Endocytosis agents

Abstract

This specification discloses compounds comprising a drug or probe linked to a chemical arm via cleavable or incleavable chemical bonds or linker units, which have binding affinity to membrane components that mediate endocytosis. Methods for using and preparing such compounds are also disclosed.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 420,326, filed on 28 October 2022, the contents of which are incorporated in their entirety by reference. [Background technology]

[0002] According to traditional guidelines, such as the "Rule of Five (Ro5, the rule of 5')" and subsequent extended guidelines (extended Ro5; eRo5 and beyond Ro5; bRo5), many therapeutic agents suffer from poor drug characteristics due to undesirable stability, water solubility, and / or membrane permeability, directly leading to a high rate of failure in drug development. Of these factors, poor permeability is recognized as the most important problem, as drugs must pass through many membrane barriers to reach the site of action after administration. Based on passive permeability, stability, water solubility, and membrane permeability are highly interrelated and sensitive to structural changes; structural modifications to adjust one property inevitably affect one or more other properties. A common method to increase the passive permeability of a drug is to increase the lipophilicity of the compound through structural optimization on a case-by-case basis, making it easily soluble in membrane phospholipids and allowing it to permeate cell membranes and diffuse rapidly, but at the cost of reduced solubility (due to decreased hydrophilicity), stability, and / or toxicity. For most therapeutic agents, desirable stability and water solubility can be achieved through structural modification and salting-out techniques. Therefore, the ultimate challenge in conventional drug discovery and development campaigns remains how to improve the membrane permeability of therapeutic agents without sacrificing solubility and stability. Evidence for this is that passive drug permeability decreases sharply as molecular size increases. Unfortunately, the biological activity of eRo5 and bRo5 molecules, such as chimeric molecules, is thought to be due to passive permeability, and the optimization of eRo5 and bRo5 molecules is based on passive diffusion. However, according to passive diffusion theory, it is difficult, or even impossible, to modify large molecules such as PROTAC molecules to improve membrane passive permeability while maintaining a good balance between metabolic stability and solubility. Therefore, there is a need in this field for a general method that can be used to accelerate therapeutic drug development and broaden the therapeutic applications of drugs by improving permeability without sacrificing solubility and stability. [Overview of the project]

[0003] This specification discloses compounds comprising a drug or probe linked to a chemical arm via a cleavable or incleavable chemical bond or linker unit, which have a binding affinity to a membrane component that mediates endocytosis. These compounds have a binding affinity KD of less than 20.0 mM to the membrane component that mediates endocytosis. In some embodiments, the compound is of formula (I):

[0004] [ka] (In the formula, m, n, and p represent integers between 0 and 100.) Represented by . Exemplary compounds may have m, n, and p equal to 1. Compounds may include drugs that are degrading agents, stabilizers, inhibitors, modulators, or activators. In some embodiments, the drug is a protein binder. In some embodiments, the compound includes a probe that is a diagnostic agent. The chemical arm may be an atom, a drug, a probe, or part of a drug or binder or probe. In some examples, the chemical arm has a binding affinity K to membrane components that mediate endocytosis less than 20.0 mM. D The membrane component mediating endocytosis is preferably a cell membrane lipid, a carbohydrate, or a protein. The membrane component mediating endocytosis may be glycolipids, glycoproteins, phospholipids, ceramides, and cholesterol. In some embodiments, the membrane component mediating endocytosis is a glycolipid or a glycoprotein containing 2 to 100 linear or branched monosaccharide units. The membrane component mediating endocytosis may be an endogenous membrane protein, a peripheral membrane protein, a lipid-anchored protein, a globular protein, or a glycoprotein.

[0005] Also disclosed are nanostructures comprising a lipid bilayer, an endocytosis-mediated membrane component, and a liquid or cytoplasm encapsulated by any of the compounds described herein. In some examples, the nanostructure has a binding affinity K to an endocytosis-mediated membrane component of less than 20.0 mM. DThey have. Exemplary nanostructures include extracellular vesicles, microvesicles, endocytosis agent-vesicle complexes, or exocytic vesicles.

[0006] Methods for preparing nanostructures are also provided. These methods may involve contacting any of the compounds disclosed herein with cells or vesicles containing membrane components that mediate endocytosis. The nanostructures may be prepared in vivo, in vitro, or ex vivo. Methods for internalizing any of the compounds disclosed herein into cells are also provided. These methods may involve contacting any of the compounds disclosed herein with cells containing membrane components that mediate endocytosis. In some embodiments, nanostructures containing the compounds are contacted with the cells. A method for isolating compounds from nanostructures is also provided. This method may include dissolving the nanostructures and separating the compounds from the dissolved material.

[0007] Methods for determining the qualitative or quantitative presence of compounds or nanostructures in cells, bodies, solutions, or media are also provided. These methods may include centrifugation of a sample containing cells, bodies, solutions, or media, and detection of compounds or nanostructures. Methods for isolating compounds or nanostructures from cells, bodies, solutions, or media are also provided. These methods may include centrifugation of a sample containing cells, bodies, solutions, or media. Methods for treating the target are also provided. These methods may include administering a compound or nanostructure to a target that requires it.

[0008] A method for identifying endocytic agents is also provided. This method may involve contacting a compound with first and second cells, wherein the presence of endocytosis-mediated membrane components in the second cells is modulated compared to that in the first cells, and the compound is determined to be an endocytic agent by comparing the activity of the compound in contact with the first cells with the activity of the compound in contact with the second cells. Methods for identifying membrane components that mediate endocytosis are also provided. These methods may involve contacting cells with a compound or nanostructure, wherein the compound includes a detectable label, and the membrane component that mediates endocytosis within the cell can be identified by determining the interaction between the endocytosis-mediating membrane component and the detectable label. A method for identifying membrane components that mediate endocytosis is also provided. This method may include comparing the sensitivity of a first cell to a second cell to treatment with a compound or nanostructure, and comparing the genomic expression or protein abundance of membrane components in the first and second cells, thereby identifying the membrane component that mediates endocytosis by an increase in the sensitivity of the first or second cell to the compound or nanostructure. A method is also provided for selecting a target for treatment with a compound or nanostructure having binding affinity to endocytosis-mediated membrane components. This method may include determining the qualitative or quantitative presence of endocytosis-mediated membrane components in a sample obtained from a prospective subject, and, if endocytosis-mediated membrane components are present in the sample, administering the compound or nanostructure to the prospective subject.

[0009] Non-limiting embodiments of the present invention are described illustratively with reference to the accompanying drawings, which are schematic and not intended to be drawn to scale. In the drawings, each identical or substantially identical component illustrated is typically represented by a single number. For clarity, not all components are labeled in every drawing, nor are all components of each embodiment of the present invention shown where not an illustration is necessary for those skilled in the art to understand the invention. [Brief explanation of the drawing]

[0010] [Figure 1A] Figures 1A-1E illustrate endocytosis agents and their applications. Figure 1A shows the mechanism and usefulness of cell pickup by endocytosis agents, as well as methods for producing endocytosis agents or polypharmacological endocytosis agents with increased binding affinity and / or valency to endocytosis-mediated membrane components in order to enhance the efficacy and / or efficiency of endocytosis. [Figure 1B] Figure 1B illustrates the mechanism of absorption of endocytic agents into the bloodstream after oral, inhalation, or topical administration. Endocytic agents can be encapsulated in intracellular vesicles via multiple pathways and released into the bloodstream in the form of free endocytic agents and / or exocytic vesicles. Cytoplasmic proteins include, but are not limited to, fatty acid-binding proteins (FABPs). [Figure 1C] Figure 1C illustrates the mechanism by which endocytic agents are absorbed into brain tissue from the bloodstream across the blood-brain barrier. Endocytic agents can be encapsulated in intracellular vesicles via multiple pathways and released into the bloodstream in the form of free endocytic agents and / or exocytic vesicles. [Figure 1D] Figure 1D illustrates the mechanisms of cellular pickup, release, and utilization of endocytosis agents and exocytosis vesicles for biological function. [Figure 1E]Figure 1E shows the relative permeability of drugs with a certain molecular weight range via passive diffusion (upper curve for low molecular weights) or endocytosis (lower curve for low molecular weights). [Figure 2A] Figures 2A-2E show that endocytosis agents bind to the human CD36 protein. Figure 2A shows an immunoblotting assay demonstrating the expression of CD36 protein in the membrane and cytoplasmic compartments of LNCaP PCa cells. [Figure 2B] Figure 2B shows a pull-down assay demonstrating that the CD36 protein in the LNCaP cell membrane is one of the targets of a multipharmacological endocytosis agent, as illustrated in Example 1. Mass spectrometry (MS) determines the binding of CD36, a protein of the bromodomain and extraterminal (BET) family, to proteins involved in the formation, anchoring, fusion, transport, and recycling of membrane components of endocytic vesicles, such as CLH1, UBR4, AP1B1, RAB5A, BIG1, AP2B1, AP3D1, ARP2, and AP3B1, as in Example 1. [Figure 2C] Figure 2C shows that the endocytosis agent binds to the hCD36 protein, as illustrated by Example 1, Example 3, or Example 4. Immunoblots for CD36 after treatment of purified his-tagged hCD36 protein with a biotin control or an endocytosis agent. [Figure 2D] Figure 2D shows that multipharmacological endocytosis agents bind to the hCD36 protein, as illustrated in Example 6 or Example 7. Immunoblots for CD36 after treatment of purified his-tagged hCD36 protein with a biotin control or with an endocytosis agent. [Figure 2E] Figure 2E shows that Example 8, a representative endocytosis agent, binds to human CD36 protein as determined by ITC assay. The titration consisted of injecting the solution of Example 8 from an injection syringe into the human CD36 protein (hCD36) solution in the sample cell at a rate of 0.5 μl / second at 150-second intervals. The KD value was determined using a sequential binding site model. [Figure 3A] Figures 3A-3O illustrate that endocytosis is the dominant pathway for drug uptake. Figure 3A shows the levels of CD36 protein in Luc control or LNCaP PCa cells transfected with shRNA containing the CD36 target sequence. [Figure 3B] Figure 3B shows the levels of EEA1 protein in LNCaP cells transfected with Luc control or shRNA containing the EEA1 target sequence. [Figure 3C] Figure 3C shows the levels of Rab5 protein in LNCaP cells transfected with Luc control or shRNA containing a Rab5 target sequence. [Figure 3D] Figure 3D shows the levels of CD36 protein in 22Rv1 PCa cells transfected with scrambled or CD36-targeted siRNA. [Figure 3E] Figure 3E shows that the EEA1 / Rab5 protein is required for endocytosis and promotes transferrin uptake. Representative confocal images of morphology mediated by DAPI and transferrin in stable LNCaP cells containing shLuc, shEEA1, or shRab5 treated for a specified period. Scale bar: 100 μm. [Figure 3F] Figure 3F demonstrates, by co-localization assay, that in LNCaP cells, the uptake of multipharmacological endocytosis agents is dependent on CD36-mediated endocytosis, as illustrated in Example 10. After treating LNCaP cells with Example 10 for the specified period, fluorescence of the endocytosis agents Example 10 and DAPI from the cells was imaged using a fluorescence microscope. Scale bar: 200 μm. [Figure 3G]Figure 3G demonstrates, by co-localization assay, that in LNCaP cells, the uptake of trivalent multipharmacological endocytosis agents is dependent on CD36-mediated endocytosis, as illustrated in Example 12. After treating LNCaP cells with Example 12 for the specified period, fluorescence of the endocytosis agent Example 12 and DAPI from the cells was imaged using a fluorescence microscope. Scale bar: 100 μm. [Figure 3H] Figure 3H shows that co-localization assays demonstrate that, in LNCaP cells, the uptake of trivalent multipharmacological endocytosis agents is dependent on CD36-mediated endocytosis, as illustrated in Example 8. After treating LNCaP cells with the specified period in Example 8, fluorescence of the endocytosis agents Example 8 and DAPI from the cells was imaged using a fluorescence microscope. Scale bar: 100 μm. [Figure 3I] Figure 3I shows that co-localization assays demonstrate that in 22Rv1 cells, the uptake of trivalent multipharmacological endocytosis agents is dependent on CD36-mediated endocytosis, as illustrated in Example 8. After treating 22Rv1 cells with 500 nM of Example 8 for 0.5 hours, fluorescence of the endocytosis agent Example 8, CD36 protein, EEA1 protein, and DAPI from the cells was imaged using a fluorescence microscope. [Figure 3J] Figure 3J demonstrates, by co-localization assay, that in LNCaP cells, the uptake of multipharmacological endocytosis agents is dependent on CD36-mediated endocytosis, as illustrated in Example 13. After treating LNCaP cells with Example 13 for the specified period, fluorescence of the endocytosis agent Example 13 and DAPI from the cells was imaged using a fluorescence microscope. Scale bar: 200 μm. [Figure 3K]Figure 3K demonstrates, by co-localization assay, that in LNCaP cells, the uptake of the divalent multipharmacological endocytosis agent is dependent on CD36-mediated endocytosis, as illustrated in Example 14. After treating LNCaP cells with Example 14 for the specified period, fluorescence of the endocytosis agent Example 14 and DAPI from the cells was imaged using a fluorescence microscope. Scale bar: 100 μm. [Figure 3L] Figure 3L demonstrates, by co-localization assay, that in LNCaP cells, the uptake of the divalent multipharmacological endocytosis agent is dependent on CD36-mediated endocytosis, as illustrated in Example 15. After treating LNCaP cells with the agent in Example 15 for the specified period, the fluorescence of the endocytosis agent in Example 15 and DAPI from the cells was imaged using a fluorescence microscope. Scale bar: 100 μm. [Figure 3M] Figure 3M demonstrates, by co-localization assay, that in 22Rv1 cells, the uptake of a divalent multipharmacological endocytosis agent is dependent on CD36-mediated endocytosis, as illustrated in Example 15. After treating 22Rv1 cells with 500 nM of Example 15 for 0.5 hours, fluorescence of the endocytosis agent Example 15, CD36 protein, EEA1 protein, and DAPI from the cells was imaged using a fluorescence microscope. Scale bar: 100 μm. [Figure 3N] Figure 3N demonstrates, by co-localization assay, that in 22Rv1 cells, the uptake of the divalent multipharmacological endocytosis agent is dependent on CD36-mediated endocytosis, as illustrated in Example 16. After treating 22Rv1 cells with Example 16 for the specified period, fluorescence of the endocytosis agent Example 16 and DAPI from the cells was imaged using a fluorescence microscope. Scale bar: 100 μm. [Figure 3O]Figure 3O shows that the trivalent polyvalent pharmacological endocytosis agent in Example 8 or Example 12 and the divalent polyvalent pharmacological endocytosis agent in Example 14 or Example 15 had little to no visible toxicity to the viability of LNCaP cells or 22Rv1 cells. (n=3) [Figure 4A] Figures 4A-4F illustrate how CD36-mediated endocytosis determines drug uptake and biological function. Figure 4A shows that Trivalent-BETD1 (Example 17), a multipharmacological endocytosis agent, induces BRD4 degradation in LNCaP or 22Rv1 PCa cells. BRD4 protein levels in LNCaP or 22Rv1 cells treated with Example 17, either pre-treated with MG132 or unpre-treated, were determined by Western blotting assay. [Figure 4B] Figure 4B shows that shCD36 in LNCaP or 22Rv1 PCa cells reverses BRD4 degradation induced by the multipharmacological endocytosis agent Trivalent-BETD1 (Example 17). LNCaP or 22Rv1 cells stably transfected with shLuc or shCD36 were treated with Example 17, and the protein levels of BRD4 and CD36 were then determined by Western blotting assay. [Figure 4C] Figure 4C shows that ARV110 (Example 19), a multipharmacologically divalent endocytosis agent, induces AR degradation in LNCaP or 22Rv1 PCa cells. AR protein levels in LNCaP or 22Rv1 cells treated with Example 19, either pre-treated with MG132 or unpre-treated, were determined by Western blotting assay. [Figure 4D]Figure 4D shows that shCD36 reverses AR degradation induced by the multipharmacologically divalent endocytosis agent ARV110 (Example 19) in LNCaP or 22Rv1 PCa cells. LNCaP or 22Rv1 cells stably transfected with shLuc or shCD36 were treated with Example 19, and the protein levels of AR and CD36 were then determined by Western blotting assay. [Figure 4E] Figures 4E and 4F show that shCD36 antagonizes the antitumor effect of the multipharmacological endocytosis agent ARV110 (Example 19) in the LNCaP-xenograft mouse model (n=5). In Figure 4E, the top curve represents shCD36 number 2 + ARV110, the second curve from the top represents shCD36 number 2, the third curve from the top represents shLuc + vehicle, the fourth curve from the top represents shCD36 number 1 + ARV110, the fifth curve from the top represents shCD36 number 1, and the bottom curve represents shLuc + ARV110. [Figure 4F] In an LNCaP-xenograft mouse model, we demonstrate that shCD36 antagonizes the antitumor effect of ARV110 (Example 19), a multipharmacological endocytosis agent. [Figure 5A] Figures 5A-5D illustrate how conjugating a drug with a chemical arm promotes cellular uptake via CD36-mediated endocytosis. Figure 5A shows that conjugating the drug (Example 20) with a dicarboxylic acid chemical arm promotes and speeds up cellular uptake of the drug. After treating HCC1806 cells with 500 nM of Example 20 or Example 21 for the specified period, the green fluorescence of the endocytosis agents Bivalent-BDP-FL1 (Example 20) or Trivalent BDP-FL1-C12Na (Example 21) from the cells was imaged using a fluorescence microscope. Scale bar: 100 μm. [Figure 5B]Figure 5B demonstrates, by co-localization assay, that CD36-mediated endocytosis is the dominant pathway for the uptake of the endocytosis agents Bivalent-BDP-FL1 (Example 20), Trivalent-BDP-FL1-C12Na (Example 21), or Trivalent-BDP-FL1-C14Na (Example 22) in HCC1806 cells. After treating HCC1806 cells with 500 nM of Example 20 or Example 21 for 0.5 hours, fluorescence imaging of the endocytosis agents Example 20, Example 21, or Example 22, CD36 protein, EEA1 protein, and DAPI from the cells was performed using a fluorescence microscope. Examples 21 and 22, with higher binding affinity and binding arms (valences) greater than 20, promote and speed up cellular uptake. Scale bar: 100 μm. [Figure 5C] Figure 5C demonstrates that co-localization assays show that CD36-mediated endocytosis is the dominant pathway for the uptake of the endocytosis agents Bivalent-BDP-FL1 (Example 20), Trivalent-BDP-FL1-C12Na (Example 21), or Trivalent-BDP-FL1-C14Na (Example 22) in shCD36 HCC1806 cells. After treating shCD36 HCC1806 cells with 500 nM of Example 20 or Example 21 for 0.5 hours, fluorescence of the endocytosis agents Example 20, Example 21, or Example 22, CD36 protein, EEA1 protein, and DAPI from the cells was imaged using a fluorescence microscope. Scale bar: 100 μm. [Figure 5D] Figure 5D shows that Bivalent-BDP-FL1 (Example 20), Trivalent-BDP-FL1-C12Na (Example 21), or Trivalent-BDP-FL1-C14Na (Example 22) exhibited little to no cytotoxicity in HCC1806 cells. On the other hand, in HCC1806 TNBC cells, the trivalent endocytosis agents Examples 28 and 29 exhibited stronger cytotoxicity than the divalent endocytosis agent Example 23. (n=2) [Figure 6A]Figures 6A–6D illustrate how CACO2 cells take up endocytic agents via endocytosis and release free endocytic agents and exocytic vesicles. Figure 6A shows a typical method in this disclosure for isolating and / or detecting endocytic agents and / or exocytic vesicles in a medium containing cells after treatment with an endocytic agent. [Figure 6B] Figure 6B shows a typical method in this disclosure for isolating and / or detecting endocytic agents and / or exocytotic vesicles in the blood and / or tissues of animals after treating them with an endocytic agent by oral administration. [Figure 6C] Figure 6C shows the detection of the polypharmacologically divalent endocytosis agent ARV110 (Example 19) in the supernatant or exosome lysate isolated from CACO2 cell-containing culture medium after treatment with Example 19. Compared to the exosome lysate sample, Example 19 was completely or almost undetectable in the supernatant sample. The endocytosis agent in the total sample was detected by LC-mass spectroscopy. [Figure 6D] Figure 6D shows the levels of CD36, CD9, HSP70, and TSG101 proteins in exocytosis vesicles released by CACO2 cells after treatment with DMSO control or the representative multipharmacological divalent endocytosis agent ARV110 (Example 19). Immunoblots for CD36, CD9, HSP70, and TSG101 proteins in exosomes isolated from CACO2 cells after treatment with DMSO vehicle control or Example 19. Compared to the control vehicle, treatment with Example 19 enhances CD36 expression in the exocytosis vehicle. [Figure 6E]Figure 6E shows the levels of CD36, CD9, HSP70, and TSG101 proteins in exosomes isolated from rat plasma after oral treatment with a specified dose of vehicle or a representative polypharmacologically divalent endocytosis agent ARV110 (Example 19). Compared to the control vehicle, treatment with Example 19 enhances CD36 expression in the exocytosis vehicle in vivo. On the other hand, compared to supernatant samples from plasma of rats treated with ARV110, LC-mass spectroscopy revealed higher concentrations of Example 19 in all corresponding exosome lysates. [Figure 7A] Figures 7A-7K show that increasing the binding affinity (see Table 2) and / or binding valency (see Table 1) to CD36 leads to increased drug uptake and therapeutic effect. Figure 7A shows that under the same conditions, some trivalent endocytosis agents (Examples 24-31) with higher binding affinity and more binding arms (valencies) induce deeper BRD4 degradation in HCC1806 TNBC or 22Rv1 PCa cells than a divalent endocytosis agent (Example 23). Immunoblots for BRD4 after treatment of HCC1806 or 22Rv1 cells with a DMSO vehicle control or endocytosis agent. [Figure 7B] Figure 7B shows that, under the same conditions, the trivalent endocytosis agent Example 29, which has higher binding affinity and more binding arms (valences), induces deeper BRD4 degradation in HCC1806 TNBC cells than the divalent endocytosis agent (Example 23). BRD4 protein was examined by immunoblotting, and BRD4 protein levels were quantified by densitometry and normalized against the density of the corresponding β-tubulin protein. [Figure 7C]Figure 7C shows that, under the same conditions, the trivalent endocytosis agent Example 29, which has higher binding affinity and more binding arms (valences), induces faster BRD4 degradation in HCC1806 TNBC cells than the divalent endocytosis agent (Example 23). Immunoblots for BRD4 after HCC1806 cells were treated with a DMSO vehicle control or the endocytosis agent for a specified period. [Figure 7D] Figure 7D shows that in HCC1806 cells, pretreatment with MG132 reverses BRD4 degradation induced by endocytosis agent Example 23 or Example 29. BRD4 protein levels in HCC1806 cells treated with the endocytosis agent examples, with or without pretreatment with the specified concentrations of MG132, were determined by Western blotting assay. [Figure 7E] Figure 7E shows that shCD36 reverses BRD4 degradation induced by endocytosis agent Example 23 or Example 29 in HCC1806 cells. After treating HCC1806 cells stably transfected with shCD36 or untransfected with the specified concentrations of endocytosis agent in the examples, BRD4 and CD36 protein levels were determined by Western blotting assay. [Figure 7F] Figure 7F shows that pretreatment with a SYK inhibitor, known to block endocytosis, reverses BRD4 degradation induced by endocytosis agents in Examples 23 or 29 in HCC1806 cells. BRD4 protein levels in HCC1806 cells treated with the specified concentrations of entospretinib, or not, were determined by Western blotting assays. [Figure 7G]Figure 7G shows that, under the same conditions, some trivalent endocytosis agents (Examples 32-37) with higher binding affinity and more binding arms (valencies) induce deeper BRD4 degradation in HCC1806 TNBC or 22Rv1 PCa cells than a divalent endocytosis agent (Example 23). Immunoblots for BRD4 after treatment of HCC1806 or 22Rv1 cells with a DMSO vehicle control or endocytosis agent. [Figure 7H] Figure 7H shows that, under the same conditions, in 22Rv1 PCa cells, the trivalent endocytosis agent Example 35, which has higher binding affinity and more binding arms (valence), induces deeper BRD4 degradation than the divalent endocytosis agent (Example 23). BRD4 protein was examined by immunoblotting, and BRD4 protein levels were quantified by densitometry and normalized against the density of the corresponding β-tubulin protein. [Figure 7I] Figure 7I shows that in 22Rv1 PCa cells, the trivalent endocytosis agent Example 35, which has higher binding affinity and more binding arms (valence), induces faster BRD4 degradation than the divalent endocytosis agent (Example 23). Immunoblots for BRD4 after 22Rv1 cells were treated with a DMSO vehicle control or the endocytosis agent for a specified period. [Figure 7J] Figure 7J shows that in 22Rv1 PCa cells, pretreatment with MG132 reverses BRD4 degradation induced by endocytosis agents Examples 23, 35, or 36. BRD4 protein levels in 22Rv1 cells treated with the specified concentrations of MG132, with or without pretreatment, were determined by Western blotting assays. [Figure 7K]Figure 7K shows that shCD36 reverses BRD4 degradation induced by endocytosis agents in Examples 23 or 35 in 22Rv1 PCa cells. After treating 22Rv1 cells stably transfected with shCD36 or untransfected with the specified concentrations of endocytosis agents in the examples, BRD4 protein levels were determined by Western blotting assay. [Figure 8A] Figures 8A–8H illustrate how introducing chemical arms via cleavable bonds or moieties enhances drug uptake and therapeutic efficacy. Figure 8A shows that, under the same conditions, several endocytosis agents conjugated with chemical arms via cleavable bonds (Examples 44–51) induced deeper AR proteolysis in MDA-MB-453 TNBC cells than the endocytosis agent ARV110 (Example 19). Unlike the data in MDA-MB-453 cells, Examples 44–51 were unable to induce AR degradation in C4-2PCa cells, indicating the existence of isoform and / or conformational diversity of membrane proteins in different cancer cell lines. Immunoblots for AR after treatment of MDA-MB-453 or C4-2 cells with DMSO vehicle control, enzalutamide, or Example 19, or Examples 44–51. [Figure 8B] Figure 8B shows that, under the same conditions, several endocytosis agents conjugated with diverse chemical arms via cleavable bonds (Examples 46-4, 46, 52, or 53) induce deeper AR proteolysis in MDA-MB-453 TNBC cells than the endocytosis agent ARV110 (Example 19). Immunoblots for AR after treatment of MDA-MB-453 cells with DMSO vehicle control, enzalutamide, or the endocytosis agents in the examples. [Figure 8C]Figure 8C shows that, under the same conditions, an endocytosis agent conjugated to a chemical arm via a cleavable bond (Example 46) induced deeper AR degradation than the endocytosis agent ARV110 (Example 19) in MDA-MB-453 TNBC cells. AR proteins were examined by immunoblotting, and AR protein levels were quantified by densitometry and normalized against the density of the corresponding GAPDH protein. [Figure 8D] Figure 8D shows that, under the same conditions, an endocytosis agent conjugated to a chemical arm via a cleavable bond (Example 46) induces faster AR degradation in MDA-MB-453 TNBC cells than the endocytosis agent ARV110 (Example 19). Immunoblots for AR after MDA-MB-453 cells treated with a DMSO vehicle control, Example 19, or Example 46 for the specified period. [Figure 8E] Figure 8E shows that shCD36 partially reverses AR degradation by endocytosis agent Example 19 or Example 46 in MDA-MB-453 TNBC cells, suggesting that the CD36 isoform or other endocytosis-mediated membrane components are involved in the pickup of endocytosis agents. After treating shCD36-transfected or untransfected MDA-MB-453 cells with the specified concentrations of endocytosis agents in the examples, AR and CD36 protein levels were determined by Western blotting assays. [Figure 8F] Figure 8F shows that pretreatment with MG132 reverses AR degradation induced by endocytosis agent Example 19 or Example 46 in MDA-MB-453 TNBC cells. AR protein levels in MDA-MB-453 cells treated with the specified concentrations of MG132, or not, were determined by Western blotting assays. [Figure 8G]Figure 8G shows that pretreatment with a SYK inhibitor reverses AR degradation in MDA-MB-453 TNBC cells as stipulated in Example 19 or Example 46. AR protein levels in MDA-MB-453 cells treated with or not treated with specified concentrations of entospretinib, as in the endocytosis agent examples, were determined by Western blotting assay. [Figure 8H] Figure 8H shows that under the same conditions, several endocytosis agents conjugated with chemical arms via cleavable binding (Examples 54-61) induce deeper BRD4 proteolysis than the divalent endocytosis agent Example 23 in HCC1806 TNBC or 22Rv1 PCa cells. Examples 58 and 59 (see Table 2), which have additional binding arms but no improved binding affinity, induce deeper BRD4 degradation. Several endocytosis agents exhibit different potencies in inducing BRD4 degradation in HCC1806 and 22Rv1 cells, indicating the existence of isoform and / or conformational diversity of membrane proteins in different cancer cell lines. Immunoblots for BRD4 after 22Rv1 cells treated with DMSO vehicle control, Example 23, or Examples 54-61. [Figure 9A] Figures 9A-9G illustrate how introducing chemical arms via relatively stable bindings or moieties enhances drug uptake and therapeutic efficacy. Figure 9A shows that under the same conditions, some of the polypharmacological trivalent endocytosis agents Examples 63-70 induce deeper BRD4 proteolysis than the polypharmacological divalent endocytosis agent Example 62 in HCC1806 TNBC or 22Rv1 PCa cells. In HCC1806 and 22Rv1 cells, some endocytosis agents exhibit different potencies in inducing BRD4 degradation, indicating the existence of diversity in membrane protein isoforms and / or conformations across different cancer cell lines. Immunoblots for BRD4 after treatment of HCC1806 or 22Rv1 cells with a DMSO vehicle control or Examples 62-70. [Figure 9B] Figure 9B shows that under the same conditions, some of the polypharmacological trivalent endocytosis agents Examples 71-77, which have higher binding affinity and more binding arms (valencies), induce deeper BRD4 proteolysis in HCC1806 TNBC, 22Rv1, or LNCaP PCa cells than the polypharmacological divalent endocytosis agent Example 62. Immunoblots for BRD4 after treatment of HCC1806, 22Rv1, or LNCaP cells with DMSO vehicle control, Example 62, or Examples 71-77. [Figure 9C] Figure 9C shows that, under the same conditions, some of the polypharmacological trivalent endocytosis agents from Examples 78–85 induced deeper BRD4 proteolysis in 22Rv1 PCa cells than the polypharmacological divalent endocytosis agent from Example 62. Immunoblots for BRD4 after treating 22Rv1 cells with DMSO vehicle control, Example 62, or Examples 78–85. [Figure 9D] Figure 9D shows that, under the same conditions, in 22Rv1 cells, Example 71, a polypharmacological trivalent endocytosis agent with higher binding affinity and more binding arms (valences), induced deeper BRD4 degradation than Example 62, a polypharmacological divalent endocytosis agent. BRD4 protein was examined by immunoblotting, and BRD4 protein levels were quantified by densitometry and normalized against the density of the corresponding β-tubulin protein. [Figure 9E] Figure 9E shows that, under the same conditions, the polypharmacological trivalent endocytosis agent Example 71 induces faster BRD4 degradation in 22Rv1 cells than the polypharmacological divalent endocytosis agent Example 62. Immunoblots for BRD4 after treating 22Rv1 cells with the indicated concentration of DMSO vehicle control, Example 62, or Example 71. [Figure 9F]Figure 9F shows that in 22Rv1 cells, pretreatment with MG132 reverses BRD4 degradation induced by multipharmacological endocytosis agents in Examples 62, 71, or 79. BRD4 protein levels in 22Rv1 cells treated with or not treated with specified concentrations of MG132 in the multipharmacological endocytosis agent examples were determined by Western blotting assay. [Figure 9G] Figure 9G shows that shCD36 in 22Rv1 cells reverses BRD4 degradation in 22Rv1 cells induced by multipharmacological endocytosis agents in Examples 62 or 79. After treating 22Rv1 cells stably transfected with shCD36 or untransfected with the specified concentrations of multipharmacological endocytosis agents in the examples, BRD4 and CD36 protein levels were determined by Western blotting assay. [Figure 10A] Figures 10A-10D illustrate how the introduction of a chemical arm increases drug uptake, expanding the therapeutic efficacy and safety window of the drug. Figure 10A shows that in a breast cancer HCC1806-xenograft mouse model, polypharmacological trivalent endocytosis agent example 28 or 29 exhibits a higher antitumor effect than polypharmacological divalent endocytosis agent example 23. In the graph, the top curve represents the vehicle, the second curve from the top represents example 23, the third curve from the top represents example 28, and the bottom curve represents example 29. [Figure 10B] Figure 10B shows that in the HCC1806 xenograft mouse model, the polypharmacological trivalent endocytosis agents in Examples 28 or 29 exhibit no visible toxicity. The top curve represents the vehicle, the second curve from the top represents Example 23, the third curve from the top represents Example 28, and the bottom curve represents Example 29. [Figure 10C]Figure 10C shows that the polypharmacological trivalent endocytosis agent Example 28 or 29 is more potent than the polypharmacological divalent endocytosis agent Example 23 in reducing BRD4 protein levels in breast cancer HCC1806 xenografts. Immunohistochemical examination of representative tumors of mice treated with the vehicle, Example 23, Example 28, or Example 29 for BRD4 (brown to black). Scale bar: 20 μm. [Figure 10D] Figure 10D shows that in a 22Rv1 xenograft mouse model of prostate cancer, Example 36 of the polypharmacologically active trivalent endocytosis agent showed a higher antitumor effect than Example 23 of the polypharmacologically active divalent endocytosis agent. On the other hand, in a 22Rv1 xenograft mouse model of prostate cancer, Examples 71 or 79 of the polypharmacologically active trivalent endocytosis agent showed a higher antitumor effect than Example 62 of the polypharmacologically active divalent endocytosis agent. In the graph, the top curve represents the vehicle, the second curve from the top represents Example 23, the third curve from the top represents Example 62, the fourth curve from the top represents Example 36, the fifth curve from the top represents Example 79, and the bottom curve represents Example 71. [Figure 10E] Figure 10E shows that in the 22Rv1 xenograft mouse model, multipharmacological trivalent endocytosis agents in Examples 23, 36, 62, or 71 did not exhibit visible toxicity. [Figure 11] This figure shows that the endocytosis agent conjugated with a chemical arm (Example 29) exhibits higher solubility in water than the endocytosis agent Example 23. After standing for 24 hours, no precipitate was observed in the aqueous solution of Example 29, and the stability of Example 29 in aqueous solution was confirmed by LC mass spectrometry. [Modes for carrying out the invention]

[0011] This specification discloses compounds comprising an endocytosis agent or probe linked to / or fused with an additional membrane-binding moiety. The additional membrane-binding moiety is selected to enhance the efficacy and / or efficiency of endocytosis by a membrane component that mediates endocytosis. Linking or fusion of the additional membrane-binding moiety with the endocytosis agent or probe allows for greater uptake across the membrane than the original agent or probe. This specification also discloses methods for screening and identifying endocytic agents, including known chemicals having an unknown endocytic mechanism for cell permeation.

