Pamam-peg-based nanoparticles for targeted protein degradation

Abstract

Disclosed are a substance represented by general formula (I), a substance represented by general formula (II), and a substance represented by general formula (III) for degrading a protein of interest (POI) in a ubiquitin-proteasome system (UPS): PEG-Ra general formula (I) PEG-Rb general formula (II) (Ra-PEG)m-PAMAM-(PEG-Rb)n general formula (III), wherein Ra, Rb, m, and n are as defined in the present disclosure.

Description

PAMAM-PEG-based nanoparticles for targeted protein degradation

[0001] Citation of relevant applications

[0002] This disclosure claims the full benefits of Chinese Patent Application No. 202410956847.0, filed on July 16, 2024 with the State Intellectual Property Office of the People's Republic of China, entitled "Polymer Nanoparticles for Protein Degradation Targeted Chimeras", the entire contents of which are incorporated herein by reference.

[0003] field

[0004] This disclosure generally relates to the biomedical field, and more specifically, to the field of targeted protein degradation.

[0005] background

[0006] Since its first report in 2001, targeted protein degradation (TPD) technology has attracted widespread attention and research, and several TPD entities have now entered the clinical research stage.

[0007] Overview

[0008] On the one hand, this disclosure relates to a substance of general formula (IV) for the degradation of target proteins (POIs) in lysosomes: PEG-R c General Formula (IV)

[0009] Wherein, PEG is polyethylene glycol, and R c The receptors were selected from cell surface transmembrane receptors (CI-M6PR), desialyl glycoprotein receptors (ASGPR), LC3, autophagy cargo receptor p62, heat shock proteins, integrins, and ADRM1-Halotag7.

[0010] On the other hand, this disclosure relates to a substance of general formula (V) for the degradation of target proteins (POIs) in lysosomes: PEG-R d General formula (V)

[0011] Wherein, PEG is polyethylene glycol, and R d It is a ligand for the target protein (POI).

[0012] Furthermore, this disclosure relates to a substance of general formula (VI) for the degradation of a target protein (POI) in lysosomes: (R c -PEG) m-PAMAM-(PEG-R d ) n General Formula (VI)

[0013] Wherein, PEG is polyethylene glycol, R c The receptors include cell surface transmembrane receptor (CI-M6PR), desialyl glycoprotein receptor (ASGPR), LC3, autophagy cargo receptor p62, heat shock protein, integrin, and ADRM1-Halotag7. d The target protein (POI) ligand is PAMAM, which is a poly(amidoamine)dendrimer, and m and n are each independently selected from real numbers from 1 to 10.

[0014] In another aspect, this disclosure relates to nanoparticles comprising a core and an outer layer, wherein the core is PAMAM and the outer layer comprises a substance represented by formula (IV) and a substance represented by formula (V) of this disclosure.

[0015] On the other hand, this disclosure relates to pharmaceutical compositions comprising the nanoparticles described herein, and pharmaceutically acceptable excipients.

[0016] Furthermore, this disclosure relates to a substance of general formula (I) for the degradation of a target protein (POI) in a ubiquitin-proteasome system (UPS): PEG-R a General Formula (I)

[0017] Wherein, PEG is polyethylene glycol, and R a It is an E3 ubiquitin ligand.

[0018] In another aspect, this disclosure relates to a substance of general formula (II) for the degradation of a target protein (POI) in a ubiquitin-proteasome system (UPS): PEG-R b General Formula (II)

[0019] Wherein, PEG is polyethylene glycol, and R b It is a ligand for the target protein (POI).

[0020] On the other hand, this disclosure relates to a substance of general formula (III) for the degradation of a target protein (POI) in a ubiquitin-proteasome system (UPS): (R a -PEG) m -PAMAM-(PEG-R b ) n General Formula (III)

[0021] Wherein, PEG is polyethylene glycol, R a For E3 ubiquitin ligase ligand, R b The target protein (POI) ligand is PAMAM, which is a poly(amidoamine)dendrimer, and m and n are each independently selected from real numbers from 1 to 10.

[0022] In another aspect, this disclosure relates to nanoparticles comprising a core and an outer layer, wherein the core is PAMAM and the outer layer comprises a substance represented by formula (I) and a substance represented by formula (II) of this disclosure.

[0023] In another aspect, this disclosure relates to pharmaceutical compositions comprising the nanoparticles described herein, and pharmaceutically acceptable excipients.

[0024] On the other hand, this disclosure relates to a method for preparing nanoparticles, comprising:

[0025] The substance represented by general formula (I) of this disclosure is dissolved in a first polar solvent to obtain a first stock solution;

[0026] The substance of general formula (II) described herein is dissolved in a second polar solvent to obtain a second stock solution; and

[0027] The first and second stock solutions are dissolved in an organic solvent, and the nanoparticles are obtained through a chemical reaction.

[0028] Furthermore, this disclosure relates to a method for preparing a substance of general formula (III) for the degradation of a target protein (POI) in a ubiquitin-proteasome system (UPS): (R a -PEG) m -PAMAM-(PEG-R b ) n General Formula (III)

[0029] The method includes processing the substance PEG-R represented by general formula (I). a General Formula (I)

[0030] The substance PEG-R represented by general formula (II) b General Formula (II)

[0031] A chemical reaction is carried out with PAMAM to obtain the substance represented by general formula (III).

[0032] Wherein, PAMAM is a poly(amidoamine) dendrimer, PEG is polyethylene glycol, and R... a For E3 ubiquitin ligase ligand, R b The target protein (POI) ligand, and m and n are each independently selected from real numbers from 1 to 10.

[0033] In another aspect, this disclosure relates to a method for degrading a target protein (POI), comprising administering the nanoparticles and E3 ubiquitin ligase described in this disclosure to the target protein (POI).

[0034] On the other hand, this disclosure relates to a method of treating tumors, comprising administering to an individual in need of the method a therapeutically effective amount of the nanoparticles of this disclosure, or a therapeutically effective amount of the pharmaceutical composition of this disclosure.

[0035] Brief description of the attached figures

[0036] Figure 1 shows a schematic diagram of the synthesis of the substance represented by general formula (III);

[0037] Figure 2 shows COOH-PEG-VHL 1 H spectrum;

[0038] Figure 3 shows COOH-PEG-TMXF 1 H spectrum;

[0039] Figure 4 shows the PAMAM-PEG5K-JQ1_VHL. 1 H spectrum;

[0040] Figure 5 shows the morphology of PAMAM-PEG-R nanoparticles under SEM.

[0041] Figure 6 shows the half-maximal inhibitory concentration (WMC) curves of nanoparticles on different cell lines: (a) growth inhibition curves of PAMAM-PEG-TMXF_VHL and PAMAM-PEG-TMXF_CRBN on T47D cell line; (b) growth inhibition curves of PAMAM-PEG-JQ1_VHL and PAMAM-PEG-JQ1_CRBN on HCT116 cell line.

[0042] Figure 7 shows the membrane penetration effects of the positive control (TAT peptide) and polymers as presented by flow cytometry. From top to bottom, they are: TAT peptide; PLA-PEG-FITC nanoparticles; PAMAM-PEG-FITC; COOH-PEG-FITC; PLA-PEG-FITC; blank control (three replicates per group).

[0043] Figure 8 shows the degradation of target proteins by some polymer nanoparticles: (a) the degradation of ER in T47D cell line by PAMAM-PEG-TMXF_VHL and PAMAM-PEG-TMXF_CRBN; (b) the degradation of BRD4 in HCT116 cell line by PAMAM-PEG-JQ1_VHL and PAMAM-PEG-JQ1_CRBN.

[0044] Figure 9 shows the recovery of target proteins after degradation by some polymers: the recovery of BRD4 in HCT116 cell line after degradation by PAMAM-PEG-JQ1_CRBN.

[0045] Figure 10 shows the degradation of BRD4 in HCT116 by some polymers at different times: (a) degradation of BRD4 by PAMAM-PEG-JQ1_VHL at different times; (b) degradation of BRD4 by PAMAM-PEG-JQ1_CRBN at different times.

[0046] Figure 11 shows the effect of not using a negative control on HCT116 cells and the effect on BRD4 protein in HCT116, highlighting the significance of PAMAM-PEG-JQ1_VHL and PAMAM-PEG-JQ1_CRBN nanoparticles.

