding and nitrogen atmosphere. The reaction mixture is dialyzed against methanol and distilled water (membrane fractional molecular weight = 2000 Da). Next, the solution is lyophilized to obtain a lyophilized powder. To avoid self-reaction of the peptide, the C-terminus of the CNS-targeting peptide may be protected, for example, by a resin during the reaction. Merrifield, hydroxymethylpolystyrene, PAM, and MBHA resins are generally used to prevent the ligation of unwanted peptides. After the reaction, the resin can be removed under acidic conditions.

[0041] In one embodiment, HO-dextran-CHO is linked to EGCG, EC, EGC, or ECG according to WO 2006 / 124000 pamphlet and WO 2009 / 054813 pamphlet. The HO-dextran-flavonoid has an OH group and reacts with the C-terminus of the CNS-targeting peptide. Generally, the peptide is incubated with HO-dextran-EGCG and N,N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction is carried out with stirring at room temperature under light shielding and nitrogen atmosphere. The reaction mixture is dialyzed against methanol-distilled water (membrane fractionation molecular weight = 2000 Da). Next, the solution is lyophilized to obtain a lyophilized powder. To avoid self-reaction of the peptide, the N-terminus of the CNS-targeting peptide may be protected by, for example, a resin during the reaction. Merrifield, hydroxymethylpolystyrene, PAM, and MBHA resins are generally used to prevent the ligation of unwanted peptides. After the reaction, the resin can be removed under acidic conditions. In this reaction, the COOH group on the peptide selectively reacts with the OH of the CH2OH terminus of dextran. This is because this is the only primary OH group in dextran and is more reactive than the other secondary OHs in dextran and the tertiary OHs of the aromatic ring of the flavonoid.

[0042] The CNS-targeting ligand in the present invention is a ligand selected to target receptors on the surface of CNS cells (neurons) or in the CNS environment. The CNS-targeting ligand of the present invention targets, for example, the following receptors of neurons or the CNS environment.

[0043] (i) Receptors on nerve cells: This group includes, but is not limited to, glutamate-activated neuronal receptors, GABA-activated neuronal receptors, dopamine-activated neuronal receptors, serotonin-activated 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, NMDAR2B, GAT1, DAT, SERT, Pet1, and VAChT.

[0044] (ii) Receptors present in the CNS environment: This group includes, but is not limited to, oligodendrocyte receptors, oligodendrocyte progenitor cell receptors, intermediate progenitor cell receptors, neuroepithelial cell receptors, Schwann cell receptors, radial glia receptors, astrocyte receptors, microglia receptors, pericytes receptors, B cell receptors, T cell receptors, RAGE receptors, Fc receptors, toll-like receptors, 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.

[0045] In one embodiment, the CNS-targeting ligand is a TfR peptide having the amino acid sequence THRPPMWSPVWP (SEQ ID NO: 1) that targets the transferrin receptor on or in the microenvironment of nerve cells in the CNS.

[0046] In one embodiment, the CNS-targeting ligand is a Tet1 peptide having the amino acid sequence HLNILSTLWKYRC (SEQ ID NO: 2) that targets the GT1b receptor on or in the microenvironment of nerve cells in the CNS.

[0047] In one embodiment, the CNS-targeting ligand is the TC13(TGN) peptide having the amino acid sequence TGNYKALHPHNGC (SEQ ID NO: 3), which targets neurons or the microenvironment in the CNS.

[0048] In one embodiment, the CNS-targeting ligand is the apamin peptide having the amino acid sequence CNCKAPETALCARRCQQH (SEQ ID NO: 4), which targets the apamin receptor on neurons or the microenvironment in the CNS.

[0049] In one embodiment, the CNS-targeting ligand is the regulon polypeptide having the amino acid sequence PTVIHGKREVTLHL (SEQ ID NO: 5), which targets the low-density lipoprotein (LDL) receptor on neurons or the microenvironment in the CNS.

[0050] In one embodiment, the CNS-targeting ligand is the RAP peptide having the amino acid sequence ELKHFEAKIEKHNHYQKQLE (SEQ ID NO: 6), which targets the LDL receptor on neurons or the microenvironment in the CNS.

[0051] In one embodiment, the CNS-targeting ligand is the Angiopep-2 peptide having the amino acid sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 7), which targets the LDL receptor on neurons or the microenvironment in the CNS.

[0052] In one embodiment, the CNS-targeting ligand is the TAT peptide having the amino acid sequence GGGGYGRKKRRQRRR (SEQ ID NO: 8), which targets neurons or the microenvironment in the CNS.

[0053] In one embodiment, the CNS-targeting ligand is the SynB1 peptide having the amino acid sequence RGGRLSYSRRRFSTSTGR (SEQ ID NO: 9), which targets neurons or the microenvironment in the CNS.

[0054] In one embodiment, the CNS-targeting ligand is a leptin 30 peptide having the amino acid sequence YQQVLTSLPSQNVLQIANDLENLRDLLHLLC (SEQ ID NO: 10), which targets the leptin receptor on neurons or in the microenvironment in the CNS.

[0055] In one embodiment, the CNS-targeting ligand is an LNP peptide having the amino acid sequence KKRTLRKNDRKKRC (SEQ ID NO: 11), which targets caveolae-mediated endocytosis and macropinocytosis on neurons or in the microenvironment in the CNS.

[0056] In one embodiment, the CNS-targeting ligand is an ApoB peptide having the amino acid sequence SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS (SEQ ID NO: 12), which targets the LRP2 receptor on neurons or in the microenvironment in the CNS.

[0057] In one embodiment, the CNS-targeting ligand is an RVG-29 peptide having the amino acid sequence YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 13), which targets the nAChR receptor on neurons or in the microenvironment in the CNS.

[0058] In one embodiment, the CNS-targeting ligand is a T7 peptide having the amino acid sequence HAIYPRH (SEQ ID NO: 14), which targets the transferrin receptor on neurons or in the microenvironment in the CNS.

[0059] In one embodiment, the CNS-targeting ligand is a GSH (glutathione) peptide having the amino acid sequence ECG, which targets neurons or the microenvironment in the CNS.

[0060] In one embodiment, the CNS-targeting ligand is a CRT peptide having the amino acid sequence CRTIGPSVC (SEQ ID NO: 15), which targets the transferrin receptor on neurons or in the microenvironment in the CNS.