[0012] The advantage of this technology is that it provides a fundamental mechanism and method for an alternative and simple drug discovery and development process based on chemical endocytosis, compared to classical drug discovery and development based on passive diffusion. Endocytosis is a biological process in which all cells absorb large, volume-fractional external substances such as nutrients, viruses, and bacteria by encapsulating them in the cell membrane. This process is initiated by the recruitment of substances (e.g., proteins, fatty acids, and exosome particles) that bind to endocytosis-mediated membrane components, such as membrane receptors including transporters. Binding to the endocytosis-mediated membrane components forms an endocytosis pit or infiltration of the cell membrane. Membrane internalization is completed by a division process that releases the initial endosome into the cell. After internalization, the substance and the contents of the endosome can be released into the cell, and the endocytosis-mediated membrane components can be recycled to the cell surface for another delivery round. Furthermore, substances and membrane components that mediate endocytosis can be released by cells into the extracellular compartment in the form of extracellular vesicles (EVs), such as exosomes, through extracellular propagation. Compared to passive diffusion driven by a concentration gradient from high to low concentration until reaching an equilibrium plateau, the higher efficacy and efficiency of the endocytosis process is demonstrated by the fact that over 95% of some nutrients in the daily diet are transported via endocytosis and enter the bloodstream (Deanna M. Minich, et al., Journal of Lipid Research, 38, 1709-1721, 1997). For example, as a multifunctional scavenger receptor with multiple ligands, CD36 is abundantly expressed on the luminal surface of intestinal cells in the gut and promotes CD36-mediated endocytosis as the dominant pathway for the highly efficient absorption of large amounts of fatty acids in food (Vincenza Cifarelli, et al., Comprehensive Physiology, (8)2, 493-507, 2018; Helene Poirier, et al., European Journal of Biochemistry, 238(2), 368-373, 1996).When fatty acids bind to CD36, downstream kinases are activated, initiating endocytosis and efficiently promoting uptake of the supply across the membrane barrier (Jian-Wei Hao, et al, Nature Communications, 11(1), 4765, 2020; Richard F. Collins, et al., Journal of Biological Chemistry, 284(44), 30288-30297, 2009). As revealed by crystallization studies, a prominent feature of CD36 is a large hydrophobic groove (residues 127-279) that extends for most of the length of the protein ectodomain, enabling the binding and accommodation of fatty acids such as stearic acid (C18:0) and kinked docosahexaenoic acid (C22:6) (Fu-Lien Hsieh, et al., Nature Communications, 7, 12837, 2016; Zineb Tarhda, et al., Bioinformatics and Biology Insights, 7, 369-373, 2013). Furthermore, endocytosis plays many important roles not only in normal cell physiology but also in pathology, such as recycling membrane receptors for dysregulated signaling and steadily increasing the influx of exogenous nutrients for homeoanabolism in cancer cells. For example, to grow under stress, cancer cells upregulate CD36 expression, increasing lipid mass production and generating oncogenic signaling lipids within the cancer cell.Increased CD36 expression is associated with poor prognosis in various cancer cells, including lung squamous epithelial cells, glioblastoma, leukemia, prostate cancer, bladder cancer, and breast cancer (Gloria Pascual, et al., Nature, 541, 41-45, 2017; Tian Zhang, et al., Blood Cancer Discovery, 1(2), 198-213, 2020; Matthew J. Watt, et al., Science Translational Medicine, 11(478), eaau5758, 2019; Aritro Nath, et al., Scientific Reports, 5, 14752, 2015; James S. Hale, et al., Stem Cell, 32(7), 1746-1758, 2014). However, the intracellular endocytosis of chemicals by targeting endocytosis-mediated membrane components for effective and efficient uptake of therapeutic agents remained unclear. Chemical endocytosis approaches can accelerate therapeutic drug development and broaden the range of therapeutic agents that can be effectively used for targeted research, diagnosis, prevention, and treatment. Broadly speaking, compared to current methods that seek to balance conflicting parameters of a drug (stability, water solubility, and membrane permeability), the medicinal chemistry methods based on chemical endocytosis in this technology can simplify and innovate the drug discovery and development process by improving permeability by enhancing the efficacy and / or efficiency of endocytosis, thereby adjusting the metabolic stability of the drug while maintaining biological activity, because solubility can be improved relatively easily by salting-out or charge formation techniques in this technology. Furthermore, precision healthcare methods based on differential expression of endocytosis-mediated membrane components can be used for patient stratification for individualized selection of drugs and drug administration routes.

[0013] The advantage of this technology is that endocytosis enables drug uptake across the membrane. In particular, this technology is useful for enabling or increasing the uptake of large and / or polar drugs, which can be effectively utilized in targeted research, diagnosis, and treatment. As predicted by the "Rule of Five (Ro5)" (Christopher A. Lipinski, et al., Advanced Drug Delivery Reviews, 46, 3-26, 2001), the most influential framework for correlating the physicochemical properties of a given compound with membrane permeability, drugs that are smaller in size (less than 500 Da), less polar (topological polar surface area (tPSA) less than or equal to 140 Å), and more lipophilic (but with a LogP of less than or equal to 5) pass through the cell membrane more easily by passive diffusion. As molecular size increases (especially when MW exceeds 700 Da), drugs exhibiting higher polarity (higher tPSA values) or lower lipophilicity (lower LogP values) may face a sharp decrease in passive diffusion across cell membranes (Par Matsson, et al., Journal of Medicinal Chemistry, 60(5), 1662-1664, 2017; Cameron R. Pye, et al., Journal of Medicinal Chemistry, 60(5), 1665-1672, 2017).As disclosed herein, 1) endocytic agents that increase binding affinity and / or titer to endocytosis-mediated membrane components, 2) endocytic agents that enable or increase dimerization or clustering of endocytosis-mediated membrane components (Laura Salavessa, et al., Proceedings of the National Academy of Sciences of the United States of America, 118(37), e2024893118, 2021; John Maringa Githaka, et al., Journal of Cell Science, 129(22), 4175-4189, 2016), or 3) endocytic agents that enable or increase conformational changes of endocytosis-mediated membrane components can be better absorbed by cells via endocytosis.

[0014] In the context of this technology, "clustering" or "cluster" refers to the process by which endocytosis-mediated membrane components assemble into nanoscale and microscale domains that control biological processes including, but not limited to, cell adhesion, endocytosis, and immune responses. For example, upon ligand binding, endocytosis-mediated membrane proteins cluster, leading to localized protein crowding and / or changes in membrane conformation, promoting membrane bending and the initiation of the endocytosis process. "Clustering" or "cluster" of endocytosis-mediated membrane components is understood to include dimerization, oligomerization, and polymerization of membrane components. "Clustering" or "cluster" of endocytosis-mediated membrane components may be homogeneous, heterogeneous, or a combination of any ratio of homogeneous and heterogeneous mixtures. It is understood that the conformational changes of membrane components that mediate endocytosis may be independent of, and / or related to, the "clustering" or "clustering" of membrane components that mediate endocytosis.

[0015] Upon receiving input signals such as ligand binding, chemical modification, or environmental changes, membrane proteins undergo conformational changes, and proteins with different conformations can exert diverse or significant biological functions. For example, after receiving a binding stimulus, membrane proteins can switch conformations, influencing the growth, bending, and endocytosis of the clathrin lattice within cells (Kazuki Obashi, et al., 14, 732, Nature Communication, 2023). Conformational changes of membrane proteins induced by ligand binding depend on factors including binding affinity and binding domains (Anna Vangone, et al., eLife, 4, e07454, 2015). Even without increasing binding affinity, increasing the binding titer to pick up additional binding domains can induce conformational changes and further promote endocytosis. The advantage of this technology is that it enables or enhances the conformational changes of endocytosis-mediated membrane components through structural modification of the endocytic agent in order to increase the efficacy and / or efficiency of endocytosis, and structural modification of the endocytic agent includes, but is not limited to, the creation of charged molecules by structural modification using medicinal chemistry strategies in the art, including by forming a polyvalent endocytic agent by linking charged, chargeable and other hydrophilic chemical arms, or by introducing reversible or irreversible covalent bonds or moieties, or by salt formation techniques in the art. Conformational changes of endocytosis-mediated membrane components to increase the efficiency and / or efficacy of endocytosis can be achieved by 1) increasing the binding affinity and / or titer of the endocytic agent to the endocytosis-mediated membrane components, and / or by 2) increasing the dimerization or clustering of the endocytosis-mediated membrane components through structural modification of the endocytic agent.Furthermore, conformational changes of endocytosis-mediated membrane components for enhanced endocytosis efficiency and / or efficacy can be achieved by adapting 1) environmental factors of endocytosis-mediated membrane components, such as pH, salt concentration, oxygen gradient, carbon dioxide gradient, H2O2 gradient, nutrient gradient, and therapeutic compound gradient; 2) interactions with membrane cofactor proteins; and / or 3) post-translational modifications of endocytosis-mediated membrane components and membrane cofactor proteins, such as ubiquitination, phosphorylation, palmitoylation, glycosylation, acetylation, and lipidation, through the use of endocytic agents or structural modifications.

[0016] Another advantage of this technology is that it enables or enhances drug permeability without sacrificing solubility and stability. Based on passive diffusion theory, a common method for enabling or enhancing drug permeability is to increase the lipophilicity of the compound through structural optimization on a case-by-case basis, allowing it to readily dissolve in membrane phospholipids and rapidly diffuse through the cell membrane, but at the cost of reduced solubility (due to decreased hydrophilicity), stability, and / or intrinsic biological activity. However, evidence has shown that structural optimization of bRo5 agents to enable or enhance passive membrane permeability while maintaining a desirable balance of metabolic stability and solubility is difficult, or even impossible (Par Matsson, et al., Journal of Medicinal Chemistry, 60(5), 1662-1664, 2017; Victoria G. Klein, et al., Journal of Medicinal Chemistry, 64(24), 18082-18101, 2021). Compared to current methods that seek to balance conflicting parameters of therapeutic agents (stability, water solubility, and membrane permeability), this technology simplifies and innovates the drug discovery and development process by improving the metabolic stability and endocytosis efficiency of chemical molecules, since water solubility can be achieved relatively easily, for example, by salt formation technology. Salt or charge formation not only improves solubility but also increases binding affinity and / or titer to membrane components that mediate endocytosis, potentially increasing cellular uptake spontaneously. On the other hand, increasing water solubility reduces metabolism in the first pathway and also decreases the tendency toward stability. This technology makes it possible to improve the solubility and stability profile by increasing permeability through improved endocytosis efficiency and / or efficacy.

[0017] Another advantage of this technology is that, in the art, by using charged molecules or salt formation techniques to form a polyvalent endocytosis agent by linking charged, chargeable, and other hydrophilic chemical arms, or by structural modifications using medicinal chemistry strategies in the art, including the introduction of reversible or irreversible covalent bonds or moieties, it is possible to increase the solubility and / or binding affinity and / or titration of the endocytosis-mediating membrane component, dimerization or clustering of the endocytosis-mediating membrane component, and / or conformational changes of the endocytosis-mediating membrane component to enhance the efficacy and / or efficiency of drug endocytosis. The resulting endocytosis agent may exist in aqueous solution at a specific pH value in the form of cations or anions, and both the solubility of the endocytosis agent and the binding affinity and / or titration for picking up salt bridges, hydrogen bond interactions, or reversible / irreversible covalent bond formation with the endocytosis-mediating membrane component are increased to enhance the efficacy and / or efficiency of endocytosis.Chargeable or hydrogen-bonding moieties such as lysine, arginine, cysteine, serine, threonine, lipids, and carbohydrates are often found in membrane components such as transmembrane proteins, and are more readily bound to ionic drugs or drugs with ionic bonds via salt bridges, hydrogen bond interactions, or reversible covalent bond formation (Ondrej Kuda, et al., Journal of Biological Chemistry, 288(22), 15547-15555, 2013; Dante Necukai, et al., Nature, 504(7478), 172-176, 2013; Joanna SG Slusky, et al., Bioinformatics, 29(17), 2122-2128, 2013; Yibo Wang, et al., Journal of Physical Chemistry B, 125(8), 2124-2133, 2021; Anupam Bandyopadhyay, et al., Current Opinion in Chemistry Biology, 34, 110-116, 2016). Therefore, by structural modifications using reversible covalent chemical strategies in the art, such as joining or substituting any chemical bond or portion in a drug having a chemical bond or portion that can form a reversible covalent bond with any nucleophile in biology (including lysine, arginine, cysteine, serine, threonine, and carbohydrates) (Anupam Bandyopadhyay, et al., Current Opinion in Chemistry Biology, 34, 110-116, 2016), the resulting endocytosis agent can increase its binding affinity and / or titer to endocytosis-mediated membrane components, enable or increase dimerization or clustering of endocytosis-mediated membrane components, and / or enable or increase conformational changes of endocytosis-mediated membrane components, thereby enhancing the efficacy and / or efficiency of endocytosis.Alternatively, by structural modifications including joining or substituting any chemical bond or moiety in a drug having a cleavable chemical bond or moiety, in combination with strategies including joining or substituting any chemical bond or moiety in a drug having a chemical bond or moiety that can form a reversible or irreversible covalent bond with any nucleophile in biology, the resulting endocytic agent can increase its binding affinity and / or titer to endocytosis-mediated membrane components, enable or increase dimerization or clustering of endocytosis-mediated membrane components, and / or enable or increase conformational changes of endocytosis-mediated membrane components, thereby enhancing the efficacy and / or efficiency of endocytosis. The cleavable chemical bond or moiety in the resulting endocytic agent can be cleaved in cells or in the body, resulting in the release of a biologically active endocytic agent from the endocytosis-mediated membrane components, which can exert any biological function. Therefore, this technology enables a spontaneous increase in the solubility and / or permeability of endocytic agents by endocytosis for the diagnosis and treatment of diseases.

[0018] Another advantage of this technology is that it allows for the modification of the metabolic stability of endocytic agents. To meet the stability requirements of the drugs needed for the treatment and diagnosis of the target, the methodologies of this disclosure include, but are not limited to, the addition or removal of substituents, fragment substitution, cyclization, scaffold hopping, bioisosterism, linkerology (linker-activity relationship studies), prodrugs, or reduction of the overall LogP value. In certain embodiments, the method refers to the substitution of any hydrogen atom on the endocytic agent with deuterium and / or fluorine. In certain embodiments, the method refers to the introduction of substituents to reduce metabolism at soft spots in the drug. These methods allow for a balance between the desired functionality of the endocytic agent, its binding affinity to membrane components that mediate endocytosis, and its metabolic stability for disease treatment.

[0019] In conventional drug discovery and development campaigns, highly polar or hydrophilic forge compounds were determined to have low permeability because they hardly dissolve in the membrane lipid bilayer and do not permeate and diffuse across the cell membrane. As polarity increases, the permeability of polar or hydrophilic compounds decreases sharply. However, the surface of proteins, especially membrane proteins, generally contains hydrophilic outward-facing amino acids, and some post-translational modification (PTM) moieties, such as glycan groups, tend to be located on the surface of membrane proteins that can bind to polar compounds (including, but not limited to, inorganic compounds, chelate compounds, metal-based compounds, and / or polar organic compounds such as phosphatase inhibitors and peptide-based compounds) via salt bridges or hydrogen bond formation. Furthermore, membrane proteins often possess small molecule binding regions. Therefore, the advantage of this technology is that endocytosis is a general pathway for the uptake of endocytic agents, regardless of their molecular weight, polarity, and hydrophilicity. In particular, a key advantage of this technology is that endocytosis can facilitate the cellular uptake of hydrophilic agents, including but not limited to polar organic compounds such as inorganic compounds, chelating compounds, metal-based compounds, and / or phosphatase inhibitors and peptide-based compounds. For example, by appropriately linking a low molecular weight membrane protein binder to a phosphatase inhibitor or insulin, salt bridges or hydrogen bonds can be formed between the phosphatase inhibitor or insulin and the surface amino acids of the membrane protein, inducing conformational changes in the endocytosis process. This technology allows polar compounds to be absorbed and used by oral administration.Endocytosis phosphatase inhibitors obtained by introducing a diffusion-facilitating glucose transporter (GLUT) binding moiety onto a phosphatase inhibitor having a cleavable or incleavable bond or moiety can be used orally for endocytosis by increasing the binding affinity and / or titer to endocytosis-mediated membrane components, and / or enhancing the dimerization or clustering of endocytosis-mediated membrane components, and / or altering the conformation of endocytosis-mediated membrane components. Endocytosis insulins obtained by introducing a CD36 binding moiety onto an insulin molecule having a cleavable or incleavable bond or moiety can be used orally for endocytosis by increasing the binding affinity and / or titer to endocytosis-mediated membrane components, and / or enhancing the dimerization or clustering of endocytosis-mediated membrane components, and / or altering the conformation of endocytosis-mediated membrane components. The stability of insulin may be increased from endocytosis and exosome formation after endosome evasion.

[0020] The driving force behind drug uptake by endocytosis is the binding between the endocytic agent and the membrane component mediating endocytosis. Since endocytosis is a process that can be independent of passive diffusion, the advantage of this technology is that it is useful for enabling endocytic agents to permeate the cell membrane and / or body barrier, either through endocytosis alone or in combination with other mechanisms including, but not limited to, passive diffusion, facilitated diffusion, transporter-mediated influx and / or efflux, and intercellular transport.

[0021] The advantage of this technology is that it expands the current concept of polypharmacology from seeking combinations or synergistic effects of the intrinsic activity of drugs against multiple disease-related targets or pathways (Rajan Chaudhari, et al., Expert Opinion on Drug Discovery, 15(9), 1025-1044, 2020) to combinations or synergistic effects of binding properties between disease-related targets or pathways and membrane components that mediate endocytosis. Synergistic or additive effects between disease-related targets or pathways and endocytosis-mediating membrane components may reduce the dose requirements of endocytic agents for therapeutic purposes; increased binding affinity and / or titer of endocytic agents to endocytosis-mediating membrane components, dimerization or clustering of endocytosis-mediating membrane components, and / or conformational changes of endocytosis-mediating membrane components may enhance the efficacy and / or efficiency of endocytosis, allowing for better absorption via endocytosis. This can compensate for the insufficient inherent activity of some endocytic agents against disease-related targets or pathways for the diagnosis and treatment of diseases.

[0022] The binding affinity of an endocytosis agent to the membrane component that mediates endocytosis may be independent of its binding ability to the original pharmacological target. Therefore, an endocytosis agent can be a polypharmacological compound that spontaneously possesses binding affinity to both the membrane component that mediates endocytosis for endocytosis and the pharmacological target for the original pharmacological activity. Thus, another advantage of this technology is a method for spontaneously identifying or generating novel endocytosis agents through polypharmacological endocytosis and functional targeting. One method is to directly modify the structure of the drug to readily bind to both the membrane component that mediates endocytosis for endocytosis and the pharmacological target for the original pharmacological activity. Here, the process of identifying or generating novel polypharmacological endocytosis and functional targeting agents utilizes classical medicinal chemistry in the art by spontaneously monitoring the activity against the modulation efficiency of both endocytosis and the biological target. For example, to develop endocytosis BCL-2 inhibitors, assays measuring CD36 binding affinity by SPR and assays measuring BCL-2 targeting efficiency by SPR can be used in a classical medicinal chemistry optimization process in the art to identify or generate endocytosis and BCL-2 dual targeting agents. Multiple chemical arms in the ligand are helpful in improving the binding affinity of both CD36 and BCL2, and / or efficient uptake. Furthermore, polyvalent agents or agents with increased binding titers can induce conformational changes in membrane components to enhance dimerization or clustering of membrane components and / or activate the endocytosis cascade. In addition, as revealed by crystallization studies and molecular modeling, bell-shaped ligands or macroligands containing multiple side chains were able to provide good alignment with the CD36 binding pocket (residues 127-279).Therefore, another method of this disclosure for identifying or generating novel endocytic agents with enhanced polypharmacological properties, including the efficacy and / or efficiency of endocytosis and the biological function of the target, is simply to link or fuse an additional chemical arm having binding affinity to endocytosis-mediating membrane components and / or target proteins to the endocytic agent via cleavable or incleavable chemical bonds or linker units, either via surface or within pocket interactions, to form a polyvalent compound, the resulting polyvalent endocytic agent may have, to enhance the efficacy and / or efficiency of endocytosis, 1) increased affinity and / or binding titer to endocytosis-mediating membrane components and / or increased activity to the biological target, 2) enable or increase dimerization or clustering of endocytosis-mediating membrane components, or 3) enable or increase conformational changes of endocytosis-mediating membrane components, thereby being better absorbed via endocytosis and further maintaining or increasing the biological function of the target. Chemical arms with affinity for CD36 and biological targets can be constructed by library synthesis and tracked by screening. Compared to current medicinal chemistry methods that modify one inherent property of a drug through structural alteration, inevitably affecting other properties, this technique allows for increased permeability of endocytosis agents without sacrificing, or even improving, solubility, stability, and the inherent pharmacological activity for disease research, diagnosis, prevention, and treatment.

[0023] As a result of endocytosis, foreign bodies can be picked up by cells and released from (donor) cells via exocytosis or membrane fusion in the form of extracellular vesicles (EVs), including but not limited to exosomes, apoptotic bodies, and microvesicles (MVs) or ectosomes, although foreign bodies can reside at any site within the extracellular vesicles. Endogenous extracellular vesicles are taken up by any (acceptor) cell via endocytosis, membrane fusion, or transcytosis, resulting in the transport of foreign bodies across two or more cell layers (Ravi Shah, et al., The New England Journal of Medicine, 8(379), 958-966, 2018; Oscar PB Wiklander, et al., Science Translational Medicine, 11(492), eaav8521, 2019; Raghu Kalluri, et al., Science, 367(6478), eaau6977, 2020). Therefore, another advantage of this technology is that it is a method for transporting endocytic agents across two or more cell layers in the body, and the endocytic agents can be taken up by cells via endocytosis and released in the form of free endocytic agent molecules and / or endocytic agent-vesicle complexes (i.e., exocytosis vesicles), which contain lipid bilayer vesicles containing the endocytic agent and extracellular vesicles, and the exocytosis vesicles can be taken up by any (acceptor) cell for action via endocytosis or membrane fusion for any purpose of use. In particular, a further advantage of this technology is that it is a "one-step" method for generating and using exocytosis vesicles in humans and animals, without further processes including isolation of the drug or vesicles, the cell body picks up the endocytic agent by endocytosis and sequentially secretes exocytosis vesicles by exocytosis, and the resulting endogenous exocytosis vesicles can be directly distributed to any tissue and used by the body, including the brain, for any purpose.

[0024] Another advantage of this technology is the method for producing, isolating, and using exocytosis vesicles in vitro and / or in vivo for any purpose. In particular, one method for generating exocytosis vesicles is that cells in an animal body can take up an endocytic agent by endocytosis and then spontaneously secrete exocytosis vesicles by exocytosis. Another method for generating functional exocytosis vesicles is that the endocytic agent molecule can be linked to extracellular vesicles and / or exocytosis vesicles in situ via covalent or noncovalent bonds. Another method for generating exocytosis vesicles is to load the endocytic agent onto exocytosis vesicles in vitro by any technique in the art. Another method is to isolate exocytosis vesicles from cells, body fluids, tissues, organs, products, or culture media by any extracellular vesicle isolation technique in the art.

[0025] Extracellular vesicles (EVs) can carry soluble substances such as soluble cytokines, and therefore EVs can be sufficiently dissolved in aqueous solutions (Ana Paula Ramos, et al., Journal of Extracellular Biology, 1(1), e34, 2022). Therefore, another advantage of this technique is the method of producing and using exocytosis vesicles in vitro or in vivo to enhance the water solubility of endocytic agents for any purpose. For example, a method to increase the water solubility of a drug in order to have water solubility in an animal body is to enable or increase the uptake of the endocytosis agent by the method described above, resulting in increased loading of the endocytosis agent into exocytosis vesicles and / or release of exocytosis vesicles via the cellular endocytosis / exocytosis process. Endocytosis agents can be dissolved in aqueous solutions in the form of exocytosis vesicles.

[0026] Another advantage of this technology is that it provides a method for modulating the absorption, distribution, metabolism, and excretion (ADME) characteristics of endocytic agents in animal bodies. For example, to extend the residence time and / or half-life of a given endocytic agent in an animal body, the method of this technology enables or increases the uptake of the endocytic agent by endocytosis via the method described above, and enables or increases the loading of the endocytic agent into exocytic vesicles and the release of exocytic vesicles by cellular endocytosis, thereby reducing or preventing the first-pass metabolism and / or excretion of the endocytic agent. To improve the permeability of the drug to the brain, the method of this technology enables or increases the uptake of the endocytic agent by endocytosis via the method described above, and enables or increases the loading of the endocytic agent into exocytic vesicles and the release of exocytic vesicles by cellular endocytosis, thereby reducing or preventing the efflux transport of the endocytic agent across the blood-brain barrier.

[0027] Another advantage of this technology is a method for evaluating and / or determining the ADME properties of an endocytosis agent in a drug discovery and development process, the method comprising a process or step of isolating and / or lysing exocytosis vesicles from a sample. For example, in a particular embodiment, the endocytosis agent concentration in blood or tissue after administration of the endocytosis agent includes free endocytosis agent molecules in the blood or tissue, and endocytosis agent molecules encapsulated in exocytosis vesicles such as exosomes in the blood or tissue. Any techniques and skills in the art for isolating and / or lysing EVs to release the endocytosis agent from the exocytosis vesicles for endocytosis agent detection are applicable to isolating and / or lysing exocytosis vesicles in this disclosure.

[0028] Another advantage of this technology is that it is a method for reducing drug toxicity by creating and using exocytosis vesicles containing endocytosis agents in vitro or in vivo. Hematological toxicity is the primary toxicity of cytotoxic drugs (Etienne Chatelut, et al., Investigational New Drugs, 21, 141-148, 2003). For example, most PARP inhibitors in clinics have been observed to cause hematological toxicity, including neutropenia, anemia, thrombocytopenia, fatigue, and bleeding, due to the toxicity of PARP inhibitors to platelets, leukocytes, and erythrocytes (Yamin Shu, et al., Cancer Medicine, 12(3), 3365-3375, 2023). As disclosed herein, endocytic agents can be taken up by cells in the form of exocytotic vesicles, such as endocytic agent-carrying exosomes, and released from cells. However, the endocytic agent resides within the exosome, which can prevent or reduce direct contact between free endocytic agent molecules and blood cells. Furthermore, as natural particles, exosomes possess unique properties such as innate stability and low immunogenicity (Raghu Kalluri, et al., Science, 367, eaau6977, 2020). Therefore, another advantage of this technology is that it is a method of reducing drug toxicity by using membrane components and exocytosis processes that mediate endocytosis within cells and tissues to release exocytotic vesicles and prevent or reduce drug toxicity. For example, after oral administration of an endocytosis agent, cells in the gastrointestinal tract can pick up the endocytosis agent via endocytosis and sequentially secrete exocytosis vesicles into the circulatory system. The resulting exocytosis vesicles have reduced hematological toxicity compared to free endocytosis agent molecules (Ashish K. Agrawal, et al., Nanomedicine, 13, 1627-1636, 2017).In particular, an additional advantage of this technology is that any structural modification method disclosed herein enables or modulates the efficacy and / or efficiency of endocytosis, and reduces drug toxicity by modulating the efficacy and / or efficiency of endocytosis, the loading of endocytic agents onto exocytic vesicles, and / or the release of exocytic vesicles in different cells and tissues.

[0029] Another advantage of this technology is that it can enhance the efficacy and reduce the toxicity of endocytic agents by utilizing the different expression of membrane components that mediate endocytosis in cells and tissues. Alterations in metabolic activity have been shown to support the malignant characteristics of cancer cells (Ralph J. DeBeradinis, et al., Science Advances, 2(5), e1600200, 2016). For example, recent studies have shown that high expression of CD36 plays a crucial role in tumor development, growth, invasion, and metastasis by increasing the supply of nutrients such as fatty acids and lipids, thereby fueling tumor cells (Gloria Pascual, et al., Nature, 541, 41-45, 2017; Matthew J. Watt, et al., Science Translational Medicine, 11(478), eaau5758, 2019). Therefore, targeted drug delivery by overexpression of endocytosis-mediated membrane components in cancer cells, or by upregulation of nutrient uptake by endocytosis in cancerous cells, can minimize potential toxicity to normal cells and extend the therapeutic window of drugs. While the examples of this disclosure demonstrate that CD36-mediated endocytosis mediates the intracellular absorption of endocytic agents, other endocytosis-mediated membrane components described in (Sara Sigismund, et al., Nature Review Molecular Cell Biology, 22, 625-643, 2021), such as scavenger receptors (SR), endothelial protein C receptors (EPCR), fatty acid-binding proteins (FABPpm), fatty acid transport proteins (FATP), free fatty acid receptor 1 (GPR40), and epidermal growth factor receptor (EGFR), and facilitating glucose transporter (GLUT) (including all variants, mutations, splice variants, indels, and fusions) can be used for endocytic drug uptake.Furthermore, other chemical modifications to endocytosis agents may confer stronger binding affinity or titer to endocytosis-mediated membrane components, enable or increase dimerization or clustering of endocytosis-mediated membrane components, and / or enable or increase conformational changes of endocytosis-mediated membrane components, thereby improving the efficacy and / or efficiency of endocytosis and potentially functioning as a platform for the uptake of enhanced efficacy and / or efficiency, particularly eRo5 and bRo5 drugs. Thus, differences in the expression of endocytosis membrane proteins can be used for patient stratification of endocytosis drugs.

[0030] Another advantage of this technology is that the different expressions of endocytosis-mediated membrane components in cells and tissues can be utilized in specialized delivery systems such as topical, inhalation, intraperitoneal, intravenous, microinjection, and oral delivery. For example, since endocytosis-mediated membrane components such as CD36 or GLUT are abundantly expressed on the luminal surface of intestinal epithelial cells in the intestine (Fatiha Nassir, et al., Journal of Biological Chemistry, 282(27), 19493-19501, 2007), by appropriately optimizing the affinity and metabolic stability to endocytosis-mediated membrane components, endocytic agents, particularly those present in the chemical space of eRo5 and bRo5, can have acceptable oral bioavailability. Another advantage of this technology is the administration of endocytosis agents via any suitable route, including oral (including buccal or sublingual), topical (including buccal, sublingual, transdermal, or ophthalmic), inhalation, parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) routes, intraperitoneal injection, and microneedle patches.

[0031] Another advantage of this technology is that the structural diversity of endocytosis-mediated membrane components can be used for the structural design and modification of endocytic agents. Membrane proteins have diverse structural states in a variety of cells or under various conditions, including but not limited to cell size and volume, the cellular microenvironment, and the status of post-translational modifications (Raghavendar Reddy Sanganna Gari, et al., Nature Communications, 12, 4363, 2021; Diego del Alamo, et al., eLife, 11, e75751, 2022; Alex R Terry, et al., Cell Metabolism, 35, 1-7, 2023). Different three-dimensional structures of membrane proteins exhibit significant sensitivity to ligands (Dante Necukai, et al., Nature, 504(7478), 172-176, 2013; Fu-Lien Hsieh, et al., Nature Communications, 7, 12837, 2016). Therefore, the advantage of this technology lies in the design or structural modification of endocytosis agents using medicinal chemistry strategies in the art, or in the use of endocytosis agents that target the specific three-dimensional structures of membrane components that mediate endocytosis for any purpose. For example, the CD36 protein located on the membranes of normal cells and cancer cells can adopt different three-dimensional structures. It is reasonable to utilize the diversity of three-dimensional structures of CD36 on normal cells and cancer cells to design or modify endocytosis agents using medicinal chemistry strategies in the art, or to select endocytosis agents that target the specific three-dimensional structures of CD36 on cancer cells in order to enhance antitumor effects and reduce toxicity.Protein structural switches change their shape and biological function in response to input signals such as ligand binding, chemical modification, or environmental changes (Matin Dutertre, et al., Perspective in Pharmacology, 295(2), 431-437, 2000). The advantage of this technology is that it can alter the conformation of endocytosis-mediated membrane components by utilizing any approach in the art, such as the application of mechanical, electrical, thermal, cooling, light, or radiation stimulation, and / or the presence of endocytic agents, thereby resulting in changes in biological events in cells and / or changes in the sensitivity of endocytosis-mediated membrane components to endocytic agents.

[0032] Another advantage of this technology is that the differential expression and / or conformation of endocytosis-mediated membrane components can be used for precision medicine in individuals receiving this endocytic agent. Meanwhile, feedback from endocytic agent treatments in clinics can be used to adjust the administration route of the endocytic agent and / or improve the outcomes of endocytic agent treatments. Studies have demonstrated differential expression of membrane components between individuals, between cells and tissues within a single individual, and the existence of different disease stages within a single individual (Roy L. Silverstein, et al., Science Signaling, 2(72), re3, 2009; Aritro Nath, et al., Scientific Reports, 4(6), 18669, 2016; Matthew J. Watt, et al., Science Translational Medicine, 11(478), eaau5758, 2019). In addition, membrane proteins have diverse conformational and / or post-translational states in different cells or under different conditions, resulting in significant sensitivity of membrane proteins to ligands. This disclosure shows that endocytosis is a pathway for cellular uptake of endocytic agents, and that endocytosis-mediated membrane components such as CD36 have different expression and / or structural conformations in cells and / or tissues of different patients (Vincenza Cifarelli, et al., Comprehensive Physiology, (8)2, 493-507, 2018; Helene Poirier, et al., European Journal of Biochemistry, 238(2), 368-373, 1996).Therefore, the advantage of this technology is that it stratifies patients for the individualized selection of endocytic agents and routes of administration. Endocytosis-mediated membrane components can be used as biomarkers in cells and / or tissues, and the different expression and / or conformation of endocytosis-mediated membrane components in a patient's cells and / or tissues can be used for patient stratification, selection of administration routes and dosages, and selection and / or administration of endocytic agents to treat the target. The diversity of gene profiling is widespread in various cells, tissues, and bodies. It is reasonable to leverage the differences in gene profiling in cells, tissues, and bodies to move biological agents within the drug discovery and development value chain. As the first PROTAC targeting the androgen receptor (AR) in clinical trials, ARV-110 was able to completely degrade AR and most of its point variants in preclinical studies. In clinical studies, patients with tumors bearing AR T878 or H875 point mutations were found to be particularly sensitive to ARV110 treatment (Xin Gao, et al, Journal of Clinical Oncology, 40(6,) suppl. 017, 2022). All of these agents / drugs / vesicles, including but not limited to those disclosed in the examples herein (Table 1), can be enhanced in terms of their clinical benefit to patients by appropriately selecting patents that have high expression of endocytosis-mediated membrane components such as CD36 and / or ligand-sensitive conformations in diseased tissue and / or absorptive tissues such as the intestine, as demonstrated by the inventors as endocytic agents. Another advantage of this technology is that endocytosis agents can be used for the study, diagnosis, prevention, and treatment of any subject, including but not limited to aging and age-related diseases and conditions, weight management, cancer, central nervous system (CNS) diseases and conditions, cardiovascular diseases (CVD), diabetes mellitus, eye diseases and conditions, hypertension, immune system diseases, infections, inflammation and inflammation-related diseases and conditions, obesity and obesity-related diseases and conditions, respiratory diseases and conditions, and skin diseases and conditions. The human body has a barrier against foreign substances, making drug discovery for the treatment of subjects, particularly CNS or skin diseases and conditions, challenging (Sung Min Pyo, et al., Skin Pharmacology and Physiology, 32, 283-293, 2019; William M. Pardridge, NeuroRX, 2(1), 3-14, 2005).Following endocytosis and exocytosis, the exocytosis process releases extracellular vesicles (EVs) and free nutrients (Marcel Grapp, et al., Nature Communications, 4, 2123, 2013; Raghu Kalluri, et al., Science, 367(640), 2020), the blood-retinal barrier (Monica Diaz-Coranguez, et al., Vision Research, 139, 123-137, 2017), the lung endothelial and epithelial barriers (Mikihisa Takano, et al., Expert Opinion on Drug Delivery, 12(5), 813-825, 2015; Julia Voigt, et al., Proceedings of the National Academy of Sciences, 111(8), 2942-2947, 2014), and the skin barrier (Noriaki Nagai, et al., International Journal of It facilitates the permeation of free-form nutrients and / or extravasation molecules across membrane barriers such as the cerebral blood barrier (Molecular Sciences, 19(7), 2138, 2018) and the cerebral blood barrier (Mathew W. Smith, et al., Journal of Drug Targeting, 14(4), 191-214, 2006).For example, by binding to receptors or proteins expressed in the cerebral blood barrier, such as scavenger receptors (e.g., CD36), major facilitator superfamily domain-containing protein 2 (Mfsd2a), flotilin-1, flotilin-2, glucose transporter 1 (GLUT1), glutathione transporter, amino acid transporters (e.g., L-type amino acid transporter 1, LAT1), transferrin receptor, lactoferrin receptor, low-density lipoprotein receptor, nicotinic acetylcholine receptor, insulin receptor, insulin-like growth factor receptor, integrins (e.g., αVβ3 integrin), and / or CD13 / APN receptor, endocytotic agents can be absorbed by cerebral endothelial cells, transported across the cerebral blood barrier via endocytosis, and reach the brain parenchyma via the endocytosis / exocytosis pathway in the form of free endocytotic agents, exocytotic vesicles, or mixtures of free endocytotic agents and exocytotic vesicles. Furthermore, endocytosis allows endocytic agents to be absorbed into cells and located in intracellular organelles and / or vesicles such as endosomes and polyvesicles, reducing or preventing the pumping of endocytic agents out of cells by efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-related proteins MRP1, MRP3, MRP4, and MRP6. It is understood that by utilizing the expression of endocytosis-mediated membrane components in the membrane barrier, and / or any structural modifications of the agents in this disclosure, the efficacy and / or efficiency of endocytic agents endocytosis and the transport of endocytic agents across the membrane barrier for any purpose may be improved. Therefore, a special advantage of this technology is that, after membrane component-mediated endocytosis, endocytic agents can be transported across the membrane barrier via the endocytosis / exocytosis pathway in the form of free endocytic agents, exocytic vesicles, or mixtures of free endocytic agents and exocytic vesicles in any ratio, thus enabling the use of endocytic agents in the study, diagnosis, prevention, and treatment of conditions and diseases of the eye, respiratory system, skin, and CNS.