[0047] Figure 12 shows COOH-PEG-CRBN 1 H spectrum;

[0048] Figure 13 shows COOH-PEG-JQ1 1 H spectrum;

[0049] Figure 14 shows PAMAM-PEG-JQ1_CRBN 1 H spectrum;

[0050] Figure 15 shows the degradation of BRD4 and GAPDH in HeLa cell lines by PAMAM-PEG-JQ1_VHL and PAMAM-PEG-JQ1_CRBN;

[0051] Figure 16 shows the degradation of BRD4 and GAPDH in the MCF-7 cell line by PAMAM-PEG-JQ1_VHL and PAMAM-PEG-JQ1_CRBN;

[0052] Figure 17 shows the drug administration regimen in Balb / c mice. A CDX model was constructed using the HCT116 cell line, and the drug was administered intraperitoneally to the mice every 7 days.

[0053] Figure 18 shows the body weight (Figure 18A), tumor volume (Figure 18B), tumor weight (Figure 18C), tumor images (Figure 18D), and tumor growth inhibition rate in different groups of mice (Figure 18E). The treatment groups were: ① control group (PBS); ② PAMAM-PEG-JQ1 (20 mg / kg) + PAMAM-PEG-CRBN (20 mg / kg); ③ ARV-825 (20 mg / kg); ④ PAMAM-PEG-JQ1_CRBN (10 mg / kg); ⑤ PAMAM-PEG-JQ1_CRBN (20 mg / kg).

[0054] Figure 19 shows hematoxylin-eosin (HE) staining images of the heart, liver, spleen, lungs, kidneys and brain after treatment with different drugs;

[0055] Figure 20 shows the drug administration regimen for Balb / c mice. A CDX model was constructed using the HCT116 cell line, and the drugs were administered orally to the mice every 3 or 7 days.

[0056] Figure 21 shows the drug administration regimen for Balb / c mice. A CDX model was constructed using the HCT116 cell line, and the mice were orally administered the drug daily.

[0057] Figure 22 shows the changes in mouse body weight throughout the treatment process (Figure 21A), where NS indicates no significant change; the mean tumor growth curve for each group throughout the treatment process (Figure 21B); the tumor tissue weight statistics at the experimental endpoint (Figure 21C); an image of tumor tissue at the experimental endpoint using a 10mm ruler (Figure 21D); and the calculated tumor growth inhibition rate in different groups of mice (Figure 21E). Tumor growth inhibition rate (tumor weight in the PBS group - tumor weight in each group) / tumor weight in the PBS group × 100%. Data were analyzed using GraphPad Prism 8 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001), with the following treatment groups: ⑥ Control group (PBS); ⑦ PAMAM-PEG-JQ1 (20mg / kg); ⑧ ARV-825 (20mg / kg); ⑨ PAMAM-PEG-JQ1_CRBN (10mg / kg); ⑩ PAMAM-PEG-JQ1_CRBN (20mg / kg); and

[0058] Figure 23 shows hematoxylin-eosin (HE) staining images of the heart, liver, spleen, lungs, kidneys, and brain after treatment with different drugs.

[0059] Detailed Explanation

[0060] The following description includes certain specific details to provide a comprehensive understanding of the various disclosed embodiments. However, those skilled in the art will recognize that the embodiments can still be implemented without employing one or more of these specific details, but using other methods, components, materials, etc.

[0061] Unless otherwise required in this application, throughout the specification and the appended claims, the words “comprising,” “including,” “containing,” and “having” shall be interpreted in an open-ended, inclusive sense, meaning “including but not limited to.”

[0062] When used in this disclosure and the appended claims, a singular designation without a quantity indication includes a plural designation unless the context clearly specifies otherwise.

[0063] Throughout this specification, the terms "an embodiment," "an embodiment," "in another embodiment," or "in some embodiments" refer to including, in at least one embodiment, a specific reference element, structure, or feature related to that embodiment. Therefore, the phrases "in an embodiment," "in another embodiment," or "in some embodiments" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, specific elements, structures, or features may be combined in one or more embodiments in any suitable manner.

[0064] It should be understood that the singular article “a” (corresponding to the English words “a,” “an,” and “the”) used in this disclosure and the appended claims includes plural objects unless otherwise expressly stated herein. Thus, for example, a microsphere comprising “a compound of formula (I) and a compound of formula (II)” includes one compound of formula (I) or one compound of formula (II), or two or more compounds of formula (I) or two or more compounds of formula (II).

[0065] definition

[0066] Therefore, unless otherwise stated, the following terms used in the specification and appended claims shall have the following meanings:

[0067] In this disclosure, the term "Ubiquitin-Proteasome System (UPS)" refers to a system composed of ubiquitin (Ub), ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), ubiquitin protein ligase (E3), proteasome, and their substrates (proteins). This system is the main pathway for intracellular protein degradation and is involved in the degradation of more than 80% of intracellular proteins.

[0068] In this disclosure, the term "ubiquitin ligase E3" refers to an enzyme capable of attaching a ubiquitin molecule to a lysine residue of a target protein.

[0069] In this disclosure, the term "target protein (POI)" refers to a protein in the body that has pharmacological function and can be acted upon by drugs.

[0070] In this disclosure, the term "ubiquitin" refers to a small protein found in most eukaryotic cells. Its primary function is to mark proteins that need to be broken down, causing them to be hydrolyzed.

[0071] In this disclosure, the term "proteasome" refers to a multi-subunit complex widely distributed in the cytoplasm and nucleus. It is composed of approximately 50 protein subunits. It possesses various proteolytic enzyme activities and can degrade ubiquitin-labeled target proteins.

[0072] In this disclosure, the term "protein degradation targeting chimera (PROTAC)" refers to a POI and E3 recruitment module formed by linking a target protein (POI) ligand molecule and a ubiquitin ligase E3 ligand molecule to the core motif PAMAM. After entering the cell, this nanoparticle can recruit the target protein (POI) and ubiquitin ligase E3, so that the target protein (POI) is labeled with ubiquitin molecules, thereby being recognized and degraded by the proteasome.

[0073] In this disclosure, the term "lysosome" generally refers to an organelle in eukaryotic cells; it is a sac-like structure enclosed by a single membrane, with a diameter of approximately 0.025 to 0.8 micrometers (mostly spherical under an electron microscope, but some are oval); it contains a variety of hydrolytic enzymes specifically designed to break down various exogenous and endogenous macromolecules.

[0074] In this disclosure, the term "cell surface transmembrane receptor" refers to key signal transduction molecules on the cell membrane, such as ion channel receptors, G protein-coupled receptors, enzyme-linked receptors, and integrin receptors.

[0075] In this disclosure, the term "desialidylglycoprotein receptor" refers to a receptor that is primarily expressed on the surface of cells in the sinusoidal space and basolateral lateral basolateral region of the liver, which specifically recognizes, binds to, and mediates the endocytosis of desialyl glycoproteins with galactose or acetylgalactose residues at the terminal end.

[0076] In this disclosure, the term "LC3" refers to microtubule-associated protein 1A / 1B-light chain 3 (MAP1LC3), a soluble protein with a molecular weight of approximately 17 kDa. LC3 is ubiquitous in mammalian tissues and cultured cells and is a key component of autophagy (the recycling system in eukaryotic cells). It is incorporated into the inner and outer membranes of autophagosomes during autophagosome biosynthesis. Therefore, LC3 is a specific marker of autophagy, particularly autophagosome formation.

[0077] In this disclosure, the term "autophagy cargo receptor" refers to a adaptor protein that plays a key role in selective autophagy and is responsible for specifically recognizing substrates to be degraded (protein aggregates, damaged organelles, or pathogens).

[0078] In this disclosure, the term "heat shock proteins (HSPs)" refers to a class of functionally related proteins whose expression increases when cells are subjected to elevated temperatures or other stresses, and which assist in the normal folding of proteins.

[0079] In this disclosure, the term "integrin" refers to a transmembrane receptor that mediates the connection between a cell and its external environment (such as the extracellular matrix) and is ubiquitous on the cell surface of vertebrates.