[0061] In one embodiment, the CNS-targeting ligand is the CAQK peptide having the amino acid sequence CAQK (SEQ ID NO: 16), which targets the proteoglycan complex on neurons or in the microenvironment in the CNS.

[0062] In one embodiment, the CNS-targeting ligand is the TACL05 peptide having the amino acid sequence SACPSHLTKMCGGG (SEQ ID NO: 17), which targets neurons or the microenvironment in the CNS.

[0063] In one embodiment, the CNS-targeting ligand is adenosine, which targets the adenosine A1 receptor.

[0064] In one embodiment, the CNS-targeting ligand is 5'-N-ethylcarboxamidoadenosine (NECA), which targets the adenosine A2A receptor.

[0065] In one embodiment, the CNS-targeting ligand is glutamate, which targets glutamate receptors.

[0066] In one embodiment, the CNS-targeting ligand is γ-aminobutyric acid (GABA), which targets the 5-HT4 receptor.

[0067] Nanoparticle composition The term "MINC" (Multi-pathway Immune-modulating Nanocomplex Combination therapy) refers to one of the platform technologies. The present invention provides a nanoparticle micelle (MINC) composition having an outer shell containing one or more of the CNS-targeting complexes of the present invention; an inner shell optionally containing one or more flavonoid oligomers; and optionally, a drug encapsulated within said shell.

[0068] In one embodiment, the micelle composition includes both an outer shell and an inner shell as described above; and the composition optionally has a drug encapsulated within those shells.

[0069] In one embodiment, the micelle composition includes an outer shell as described above and does not include an inner shell; optionally, the composition has a drug encapsulated within the shell.

[0070] In one embodiment, the micelle composition includes an outer shell CNS-targeting ligand-polymer-flavonoid complex and an inner shell oligomeric flavonoid, wherein the flavonoid of the outer shell and the flavonoid of the inner shell are independently EGCG, EC, EGC, or ECG, and the polymer is PEG, PLA, PLGA, or dextran. A preferred polymer is PEG. A preferred flavonoid is EGCG. Figure 1 shows a preferred micelle composition. The CNS-targeting ligand enables the nanoparticle composition to specifically target CNS tissue.

[0071] In one embodiment, the micelle outer shell further includes a naked PEG-flavonoid complex such as PEG-EGCG that does not have a CNS-targeting ligand linked to the PEG-flavonoid. See Figure 2. In such a micelle outer shell, the ratio of ligand-PEG-EGCG to PEG-EGCG is generally greater than 10%, or greater than 20%, or greater than 30%, or greater than 50%, up to 100%. In one embodiment, the ratio of ligand-PEG-EGCG to PEG-EGCG is 10 - 90%, or 20 - 80%, or 40 - 60%.

[0072] The micelle optionally includes a CNS therapeutic molecule (drug or agent) encapsulated within the micelle (MINC-agent).

[0073] In one embodiment, the MINC-drug composition contains three active ingredients, which are complementary in terms of the function of addressing both the immune response and the signal transduction pathway by the skeletal elements of the composition (PEG-flavonoid / OEGCG), and the function of addressing additional signal transduction pathways by selected drug molecules for treating CNS diseases. Each nanoparticle is a fixed-dose combination drug formulated with those three active ingredients in a certain molar ratio.

[0074] The present invention delivers the MINC-drug to the target CNS tissue by actively delivering micelles to the brain having specific receptors via a CNS-targeting ligand.

[0075] The nanocomposite of the present invention contains, in the backbone of the micelle composition, the first two active ingredients, such as flavonoids like OEGCG and PEG-flavonoids like PEG-EGCG. These are derivatives of EGCG and are potent immunomodulators that control a wide range of disease signal transduction pathways. EGCG controls both innate and adaptive immunity. However, the bioavailability of EGCG is low, and EGCG is not stable. The nanocomposite composition of the present invention overcomes the problem of the bioavailability of EGCG by forming a nanocarrier that transports EGCG to the target site for treatment, and also overcomes the problem of the stability of EGCG by forming OEGCG and PEG-EGCG complexes, thereby effectively making EGCG a highly effective therapeutic agent.

[0076] The nanocomposite of the present invention optionally contains a third active ingredient, which is a drug molecule encapsulated in nanoparticles for treating 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, anti-tau, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, GDNF, NRTN, PDGF-BB, CDNF, or BDNF.

[0077] 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.

[0078] In one embodiment, 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, anti-α synuclein, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, GDNF, NRTN, PDGF-BB, CDNF, or BDNF.

[0079] In one embodiment, the CNS disease is a brain tumor, and the drug is doxorubicin, disulfiram, celecoxib, temsirolimus, everolimus, vorinostat, cabozantinib, marizomib, fimibrostatin, 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.

[0080] In one embodiment, the CNS disease is a stroke, and the drug is an 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.

[0081] 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, bevantrol, pridopidine, branaplam, nilotinib, mitoconix, or azathioprine.

[0082] 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.

[0083] 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-hnRNPs, a CK-1 inhibitor, anti-FET or an HDAC inhibitor, EPO, or IL-2.

[0084] In one embodiment, the CNS disease is acute spinal cord injury, and the drug is an extracellular domain of the Nogo receptor, 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.

[0085] 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.

[0086] In one embodiment, the CNS disease is epilepsy or spasm, and the drug is an mTOR inhibitor, PI3K inhibitor, GABA inhibitor, anti-Glu3B peptide antibody, anti-NR1 antibody, anti-CASPR2, or anti-LGI-1.

[0087] In one embodiment, the CNS disease is meningitis, and the drug is C1 inhibitor, anti-C5, anti-MASP-2, anti-PD-L1, anti-CTLA-4, or anti-PD-1.

[0088] In one embodiment, the CNS disease is motor neuron disease (MND), and the drug is anti-SOD1, anti-TDP-43, anti-C90RF72, 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.

[0089] The nanoparticles are stable in a hydrophilic environment such as blood circulation and dissociate in a hydrophobic environment such as CNS tissue.