[0033] Another advantage of this technology is that it is a method for identifying literature-known bioactive compounds that can be taken up by cells via endocytosis. This method involves modifying (e.g., increasing, decreasing, or deleting) the expression of endocytosis-mediated membrane components in cells, tissues, and / or bodies by gene editing, knockdown, or silencing of the endocytosis-mediated membrane components, and then comparing the activity of bioactive compounds in cells, tissues, and / or bodies with or without gene editing, knockdown, or silencing of the endocytosis-mediated membrane components. Compared to the activity of compounds in cells, tissues, and / or bodies without gene editing, knockdown, or silencing of the endocytosis-mediated membrane components, compounds taken up by endocytosis show significantly increased or decreased bioactivity in cells, tissues, and / or bodies with editing, knockdown, or silencing of the endocytosis-mediated membrane components.

[0034] Another advantage of this technology is the method of using identified endocytosis agents as probes for identifying cellular and intracellular biological targets by any technology and skill in the art. For example, identified endocytosis agents having chemical bonds or moieties that can form covalent bonds with any biological target, such as biotin, fluorescence, halo-tag ligands, SNAP-tag ligands, CLIP-tag ligands, or any biological target, are particularly suitable for identifying the biological targets of endocytosis agents and derivatives in combination with technologies and skills including microscopy imaging, immunophenotyping, immunoprecipitation, flow cytometry, fluorescence-activated cell sorting (FACS), fluorescence resonance energy transfer (FRET), and / or omics (including genomics, epigenomics, transcriptomics, proteomics, and metabolomics) analysis. In particular, an advantage of this technology is the method for identifying membrane targets that mediate endocytosis. A method for identifying membrane targets that mediate endocytosis may involve using a labeled endocytosis agent followed by drug administration. Detection and identification techniques include microscopy, immunoprecipitation, immunophenotyping, flow cytometry, fluorescence-activated cell sorting (FACS), fluorescence resonance energy transfer (FRET), omics (including genomics, epigenomics, transcriptomics, proteomics, and metabolomics) analysis, and / or positron emission tomography (PET) scans using computed tomography (CT) or magnetic resonance imaging (MRI). For example, a biotin-labeled endocytosis agent can be obtained by ligating a labeled probe, such as biotin, to an endocytosis agent at an appropriate site. Subsequently, cells can be cultured with the biotin-labeled endocytosis agent, membrane proteins can be isolated, and then identified by immunoprecipitation, FACS, omics analysis, and Western blotting to target the membrane components that mediate endocytosis of the endocytosis agent. Another method involves using genome scanning techniques in the art to identify membrane targets that mediate endocytosis.For example, comparing gene expression between cells, tissues, or bodies with different sensitivities to a particular endocytosis agent can be used to identify membrane targets that mediate endocytosis. Another method involves using any gene editing technique in the art to identify membrane targets that mediate endocytosis, where the gene-edited cells, tissues, or bodies may be more sensitive or more resistant to endocytosis treatment. For example, cells with gene expression inhibition and activation by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa), respectively, may act separately or complementarily to a particular endocytosis treatment, and this can be used to identify membrane targets that mediate endocytosis.

[0035] As used herein, “gene editing” refers to the process of artificially introducing genetic modification. Genetic manipulation can be performed at the DNA, RNA, or epigenetic level. Genetic recombination includes: (i) deletion of an endogenous gene; (ii) introduction of recombinant nucleic acids encoding wild-type or mutant versions of endogenous or exogenous proteins; (iii) introduction of RNA molecules that interfere with the functional expression of a protein (e.g., RNA molecules that inhibit the functional expression of a protein (e.g., small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense RNA, and microRNA (miRNA)); or (iv) modification of the promoter or enhancer element (i.e., regulatory element) of one or more endogenous genes. Item (ii) includes the replacement of an endogenous gene (e.g., by homologous recombination) with a gene encoding a modified or entirely different protein, and item (iv) includes the modification or manipulation of a regulatory region of a target gene, or any region contiguous with the target gene (e.g., up to 5KB bilaterally of the target sequence). It is understood that genetic engineering includes modifying endogenous genes to produce proteins with additions (e.g., heterologous sequences), deletions, or substitutions (e.g., point mutations or other mutations; conserved or non-conserved mutations). Mutations can be introduced specifically (e.g., by site-directed mutagenesis or homologous recombination) or randomly (e.g., by chemical mutagenesis). Thus, genetic engineering can modulate genes in several ways, including increased expression, increased function, decreased expression, decreased function, and gene knockout. Exemplary methods include, but are not limited to, RNA-based RNA interference, including small interfering RNAs (siRNAs) and small hairpin RNAs (shRNAs), DNA-based RNA interference, including antisense oligonucleotides, and CRISP-mediated gene genome editing techniques.

[0036] Another advantage of this technology is that when using or selecting the endocytosis agents of the present invention for any purpose, different cellular and tissue microenvironmental factors can be used as modulators, which include, but are not limited to, pH values, salt concentrations, oxygen gradients, carbon dioxide gradients, H2O2 gradients, nutrient gradients, and therapeutic compound gradients. For example, because cancer cells are in an acidic environment, using endocytosis agents with basic groups for tumor-targeted delivery can enhance therapeutic function and reduce toxicity. Similarly, endocytosis agents with basic amino groups can be used to enhance localization to the blood-brain barrier (BBB), thereby enhancing therapeutic function and reducing toxicity.

[0037] Another advantage of this technology is that the expression of endocytosis-mediated membrane components with diverse three-dimensional structures, isoforms (or variants), and / or post-translational modifications can be used as modulators when using or selecting this endocytosis agent for any purpose. Another advantage of this technology is that the expression of cofactors that form complexes with endocytosis-mediated membrane components can be used as modulators when using or selecting current endocytosis agents for any purpose. Membrane proteins adopt diverse three-dimensional structures under various conditions, and proteins with diverse three-dimensional structures exhibit various affinities and biological functions to ligands and other proteins. Isoforms of membrane proteins may have unique expression differences between cells, between individuals, or between functions. Furthermore, the diversity of glycosylation states in membrane proteins can lead to complex pleiotropy, where a unique modification at one glycosylation site may alter function or recognition in certain cellular conditions, but produce different effects or functional silence in other conditions. Therefore, endocytosis agents can be used to enhance therapeutic function and reduce toxicity in cancer treatment by targeting, for example, the specific three-dimensional structure, isoform, glycosylation, palmitoylation, or phosphorylation state of membrane components or cofactors that mediate endocytosis in cancer cells.

[0038] Another advantage of this technology is that the endocytosis agent binds to endocytosis-mediated membrane components, inducing membrane internalization via the endosomal / lysosomal pathway for digestion. This technology is useful when utilizing endocytosis agents to deliver disease-related endocytosis-mediated membrane components or extracellular materials into cells via endocytosis, and to disrupt or degrade disease-related endocytosis-mediated membrane components or extracellular materials via the endosomal / lysosomal system. Disease-related endocytosis-mediated membrane components or extracellular materials include, but are not limited to, membrane proteins or extracellular proteins, carbohydrates, lipids, pathogens, particles, viruses, fungi, protozoa, bacteria, vectors, cellular debris, and other cells.

[0039] Another advantage of this technology is that, in order to enhance the endocytic absorption of endocytic agents, the endocytic process can be activated or the expression of endocytosis-mediated membrane components can be increased by modulating input signals, such as the binding of endogenous or exogenous substances to membrane proteins. For example, the binding of insulin to insulin receptors on the membrane activates the endocytic cycling of endocytosis-mediated membrane components and / or increases the expression / or rearrangement of endocytosis-mediated membrane components such as GLUT, resulting in enhanced absorption of endocytic agents via endocytosis. It can be understood that endocytic agents can enhance endocytosis-mediated absorption by activating the endocytic process or increasing the expression of endocytosis-mediated membrane components by simultaneously binding to endocytosis-mediated and non-endocytosis-mediated membrane components.

[0040] As shown in Figure 1A, this specification discloses endocytic agents or salts thereof that bind to membrane components mediating endocytosis and can be absorbed into cells by endocytosis. Also disclosed in Figure 1A are drug discovery methods that enable or modify the binding affinity and / or titer of a drug to membrane components mediating endocytosis in order to enhance the efficacy and / or efficiency of endocytosis. Figure 1B discloses the absorption mechanisms, sequential distribution, metabolism, and excretion (ADME) of endocytosis agents across various administration routes (including oral, topical, and inhalation). Methods for evaluating or determining the ADME characteristics of endocytosis agents in the drug discovery and development process are also disclosed. As shown in Figure 1C, methods for administering endocytotic agents or exocytotic vesicles orally or intravenously are disclosed for the study, diagnosis, prevention, and treatment of all subjects, particularly CNS conditions and diseases. Methods for using endocytotic agents in combination with extracellular vesicles (EVs) are also disclosed for the study, diagnosis, prevention, and treatment of all diseases. As shown in Figure 1D, the mechanism of action of the endocytosis agent is disclosed. After absorption, the endocytosis agent or extracellular vesicles, or exocytosis vesicles containing the endocytosis agent, are absorbed into the cell via endocytosis or membrane fusion and can act for any purpose.

[0041] Furthermore, a “one-step” method for generating and using exocytosis vesicles in humans or animals is disclosed, which allows cells in the body to take up an endocytic agent via endocytosis, sequentially secrete exocytosis vesicles, and directly use the resulting endogenous exocytosis vesicles for any purpose in the body, without further processes including the isolation of drugs or vesicles. A method for isolating exocytosis vesicles is also disclosed. Here, a method for producing exocytosis vesicles is one in which cells take up an endocytic agent via endocytosis and then secrete exocytosis vesicles via exocytosis. Another method for forming functional exocytosis vesicles is to covalently or noncovalently bind endocytic agent molecules to extracellular vesicles and / or exocytosis vesicles in situ. Methods for producing and using them are disclosed.

[0042] Endocytosis agents An endocytosis agent, or drug, refers to any compound, part thereof, or conjugate, that binds to membrane components that mediate endocytosis and can be absorbed by a cell by endocytosis. In some embodiments, a drug is a therapeutic agent, a diagnostic agent, a conjugate, a conjugate, a nanoparticle, or a vesicle. A therapeutic agent is any compound useful for treating the target. A diagnostic agent is any compound useful for providing qualitative or quantitative information about a biomolecular target or a biological environment of interest. A conjugate is any compound that binds to membrane components or extracellular material that mediates endocytosis, and includes membrane proteins or extracellular proteins, carbohydrates, lipids, pathogens, particles, viruses, bacteria, fungi, protozoa, vectors, cell debris, and cells. A conjugate is a compound consisting of one or more drugs conjugated to one or more chemical arms via cleavable or incleavable chemical bonds or linker units. A vesicle is an extracellular or extracellular structure consisting of fluid or cytoplasm encapsulated in a lipid bilayer.

[0043] As described below, the endocytosis agents of this disclosure include, but are not limited to, neutral compounds, free bases or acids, their salts, solvates, and prodrugs, and, although not expressly stated or shown, their structures, particularly their pharmaceutically acceptable forms, may contain sulfur oxide atoms or nitrogen quaternide atoms. Such forms, particularly pharmaceutically acceptable forms, are intended to be covered by the appended claims.

[0044] In some embodiments, the endocytosis agent has a binding affinity of less than 20.0 mM K D It has binding affinity to membrane components that mediate endocytosis, which has a value. In some embodiments, the endocytosis agent has a molecular weight greater than 200 Da. In certain embodiments, the endocytosis agent has a molecular weight greater than 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, and 3000 Da. In some embodiments, the endocytosis agent has a molecular weight between 200 and 10000 Da. In some embodiments, endocytosis agents are therapeutic agents. Exemplary therapeutic agents include, but are not limited to, drugs, protein inhibitors or antagonists, protein activators or agonists, protein modulators, molecular adhesives that induce or stabilize protein-protein interactions, proteolytic agents, or polyvalent agents, protein binders, diagnostic agents or chemical probes, or vesicles containing deuterium / or fluorine-substituted derivatives, analogs and chelates thereof, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers. Therapeutic agents are also protein, DNA, or RNA modifiers.

[0045] In certain embodiments, the therapeutic agent is a drug that can bind to membrane receptors involved in the cellular endocytosis process. Exemplary drugs include, but are not limited to, any of the following compounds: 68Ga-DOTA-FAPI-46, 68Ga-DOTA-2P9FAPI)2, ABT-737, acarbose, acetaminosarol, actinomycin D, adenosine A1 receptor (A1R) agonists, aliskiren, AMG-131, argatroban, ascomycin, asukamycin, asunaprevir, atazanavir, atorvastatin, AZD5153, azithromycin, B-cell lymphoma 2 (BCL-2 family protein inhibitors), e.g., venetoclax (ABT-119), navitoclax (ABT-263), APG-2575 (Lisaftoclax), UBX1325 (CAS number: 2271269-01-1), APG-1252 (Pelcitoclax), APG-2575 (Lisaftoclax), AMG176, AMG397, AZD4606 (CAS: 2241039-81-4), AZD5991, AZD4747 (CAS: 2489226-14-2), BGJ-398, Virinapant, Bis(7)-Taclin, BLU-945, BMS-777607, BM S-791325, BMS-986165 (CAS: 1609392-27-9), Brigatinib (CAS: 1197953-54-0), Brigimadlin, Camdronate, Camptothecin, Candoxatril, Capreomycin, Ceritinib, CHF-6366, Clarithromycin, Cobimetinib, Combretatropone, Compound 28 (Fedor Romanov-Michailidis, et al., Journal of Medicinal Chemistry, 66, 6122-6148, 2023), Cosmomycin D, CPT-Gly-PEG-Folate, CUDC-101, CUDC-907, Cyclosporine A, Daclatasvir, Dactinomycin, Dalfopristine, Danamide F, Danaprevir, Danoprevir (CAS: 850876-88-9), Danuglipron (CAS: 2230198-02-2), DHP1808, Diaspirin, Digoxin, Diprobosim-X,Divalisib (CAS: 2417987-45-0), Diaminoallose phosphate (DAP), DNL343, Doxorubicin, Dasatinib, DU1301, ECPU-0001, EDO-S101, EML981, Epcolitamab (CAS: 2134641-34-0), Eptifivatide, EPZ-5676, Ergotamine, Eritoran, Erythromycin A, Erythronolide, Etoposide, Erythromycin A, Estramustine, Etaclaplatin, Everolimus, EZN-2208 (CAS: 946062-05-1), FAP I-46, FAPI dimer, Fenebrutinib (CAS: 1434048-34-6), Fedratinib, Fosinopril (CAS: 98048-97-6), Hostamatinib, G protein-coupled receptor (GPCR) inhibitor, G protein-coupled receptor (GPCR) agonist, Galticertive, Glecaprevir (CAS: 1365970-03-1), Himeate A, Histone deacetylase (HDAC) inhibitor, Homohalintin, Inhibitor of Apoptosis (IAP) protein inhibitor, e.g., AZD5582 (CAS :1258392-53-8), SM-164 (CAS number: 957135-43-2) and xebinapant, indoleamine 2,3-dioxygenase (IDO) inhibitor, indobufen, IR820-SS-CPT, IT-101, itraconazole, ivermectin, JNJ78394355, JS230, KX2-361, radostigil, lapatinib, leucomycin, Lipitor (atorvastatin), ronafarnib, Lumakras (CAS: 2296729-00-3), LUNA18 (CAS: 22676177- 63-0), LY3502970 (CAS:22212020-52-3), Mammalian target of rapamycin (mTOR) and / or FK506-binding protein (FKBP) protein inhibitors, medoxomil, mirademethan (CAS:1398568-47-2), MIP-1404, MK-1468, MK-8768 (CAS:1432729-22-0), mometasone furoate, motixafortide (CAS:664334-36-5), MPI8 (CAS:856242-63-2), MRT-2359 (CAS:2803881-11-8),MRTX-849 (CAS: 2326521-71-3), Mubaraprine (CAS: 2565656-70-2), NDI-034858 (CAS: 2272904-53-5), Nilotinib, Nintedanib, NOSH-Aspirin (NBS-1120), NKTR-102 (CAS: 1193151-09-5), NKTR-105, Nilmatrellvir ODDA-PTX, Omaberoxolone, Anovinost, Ortataxel, Ouabain, PAANIB-1, Paclitaxel, Pacritinib (CAS: 937272-79-2), Perabrecib, Pevonedistat (CAS: 905579-51-3), PF-03715455, Faceristatin, Pictilisib, Piflufolastat F-18 injection, Pulvicto (CAS number: 1703749-62-5), peroxisome proliferator-activated receptor (PPAR) agnoist, PRMT inhibitor, protein phosphatase inhibitor, protein arginine methyltransferase (PRMT) inhibitor, pralcetinib, pseudomonate A, PSMA-11, PSMA-617, PSMA-1007, PSMA I&S, PSMA I&T, PSMA SMOL-TTC monomer, PSMA SMOL-TTC dimer, PSMA SMOL-TTC trimer, PSMA SMOL-TTC tetramer, PU-H71, pyrilutamide, quizartinib (CAS: 950769-58-1), quinupristin, rapamycin, Rapalink-1, REC-3599, rifampicin, rifapentin, rifabutin, rifaximin, Rilzabrutinib (CAS: 1575596-29-0), ritonavir, rivaroxaban, RMC-4998 (CAS: 2642037-07-6), RMC-6291 (CAS: 2641998-63-0), RMC-62 36 (CAS:2765081-21-6), RMC-4998 (CAS:2642037-07-6), Roxithromycin, RPT193 (CAS:2366152-15-8), Sanguinamide A, S63845, S64315, Saquinavir, Scavenger receptor (SR) inhibitors, activators, or binders, Endothelial cell protein C receptor (EPCR) inhibitors, activators, or binders, Cetileuton, SHP-1971, Simeprevir, Sirolimus, Solute carrier (SLC) transporter inhibitors,An activator, or a binder, Simeprevir (CAS: 923604-59-5), Sparsentan (CAS: 254740-64-2), Spiramycin, Staurosporine, Interferon gene stimulator (STING) agonist, Sug-HisVal-CPT, Tacrolimus, Taledegib, Tapotoclax (CAS: 1883727-34-1), Tat-P4-(C5)2 (doi.org / 10.15252 / emmm.201911248), TNG348, Tryptophan 2,3-dioxygenase (TDO) inhibitor, Telithromycin, Thiosptrepton, Chinostamstin, Tirbanibulin (KX2-391), Trioxacine, Tripdronate, Tubacin, Tubocurarin, VCP746 (CAS number: 1582751-84-5), Bora selcib, Bazepant (CAS: 1337918-83-8), VX-548 (CAS: 2649467-58-1), Zatebradine, Zotarolimus, deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.,

[0046] In certain embodiments, the therapeutic agent is a protein inhibitor or antagonist that includes isotopes thereof, such as deuterium-substituted derivatives.,

[0047] The protein inhibitor or antagonist can target any suitable protein (including all variants, mutants, splice variants, indels, and fusions of these listed target proteins). Examples include the 5HT2c receptor, α 1A-AR, α-2 adrenergic receptor, α-synuclein, AAK1, ATP-binding cassette (ABC) transporter, e.g., MDR1 / 2 / 3 / 4 / 5 and ABCG2, ABL, cholesterol acyltransferase (ACAT), ACE protein, ACK, ACTR2, ACTR2B, ADCK1 / 2 / 3 / 4 / 5, protein kinase B (AKT), AlphaK1 / 2 / 3, anaplastic lymphoma kinase (ALK), ALIX, amnionless, AMPA receptor (AMPAR), AMP-related protein kinase (AMPK), amyloid beta, angiotensin II receptor, ANKRD3, Apaf-1, apolipoprotein E receptor (ApoER), amyloid precursor protein (APP), androgen receptor (AR), ARAF, ARG, AR-V7, ileal tip sodium / bile acid cotransporter (ASBT), V-type proton ATPase 6 (ATP6 V), ATP6V1H, avidin, amino acid transporter, alanine serine cysteine ​​transporter (ASCT), ASGPR, ASK1 / 2, ataxin-1, ataxia telangiectasia mutant protein (ATM), ATM and Rad3-related protein (ATR), aurora kinase, AXL, β-site amyloid precursor protein cleavage enzyme 1 (BACE1), β2-adrenergic receptor, BAD, BARK1 / 2, Bax, BCKDK, B-cell leukemia / lymphoma (BCL) family proteins, e.g., BCL2, BCL-XL, and MCL-1, BCR-ABL; bromodomain and extraterminal domain family proteins (BET), e.g., BRD2 / 3 / 4 / T, beta-catenin, BIKE / BMP2K, BLK, BMPR1A, BMPR1B, BMPR2, BMX / ETX, CDO sibling (Boc), BRD9, BMI1, BRAF, BRAF V600EBrassinosterone-resistant 1 (BRI1), BRK, BRM, BRSK1, BRSK2, Bruton's tyrosine kinase (BTK), BUB1, C3G, calreticrin, cell adhesion molecule (CAM) receptor, CAMK1α / β / γ / δ, CAMK2α / β / γ / δ, CAMK4, caMLCK, cannabinoid-1 receptor, coxsackievirus-adenovirus receptor (CAR), CBL-B, CRISPR-related protein (Cas), CASK, caspase-3, caspase-6, caspase-7, caspase- 9, CBFβ, CBL, CBP, chemokine receptors, e.g., CC chemokine receptor (CCR) and CXC chemokine receptor (CXCR), e.g., CXCR2, CXCR4, and CXCR7, CCK4 / PTK7, CCR2, CCR9, CCRK, differentiation antigens (CD), e.g., CD4, CD9, CD13 / APN receptor, CD25, CD26 (DPP4), CD30, CD33, CD36, CD38, CD46, CD56, CD63, CD71, CD74, CD80, CD81, CD82, CD83, CD86, CD123, CD138, CD147, C D152, CD152 (CTLA4), CD166, CD174, CD197, CD205, CD227, CD228, CD269, CD276, and CD326, CDC7, CDC20, CDC25, CDC37, CDK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18, CDKL1 / 2 / 3 / 4 / 5, CDON, CEACAM5, Cholesteryl ester transfer protein (CETP), c-Fos, Cystic fibrosis transmembrane conduction regulator (CFTR), Cyclic GMP-AMP synthase (cGas), Chak1 / 2, CHK1 / 2, CI-M6PR, CK1α, CK1α2, CK1δ, CK1ε, CK1γ1 / 2 / 3, CK2α1 / 2, CLIK1, CLIK1L, Clip, CLK1 / 2 / 3 / 4, Human Collectin Placenta 1 (CL-P1), CMYC, Cone Opsin, COT / TPL2, Cell Permeation Peptide (CPP), Connexin, Coronavirus Protease, CRABP, C-RAF, Cereblon (CRBN), CREB, CRK, CRIK, CRIPTO, CSF2R, CSF1R / FMS, CSK, C-TAK1, CTK, Cubilin, Cyclin D,Cyclin E, CytoC, DAPK1 / 2 / 3, DCAMKL1 / 2 / 3, DDR1 / 2, Diglyceride syltransferase (DGAT), DLK, DMPK1 / 2, DNA-PK, DP-1, DPP-4, DRAK1 / 2, Deubiquitinating enzyme (DUB), DYRK1A / 1B / / 2 / 3 / 4, E2F, Excitatory amino acid carrier 1 (EAAC1), E-cadherin, Endothelin-converting enzyme (ECE), ED-B, EED, EF2K, eEF2K, EIF2A3, EIF4E, EIF4G, ELK, ENL, Endothelin B receptor, Epithelial cell adhesion molecule (EpCAM), Endothelial cell protein C receptor (EPCR), Ephrin receptor (EphR), Estrogen receptor (ER), ER81, ErbB2, ErbB3, ETS transcription Factors (ERG), ERK1 / 2 / 3 / 4 / 5 / 7 / 8, ERRa, EZH2, fatty acid binding protein (FABP), FADD, FAK, FAP, fatty acid transport protein (FATP), neonatal Fc receptor (FCRN), FER, FKBP, FLIP, folate receptors, e.g., reduced folate carriers, FOLT1 / 2 / 3 and proton-bound folate transporters (PCFT), FOXM1, FOXO1 / 2 / 3 / 4, FRK, Frizzled4, fructose-1,6-bisphosphatase, FYN, γ-aminobutyric acid type A receptor (GABAAR), GAK, Gap-1, growth arrest specific 1 (Gas1), GCC, GCK, GCN2, GD2, ghrelin receptor (ghrelinR), glucose-dependent insulinopromoting polypeptide (GIP), Globo H, glucagon receptor, glucagon-like peptide 1 (GLP-1), glutamate transporter (GLT), glucokinase, ion channel type AMPA glutamate receptor (GluR), glucose-6-phosphatase, glucose transporter (GLUT), glutathione transporter, glycogen phosphorylase, glycogen synthase kinase, glycoproteins, e.g., p18, gp31, and gp60, G protein-coupled receptors (GPCRs), e.g., GPR20, GPR20, free fatty acid receptor 1 (GPR40), GPR119, and GPR120, GPNMB, GPRK4 / 5 / 6 / 7, GRB2, GSK-3, GSPT1, Haspin kinase, HCK, hepatitis C virus (HCV) NS3 / 4A, histone deacetylase (HDAC),HDL receptor (HDLR), HectH9, HH498, HIF1, HIPK1 / 2 / 3 / 4, HMGN1, HMG-CoA reductase, hnRNP, H-PGDS, HPGCR, hematopoietic precursor kinase 1 (HPK1), HRAS, HRI, hRpn13, Pru HSD-11β (11β-hydroxysteroid dehydrogenase), heat shock protein (HSP), huntingtin protein (HTT), HUNK, ICK, intermediate-density lipoprotein receptor (IDLR), IDO-1, IFN-γR, IGF-1R, IGF-2R, IKK-alpha, IKK-beta, IKK-gamma, IKK-epsilon, Ikaros (IKZF1), Helios (IKZF2), Aiolos (IKZF3), Apoptosis inhibitor (IAP) proteins, e.g., cIAP and XIAP, IKZF4, IL-4R, IL-10R, ILK; integrins, e.g., αVβ3, α4β1 and α5β1 integrins; insulin receptor (IR), insulin-like growth factor receptor, INSR, IRE1 / 2, IRR, ITKIMP; integrin receptor (IR), IRA2, IRAK1 / 2 / 3 / 4, IRE1, IRR; Janus tyrosine kinase (JAK), c-Jun N-terminal kinase (JNK); potassium-chloride cotransporter 2 (KCC2), KDM5C, KHS1 / 2; inward-rectifying potassium channels (Kir2,3), KIS, KIT; Kelch domain-containing protein 2 (KLHDC2), KLF4, KRAS, KRAS G12C, KSR1 / 2, lamin, lysosome-associated membrane protein (Lamp), LAMTOR2, LANA, lactoferrin receptor, L-amino acid transporter (LAT), LATS1 / 2, LCK, low density lipoprotein receptor (LDLR), LEF, LIMK1 / 2, LKB1, LMR1 / AATK, LMR2 / 3, LOK, low density lipoprotein receptor-related protein (LRP), LRRK1 / 2, LTK, LXR-β, LYN, LZK, LZTFL1, m5C, m6A, MAK, MALT, MAP3K4 / 8, MAP4K3, MAP4K5, MAPK, MAPKlb, MAPKAPK2 / 3 / 5, MARK1 / 2 / 3 / 4, MAST1 / 2 / 3 / 4 / L, MC4 receptor, MCAK, MCH-1 receptor, monocarboxylate transporter (MCT), mouse double minute 2 homolog (MDM2), MDMx, megalin, mitogen-activated protein kinase (MEK), MEKK1 / 2 / 3, MEL, MELK, MER, MERTK, mesothelin, MET, Mfsd2a, metabotropic glutamate receptor (mGlu1), major histocompatibility complex class I protein (MHC-I), MHC-II, MIF, MINCLE, MINK1, MISR2, MITF, MKK1 / 2 / 3 / 4 / 6 / 7, MKP3, MLK1 / 2 / 3 / 4, MLKL, MLN1 / 3, MNK1 / 2, MOK, MOS, MPK, MPSK1, MRCKα / β, multidrug resistance protein (MRP), MSK1 / 2, MSSK1, MST1 / 2 / 3 / 4, mammalian target of rapamycin (mTOR), MUCL, MUSK, Myc protein, MYO3A / 3B, MYT1, NAMPT, N-cadherin, K + -dependent Na + / Ca 2+Exchanger 2 (NCKX2), NDC80, NDR1 / 2, NEK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11, NF-Kappa B, Nicastrin, Nicotinic Acetylcholine Receptor, NIK, NLK, NOTCH Receptor, Niemann-Pick C1-like 1 (NPC1L1), N-Methyl D-Aspartate Receptor (NR), NRAS, NRBP1 / 2, NRF2, NSD2, NSD3, Nucleoside Transporter (NT), Sodium / Taurocholic Acid Cotransporter Peptide (N TCP), NuaK1 / 2, NUAK1, organic anion transporter (OAT), organic anion transport polypeptide (OATP, OATP1B1, OATP2B1, OATP4C1, etc.), obsculin, organic cation transporter (OCT), OSR1, organic solute transporter (OST), otoferlin, P2X prinoceptor 4 receptor (P2x4R), purinergic receptor P2Y12 (P2Y12), p16INK4A, p18INK4, p19INK4D, p21, p27 kip1p38α, p38β, p38γ, p38δ, p53, p65 / RELA, p70S6K, p70S6Kb, p90, p300 proteins, PAK1 / 2 / 4 / 5 / 6, pancoronavirus antiviral, protease-activated receptor (PAR), PARP, PASK, PAX, PBRM1, PCSK9, PD-1, phosphodiesterase (PDE), PDE4, PDEδ, PDGFRα / β, pdhk1 / 2 / 3 / 4, PDK1, PD-L1, PEA-15, PEK, peptide transporter (PEPT ), P-glycoprotein (P-gp), PHKy1 / 2, phosphoenolpyruvate carboxykinase, phosphoinositide 3-kinase (PI3K), PIM1 / 2 / 3, PINK1, PITSLRE, PKA, PKACα / β / γ, PKBα / β, PKC, PKD1 / 2 / 3, PKG1 / 2, PKN1 / 2 / 3, PKR, PLC, PLDL, PLK1 / 2 / 3 / 4, PMEL17, PRAK, peroxisome proliferator-activated receptor (PPAR), PRC2, PRK2, PRKX, PRKY, PRMT5, protein phosphatase, protein tyrosine phosphatase (PTP), PRP4, PRPK, PSKH1 / 2, prostate-specific membrane antigen (PSMA), receptor patched 1 (PTCH1), PTEN, protein tyrosine kinase (PTK), PVRL4, pyruvate dehydrokinase, PYK2, QIK, QSK, R1P1, RACK-1, Rac protein, RAF protein, RAPl, Raptor, RAR, Ras protein family (RAS), Rb, retinol Synthetic protein (RBP), RET, riboflavin transport protein (RFVT), RFX1, RHAU, RHODK, RIOK1 / 2, receptor-interacting protein kinase (RIPK), rhodopsin, ribonuclease K (RNASEK), RNAseL, ring finger protein (RNF), ROCK1 / 2, extrarenal medulla potassium channel (ROMK), RON, ROR1 / 2, ROS, RPN11, RPN13, ribosomal s6 kinase, receptor serine / threonine kinase (RSK),For example, the transforming growth factor β (TGF-β) receptor and the proteins listed in Aristidis Moustakas, et al, Receptor Serine / Threonine Kinases (ISBN: 978-3-540-44244-8), receptor tyrosine kinases (RTKs), such as fibroblast growth factor receptor (FGFR), FLT1 / 2 / 3 / 4, FGFR1 / 2 / 3 / 4, RET, tropomyosin-related kinase (TRK), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER), vascular endothelial growth factor receptor (VEGFR), TYRO3, and publications Mark A. Lemmon and Joseph Schlessinger, Cell, 141(7) 1117-1134. Other proteins listed in 2010, RUNX1 / 2 / 3, RYK, spindle assembly checkpoint (SAC), Sanpodo / Notch, SAP, SARS-CoV-2, SBK, solute carrier (SC), SCF, SCYL1 / 2 / 3, SF3B1, serine / threonine protein kinase (SGK), SGLT, SHC, SHP2, SIK2 / 3, SIRT2, SIX1, skMLCK, S-phase kinase-related protein 1 (SKP1), S-phase kinase-related protein 2 (SKP2), solute carrier (SLC, e.g., SCL19A1) transporter, e.g., hMATE1, and publication Enrico Girardi, et al., Nature Chemical Biology, 16, 469-478. The proteins listed in 2020 include SLK, SLOB, Smac, Smad proteins, SMARCA, sodium-bound monocarboxylic acid transporter (SMCT), SMG1, smMLCK, sodium-dependent multivitamin transporter (SMVT), synaptosome-associated protein (SNAP), SNRK, sortilin-associated CNS expression 1a (SorCS1a), SorCS1c, son of sevenless protein (SOS), SOS1, SOX2, acidic cysteine-rich secretory protein (SPARC), SPEG, speckled BTB / POZ protein (SPOP), SPRED, SPRY, SPv-NK1R, scavenger receptors (SR), e.g., CD36, LAMP1, and LAMP2, Src proteins, SRF, SRM, SRPK1 / 2,SSTK, StaO, Activating Signal Transduction and Transcription Protein (STAT), STING, Serine / Threonine Kinase (STK), STLK3 / 5 / 6, Syntaxin (STX), SuRTK106, SUV39HI, SUZ12, Sodium-Vitamin C Cotransporter (SVCT), Synaptotagmin (SYT), Triiodothyronine (T3), TAB, TAK1, TAL, TAO1 / 2 / 3, Tau Protein, TBCK, TBK1, T Cell Factor / Lymphoid Enhancer Binding Factor (TCF / LEF Famin Lee), TEC, TESK1 / 2, TFAM, transcription factor EB (TFEB), transferrin receptor (TfR), TGF-β, trans Golgi network (TGN38), thiamine transporter (THTR), TIE1, TIE2 / TEK, TIF1a / β / γ, titin / TTN, TLK1 / 2, Toll-like receptor (TLR), e.g., TLR4, TNK1, TOPK, TPL2 / COT, TPN1p, TRAD, TRAF protein, Trb1 / 2 / 3, tri-motif family protein (TRIM), Trio, TRRAP, TSC2, TSG101, TSSK1 / 2 / 3 / 4, TTBK1 / 2, TTK, TXK, TYK2, tyrosinase, tubulin, UBF, UCHL5, ULK1 / 2 / 3 / 4, ubiquitin-specific peptidases (USP), e.g., USP7, USP11, and USP14, VACAMKL protein, vesicle-associated membrane protein (VAMP), vitamin D receptor (VDR), vesicle-mediated GABA transporter (VGAT), vesicle-mediated glutamate transporter (VGLUT), very low-density lipoprotein receptor Examples include, but are not limited to, the following target proteins: uric acid transporter 1 (VLDLR), uric acid transporter 1 (URAT1), VRK1 / 2 / 3, WAVE-2, WDR5, WEE-1, WEE-1B, WNK1 / 2 / 3 / 4, Wnt, XBP1, YANK1 / 2 / 3, YBX1, YES, YSK1, ZAK, ZAP70, ZC1 / 2 / 3 / 4, ZFP91, and zinc ring finger protein (ZRNF) (including all variants, mutants, splice variants, indels, and fusions of these listed target proteins).