[0080] In this disclosure, the term "ADRM1-Halotag7" refers to a fusion protein produced by fusing the HaloTag7 protein with the ADRM1 protein.

[0081] In this disclosure, the term "poly(amidoamine)dendrimer" refers to a class of dendritic macromolecules composed of repeating branched subunits of amide and amine functional groups.

[0082] In this disclosure, the term "polyethylene glycol (PEG)" refers to a general term for ethylene glycol polymers containing α,ω-terminated hydroxyl groups.

[0083] In this disclosure, the term "ligand" refers to a substance that has the ability to recognize and bind to a receptor.

[0084] In this disclosure, the term "drug-acceptable" means a carrier, delivery unit, diluent, excipient, and / or salt that must be compatible with other components of the formulation and not be harmful to the recipient.

[0085] In this disclosure, the term "pharmaceutical acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the U.S. Food and Drug Administration for use in humans or animals and has no adverse effects on the composition of the pharmaceutical composition.

[0086] In this disclosure, the terms “arbitrary” or “optionally” mean that the event or condition described below may or may not occur, and the description includes both the occurrence and non-occurrence of the event or condition.

[0087] In this disclosure, the term "mammal" refers to animals including, for example, dogs, cats, cattle, sheep, horses, and humans. In some embodiments, mammals include humans.

[0088] In this disclosure, the term "patient" refers to animals (e.g., humans), companion animals (e.g., dogs, cats, or horses), and livestock (e.g., cattle, pigs, and sheep). In some embodiments, the patient is a mammal that includes both males and females. In some embodiments, the patient is a human.

[0089] In this disclosure, the term "pharmaceutical composition" refers to an formulation formed by the compound described herein with a medium generally accepted in the art for delivering a bioactive compound to a mammal such as a human. Such a medium includes all pharmaceutically acceptable carriers, diluents, or excipients.

[0090] In this disclosure, the term "therapeuticly effective amount" refers to the amount of a compound or combination of compounds that improves, reduces, or eliminates a particular disease or condition and its symptoms, or avoids or delays the onset of a particular disease or condition or its symptoms. The amount of the compound constituting a "therapeuticly effective amount" described in this disclosure will vary depending on the compound, the disease state and its severity, and the age, weight, etc., of the mammal to be treated; however, those skilled in the art can determine the amount of the compound described in this disclosure conventionally based on their own knowledge and this disclosure.

[0091] As used in this disclosure, "to treat" or "to treat" encompasses the treatment of a related disease or condition in mammals, such as humans, suffering from a related disease or ailment, and includes:

[0092] (i) To prevent the occurrence of disease or disease state in mammals, especially when the mammal is susceptible to the disease state but has not yet been diagnosed with the disease state;

[0093] (ii) Suppress the disease or disease state, that is, prevent it from occurring; or

[0094] (iii) Alleviate the disease or disease state, even if the disease or disease state subsides or does not progress.

[0095] As used in this disclosure, the terms “disease” and “disease state” may be used interchangeably or may be different, because a particular disease or disease state may not have a known causative agent (and therefore cannot be explained by etiology), and thus is not recognized as a disease, but rather as an undesirable disease state or symptom in which a clinician has identified a more or less specific set of symptoms. Detailed Implementation

[0096] On the one hand, this disclosure relates to a substance of general formula (IV) for the degradation of target proteins (POIs) in lysosomes: PEG-R c General Formula (IV)

[0097] Wherein, PEG is polyethylene glycol, and R c The receptors were selected from cell surface transmembrane receptors (CI-M6PR), desialyl glycoprotein receptors (ASGPR), LC3, autophagy cargo receptor p62, heat shock proteins, integrins, and ADRM1-Halotag7.

[0098] In some embodiments, the molecular weight of the exemplary PEG that can be used in this disclosure is from 5,000 to 45,000.

[0099] In some implementations, exemplary examples of PEGs that can be used in this disclosure include, but are not limited to, PEG5000, PEG6000, PEG8000, PEG10000, PEG20000, PEG30000 and PEG45000.

[0100] In some implementations, exemplary instances R that can be used in this disclosure c Including but not limited to:

[0101] In some embodiments, the substance represented by general formula (IV) is selected from...

[0102] Where n ranges from 116 to 1047.

[0103] On the other hand, this disclosure relates to a substance of general formula (V) for the degradation of target proteins (POIs) in lysosomes: PEG-R d General formula (V)

[0104] Wherein, PEG is polyethylene glycol, and R d It is a ligand for the target protein (POI).

[0105] In some embodiments, the molecular weight of the exemplary PEG that can be used in this disclosure is from 5,000 to 45,000.

[0106] In some implementations, examples of exemplary PEGs that can be used in this disclosure include, but are not limited to, PEG5000, PEG6000, PEG8000, PEG10000, PEG20000, PEG30000 and PEG45000.

[0107] In some embodiments, exemplary examples of target protein (POI) ligands that can be used in this disclosure include, but are not limited to, estrogen receptor (ERα) ligands, androgen receptor (AR) ligands, epidermal growth factor receptor (EGFR) ligands, human bromine-containing protein 2 / 4 (BRD2 / 4) ligands, mitogen-activated protein kinase kinase 1 and 2 (MEK1 / 2) ligands, cyclin-dependent kinase 4 / 6 (CDK4 / 6) ligands, and BCR-ABL ligands.

[0108] In some embodiments, exemplary examples of estrogen receptor (ERα) ligands that can be used in this disclosure include, but are not limited to:

[0109] In some embodiments, exemplary examples of androgen receptor (AR) ligands that can be used in this disclosure include, but are not limited to:

[0110] In some embodiments, exemplary examples of epidermal growth factor receptor (EGFR) ligands that can be used in this disclosure include, but are not limited to:

[0111] In some embodiments, exemplary examples of human bromine-containing region protein 2 / 4 (BRD2 / 4) ligands that can be used in this disclosure include, but are not limited to:

[0112] In some embodiments, exemplary examples of mitogen-activated protein kinase kinase 1 and 2 (MEK1 / 2) ligands that can be used in this disclosure include, but are not limited to:

[0113] In some embodiments, exemplary examples of cyclin-dependent kinase 4 / 6 (CDK4 / 6) ligands that can be used in this disclosure include, but are not limited to:

[0114] In some implementations, exemplary examples of BCR-ABL ligands that can be used in this disclosure include, but are not limited to:

[0115] In some embodiments, exemplary examples of target protein (POI) ligands that can be used in this disclosure include, but are not limited to:

[0116] In some embodiments, the substance represented by general formula (V) is selected from...

[0117] Where n ranges from 116 to 1047.

[0118] Furthermore, this disclosure relates to a substance of general formula (VI) for the degradation of a target protein (POI) in lysosomes: (R c -PEG) m -PAMAM-(PEG-R d ) n General Formula (VI)

[0119] Wherein, PEG is polyethylene glycol, R cThe receptors include cell surface transmembrane receptor (CI-M6PR), desialyl glycoprotein receptor (ASGPR), LC3, autophagy cargo receptor p62, heat shock protein, integrin, and ADRM1-Halotag7. d The target protein (POI) ligand is PAMAM, which is a poly(amidoamine)dendrimer, and m and n are each independently selected from real numbers from 1 to 10.

[0120] In some implementations, the PAMAM component is between 14,000 and 60,000.

[0121] In some embodiments, exemplary examples of PAMAMs that can be used in this disclosure include, but are not limited to, those selected from fourth-generation polyamide-amine dendrimers (G4-PAMAM), fifth-generation polyamide-amine dendrimers (G5-PAMAM), sixth-generation polyamide-amine dendrimers (G6-PAMAM), and seventh-generation polyamide-amine dendrimers (G7-PAMAM).

[0122] In some embodiments, the molecular weight of the exemplary PEG that can be used in this disclosure is about 5,000 to 45,000.

[0123] In some implementations, exemplary examples of PEGs that can be used in this disclosure include, but are not limited to, PEG5000, PEG6000, PEG8000, PEG10000, PEG20000, PEG30000 and PEG45000.

[0124] In some implementations, exemplary instances R that can be used in this disclosure c Including but not limited to:

[0125] In some embodiments, the substance represented by general formula (IV) is selected from...

[0126] Where n ranges from 116 to 1047.