[0090] Method for preparing the nanoparticle composition The nanoparticle composition of the present invention can be prepared by a process comprising the following steps: (a) mixing a drug molecule with a flavonoid oligomer (e.g., OEGCG) and the CNS-targeting ligand complex of the present invention in an aqueous solution; and (b) filtering the mixture through a membrane with a fractional molecular weight of 8,000 to 300,000 daltons to remove low molecular weight molecules and retain high molecular weight molecules.

[0091] In a preferred embodiment, this process further includes step (c) of filtering high molecular weight molecules through a 0.2 - 0.3 μm membrane and recovering the filtrate.

[0092] In step (a), the drug molecule is dissolved in an aqueous solvent such as phosphate buffered saline, saline, water, bicarbonate buffer, oxyhemoglobin buffer, bis - tris alkane, 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 - 50 mg / ml, preferably 0.05 - 10 mg / ml, more preferably 0.1 - 5 mg / ml.

[0093] The flavonoid oligomer and CNS - targeted ligand complex are dissolved in ketone, acetonitrile, alcohol, aldehyde, ether, acetate, sulfoxide, benzene, organic acid, amide, aqueous buffer, and any combination thereof. Preferred solvents are alcohol, acetonitrile, sulfoxide, amide, and any combination thereof. For example, the OEGCG / EGCG and PEG - EGCG concentrations are generally, independently, 0.001 - 10 mg / ml, preferably 0.005 - 1 mg / ml, or 0.1 - 5 mg / ml.

[0094] It is important that OEGCG is in molar excess with respect to the drug agent. Generally, the molar ratio of EGCG in OEGCG to the drug molecule is between 1 to 1 - 500, 1 to 2 - 500, 1 to 3 - 500, or 1 to 5 - 500, preferably 1 to 3 - 100, 1 to 5 - 100, or 1 to 10 - 50. The molar ratio is calculated by the number of moles of monomeric EGCG in OEGCG with respect to the number of moles of the drug molecule. By having EGCG in molar excess, it is ensured that most or all of the drug agent is encapsulated by OEGCG molecules. Unencapsulated drug agent may not be selectively distributed to the target tissue and can cause a decrease in efficacy and safety issues. In this process, this is avoided by controlling the molar ratio of OEGCG to the protein.

[0095] In step (b), the above mixture is filtered through a membrane with a molecular weight cut-off of 8000 - 300,000 Daltons, preferably 8000 - 200,000 Daltons, 8000 - 150,000 Daltons, or 8000 - 12,000 Daltons to remove low molecular weight molecules and retain high molecular weight molecules. The ultrafiltration membrane material is selected from the group consisting of cellulose (and its derivatives), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, polyvinylidene fluoride or polyvinylidene difluoride (PVDF), and polypropylene (PP); preferably, cellulose (and its derivatives), PTFE, and PVDF.

[0096] This mixture is optionally diluted with an aqueous solvent as described above, for example, in step (a), prior to ultrafiltration.

[0097] In the ultrafiltration step (b), unwanted low molecular weight impurities such as unreacted OEGCG or EGCG, or reaction by-products are removed. These impurities may reduce the drug efficacy and safety. Also, if the unreacted OEGCG or EGCG is in excess, individual nanoparticles may aggregate into particles of about 1000 nm in size, which can reduce the efficacy and cause toxicity.

[0098] In step (c), the retained high molecular weight molecules are filtered through a membrane having a pore size of about 0.2 to 0.3 μm, for example 0.22 μm, and the filtrate is recovered. This is to remove unnecessary impurities of polymer size, such as mega-aggregates. Due to their large size, these aggregates may be excreted from the entry tissue. These aggregates reduce the overall efficacy / safety and are likely to induce immunogenicity in patients. Also, large-sized nanoparticles are easily taken up by the RES in unwanted organs such as the liver and lungs.

[0099] The membrane material for 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.

[0100] In one embodiment, before step (d), steps (b) and (c) are repeated at least once, for example 1, 2, 3, or 4 times, thereby effectively removing unnecessary low molecular weight impurities and large aggregates.

[0101] After step (c), the filtrate is stored at 2 - 8°C and is stable for at least 100 days.

[0102] Optionally, this process further includes a lyophilization step (d) after step (c) to provide long-term stability of the nanoparticle composition.

[0103] Pharmaceutical composition The present invention provides a pharmaceutical composition comprising the nanoparticle composition of the present invention and optionally one or more pharmaceutically acceptable carriers. Generally, in the case of tablets, powders, or parenteral preparations, the content of nanoparticles in the pharmaceutical composition is about 1 to 90%, preferably 20 to 90%, or 30 to 80%. Generally, in the case of capsule preparations, the content of nanoparticles in the pharmaceutical composition is 1 to 100%, preferably 20 to 100%, 50 to 100%, or 70 to 100%. Generally, in the case of suspension preparations, the content of nanoparticles in the pharmaceutical composition is 1 to 50%, 5 to 50%, or 10 to 40%.

[0104] In one embodiment, the pharmaceutical composition can be in the form of tablets, capsules, granules, fine granules, powders, suspensions, patches, parenterals, injections, or the like. The above pharmaceutical composition can be prepared by conventional methods.

[0105] Pharmaceutically acceptable carriers that are inactive ingredients can be selected by those skilled in the art using conventional criteria. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and dextrose; pH adjusters and buffers such as salts of hydroxides, phosphoric acid, citric acid, acetic acid, boric acid, and trolamine; antioxidants such as bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, and salts, acids, and / or bases of ascorbyl palmitate; surfactants such as lecithin and phospholipids including, but not limited to, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol; poloxamers and poloxamines; polysorbates such as polysorbate 80, polysorbate 60, and polysorbate 20; polyethers such as polyethylene glycol and polypropylene glycol; polyvinyls such as polyvinyl alcohol and polyvinylpyrrolidone (PVP, povidone); cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose, and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, and soybean oil; mono-, di-, and triglycerides; polysaccharides such as dextran; and glycosaminoglycans such as sodium hyaluronate; and the like. Such pharmaceutically acceptable carriers may be preserved against bacterial contamination using well-known preservatives such as benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenyl ethyl alcohol, among others, or formulated as non-preserved formulations for single or multiple use.

[0106] For example, tablets, capsules, or parenteral formulations of the active compound may contain other excipients that are biologically inactive and do not react with the active compound. Excipients for tablets or capsules include fillers, binders, lubricants and glidants, disintegrants, wetting agents, and release rate regulators, among others. Examples of excipients for 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.