[0048] In certain embodiments, the protein inhibitor or antagonist is a molecule that targets B-cell lymphoma 2 (BCL-2) family proteins. In certain embodiments, the protein inhibitor or antagonist that targets BCL-2 family proteins is selected from ABT737 (CAS: 852808-04-9), ABT263 (CAS: 923564-51-6), ABT199 (CAS: 1257044-40-8), GX15-070 (CAS: 803712-79-0), UBX1325 (Lisaftoclax), APG-1252 (Peltoclax), APG-2575 (Lisaftoclax), or (R)-(-)-Gossypol (CAS: 90141-22-3), any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0049] In certain embodiments, the protein inhibitor or antagonist is a molecule that targets proprotein convertase subtilisin / kexin type 9 (PCSK9). In certain embodiments, the protein inhibitor or antagonist that targets PCSK9 protein is selected from CVI-LM001, PF-06815345, MK-0616 or efinicitide chloride (CAS: 2407527-16-4), NN6434, 13PCSK9i, and any molecule listed in the publication Shakir Ahamad, et al., Journal of Medicinal Chemistry, 65(23), 15513-15539, 2022, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier.

[0050] In certain embodiments, the protein inhibitor or antagonist is a molecule that targets a protein phosphatase. In certain embodiments, the protein inhibitor or antagonist targeting a protein phosphatase is selected from TNO155 (CAS: 1801765-04-7), RMC-4630, RMC-4550 (CAS: 2172651-73-7), IFB-088 (CAS: 951441-04-6), SHP1 inhibitor (CAS: 56932-43-5), SHP009 (CAS: 1801747-42-1), DPM-1001 (1471172-27-6), AKB-9778 (CAS: 1008510-37-9), LB-100 (CAS: 1026680-07-8), any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, as well as pharmaceutically acceptable carriers.

[0051] In certain embodiments, the protein inhibitor or antagonist is a molecule that targets histone deacetylase (HDAC). In certain embodiments, the HDAC inhibitor or antagonist is vorinostat, romidepsin, bellinostat (PXD101), panobinostat (LBH589), valproic acid, entinostat (MS275), butyrate, trichostatin A, dibinostat (ITF2357), sitalinostat (ACY-241), mosetinostat (MGCD0103), prasinostat (SB939), resminostat, RGFP966, CUDC-1 01, Abexinostat (PCI-24781), Nocetinostat, Phenylbutyrate, Tasejinarin, Tubasin, Tuvastatin A, R306464, SE-7552, MPT0B451, Dasinostat (LAQ824), HDAC10-IN-1, HDAC10-IN-2, AR-42, GSK3117391, MC1568, Xinostat (JNJ-26481585), PCI-34051, Droxinostat, RGFP966, Licorino Stat (ACY-1215), Tasejinarin (CI994), Fimepinostat (CUDC-907), M344, RG2833 (RGFP109), Scriptide, TMP269, TMP195, Santa Cruzamate A (CAY10683), SKLB-23bb, ACY-775, BRD73954, CXD101, Suberohydroxamic acid, BRD3308, HPOB, LMK-235, Nexturastat A, BML-210 (CAY1043 3) Selected from, but not limited to, KA2507, TC-H106, Tucidinostat (Chidamide), SIS17, WT161, CAY10603, ACY-738, tinostamstine (EDO-S101), domatinostat (4SC-202), BG45, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0052] In certain embodiments, the protein inhibitor or antagonist is a molecule that targets the mammalian target of rapamycin (mTOR) and / or FK506-binding protein (FKBP) protein. In certain embodiments, the mTOR and / or FKBP (or 4EBO1) inhibitor or antagonist is rapamycin, Rapalink-1 (CAS: 1887095-82-0), or RMC-5552 (CAS: 2382768-62-7), any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and a pharmaceutically acceptable carrier. In certain embodiments, the protein inhibitor or antagonist is a molecule that targets a protein methyltransferase, such as arginine methyltransferase (PRMT). In certain embodiments, the PRMT inhibitor or antagonist is selected from, but is not limited to, S-adenosylmethionine (SAM), S-adenosylhomocysteine ​​(SAH), AMI-1, synefungin, homosynefungin, GSK3326595, JNJ-63619178, GSK3368715, EML108, EPZ004777, and EML981, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers. In certain embodiments, the protein inhibitor or antagonist is a molecule that targets signaling and transcription-activating proteins (STATs).

[0053] In certain embodiments, the therapeutic agent is a protein activator or agonist and modulator, including isotopes such as deuterium-substituted derivatives. The protein activator or agonist may target any suitable protein. Examples include the adenosine A1 receptor (A1R), A 2A Receptor, A 2BReceptors, including but not limited to A3 receptors, AMPK, cGAS, chemokine receptors, FXR, GCK, glucagon-like peptide 1 (GLP-1), G protein-coupled receptors (GPCRs), such as GPR20, GPR20, free fatty acid receptor 1 (GPR40), GPR119, and GPR120, integrins, peroxisome proliferator-activated receptors (PPARs), ring finger proteins (RNFs), interferon gene stimulators (STINGs), Toll-like receptors (TLRs) such as TLR4, and zinc ring finger proteins (ZRNFs) (including all variants, mutants, splice variants, indels, and fusions of these listed target proteins).

[0054] In some embodiments, the activator or agonist is a molecule that targets the adenosine A1 receptor (A1R). In certain embodiments, the A1R activator or agonist is selected from, but is not limited to, CPA (CAS: 41552-82-3), BnOCPA, LUF6258 and VCP746 (CAS: 1582751-84-5), any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0055] In certain embodiments, the activator or agonist is a molecule that targets glucagon-like peptide 1 (GLP-1). In certain embodiments, the GLP1 activator or agonist is Lixisenatide / AVE0010 / ZP10 / Lyxumia, Exenatide / Exendin-4 / Byetta / Bydureon / ITCA650 / AC-2993, Liraglutide / Victoza, Tirzepatide, Semaglutide, Taspoglutide, Syncria / Albiglutide, Dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, Efpeglenatide / HM-11260C, CM-3, GLP-1Eligen, ORMD-0901, NN-9924, NN-9926, NN-9927, Nodexen, Vi Selected from, but not limited to, ador-GLP-1, CVX-096, ZYOG-1, ZYD-1, GSK-2374697, DA-3091, MAR-701, MAR709, ZP-2929, ZP-3022, ZP-DI-70, TT-401, MK-8521, MED10382, BHM-034, HM12525A, MOD-6030, CAM-2036, DA-15864, ARI-2651, ARI-2255, LY3298176, NN1177, Exenatide-XTEN and Glucagon-XTEN, NN9030, and any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0056] In certain embodiments, the activator or agonist and modulator is a molecule that targets integrins. In certain embodiments, the integrin activator or agonist and modulator is selected from, but is not limited to, SAR-1118, BMS-587101, 1,2,3,4-tetrahydroquinoline-6-carboxylic acid, HC-0303, Compactin, AJM-300, HMR-1031, Firategrast, Tirofiban, Eptifibatide, MK-0429, ATN-161, JSM-6427, and any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0057] In certain embodiments, the activator or agonist is a molecule that targets peroxisome proliferator-activated receptors (PPARs). In certain embodiments, the PPAR activator or agonist is selected from, but is not limited to, GW0742, L-165041, MA-0211, KD-3010, CER-002, SAR351034, Oxeglitazar, LY518674, ZYH7, K111, Macuneos, Efatutazone, CHS-131, OMS-405, GED0507-34-Levo, T2D959, Lanifibranor, Gemfibrozil, Rosiglitazone, Ciprofibrate, Piolitazone, Bezafibrate, Lobeglitazone, Fenofibrate, Saroglitazar, Pemafibrate, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0058] In certain embodiments, the activator or agonist is a molecule that targets a stimulant of the interferon gene (STING) protein. In certain embodiments, the STING activator or agonist is c(di-GMP), 3',3'-cGAMP, 2',3'-cGAMP, ML-RR-S2-cGAMP, ADU-S100, ML-RR-S2-CDG, DMXAA, aminobenzimidazole, ExoSTING, MV-626, SB11285, STACT-TREX1, SYN-STING (SYNB1891), E7766, GSK37 Selected from, but not limited to, 45417, MK-1454, MK-2118, BMS-986301, SB-11285, IMSA-101, BI-1387446, TAK676, SNX281, HG-381, DN-015089, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0059] In certain embodiments, the therapeutic agent is a pyrophosphate or bisphosphonate-containing agent capable of binding to bone minerals and being taken up by osteocytes via membrane receptors such as SCL37A3. In certain embodiments, the pyrophosphate or bisphosphonate-containing agent is selected from, but is not limited to, pamidronic acid, risedronic acid, alendronic acid, zoledronic acid, ibandronic acid, minodronic acid, compounds in the publications Jaeok Park, et al., Frontiers in Chemistry, 8, 612728, 2021 and Zhou Yu, et al., eLife, e36620, 2018, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0060] In certain embodiments, the therapeutic agent is a molecular adhesive or degrading agent that includes their isotopes such as deuterium-substituted derivatives. A molecular adhesive can be any compound that can stabilize the interaction between two or more proteins. A degrading agent can be any compound that binds to a protein of interest (POI) and induces the degradation of the POI through direct regulation of the POI such as modification of the surface topology of the POI. In some embodiments, the therapeutic agent is a multivalent endocytosis agent. The multivalent endocytosis agents herein are agents or probes conjugated with chemical arms through cleavable or non-cleavable chemical bonds or linker units

Chemical formula

Chemical formula

Chemical formula

[0061] In certain embodiments, the multivalent endocytosis agent includes, but is not limited to, the general formula (I):

Chemical formula

[0062] ​In certain embodiments, the polyvalent endocytosis agent itself can exert biological functions in bacteria, viruses, fungi, protozoa, vectors, cells, tissues, and animal bodies. In certain embodiments, the chemical bonds or linker units within the polyvalent endocytosis agent [ka] These can be cleaved by bacteria, viruses, fungi, protozoa, vectors, cells, tissues, and enzymes (or proteins) within the animal body, releasing a portion of the multivalent endocytotic agent and allowing it to exert any biological function. Internalized membrane components and extracellular materials such as proteins, carbohydrates, lipids, pathogens, particles, viruses, bacteria, fungi, protozoa, vectors, cellular debris, and other cells can also be degraded by lysosomes.

[0063] Drug and chemical arms are chemically bonded or linker units. [ka] They are conjugated by covalent bonds. In certain embodiments, they are conjugated by chemical bonds or linker units. [ka] is a drug or probe [ka] and chemical arm [ka] It can be attached to any part of it.

[0064] In certain embodiments, the conjugate site between the drug or probe and the chemical arm may be selected to enable, improve, or enhance the intended functionality of the drug, while enabling, improving, or enhancing the binding affinity to endocytosis-mediated membrane components to increase the efficacy and / or efficiency of endocytosis. For example, the conjugation between the drug or probe and the chemical arm may result in a polyvalent endocytogenic agent.

[0065] The conjugate site between the drug or probe and the chemical arm may be selected to impair, reduce, or eliminate the intended function of the drug. In certain embodiments, chemical bonds or linker units

[0066] [ka] are -H, -D, -O(R), =O, -S(R), =S, =NR, =N(R), -N(R)(R'), -Se(R), =Se, -F, -Cl, -Br, -I, -F, - 18F, -C(R)(R')(R''), =C(R)(R'), ≡C(R), -Si(R)(R')(R''), =Si(R)(R'), ≡Si(R), -OS(O)2R, -OS(O)R, -S(O)R, -S(O)2R, -P( O)(R)(R'), -P(R)(R'), -P(O)(OH)O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(S)O-, -OC(S)-, -OC (S)O-, -C(NOR)-, -C(O)N(R')-, -C(O)N(R)C(O)-, -C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R')C(O)N(R)-, -N(R)C(O)O-, - OC(O)N(R)-, -C(NR')-, -N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2- -S(O)2O-, -N(R)S(O)2-, -S(O)2N(R)-, -N(R')S(O)-, -S(O)N(R)-, -N(R)S(O)2N(R')-, -N(R')S(O)N(R')-, -SS-, -O-Si(R)(R')-O-, -C(=N)N(R)(R')-, -C(R)=C(R')-, -C≡C-, C3-12 cycloalkane, C3-12 cycloalkene, C3-12 cycloalkyne, 3-membered to 12-membered heterocycle, 5-membered to 12-membered aryl, 5-membered to 12-membered heteroaryl, any deuterium-substituted derivative, or any combination thereof that can terminate at least one of these (at either or both ends) polyvalent polyethylene The end groups may be polyethylene glycol, alkoxy, alkyl, alkylene, alkyne, cycloalkyl, cycloalkylene, cycloalkyne, heterocyclyl, aryl, or heteroaryl, where R, R', or R'' are H, D, 1-100 polyethylene glycol, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkylene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or C1-C100 heteroaryl, and one or both end groups may be the same or different.

[0067] In certain embodiments, chemical bonds or linker units [ka] is a polyvalent chain unit [ka] Even if that is the case, This refers to one or more chemical bonds or linker units in any equivalent and arrangement.

[0068] [ka] One or more cores coupled with

[0069] [ka] It contains, and m and n represent integers from 0 to 100. In a particular embodiment, the core

[0070] [ka] This includes, but is not limited to, polyethylene glycols of atoms H, C, Si, N, P, B, O, S, Se, 1 to 100, C1-C100 alkoxys, C1-C100 alkyls, C2-C100 alkylenes, C2-C100 alkynes, C3-C100 cycloalkyls, C3-C100 cycloalkienes, C3-C100 cycloalkynes, C3-C100 heterocyclyls, C6-C100 aryls, or C1-C100 heteroaryls, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers. In certain embodiments, chemical bonds or linker units

[0071] [ka] are -H, -D, -O(R), =O, -S(R), =S, =NR, =N(R), -N(R)(R'), -Se(R), =Se, -F, -Cl, -Br, -I, -F, - 18F, -C(R)(R')(R''), =C(R)(R'), ≡C(R), -Si(R)(R')(R''), =Si(R)(R'), ≡Si(R), -OS(O)2R, -OS(O)R, -S(O)R, -S(O)2R, -P( O)(R)(R'), -P(R)(R'), -P(O)(OH)O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(S)O-, -OC(S)-, -OC (S)O-, -C(NOR)-, -C(O)N(R')-, -C(O)N(R)C(O)-, -C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R')C(O)N(R)-, -N(R)C(O)O-, - OC(O)N(R)-, -C(NR')-, -N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2- -S(O)2O-, -N(R)S(O)2-, -S(O)2N(R)-, -N(R')S(O)-, -S(O)N(R)-, -N(R)S(O)2N(R')-, -N(R')S(O)N(R')-, -SS-, -O-Si(R)(R')-O-,)-, -C(=N)N(R)(R')-, -C(R)=C(R')-, -C≡C-, C3-12 cycloalkane, C3-12 cycloalkene, C3-12 cycloalkyne, 3-membered to 12-membered heterocycle, 5-membered to 12-membered aryl, 5-membered to 12-membered heteroaryl, any deuterium-substituted derivative, or any combination thereof that can terminate at least one of these (at either end or both ends) divalent or trivalent Polyethylene glycol, alkoxy, alkyl, alkylene, alkyne, cycloalkyl, cycloalkylene, cycloalkyne, heterocyclyl, aryl, heteroaryl, where R, R', or R'' is polyethylene glycol with H, D, 1-100, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkylene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or C1-C100 heteroaryl, where one or both terminal groups may be the same or different.In some embodiments, m or n is an integer between 0 and 50.

[0072] In certain embodiments, the linker is a cleavable bond. Cleavable bonds include phosphate esters, amides, esters, dialkyl or diaryldialkoxysilanes, cyanoethyl groups, sulfones, ethylene, glycolyl disuccinates, cyclic acetals, 2-N-acylnitrobenzenesulfonamides, α-thiophenyl esters, unsaturated vinyl sulfides, sulfonamides, malondialdehyde (MDA)-indole derivatives, levulinoyl esters, hydrazones, oximes, imines, acylhydrazones, alkylthioesters, thioesters, disulfide crosslinks, and azotization. This includes, but is not limited to, compounds, 2-nitrobenzyl derivatives, phenacyl esters, 8-quinolinylbenzenesulfonic acid, coumarin, bisarylhydrazone, bimannbitiopropionic acid derivatives, paramethoxybenzyl derivatives, tert-butylcarbamate analogs, orthoesters, acetals, aconityl, silyl ethers, β-thiopropionates, phosphoramidates, disulfides, vinyl ethers, polyketals, allyl esters, picolinic acid esters, vicinaldiols, and selenium compounds.

[0073] In certain embodiments, the drug or probe [ka] This includes a part of a drug, therapeutic agent, diagnostic agent, or chemical probe, as well as any deuterium-substituted derivative, analogue, and chelate, or pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and a pharmaceutically acceptable carrier.

[0074] In a particular embodiment, chemical arm [ka] A polyvalent endocytosis agent is an atom or group of atoms, compound, or part of a compound that can bind to membrane components or extracellular substances, including membrane proteins, extracellular proteins, carbohydrates, lipids, pathogens, particles, viruses, bacteria, fungi, protozoa, vectors, cell debris, and other cells. Therefore, a polyvalent endocytosis agent can be used to internalize the cell membrane to form endosomes, and then to transport the polyvalent endocytosis agent itself, membrane proteins, extracellular proteins, carbohydrates, lipids, pathogens, particles, bacteria, viruses, fungi, protozoa, vectors, cell debris, and other cells into cells or cellular lysosomes.

[0075] In a particular embodiment, chemical arm [ka] A chemical arm is a binding site capable of binding to a membrane component that mediates endocytosis, and the chemical arm is selected from, but is not limited to, atoms, chemical bonds including reversible or irreversible covalent bonds, charged or chargeable chemical moieties (at a specific pH value), hydrophilic moieties, lipophilic moieties, chemical moieties containing reversible or irreversible covalent bonds, drugs, parts of drugs, membrane-bound chemical fragments (MBCFs), substituted mono or dicarboxylic acid derivatives (SMDAs), lipids and derivatives (LAs), substituted phosphate derivatives, glycers and derivatives (GAs), phospholipids and derivatives (PPAs), steroids, vitamins and derivatives (VtAs), amino acids and peptides and derivatives (AAPs), monosaccharides, sugars and derivatives (SCAs), nucleic acid bases and nucleosides and nucleotide derivatives (NNNAs), reported membrane protein binders and derivatives (RMPBs), any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0076] In certain embodiments for membrane-bound chemical fragments, substituted mono- or dicarboxylic acid derivatives (SMDAs) include, but are not limited to, saturated or unsaturated alkyl or heteroalkyl chains, and also include chains having 1 to 50 terminal carboxylic acid moieties. Alkyl or heteroalkyls having terminal carboxylic acid moieties include, but are not limited to, propionic acid, butyric acid, valeric acid, hexanoic acid, enanthic acid, octanoic acid, nonanoic acid, decanoic acid, undecylenic acid, lauric acid, tridecanoic acid, myristic acid, palmitic acid, palmitic acid, hexadecanoic acid, heptadecanoic acid, stearic acid, nonadecanoic acid, arachidic acid, heneicosanoic acid, docosanoic acid, tricosanoic acid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, tetradecenoic acid, pentadecanoic acid, palmitoleic acid, and Represented by leic acid, eicosenoic acid, docosenoic acid, tetracosanoic acid, eicosapentaenoic acid, docosatrienoic acid, docosahexaenoic acid, octadecadienoic acid, octadecadienoic acid, eicosadienoic acid, eicosatrienoic acid, eicosatetraenoic acid, mevalonic acid, carotenic acid, retinoic acid, dihydroretinoic acid, fenofibric acid, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0077] In certain embodiments for membrane-bound chemical fragments, the terminal carboxylic acid moiety can be replaced with, but are not limited to, hydroxamic acid, hydroxamic acid ester, hydroxamic acid amide, carbonic acid, carbonate ester, carbonate amide, sulfonic acid, sulfonic acid ester, sulfonic acid amide, sulfite, sulfite ester, sulfite amide, nitric acid, nitric acid ester, nitric acid amide, nitrite, nitrite ester, nitrite amide, nitrite, nitrite ester, nitrite amide, boronic acid, boronic acid ester, boronic acid amide, phosphoric acid, phosphate ester, phosphate amide, phosphorous acid, phosphorous acid ester, phosphorous acid amide, phosphinic acid, phosphinic acid ester, phosphinic acid amide, pyrophosphate, pyrophosphate ester, pyrophosphate amide, alcohol, aldehyde, anime, any deuterium-substituted derivative, analogue and chelate, or pharmaceutically acceptable salt or stereoisomer, or any combination thereof, and a pharmaceutically acceptable carrier.

[0078] In certain embodiments for membrane-bound chemical fragments, lipids and derivatives (LA) have 0-6 R 1 Saturated or unsaturated C4-C groups may be substituted with a group. 100 Alkyl chain, saturated or unsaturated C4-C, which may be substituted with 0 to 6 groups. 100 This includes, but is not limited to, heteroalkyl chains, glycerides, phospholipids, ceramides, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers thereof, or any combination thereof, and pharmaceutically acceptable carriers.

[0079] In certain embodiments for membrane-bound chemical fragments, saturated or unsaturated alkyl or heteroalkyl chains include phytoene, phytofluene, neurosporene, lycopene, didehydrolycopene, apolicopene, aponeurosporene, diaponeurosporene, cryptoxanthin, lutein, zeaxanthin, phytoene, carotenal, retinal, squalene, squalane, squalene 2,3-oxide, squalene 2,3:22,23-dioxide, polypodatetraene, isodamara-20(21),24-diene, isodamara-12,24-diene, damara-13(17),2 This includes, but is not limited to, 4-dienes, eupha-7,24-dienes, damara-20(21),24-dienes, oxidesqualenes, farnesol, farnesyl acetates, 11-hydroxy-10,11-dihydrofarnesyl acetates, 10-bromo-11-hydroxy-10,11-dihydrofarnesol, 10,11-epoxyfarnesyl acetates, 10,11-epoxyfarnesol, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0080] In certain embodiments for membrane-bound chemical fragments, glycerides and derivatives (GA) include monoerucine, monolaurin, monomyristaten, monopalmitin, monostearin, 1,3-dioleoyl-2-palmitoylglycerol, 1,3-dipalmitolein, 1,2-diolein, 1,3-diarachidonine, 1,3-dipalmitin, 1,2-dipalmitin, 1,3-distearin, tripalmitolein, triellidine, tripetroseridin, trilinolein, trimiristin, tripalmitin, tristearin, 1,3-dipalmiteridine, 2-acetyl-1,3-dicaffeoylglycerol, 2-acetyl-1-caffeoyl-3-coumaroylglycerol, 2-acetyl-1-feruroyl-3-caffeoylglycerol, 2-acetyl-1-feruroyl-3-coumaroylglycerol This includes, but is not limited to, lycerol, 2-acetyl-1,3-diferuloylglycerol, 2-acetyl-1-caffeoyl-3-cinnamoylglycerol, 2-acetyl-1,3-dicumaroylglycerol, 2-acetyl-1-coumaroyl-3-feruloylglycerol, acetylcoumaroylglycerol, coumaroylglycerol, 1,3-dicumaroylglycerol, 1-coumaroyl-3-caffeoylglycerol, caffeoylglycerol, tricoumaroylglycerol, coumaroylferuloylglycerol, dicaffeoylcoumaroylglycerol, dicaffeoylferuloylglycerol, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0081] In certain embodiments for membrane-bound chemical fragments, phospholipids and derivatives (PPAs) include, but are not limited to, phosphatidic acid, cardiolipin (CL), lysobisphosphatidic acid (LBPA), lysophosphatidic acid (LPA), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylserine (PtSer), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidicerine (PS), phosphatidylinositol (PI), phosphatidylinositol, sphingomyelin, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0082] In certain embodiments for membrane-bound chemical fragments, the steroid is cholesterol, ergosterol, lithocholic acid, 7-dehydrocholesterol, 22,23-dihydroergosterol, 7-dehydrositosterol, 7-dehydrostigmasterol, 7-dehydrocamperterol, pregnenolone, 17α-hydroxypregnenolone, 16α-hydroxypregnenolone, 20α-dihydropregnenolone, dehydroepiandrosterone (DHEA), 7α-H Droxy-DHEA, 7-oxo-DHEA, 7β-hydroxy-DHEA, 5-androsten-3β,17β-diol, 5-androsten-3β,7α,17β-triol, 5-androsten-3β,7β,17β-triol, 5-androsten-3β,16α,17β-triol, progesterone, 17α-hydroxyprogesterone, 17α,20α-dihydroxy-4-pregnen-3-one, 16α-hydroxyprogesterone, 20α-dihydroprogesterone, androstenedione, testosterone Lon, 16α-hydroxytestosterone, 5α-dihydrotestosterone, estrone, estradiol, estriol, 5α-dihydroprogesterone, allopregnanolone, isopregnanolone, 5β-dihydroprogesterone, pregnanolonone, epipregnanolonone, 5α,20α-tetrahydroprogesterone, 5α-pregnan-3α,20α-diol, 5α-pregnan-3β,20α-diol, 5β,20α-tetrahydroprogesterone, 5β-pregnan-3α,20α-diol, 5β-pregnana n-3β,20α-diol, 17α-hydroxyalopregnanolon, 17α-hydroxypregnanolon, 5α-pregnane-3α,17α,20α-triol, 5α-pregnane-3β,17α,20α-triol, 5β-pregnane-3α,17α,20α-triol, 5α-androstan-3,17-dione, androsterone, epiandrosterone, etiocolanolonone, 5α-androstan-3α,17β-diol, 5α-androstan-3β,17β-diol, 5α-androstan-3α,17β-diol, cortisol, cortisone, corticosterone, 21-deoxycortisol, 11-deoxycorticosterone, 3α,5α-tetrahydrocorticosterone, 3α,5β-tetrahydrocorticosterone, 11β-hydroxyandrostenedione, tetrahimanol, 11β-hydroxyandrosterone, 11β-hydroxyepiandrosterone, 11β-hydroxyethiocolonolone, hopen, hopanol, hop-22(29)-ene, hopan-22-ol, dipropterol L, tetrahimanol, bacteriohopaneteirol, aminobacteriohopaneteirol, lanosterol, 24,25-hydroxydranosterol, 24,25-epoxycholesterol, pregnenolone sulfate, 17α-hydroxypregnenolone sulfate, 20α-dihydropregnenolone sulfate, DHEA sulfate, androstenediol sulfate, 5-androsten-3β,16α,17β-triol sulfate, Conjugate 17α,20α-dihydroxy-4-pregnen-3-one, conjugate 20α-dihydroprogesterone, conjugate testosterone, conjugate epitestosterone, estrone sulfate, estradiol sulfate, estriol sulfate, allopregnanolone sulfate, isopregnanolone sulfate, conjugate pregnanolonone, conjugate epipregnanolonone, conjugate 5α,20α-tetrahydroprogesterone, conjugate 5α-pregnan-3α,20α-diol , conjugate 5α-pregnane-3β,20α-diol, conjugate 5β,20α-tetrahydroprogesterone, conjugate 5β-pregnane-3α,20α-diol, conjugate 5β-pregnane-3β,20α-diol, 17α-hydroxyalopregnanolone sulfate, conjugate 17α-hydroxypregnanolonone, 5α-pregnane-3α,17α,20α-triol, 5α-pregnane-3β,17α,20α-triol, 5β-pregnane-3α,17α,20α-triol, androsterone sulfate, epiandrosterone sulfate, etiocholanolone sulfate, epiethiocholanolone sulfate, conjugate 5α-androstan-3α,17β-diol, conjugate 5α-androstan-3β,17β-diol, conjugate 5β-androstan-3α,17β-diol, conjugate 5β-androstan-3β,17β-diol, conjugate 3α,5α -This includes, but is not limited to, tetrahydrocorticosterone, conjugate 3α,5β-tetrahydrocorticosterone, 11β-hydroxyandrosterone sulfate, 11β-hydroxyepiandrosterone sulfate, 11β-hydroxyethiocolanolone sulfate, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0083] In certain embodiments for membrane-bound chemical fragments, vitamins and derivatives (VtA) include coenzyme Q10, vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B8 (inositol), vitamin B9 (folic acid), vitamin B12 (cobalamin or methylcobalamin), vitamin C, vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol) This includes, but is not limited to, vitamin D4 (22-dihydroergocalciferol), vitamin D5 (citocalciferol), vitamin D6 (calciferol), vitamin D7, vitamin E, vitamin K1 (phylloquinone), vitamin K1O (phylloquinone epoxide), vitamin K2 (menaquinone), vitamin K3 (menadione), any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0084] In certain embodiments for membrane-bound chemical fragments, amino acids, peptides, and derivatives (AAPs) include, but are not limited to, glutamic acid (Glu), glutamine (Gln), glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp), tyrosine (Tyr), aspartic acid (Asp), histidine (His), asparagine (Asn), lysine (Lys), methionine (Met), arginine (Arg), serine (Ser), threonine (Thr), cysteine ​​(Cys), proline (Pro), peptides containing 2 to 60 amino acids, any deuterium-substituted derivatives, analogs, and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0085] In certain embodiments for membrane-bound chemical fragments, sugars and derivatives (SCAs) include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides, glycosides, glycoproteins, and glycolipids.

[0086] In certain embodiments for membrane-bound chemical fragments, sugars and derivatives (SCAs) include D-ribose, L-ribose, D-arabinose, L-arabinose, D-xylose, L-xylose, D-lyxose, L-lyxose, D-allose, L-allose, D-altrose, L-altrose, D-glucose, L-glucose, D-mannose, L-mannose, D-growth, L-growth, D-ldose, L-ldose, D-galactose, L-galactose D-talose, L-talose, D-fucose, L-fucose, N-acetylneuraminic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, trehalose, maltose, sucrose, cellobiose, kestose, raffinose, nystose, fructosylnystose, glucan, arabinoxylan, apigenin-7-O-glucoside, quercetin-3-O-glucoside, isorhamnetin-3-O-rutinoside, kaempferol-p-coumaroyl rhamnoside, quercetin-3- O-rutinoside, quercetin-3-O-glucuronide, kaempferol-3-O-rutinoside, isorhamnetin-O-pentoside, quercetin-3-O-rhamnoside, isorhamnetin-O-glucuronide, kaempferol-methyl ether-O-glucoside, isorhamnetin-O-acetylrutinoside, rhamnetin-O-glucuronide, quercetin-dimethyl ether-O-rutinoside, quercetin-dimethyl ether-O-glucuronide, kaempferol-Op-coumaroyl rhamnoside, quercetin-7 This includes, but is not limited to, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, containing 2 to 100 units of monosaccharides and / or derivatives, and which may be linear or branched, as well as pharmaceutically acceptable carriers.

[0087] In certain embodiments for membrane-bound chemical fragments, nucleic acid bases and nucleosides and nucleotide derivatives (NNNAs) include, but are not limited to, adenine, guanine, thymine, cytosine, uracil, hypoxanthine, xanthine, epiguanine, dihydrouracil, adenosine, guanosine, thymidine, cytidine, uridine, inosine, xanthosine, 7-methylguanosine, dihydroiridine, cAMP, pppGpp, NADP, FAD, ATP, ADP, AMP, GTP, GDP, GMP, UTP, UDP, UMP, CTP, CDP, CMP, TTP, TDP, TMP, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0088] In certain embodiments, the reported membrane protein binders and derivatives (MPBDs) include, but are not limited to, atoms, drugs, parts of drugs, or derivatives, as well as chelates, any deuterium-substituted derivatives, pharmaceutically acceptable salts or stereoisomers, or any combination thereof, that target membrane components that mediate endocytosis.