[0127] In some embodiments, exemplary examples of target protein (POI) ligands that can be used in this disclosure include, but are not limited to, estrogen receptor (ERα) ligands, androgen receptor (AR) ligands, epidermal growth factor receptor (EGFR) ligands, human bromine-containing protein 2 / 4 (BRD2 / 4) ligands, mitogen-activated protein kinase kinase 1 and 2 (MEK1 / 2) ligands, cyclin-dependent kinase 4 / 6 (CDK4 / 6) ligands, and BCR-ABL ligands.

[0128] In some embodiments, exemplary examples of estrogen receptor (ERα) ligands that can be used in this disclosure include, but are not limited to:

[0129] In some embodiments, exemplary examples of androgen receptor (AR) ligands that can be used in this disclosure include, but are not limited to:

[0130] In some embodiments, exemplary examples of epidermal growth factor receptor (EGFR) ligands that can be used in this disclosure include, but are not limited to:

[0131] In some embodiments, exemplary examples of human bromine-containing region protein 2 / 4 (BRD2 / 4) ligands that can be used in this disclosure include, but are not limited to:

[0132] In some embodiments, exemplary examples of mitogen-activated protein kinase kinase 1 and 2 (MEK1 / 2) ligands that can be used in this disclosure include, but are not limited to:

[0133] In some embodiments, exemplary examples of cyclin-dependent kinase 4 / 6 (CDK4 / 6) ligands that can be used in this disclosure include, but are not limited to:

[0134] In some implementations, exemplary examples of BCR-ABL ligands that can be used in this disclosure include, but are not limited to:

[0135] In some embodiments, exemplary examples of target protein (POI) ligands that can be used in this disclosure include, but are not limited to:

[0136] In some embodiments, the substance represented by general formula (V) is selected from...

[0137] In another aspect, this disclosure relates to nanoparticles comprising a core and an outer layer, wherein the core is PAMAM and the outer layer comprises a substance represented by formula (IV) and a substance represented by formula (V) of this disclosure.

[0138] In some embodiments, in the nanoparticles described in this disclosure, the molar ratio of the substance represented by general formula (IV) to the substance represented by general formula (V) is m:n, wherein m and n are each independently selected from real numbers from 1 to 10.

[0139] In some embodiments, the molar ratio of the substance represented by formula (IV) to the substance represented by formula (V) in the nanoparticles described in this disclosure is 1:1.

[0140] In some implementations, the PAMAM component is between 14,000 and 60,000.

[0141] In some embodiments, exemplary examples of PAMAMs that can be used in this disclosure include, but are not limited to, those selected from fourth-generation polyamide-amine dendrimers (G4-PAMAM), fifth-generation polyamide-amine dendrimers (G5-PAMAM), sixth-generation polyamide-amine dendrimers (G6-PAMAM), and seventh-generation polyamide-amine dendrimers (G7-PAMAM).

[0142] In some embodiments, the nanoparticles of this disclosure effectively combine the advantages of protein degradation targeting chimeras and nanoparticle delivery systems, circumventing some of the shortcomings of existing small molecule protein degradation targeting chimeras.

[0143] In some embodiments, the nanoparticles of this disclosure have high bioavailability.

[0144] In some embodiments, the nanoparticles of this disclosure exhibit superior membrane-penetrating properties.

[0145] In some embodiments, the nanoparticles of this disclosure have a low probability of off-target aggregation.

[0146] In some embodiments, the nanoparticles of this disclosure have high safety.

[0147] In some embodiments, the nanoparticles of this disclosure are non-immunogenic.

[0148] In some embodiments, the nanoparticles of this disclosure are obtained by chemical coupling of the substance represented by general formula (IV) and the substance represented by general formula (V) with PAMAM.

[0149] On the other hand, this disclosure relates to pharmaceutical compositions comprising the nanoparticles described herein, and pharmaceutically acceptable excipients.

[0150] Furthermore, this disclosure relates to a substance of general formula (I) for the degradation of a target protein (POI) in a ubiquitin-proteasome system (UPS): PEG-R a General Formula (I)

[0151] Wherein, PEG is polyethylene glycol, and R aIt is an E3 ubiquitin ligand.

[0152] In some embodiments, the molecular weight of the exemplary PEG that can be used in this disclosure is from 5,000 to 45,000.

[0153] In some implementations, exemplary examples of PEGs that can be used in this disclosure include, but are not limited to, PEG5000, PEG6000, PEG8000, PEG10000, PEG20000, PEG30000 and PEG45000.

[0154] In some embodiments, exemplary examples of E3 ubiquitin ligands that can be used in this disclosure include, but are not limited to, VHL ligands, MDM2 ligands, clAP1 ligands, and CRBN ligands.

[0155] In some implementations, exemplary examples of VHL ligands that can be used in this disclosure include, but are not limited to:

[0156] In some implementations, exemplary examples of the MDM2 ligands that can be used in this disclosure include, but are not limited to:

[0157] In some implementations, exemplary examples of clAP1 ligands that can be used in this disclosure include, but are not limited to:

[0158] In some implementations, exemplary examples of CRBN ligands that can be used in this disclosure include, but are not limited to:

[0159] In some embodiments, exemplary examples of E3 ubiquitin ligase ligands that can be used in this disclosure include, but are not limited to:

[0160] In some embodiments, the substance represented by general formula (I) is selected from...

[0161] Where n ranges from 116 to 1047.

[0162] In another aspect, this disclosure relates to a substance of general formula (II) for the degradation of a target protein (POI) in a ubiquitin-proteasome system (UPS): PEG-R b General Formula (II)

[0163] Wherein, PEG is polyethylene glycol, and R b It is a ligand for the target protein (POI).

[0164] In some embodiments, the molecular weight of the exemplary PEG that can be used in this disclosure is from 5,000 to 45,000.

[0165] In some implementations, examples of exemplary PEGs that can be used in this disclosure include, but are not limited to, PEG5000, PEG6000, PEG8000, PEG10000, PEG20000, PEG30000 and PEG45000.

[0166] In some embodiments, exemplary examples of target protein (POI) ligands that can be used in this disclosure include, but are not limited to, estrogen receptor (ERα) ligands, androgen receptor (AR) ligands, epidermal growth factor receptor (EGFR) ligands, human bromine-containing protein 2 / 4 (BRD2 / 4) ligands, mitogen-activated protein kinase kinase 1 and 2 (MEK1 / 2) ligands, cyclin-dependent kinase 4 / 6 (CDK4 / 6) ligands, and BCR-ABL ligands.

[0167] In some embodiments, exemplary examples of estrogen receptor (ERα) ligands that can be used in this disclosure include, but are not limited to:

[0168] In some embodiments, exemplary examples of androgen receptor (AR) ligands that can be used in this disclosure include, but are not limited to:

[0169] In some embodiments, exemplary examples of epidermal growth factor receptor (EGFR) ligands that can be used in this disclosure include, but are not limited to:

[0170] In some embodiments, exemplary examples of human bromine-containing region protein 2 / 4 (BRD2 / 4) ligands that can be used in this disclosure include, but are not limited to:

[0171] In some embodiments, exemplary examples of mitogen-activated protein kinase kinase 1 and 2 (MEK1 / 2) ligands that can be used in this disclosure include, but are not limited to:

[0172] In some embodiments, exemplary examples of cyclin-dependent kinase 4 / 6 (CDK4 / 6) ligands that can be used in this disclosure include, but are not limited to:

[0173] In some implementations, exemplary examples of BCR-ABL ligands that can be used in this disclosure include, but are not limited to:

[0174] In some embodiments, exemplary examples of target protein (POI) ligands that can be used in this disclosure include, but are not limited to:

[0175] In some embodiments, the substance represented by general formula (II) is selected from...

[0176] Where n ranges from 116 to 1047.

[0177] On the other hand, this disclosure relates to a substance of general formula (III) for the degradation of a target protein (POI) in a ubiquitin-proteasome system (UPS): (R a -PEG) m -PAMAM-(PEG-R b ) n General Formula (III)

[0178] Wherein, PEG is polyethylene glycol, R a For E3 ubiquitin ligase ligand, R b The target protein (POI) ligand is PAMAM, which is a poly(amidoamine)dendrimer, and m and n are each independently selected from real numbers from 1 to 10.