[0107] 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. Capsules 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.

[0108] Treatment method The present invention is directed to a method for treating CNS diseases, which includes the step of administering an effective amount of the nanoparticle composition of the present invention to a subject in need thereof. Suitable CNS diseases treatable 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, and the like.

[0109] As used in this application, "effective amount" means an amount effective to treat a disease by alleviating the medical condition or reducing the symptoms of the disease.

[0110] The dosage of the ligand-polymer-flavonoid for injection, for example, ligand-polymer-EGGC, is generally 0.01 - 100 mg / kg (total weight of polymer-flavonoid / body weight of subject), or 0.001 - 1000 mg / kg.

[0111] In one embodiment, the method includes administering to a subject in need thereof an effective amount of MINC having a shell, wherein the shell is formed by one or more ligand-hydrophilic polymer-flavonoid complexes and optionally naked polymer-flavonoid complexes, which may or may not contain flavonoid oligomers, and optionally has a drug encapsulated therein.

[0112] In one embodiment, the shell is formed of ligand-PEG-EGCG and optionally PEG-EGCG.

[0113] In one embodiment, the shell is formed of ligand-PEG-EGCG and OEGCG and optionally PEG-EGCG.

[0114] The ligand-polymer-flavonoid complex of the present invention can pass through the blood-brain barrier (BBB) from the circulating blood vessels to the brain. The ligand-polymer-flavonoid complex of the present invention can target CNS lesions after passing through the BBB. The ligand-polymer-flavonoid complex of the present invention further has the activity of repairing or regenerating nerve cells for treating CNS diseases, and also has immune and disease-regulating functions.

[0115] The flavonoid oligomer used in the method of the present invention can pass through the BBB from the circulating blood vessels to the brain, and has immune and disease-regulating functions for treating CNS diseases. The flavonoid oligomer of the present invention further has the activity of repairing or regenerating nerve cells for treating CNS diseases.

[0116] The dosage of the MINC-agent is based on the known dosage of the agent for treating a specific disease and the condition of the subject. The dosage may be the dosage approved by the Food and Drug Administration (FDA) or the dosage used in clinical trials.

[0117] In the MINC-agent, the total weight of the ligand-PEG-EGCG and PEG-EGCG (if present) is close to that of the encapsulated drug substance. The weight of OEGCG (if present) varies. Generally, the dosage of the ligand-PEG-EGCG and PEG-EGCG (if present) in combination with OEGCG is 0.01 - 1000 mg / kg.

[0118] The concentration of the encapsulated drug substance can be as low as 0.01 mg / kg (such as rhBDNF in cytokine drugs) or as high as 100 mg / kg (such as the anti-α-synuclein antibody in antibody drugs at this concentration).

[0119] For the treatment of Alzheimer's disease, anti-β-amyloid (Aducanumab, Solanezumab, Crenezumab, Gantenerumab, Donanemab, or Lecanemab) is intravenously administered at 0.01 - 100 mg / kg or 0.01 - 1000 mg / kg every 1 - 4 weeks. An effective amount of MINC-anti-β-amyloid within a similar dosage range can be used for the treatment of Alzheimer's disease.

[0120] For the treatment of Parkinson's disease, anti-α-synuclein (Prasinezumab or Cinpanemab) is intravenously administered at 0.01 - 100 mg / kg or 0.01 - 1000 mg / kg every 1 - 4 weeks. An effective amount of MINC-anti-α-synuclein within a similar dosage range can be used for the treatment of Parkinson's disease.

[0121] For the treatment of Huntington's disease, anti-mHtt (C6 / 17) is intravenously administered at 0.01 to 100 mg / kg or 0.01 to 1000 mg / kg every 1 to 4 weeks. An effective amount of MINC-anti-mHtt within the same dosage range can be used for the treatment of Huntington's disease.

[0122] For the treatment of migraine, anti-CGRP (eptinezumab, fremanezumab, galcanezumab, erenumab) is intravenously administered at 0.01 to 100 mg / kg or 0.01 to 1000 mg / kg every 1 to 4 weeks. An effective amount of MINC-anti-CGRP within the same dosage range can be used for the treatment of migraine.

[0123] For the treatment of multiple sclerosis, anti-CD20 (ocrelizumab, rituximab, ofatumumab, or ublituximab) is intravenously administered at 0.01 to 100 mg / kg or 0.01 to 1000 mg / kg 1 to 4 times a year. An effective amount of MINC-anti-CD20 within the same dosage range can be used for the treatment of multiple sclerosis.

[0124] The present invention is useful for the treatment of humans and non-human animals. For example, the present invention is useful for treating mammalian subjects such as humans, horses, pigs, cats, and dogs.

[0125] The following examples further illustrate the present invention. These examples are merely intended to illustrate the present invention and should not be construed in a limiting sense.

Examples

[0126] Example 1: Conjugation of TfR Peptide to HOOC-PEG-EGCG Materials The TfR peptide was purchased from Hangzhou Xinbosi Biomedical. HOOC-PEG-CHO was purchased from NBC chemical.

[0127] Method HOOC-PEG-CHO was linked to EGCG according to WO 2006 / 124000 and WO 2009 / 054813; the TfR peptide was linked to HOOC-PEG-EGCG via a linkage between the COOH group on PEG and the NH2 group on TfR to form TfR-PEG-EGCG (N'-linked) (Figure 3).

[0128] Specifically, the PEGylation of 1 - 1000 mg of TfR was carried out by incubating it with 1 - 1000 mg of HOOC-PEG-EGCG, 1 - 1000 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 1 - 1000 mg of N-hydroxysuccinimide (NHS) in DMSO. The reaction was carried out with stirring at room temperature for 24 hours under light-shielding and nitrogen atmosphere. The reaction mixture was dialyzed against methanol and distilled water for 3 days (membrane molecular weight cut-off = 2000 Da). Then, the solution was lyophilized to obtain a lyophilized powder.

[0129] Results HPLC was used to confirm the formulation of TfR-PEG-EGCG. HPLC was performed under the following conditions: column: C18, 4.6×150 mm, 4 μm; elution: A = 0.1% TFA in H2O, B = 0.1% TFA in ACN; oven temperature: 40 °C; flow rate: 1 ml / min; autosampler temperature: 15 °C; measurement: UV280.