[0089] In a particular embodiment, the chelate atom is Ag + Cu + Au + Hg 2+ Pb 2+ Cu 2+ , Cd 2+ Zn 2+ , Se 2+ , Se 4+ , Se 6+ , and Sb 3+ This includes, but is not limited to, the following:

[0090] In certain embodiments, selected drugs, parts of drugs, or drug derivatives, and chelates (ADDs), any deuterium-substituted derivatives, pharmaceutically acceptable salts or stereoisomers, or any combination thereof have been reported to target proteins. In certain embodiments, the target proteins include, but are not limited to, the target proteins listed in U.S. Patent Application Publication No. 20210002296 (including all variants, mutants, splice variants, indels, and fusions of these listed target proteins). Examples include the 5HT2c receptor, α 1A-AR, α-2 adrenergic receptor, α-synuclein, AAK1, ATP-binding cassette (ABC) transporter, e.g., MDR1 / 2 / 3 / 4 / 5 and ABCG2, ABL, cholesterol acyltransferase (ACAT), ACE protein, ACK, ACTR2, ACTR2B, ADCK1 / 2 / 3 / 4 / 5, protein kinase B (AKT), AlphaK1 / 2 / 3, amino acid transporter, ALIX, anaplastic lymphoma kinase (ALK), amnionless, AMPA receptor (AMPAR), AMP-related protein kinase (AMPK), amyloid beta, angiotensin II receptor, ANKRD3, Apaf-1, apolipoprotein E receptor (ApoER), amyloid precursor protein (APP), androgen receptor (AR), ARAF, ARG, AR-V7, ileal tip sodium / bile acid cotransporter (ASBT), type V proton ATPase 6( ATP6V), ATP6V1H, avidin, alanine serine cysteine ​​transporter (ASCT), ASGPR, ASK1 / 2, ataxin-1, ataxia telangiectasia mutant protein (ATM), ATM and Rad3-related protein (ATR), aurora kinase, AXL, β-site amyloid precursor protein cleavage enzyme 1 (BACE1), β2-adrenergic receptor, BAD, BARK1 / 2, Bax, BCKDK B-cell leukemia / lymphoma (BCL) family proteins, e.g., BCL2, BCL-XL, and MCL-1, BCR-ABL; bromodomain and extraterminal domain family proteins (BET), e.g., BRD2 / 3 / 4 / T, beta-catenin, BIKE / BMP2K, BLK, BMPR1A, BMPR1B, BMPR2, BMX / ETX, CDO sibling (Boc), BRD9, BMI1, BRAF, BRAF V600EBrassinosteroid-refractory 1 (BRI1), BRK, BRM, BRSK1, BRSK2, Bruton's tyrosine kinase (BTK), BUB1, C3G, calreticrin, cell adhesion molecule (CAM) receptors, CAMK1α / β / γ / δ, CAMK2α / β / γ / δ, CAMK4, caMLCK, cannabinoid-1 receptor, coxsackievirus-adenovirus receptor (CAR), CRISPR-related protein (Cas), CASK, caspase-3, caspase-6, caspase-7, caspase-9, CBFβ, CBL-B, CBP, chemokine receptors The receptors include, for example, CC chemokine receptors (CCR) and CXC chemokine receptors (CXCR), for example, CXCR2, CXCR4, and CXCR7, CCK4 / PTK7, CCR2, CCR9, CCRK, and differentiation antigens (CDs), for example, CD4, CD9, CD13 / APN receptor, CD25, CD26 (DPP4), CD30, CD33, CD36, CD38, CD46, CD56, CD63, CD71, CD74, CD80, CD81, CD82, CD86, CD123, CD138, CD147, CD152, CD152 (CTLA4), CD166, C D174, CD197, CD205, CD227, CD228, CD269, CD276, and CD326, CDC7, CDC20, CDC25, CDC37, CDK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18, CDKL1 / 2 / 3 / 4 / 5, CDON, CEACAM5, Cholesteryl ester transfer protein (CETP), c-Fos, Cystic fibrosis transmembrane conduction regulator (CFTR), Cyclic GMP-AMP synthase (cGas), Chak1 / 2, CHK1 / 2, CI-M6PR, CK1α, C K1α2, CK1δ, CK1ε, CK1γ1 / 2 / 3, CK2α1 / 2, CLIK1, CLIK1L, Clip, CLK1 / 2 / 3 / 4, Human Collectin Placenta 1 (CL-P1), CMYC, Cone Opsin, COT / TPL2, Cell Permeation Peptide (CPP), Connexin, Coronavirus Protease, CRABP, C-RAF, Cereblon (CRBN), CREB, CRK, CRIK, CRIPTO, CSF2R, CSF1R / FMS, CSK, C-TAK1, CTK, Cubilin, Cyclin D, Cyclin E, CytoC, DAPK1 / 2 / 3,DCAMKL1 / 2 / 3, DDR1 / 2, Diglyceride syltransferase (DGAT), DLK, DMPK1 / 2, DNA-PK, DP-1, DPP-4, DRAK1 / 2, Deubiquitinating enzyme (DUB), DYRK1A / 1B / / 2 / 3 / 4, E2F, Excitatory amino acid carrier 1 (EAAC1), E-cadherin, Endothelin-converting enzyme (ECE), ED-B, EED, EF2K, eEF2K, EIF2A3, EIF4E, EIF4G, ELK, ENL, Endothelin B receptor, Epithelial cell adhesion molecule (EpCAM), Endothelial cell protein C receptor (EPCR), Ephrin receptor (EphR), Estrogen receptor (ER), ER81, ErbB2, ErbB3, ETS transcription factor (ERG), ERK1 / 2 / 3 / 4 / 5 / 7 / 8, ERRa, EZH2, fatty acid binding protein (FABP), FADD, FAK, FAP, fatty acid transport protein (FATP), neonatal Fc receptor (FCRN), FER, FKBP, FLIP, Flotilin-1, Flotilin-2, folate receptor, e.g. reduced folate carrier, FOLT1 / 2 / 3 and proton-bound folate transporter (PCFT), FOXM1, FOXO1 / 2 / 3 / 4, FRK, Frizzled4, fructose-1,6-bisphosphatase, FYN, γ-aminobutyric acid type A receptor (GABAAR), GAK, Gap-1, growth arrest specific 1 (Gas1), GCC, GCK, GCN2, GD2, ghrelin receptor (ghrelinR), glucose-dependent insulinopromoting polypeptide (GIP), Globo H, glucagon receptor, glucagon-like peptide 1 (GLP-1), glutamate transporter (GLT), glucokinase, ion channel type AMPA glutamate receptor (GluR), glucose-6-phosphatase, glucose transporter (GLUT), glutathione transporter, glycogen phosphorylase, glycogen synthase kinase, glycoproteins, e.g., gp18, gp31, and gp60, G protein-coupled receptors (GPCRs), e.g., GPR20, GPR20, free fatty acid receptor 1 (GPR40), GPR119, and GPR120, GPNMB, GPRK4 / 5 / 6 / 7, GRB2, GSK-3, GSPT1, Haspin kinase, HCK, hepatitis C virus (HCV) NS3 / 4A, histone deacetylase (HDAC),HDL receptor (HDLR), HectH9, HH498, HIF1, HIPK1 / 2 / 3 / 4, HMGN1, HMG-CoA reductase, hnRNP, H-PGDS, HPGCR, hematopoietic precursor kinase 1 (HPK1), HRAS, HRI, hRpn13, Pru HSD-11β (11β-hydroxysteroid dehydrogenase), heat shock protein (HSP), huntingtin protein (HTT), HUNK, Apoptosis inhibitor (IAP) proteins, e.g., cIAP and XIAP, ICK, intermediate-density lipoprotein receptor (IDLR), IDO-1, IFN-γR, IGF-1R, IGF-2r, IKK-alpha, IKK-beta, IKK-gamma, IKK-epsilon, Ikaros (IKZF1), Helios (IKZF2), Aiolos (IKZF3), IKZF4, IL-4R, IL-10R, ILK, integrins, e.g., αVβ3, α4β1 and α5β1 integrins, insulin receptor (IR), INSR, IRE1 / 2, IRR, ITKIMP, integrin receptor (IR), insulin-like growth factor receptor, IRA2, IRAK1 / 2 / 3 / 4, IRE1, IRR, Janus tyrosine kinase (JAK), c-Jun N-terminal kinase (JNK), potassium-chloride cotransporter 2 (KCC2), KDM5C, KHS1 / 2, inward-rectifying potassium channel (Kir2,3), KIS, KIT, Kelch domain-containing protein 2 (KLHDC2), KLF4, KRAS, KRAS G12CKSR1 / 2, lamin, lysosome-associated membrane protein (Lamp), LAMTOR2, LANA, lactoferrin receptor, L-amino acid transporter (LAT), LATS1 / 2, LCK, low-density lipoprotein receptor (LDLR), LEF, LIMK1 / 2, LKB1, LMR1 / AATK, LMR2 / 3, LOK, low-density lipoprotein receptor-related protein (LRP), LRRK1 / 2, LTK, LXR-β, LYN, LZK, LZTFL1, m5C, m6A, MAK, MALT, MAP3K4 / 8, MAP4K3, MAP4K5, MAPK, MAPKlb, MAPKAPK2 / 3 / 5, MARK1 / 2 / 3 / 4, MAST1 / 2 / 3 / 4 / L, MC4 receptor, MCAK, MCH-1 receptor, monocarboxylic acid transporter (MCT), mouse double minute 2 homolog (MDM) 2) MDMx, Megalin, Mitogen-Activated Protein Kinase (MEK), MEKK1 / 2 / 3, MEL, MELK, MER, MERTK, Mesothelin, MET, Mfsd2a, Metabotropic Glutamate Receptor (mGlu1), Major Histocompatibility Complex Class I Protein (MHC-I), MHC-II, MIF, MINCLE, MINK1, MISR2, MITF, MKK1 / 2 / 3 / 4 / 6 / 7, MKP3, MLK1 / 2 / 3 / 4, MLKL, MLN1 / 3, MNK1 / 2, MOK, MOS, MPK, MPSK1, MRCKα / β, Multidrug Resistance Protein (MRP), MSK1 / 2, MSSK1, MST1 / 2 / 3 / 4, Mammalian Rapamycin Target (mTOR), MUCL, MUSK, Myc Protein, MYO3A / 3B, MYT1, NAMPT, N-Cadherin, K + Dependency Na + / Ca 2+Exchanger 2 (NCKX2), NDC80, NDR1 / 2, NEK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11, NF-Kappa B, nicatrin, nicotinic acetylcholine receptor, NIK, NLK, NOTCH receptor, Niemann-Pick C1-like 1 (NPC1L1), N-methyl D-aspartate receptor (NR), NRAS, NRBP1 / 2, NRF2, NSD2, NSD3, nucleoside transporter (NT, e.g., hENT1, hCNT1~3, etc.), sodium / taurochol Acid cotransport peptides (NTCP), NuaK1 / 2, NUAK1, organic anion transporters (OAT), organic anion transport polypeptides (OATP, OATP1B1, OATP2B1, OATP4C1, etc.), obsculin, organic cation transporters (OCT), OSR1, organic solute transporters (OST), otoferlin, P2X prinoceptor 4 receptor (P2x4R), purinergic receptor P2Y12 (P2Y12), p16INK4A, p18INK4, p19INK4D, p21, p27 kip1p38α, p38β, p38γ, p38δ, p53, p65 / RELA, p70S6K, p70S6Kb, p90, p300 proteins, PAK1 / 2 / 4 / 5 / 6, pancoronavirus antiviral, protease-activated receptor (PAR), PARP, PASK, PAX, PBRM1, PCSK9, PD-1, phosphodiesterase (PDE), PDE4, PDEδ, PDGFRα / β, PDHK1 / 2 / 3 / 4, PDK1, PD-L1, PEA-15, PEK, peptide transporter (PEPT ), P-glycoprotein (P-gp), PHKy1 / 2, phosphoenolpyruvate carboxykinase, phosphoinositide 3-kinase (PI3K), PIM1 / 2 / 3, PINK1, PITSLRE, PKA, PKACα / β / γ, PKBα / β, PKC, PKD1 / 2 / 3, PKG1 / 2, PKN1 / 2 / 3, PKR, PLC, PLDL, PLK1 / 2 / 3 / 4, PMEL17, PRAK, peroxisome proliferator-activated receptor (PPAR), PRC2, PRK2, PRKX, PRKY, PRMT5, protein phosphatase, protein tyrosine phosphatase (PTP), PRP4, PRPK, PSKH1 / 2, prostate-specific membrane antigen (PSMA), receptor patched 1 (PTCH1), PTEN, protein tyrosine kinase (PTK), PVRL4, pyruvate dehydrokinase, PYK2, QIK, QSK, R1P1, RACK-1, Rac protein, RAF protein, RAPl, Raptor, RAR, Ras protein family (RAS), Rb, retinol Synthetic protein (RBP), RET, riboflavin transport protein (RFVT), RFX1, RHAU, RHODK, RIOK1 / 2, receptor-interacting protein kinase (RIPK), rhodopsin, ribonuclease K (RNASEK), RNAseL, ring finger protein (RNF), ROCK1 / 2, extrarenal medulla potassium channel (ROMK), RON, ROR1 / 2, ROS, RPN11, RPN13, ribosomal s6 kinase, receptor serine / threonine kinase (RSK),For example, the transforming growth factor β (TGF-β) receptor and the proteins listed in Aristidis Moustakas, et al, Receptor Serine / Threonine Kinases (ISBN: 978-3-540-44244-8), receptor tyrosine kinases (RTKs), such as fibroblast growth factor receptor (FGFR), FLT1 / 2 / 3 / 4, FGFR1 / 2 / 3 / 4, RET, tropomyosin-related kinase (TRK), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER), vascular endothelial growth factor receptor (VEGFR), TYRO3, and publications Mark A. Lemmon and Joseph Schlessinger, Cell, 141(7)1117-1134. Other proteins listed in 2010, RUNX1 / 2 / 3, RYK, spindle assembly checkpoint (SAC), Sanpodo / Notch, SAP, SARS-CoV-2, SBK, solute carrier (SC), SCF, SCYL1 / 2 / 3, SF3B1, serine / threonine protein kinase (SGK), SGLT, SHC, SHP2, SIK2 / 3, SIRT2, SIX1, skMLCK, S-phase kinase-related protein 1 (SKP1), S-phase kinase-related protein 2 (SKP2), solute carrier (SLC, e.g., SCL19A1) transporter, e.g., hMATE1, and publication Enrico Girardi, et al., Nature Chemical Biology, 16, 469-478. The proteins listed in 2020 include SLK, SLOB, Smac, Smad proteins, SMARCA, sodium-bound monocarboxylic acid transporter (SMCT), SMG1, smMLCK, sodium-dependent multivitamin transporter (SMVT), synaptosome-associated protein (SNAP), SNRK, sortilin-associated CNS expression 1a (SorCS1a), SorCS1c, son of sevenless protein (SOS), SOS1, SOX2, acidic cysteine-rich secretory protein (SPARC), SPEG, speckled BTB / POZ protein (SPOP), SPRED, SPRY, SPv-NK1R, scavenger receptors (SR), e.g., CD36, LAMP1, and LAMP2, Src proteins, SRF, SRM, SRPK1 / 2, SSTK,StaO, signaling and transcriptional activation protein (STAT), STING, serine / threonine kinase (STK), STLK3 / 5 / 6, syntaxin (STX), SuRTK106, SUV39HI, SUZ12, sodium-vitamin C cotransporter (SVCT), synaptotagmin (SYT), triiodothyronine (T3), TAB, TAK1, TAL, TAO1 / 2 / 3, Tau protein, TBCK, TBK1, T cell factor / lymphoid enhancer binding factor (TCF / LEF family), TEC, TESK1 / 2, TFAM, transcription factor EB (TFEB), transferrin receptor (TfR), TGF-β, trans Golgi network (TGN38), thiamine transporter (THTR), TIE1, TIE2 / TEK, TIF1a / β / γ, titin / TTN, TLK1 / 2, Toll-like receptor (TLR), e.g., TLR4, TNK1, TOPK, TPL2 / COT, TPN1p, TRAD, TRAF protein, Trb1 / 2 / 3, tri-motif family protein (TRIM), Tri o, TRRAP, TSC2, TSG101, TSSK1 / 2 / 3 / 4, TTBK1 / 2, TTK, TXK, TYK2, tyrosinase, tubulin, UBF, UCHL5, ULK1 / 2 / 3 / 4, ubiquitin-specific peptidases (USP), e.g., USP7, USP11, and USP14, VACAMKL protein, vesicle-associated membrane protein (VAMP), vitamin D receptor (VDR), vesicle-mediated GABA transporter (VGAT), vesicle-mediated glutamate transporter (VGLUT), very low-density lipoprotein receptor ( Examples include, but are not limited to, VLDLR, uric acid transporter 1 (URAT1), VRK1 / 2 / 3, WAVE-2, WDR5, WEE-1, WEE-1B, WNK1 / 2 / 3 / 4, Wnt, XBP1, YANK1 / 2 / 3, YBX1, YES, YSK1, ZAK, ZAP70, ZC1 / 2 / 3 / 4, ZFP91, and zinc ring finger protein (ZRNF) (including all variants, mutants, splice variants, indels, and fusions of these listed target proteins).

[0091] In certain embodiments, the polyvalent endocytosis agent is a bivalent or polyvalent agent for protein inhibition and chemical modification of proteins, DNA, or RNA, including but not limited to protein ubiquitination, protein deubiquitination, protein phosphorylation, protein methylation, protein acetylation, protein folding or unfolding, and DNA / RNA degradation.

[0092] In certain embodiments, the divalent or polyvalent agents are proteolytic agents, including, but not limited to, proteolytic target chimeras (PROTACs). PROTAC degradation products of proteins after ubiquitination are compounds that physically degrade target proteins by cleaving one or more bonds of the target protein through proteolysis. PROTACs also include all PROTAC variations, such as photocage PROTACs and tag-based PROTACs. Exemplary proteolytic agents with other mechanisms include, but are not limited to, proteolytic agents, autophagosome tethering compounds (ATTECs), autophagy-targeted chimeras (AUTACs), chaperone-mediated proteolytic agents (CHAMPs), BacPROTACs, ASGPR-targeted chimeras (ATACs), lysosome-targeted chimeras (LYTACs), and mitochondrial protease-targeted chimeras (MtPTACs). Divalent or polyvalent agents include dubiquitinase-targeted chimeras (DUBTAC), RESTORAC, enhancer-targeted chimeras (ENTAC), phosphorylation-inducing small molecule chimeras (PHICS), ribonuclease-targeted chimeras (RIBOTAC), phosphatase-recruiting chimeras (PhoRC), dephosphorylation-targeted chimeras (DEPTAC), phosphorylation-targeted chimeras (PhosTAC), phosphorylation-inducing small molecule chimeras (PHICS), acetylation-tagged molecules (AceTAG), regulated induction proximity-targeted chimeras (RIPTAC), transcriptional / epigenetic chemical proximity inducers (TCIP), caspase-cleavage-targeted chimeras (CACTAC), their deuterium-substituted derivatives, their analogues, or combinations thereof.

[0093] In certain embodiments, the functional group in a divalent or polyvalent drug, or the divalent or polyvalent drug itself, is represented by one of the following compounds: 3-aminophthalic acid (CAS: 5434-20-8), 4-aminoisoindoline-1,3-dione (CAS: 2518-24-3), 4-aminoisobenzofuran-1,3-dione (CAS: 17395-99-2), 5-aminoisoindoline-1,3-dione (CAS: 3676-85-5), 5-aminoisobenzofuran-1,3-Shell (CAS:17011-53-9) 4-Shell (CAS:5434-21-9) 753b, A7, A1 6, A031, A1874(CAS:2064292-12-0), ABBV-101, Ab-PROTAC3, ACBI1( CAS:2375564-55-7) ACBI3 AC-0682 AC-0716 AC-0676 ACBI2(CAS :2913161-19-8) AGB1 AM-A3 AMG232(CAS:1352066-68-2) AP-01- 104. APG-265, APR-Cy3, AR-VHL-SF2, AR2-VHL-SF2, ARCC-4(CAS:1973403-00-7), ARD-69(CAS:2316837-10-0), ARD-61(CAS:2316837-08). -6) ARD-69, ARD-2128(CAS:2222111-87-5), ARD1676, GT20029, ARD-266, ARD-2051(CAS:2632305-17-8), ARD-2585(CAS:2757422-79-8). )、AR-ARL、AR-LDD、ARV110(CAS:2222112-77-6)、ARV-766(CAS:2750830-09-0)、ARV-471(CAS:2229711-68-4), ARV-771(CAS:1949837-1). 2-0), ARV-825(1818885-28-7), ARV-763, ASP-3082, AT-1(CAS:2098). 836-45-2) ATTEC AU-15330 AUTAC4 N112979747A) Azo-PROTAC-4C β-NF-ATRA BC5P BCPyr BETd-260(CAS:2093388-62-4) B03 BGB-16673 BI-3663(CAS:2341740-84-7) BI3802(CAS:2166387-65-9), BP3, BRD4-SF2, BSJ-4-116(CAS:2519). 823-34-6) BT1 BTX-1188 BTX-9341 BWA-522 C004019 C13(Jingyu Zhang, et al., Journal of Medicinal Chemistry, 65, 13, 9096-9125, 2013-2014.2022, CC885(CAS:1010100-07-8), CC-90009(CAS:1860875-51-9), CC-92480(CAS:2259648-80-9)(Joshua D. Hansen, et al., Journal of Medicinal Chemistry, 63, 2022). 6648-6676, 2020) CC-94676, CC-99282, CCR9-PROTAC, CCT369260, CCW28-3, CDDO-JQ1, CDDO-Me, CFT7455(NCT04756726), CFT8634(CAS). :2704617-96-7)、CFT8919、CFT1946(CAS:2882165-79-7)、CG416、CG428、CG001419、CL1-YL2、CLL1-5、CM11、Introduction3(Mingming Wei, et al., European Journal of Medicinal Chemistry, 209, 112903, 2021); Peng, et al., ACS Medicinal Chemistry Letters, 10, 767-772, 2019); Luo, et al., Acta Pharmaceutica Sinica B, 11(5), 1300-1314, 2021); 2023) CP5V(CAS:2509359-75-3) CP-10(CAS:2366268-80-4) CPD-12 24(CAS:2891620-68-9)、CPR3、CPR4、CPS2、CRBN-6-5-VHL、D15(Pengyun Li, et al., Acta Pharmaceutica Sinica B, 13(6),2715-2735, 2023)、d4E-4、d4E-6、d9A-2、dBET1(CAS:1799711-21-9)、dBET6(CAS:1950634-92-0)、DBt-10(Martin Schroder, et al., bioRxiv preprint, 2023、DOI: 10.1101 / 2023.04.09.536153)、dCBP-1(CAS:2484739-25-3)、DCY-09-192、DD-03-171(CAS:2366132-45-6)、DD-04-015、dFKBP-1(CAS:1799711-22-0)、DGY-04-035、DGY-06-177、DGY-06-177-pk1、DGY-06-177-pk2、DGY-09-192、DKY709(NCT03891953)、dMCL1-2(CAS:2351218-88-5)、DP1、DP-C-1、DP-C-4、DP-V-4、DS-3032(CAS:1398568-47-2)、DT2216(CAS:2365172-42-3)、dTRIM24(CAS:2170695-14-2)、ERD-148、ERD-308(CAS:2320561-35-9)、ERD-3111(CAS:2832865-25-3)、ERG OP-C1、ER PROTAC(ES2717436T3)、FA-S2-POMA、FA-S2-MS4048、FHD-609(CAS:2676211-64-4)、Folate-ARV-771、Folate-MS432、Folate-MS99、, Packaging G4-PROTAC, GBD-9, GBM-475, GMB-475 (CAS:2490599-18-1), GNE-987, GSK215 GT20029, GT19630, GT19715, GW3965-PEG5-VH032, HaloPROTAC-3, HBL-4, HC-X029, HC-X0 35. HD-TAC7, HJM-561, HER2-14, HP14, HP17, HP518, HP568, HPB-143, HRS-1358, HRS-5041 HSK29116, I-6, I-685, INY-03-041, ITRI-90, ITRI-125, and ITRI-126 (Chiu-Lien Hung, et al., eBioMedicine, 90, 104500, 2013-2014. 2023) JB170 JH-XI-10-02(CAS:2209085-22-1) JP-1 JP-2 JP-3, JP-4, JP-5, JP-6, JPX-0802, JPX-1185, JPX1188, JNJ- 1013, JMKX000623, KB02-JQ-1(CAS:2384184-44-3), KB02-SL F(CAS:2384184-40-9) and KP-14 are listed in the Airport Number 2022173032. KRAS PROTAC(KRAS G12D Zone 17(CAS:2821793-99-9) KT-333, KT-413, KT-253, KT-474(CAS:2432994-31). -3) KTX-335, KTX-652, KTX-959, KTX-978, KTX-214, KYH1872, L18i, LC-2, LC-BM12, LEF1 OP-V1, LC-MB12(CAS:828438-38-4), LG1188, LT-002, macroPROTAC-1, MK-8242 (CAS:828438-38-4). :147-94-4) MD13 MD-222(CAS:2136246-72-3) MD-224(CAS:2136247-12-4) MDEG-541 MEK PROTAC3(Stefan Vollmer, et al., Journal of Medicinal Chemistry, 63, 157-162, 2020), MG-277(CAS:2411085-89-5), MM-02-08, MM-03-75, and MM-04-09(Margot Meyers, et al., bioRxiv Preprint, 2023, DOI: 10.1101 / 2023.08.11.553046), MS-170(CAS:2376136-61-5), MR837(CAS:1210906-48-1), MS28(CAS:20 93386-22-0), MS33, MS39, MS67, MS83, MS98, MS154, MS170, MS4077(CAS:2230077-10-6), MS432, MS910, MS 928, MS934, MS1943, MS4332, MS9715, MT-802 (CAS: 2231744-29-7), MTX-23 (CAS: 2488296-74-6), MZ1, N3-NF-κB-ODN, dNF-κB number 15, dNF-κB number 16, N3-E2F-ODN, dE2F number 16, dE2F number 17, NH2, NJH-04-086, NJH-04-087, N JH-04-098, NR-6a, NR-7h, Natlin 3a, Natlin 3, NVP-CGM097 (CAS: 1313363-54-0), NRX-0492 (CAS: 2416130-57-7), NX-0479, NX-2127 (CAS: 2416131-46-7), NX-5948 (CAS: 2649400-34-8), Ortasidenib, ORM-5029, Obalicin , P3, P4B, P19A, P19P, P22, P22A, P22D, PAP508, pc-PROTAC1, PG21, pc-PROTAC3, P13i (CAS:2360561-66-4) , PF15, Pomalidomide, PP-C8, Pre-PROTAC, PROTAC-8, PROTAC_ERRα(CAS:1801547-15-8), PROTAC_RIPK2(Daniel P. Bondeson, et al., Nature Chemical Biology, 11, 611-617, 2015), RIPK2 PROTAC(Anh-Tuan Pham, et al., Frontiers in Pharmacology, 14, 1127722, 2023) , PRE3789 , PROTAC MDM2 Enhancer-1 (CAS:2249944-98-5) , PROTAC BRD9 Switch-1(CAS:2097971-01-0) PROTAC BET Switch 23 PROTAC-D PROTAC ER FORCE-3(CAS:2158322-29-1), PROTAC-FCPF, PROTAC-O412, PROTAC(HPGDS)-1, PROTAC(HPGDS)-7, PRTC, PZ703b, PZ 15527, QCA570(CAS:2207569-08-0), R1-5C, RBN012811, RC-1, RC-3, RG7112 (CAS:939981-39-2), RG7388 (CAS:12297). 05-06-9) RNK05047 RNK-AP6 SAR405838(CAS:1303607-60-4) SARD279 SD-36(CAS:2429877-44-9) SD-91 SHP 2-D26(CAS:2458219-65-1), SIAIS001, SIAIS056, SIAIS091, SIAIS117, SIAIS164018, SIAIS117(CAS:2353494-84-3). )、SIAIS125、SIAIS126、SIAIS178(2376047-73-1)、SIAIS629048、SIAIS629049、SIAIS629050、SIAIS629051、SIM1 SJ995973(CAS:2882065-25-8) SJF620(CAS:2376187-16-3) SJF-0628 SJF-0661 SJH1-62B SK-575 SNIPER(ER)- 3. SNIPER(ER)-87(CAS:2222354-91-6), SNIPER-BRD4-1, SNIPER-ABL-62, SP4(CAS:2624181-69-5), SPB5208, SMD-3 040, SR-1114, STEAP1-5a, STEAP1-13a, Small-Step Protection PROTAC(sr-PROTAC), T1101, STEEL (CAS:2250404-95-4), TD-004(Chung Hyo Kang, et al., Biochemical and Biophysical Research Communications, 502(2), 542-547, 2018.), TD-9, TD-165, TD-428(CAS:2334525-50-5), TD-802, Thalidomide, TAI-1(CAS:1334921-03-7), TM-P4-Thal, TMX-2172, UBX-382, UI-EP002, UNC 6852 (CAS:2688842-08-0), UNC7700, UNC7698, VHLL-X-BCN number 15, VHLL-X-BCN number 16, VHLL-X-BCN number 17, Versortrexate (VSTX), VZ185, WB214, WWL0245 , xStAx-VHLL, 7-143, XY-07-189, XZ739(CAS:2365172-19-4), XZ424, M. Adams, et al., Cancer Discovery, 13(5), 1210-1229, 2023), YZ167, YZ268, ZB-S-29, ZCY-PROTAC, ZNL-02-096, ZXH-4-130, and ZXH-4-137, all deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0094] In certain embodiments, the divalent or polyvalent drug is a PROTAC molecule. A PROTAC is a heterobifunctional molecule consisting of one or more warheads, linkers, and one or more ligands that recruit an E3 ubiquitin ligase to a protein of interest (POI). By simultaneously binding to the POI and the E3 ligase, a PROTAC can bring two or more proteins into close proximity and promote ubiquitination of the POI for sequential degradation by the proteasome. In contrast to protein inhibition, the event-driven pharmacological mechanism of PROTACs is inherently catalytic. Targeted degradation can be carried out sufficiently and for a long period of time at concentrations lower than the effective inhibitory drug concentration, thus avoiding extratarget toxicity from high-dose drugs. Unfortunately, PROTACs have the problem of low durability due to their high molecular weight, which is mostly related to metabolic instability, solubility, and poor permeability. Theoretically, metabolic fragility and insolubility can be completely resolved by blocking metabolic hotspots and forming salts on basic or acidic functional groups, respectively. Furthermore, formulation techniques can be used to enhance water solubility. However, as evidenced by numerous published studies, passive permeability of protacs has been found to be difficult or prohibited, and modifying the structure of protacs to improve cell permeability through classical medicinal chemistry is severely limited or even impossible due to the large molecular size of protac drugs.

[0095] PROTAC warheads can target any suitable target protein (POI). In certain embodiments, the target protein includes, but is not limited to, the target proteins listed in U.S. Patent Application Publication No. 20210002296 (including all variants, mutants, splice variants, indels, and fusions of these listed target proteins). Examples include the 5HT2c receptor, α 1A-AR, α-2 adrenergic receptor, α-synuclein, AAK1, ATP-binding cassette (ABC) transporter, e.g., MDR1 / 2 / 3 / 4 / 5 and ABCG2, ABL, cholesterol acyltransferase (ACAT), ACE protein, ACK, ACTR2, ACTR2B, ADCK1 / 2 / 3 / 4 / 5, protein kinase B (AKT), AlphaK1 / 2 / 3, amino acid transporter, anaplastic lymphoma kinase (ALK), ALIX, amnionless, AMPA receptor (AMPAR), AMP-related protein kinase (AMPK), amyloid beta, angiotensin II receptor, ANKRD3, Apaf-1, apolipoprotein E receptor (ApoER), amyloid precursor protein (APP), androgen receptor (AR), ARAF, ARG, AR-V7, ileal tip sodium / bile acid cotransporter (ASBT), type V proton ATPa se6(ATP6V), ATP6V1H, avidin, alanine serine cysteine ​​transporter (ASCT), ASGPR, ASK1 / 2, ataxin-1, ataxia telangiectasia mutant protein (ATM), ATM and Rad3-related protein (ATR), aurora kinase, AXL, β-site amyloid precursor protein cleavage enzyme 1 (BACE1), β2-adrenergic receptor, BAD, BARK1 / 2, Bax, BCKDK, B-cell leukemia / lymphoma (BCL) family proteins, e.g., BCL2, BCL-XL, and MCL-1, BCR-ABL; bromodomain and extraterminal domain family proteins (BET), e.g., BRD2 / 3 / 4 / T, beta-catenin, BIKE / BMP2K, BLK, BMPR1A, BMPR1B, BMPR2, BMX / ETX, CDO sibling (Boc), BRD9, BMI1, BRAF, BRAF V600EBrassinosteroid-refractory 1 (BRI1), BRK, BRM, BRSK1, BRSK2, Bruton's tyrosine kinase (BTK), BUB1, C3G, calreticrin, cell adhesion molecule (CAM) receptor, CAMK1α / β / γ / δ, CAMK2α / β / γ / δ, CAMK4, caMLCK, cannabinoid-1 receptor, coxsackievirus-adenovirus receptor (CAR), CRISPR-related protein (Cas), CASK, caspase-3, caspase-6, caspase-7, caspase-9, CBFβ, CBL-B, CBP, chemokine Receptors, e.g., CC chemokine receptors (CCR) and CXC chemokine receptors (CXCR), e.g., CXCR2, CXCR4, and CXCR7, CCK4 / PTK7, CCR2, CCR9, CCRK; Differentiation antigens (CDs), e.g., CD4, CD9, CD13 / APN receptor, CD25, CD26 (DPP4), CD30, CD33, CD36, CD38, CD46, CD56, CD63, CD71, CD74, CD80, CD81, CD83, CD86, CD123, CD138, CD147, CD152, CD152 (CTLA4), CD166, C D174, CD197, CD205, CD227, CD228, CD269, CD276, and CD326, CDC7, CDC20, CDC25, CDC37, CDK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18, CDKL1 / 2 / 3 / 4 / 5, CDON, CEACAM5, Cholesteryl ester transfer protein (CETP), c-Fos, Cystic fibrosis transmembrane conduction regulator (CFTR), Cyclic GMP-AMP synthase (cGas), Chak1 / 2, CHK1 / 2, CI-M6PR, CK1α, C K1α2, CK1δ, CK1ε, CK1γ1 / 2 / 3, CK2α1 / 2, CLIK1, CLIK1L, Clip, CLK1 / 2 / 3 / 4, Human Collectin Placenta 1 (CL-P1), CMYC, Cone Opsin, COT / TPL2, Cell Permeation Peptide (CPP), Connexin, Coronavirus Protease, CRABP, C-RAF, Cereblon (CRBN), CREB, CRK, CRIK, CRIPTO, CSF2R, CSF1R / FMS, CSK, C-TAK1, CTK, Cubilin, Cyclin D, Cyclin E, CytoC, DAPK1 / 2 / 3,DCAMKL1 / 2 / 3, DDR1 / 2, Diglyceride syltransferase (DGAT), DLK, DMPK1 / 2, DNA-PK, DP-1, DPP-4, DRAK1 / 2, Deubiquitinating enzyme (DUB), DYRK1A / 1B / / 2 / 3 / 4, E2F, Excitatory amino acid carrier 1 (EAAC1), E-cadherin, Endothelin-converting enzyme (ECE), ED-B, EED, EF2K, eEF2K, EIF2A3, EIF4E, EIF4G, ELK, ENL, Endothelin B receptor, Epithelial cell adhesion molecule (EpCAM), Endothelial cell protein C receptor (EPCR), Ephrin receptor (EphR), Estrogen receptor (ER), ER81, ErbB2, ErbB3, ETS transcription factor (ERG), ERK1 / 2 / 3 / 4 / 5 / 7 / 8, ERRa, EZH2, fatty acid binding protein (FABP), FADD, FAK, FAP, fatty acid transport protein (FATP), neonatal Fc receptor (FCRN), FER, FKBP, FLIP, Flotilin-1, Flotilin-2, folate receptor, e.g. reduced folate carrier, FOLT1 / 2 / 3 and proton-bound folate transporter (PCFT), FOXM1, FOXO1 / 2 / 3 / 4, FRK, Frizzled4, fructose-1,6-bisphosphatase, FYN, γ-aminobutyric acid type A receptor (GABAAR), GAK, Gap-1, growth arrest specific 1 (Gas1), GCC, GCK, GCN2, GD2, ghrelin receptor (ghrelinR), glucose-dependent insulinopromoting polypeptide (GIP), Globo H, glucagon receptor, glucagon-like peptide 1 (GLP-1), glutamate transporter (GLT), glucokinase, ion channel type AMPA glutamate receptor (GluR), glucose-6-phosphatase, glucose transporter (GLUT), glutathione transporter, glycogen phosphorylase, glycogen synthase kinase, glycoproteins, e.g., gp18, gp31, and gp60, G protein-coupled receptors (GPCRs), e.g., GPR20, GPR20, free fatty acid receptor 1 (GPR40), GPR119, and GPR120, GPNMB, GPRK4 / 5 / 6 / 7, GRB2, GSK-3, GSPT1, Haspin kinase, HCK, hepatitis C virus (HCV) NS3 / 4A, histone deacetylase (HDAC),HDL receptor (HDLR), HectH9, HH498, HIF1, HIPK1 / 2 / 3 / 4, HMGN1, HMG-CoA reductase, hnRNP, H-PGDS, HPGCR, hematopoietic precursor kinase 1 (HPK1), HRAS, HRI, hRpn13, Pru HSD-11β (11β-hydroxysteroid dehydrogenase), heat shock protein (HSP), huntingtin protein (HTT), HUNK, Apoptosis inhibitor (IAP) proteins, e.g., cIAP and XIAP, ICK, intermediate-density lipoprotein receptor (IDLR), IDO-1, IFN-γR, IGF-1R, IGF-2r, IKK-alpha, IKK-beta, IKK-gamma, IKK-epsilon, Ikaros (IKZF1), Helios (IKZF2), Aiolos (IKZF3), IKZF4, IL-4R, IL-10R, ILK, integrins, e.g., αVβ3, α4β1 and α5β1 integrins, insulin receptor (IR), insulin-like growth factor receptor, INSR, IRE1 / 2, IRR, ITKIMP, integrin receptor (IR), IRA2, IRAK1 / 2 / 3 / 4, IRE1, IRR, Janus tyrosine kinase (JAK), c-Jun N-terminal kinase (JNK), potassium-chloride cotransporter 2 (KCC2), KDM5C, KHS1 / 2, inward-rectifying potassium channel (Kir2,3), KIS, KIT, Kelch domain-containing protein 2 (KLHDC2), KLF4, KRAS, KRAS G12CKSR1 / 2, lamin, lysosome-associated membrane protein (Lamp), LAMTOR2, LANA, lactoferrin receptor, L-amino acid transporter (LAT), LATS1 / 2, LCK, low-density lipoprotein receptor (LDLR), LEF, LIMK1 / 2, LKB1, LMR1 / AATK, LMR2 / 3, LOK, low-density lipoprotein receptor-related protein (LRP), LRRK1 / 2, LTK, LXR-β, LYN, LZK, LZTFL1, m5C, m6A, MAK, MALT, MAP3K4 / 8, MAP4K3, MAP4K5, MAPK, MAPKlb, MAPKAPK2 / 3 / 5, MARK1 / 2 / 3 / 4, MAST1 / 2 / 3 / 4 / L, MC4 receptor, MCAK, MCH-1 receptor, monocarboxylic acid transporter (MCT), mouse double-minute 2 homolog (MDM2) MDMx, Megalin, Mitogen-Activated Protein Kinase Kinase (MEK), MEKK1 / 2 / 3, MEL, MELK, MER, MERTK, Mesothelin, MET, Metabotropic Glutamate Receptor (mGlu1), Mfsd2a, Major Histocompatibility Complex Class I Protein (MHC-I), MHC-II, MIF, MINCLE, MINK1, MISR2, MITF, MKK1 / 2 / 3 / 4 / 6 / 7, MKP3, MLK1 / 2 / 3 / 4, MLKL, MLN1 / 3, MNK1 / 2, MOK, MOS, MPK, MPSK1, MRCKα / β, Multidrug Resistance Protein (MRP), MSK1 / 2, MSSK1, MST1 / 2 / 3 / 4, Mammalian Rapamycin Target (mTOR), MUCL, MUSK, Myc Protein, MYO3A / 3B, MYT1, NAMPT, N-Cadherin, K + Dependency Na + / Ca 2+Exchanger 2 (NCKX2), NDC80, NDR1 / 2, NEK1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11, NF-Kappa B, Nicastrin, Nicotinic Acetylcholine Receptor, NIK, NLK, NOTCH Receptor, Niemann-Pick C1-like 1 (NPC1L1), N-Methyl D-Aspartate Receptor (NR), NRAS, NRBP1 / 2, NRF2, NSD2, NSD3, Nucleoside Transporter (NT), Sodium / Tauro Cholic acid cotransport peptide (NTCP), NuaK1 / 2, NUAK1, organic anion transporter (OAT), organic anion transport polypeptide (OATP), obsculin, organic cation transporter (OCT), OSR1, organic solute transporter (OST), otoferlin, P2X prinoceptor 4 receptor (P2x4R), purinergic receptor P2Y12 (P2Y12), p16INK4A, p18INK4, p19INK4D, p21, p27 kip1p38α, p38β, p38γ, p38δ, p53, p65 / RELA, p70S6K, p70S6Kb, p90, p300 proteins, PAK1 / 2 / 4 / 5 / 6, pancoronavirus antiviral, protease-activated receptor (PAR), PARP, PASK, PAX, PBRM1, PCSK9, PD-1, phosphodiesterase (PDE), PDE4, PDEδ, PDGFRα / β, pdhk1 / 2 / 3 / 4, PDK1, PD-L1, PEA-15, PEK, peptide transporter (PEPT ), P-glycoprotein (P-gp), PHKy1 / 2, phosphoenolpyruvate carboxykinase, phosphoinositide 3-kinase (PI3K), PIM1 / 2 / 3, PINK1, PITSLRE, PKA, PKACα / β / γ, PKBα / β, PKC, PKD1 / 2 / 3, PKG1 / 2, PKN1 / 2 / 3, PKR, PLC, PLDL, PLK1 / 2 / 3 / 4, PMEL17, PRAK, peroxisome proliferator-activated receptor (PPAR), PRC2, PRK2, PRKX, PRKY, PRMT5, protein phosphatase, protein tyrosine phosphatase (PTP), PRP4, PRPK, PSKH1 / 2, prostate-specific membrane antigen (PSMA), receptor patched 1 (PTCH1), PTEN, protein tyrosine kinase (PTK), PVRL4, pyruvate dehydrokinase, PYK2, QIK, QSK, R1P1, RACK-1, Rac protein, RAF protein, RAPl, Raptor, RAR, Ras protein family (RAS), Rb, retinol Synthetic protein (RBP), RET, riboflavin transport protein (RFVT), RFX1, RHAU, RHODK, RIOK1 / 2, receptor-interacting protein kinase (RIPK), rhodopsin, ribonuclease K (RNASEK), RNAseL, ring finger protein (RNF), ROCK1 / 2, extrarenal medulla potassium channel (ROMK), RON, ROR1 / 2, ROS, RPN11, RPN13, ribosomal s6 kinase, receptor serine / threonine kinase (RSK),For example, the transforming growth factor β (TGF-β) receptor and the proteins listed in Aristidis Moustakas, et al, Receptor Serine / Threonine Kinases (ISBN: 978-3-540-44244-8), receptor tyrosine kinases (RTKs), such as fibroblast growth factor receptor (FGFR), FLT1 / 2 / 3 / 4, FGFR1 / 2 / 3 / 4, RET, tropomyosin-related kinase (TRK), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER), vascular endothelial growth factor receptor (VEGFR), TYRO3, and publications Mark A. Lemmon and Joseph Schlessinger, Cell, 141(7) 1117-1134. Other proteins listed in 2010, RUNX1 / 2 / 3, RYK, spindle assembly checkpoint (SAC), Sanpodo / Notch, SAP, SARS-CoV-2, SBK, solute carrier (SC), SCF, SCYL1 / 2 / 3, SF3B1, serine / threonine protein kinase (SGK), SGLT, SHC, SHP2, SIK2 / 3, SIRT2, SIX1, skMLCK, S-phase kinase-related protein 1 (SKP1), S-phase kinase-related protein 2 (SKP2), solute carrier (SLC) transporters, e.g., hMATE1, and publications such as Enrico Girardi, et al., Nature Chemical Biology, 16, 469-478. The proteins listed in 2020 include SLK, SLOB, Smac, Smad proteins, SMARCA, sodium-bound monocarboxylic acid transporter (SMCT), SMG1, smMLCK, sodium-dependent multivitamin transporter (SMVT), synaptosome-associated protein (SNAP), SNRK, sortilin-associated CNS expression 1a (SorCS1a), SorCS1c, son of sevenless protein (SOS), SOS1, SOX2, acidic cysteine-rich secretory protein (SPARC), SPEG, speckled BTB / POZ protein (SPOP), SPRED, SPRY, SPv-NK1R, scavenger receptors (SR), e.g., CD36, LAMP1, and LAMP2, SR-Bs, Src proteins, SRF, SRM, SRPK1 / 2, SSTK,StaO, signaling and transcriptional activation protein (STAT), STING, serine / threonine kinase (STK), STLK3 / 5 / 6, syntaxin (STX), SuRTK106, SUV39HI, SUZ12, sodium-vitamin C cotransporter (SVCT), synaptotagmin (SYT), triiodothyronine (T3), TAB, TAK1, TAL, TAO1 / 2 / 3, Tau protein, TBCK, TBK1, T cell factor / lymphoid enhancer binding factor (TCF / LEF family), TEC, TESK1 / 2, TFAM, transcription factor EB (TFEB), transferrin receptor (TfR), TGF-β, trans Golgi network (TGN38), thiamine transporter (THTR), TIE1, TIE2 / TEK, TIF1a / β / γ, titin / TTN, TLK1 / 2, Toll-like receptor (TLR), e.g., TLR4, TNK1, TOPK, TPL2 / COT, TPN1p, TRAD, TRAF protein, Trb1 / 2 / 3, tri-motif family protein (TRIM), Tri o, TRRAP, TSC2, TSG101, TSSK1 / 2 / 3 / 4, TTBK1 / 2, TTK, TXK, TYK2, tyrosinase, tubulin, UBF, UCHL5, ULK1 / 2 / 3 / 4, ubiquitin-specific peptidases (USP), e.g., USP7, USP11, and USP14, VACAMKL protein, vesicle-associated membrane protein (VAMP), vitamin D receptor (VDR), vesicle-mediated GABA transporter (VGAT), vesicle-mediated glutamate transporter (VGLUT), very low-density lipoprotein receptor ( Examples include, but are not limited to, VLDLR, uric acid transporter 1 (URAT1), VRK1 / 2 / 3, WAVE-2, WDR5, WEE-1, WEE-1B, WNK1 / 2 / 3 / 4, Wnt, XBP1, YANK1 / 2 / 3, YBX1, YES, YSK1, ZAK, ZAP70, ZC1 / 2 / 3 / 4, ZFP91, and zinc ring finger protein (ZRNF) (including all variants, mutants, splice variants, indels, and fusions of these listed target proteins).