[0179] In some implementations, the PAMAM component is between 14,000 and 60,000.

[0180] In some embodiments, exemplary examples of PAMAMs that can be used in this disclosure include, but are not limited to, those selected from fourth-generation polyamide-amine dendrimers (G4-PAMAM), fifth-generation polyamide-amine dendrimers (G5-PAMAM), sixth-generation polyamide-amine dendrimers (G6-PAMAM), and seventh-generation polyamide-amine dendrimers (G7-PAMAM).

[0181] In some embodiments, the molecular weight of the exemplary PEG that can be used in this disclosure is about 5,000 to 45,000.

[0182] In some implementations, exemplary examples of PEGs that can be used in this disclosure include, but are not limited to, PEG5000, PEG6000, PEG8000, PEG10000, PEG20000, PEG30000 and PEG45000.

[0183] In some embodiments, exemplary examples of E3 ubiquitin ligands that can be used in this disclosure include, but are not limited to, VHL ligands, MDM2 ligands, clAP1 ligands, and CRBN ligands.

[0184] In some implementations, exemplary examples of VHL ligands that can be used in this disclosure include, but are not limited to:

[0185] In some implementations, exemplary examples of the MDM2 ligands that can be used in this disclosure include, but are not limited to:

[0186] In some implementations, exemplary examples of clAP1 ligands that can be used in this disclosure include, but are not limited to:

[0187] In some implementations, exemplary examples of CRBN ligands that can be used in this disclosure include, but are not limited to:

[0188] In some embodiments, exemplary examples of E3 ubiquitin ligase ligands that can be used in this disclosure include, but are not limited to:

[0189] In some embodiments, the substance represented by general formula (I) is selected from...

[0190] Where n ranges from 116 to 1047.

[0191] In some embodiments, exemplary examples of target protein (POI) ligands that can be used in this disclosure include, but are not limited to, estrogen receptor (ERα) ligands, androgen receptor (AR) ligands, epidermal growth factor receptor (EGFR) ligands, human bromine-containing protein 2 / 4 (BRD2 / 4) ligands, mitogen-activated protein kinase kinase 1 and 2 (MEK1 / 2) ligands, cyclin-dependent kinase 4 / 6 (CDK4 / 6) ligands, and BCR-ABL ligands.

[0192] In some embodiments, exemplary examples of estrogen receptor (ERα) ligands that can be used in this disclosure include, but are not limited to:

[0193] In some embodiments, exemplary examples of androgen receptor (AR) ligands that can be used in this disclosure include, but are not limited to:

[0194] In some embodiments, exemplary examples of epidermal growth factor receptor (EGFR) ligands that can be used in this disclosure include, but are not limited to:

[0195] In some embodiments, exemplary examples of human bromine-containing region protein 2 / 4 (BRD2 / 4) ligands that can be used in this disclosure include, but are not limited to:

[0196] In some embodiments, exemplary examples of mitogen-activated protein kinase kinase 1 and 2 (MEK1 / 2) ligands that can be used in this disclosure include, but are not limited to:

[0197] In some embodiments, exemplary examples of cyclin-dependent kinase 4 / 6 (CDK4 / 6) ligands that can be used in this disclosure include, but are not limited to:

[0198] In some implementations, exemplary examples of BCR-ABL ligands that can be used in this disclosure include, but are not limited to:

[0199] In some embodiments, exemplary examples of target protein (POI) ligands that can be used in this disclosure include, but are not limited to:

[0200] In some embodiments, the substance represented by general formula (II) is selected from...

[0201] In another aspect, this disclosure relates to nanoparticles comprising a core and an outer layer, wherein the core is PAMAM and the outer layer comprises a substance represented by formula (I) and a substance represented by formula (II) of this disclosure.

[0202] In some embodiments, in the nanoparticles described in this disclosure, the molar ratio of the substance represented by general formula (I) to the substance represented by general formula (II) is m:n, wherein m and n are each independently selected from real numbers from 1 to 10.

[0203] In some embodiments, the molar ratio of the substance represented by general formula (I) to the substance represented by general formula (II) in the nanoparticles described in this disclosure is 1:1.

[0204] In some implementations, the PAMAM component is between 14,000 and 60,000.

[0205] In some embodiments, exemplary examples of PAMAMs that can be used in this disclosure include, but are not limited to, those selected from fourth-generation polyamide-amine dendrimers (G4-PAMAM), fifth-generation polyamide-amine dendrimers (G5-PAMAM), sixth-generation polyamide-amine dendrimers (G6-PAMAM), and seventh-generation polyamide-amine dendrimers (G7-PAMAM).

[0206] In some embodiments, the nanoparticles of this disclosure effectively combine the advantages of protein degradation targeting chimeras and nanoparticle delivery systems, circumventing some of the shortcomings of existing small molecule protein degradation targeting chimeras.

[0207] In some embodiments, the nanoparticles of this disclosure have high bioavailability.

[0208] In some embodiments, the nanoparticles of this disclosure exhibit superior membrane-penetrating properties.

[0209] In some embodiments, the nanoparticles of this disclosure have a low probability of off-target aggregation.

[0210] In some embodiments, the nanoparticles of this disclosure have high safety.

[0211] In some embodiments, the nanoparticles of this disclosure are non-immunogenic.

[0212] In some embodiments, the nanoparticles of this disclosure are obtained by chemical coupling of the substance represented by general formula (I) and the substance represented by general formula (II) with PAMAM.

[0213] In another aspect, this disclosure relates to pharmaceutical compositions comprising the nanoparticles described herein, and pharmaceutically acceptable excipients.

[0214] On the other hand, this disclosure relates to a method for preparing nanoparticles, comprising:

[0215] The substance represented by general formula (I) of this disclosure is dissolved in a first polar solvent to obtain a first stock solution;

[0216] The substance of general formula (II) described herein is dissolved in a second polar solvent to obtain a second stock solution; and

[0217] The first and second stock solutions are dissolved in an organic solvent, and the nanoparticles are obtained through a chemical reaction.

[0218] In some embodiments, exemplary examples of the first polar solvents that can be used in this disclosure include, but are not limited to, acetonitrile, tetrahydrofuran, acetone, N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, and any mixtures thereof.

[0219] In some embodiments, exemplary examples of second polar solvents that can be used in this disclosure include, but are not limited to, acetonitrile, tetrahydrofuran, acetone, N,N-dimethylformamide, dimethyl sulfoxide, methanol, and any mixture thereof.

[0220] In some implementations, the chemical reaction is an amide condensation reaction.

[0221] Furthermore, this disclosure relates to a method for preparing a substance of general formula (I) for the degradation of a target protein (POI) in a ubiquitin-proteasome system (UPS): (R a -PEG) m -PAMAM-(PEG-R b ) n General Formula (III)

[0222] The method includes processing the substance PEG-R represented by general formula (I). a General Formula (I)

[0223] The substance PEG-R represented by general formula (II) b General Formula (II)

[0224] A chemical reaction is carried out with PAMAM to obtain the substance represented by general formula (III).

[0225] Wherein, PAMAM is a poly(amidoamine) dendrimer, PEG is polyethylene glycol, and R... a For E3 ubiquitin ligase ligand, R b The target protein (POI) ligand, and m and n are each independently selected from real numbers from 1 to 10.

[0226] In some embodiments, the method for preparing nanoparticles disclosed herein is simple.

[0227] In another aspect, this disclosure relates to a method for degrading a target protein (POI), comprising administering the nanoparticles and E3 ubiquitin ligase described in this disclosure to the target protein (POI).

[0228] On the other hand, this disclosure relates to a method of treating tumors, comprising administering to an individual in need of the method a therapeutically effective amount of the nanoparticles of this disclosure, or a therapeutically effective amount of the pharmaceutical composition of this disclosure.

[0229] In some implementations, exemplary examples of mammals that can be used in this disclosure include, but are not limited to, humans.

[0230] In some implementations, exemplary examples of tumors that can be used in this disclosure include, but are not limited to, lung cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), low-grade serous ovarian cancer, neurofibroma, liver cancer, and esophageal cancer.

[0231] In some implementations, exemplary examples of individuals that can be used for the purposes of this disclosure include, but are not limited to, mammals.