[0130] The retention time of HOOC-PEG-EGCG was 6.04 minutes, but after the ligation of TfR, a new peak with a retention time of 6.46 minutes was present. The HPLC results indicate that the ligation of TfR-PEG-EGCG was successful.

[0131] Example 2: Ligation of Tet1 Peptide to HOOC-PEG-EGCG Materials The Tet1 peptide was purchased from Hangzhou Xinbosi Biomedical Co., Ltd. HOOC-PEG-CHO was purchased from NBC chemical.

[0132] Method HOOC-PEG-CHO was conjugated to EGCG according to WO2006 / 124000 and WO2009 / 054813; the Tet1 peptide was conjugated to HOOC-PEG-EGCG via a linkage between the COOH group on PEG and the NH2 group on Tet1 to form Tet1-PEG-EGCG (N'-linkage) (Figure 4).

[0133] Specifically, the PEGylation of 1 - 1000 mg of Tet1 was carried out by incubating with 1 - 1000 mg of HOOC-PEG-EGCG, 1 - 1000 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 1 - 1000 mg of N-hydroxysuccinimide (NHS) in DMSO. The reaction was stirred at room temperature for 24 hours under light-shielding and nitrogen atmosphere. The reaction mixture was dialyzed against methanol and distilled water for 3 days (membrane molecular weight cut-off = 2000 Da). Then, the solution was lyophilized to obtain a lyophilized powder.

[0134] Results The formulation of Tet1-PEG-EGCG was confirmed using HPLC. HPLC was performed under the following conditions: column: C18, 4.6×150 mm, 4 μm; elution: A = 0.1% TFA in H2O, B = 0.1% TFA in ACN; oven temperature: 40 °C; flow rate: 1 ml / min; autosampler temperature: 15 °C; measurement: UV280.

[0135] The retention time of HOOC-PEG-EGCG was 6.04 minutes, but after the conjugation of Tet1, a new peak with a retention time of 7.54 minutes was present. The HPLC results indicate that the conjugation of Tet1-PEG-EGCG was successful.

[0136] Example 3: Conjugation of the TC13 peptide to HOOC-PEG-EGCG Materials The TC13 peptide was purchased from Hangzhou Xinbosi Biomedical Co., Ltd. HOOC-PEG-CHO was purchased from NBC chemical Co., Ltd.

[0137] Methods HOOC-PEG-CHO was conjugated to EGCG according to the International Publication No. WO2006 / 124000 and International Publication No. WO2009 / 054813; the TC13 peptide was conjugated to HOOC-PEG-EGCG via a linkage between the COOH group on PEG and the NH2 group on TC13 to form TC13-PEG-EGCG (N'-linked) (Figure 5).

[0138] Specifically, the PEGylation of 1-1000 mg of TC13 was carried out by incubating it with 1-1000 mg of HOOC-PEG-EGCG, 1-1000 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 1-1000 mg of N-hydroxysuccinimide (NHS) in DMSO. The reaction was carried out with stirring at room temperature for 24 hours under light shielding and nitrogen atmosphere. The reaction mixture was dialyzed against methanol and distilled water for 3 days (membrane molecular weight cut-off = 2000 Da). Next, the solution was lyophilized to obtain a lyophilized powder.

[0139] Results The formulation of TC13-PEG-EGCG was confirmed using HPLC. HPLC was performed under the following conditions: column: C18, 4.6×150 mm, 4 μm; eluent: A = 0.1% TFA in H2O, B = 0.1% TFA in ACN; oven temperature: 40 °C; flow rate: 1 ml / min; autosampler temperature: 15 °C; measurement: UV280.

[0140] The retention time of HOOC-PEG-EGCG was 6.04 minutes, but after the conjugation of TC13, a new peak with a retention time of 6.54 minutes was present. The results of HPLC indicate that the conjugation of TC13-PEG-EGCG was successful.

[0141] Example 4: Preparation of TfR-MINC-Doxorubicin, Tet1-MINC-Doxorubicin, and TC13-MINC-Doxorubicin Materials TfR-PEG-EGCG was prepared according to Example 1. Tet1-PEG-EGCG was prepared according to Example 2. TC13-PEG-EGCG was prepared according to Example 3. Doxorubicin was purchased from Sigma-Aldrich.

[0142] Methods TfR-MINC-Doxorubicin nanoparticles, Tet1-MINC-Doxorubicin nanoparticles, and TC13-MINC-Doxorubicin nanoparticles were prepared according to the following protocol:

[0143] 1. Incubate 5 - 500 μg of doxorubicin in 1 mL of DMSO for 15 minutes to 1 hour. 2. Add 1 - 100 μg of OEGCG and 1 - 10,000 μg of TfR-PEG-EGCG, Tet1-PEG-EGCG, or TC13-PEG-EGCG. Incubate the mixture at 25°C for 3 hours. 3. Filter the liquid through a 10K MWCO filter unit. Wash the filter three times with 0.9% NaCl. 4. Lyophilize to obtain a dry powder.

[0144] Results The nanoparticle size was measured by DLS (Anton Paar, Litesizer 500). Figure 6 shows the successful formulation of TfR-MINC-Doxorubicin (A), Tet1-MINC-Doxorubicin (B), and TC13-MINC-Doxorubicin (C).

[0145] Example 5: Various Ligand-MINC-Doxorubicin in Drug Delivery to Brain Endothelial Cells Materials TfR-MINC-doxorubicin, Tet1-MINC-doxorubicin, TC13-MINC-doxorubicin, or MINC-doxorubicin was formulated according to Example 4.

[0146] Method The brain endothelial cell line bEnd.3 was seeded in a 96-well plate 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 delivery efficiency was evaluated by measuring the fluorescence intensity with a Molecular Devices Gemini XPS fluorescence microplate reader. The data are shown as mean ± SD and were statistically analyzed by GraphPad Prism 7. Statistical significance was calculated by one-way ANOVA, * : p < 0.05, * * : p < 0.01, *** : and a significant difference was considered at p < 0.001.