[0096] In certain embodiments, the E3 ligase is selected from, but is not limited to, the following proteins: an inhibitor of apoptosis (IAP) protein, an X-linking inhibitor of apoptosis (XIAP) protein, an aryl hydrocarbon receptor (AhR), ring finger protein 4 (RNF4), ring finger protein 114 (RNF114), Fem-1 homolog B (FEM1B) protein, von Hippel-Lindau (VHL), and casitas. B-lineage lymphoma proto-oncogene B (CBL-B) protein, cereblon (CRBN), damage-specific DNA-binding protein 1 (DDB1), mouse double-minute 2 homolog (MDM2) protein, Kelch-like ECH-related protein 1 (KEAP1), Kelch domain-containing protein 2 (KLHDC2), S-phase kinase-related protein 1 (SKP1), S-phase kinase-related protein 2 (SKP2), ubiquitin-protein ligase N-recogninin 5 (UBR5), DDB1 and CUL4-related factor 1 (DCAF1) protein, DDB1 and CUL4-related factor 11 (DCAF11) protein, DDB1 and CUL4-related factor 15 (DCAF15) protein, and DDB1 and CUL4-related factor 16 (DCAF16) (including all variants, mutants, splice variants, indels, and fusions of these listed target proteins).

[0097] PROTAC may contain one or more E3 ligase ligands that target IAP, VHL, CBL-B, CRBN, DDB1, MDM2, KEAP1, KLHDC2, SKP1, SKP2, DCAF1, DCAF15, DCAF16, UBR5, or any combination thereof. POI-binding ligands and E3 ligase ligands are chemically bonded or linker units. [ka] They can be chemically linked or bonded via a chemical bond or linker unit.

[0098] [ka] The linker can bind to any site of the POI-binding ligand and the E3 ligase ligand. The linker must be capable of properly forming the target protein-ligase complex. The linker group consists of one or more structural units. [ka] It may include.

[0099] In certain embodiments, chemical bonds or linker units [ka] are -H, -D, -O(R), =O, -S(R), =S, =NR, =N(R), -N(R)(R'), -Se(R), =Se, -F, -Cl, -Br, -I, -F, - 18F, -C(R)(R')(R''), =C(R)(R'), ≡C(R), -Si(R)(R')(R''), =Si(R)(R'), ≡Si(R), -OS(O)2R, -OS(O)R, -S(O)R, -S(O)2R, -P( O)(R)(R'), -P(R)(R'), -P(O)(OH)O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(S)O-, -OC(S)-, -OC (S)O-, -C(NOR)-, -C(O)N(R')-, -C(O)N(R)C(O)-, -C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R')C(O)N(R)-, -N(R)C(O)O-, - OC(O)N(R)-, -C(NR')-, -N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2- -S(O)2O-, -N(R)S(O)2-, -S(O)2N(R)-, -N(R')S(O)-, -S(O)N(R)-, -N(R)S(O)2N(R')-, -N(R')S(O)N(R')-, -SS-, -O-Si(R)(R')-O-, -C(=N)N(R)(R')-, -C(R)=C(R')-, -C≡C-, C3-12 cycloalkane, C3-12 cycloalkene, C3-12 cycloalkyne, 3-membered to 12-membered heterocycle, 5-membered to 12-membered aryl, 5-membered to 12-membered heteroaryl, any deuterium-substituted derivative, or any combination thereof that can terminate at least one of these (at either or both ends) polyvalent polyethylene The end groups may be polyethylene glycol, alkoxy, alkyl, alkylene, alkyne, cycloalkyl, cycloalkylene, cycloalkyne, heterocyclyl, aryl, or heteroaryl, where R, R', or R'' are polyethylene glycol with H, D, 1-100, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkylene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or C1-C100 heteroaryl, and one or both end groups may be the same or different.

[0100] In certain embodiments, chemical bonds or linker units [ka] is a polyvalent chain unit [ka] Even if that is the case, This refers to one or more chemical bonds or linker units in any equivalent and arrangement. [ka] One or more cores coupled with [ka] It contains, and m and n represent integers from 0 to 100.

[0101] In a particular embodiment, the core [ka] This includes, but is not limited to, polyethylene glycols of atoms H, C, Si, N, P, B, O, S, Se, 1 to 100, C1-C100 alkoxys, C1-C100 alkyls, C2-C100 alkylenes, C2-C100 alkynes, C3-C100 cycloalkyls, C3-C100 cycloalkienes, C3-C100 cycloalkynes, C3-C100 heterocyclyls, C6-C100 aryls, or C1-C100 heteroaryls, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0102] In certain embodiments, chemical bonds or linker units [ka] are -H, -D, -O(R), =O, -S(R), =S, =NR, =N(R), -N(R)(R'), -Se(R), =Se, -F, -Cl, -Br, -I, -F, - 18F, -C(R)(R')(R''), =C(R)(R'), ≡C(R), -Si(R)(R')(R''), =Si(R)(R'), ≡Si(R), -OS(O)2R, -OS(O)R, -S(O)R, -S(O)2R, -P( O)(R)(R'), -P(R)(R'), -P(O)(OH)O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(S)-, -C(S)O-, -OC(S)-, -OC (S)O-, -C(NOR)-, -C(O)N(R')-, -C(O)N(R)C(O)-, -C(O)N(R)C(O)N(R')-, -N(R)C(O)-, -N(R')C(O)N(R)-, -N(R)C(O)O-, - OC(O)N(R)-, -C(NR')-, -N(R)C(NR')-, -C(NR')N(R)-, -N(R)C(NR')N(R)-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2- -S(O)2O-, -N(R)S(O)2-, -S(O)2N(R)-, -N(R')S(O)-, -S(O)N(R)-, -N(R)S(O)2N(R')-, -N(R')S(O)N(R')-, -SS-, -O-Si(R)(R')-O-,)-, -C(=N)N(R)(R')-, -C(R)=C(R')-, -C≡C-, C3-12 cycloalkane, C3-12 cycloalkene, C3-12 cycloalkyne, 3-membered to 12-membered heterocycle, 5-membered to 12-membered aryl, 5-membered to 12-membered heteroaryl, any deuterium-substituted derivative, or any combination thereof that can terminate at least one of these (at either end or both ends) divalent or trivalent Polyethylene glycol, alkoxy, alkyl, alkylene, alkyne, cycloalkyl, cycloalkylene, cycloalkyne, heterocyclyl, aryl, heteroaryl, where R, R', or R'' is polyethylene glycol with H, D, 1-100, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkylene, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or C1-C100 heteroaryl, where one or both terminal groups may be the same or different.In some embodiments, m or n is an integer between 0 and 50.

[0103] In certain embodiments, PROTAC targets androgen receptor (AR), B-cell lymphoma-2 (BCL-2) family proteins, bromodomain and extraterminal (extrateminal) (BET) family proteins, bromodomain-containing 9 (BRD9) protein, Bruton's tyrosine kinase (BTK), CREB-binding protein (CBP) and / or p300 protein, cyclin-dependent kinase (CDK), epidermal growth factor receptor (EGFR), estrogen receptor (ER), interleukin-1 receptor-related kinase (IRAK), Janus kinase (JAK) family proteins, Myc protein, RAF protein, RAS protein, SMARCA protein, signaling and transcriptional activation protein (STAT), Tau protein, tropomyosin receptor kinase (TRK), or any combination thereof.

[0104] In certain embodiments, the bivalent or polyvalent agent is ATAC or LYTAC. ATAC or LYTAC is a heterobifunctional molecule comprising a POI ligand, an endocytosis agent, and a linker between two parts. ATAC or LYTAC can induce lysosomal degradation of membrane POIs or extracellular POIs. In certain embodiments, ATAC or LYTAC as described herein, or a structural part of LYTAC, is an agent that can bind to endocytosis-mediated membrane components such as CD36 or GLUT and be taken up into cells via endocytosis.

[0105] In certain embodiments, the divalent or polyvalent agent is MtPTAC. MtPTAC refers to a heterobifunctional molecule consisting of a POI ligand, a mitochondrial casein-soluble protease P (ClpP) ligand, and a linker between the two parts. MtPTAC activates the hydrolase activity of ClpP while simultaneously bringing POI closer to ClpP for degradation. In some embodiments, MtPTAC is selected from all compounds, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, as well as pharmaceutically acceptable carriers, as described in the publication Dachi Wang, et al., Journal of American Chemistry Society, 145(23), 12861-12869, 2023.

[0106] In certain embodiments, the bivalent or polyvalent agent is AUTAC or AUTOTAC. AUTAC is a heterobifunctional molecule consisting of a POI ligand, an autophagosome recruiting motif, and a linker between the two parts. By binding to POI, the AUTAC molecule can induce the degradation of POI by recruiting autophagosomes. POI can be any suitable protein of interest. AUTOTAC consists of an autophagy-targeting ligand, a POI ligand, and a linker. AUTOTAC can directly anchor the receptor p62 to POI and induce autophagy of POI.

[0107] In certain embodiments, AUTAC and AUTOTAC are represented by any of the following compounds: AUTAC1, AUTAC2, AUTAC3, AUTAC4, PHTPP-1304, Vinclozoline M2-2204, Fumagilin-105, PBA-1105, PBA-1106, Anle138b-F105, and PBA-1105b, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0108] In certain embodiments, the divalent or polyvalent agent is a nucleolytic agent. Examples include, but are not limited to, ribonuclease-targeting chimeras (RIBOTACs), proximity-inducing nucleolytic agents (PINADs), or RNA-degrading chimeras. A RIBOTAC is a heterobifunctional molecule consisting of an RNA-targeting ligand, an RNase recruiter or binder, and a linker between the two parts. RIBOTACs function by recruiting endogenous RNase to a specific RNA, activating the RNase, and inducing cleavage of the target RNA. Examples of RNases include, but are not limited to, RNaseA, RNaseH, RNaseIII, RNaseL, RNaseP, RNasePhyM, RNaseT1, RNaseT2, RNaseU2, RNaseV, RNaseE, RNaseG, polynucleotide phosphorylase (PNPase), RNasePH, RNaseR, RNaseD, RNaseT, oligoribonuclease, exoribonuclease I, and exoribonuclease II (including all variants, mutants, splice variants, indels, and fusions of these listed proteins).

[0109] In certain embodiments, RIBOTAC is represented by any of the following compounds: Compounds 2 and 5 in the publication Matthew G. Costales, et al., Proceedings of the National Academy of Sciences, 117(5), 2406-2411, 2020; C5-RIBOTAC in the publication Hafeez S. Haniff, et al., ACS Central Science, 6(10), 1713-1721, 2020; any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof; and pharmaceutically acceptable carriers.

[0110] In certain embodiments, the divalent or polyvalent agent is a chimera (DUBTAC), RESTORAC, or ENTAC that targets deubiquitinase. DUBTAC, RESTORAC, or ENTAC is a heterobifunctional molecule consisting of a recruiter or binder of a deubiquitinating enzyme or deubiquitinase (DUB) linked to a POI ligand via a linker to stabilize the level of ubiquitin-dependently degraded POI. POI can be any suitable protein of interest. DUBs include, but are not limited to, ubiquitin-specific protease (USP / UBP) superfamily proteins, such as USP1, USP2, USP3, USP4, USP5, USP6, USP7, USP8, USP9X, USP9Y, USP10, USP11, USP12, USP13, USP14, USP15, USP16, USP17, USP17L2, and USP1 7L3, USP17L4, USP17L5, USP17L7, USP17L8, USP18, USP19, USP20, USP21, USP22, USP23, USP24, USP25, U SP26, USP27X, USP28, USP29, USP30, USP31, USP32, USP33, USP34, USP35, USP36, USP37, USP38, USP39, U Examples include SP40, USP41, USP42, USP43, USP44, USP45, USP46; ovarian tumor (OTU) superfamily proteins, e.g., OTUB1, OTUB2; Machado-Josephin domain (MJD) superfamily proteins, e.g., ATXN3, ATXN3L; ubiquitin C-terminal hydrolase (UCH) superfamily proteins, e.g., BAP1, UCHL1, UCHL3, UCHL5; K48-specific deubiquitinases of the MINDY family, e.g., MINDY1, MINDY2, MINDY3, MINDY4; and ZUFSP family proteins, e.g., ZUP (including all variants, mutants, splice variants, indels, and fusions of these listed proteins).

[0111] In certain embodiments, DUBTAC is represented by one of the following compounds: 05IB9, 11JQ15, 11JQ16, NJH-2-075 (CAS: 2858812-70-9), NJH-2-056 (CAS: 2858812-69-6), NJH-2-057 (CAS: 2858812-70-9), LEB-03-153 (CAS: 2858812-88-9), LEB-03-144 (CAS: 2858812-89-0), LEB-03-145 (CAS: 2858812-90-3), LEB-03-146 (CAS: 2858812-91-4). Publication: Jing Liu, et al., Journal of American Chemistry Society, 144. FOXO-DUBTAC No. 6, p53-DUBTAC No. 6, p53-DUBTAC No. 7, and IRF-DUBTAC No. 7 in 12934-12941, 2022, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0112] In certain embodiments, the divalent or polyvalent agent is a phosphatase recruiting chimera (PhoRC), a dephosphorylation-targeting chimera (DEPTAC), or a phosphorylation-targeting chimera (PhosTAC). PhoRC, DEPTAC, or PhosTAC is a heterobifunctional molecule consisting of a phosphatase recruiter or binder linked to a POI ligand via a linker. PhoRC, DEPTAC, or PhosTAC can induce dephosphorylation of POI by the function of a protein phosphatase (PP). POI can be any suitable protein of interest. PPs include, but are not limited to, tyrosine-specific phosphatases, serine / threonine-specific phosphatases, bispecific phosphatases, and histidine phosphatases (including all variants, mutants, splice variants, indels, and fusions of these listed target proteins).

[0113] In certain embodiments, PhoRC, DEPTAC, or PhosTAC is represented by one of the following compounds: Compound 1, Compound 3, Compound 4a, Compound 5a, and Compound 7 in the publication Sayumi Yamazoe, et al., Journal of Medicinal Chemistry, 63, 2807-2813, 2020; DDO-3709R8, DDO-3710, and DDO-3711 in the publication Zhang Qiuyue, et al., Journal of the American Chemistry Society, 145, 1118-1128, 2023; and Chen P. H, et al., ACS Chemical Biology, 16, 2808-2815. PhosTAC7 in 2021, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0114] In certain embodiments, the bivalent or polyvalent agent is a regulated induced proximity targeting chimera (RIPTAC). RIPTAC is a heterobifunctional small molecule that induces a stable ternary complex between a target protein selectively expressed in cancer tissue and a generalized protein essential for cell survival. As a result, cooperative protein-protein interactions (PPIs) inhibit the function of the essential protein, thus selectively inducing cell death in cells expressing the target protein.

[0115] In certain embodiments, RIPTAC is represented by any of the following compounds listed in publication Kanak Raina, et al., bioRxiv preprint, 2023, https: / / doi.org / 10.1101 / 2023.01.01.522436, publication Zonghui Ma, et al., Drug Discovery Today, 2023, https: / / doi.org / 10.1016 / j.drudis.2023.103774, and patent International Patent No. 2023059581, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and any pharmaceutically acceptable carrier. In certain embodiments, the proximity-derived modality is represented by any compound listed in Chem. Soc. Rev. 52, 5485-5515, 2023, any deuterium-substituted derivative, analogue and chelate, or pharmaceutically acceptable salt or stereoisomer, or any combination thereof, as well as a pharmaceutically acceptable carrier. In certain embodiments, the bivalent or polyvalent agent is a transcriptional / epigenetic chemical proximity inducer (TCIP). TCIPs are heterobifunctional small molecules that recruit endogenous cancer drivers, or their downstream transcription factors, to the promoters of cell death genes, thereby activating their expression. In certain embodiments, TCIP is represented by any of the following compounds listed in the publication Sai Gourisankar, et al., Nature, 620, 417-425, 2023, and in International Patent No. 2022098989, any deuterium-substituted derivatives, analogs and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and any pharmaceutically acceptable carrier.

[0116] In certain embodiments, the divalent or polyvalent agent is a phosphorylation-inducing chimeric small molecule (PHICS). PHICS is a heterobifunctional molecule comprising a kinase activator, a POI conjugate, and a linker, which recruits the kinase to phosphorylate the POI by inducing substrate rearrangement to bring the POI into proximity. The POI can be any suitable protein of interest. The kinases include, but are not limited to, AMPK and PKFC (including all variants, mutants, splice variants, indels, and fusions of these enumerated target proteins). In certain embodiments, PHICS is represented by any of the following compounds: PHICS1, PHICS2, and PHICS3 in the publication Sachini U. Siriwardena, et al., Journal of American Chemistry Society, 142, 14052-14057, 2020, any deuterium-substituted derivatives, analogs, and chelates, or pharmaceutically acceptable salts or stereoisomers, or any combination thereof, and pharmaceutically acceptable carriers.

[0117] In certain embodiments, the divalent or polyvalent agent is an acetylation tag molecule (AceTAG). An AceTAG heterobifunctional molecule consists of lysine acetyltransferase, a POI-binding ligand, and a linker. By binding to lysine acetyltransferase and POI, AceTAG can modulate the distance between lysine acetyltransferase and POI, thereby inducing acetylation of POI. POI can be any suitable protein of interest. In certain embodiments, AceTAG is represented by AceTAG-1, any deuterium-substituted derivative, analogue and chelate, or pharmaceutically acceptable salt or stereoisomer, or any combination thereof, as described in the publication Wesley W. Wang, et al., Journal of the American Chemistry Society, 143, 16700-16708, 2021, as well as any pharmaceutically acceptable carrier.

[0118] In certain embodiments, the divalent or polyvalent agent is a chaperone-mediated proteolytic agent (CHAMP). CHAMP is a heterobifunctional molecule comprising a chaperone protein-binding ligand, a POI-binding ligand, and a linker. By binding to the chaperone protein and POI, CHAMP can modulate the distance between the chaperone protein and POI, thereby inducing the degradation of POI. POI can be any suitable protein of interest. In certain embodiments, the bivalent or polyvalent agent is BacPROTAC as an endocytosis agent mediated by bacterial cell membrane proteins such as CD36. BacPROTAC is a heterobifunctional molecule comprising a bacterial CIpCP protease-binding ligand, a POI-binding ligand, and a linker. By binding to bacterial CIpCP protease and POI, BacPROTAC can modulate the distance between the CIpCP protease and POI, thereby inducing the degradation of POI. POI can be any suitable protein of interest. In certain embodiments, the divalent or polyvalent agent is a caspase-cleavage-targeted chimera (CACTAC) as an endocytosis agent. CACTAC is a heterobifunctional molecule comprising a caspase-binding ligand, a POI-binding ligand, and a linker. By binding to the caspase and POI, CACTAC can modulate the distance between the caspase and POI, thereby inducing cleavage of the POI. The POI can be any suitable protein of interest.

[0119] In some embodiments, the therapeutic agent is a binder. A binder is any compound that binds to a membrane or extracellular material, including membrane proteins or extracellular proteins, carbohydrates, lipids, pathogens, particles, viruses, bacteria, fungi, protozoa, vectors, cell debris, and other cells. Thus, a binder can be used to internalize a cell membrane to form an endosome, and then to transport transport membrane proteins, extracellular proteins, carbohydrates, lipids, pathogens, particles, viruses, bacteria, fungi, protozoa, vectors, cell debris, and other cells to the (recipient) cell via endocytosis. Components in the internalized membrane or extracellular material, such as proteins, carbohydrates, lipids, pathogens, particles, viruses, bacteria, fungi, protozoa, vectors, cell debris, and other cells, can ultimately be degraded by the lysosomes of the (recipient) cell.

[0120] In some embodiments, the therapeutic agent is a DNA groove binder or an RNA binder. DNA groove binders are crescent-shaped molecules that selectively non-covalently bind to the grooves of DNA or the grooves of the DNA helix (Sayantan Bhaduri, et al., Beilstein Journal of Organic Chemistry, 14, 1051-1086, 2017). RNA binders are molecules that can specifically bind to RNA (Jessica L. Childs-Disney, et al., Nature Reviews Drug Discovery, 21, 736-762, 2022). Binding to DNA or RNA with a specific sequence usually occurs through a combination of directional hydrogen bonds to the base pair edges. By binding to DNA or RNA, the molecule can influence the function of the DNA or RNA.

[0121] In some embodiments, the therapeutic agent is a diagnostic agent or chemical probe. The diagnostic agent or chemical probe includes a detectable label. Detectable labels include, but are not limited to, binding labels, chromophores, enzyme labels, bioluminescent labels, fluorescent labels, quenchers, radioactive labels, or any other labels suitable for detection. Binding labels provide a detectable signal via a binding event. In some embodiments, the binding label may be biotin, a compound used in HaloTag, CLIP-Tag, or SNAP-Tag technology (Jonas Wilhelm et al., Biochemistry, 60(3), 2560-2575, 2021), an antibody, an antigen, or any other label that can provide a detectable signal via a binding event. Chromophores provide a detectable signal via the absorption and emission of photons. In some embodiments, the chromophore is a fluorophore, a phosphor, a dye, a quantum dot, or any other chromophore capable of absorbing and emitting detectable photons. Enzyme labels provide a detectable signal via reaction with a substrate. Bioluminescent labels provide a detectable signal through the emission of light from proteins. Quenchers or fluorescent labels provide a detectable signal through the modulation of photon emission from chromophores. Radioactive labels provide a detectable signal through radioactive decay. As shown in the examples, conjugated diagnostics containing fluorophores passed through the membrane successfully, but other detectable labels may be incorporated into the cargo.Examples of diagnostic agents or chemical probes include, but are not limited to, the following: Alexa Fluor dyes (Alexa Fluor350, Alexa Fluor488, Alexa Fluor532, Alexa Fluor546, Alexa Fluor568, Alexa Fluor594, Alexa Fluor633, Alexa Fluor660, Alexa Fluor680, AMCA, AMCA-S), BODIPY dyes (BODIPY FL, BODIPY ROG, BODIPY TMR, BODIPY TR, BODIPY530 / 550, BODIPY558 / 568, BODIPY564 / 570, BODIPY576 / 589, BODIPY581 / 591, BODIPY630 / 650, BODIPY650 / 665), Carboxyrhodamine 6G, Carboxy-X-Rhodamine (ROX), Cascade Blue, Cascade Yellow, Coumarin 343, Cyanine Pigments (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin, 4',5-Dichloro-2',7'-Dimethoxyfluorescein, DM-NeRF, Eosin, Erythrosine, Fluorescein, FAM Hydroxycoumarin, IRDyes (IRD40, IRD700, IRD800), JOE, Lisamin Rhodamine B, Marina Blue, Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol Green, 2',4,5',7'-Tetra-Bromosulfone-Fluorescein, Tetramethyl-Rhodamine (TMR), Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X.

[0122] In certain embodiments, the diagnostic or tracking agent is a polyvalent compound containing one or more fluorescent chemical fragments as a detectable label. The emission and excitation wavelengths of the diagnostic agent are 200 nm to 900 nm, but are not limited to this range. The diagnostic or tracking agent can be used for in vitro and in vivo imaging.

[0123] In certain embodiments, the endocytosis agent is an exocytosis vesicle (or endocytosis agent-vesicle complex). An exocytosis vesicle refers to a vesicle or vesicle composition comprising vesicle-forming components or particles and one or more endocytosis agents associated with the vesicle-forming components or particles, the vesicle-forming components including all cytoplasmic and endocytosis-mediating membrane components of a cell, such as nucleic acids, proteins, lipids, carbohydrates, and metabolites. The endocytosis agent can be located at any site of the vesicle, such as encapsulated or enclosed within the lipid-based vesicle-forming components, attached to membrane components (inward and / or outward), inserted into membrane components, or inserted across membrane components. The endocytosis agent has a binding affinity K D It can bind to any component of the vesicle via covalent or non-covalent bonds of less than 20.0 mM.

[0124] Exocytosis vesicles have a diameter of less than 10,000 nm. In certain embodiments, exocytosis vesicles have a diameter of 10 to 10,000 nm. In certain embodiments, exocytosis vesicles have a diameter of 10 to 1,000 nm.

[0125] In certain embodiments, the exocytosis vesicle may be an extracellular vesicle (EV), which contains or binds to an endocytosis agent in any equivalent. EVs include, but are not limited to, exosomes, microvesicles, microparticles, apoptotic bodies, oncosomes, ectosomes, synaptic vesicles, prostasomes, and matrix vesicles. The endocytosis agent can be selected from any endocytosis agent as defined herein. In the exocytosis vesicle, the endocytosis agent may be present at any site of the EV and / or may bind to any component of the EV via covalent or noncovalent bonding.

[0126] In certain embodiments, exocytotic vesicles may be produced by any cell and secreted from the cell into the extracellular space via exocytosis, transcytosis, and / or membrane fusion. In certain embodiments, exocytotic vesicles may be produced in vitro and / or in vivo by mixing vesicle-forming components or particles (including EVs) with an endocytic agent in any suitable solution, medium, or body fluid, in any equivalent, and the endocytic agent may form covalent or noncovalent bonds with the vesicle-forming components or particles (including EVs).

[0127] Based on the fact that foreign substances can be taken up by cells and released from (donor) cells in the form of extracellular vesicles (EVs), and that these extracellular vesicles can be taken up by either the same cell or another cell via exocytosis or membrane fusion (Ravi Shah, et al., The New England Journal of Medicine, 8(379), 958-966, 2018; Oscar PB Wiklander, et al., Science Translational Medicine, 11(492), eaav8521, 2019; Raghu Kalluri, et al., Science, 367(6478), eaau6977, 2020), these exocytolytic vesicles can be used to transport any endocytic agent as defined in this disclosure into cells. In addition, these exocytosis vesicles can be used to cross membrane barriers (including the blood-retinal barrier, lung endothelial and epithelial barrier, skin barrier, and cerebral blood barrier) and / or to deliver any endocytic agent as defined herein to any target tissue and organ in an animal body. In particular, the exocytosis vesicles of the present invention are suitable for the delivery of endocytic agents for studying, diagnosing, preventing and treating conditions and diseases of the eye, respiratory system, skin, and CNS. In this disclosure, exocytosis vesicles are referred to as a type of endocytosis agent. All the advantages and methods associated with endocytosis agents can be applied to exocytosis vesicles.

[0128] Endocytosis-mediated membrane components Endocytosis-mediated membrane components include proteins, lipids, and carbohydrates, which are membrane components, including the cell membrane, that mediate binding that enables the endocytosis process, or increased uptake via endocytosis. In some embodiments, the lipids include glycolipids, phospholipids, ceramides, and cholesterol. In some embodiments, the carbohydrates are molecules consisting of monosaccharides or monosaccharides and / or derivatives of 2 to 100 units, and may be linear or branched, and these bind to proteins to form glycoproteins, or bind to lipids to form glycolipids. In certain embodiments, the carbohydrates include, but are not limited to, N-acetylgalactosamine (GalNAc), chondroitin sulfate (CS), dermatan sulfate (DS), heparin sulfate (HS), keratan sulfate (KS), hyaluronic acid, and sialic acid (SA).

[0129] In some embodiments, the protein includes, but is not limited to, endogenous membrane proteins, peripheral membrane proteins, lipid-anchored proteins, globular proteins, and glycoproteins (including all variants, mutants, splice variants, substitutions, and fusions).