[0232] In the following sections, this disclosure will be explained in detail through the following embodiments to provide a better understanding of the various aspects of this application and its advantages. However, it should be understood that the following embodiments are non-limiting and are only used to illustrate certain implementations of this disclosure.

[0233] Example

[0234] The reagents and equipment used in the embodiments of this disclosure are all conventional and commercially available. For example:

[0235] Example 1

[0236] 50 mg of COOH-PEG5000-NHS and 10 μL of N,N-diisopropylethylamine (DIPEA) were dissolved in dichloromethane, followed by the addition of 20 mg of ligand (JQ1, TMXF, VHL, or CRBN) to ensure complete dissolution. The mixture was stirred at room temperature for 48 hours. The dichloromethane was then evaporated to dryness, and ethanol was added to dissolve the reactants completely. The solution was then transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed for 72 hours. The results were identified using 1H NMR spectroscopy (see Figures 2, 3, 12, and 13). It is important to note that water was replaced every 20 minutes for the first two hours of dialysis.

[0237] Example 2

[0238] Equal amounts of compounds COOH-PEG-JQ1 and COOH-PEG-CRBN (denoted by ligand content) were weighed and dissolved in a corresponding volume of organic solvent. Then, appropriate amounts of condensing agents, such as HATU, HCTU, and PyBOP, and acid-binding agents, such as DIPEA and TEA, were added. The reaction mixture was stirred for 24 to 48 hours, then transferred to a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed for 72 hours. The final results were identified using 1H NMR spectroscopy. It is important to note that water was replaced every 20 minutes for the first two hours of dialysis. The reaction process and the resulting compounds are shown in Figures 1, 4, and 14.

[0239] Example 3

[0240] The morphology of nanoparticles was detected using scanning electron microscopy (SEM).

[0241] A small amount of the nanoparticles obtained in Example 2 was redissolved in ethanol and then dropped onto a silicon wafer preheated to 40°C. The wafer was allowed to cool naturally until the ethanol completely evaporated. Before SEM observation, a thin layer of platinum was deposited onto the sample using a low-vacuum coating apparatus (processing time 5 s). The morphology of the sample was then observed at 5K voltage. See Figure 5 for details.

[0242] Example 4

[0243] CCK-8 assay for the toxicity of polymer nanoparticles to cells (using adherent cells as an example)

[0244] Cells in good proliferative condition were seeded into 96-well plates, with 6000-8000 cells per well. After complete cell adhesion, nanoparticles were serially diluted to nine aliquots (maximum concentration 100 μM), and added to the 96-well plates in triplicate. Depending on the cell type, the cells were cultured for 24 to 72 hours. Then, 10 μL of commercially available CCK-8 solution was added to each well. After 2 to 4 hours, the absorbance was measured at 450 nm. The results were processed using GraphPad software to determine the half-maximal inhibitory concentration (IC50). 50 Value. The specific results are shown in Figure 6.

[0245] Example 5

[0246] Detection of the membrane-penetrating ability of nanoparticles using flow cytometry

[0247] Healthy cells were seeded into 24-well plates at approximately 60,000 cells per well and cultured for 24 hours to allow for stable growth. Then, the cells were co-incubated with 10 μM of a FITC-labeled drug for 4 hours. The culture medium was discarded, and the cells were washed three times with phosphate-buffered saline. Trypsin was added to digest the cells, which were then resuspended. The cells were treated with 0.25% trypan blue for 10 minutes, followed by flow cytometry analysis. The cell count was consistently at least 10,000 cells per well. The specific results are shown in Figure 7.

[0248] Example 6

[0249] Western blotting to verify the degradation of target proteins by polymers

[0250] Healthy cells were seeded into 24-well plates at a density of 60% coverage of the bottom area per well. Cells were cultured until stable. Then, polymer nanoparticles were serially diluted with 5% FBS medium and added to each well. After incubation for 24 hours, the medium was discarded, cell lysis buffer was added, and cells were scraped off. The lysis buffer was treated at 99°C for 40 minutes, and protein degradation was verified using Western blotting. Some degradation examples are shown in Figure 8.

[0251] Example 7

[0252] Western blotting validates that the degradation of target proteins by polymers depends on the ubiquitin-proteasome system.

[0253] Healthy cells were seeded into 24-well plates at a density of 60% coverage of the bottom area per well. Cells were cultured until stable. Then, polymer nanoparticles were serially diluted with 5% FBS medium and added to each well. After incubation for 20 hours, 10 μM of proteasome inhibitors MG132 and MLN7243 were added to the control wells, followed by incubation for 4 hours. The culture medium was discarded, cell lysis buffer was added, and cells were scraped off. The lysis buffer was treated at 99°C for 40 minutes, and protein degradation was verified using Western blotting. Some examples are shown in Figure 9.

[0254] Example 8

[0255] Western blotting to verify the degradation of target proteins by polymers at different time points

[0256] Healthy cells were seeded into 24-well plates at a density of 60% cell density per well. Cells were cultured until stable. Then, polymer nanoparticles were serially diluted with 5% FBS medium and added to each well. After incubation for different times, the medium was discarded, cell lysis buffer was added, and cells were scraped off. Samples were collected at different time points, and the lysis buffer was treated at 99°C for 40 minutes. Western blotting was then used to verify protein degradation. Some degradation examples are shown in Figure 10.

[0257] Example 9

[0258] Following the methods described in Examples 4 and 6, cells were treated with different negative control drugs, and the results shown in Figure 11 were obtained. The different samples were: JQ1 PAMAM-PEG-JQ1 PAMAM-PEG-VHL+PAMAM-PEG-JQ1 PAMAM-PEG-CRBN+PAMAM-PEG-JQ1 PAMAM-PEG-JQ1_VHL PAMAM-PEG-JQ1_CRBN.

[0259] Example 10

[0260] Western blotting to verify the degradation of target proteins by polymers

[0261] Healthy HeLa cells were seeded into 24-well plates at a density of 60% coverage of the bottom area per well. Cells were cultured until stable. Then, polymer nanoparticles were serially diluted with 5% FBS medium and added to each well. After incubation for 24 hours, the medium was discarded, cell lysis buffer was added, and cells were scraped off. The lysis buffer was treated at 99°C for 40 minutes, and protein degradation was verified using Western blotting. Some degradation examples are shown in Figure 15.

[0262] Example 11

[0263] Western blotting to verify the degradation of target proteins by polymers

[0264] MCF-7 cells in good growth condition were seeded into 24-well plates at a cell density of 60% coverage of the bottom area per well. Cells were cultured until stable. Then, polymer nanoparticles were serially diluted with 5% FBS medium and added to each well. After incubation for 24 hours, the medium was discarded, cell lysis buffer was added, and cells were scraped off. The lysis buffer was treated at 99°C for 40 minutes, and protein degradation was verified using Western blotting. Some degradation examples are shown in Figure 16.

[0265] Example 12

[0266] Evaluation of antitumor activity

[0267] The antitumor activity of PAMAM-PEG nanoparticles was evaluated in vivo in BALB / c nude mice (19-20g, 5-6 weeks old, purchased from Jiangsu Xishan Biotechnology Co., Ltd.). 6-week-old female Balb / c nude mice were injected with 100 μL of HCT116 cell suspension (2 × 10⁻⁶ cells / mL). 6 / 100μL, resuspended in PBS). When the tumor volume reaches approximately 50 to 100 mm... 3Mice were randomly divided into 7 groups (n=5) and injected with different drugs. Tumor-carrying mice were administered the drugs via intravenous injection or oral administration. Tumor volume and body weight were recorded every 3 days and expressed as V(mm²) using the equation. 3 The tumor volume was calculated as (a × b × b) (where a and b represent the longest and shortest dimensions of the tumor tissue). At the end of treatment, tumors and major organs (heart, liver, spleen, lung, kidney, and brain) were collected from the mice. Tumor weights and microscopic images were collected, and organs were fixed with 4% paraformaldehyde (Biosharp, BL539A) for hematoxylin-eosin (HE) staining (as shown in Figure 19).