[0147] Results Doxorubicin is a red fluorescent compound, and its delivery into cells was observed using a fluorescence microscope. The higher the fluorescence intensity, the more doxorubicin was delivered into the cells. In Figure 7, compared with MINC-doxorubicin, the fluorescence signals of TfR-MINC-doxorubicin and Tet1-MINC-doxorubicin were significantly stronger in bEnd.3 cells. Also, a tendency for a high fluorescence signal was observed in TC13-MINC-doxorubicin-treated cells. These results indicate that the CNS-targeting peptide increased the specific drug delivery to brain endothelial cells.

[0148] Example 6: Ligation of the TfR Peptide to HO-PEG-EGCG (Predictive Example) Objective This experiment aims to illustrate the conjugation of the TfR peptide to HO-PEG-EGCG. Using HPLC, the formation of a new product (TfR-PEG-EGCG) with a retention time different from that of HO-PEG-EGCG is detected. The structure can be confirmed using NMR.

[0149] Materials The TfR peptide is purchased from Hangzhou Xinbosi Biomedical. HO-PEG-CHO is purchased from Huanteng pharma.

[0150] Methods HO-PEG-EGCG is prepared by conjugating HO-PEG-CHO to EGCG according to WO 2006 / 124000 pamphlet and WO 2009 / 054813 pamphlet.

[0151] The TfR peptide is conjugated to HO-PEG-EGCG via the linkage between the OH group on PEG and the COOH group on TfR to form TfR-PEG-EGCG (C’ linkage). Refer to Figure 8.

[0152] Specifically, the PEGylation of 1 - 1000 mg of TfR is carried out by incubating with 1 - 1000 mg of HO-PEG-EGCG and 1 - 1000 mg of N,N’-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction is carried out with stirring at room temperature for 24 hours under light shielding and nitrogen atmosphere. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cut-off = 2000 Da). Then, the solution is lyophilized to obtain a lyophilized powder.

[0153] Example 7: Conjugation of Tet1 Peptide to HO-PEG-EGCG (Predictive Example) Purpose This experiment aims to illustrate the conjugation of Tet1 peptide to HO-PEG-EGCG. Using HPLC, the formation of a new product (Tet1-PEG-EGCG) with a retention time different from that of HO-PEG-EGCG is detected. The structure can be confirmed using NMR.

[0154] Materials The Tet1 peptide is purchased from Hangzhou Xinbosi Biomedical. HO-PEG-CHO is purchased from Huanteng pharma.

[0155] Methods HO-PEG-EGCG is prepared by conjugating HO-PEG-CHO to EGCG according to WO 2006 / 124000 pamphlet and WO 2009 / 054813 pamphlet.

[0156] The Tet1 peptide is conjugated to HO-PEG-EGCG via the linkage between the OH group on PEG and the COOH group on Tet1 to form Tet1-PEG-EGCG (C’ linkage). Refer to Figure 9.

[0157] Specifically, the PEGylation of 1 - 1000 mg of Tet1 is carried out by incubating it with 1 - 1000 mg of HO-PEG-EGCG and 1 - 1000 mg of N,N’-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction is stirred at room temperature for 24 hours under light-shielding and nitrogen atmosphere. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cut-off = 2000 Da). Then, the solution is lyophilized to obtain a lyophilized powder.

[0158] Example 8: Conjugation of TC13 Peptide to HO-PEG-EGCG (Predictive Example) Objective This experiment aims to illustrate the conjugation of the TC13 peptide to HO-PEG-EGCG. Using HPLC, the formation of a new product (TC13-PEG-EGCG) with a retention time different from that of HO-PEG-EGCG is detected. The structure can be confirmed using NMR.

[0159] Materials The TC13 peptide is purchased from Hangzhou Xinbosi Biomedical. HO-PEG-CHO is purchased from Huanteng pharma.

[0160] Methods HO-PEG-EGCG is prepared by conjugating HO-PEG-CHO to EGCG according to WO 2006 / 124000 pamphlet and WO 2009 / 054813 pamphlet.

[0161] The TC13 peptide is conjugated to HO-PEG-EGCG via a linkage between the OH group on PEG and the COOH group on TC13 to form TC13-PEG-EGCG (C’ linkage). Refer to Figure 10.

[0162] Specifically, the PEGylation of 1 - 1000 mg of TC13 is carried out by incubating it with 1 - 1000 mg of HO-PEG-EGCG and 1 - 1000 mg of N,N’-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction is carried out with stirring at room temperature for 24 hours under a light-shielded and nitrogen atmosphere. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cut-off = 2000 Da). Next, the solution is lyophilized to obtain a lyophilized powder.

[0163] Example 9: Conjugation of Adenosine to HOOC-PEG-EGCG (Predictive Example) Objective This experiment aims to illustrate the conjugation of adenosine to HOOC-PEG-EGCG. Using HPLC, the formation of a new product (adenosine-PEG-EGCG) with a retention time different from that of HO-PEG-EGCG is detected. The structure can be confirmed using NMR.

[0164] Materials Adenosine is purchased from Sigma Aldrich. HOOC-PEG-CHO is purchased from NBC chemical.

[0165] Methods HOOC-PEG-CHO is conjugated to EGCG according to WO 2006 / 124000 pamphlet and WO 2009 / 054813 pamphlet.

[0166] Adenosine is conjugated to HOOC-PEG-EGCG via the linkage between the COOH group on PEG and the COOH group on adenosine to form adenosine-PEG-EGCG. Refer to Figure 11.

[0167] Specifically, the PEGylation of 1 - 1000 mg of adenosine is carried out by incubating it with 1 - 1000 mg of HOOC-PEG-EGCG and 1 - 1000 mg of N,N'-dicyclohexylcarbodiimide (DCC) in DMSO. The reaction is carried out with stirring at room temperature for 24 hours under light-shielding and nitrogen atmosphere. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cut-off = 2000 Da). Then, the solution is lyophilized to obtain a lyophilized powder.

[0168] Example 10: Conjugation of TfR peptide to HOOC-PLA-EGCG (predictive example) Objective This experiment aims to illustrate the conjugation of the TfR peptide to HOOC-PLA-EGCG. Using HPLC, the formation of a new product (TfR-PLA-EGCG) with a retention time different from that of HOOC-PLA-EGCG is detected. The structure can be confirmed using NMR.