[0130] In certain embodiments, the proteins include ATP-binding cassette (ABC) transporters such as MDR1 / 2 / 3 / 4 / 5 and ABCG2, anaplastic lymphoma kinase (ALK), amnionless, AMPA receptor (AMPAR) (Maria Fiuza, et al., Journal of Cell Biology, 216(10), 3323-3338, 2017), apolipoprotein E receptor (ApoER), amino acid transporters, amyloid precursor protein (APP), ileal apical sodium / bile acid cotransporter (ASBT), alanine serine cysteine ​​transporter (ASCT), asialogycoprotein receptor (ASPGPR), V-type proton ATPase 6 (ATP6V), ATP6V1H, avidin, β-site amyloid precursor protein cleavage enzyme 1 (BACE1), β2-adrenergic receptor, CDO sibling (Boc), brassinosteroid-refractory-1 (BRI1), cell adhesion molecule (CAM) receptor, coxsackievirus-adenovirus receptor (CAR), chemokine receptors, e.g., CC chemokine receptor (CCR) and CXC chemokine receptor (CXCR), e.g., CXCR2, CXCR4, and CXCR7, differentiation antigens (CD), e.g., CD4, CD9, CD13 / APN receptor, CD25, CD26 (DPP4), CD30, CD33, CD36, CD38, CD46, CD56, CD63, CD71, CD74, CD80, CD81, CD82, CD86, CD123, CD138, CD147, CD152, CD152 (CTLA4), CD166, CD174, CD197, CD205, CD227, CD228, CD269, CD276, and CD326, cation-dependent MPR (CD-MPR), Cdon, cystic fibrosis transmembrane conduction regulator (CFTR), catethio Independent MPR / insulin-like growth factor-II (IGF-II) receptor (CI-MPR), IGF-2R, chemokine receptor, human collectin placenta 1 (CL-P1), pyramidal opsin, connexin, cell permeable peptide (CPP), CSF2R, CSF1R / FM, excitatory amino acid carrier 1 (EAAC1), E-cadherin, early endosomal autoantigen 1 (EEA1), epithelial cell adhesion molecule (EpCAM), erythropoietin-producing hepatocellular carcinoma receptor (EphR),Endothelial cell protein C receptor (EPCR), fatty acid binding protein (FABP), fatty acid transport protein (FATP), major facilitator superfamily domain-containing protein 2 (mfsd2), neonatal Fc receptor (FCRN), free fatty acid receptor (FFAR), flotilin-1, flotilin-2, folate receptors, e.g., reduced folate carriers, FOLT1 / 2 / 3 and proton-bound folate transporter (PCFT), Frizzled4, γ-A Minobutyrate A receptor (GABAAR), growth arrest specific 1 (Gas1), glutamate transporter (GLT), glucose transporter (GLUT), glutathione transporter, ion channel type AMPA glutamate receptor (GluR), GLP1, glycoproteins, e.g., gp18, gp31, and gp60, glucokinase, G protein-coupled receptors (GPCRs), e.g., GPR20, GPR20, free fatty acid receptor 1 (GPR40), GPR119, and GP R120, HDLR, IFN-γR, intermediate-density lipoprotein receptor (IDLR), IL-10R, IL-4R, integrins, e.g., αVβ3, α4β1 and α5β1 integrins, insulin receptor (IR), insulin-like growth factor receptor, potassium-chloride cotransporter 2 (KCC2), inward-rectifying potassium channel (Kir2,3), lactoferrin receptor, L-amino acid transporter (LAT), lysosome-associated membrane protein (LAMP), low-density lipoprotein receptor (LDLR), low-density lipoprotein receptor-associated protein (LRP), major facilitator superfamily domain-containing protein 2 (Mfsd2a), monocarboxylic acid transporter (MCT), multidrug resistance protein (MRP), MET, metabotropic glutamate receptor (mGlu1), major histocompatibility complex class II molecule (MHC-II), MHC-I, MINCLE, multidrug resistance protein (MRP), N-cadherin, K, + Dependency Na + / Ca 2+Exchanger 2 (NCKX2), nicatrin, nicotinic acetylcholine receptor, NOTCH receptor, Niemann-Pick C1-like 1 (NPC1L1), N-methyl D-aspartate receptor (NR), nucleoside transporter (NT), sodium / taurocholic acid cotransport peptide (NTCP), organic anion transporter (OAT), organic anion transport polypeptide (OATP), organic cation transporter (OCT), organic solute transporter (OST), otoferlin, P2X prinoceptor 4 receptor (P2x4R), purinergic receptor P2Y12 (P2Y12), protease-activated receptor (PAR), PE IZO1, PEIZO2, peptide transporter (PEPT), p-glycoprotein (P-gp), peroxisome proliferator-activated receptor (PPAR), prostate-specific membrane antigen (PSMA), receptor patched 1 (PTCH1), RAS, retinol-binding protein (RBP), riboflavin transporter protein (RFVT), rhodopsin, ribonuclease K (RNASEK), ring finger protein (RNF), extrarenal medulla potassium channel (ROMK), receptor serine / threonine kinase (RSK), e.g., transforming growth factor β (TGF-β) receptor and Aristidis Proteins listed in Moustakas, et al, Receptor Serine / Threonine Kinases (ISBN: 978-3-540-44244-8), receptor tyrosine kinases (RTKs), e.g., fibroblast growth factor receptor (FGFR), FLT1 / 2 / 3 / 4, FGFR1 / 2 / 3 / 4, RET, tropomyosin-related kinases (TRKs), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER), vascular endothelial growth factor receptor (VEGFR), TYRO3, and other proteins listed in the publication Mark A. Lemmon and Joseph Schlessinger, Cell, 141(7)1117-1134, 2010, Sanpodo / Notch, solute carrier (SLC) transporters, e.g., hMATE1, and publication Enrico Girardi, et al.The proteins listed in Nature Chemical Biology, 16, 469-478, 2020, include solute carrier family 37 member A3 (SLC37A3), SGLT, sodium-bound monocarboxylic acid transporter (SMCT), sodium-dependent multivitamin transporter (SMVT), synaptosome-associated protein (SNAP), sortilin-associated CNS expression 1a (SorCS1a), acidic, cysteine-rich SorCS1c secretory protein (SPARC), SPv-NK1R, SRC, scavenger receptor (SR), e.g., CD36, LAMP1, and LAMP2, syntaxin (STX), sodium-vitamin C cotransporter (SVCT), synaptotagmin (SYT), triiodothyronine (T3), and transferin This includes, but is not limited to, the following target proteins expressed on microbial cell membranes: β-transceptors (TfR), transforming growth factor β (TGF-β), trans-Golgi network (TGN38), thiamine transporter (THTR), Toll-like receptors (TLRs), such as TLR4 and TPN1p, vesicle-associated membrane proteins (VAMP), vitamin D receptor (VDR), vesicle-mediated GABA transporter (VGAT), vesicle-mediated glutamate transporter (VGLUT), very low-density lipoprotein receptor (VLDLR), uric acid transporter 1 (URAT1), and zinc ring finger protein (ZRNF) (including all variants, mutants, splice variants, indels, and fusions of these listed target proteins). Similar transporters or receptors expressed on microbial cell membranes have also been applied to BacPROTAC, etc.

[0131] Chemical substances and terminology When used in this specification, [ka] The symbol represents the binding site;

[0132] [ka] It is either a single bond or a double bond; R, R', and R'' are, independently, H, D, O, =O, S, =S, SS, =NH, =N(OH), N(OH), N(H)O, CH2F, CHF2, CF3, polyethylene glycol 1-100, C1-C100 alkoxy, C1-C100 alkyl, C2-C100 alkylene, C2-C100 alkyne, C3-C100 cycloalkyl, C3-C100 cycloalkie, C3-C100 cycloalkyne, C3-C100 heterocyclyl, C6-C100 aryl, or C1-C100 heteroaryl, polyethylene glycol-R a C1-C100 alkyl-R a C2-C100 Alkilen-R a C2-C100 Alkyne-R a C3-C100 Cycloalkyl-R a C3-C100 Cycloalkylene-R a C3-C100 Cycloalkyne-R a C3-C100 heterocyclyl-R a C6-C100 aryl-R a , or C1-C100 heteroaryl-R a , B(OR a 2), Si(OR a 3) C≡C, C≡CR a CH=CH 2、 CH=CHR a 、 CH=C(R a ) 2、 CR a =CHR a 、 CR a =CR a 、 C(O), CR a =C(R a )2, COR a 、 CONH2, C(O)OR a ,OC(O)R a , OC(O)OR a , OCN(R) a )2, CONHR a CON(R a )2, NH2, N(H)R a , N(R a)2、N + (R a )3、NHNH2、NHN(H)R a ,-NHN(R a )2、NHC(O)R a 、 NHC(O)OR a 、 NHC(O)NH2、NHC(O)NHR a 、NHC(O)N(R a )2、NR a C(O)NH2、NR a C(O)NHR a 、NR a C(O)N(R a )2、NR a C(S)N(R a )2、NHC(NH)NH2、NHC(NH)NHR a 、NHC(NH)N(R a )2、NR a C(NH)NH2、NR a C(NH)NHR a 、NR a C(NH)N(R a )2、NR a C(NR a )N(R a )2、NHS(O)R a 、NHS(O)NH2、NHS(O)NHR a 、NHS(O)N(R a )2、NR a S(O)NH2、NR a S(O)NHR a 、NR a S(O)N(R a )2、NHS(O)2R a 、NHS(O)2NH2、NHS(O)2N(H)R a 、NHS(O)2N(R a )2、NR a S(O)2NH2、NR a S(O)2NHR a 、NR a S(O)2N(R a )2、OH、OR a 、OS(O)2OH、OS(O)2OR a 、OS(O)OH、OS(O)OR a, OS(O)2NH2, OS(O)2N(H)R a , OS(O)2N(R a )2, OS(O)NH2, OS(O)N(H)R a , OS(O)N(R a )2, SH, SR a , S(O)R a , S(O)2NH2, S(O)2NHR a , S(O)2N(R a )2, S(O)N(H)R a , S(O)N(R a )2, SON(R a )3, SO2H, SO2R a , P(O)(OH)2, P(O)(OH)(OR a ), P(O)(OR a )2, P(O)(OH)OP(O)(OH)2, P(O)(OH)OP(O)(OH)(OR a ), P(O)(OH)OP(O)(OR a )2, P(O)(OH)OP(O)(OH)OP(O)(OH)2, P(O)(OH)OP(O)(OH)OP(O)(OH)(OR a ), P(O)(OH)OP(O)(OH)OP(O)(OR a )2, OP(O)(OR a ), OP(O)OP(O)(OH)2, OP(O)OP(O)(OH)(OR a ), OP(O)OP(O)(OR a )2, OP(O)OP(O)(OH)OP(O)(OH)2, OP(O)OP(O)(OH)OP(O)(OH)(OR a ), OP(O)OP(O)(OH)OP(O)(OR a )2,

[0133]

Chem.

Chem.

Chem.

[0134] Chemical bond or linker unit [ka] is a chemical bond or linker unit [ka] It can share the same structure and bind to any of the following sites: ligand, E3 ligase ligand, drug, chemical arm, SMDA, LA, Gas, PPA, VtA, AAP, SCA, NNNA, RMPB, or ADD; m, n, and q are independently selected from 0 to 100 and include any number in between.

[0135] The term "substituted" means that a particular group or part has one or more suitable substituents, and that the substituents can be attached to the particular group or part at one or more positions. For example, a cycloalkyl-substituted aryl indicates that the cycloalkyl is either attached to one atom of the aryl or fused with the aryl to share two or more common atoms.

[0136] Unless otherwise defined, "aryl" means a cyclic aromatic hydrocarbon group having 1 to 100 aromatic rings, including monocyclic or bicyclic groups such as phenyl, biphenyl, and naphthyl. If two aromatic rings are present (e.g., bicyclo), the aromatic rings of the aryl group may be bonded at one point (e.g., biphenyl) or fused together (e.g., naphthyl). The aryl group may be substituted with one or more substituents, e.g., 1 to 100 substituents, at any bonding point. Examples of substituents include -H, -halogen, -CN, -O(C1-C100)alkyl, -(C1-C100)alkyl, -O(C2-C100)alkenyl, -O(C2-C100)alkynyl, -(C2-C100)alkenyl, -(C2-C100)alkynyl, -OH, -OP(O)(OH)2, -OC(O)(C1-C100)alkyl, and -C(O) (C1-C100)alkyl, -OC(O)O(C1-C100)alkyl, -NH2, -NH((C1-C100)alkyl), -N(C1-C100)alkyl)2, -S(O)2-, -(C1-C100)alkyl, -S(O)NH(C1-C100)alkyl, and -S(O)N((C1-C100)alkyl)2 are included but not limited to these. Substituents may be substituted themselves. Furthermore, if two fused rings are present, the aryl group may have an unsaturated or partially saturated ring fused with a fully saturated ring. Exemplary ring systems of these aryl groups include but are not limited to phenyl, biphenyl, naphthyl, anthracenyl, phenalenyl, phenantrenyl, indanyl, indenyl, tetrahydronaphthalenyl, and tetrahydrobenzoanurenyl.

[0137] Unless otherwise defined, “heteroaryl” means a monovalent monocyclic or polycyclic aromatic radical having 3 to 100 ring atoms, containing one or more ring heteroatoms selected from N, O, or S, with the remaining ring atoms being C. The heteroaryl as defined herein also means a bicyclic heteroaromatic group in which the heteroatoms are selected from N, O, or S. Aromatic radicals may be independently substituted with one or more substituents as described herein. Examples include, but are not limited to, furyl, thienyl, pyrrolyl, pyridyl, pyrazolyl, pyrimidinil, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, pyrazinil, indolyl, thiophen-2-yl, quinolyl, benzopyranil, isothiazolyl, thiazolyl, thiadiazole, indazole, benzimidazolyl, thieno[3,2-b]thiophene, triazolyl, triazinil, imidazo[1,2-b]pyrazolyl, fluo[2,3-c]pyridinil, imidazo[1,2-a]pyridinil, indazolyl, pyrrolo[2,3-c]pyridinil, pyrrolo[3,2-c]pyridinil, pyrazolo[3,4 -c]pyridinyl, thieno[3,2-c]pyridinyl, thieno[2,3-c]pyridinyl, thieno[2,3-b]pyridinyl, benzothiazolyl, indolyl, indolinyl, indolinoneyl, dihydrobenzothiophenyl, dihydrobenzofluanil, benzofluan, chromanil, thiochromanil, tetrahydroquinolineyl, dihydrobenzothiazine, dihydrobenzooxanil, quinolineyl, isoquinolineyl, 1,6-naphthyridineyl, benzo[de]isoquinolineyl, pyrido[4,3-b][1,6]naphthyridineyl, thieno[2,3-b]pyradinyl, quinazolinyl, tetrazolo[1,5-a]pyridinyl, [1,2,4]triazolo[4,3-a]pyridinyl, isoindolyl, pyrrolo[2,3-b]pyridinyl, pyrrolo[3,4-b]pyridinyl, pyrrolo[3,2-b]pyridinyl, imidazo[5,4-b]pyridinyl, pyrrolo[1,2-a]pyrimidinyl, tetrahydropyrrolo[1,2-a]pyrimidinyl, 3,4-dihydro-2H-1Δ2-pyrrolo[2 ,1-b]pyrimidine, dibenzo[b,d]thiophene, pyridine-2-one, fluoro[3,2-c]pyridinyl, fluoro[2,3-c]pyridinyl, 1H-pyrido[3,4-b][1,4]thiadinyl, benzoxazolyl, benzoisoxazolyl, fluoro[2,3-b]pyridinyl, benzothiophenyl, 1,5-naphthyridinyl, fluoro[3,2 Examples include pyridine, [1,2,4]triazolo[1,5-a]pyridinyl, benzo[1,2,3]triazolyl, imidazo[1,2,4]pyrimidineyl, [1,2,4]triazolo[4,3-b]pyridazineyl, benzo[c][1,2,5]thiadiazolyl, benzo[c][1,2,5]oxadiazole, 1,3-dihydro-2H-benzo[d]imidazole-2-one, 3,4-dihydro-2H-pyrazolo[1,5-b][1,2]oxazinyl, 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridinyl, thiazolo[5,4-d]thiazolyl, imidazo[2,1-b][1,3,4]thiadiazolyl, thieno[2,3-b]pyrrolyl, 3H-indolyl, and their derivatives. Furthermore, when containing two fused rings, the aryl groups as defined herein have a partially saturated ring fused with an unsaturated or fully saturated ring. Exemplary ring systems of these heteroaryl groups include indolinyl, indolinonyl, dihydrobenzothiophenyl, dihydrobenzofuran, chromanyl, thiochromanyl, tetrahydroquinolinyl, dihydrobenzothiazine, 3,4-dihydro-1H-isoquinolinyl, 2,3-dihydrobenzofuran, indolinyl, indolyl, and dihydrobenzooxanyl.

[0138] The term "halogen" refers to fluorine, chlorine, bromine, iodine, and their isotopes.

[0139] "Alkyl" refers to a linear or branched saturated hydrocarbon containing 1 to 100 carbon atoms. Examples of (C1-C100) alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, and isohexyl.

[0140] "Alkoxy" refers to a linear or branched saturated hydrocarbon containing 1 to 100 carbon atoms, including terminal "O" atoms in the chain, such as O(alkyl). Examples of alkoxy groups, though not limited to these, include methoxy, ethoxy, propoxy, butoxy, t-butoxy, or pentoxy groups.

[0141] "Alkiene" refers to a linear or branched unsaturated hydrocarbon containing 2 to 100 carbon atoms. The "alkiene" group contains at least one double bond in its chain. The double bond of the alkenyl group may be unconjugated or conjugated with another unsaturated group. Examples of alkenyl groups include ethenyl, propenyl, n-butenyl, isobutenyl, pentenyl, or hexenyl. The alkenyl group may be unsubstituted or substituted, and may be linear or branched. An "alkyne" refers to a linear or branched unsaturated hydrocarbon containing 2 to 100 carbon atoms. An "alkyne" group contains at least one triple bond in its chain. Examples of alkenyl groups include ethynyl, propargyl, n-butynyl, isobutynyl, pentynyl, or hexynyl. Alkynyl groups may be unsubstituted or substituted.

[0142] "Cycloalkyl" or "carbocyclyl" refers to a monocyclic or polycyclic saturated carbocyclic ring containing 3 to 100 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptanyl, cyclooctanyl, norboranyl, norborenyl, bicyclo[2.2.2]octanyl, or bicyclo[2.2.2]octenyl and their derivatives. (C3-C100) cycloalkyl groups are cycloalkyl groups containing 3 to 100 carbon atoms. Cycloalkyl groups may be formed by condensation (e.g., decalin) or crosslinking (e.g., norbornane). A "cycloalkylene" refers to a monocyclic or polycyclic carbocyclic ring containing 3 to 100 carbon atoms. A cycloalkylene group contains at least one double bond in its chain. The double bond of the cycloalkylene group may be unconjugated or conjugated with another unsaturated group. Examples of cycloalkylene groups include cyclopropenyl, cyclobutenyl, butadienyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptatrielinyl, cycloheptenyl, cycloheptadienyl, cycloheptatrielinyl, cyclooctenyl, cyclooctadienyl, or cyclooctatetraenyl. A "cycloalkyne" refers to a monocyclic or polycyclic carbon ring containing 3 to 100 carbon atoms. A cycloalkyne group contains at least one triple bond in its chain. The triple bond of the cycloalkyne group may be unconjugated or conjugated with other unsaturated groups. An example of a cycloalkyne group is cyclooctinyl.

[0143] "Heterocyclyl" or "heterocycloalkyl" means a saturated or partially saturated monocyclic or polycyclic ring containing carbon and at least one heteroatom selected from oxygen, nitrogen, or sulfur (O, N, or S), wherein no delocalized n electrons (aromaticity) are shared between the ring carbons or heteroatoms. The heterocycloalkyl ring structure may be substituted with one or more substituents. Substituents may be substituted themselves. Examples of heterocyclyl rings include, but are not limited to, oxetanil, azetadinil, tetrahydrofuranil, tetrahydropyranil, pyrrolidinil, oxazolinil, oxazolidinil, thiazolinil, thiazolidinil, pyranil, thiopyranil, tetrahydropyranil, dioxalinil, piperidinil, morpholinil, thiomorpholinil, thiomorpholinil S-oxide, thiomorpholinil S-dioxide, piperazinil, azepinil, oxepinil, diazepinil, tropanil, oxazolidinol, 1,4-dioxanil, dihydrofuranil, 1,3-dioxolanil, imidazolidinil, imidazolinil, dithiolanil, and homotropanil.

[0144] "Haloalkyl" refers to an alkyl group substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, pentafluoroethyl, or trichloromethyl.

[0145] "Haloalkoxy" refers to an alkoxy group substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethoxy, difluoromethoxy, pentafluoroethoxy, or trichloromethoxy. "Cyano" refers to a substituent that has a carbon atom bonded to a nitrogen atom by a triple bond, such as C≡N. "Amino" refers to a substituent that contains at least one nitrogen atom (e.g., -NH2).

[0146] An "isomer" refers to a compound that has the same number and type of atoms, and therefore the same molecular weight, but differs in the arrangement and configuration of its atoms in space. This term includes stereoisomers and geometric isomers. "Stereoisomer" or "optical isomer" means a stable isomer that has at least one chiral atom or restricted rotation that gives rise to a perpendicular asymmetric plane (e.g., certain biphenyl, allene, and spiro compounds) and can rotate the plane of plane-polarized light. Because the compounds of this disclosure have chiral centers and other chemical structures that can give rise to stereoisomerism, this disclosure intends to describe stereoisomers and mixtures thereof. Because the compounds of this disclosure and their salts contain a chiral carbon atom, they can exist as single stereoisomers, racemates, and mixtures of enantiomers and diastereomers. Typically, such compounds are prepared as racemic mixtures. However, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as concentrated mixtures of stereoisomers. As detailed below, individual stereoisomers of a compound are prepared by synthesis from optically active starting materials containing the desired chiral center, or by separation or decomposition such as separation or recrystallization, chromatography, use of chiral separation agents, or direct separation of enantiomers in a chiral chromatography column, following the preparation of a mixture of enantiomer products and conversion to a mixture of diastereomers. Starting compounds with specific stereochemistry are commercially available or can be produced by the methods described below and decomposed by techniques well known in the art.

[0147] An "enantiomer" refers to a pair of stereoisomers that are mirror images of each other but cannot be superimposed. "Diastereoisomers" or "diastereomers" refer to optical isomers that are not mirror images of each other. A "racemic mixture" or "racemic mixture" means a mixture containing equal amounts of individual enantiomers. A "non-racemic mixture" refers to a mixture containing unequal amounts of individual enantiomers.

[0148] "Geometric isomers" refer to stable isomers arising from restricted rotational freedom of double bonds (e.g., cis-2-butene and trans-2-butene) or cyclic structures (e.g., cis-1,3-dichlorocyclobutane and trans-1,3-dichlorocyclobutane). Since carbon-carbon double (olefinic) bonds, C=N double bonds, and cyclic structures may be present in the compounds of this disclosure, this disclosure intends to describe various stable geometric isomers and mixtures thereof arising from the arrangement of substituents around these double bonds and within these cyclic structures. Substituents and isomers are designated using the cis / trans convention or the E or Z system (the term "E" refers to a higher-order substituent on the opposite side of the double bond, and the term "Z" refers to a higher-order substituent on the same side of the double bond). For a thorough discussion of E and Z isomers, see J. March, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 4 th Provided in ed., John Wiley & Sons, 1992, which is incorporated herein by reference in its entirety.

[0149] Some of the compounds of this disclosure may exist in two or more tautomer forms. As stated above, the compounds of this disclosure include all such tautomers. It is well known to those skilled in the art that the biological and pharmacological activity of a compound is sensitive to its stereochemistry. Therefore, for example, enantiomers often exhibit remarkably different biological activities, including differences in pharmacokinetic properties, metabolism, solubility, and protein binding, as well as pharmacological properties such as the type, degree, and toxicity of the activity exhibited. Thus, those skilled in the art will understand that one enantiomer may be more active or exhibit beneficial effects when concentrated relative to or separated from another enantiomer. Furthermore, those skilled in the art will know, from the present disclosure and prior art, methods for separating, concentrating, or selectively preparing enantiomers of the compounds of this disclosure. Therefore, while racemic drugs can be used, they are often less effective than administering the same amount of enantiomerically pure drugs. Consequently, if one enantiomer is pharmacologically more active, less toxic, or has superior pharmacokinetic properties compared to the other, it is therapeutically more beneficial to administer that enantiomer preferentially.

[0150] The preparation of a pure enantiomer or a mixture of a desired enantiomer excess (ee) or enantiomer purity can be achieved by one or more of the following methods: (a) separation or decomposition of the enantiomer, or (b) enantioselective synthesis known to those skilled in the art, or a combination thereof. These decomposition methods generally rely on chiral recognition, and such methods are generally known as Chiral Separation Techniques: A Practical Approach (2 nd This is disclosed in (ed.), G. Subramanian, Wiley-VCH, 2000; TE Beasley and RPW Scott, Chiral Chromatography, John Wiley & Sons, 1999; and Satinder Ahuja, Chiral Separations by Chromatography, Am. Chem. Soc., 2000.

[0151] Generally, unless a specific stereochemical or isomer is specifically indicated by the compound name or structure, the scope includes all tautomers, isomers, and mixtures (individual geometric isomers, or stereoisomers, or racemic or non-racemic mixtures) of a chemical structure or compound. "Charged" refers to the ionic form of an atom or group of atoms in which the number of electrons is not equal to the number of protons. Charged atoms or groups of atoms can become anions (negatively charged) or cations (positively charged) at certain pH values. "Chargible" or "chemically chargeable" refers to the ability of an atom or group of atoms to be ionized at a certain pH value in an aqueous solution.

[0152] "A chemical bond or part that can form a reversible or irreversible covalent bond with any nucleophile in biology" means any chemical bond or part that can reversibly or irreversibly crosslink endocytotic agents and any nucleophile in biology (including lysine, arginine, cysteine, serine, threonine, and carbohydrates) via covalent bonding. In particular, "a chemical bond or part that can form a reversible covalent bond with any nucleophile in biology" means any chemical bond or part that can crosslink endocytotic agents and any nucleophile in biology via covalent bonding, provided that the dissociation of such covalent bond can be cleaved and / or reformed under any conditions. In certain embodiments, "a chemical bond or portion that can form a reversible or irreversible covalent bond with any nucleophile in biology" is described in International Patent Publication No. 2020252397 and International Publication No. 2011018611, and in publications Anupam Bandyopadhyay, et al., Current Opinion Chemical Biology, 34, 110-116, 2016, Hannah Kiely-Collins, et al., Cell Chemical Biology, 28(7), 952-968, 2021, Fandi Sutanto, et al., RSC Medicinal Chemistry, 11, 876-884, 2020, and Sijie Wang, et al., ChemRxiv preprint, 2022 (DOI: It may be selected from any of the chemical bonds, links, or parts listed in 10.26434 / chemrxiv-2022-tvgn1).In certain embodiments, "chemical bonds or moieties that can form reversible or irreversible covalent bonds with any nucleophile in biology" include substituted or unsubstituted Michael acceptors (including acrylamides and acrylates), cyclic imines, thiols, α-cyanoacrylamides (or acrylates), α-substituted acrylonitriles, α,β-diketoamides with arginine (Ziyang Zhang, et al., Journal of the American Chemistry Society, 144(35), 15916-15921, 2022), and β-lactone derivatives (Ziyang Zhang, et al., Nature Chemical Biology, 18, 1177-1183, 2022), includes, but is not limited to, benzylidine rhodanine derivatives, disulfide bonds, boronic acids, α-ketoamides, nitrile moieties, methyl esters, ketones, ortho-phenoxide moieties, aromatic aldehydes, coumarin 3-aldehydes, ortho-boronic acid-substituted benzaldehydes (or acetophenones), and any deuterium-substituted derivatives, or any combination thereof, wherein the interrupting group and one or both terminal groups may be the same or different.

[0153] Method and drug administration One aspect of this technology provides a method for influencing the uptake of drugs by cells via endocytosis.

[0154] In some embodiments, the method refers to employing structural modification strategies used in the art to enhance the efficacy and / or efficiency of endocytosis by enabling or regulating the binding affinity and / or titer of a drug to endocytosis-mediated membrane components, dimerization or clustering of endocytosis-mediated membrane components, and / or conformational changes of endocytosis-mediated membrane components. Structural modification strategies include, but are not limited to, the addition or removal of substituents, fragment substitution, cyclization, scaffold hopping, bioisosterism, linkerology (linker-activity relationship studies), and prodrugs. The method can enhance the desired biological activity and drug binding affinity of a drug to endocytosis-mediated membrane components, thereby improving the efficacy of endocytosis and / or the efficiency of disease diagnosis and treatment. In some embodiments, the method refers to generating a polyvalent endocytic agent by covalently conjugating a chemical arm onto a drug via cleavable or incleavable chemical bonds or linkers. Polyvalent endocytic agents can be better absorbed for disease diagnosis and treatment via enhanced endocytosis. In certain embodiments, the method refers to covalently conjugating a chemical arm onto a drug via a chemical bond or linker to generate a polyvalent endocytic agent, the chemical arm being selected to enable or increase conformational changes of the endocytosis-mediating membrane component to enhance the efficacy and / or efficiency of endocytosis without sacrificing the binding affinity and / or titer of the resulting polyvalent endocytic agent to the endocytosis-mediating membrane component, dimerization or clustering of the endocytosis-mediating membrane component, and / or the intrinsic pharmacological activity of the drug. In certain embodiments, the method refers to covalently conjugating a chemical arm onto a therapeutic agent via a cleavable bond or linker, the chemical arm being selected to impair, eliminate, or remove the intended functionality of the therapeutic agent until the chemical arm is cleaved from the therapeutic agent. This method enables enhanced targeted delivery to cells and tissues via endocytosis.

[0155] In some embodiments, the method refers to employing a structural modification strategy used to enable or enhance endocytosis-mediated steric changes of membrane components by structural modification of an endocytic agent, for any purpose, including, but not limited to, the construction of charged molecules or salt formation techniques by structural modification using medicinal chemistry strategies in the art, including the formation of a polyvalent endocytic agent via the linkage of charged, chargeable, and other hydrophilic chemical arms, or the introduction of reversible or irreversible covalent bonds or moieties. In certain embodiments, a method for enabling or enhancing endocytosis-mediated steric changes of membrane components to improve the efficiency of endocytosis and / or the efficacy of an endocytic agent is to increase the binding affinity and / or binding titer between the endocytosis-mediated membrane component and the endocytic agent. In certain embodiments, a method for enabling or enhancing conformational changes of endocytosis-mediated membrane components to improve the efficiency of endocytosis and / or the effectiveness of endocytic agents is to increase the dimerization or clustering of endocytosis-mediated membrane components. In certain embodiments, a method for enabling or enhancing conformational changes of endocytosis-mediated membrane components to improve the efficiency of endocytosis and / or the effectiveness of endocytic agents is to adjust the environmental factors of the endocytosis-mediated membrane components, their interactions with membrane cofactor proteins, and / or the status of post-translational modifications of the endocytosis-mediated membrane components and membrane cofactor proteins by the art-utilized or structural modification strategies of endocytic agents.

[0156] Aspects of this technology provide methods for enhancing the efficacy and / or efficiency of drug endocytosis by enabling or increasing the binding affinity and / or titer of an endocytosis agent to endocytosis-mediating membrane components, dimerization or clustering of endocytosis-mediating membrane components, and / or conformational changes of endocytosis-mediating membrane components. In some embodiments, the method refers to using medicinal chemistry strategies in the art, such as forming a polyvalent endocytosis agent or introducing reversible or irreversible covalent bonds or parts in any equivalent of any suitable part of an endocytosis agent. In certain embodiments, the strategy refers to conjugating a chemical arm containing a reversible or irreversible covalent bond or part onto a drug via a cleavable or incleavable chemical bond or linker, or replacing any bond or part on the endocytosis agent with a reversible covalent bond or part. The resulting endocytosis agent can form reversible or irreversible covalent interactions with endocytosis-mediating membrane components, enabling or increasing dimerization or clustering of endocytosis-mediating membrane components, and / or enabling or increasing conformational changes of endocytosis-mediating membrane components, thereby enhancing the efficacy and / or efficiency of endocytosis.

[0157] Aspects of this technology provide methods for increasing the solubility, binding affinity, and / or binding titer of an endocytic agent with endocytosis-mediating membrane components in order to enhance the efficacy and / or efficiency of endocytosis. In some embodiments, the method refers to forming a polyvalent endocytic agent via salting-out techniques in the art, structural modification of a drug using medicinal chemistry strategies in the art, and / or conjugate with a chargeable or charged chemical arm, thereby enabling the solubility, binding affinity, and / or binding titer of the endocytic agent with endocytosis-mediating membrane components to enhance the efficacy and / or efficiency of endocytosis. The resulting endocytic agent can exist in the form of a cationic or anionic in an aqueous solution at a specific pH value, and its solubility is increased. By this method, the endocytic agent spontaneously increases its solubility and permeability via endocytosis, enabling the diagnosis and treatment of diseases. In some embodiments, the method refers to forming exocytosis vesicles in vitro or in vivo in order to enable the formation of exocytic vesicles in order to enhance the stability and solubility of the endocytic agent.

[0158] Aspects of this technology provide a method for enabling endocytotic uptake of a drug without limitations on the molecular weight, polarity, and lipophilicity of the endocytic agent. In certain embodiments, the technology provides a method for promoting endocytotic uptake of hydrophilic agents, including but not limited to polar organic compounds such as inorganic compounds, chelate compounds, metal-based compounds, and / or peptide-based compounds. For example, highly polar compounds can be taken up into cells via endocytosis by binding to endocytosis-mediated membrane components via salt bridges and / or hydrogen bond formation. Another aspect of the present technology provides a method for increasing the binding affinity and / or binding value to endocytosis-mediated membrane components, and / or dimerization or clustering of endocytosis-mediated membrane components, and / or conformational changes of endocytosis-mediated membrane components, by utilizing charged molecule or salt formation techniques in the art to form a polyvalent endocytosis agent via linked chemical arms, or by structural modification using medicinal chemistry strategies in the art, including introducing reversible or irreversible covalent bonds or moieties, in order to enhance the efficacy and / or efficiency of endocytosis of polar or hydrophilic agents. In certain embodiments, by introducing a portion of the hydrophobic core of CD36 into, for example, a polar phosphatase inhibitor, the resulting endocytosis phosphatase inhibitor may have increased binding affinity and / or binding titer to endocytosis-mediated membrane components, dimerization and / or clustering of endocytosis-mediated membrane components, and / or conformational changes of endocytosis-mediated membrane components, thereby enhancing the efficacy and / or efficiency of endocytosis.In certain embodiments, by introducing an endocytic insulin agent into an insulin molecule having a cleavable or incleavable CD36 binding site, the resulting endocytic insulin agent can enhance the efficacy and / or efficiency of endocytosis when used orally, by increasing the binding affinity and / or titer to endocytosis-mediated membrane components, dimerization or clustering of endocytosis-mediated membrane components, and / or conformational changes of endocytosis-mediated membrane components. Encapsulation of insulin into exosomes after endocytosis and exocytosis enhances stability. Another advantage of this technology is that it provides a method for enabling an endocytic agent to cross the cell membrane and / or body barrier, either alone or in combination with other mechanisms including but not limited to passive diffusion, facilitated diffusion, transporter-mediated influx and / or efflux, and paracellular transport.

[0159] Aspects of this technology provide methods for influencing the metabolic stability of endocytic agents. To meet the stability requirements of the agents for the treatment and diagnosis of the subject, the methodologies of this disclosure include, but are not limited to, the addition or removal of substituents, fragment substitution, cyclization, scaffold hopping, bioisosterism, linkerology (linker-activity relationship studies), prodrugs, reduction of overall LogP values, or the formation of exocytotic vesicles in vitro or in vivo. In certain embodiments, the method refers to the replacement of any hydrogen atom on the endocytic agent with deuterium. In certain embodiments, the method refers to the introduction of substituents to reduce metabolism in soft spots within the agent.

[0160] Aspects of this technology provide methods for manufacturing and / or using (polypharmacological) endocytotic agents for any purpose, wherein at least one target of the (polypharmacological) endocytotic agent is an endocytosis-mediating membrane component. Since the binding affinity of an endocytotic agent to an endocytosis-mediating membrane component may be independent of its binding affinity to an endogenous pharmacological target, an endocytotic agent can be a (polypharmacological) compound that spontaneously has binding affinity for both an endocytosis-mediating membrane component for endocytosis and a pharmacological target for its intrinsic pharmacological activity, without balancing them. In some embodiments, a method for identifying or generating a (polypharmacological) endocytotic agent involves directly modifying the drug's structure to readily bind to both an endocytosis-mediating membrane component for endocytosis and a pharmacological target for its endogenous pharmacological activity. In the process of identifying or generating novel (multipharmacological) endocytosis and functional targeting agents, classical medicinal chemistry in the art involves spontaneously monitoring activity for the regulatory efficiency of both endocytosis and biological targets. Structural modifications include, but are not limited to, the structural modification of drugs using medicinal chemistry strategies in the art, the formation of multivalent endocytic agents by conjugation with charged or charged chemical arms, and the formation of exocytosis vesicles.

[0161] A part of this technology provides a method for transporting (multipharmacological) endocytosis agents and exocytosis vesicles to foreign bodies for any purpose by preparing and / or using (multipharmacological) endocytosis agents and exocytosis vesicles, through barriers within the human body, wherein the barrier is the blood-retinal barrier, the lung endothelial and epithelial barrier, the skin barrier, or the cerebral blood barrier. In some embodiments, a method for producing and / or using (multipharmacological) endocytosis agents and exocytosis vesicles involves the endocytosis agent and exocytosis vesicles binding to receptors or proteins expressed in cells within the barrier, such as scavenger receptors, Mfsd2a, GLUT1, Flotilin-1, Flotilin-2, glutathione transporters, amino acid transporters, transferrin receptors, lactoferrin receptors, low-density lipoprotein receptors, nicotinic acetylcholine receptors, insulin receptors, insulin-like growth factor receptors, integrins, and / or CD13 / APN receptors, and being transported across the barrier via the endocytosis / exocytosis pathway in the form of free endocytosis agents, exocytosis vesicles, or mixtures of free endocytosis agents and exocytosis vesicles in any ratio. In some embodiments, methods for constructing and / or using (multipharmacological) endocytotic agents and exocytotic vesicles involve the endocytotic agent being absorbed into the cell by endocytosis, encapsulated in intracellular organelles or vesicles such as endosomes and polyvesicles, and reducing or preventing the pumping of the endocytotic agent out of the cell by efflux transporters such as P-gp, BCRP, and multidrug resistance-associated proteins MRP1, MRP3, MRP4, and MRP6. In some embodiments, methods for enhancing the efficacy and / or efficiency of endocytosis of an endocytotic agent and the transport of the endocytotic agent across the membrane barrier for any purpose by using the expression of endocytosis-mediated membrane components in the membrane barrier and / or any structural modification of the agent in this disclosure.In particular, the use of (polypharmacological) endocytosis agents and exocytosis vesicles involves administering them via any route for the treatment of conditions and diseases of the eyes, respiratory system, skin, and CNS, and (polypharmacological) endocytosis agents and exocytosis vesicles can be used alone or in any ratio in combination.