[0268] No significant changes in mouse body weight were observed during treatment (Figure 18A). As shown in Figures 18B (tumor volume), 18C (tumor weight), and 18D (tumor image), mice treated with PAMAM-PEG-JQ1_CRBN exhibited good tumor growth inhibition, particularly the high-dose group (20 mg / kg). Conversely, the group (PAMAM-PEG-JQ1+PAMAM-PEG-CRBN) and the small molecule PROTAC group (ARV-825) showed no ability in tumor growth inhibition (Figure 18E).

[0269] Example 13

[0270] Evaluation of antitumor activity

[0271] Given the excellent water solubility of PAMAM-PEG-JQ1_CRBN, oral administration to mice was also chosen. As shown in Figure 20, the positive control drug ARV-825, administered every three days or every seven days, showed no significant tumor inhibition. Conversely, PAMAM-PEG-JQ1_CRBN exhibited a significant degree of tumor inhibition when administered every three days. However, the antitumor effect was very limited, mainly due to differences in the bioavailability of the drug molecule in mice. Therefore, the oral administration cycle was changed to once a day (Figure 21). Indeed, the antitumor effect of PAMAM-PEG-JQ1_CRBN has been shown to be significantly improved. As shown in Figure 22, PAMAM-PEG-JQ1_CRBN showed effective tumor growth inhibition in both low-dose and high-dose groups. These findings are consistent with those observed after intravenous administration and exceed the performance of the positive reference compound ARV-825. Notably, with increasing doses, even with oral administration, the tumor-suppressive effect of PAMAM-PEG-JQ1_CRBN can reach or exceed that achieved with intravenous administration. This highlights the potential of PAMAM-PEG-JQ1_CRBN as an effective antitumor agent with favorable pharmacokinetic properties supporting both oral and intravenous administration strategies. Although the positive control drug ARV-825 also showed better results with higher doses, it still performed worse than PAMAM-PEG-JQ1_CRBN. Conversely, all this evidence suggests potential drawbacks of ARV-825, including faster metabolism and poor membrane permeability.

[0272] Furthermore, compared with the blank control, no major organs (heart, liver, spleen, lung and kidney) showed significant morphological differences in hematoxylin and eosin (H&E) staining (Fig. 23).

[0273] In fact, for ease of operation, all drug groups in this experimental section were measured by weight. The average molecular weight of PAMAM-PEG is greater than 28,000 g / mol, while the molecular weight of ARV-825 is 923 g / mol. This means that even in the most effective group with a drug weight of 20 mg / kg PAMAM-PEG-JQ1_CRBN, the actual amount of the effective functional molecule is far lower than the actual amount of the small molecule (less than 5%). In summary, our in vivo experiments demonstrate that PAMAM-based SM-PROTACs are highly effective, low in toxicity, and durable. Utilizing the non-self-assembly properties of the PAMAM system, the PAMAM system will enhance the clinical application potential of SM-PROTAC technology and provide a more precise characterization strategy. In particular, the PAMAM system exhibits strong oral potential. This may be attributed to the conversion of small molecule PROTACs or SM-PROTACs from injectable formulations to oral formulations.

[0274] In this disclosure, relational terms such as first and second are used merely to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0275] As will be understood from the foregoing, although specific embodiments of this disclosure have been described for illustrative purposes, various modifications or alterations can be made by those skilled in the art without departing from the spirit and scope of this disclosure. All such modifications or alterations should fall within the scope of the appended claims.

Claims

1. A substance represented by the general formula (IV) for degrading a Protein of Interest (POI) in a lysosome: PEG-R c General Formula (IV) wherein PEG is polyethylene glycol, and R c selected from the group consisting of cell surface transmembrane receptors (CI-M6PR), asialoglycoprotein receptors (ASGPR), LC3, autophagic cargo receptor p62, heat shock proteins, integrins, and ADRM1-Halotag7.

2. The substance as claimed in claim 1, wherein the PEG has a molecular weight of 5000 to 45000.

3. The substance as claimed in claim 1 or 2, wherein the PEG is selected from the group consisting of PEG5000, PEG6000, PEG8000, PEG10000, PEG20000, PEG30000 and PEG45000.

4. The substance of any one of claims 1 to 3, wherein R c selected from 5. The substance of any one of claims 1 to 4, which is selected from wherein n is from 116 to 1047.

6. A substance according to general formula (V) for degradation of a Protein of Interest (POI) in lysosomes: PEG-R d General formula (V) wherein PEG is polyethylene glycol, and R d are target protein (POI) ligands.

7. The substance as claimed in claim 6, wherein the PEG has a molecular weight of 5000 to 45000.

8. The substance as claimed in claim 6 or 7, wherein the PEG is selected from the group consisting of PEG5000, PEG6000, PEG8000, PEG10000, PEG20000, PEG30000 and PEG45000.

9. The substance as claimed in any one of claims 6 to 8, wherein the target protein (POI) ligand is selected from the group consisting of an estrogen receptor (ERa) ligand, an androgen receptor (AR) ligand, an epidermal growth factor receptor (EGFR) ligand, a human bromodomain-containing protein 2 / 4 (BRD2 / 4) ligand, a mitogen-activated protein kinase kinase 1 and 2 (MEK1 / 2) ligand, a cyclin-dependent kinase 4 / 6 (CDK4 / 6) ligand and a BCR-ABL ligand.

10. The agent of any one of claims 6 to 9, wherein the target protein (POI) ligand is selected from the group consisting of:

11. The substance of any one of claims 6 to 10, which is selected from 12. A substance according to general formula (VI) for degradation of a Protein of Interest (POI) in lysosomes: (R c -PEG) m -PAMAM-(PEG-R d ) n general formula (VI) wherein, PEG is polyethylene glycol, R c is a cell surface transmembrane receptor (CI-M6PR), an asialoglycoprotein receptor (ASGPR), LC3, an autophagic cargo receptor p62, a heat shock protein, an integrin, and ADRM1-Halotag7, R d is a target protein (POI) ligand, PAMAM is a poly(amidoamine) dendrimer, and m and n are each independently selected from real numbers from 1 to 10.

13. The substance as claimed in claim 12, wherein the PAMAM has a molecular weight of 14000 to 60000.

14. The substance as claimed in claim 12 or 13, wherein the PAMAM is selected from the group consisting of a fourth generation polyamidoamine dendrimer (G4-PAMAM), a fifth generation polyamidoamine dendrimer (G5-PAMAM), a sixth generation polyamidoamine dendrimer (G6-PAMAM) and a seventh generation polyamidoamine dendrimer (G7-PAMAM).

15. The substance as claimed in any one of claims 12 to 14, wherein the PEG has a molecular weight of 5000 to 45000.

16. The substance as claimed in any one of claims 12 to 15, wherein the PEG is selected from the group consisting of PEG5000, PEG6000, PEG8000, PEG10000, PEG20000, PEG30000 and PEG45000.

17. The substance as claimed in any one of claims 12 to 16, wherein the target protein (POI) ligand is selected from the group consisting of an estrogen receptor (ERa) ligand, an androgen receptor (AR) ligand, an epidermal growth factor receptor (EGFR) ligand, a human bromodomain-containing protein 2 / 4 (BRD2 / 4) ligand, a mitogen-activated protein kinase kinase 1 and 2 (MEK1 / 2) ligand, a cyclin-dependent kinase 4 / 6 (CDK4 / 6) ligand and a BCR-ABL ligand.

18. The agent of any one of claims 12 to 17, wherein the target protein (POI) ligand is selected from:

19. A nanoparticle comprising an inner core and an outer layer, wherein the inner core is PAMAM and the outer layer comprises the substance of general formula (IV) as claimed in any one of claims 1 to 5 and the substance of general formula (V) as claimed in any one of claims 6 to 11.

20. The nanoparticle of claim 19, wherein the m:n ratio of the amount of substance of the substance of general formula (IV) to the amount of substance of the substance of general formula (V), wherein m and n are each independently selected from the real numbers 1 to 10.

21. The nanoparticle of claim 19 or 20, wherein the m:n ratio of the amount of substance of the substance of general formula (IV) to the amount of substance of the substance of general formula (V) is 1 :

1.