[0169] Materials The TfR peptide is purchased from Hangzhou Xinbosi Biomedical Co., Ltd. HOOC-PLA-CHO is purchased from Merck (Sigma-Aldrich).

[0170] Methods HOOC-PLA-CHO is conjugated to EGCG according to WO 2006 / 124000 and WO 2009 / 054813; the TfR peptide is conjugated to HOOC-PLA-EGCG via a linkage between the COOH group on PLA and the NH2 group on TfR to form TfR-PLA-EGCG (N'-linked). See Figure 12.

[0171] Specifically, 1 - 1000 mg of TfR is incubated in DMSO with 1 - 1000 mg of HOOC-PLA-EGCG, 1 - 1000 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 1 - 1000 mg of N-hydroxysuccinimide (NHS). The reaction is stirred at room temperature for 24 hours under light protection and a nitrogen atmosphere. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cut-off = 2000 Da). Next, the solution is lyophilized to obtain a lyophilized powder.

[0172] Example 11: Conjugation of TfR Peptide to HOOC-PLGA-EGCG (Predictive Example) Objective This experiment aims to illustrate the conjugation of the TfR peptide to HOOC-PLGA-EGCG. HPLC is used to detect the formation of a new product (TfR-PLGA-EGCG) with a retention time different from that of HOOC-PLGA-EGCG. The structure can be confirmed using NMR.

[0173] Materials The TfR peptide is purchased from Hangzhou Xinbosi Biomedical Co., Ltd. HOOC-PLGA-CHO is purchased from Merck (Sigma-Aldrich).

[0174] Method HOOC-PLGA-CHO is conjugated to EGCG according to WO 2006 / 124000 and WO 2009 / 054813; the TfR peptide is conjugated to HOOC-PLGA-EGCG via a linkage between the COOH group on PLGA and the NH2 group on TfR to form TfR-PLGA-EGCG (N'-linked). See Figure 13.

[0175] Specifically, 1 - 1000 mg of TfR is incubated in DMSO with 1 - 1000 mg of HOOC-PLGA-EGCG, 1 - 1000 mg of N,N'-dicyclohexylcarbodiimide (DCC), and 1 - 1000 mg of N-hydroxysuccinimide (NHS). The reaction is stirred at room temperature for 24 hours under light protection and a nitrogen atmosphere. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cut-off = 2000 Da). Next, the solution is lyophilized to obtain a lyophilized powder.

[0176] Example 12: Conjugation of TfR Peptide to HO-Dextran-EGCG (Predictive Example) Objective This experiment aims to illustrate the conjugation of TfR to HO-dextran-EGCG. Using HPLC, the formation of a new product (TfR-dextran-EGCG) with a retention time different from that of HO-dextran-EGCG is detected. The structure can be confirmed using NMR.

[0177] Materials The TfR peptide is purchased from Hangzhou Xinbosi Biomedical. HO-dextran-CHO is purchased from Merck (Sigma-Aldrich).

[0178] Method HO-dextran-CHO is linked to EGCG according to the pamphlets of International Publication No. WO2006 / 124000 and International Publication No. WO2009 / 054813 to prepare HO-dextran-EGCG.

[0179] TfR is linked to HO-dextran-EGCG via a linkage between the OH group on dextran and the COOH group on TfR to form TfR-dextran-EGCG (C' linkage). Refer to Figure 14.

[0180] Specifically, 1 to 1000 mg of TfR is incubated in DMSO together with 1 to 1000 mg of HO-dextran-EGCG and 1 to 1000 mg of N,N'-dicyclohexylcarbodiimide (DCC). The reaction is carried out by stirring at room temperature for 24 hours under light shielding and nitrogen atmosphere. The reaction mixture is dialyzed against methanol and distilled water for 3 days (membrane molecular weight cut-off = 2000 Da). Next, the solution is lyophilized to obtain a lyophilized powder.

[0181] Example 13: Formulation of Adenosine-MINC-Doxorubicin (Predictive Example) Purpose This experiment aims to illustrate the formulation of adenosine-MINC-doxorubicin. The size of the nanoparticles is measured using DLS.

[0182] Materials Adenosine-PEG-EGCG is formulated according to Example 9. Doxorubicin is purchased from Sigma-Aldrich or other suppliers.

[0183] Methods Adenosine-MINC-doxorubicin nanoparticles are prepared according to the following protocol: 1. 5 to 500 μg of doxorubicin is incubated in 1 mL of DMSO for 15 minutes to 1 hour. 2. 1 to 100 μg of OEGCG and 1 to 10,000 μg of adenosine-PEG-EGCG are added. The mixture is incubated at 25°C for 3 hours. 3. Filter the liquid with a 10K MWCO filter unit. Wash the filter three times with 0.9% NaCl. 4. Make it into a dry powder by lyophilization.

[0184] Example 14: Efficacy study (predicted example) of TfR-MINC-anti-CD3, Tet1-MINC-anti-CD3, TC13-MINC-anti-CD3, and adenosine-MINC-anti-CD3 in an Alzheimer's disease mouse model. Objective This experiment aims to illustrate the therapeutic effects of TfR, Tet1, TC13 peptide, and adenosine conjugation. Detect brain Aβ content using histological anti-Aβ staining. Evaluate spatial working memory and exploratory activity using behavioral studies.

[0185] Materials Prepare TfR-MINC-anti-CD3 nanoparticles, Tet1-MINC-anti-CD3 nanoparticles, TC13-MINC-anti-CD3 nanoparticles, adenosine-MINC-anti-CD3, and MINC-anti-CD3 nanoparticles by replacing doxorubicin with anti-CD3 in the method taught in Example 4. Purchase anti-CD3 antibody from Biolegend.

[0186] Methods Use Tg APPsw (2576 line) mice, APP / PS1 mice, or Wistar rats. Divide the mice or rats and treat them by intravenous injection once a week for 4 - 8 weeks using a vehicle (PBS or saline as an untreated control) and 5 - 125 μg (anti-CD3 / mouse) of MINC-anti-CD3, TfR-MINC-anti-CD3 nanoparticles, Tet1-MINC-anti-CD3 nanoparticles, TC13-MINC-anti-CD3, or adenosine-MINC-anti-CD3. Sacrifice these mice or rats at 6 - 24 months of age and analyze the Aβ concentration and Aβ load in the brain. Perform quantitative Aβ image analysis using anti-β amyloid (clone 4G8).