22. The nanoparticle of any one of claims 19 to 21, wherein the PAMAM has a generation of 14,000 to 60,000.

23. The nanoparticle of any one of claims 19 to 22, wherein the PAMAM is selected from the group consisting of a fourth generation polyamidoamine dendrimer (G4-PAMAM), a fifth generation polyamidoamine dendrimer (G5-PAMAM), a sixth generation polyamidoamine dendrimer (G6-PAMAM), and a seventh generation polyamidoamine dendrimer (G7-PAMAM).

24. A pharmaceutical composition comprising the nanoparticle of any one of claims 19 to 23, and a pharmaceutically acceptable excipient.

25. A substance according to general formula (I) for the degradation of a Protein of Interest (POI) in the Ubiquitin-Proteasome System (UPS): PEG-R a General Formula (I) wherein PEG is polyethylene glycol, and R a is an E3 ubiquitin ligase ligand.

26. The substance of claim 25, wherein the PEG has a molecular weight of 5,000 to 45,000.

27. The substance of claim 25 or 26, wherein the PEG is selected from the group consisting of PEG 5,000, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, PEG 30,000, and PEG 45,000.

28. The substance of any one of claims 25 to 27, wherein the E3 ubiquitin ligase ligand is selected from the group consisting of a VHL ligand, a MDM2 ligand, a clAP1 ligand, and a CRBN ligand.

29. The agent of any one of claims 25 to 28, wherein the E3 ubiquitin ligase ligand is selected from:

30. The agent of any one of claims 25 to 29, which is selected from wherein n is from 116 to 1047.

31. A substance represented by the general formula (II) for degrading a Protein of Interest (POI) in the Ubiquitin-Proteasome System (UPS): PEG-R b General Formula (II) wherein, PEG is polyethylene glycol, and R b are target protein (POI) ligands.

32. The substance of claim 31, wherein the PEG has a molecular weight of 5,000 to 45,000.

33. The substance of claim 31 or 32, wherein the PEG is selected from the group consisting of PEG 5,000, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, PEG 30,000, and PEG 45,000.

34. The substance of any one of claims 31 to 33, wherein the target protein (POI) ligand is selected from the group consisting of an estrogen receptor (ERa) ligand, an androgen receptor (AR) ligand, an epidermal growth factor receptor (EGFR) ligand, a human bromodomain-containing protein 2 / 4 (BRD2 / 4) ligand, a mitogen-activated protein kinase kinase 1 and 2 (MEK1 / 2) ligand, a cyclin-dependent kinase 4 / 6 (CDK4 / 6) ligand, and a BCR-ABL ligand.

35. The agent of any one of claims 31 to 34, wherein the target protein (POI) ligand is selected from:

36. The agent of any one of claims 31 to 35, which is selected from wherein n is from 116 to 1047.

37. A substance according to general formula (III) for degrading a Protein of Interest (POI) in the Ubiquitin-Proteasome System (UPS): ###00016### (PEG-R a -PEG) m -PAMAM-(PEG-R b ) n general formula (III) wherein PEG is polyethylene glycol, R a is an E3 ubiquitin ligase ligand, R b is a target protein (POI) ligand, PAMAM is a poly(amidoamine) dendrimer, and m and n are each independently selected from real numbers from 1 to 10.

38. The substance of claim 37, wherein the PAMAM has a generation of 14,000 to 60,000.

39. The substance of claim 37 or 38, wherein the PAMAM is selected from the group consisting of fourth generation polyamidoamine dendrimers (G4-PAMAM), fifth generation polyamidoamine dendrimers (G5-PAMAM), sixth generation polyamidoamine dendrimers (G6-PAMAM), and seventh generation polyamidoamine dendrimers (G7-PAMAM).

40. The substance of any one of claims 37 to 39, wherein the PEG has a molecular weight of 5000 to 45000.

41. The substance of any one of claims 37 to 40, wherein the PEG is selected from the group consisting of PEG5000, PEG6000, PEG8000, PEG10000, PEG20000, PEG30000, and PEG45000.

42. The substance of any one of claims 37 to 41, wherein the E3 ubiquitin ligase ligand is selected from the group consisting of VHL ligand, MDM2 ligand, clAP1 ligand, and CRBN ligand.

43. The agent of any one of claims 37 to 42, wherein the E3 ubiquitin ligase ligand is selected from:

44. The substance of any one of claims 37 to 43, wherein the target protein (POI) ligand is selected from the group consisting of estrogen receptor (ERa) ligand, androgen receptor (AR) ligand, epidermal growth factor receptor (EGFR) ligand, human bromodomain-containing protein 2 / 4 (BRD2 / 4) ligand, mitogen-activated protein kinase kinase 1 and 2 (MEK1 / 2) ligand, cyclin-dependent kinase 4 / 6 (CDK4 / 6) ligand, and BCR-ABL ligand.

45. The agent of any one of claims 37 to 44, wherein the target protein (POI) ligand is selected from:

46. A nanoparticle comprising an inner core and an outer layer, wherein the inner core is PAMAM and the outer layer comprises the substance of any one of claims 25 to 30 represented by general formula (I), and the substance of any one of claims 31 to 36 represented by general formula (II).

47. The nanoparticle of claim 46, wherein the substance of general formula (I) and the substance of general formula (II) are present in a substance amount ratio m:n, wherein m and n are each independently selected from the group consisting of real numbers from 1 to 10.

48. The nanoparticle of claim 46 or 47, wherein the substance of general formula (I) and the substance of general formula (II) are present in a substance amount ratio of 1:

1.

49. The nanoparticle of any one of claims 46 to 48, wherein the PAMAM has a generation of 14000 to 60000.

50. The nanoparticle of any one of claims 46 to 49, wherein the PAMAM is selected from the group consisting of fourth generation polyamidoamine dendrimers (G4-PAMAM), fifth generation polyamidoamine dendrimers (G5-PAMAM), sixth generation polyamidoamine dendrimers (G6-PAMAM), and seventh generation polyamidoamine dendrimers (G7-PAMAM).

51. A pharmaceutical composition comprising the nanoparticle of any one of claims 46 to 50, and a pharmaceutically acceptable excipient.

52. A method of preparing nanoparticles, comprising: dissolving a substance represented by general formula (I) as described in any one of claims 25 to 30 in a first polar solvent, thereby obtaining a first stock solution; dissolving a substance represented by general formula (II) as described in any one of claims 31 to 36 in a second polar solvent, thereby obtaining a second stock solution; and dissolving the first stock solution and the second stock solution in an organic solvent, thereby obtaining the nanoparticles through a chemical reaction.

53. The method of claim 52, wherein the first polar solvent is selected from the group consisting of acetonitrile, tetrahydrofuran, acetone, N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, and any mixture thereof.

54. The method of claim 52 or 53, wherein the second solvent is selected from the group consisting of acetonitrile, tetrahydrofuran, acetone, N,N-dimethylformamide, dimethyl sulfoxide, methanol, and any mixture thereof.

55. The method of any one of claims 52 to 54, wherein the chemical reaction is an amide condensation reaction.

56. A method for preparing a substance of general formula (III) for the degradation of a target protein (POI) in a ubiquitin-proteasome system (UPS): (R a -PEG) m -PAMAM-(PEG-R b ) n General Formula (III) the method comprising dissolving a substance represented by general formula (I) PEG-R a general formula (I) a substance represented by general formula (II) PEG-R b general formula (II) with PAMAM, thereby obtaining the substance represented by general formula (III), wherein PAMAM is a poly(amidoamine) dendrimer, PEG is a polyethylene glycol, R a is an E3 ubiquitin ligase ligand, R b is a target protein (POI) ligand, and m and n are each independently selected from real numbers from 1 to 10.

57. A method of degrading a Protein of Interest (POI), comprising administering to the Protein of Interest (POI) the nanoparticles of any one of claims 46 to 50 and an E3 ubiquitin ligase.

58. A method of treating a tumor, comprising administering to an individual in need of the method a therapeutically effective amount of the nanoparticles of any one of claims 46 to 50, or a therapeutically effective amount of the pharmaceutical composition of claim 51.

59. The method of claim 58, wherein the individual is a mammal, preferably a human.

60. The method of claim 58 or 59, wherein the tumor is selected from the group consisting of lung cancer, breast cancer, ovarian cancer, prostate cancer, bladder cancer, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), low-grade serous ovarian cancer, neurofibroma, liver cancer, and esophageal cancer.

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