[0187] To measure spatial working memory and exploratory activity, individual animals are placed in one arm of a radiating symmetric Y-maze made of opaque gray acrylic or other suitable maze, and behavioral tests are performed. The RAWM test or a suitable test is carried out to evaluate the behavior of mice or rats.

[0188] Example 15: Efficacy study (predictive example) of TfR-MINC-trastuzumab, Tet1-MINC-trastuzumab, TC13-MINC-trastuzumab, and adenosine-MINC-trastuzumab in a glioma mouse model. Objective This experiment aims to illustrate the therapeutic efficacy of TfR, Tet1, TC13 peptide, and adenosine conjugation in the treatment of glioma. An in vivo imaging system (IVIS) is used to measure tumor volume.

[0189] Materials Using the method taught in Example 4, TfR-MINC-trastuzumab nanoparticles, Tet1-MINC-trastuzumab nanoparticles, TC13-MINC-trastuzumab nanoparticles, adenosine-MINC-trastuzumab, and MINC-trastuzumab nanoparticles are prepared. Trastuzumab is purchased from EirGenix. The A172 glioma cell line is derived from ATCC (CRL1620). D-luciferin is purchased from Sigma-Aldrich.

[0190] Methods Using an orthotopic glioma mouse model, evaluate the efficacy of TfR-MINC-trastuzumab nanoparticles, Tet1-MINC-trastuzumab nanoparticles, TC13-MINC-trastuzumab nanoparticles, adenosine-MINC-trastuzumab, and MINC-trastuzumab nanoparticles in glioma treatment. To image tumor size, introduce the luciferase gene into the A172 cell line (A172-Luc). To generate the mouse model, drill a burr hole in the brain region of the right frontal lobe, and then slowly inject 1×10 6 cells of A172-Luc (suspended in 3 μl of DMEM) into the mouse brain. Two weeks after tumor transplantation, inject TfR-MINC-trastuzumab, Tet1-MINC-trastuzumab, TC13-MINC-trastuzumab, adenosine-MINC-trastuzumab, and MINC-trastuzumab intravenously twice a week at 0.1 - 10 mg / kg for 5 - 10 weeks. Examine tumor size every other week using an in vivo imaging system (IVIS). After anesthetizing the mouse, inject the luciferase substrate solution intraperitoneally, and then transfer the mouse to the IVIS chamber to acquire images.

[0191] Abbreviation Table ADO Adenosine HENECA N-Ethylcarboxamidoadenosine GDNF Glial cell line-derived neurotrophic factor PDGF Platelet-derived growth factor CDNF Cerebral dopamine neurotrophic factor TfR Transferrin receptor IR Insulin receptor LDLR Low-density lipoprotein receptor Dhh Desert hedgehog P75NTR p75 neurotrophin receptor NCAM Neural cell adhesion molecule PDGFRA Platelet-derived growth factor receptor α NG2 Neuron-glial antigen 2 MOG Myelin oligodendrocyte glycoprotein MBP Myelin basic protein TMEM119 Transmembrane protein 119 CX3CR1 CX3C chemokine receptor 1 CD45 Cluster of differentiation 45

Claims

1. (a) CNS target ligand; (b) hydrophilic polymer which is polyethylene glycol (PEG), polylactic acid (PLA), polylactic acid-co-glycolic acid (PLGA), or dextran; and (c) structure as shown below: 【Chemistry 1】 A complex comprising a flavonoid, which is EGCG, EC, EGC, or ECG, The hydrophilic polymer is covalently bonded to the flavonoid and the CNS-targeting ligand. The CNS-targeted ligand is TfR peptide having amino acid sequence THRPPMWSPWPP (SEQ ID NO: 1), Tet1 peptide having amino acid sequence HLNILSTLWKYRC (SEQ ID NO: 2), TC13 peptide having the amino acid sequence TGNYKALHPHNGC (SEQ ID NO: 3), Apamin peptide having the amino acid sequence CNCKAPETALCARRCQQH (SEQ ID NO: 4), A regulon polypeptide having the amino acid sequence PTVIHGKREVTLHL (SEQ ID NO: 5), RAP peptide having the amino acid sequence ELKHFEAKIEKHNHYQKQLE (SEQ ID NO: 6), Angiopep-2 peptide having the amino acid sequence TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 7), TAT peptide having the amino acid sequence GGGGYGRKKRRQRRR (SEQ ID NO: 8), SynB1 peptide having the amino acid sequence RGGRLSYSRRRFSTSTGR (SEQ ID NO: 9), Leptin 30 peptide having the amino acid sequence YQQVLTSLPSQNVLQIANDLENLRDLLHLLC (SEQ ID NO: 10), LNP peptide having the amino acid sequence KKRTLRKNDRKKRC (SEQ ID NO: 11), ApoB peptide having the amino acid sequence SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS (SEQ ID NO: 12), RVG-29 peptide having the amino acid sequence YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 13), T7 peptide having amino acid sequence HAIYPRH (SEQ ID NO: 14), GSH peptide having amino acid sequence ECG, CRT peptide having amino acid sequence CRTIGPSVC (SEQ ID NO: 15), CAQK peptide having the amino acid sequence CAQK (SEQ ID NO: 16), TACL05 peptide having the amino acid sequence SACPSHLTKMCGGG (SEQ ID NO: 17), Adenosine, 5'-N-ethylcarboxamide adenosine, glutamic acid or glutamate, and gamma-aminobutyric acid (GABA) Selected from the group consisting of, A complex.

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

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

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

5. The nanoparticle composition according to 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 according to claim 4, further comprising a bare hydrophilic polymer-flavonoid complex in which the outer shell is not covalently bonded to the CNS-targeting ligand.

7. A pharmaceutical composition for use in a method for treating CNS disease, comprising the nanoparticle composition described in claim 4.

8. The pharmaceutical composition according to 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 pharmaceutical composition according to 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 pharmaceutical composition according to 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, anti-α-synuclein, anti-IL6R, anti-IL-1β, anti-CD38, anti-TREM2, GDNF, NRTN, PDGF-BB, CDNF, or BDNF.

11. The pharmaceutical composition according to claim 7, wherein 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.

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