[0162] Aspects of this technology provide a method for transporting an endocytic agent across two or more cell layers in the body for any purpose, wherein the endocytic agent may be taken up by cells via endocytosis and released in the form of free endocytic agent molecules and / or endocytic agent-vesicle complexes (i.e., exocytic vesicles), which include lipid bilayer vesicles containing the endocytic agent and extracellular vesicles, and the free endocytic agent molecules and / or exocytic vesicles may be re-taken up by any (acceptor) cell for action via endocytosis or membrane fusion for any purpose of use.

[0163] Aspects of this technology provide a method for producing exocytotic vesicles in vitro and / or in vivo for any purpose. In particular, aspects of this technology provide a “one-step” method for generating and using exocytotic vesicles in an animal body, without further processes including the isolation of drugs or vesicles, in which the cell body takes up an endocytic agent via endocytosis, sequentially secretes exocytotic vesicles, and the resulting endogenous exocytotic vesicles can be directly used in the body for any purpose. In certain embodiments, “body” or “animal body” refers to the human body. In another embodiment, a method for generating exocytotic vesicles is that a free endocytic agent molecule binds to extracellular vesicles and / or exocytotic vesicles in situ via covalent or non-covalent bonding. In another embodiment, a method for generating exocytotic vesicles is that the endocytic agent is loaded onto exocytotic vesicles in vitro by any technique of the art. In another embodiment, a method for isolating exocytotic vesicles from cells, body fluids, tissues, organs, products, or culture media by any extracellular vesicle isolation technique in the art.

[0164] Aspects of this technology provide methods for modulating the absorption, distribution, metabolism, and excretion (ADME) characteristics of endocytic agents in animal bodies. In certain embodiments, there are methods that enhance the efficacy and / or efficiency of endocytosis of a given endocytic agent, increase the loading of the endocytic agent onto exocytic vesicles via cellular endocytosis or exocytic vesicle release, and reduce or prevent the rapid metabolism and / or excretion of the endocytic agent, in order to extend the residence time and / or half-life of the endocytic agent in animal bodies.

[0165] Aspects of this technology provide a method for evaluating and / or determining the ADME properties of an endocytosis agent in a drug discovery and development process, the method involving a process or step for the isolation and / or lysis of exocytosis vesicles by any technology and skill in the art. In certain embodiments, the technologies, skills, and agents that may be used in the exocytosis vesicle isolation process include, but are not limited to, any suitable methods for the isolation of extracellular vesicles (EVs) or cells listed in the publications Brennan K., et al., Scientific Reports, 10, 1039, 2020 and Thanaporn Liangsupree, et al., 1636, 461773, 2021. In certain embodiments, techniques, skills, and agents that may be used in the exocytosis vesicle lysis process include, but are not limited to, any suitable methods or agents for lysing extracellular vesicles (EVs) or cells, or for disrupting EVs or cell membranes, including freeze-thaw cycle processes, and any methods or agents listed in the publication Prabal Subedi, et al., Analytical Biochemistry, 584, 113390, 2019.

[0166] In some embodiments, this method refers to reducing the toxicity of a drug / medicine by producing and using exocytotic vesicles from an endocytotic agent in vitro or in vivo. Since the endocytotic agent can be taken up by cells in the form of exocytotic vesicles, such as endocytotic agent-exosome complexes, and released from cells, the endocytotic agent can be present in exosomes, preventing or reducing direct contact between free endocytotic agent molecules and cells in the body, such as blood cells. In some embodiments, this refers to a method in which an endocytotic agent is administered orally, and cells in the human body, such as cells in the gastrointestinal tract, liver, or tumors, take up the endocytotic agent via endocytosis, secrete exocytotic vesicles, and the resulting exocytotic vesicles have reduced toxicity compared to free endocytotic agent molecules. In certain embodiments, the method for reducing toxicity is to adjust the efficacy and / or efficiency of endocytosis of the drug by any of the above methods, to load the endocytotic agent into exocytotic vesicles, and / or to release the exocytotic vesicles. For example, by linking or fusing an endocytic agent with an additional chemical arm that has binding affinity to the membrane components mediating endocytosis via cleavable or incleavable chemical bonds or linker units to form a polyvalent compound, the resulting polyvalent endocytic agent has improved affinity to the membrane components mediating endocytosis, enhances the efficacy and / or efficiency of endocytosis, increases the loading of the endocytic agent onto extracellular vesicles and / or increases the release of exocytosis vesicles, and ultimately reduces toxicity because the formation of exocytosis vesicles prevents or reduces direct contact between free endocytic agent molecules and cells such as blood cells.

[0167] Aspects of this technology provide methods for designing or structurally modifying endocytic agents using medicinal chemistry strategies in the art, or for utilizing endocytic agents for any purpose that specifically targets endocytosis-mediated membrane components. In certain embodiments, the technology provides methods for structurally designing or modifying endocytic agents using medicinal chemistry strategies in the art, or for selecting endocytic agents that specifically target the conformation of CD36 on cancer cells for enhanced antitumor effects and reduced toxicity, by utilizing the conformational diversity of CD36 on normal and cancer cells. Another advantage of the technology is that it provides methods for altering the conformation of endocytosis-mediated membrane components by utilizing any approach in the art, such as the application of mechanical, electrical, thermal, cold, light, or radiation stimulation, and / or the presence of a single endocytic agent, thereby altering biological events in cells and / or the sensitivity of endocytosis-mediated membrane components to endocytic agents.

[0168] Aspects of this technology provide general methods for improving clinical outcomes. In some embodiments, the method refers to utilizing differences in the expression of endocytosis-mediated membrane components for patient stratification, administration route, and dosage selection. On the other hand, feedback on endocytic agent treatment in a clinic may be used to adjust the administration route of endocytic agents and / or improve the outcomes of endocytic agent treatment. In certain embodiments, the method refers to treating subjects requiring endocytic agents. In certain embodiments, the method refers to treating subjects requiring endocytic agents via specific delivery systems such as topical, inhalation, intraperitoneal, intravenous, and oral delivery by utilizing different expressions of endocytosis-mediated membrane components in cells and tissues. For example, CD36-mediated endocytosis is the dominant pathway for cellular uptake of nutrients and macromolecules (Jurgen Pohl, et al., Molecular Biology of the Cell, 16(1), 24-31, 2005; Nanxia Zhao, et al., Advanced NanoBiomed Research, 2(6), 2100120, 2022; Vincenza Cifarelli, et al., Comprehensive Physiology, 8(2), 493-507, 2018; Youchun Zeng, et al., Journal of Biological Chemistry, 278(46), 45931-45936, 2003). CD36 is highly expressed in the gut, cancer cells, and metabolism-related cells, and is upregulated in the central nervous system of patients with pathological changes (Vincenza Cifarelli, et al., Comprehensive Physiology, 8(2), 493-507, 2018; Shunjie Bai, et al., Translational Psychiatry, 11(16), 2021; Octavian Ioghen, et al., European Journal of Neuroscience, 53, 2500-2510, 2021).Furthermore, receptor-mediated endocytosis and exocytosis pathways are major pathways for transporting nutrients such as folic acid and similar substances to the brain parenchyma in the form of free nutrient compounds and extracellular vesicles carrying these compounds (Marcel Grapp, et al., Nature Communications, 4, 2123, 2013; Andong Qiu, et al., Cell, 127, 917-928, 2006). Therefore, this technology provides a general method for enhancing the efficacy and reducing the toxicity of a much broader range of endocytic agents to the central nervous system after oral or intravenous delivery for therapeutic and prophylactic administration.

[0169] Aspects of this technology provide a method for identifying literature-known bioactive compounds that can be taken up by cells via endocytosis. This method includes regulating the expression of endocytosis-mediated membrane components in cells, tissues, and / or bodies by gene editing of these membrane components, and then comparing the activity of the bioactive compounds in cells, tissues, and / or bodies with and without gene editing of the endocytosis-mediated membrane components. Compared to the activity of compounds in cells, tissues, and / or bodies without gene editing of the endocytosis-mediated membrane components, compounds taken up via endocytosis show significantly increased or decreased bioactivity in cells, tissues, and / or bodies with edited endocytosis-mediated membrane components.

[0170] Aspects of this technology provide methods for identifying cellular and intracellular biological targets using the endocytosis agent as a probe by any technique and skill in the art. For example, the endocytosis agent having a chemical bond or moiety capable of forming a covalent bond with biotin, fluorescence, halo-tag ligand, SNAP-tag ligand, CLIP-tag ligand, or any biological target is particularly suitable for identifying the biological targets of endocytosis agents and derivatives in combination with techniques and skills including microscopy imaging, immunophenotyping, immunoprecipitation, flow cytometry, fluorescence-activated cell sorting (FACS), fluorescence resonance energy transfer (FRET), and / or omics (including genomics, epigenomics, transcriptomics, proteomics, and metabolomics) analysis. In particular, aspects of this technology provide methods for identifying membrane targets that mediate endocytosis. In certain embodiments, the method for identifying membrane targets that mediate endocytosis refers to using a labeled endocytosis agent, then administering the agent, and using any detection and identification techniques in the art. For example, a biotin-labeled endocytosis agent can be obtained by ligating a labeled probe, such as biotin, to any site on the endocytosis agent. Subsequently, cells can be cultured with the biotin-labeled endocytosis agent, membrane proteins can be isolated, and then identified by immunoprecipitation, FACS, omics analysis, and Western blotting to determine the endocytosis-mediating membrane components that target the endocytosis of the endocytosis agent. In another embodiment, a method for identifying endocytosis-mediating membrane targets refers to using any genome scanning technique in the art to identify endocytosis-mediating membrane targets. For example, comparing gene expression between cells, tissues, or bodies with different sensitivities to a particular endocytosis agent can be used to identify endocytosis-mediating membrane targets.In another embodiment, a method for identifying endocytosis-mediated membrane targets refers to using any gene editing technique in the art to identify endocytosis-mediated membrane targets, and gene-edited cells, tissues, or bodies may be more sensitive or more resistant to treatment with endocytic agents. For example, cells with gene expression inhibition and activation by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa), respectively, may act differently or complementaryly to treatment with specific endocytic agents, and these can be used to identify endocytosis-mediated membrane targets.

[0171] Aspects of this technology provide methods for using and selecting endocytosis agents based on different microenvironmental factors of cells and tissues for any purpose, including, but not limited to, pH values, salt concentrations, oxygen gradients, carbon dioxide gradients, H2O2 gradients, nutrient gradients, and therapeutic compound gradients. For example, because cancer cells are in an acidic environment, using endocytosis agents with basic groups for tumor-targeted delivery can enhance therapeutic function and reduce toxicity.

[0172] Aspects of this technology provide methods for using and selecting endocytic agents based on the expression of endocytosis-mediated membrane components having diverse conformations, isoforms (or variants), and / or post-translational modifications such as glycosylation states, for any purpose. Another aspect of this technology provides methods for using and selecting endocytic agents for any purpose based on the expression of cofactors that form complexes with endocytosis-mediated membrane components. For example, endocytic agents can be used to enhance therapeutic function and reduce toxicity in cancer treatment by targeting specific conformations, isoforms, or glycosylations of endocytosis-mediated membrane components or cofactors in cancer cells.

[0173] Aspects of this technology provide a method for activating the endocytosis process or increasing the expression of endocytosis-mediated membrane components by modulating input signals, such as the binding of endogenous or exogenous substances to membrane proteins, and thereby enhancing the endocytic absorption of endocytic agents. In certain embodiments, the method refers to utilizing the binding of endogenous substances (e.g., insulin and its derivatives) to membrane proteins (e.g., insulin receptors) to activate the endocytic cycling of endocytosis-mediated membrane components such as GLUT4, and / or increasing the expression of endocytosis-mediated membrane components, thereby enhancing the absorption of endocytic agents via endocytosis. In certain embodiments, the method refers to simultaneously utilizing the binding of endocytosis-mediated and non-endocytosis-mediated membrane components to endocytic agents in order to activate the endocytosis process or to increase the expression of endocytosis-mediated membrane components and enhance endocytosis-mediated absorption.

[0174] Aspects of this technology provide a method for delivering endocytosis-mediated membrane components or extracellular materials associated with any disease into a cell via endocytosis, and for disrupting or degrading disease-related endocytosis-mediated membrane components or extracellular materials via the endosomal / lysosome system. In certain embodiments, disease-related endocytosis-mediated membrane components or extracellular materials include, but are not limited to, membrane proteins or extracellular proteins, carbohydrates, lipids, pathogens, particles, viruses, fungi, protozoa, bacteria, vectors, cellular debris, and other cells. Aspects of this technology provide methods for treating subjects requiring any of the endocytotic agents described herein. Preferably, the methods include administering an effective amount of the agent to the subject. As used herein, the terms “treating” and “to treat” mean, respectively, alleviating symptoms, eliminating the cause of symptoms that result temporarily or permanently, and / or preventing or delaying the onset of symptoms resulting from a designated disease or disorder, or reversing their progression or severity. Thus, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0175] Aspects of this technology provide a method for diagnosing a subject requiring any of the endocytotic agents described herein. Preferably, the method involves administering an effective amount of the agent to the subject. As used herein, the terms “diagnosing” and “to diagnose” mean, respectively, presenting or imaging a site of disease, alleviating symptoms, eliminating the cause of symptoms that result temporarily or permanently, and / or preventing or delaying the appearance of symptoms resulting from a designated disease or disorder, or reversing their progression or severity. Thus, the methods disclosed herein encompass both therapeutic and prophylactic administration.

[0176] "Subject" can be replaced with "patient" or "individual" and means an animal that may be a human or non-human requiring treatment. "Subject requiring treatment" includes subjects having a disease, disorder or condition that responds to treatment with the endocytosis agents disclosed herein alone or in combination with other agents. Preferably, subjects requiring treatment include, but are not limited to, diseases and conditions related to aging and age, weight management, cancer, central nervous system (CNS) diseases and conditions, cardiovascular diseases (CVD), diabetes mellitus, eye diseases and conditions, hypertension, immune system diseases, infections, inflammation and inflammation-related diseases and conditions, obesity and obesity-related diseases and conditions, respiratory diseases and conditions, or skin diseases and conditions. In certain embodiments, subjects requiring treatment include those requiring prevention, treatment, and research of aging and age-related diseases and conditions. "Aging is a physiological process mediated by biological and genetic pathways that directly impacts lifespan and drives age-related diseases. Mechanisms of aging have been elucidated and include, but are not limited to, genomic instability, telomere shortening, and cellular senescence. For example, age-related diseases include, but are not limited to, cardiovascular diseases, cancer, diabetes mellitus, immune system disorders, hearing loss, macular degeneration, and musculoskeletal disorders such as osteoarthritis."

[0177] In certain embodiments, subjects requiring treatment include those requiring cancer prevention, treatment, and research. "Cancer" refers to a pathological process that results in the formation and growth of cancerous or malignant neoplasms; that is, abnormal tissue that grows through cell proliferation, often growing more rapidly than normal and continuing to grow even after the stimulus that initiated the growth has ceased. For example, cancer includes, but is not limited to, mesothelioma, leukemia, lymphomas such as cutaneous T-cell lymphoma (CTCL), non-cutaneous peripheral T-cell lymphoma, lymphomas associated with human T-cell lymphotropic virus (HTLV) such as adult T-cell leukemia / lymphoma (ATLL), B-cell lymphoma, acute non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, lymphoma, and multiple myeloma, non-Hodgkin lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, Burkitt lymphoma, adult T-cell leukemia / lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), or hepatocellular carcinoma. Further examples include pediatric solid tumors such as myelodysplastic syndrome, brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft tissue sarcomas; common solid tumors in adults such as head and neck cancers (e.g., oral cancer, laryngeal cancer, nasopharyngeal cancer, and esophageal cancer); genitourinary cancers (e.g., prostate cancer, bladder cancer, kidney cancer, uterine cancer, ovarian cancer, and testicular cancer); lung cancers (e.g., small cell and non-small cell lung cancer); breast cancer (e.g., triple-negative breast cancer (TNBC)); pancreatic cancer; melanoma and other skin cancers; gastric cancer; brain tumors; tumors associated with Gorin syndrome (e.g., medulloblastoma or meningioma); or liver cancer.

[0178] Additional exemplary forms of cancer that can be prevented or treated by endocytosis include cancers of skeletal muscle or smooth muscle, gastric cancer, small intestine cancer, rectal cancer, salivary gland cancer, endometrial cancer, adrenal gland cancer, anal cancer, parathyroid cancer, pituitary cancer, colon cancer, familial adenomatous polyposis cancer, and hereditary nonpolyposis colorectal cancer, labial cancer, laryngeal cancer, carcinoma, tongue cancer, salivary gland cancer, gastric cancer, thyroid cancer (medullary and papillary thyroid carcinoma), renal cancer, renal parenchymal cancer, kidney cancer, cervical cancer, endometrial cancer, choriocarcinoma, testicular cancer, urinary tract cancer, glioblastoma, astrocytoma, meningioma, medulloblastoma, and peripheral nerve ectoderm tumors. This includes, but is not limited to, brain tumors, gallbladder cancer, bronchial cancer, basal cell tumor, teratoma, retinoblastoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myasthenia, liposarcoma, fibrosarcoma, Ewing's sarcoma, plasmacytoma, melanoma, nasopharyngeal carcinoma (NPC), microsatellite-stable colorectal cancer (mssCRC), thymoma, carcinoid, acute myeloid leukemia, and gastrointestinal stromal tumors (GIST), breast cancer, triple-negative breast cancer (TNBC), small cell lung cancer, non-small cell lung cancer, prostate cancer, castration-resistant prostate cancer (CRPC), or metastatic castration-resistant prostate cancer (mCRPC).

[0179] In certain embodiments, subjects requiring treatment include those requiring prevention, treatment, and research of cardiovascular diseases. "Cardiovascular diseases" refers to a group of diseases of the heart and blood vessels. For example, cardiovascular diseases include, but are not limited to, coronary heart disease, cerebrovascular disease, peripheral artery disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis, or pulmonary embolism.

[0180] In certain embodiments, subjects requiring treatment include those requiring prevention, treatment, and study of diseases and conditions of the central nervous system (CNS). "CNS diseases and conditions" refers to a broad group of neurological disorders affecting the structure or function of the brain or spinal cord that collectively form the central nervous system. For example, CNS diseases include, but are not limited to, brain tumors, neurodegenerative diseases such as Alzheimer's disease, frontotemporal dementia, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, vascular dementia, Parkinson's disease, Lewy body dementia, Huntington's disease, spinocerebellar ataxia, Friedrich's ataxia, ataxic telangiectasia, amyotrophic lateral sclerosis (ALS), bulbar spinal atrophy, or spinal muscular atrophy.

[0181] In certain embodiments, subjects requiring treatment include those requiring prevention, treatment, and research of diabetes mellitus. "Diabetes mellitus" refers to a group of diseases characterized by a reduced ability to produce or respond to insulin, resulting in abnormal carbohydrate metabolism and elevated blood and urinary glucose levels. Examples of diabetes mellitus include, but are not limited to, type 2 diabetes, gestational diabetes, prediabetes, monogenic diabetes, cystic fibrosis-associated diabetes, or drug or chemical-induced diabetes. In certain embodiments, subjects requiring treatment include those requiring prevention, treatment, and study of eye diseases and conditions. "Eye diseases and conditions" means any disease or disorder affecting the human eye. For example, eye diseases include, but are not limited to, age-related macular degeneration (AMD), amblyopia, anophthalmia and microphthalmia, astigmatism, Behçet's disease, Vietti crystalline dystrophy, blepharitis, blepharospasm, cataracts, central retinal vein occlusion (CRVO), cerebral visual impairment (CVI), coloboma, color blindness, convergence insufficiency, corneal conditions, diabetic retinopathy, dry eye, eye cancer, hyperopia, floaters, glaucoma, Graves' disease, idiopathic intracranial hypertension, amblyopia, macular edema, macular hole, macular packer, myopia, ocular histoplasmosis syndrome (OHS), pink eye, presbyopia, rare diseases, refractive errors, retinal detachment, retinitis pigmentosa, retinoblastoma, retinopathy of prematurity, Stargardt disease, Ushe syndrome, uveitis, or vitreous detachment.

[0182] In certain embodiments, subjects requiring treatment include those requiring prevention, treatment, and research of hypertension. "Hypertension" refers to a medical condition in which arterial blood pressure is persistently elevated. In most adults, hypertension is defined as a persistent resting blood pressure of 120 / 80, 130 / 80, or 140 / 90 mmHg or higher. For example, hypertension includes, but is not limited to, primary or secondary hypertension. In certain embodiments, subjects requiring treatment include those requiring prevention, treatment, and study of immune system diseases and conditions. “Immune system diseases and conditions” means immunodeficiency and autoimmune diseases, where “autoimmune disease” means a condition resulting from an abnormal immune response to a functioning body part. A functioning body part refers to any body part of an animal. For example, autoimmune diseases include, but are not limited to, rheumatoid arthritis (RA), psoriasis / psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), inflammatory bowel disease, Addison's disease, Graves' disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, or celiac disease.

[0183] In certain embodiments, subjects requiring treatment include those requiring prevention, treatment, and research of inflammation and inflammation-related diseases and conditions. "Inflammation" is defined as the immune system attacking the body's own tissues, causing inflammation. Many conditions may be associated with inflammation, particularly chronic inflammation. For example, inflammation-related diseases include, but are not limited to, Alzheimer's disease, asthma, cancer, CDV, rheumatoid arthritis (RA), ankylosing spondylitis (AS), or stroke. In certain embodiments, subjects requiring treatment include those requiring weight management, prevention, treatment, and research for obesity and obesity-related diseases and conditions. "Obesity" refers to a physical condition in which the body mass index (BMI) is 30.0 or higher. Obesity-related diseases include, but are not limited to, type 2 diabetes, CDV, fatty liver, or cancer. Weight management refers to the techniques and physiological processes that contribute to a person's ability to reach and maintain a certain weight.

[0184] In certain embodiments, the endocytosis agents described herein can be used for the prevention, treatment, and study of respiratory diseases and conditions. “Respiratory diseases” refers to diseases affecting the lungs and other parts of the respiratory system. Examples of respiratory diseases include, but are not limited to, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, idiopathic pulmonary fibrosis, or lung cancer.

[0185] In certain embodiments, subjects requiring treatment include subjects requiring prevention, treatment, and study of skin diseases and conditions. “Skin diseases and conditions” refers to any condition affecting the skin. For example, skin diseases and conditions include acanthosis nigricans, acne, keloid acne of the neck, acne scars, actinic keratosis, alopecia areata, athlete's foot, atopic dermatitis, basal cell carcinoma, bed bugs, birthmarks, boils and styes, botulinum toxin, bullous pemphigoid, cellulitis, central centrifugal scarring alopecia (CCCA), chemical peels, chickenpox, herpes simplex, contact dermatitis, infantile eczema, and cutaneous T10. Cellular lymphoma, dandruff, dermatofibrosarcoma protuberance (DFSP), diabetes-related skin diseases, diaper rash, dry skin, hyperhidrosis, epidermolysis bullosa, female pattern hair loss, folliculitis, frontal fibrous alopecia, genital herpes, genital warts, granuloma annulare, hair loss, hand, foot, and mouth disease, head lice, heart disease-related skin diseases, herpes simplex, hidradenitis suppurativa, urticaria, hyperhidrosis, ichthyosis vulgaris, imiquimod, impetigo, isotretino Infections, keloid scars, keratosis pilaris, kidney disease-related skin conditions, laser-related skin conditions, leprosy, lichen planus, lupus, Lyme disease, melanoma, melanosis, Merkel cell carcinoma, moles, molluscum contagiosum, monkeypox rash, onychomycosis, neurodermatitis, nickel allergy, alopecia areata, ocular rosacea, pemphigus, perioral dermatitis, pityriasis rosea, poison ivy, oak, and poison ivy-related skin conditions. This includes, but is not limited to, diseases, nodular prurigo, psoriasis, psoriatic arthritis, rashes, tinea, rosacea, sarcoidosis, scabies, scalp psoriasis, scars, scleroderma, sebaceous gland carcinoma, seborrheic dermatitis, seborrheic keratosis, herpes zoster, skin biopsies, skin cancer, squamous cell carcinoma, congestive dermatitis, stretch marks, syphilis, skin conditions associated with thyroid disease, tinea versicolor, vitiligo, warts, wounds, xerosis, or hyperpigmentation.

[0186] In some embodiments, subjects requiring treatment include those requiring sensitization to bioactive agents such as anti-aging, anti-aging-related diseases, anti-cancer, anti-cardiovascular diseases, anti-diabetes, anti-ocular diseases, anti-hypertension, anti-immune system diseases, anti-infective, anti-inflammatory, anti-inflammatory-related diseases, anti-CNS diseases, weight management, anti-obesity, anti-obesity-related diseases, anti-respiratory diseases, anti-skin diseases and conditions. The terms “sensitize” and “sensitizing” refer to making a subject or cell more sensitive or responsive to the biological effects of a second agent (e.g., a bioactive agent) through the administration of a first agent (e.g., promotion or delay of a mode of cellular function including, but not limited to, cell secretion, cell division, cell growth, proliferation, invasion, angiogenesis, necrosis, or apoptosis). The sensitization effect of the first agent on target cells can be measured as the difference in the intended biological effects (e.g., promotion or delay of a modality of cellular function, including but not limited to cell growth, proliferation, invasion, angiogenesis, or apoptosis) observed when the first agent is administered versus when the second agent is administered without the first agent.

[0187] In some embodiments, the endocytosis agents described herein may be administered in combination with one or more bioactive agents, including anti-aging agents, anti-aging-related disease agents, anticancer agents, anti-cardiovascular disease agents, anti-diabetic agents, anti-ocular disease agents, anti-hypertensive agents, anti-immune system disease agents, anti-infective agents, anti-inflammatory agents, anti-inflammatory-related disease agents, anti-CNS disease agents, anti-obesity agents, anti-obesity-related disease agents, anti-respiratory disease agents, anti-dermatological disease agents, and conditioning agents. The term “bioactive agent” is used to describe an agent having bioactivity that assists the effect of the therapeutic, inhibitory and / or preventive / preventive method in which the compound is intended to be used. Exemplary bioactive agents include anticancer agents. “Anticancer” means a compound or composition that, in combination with an endocytosis agent, can treat cancer, inhibit the growth or proliferation of cancer cells, or kill cancer cells. Preferably, the endocytosis agent may be administered before, during, or after administration of the bioactive agent. The endocytosis agent can sensitize the subject to the bioactive agent. This makes it possible to improve the therapeutic effectiveness of bioactive agents, reduce the effective amount of bioactive agent required to obtain the desired effect, or shorten the treatment period with bioactive agents.

[0188] This disclosure demonstrates the usefulness of the technology with various probes and therapeutic endocytosis agents, but the technology is not limited to these probes and therapeutic endocytosis agents. In some embodiments, endocytosis agents include, but are not limited to, immunoconjugates, drugs, prodrugs, cytotoxic agents, proapoptotic agents, toxins, nucleases (including DNAse and RNAse), hormones, vitamins, immunomodulators, chelating agents, boron compounds, photoactivators, radionuclides, oligonucleotides, interference agents, DNA, RNA, siRNA, RNAi, anti-angiogenic agents, protein inhibitors, protein activators, molecular adhesives, degrading agents, chemotherapeutic agents, cytokines, chemokines, amino acids, peptides, deuterium-substituted derivatives thereof, or combinations thereof.

[0189] As used herein, the term “effective dose” refers to the amount or dosage of a compound that produces the desired effect, such as a single or multiple administration to a subject. With respect to sensitization, the effective dose refers to the amount of therapeutic agent that causes sensitization of the subject or cells, as described above. As used herein, the term “constant weight” means, in terms of weight management, the weight of a person that maintains or reduces a person’s desired weight. The effective dose can be determined by a diagnostician skilled in the art, using known techniques and observing results obtained under similar circumstances.

[0190] In some embodiments, the endocytosis agents used in the methods disclosed herein may be formulated as a pharmaceutical composition comprising: (a) one or more therapeutically effective amounts of the endocytosis agents described herein, and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. Such compositions may take any pharmaceutically acceptable physical form; exemplary, they may be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain a disclosed effective amount of the endocytosis agent, the effective amount relating to the daily dose of the agent administered. Each dose unit may contain a given daily dose of the endocytosis agent, or each dose unit may contain a portion of the daily dose, such as half or one-third of the dose. The amount of each endocytosis agent contained in each dose unit may depend in part on the identity of the endocytosis agent selected for treatment and other factors such as the indication for which it is administered. The pharmaceutical compositions disclosed herein may be formulated to provide rapid, sustained, or delayed release of the endocytosis agent after administration to a patient by employing well-known procedures. Endocytotic agents for use according to the methods disclosed herein may be administered as a single compound or as a combination of compounds. For example, an endocytotic agent having anticancer activity may be administered as a single compound or in combination with another compound that also promotes anticancer activity or has different pharmacological activity.

[0191] In certain embodiments, the endocytosis agents used in the methods disclosed herein can be supported on extracellular vesicles (EVs) to form endocytosis agent-supported EVs in vitro for any purpose, and extracellular vesicles include, but are not limited to, exosomes, microvesicles, ectosomes, oncosomes, prostagosomes, and apoptotic bodies. Endocytosis agent-supported EVs can be obtained by any in vitro technique in the art for the generation, isolation, and modification of drug-supported EVs, including, but not limited to, the methods described in publications Shuang Du, et al., Journal of Nanobiotechnology, 21, 231, 2023 and Inge Katrin Herrmann, et al., Nature Nanotechnology, 16, 748-759, 2021. In some embodiments, the endocytosis agents used in the methods disclosed herein may be immobilized in exosomes as nanoparticles by microfluidic droplet-based electroporation (μDES), which may result in enhanced stability, biocompatibility, transportability, and / or targeting ability compared to free endocytosis agents.

[0192] In certain embodiments, the endocytosis agent, comprising exocytosis vesicles, used in the methods disclosed herein may be formulated in vitro or in vivo with human serum albumin. Exemplary formulations with human serum albumin are conjugate:albumin molar ratios of 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the endocytosis agent, used in the methods disclosed herein, may be formulated with albumin as nanoparticles, microparticles, albumin-coated liposomes, albumin microbubbles, and albumin nanocapsules. Exemplary nanoparticle sizes range from 1 to 100 nanometers. In further embodiments, the endocytosis agent-albumin conjugate may be used in combination with any of its nanoparticle technologies. Exemplary methods for formulation using human serum albumin include, but are not limited to, those described in the publications Ella N. Hoogenboezem, et al., Advanced Drug Delivery Reviews, 130, 73-89, 2018; Cassandra E. Callmann, et al., Journal of American Chemistry Society, 141(30) 11765-11769, 2019; and Shrawani Lamichhane, et al., Archives of Pharmacal Research, 43, 118-133, 2020. Compared to free endocytic agents, formulation of endocytic agents with albumin in vitro or in vivo can: 1) extend the residence time and / or half-life of a given endocytic agent or exocytosis vesicle in the animal body; 2) increase water solubility; 3) increase the permeability of endocytic agents across cell membranes and membrane barriers (e.g., blood-retinal barrier, lung endothelial barrier, epithelial barrier, skin barrier, cerebral blood barrier); 4) be used for delivery of endocytic agents to specific organ systems such as the liver and brain; 5) be used for delivery of endocytic agents to specific body tissues such as tumors; and / or 6) mitigate the toxicity of endocytic agents in the animal body.

[0193] In certain embodiments, an endocytosis agent comprising exocytosis vesicles, as used in the methods disclosed herein, may be formulated as a pharmaceutical composition. The term "pharmaceutical composition" means the agent of this disclosure, or a pharmaceutically acceptable salt, hydrate, solvate, prodrug, stereoisomer, or tautomer thereof, in a form suitable for oral or parenteral administration, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the endocytosis agent used in the methods disclosed herein may be formulated as a pharmaceutical composition comprising a carrier. For example, the carrier can be selected from the group consisting of proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste. In certain embodiments, the endocytosis agent in the method disclosed herein may be formulated as a pharmaceutical composition comprising one or more binders, diluents, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and foaming agents.

[0194] Suitable diluents include pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, dibasic calcium phosphate, sugars, and mixtures of any of the aforementioned. Suitable disintegrants include lightly crosslinked polyvinylpyrrolidone, corn starch, potato starch, corn starch, and modified starch, croscarmellose sodium, crospovidone, sodium starch glycolate, and mixtures thereof. Examples of foaming agents include effervescent couples such as organic acids and carbonates or bicarbonates. Alternatively, only the sodium bicarbonate component of the effervescent couple can be present.

[0195] In some embodiments, the endocytosis agents used in the methods disclosed herein may be used alone or may be formulated as pharmaceutical compositions for topical administration via any suitable route, such as oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) routes, intraperitoneal injection, microneedle patch, and eye drops. Such formulations may be prepared by any method known in the art of pharmaceuticals, for example, by conjugating the active ingredient with a carrier or excipient. Preferably, the endocytosis agent or composition is administered topically, orally, intraperitoneally, intravenously, by inhalation, or by microinjection.

[0196] In certain embodiments, the endocytosis agents used in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the endocytosis agent with a standard pharmaceutical carrier or diluent according to conventional procedures well known in the art. These procedures include mixing, granulating, and compressing or dissolving components suitable for the desired preparation. In certain embodiments, the endocytosis agent used in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, but any pharmaceutically acceptable dosage form may be used. Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, tablets, or granules, and solid dosage forms may be, for example, fast-dissolving, controlled-release, lyophilized, delayed-release, sustained-release, pulsatile-release, mixed immediate-release and controlled-release dosage forms, or combinations thereof. In certain embodiments, the endocytosis agents used in the methods disclosed herein may be compounds, or solvates of their salts, prodrugs, esters, and / or amides. Examples of solvates include ethanol solvates and hydrates. "Hydrate" means a solvate in which the solvent molecule is water.

[0197] The formulation can be provided in containers containing a single dose or multiple doses. A "salt" refers to the ionic form of a parent compound, or the reaction product of a parent compound with a suitable acid or base, forming an acid salt or base salt of the parent compound. Generally, salts are prepared by reacting a free base or acid parent compound with an inorganic or organic acid or base that forms a desired salt in a stoichiometric amount or an excess amount, in a suitable solvent or a combination of various solvents. Counterions that form part of any salt of an endocytosis agent disclosed herein may not be important to the activity of the compound, provided that the salt is pharmacokinetically acceptable and the counterions do not impart undesirable properties to the salt as a whole. Undesirable properties include undesirable solubility and toxicity.

[0198] "Prodrug" or "prodrug derivative" means a covalent derivative or carrier of a parent compound or active drug raw material that undergoes at least some biotransformation before exhibiting pharmacological effects. More detailed definitions and clinical benefits of prodrugs and their preparations are known in the art, for example, in *A Textbook of Drug Design and Development*, Krogsgaard-Larsen and H. Bundgaard (eds.), Gordon & Breach, 1991, particularly Chapter 5: “Design and Applications of Prodrugs”; *Design of Prodrugs*, H. Bundgaard (...

Claims

1. Interruptible or ininterruptible chemical bonds or linker units 【Chemistry 1】 via chemical arm 【Chemistry 2】 A drug or probe linked to it 【Transformation 3】 A compound containing, A compound that has binding affinity to membrane components that mediate endocytosis.

2. Binding affinity K to membrane components that mediate endocytosis less than 20.0 mM D The compound according to claim 1, having the following characteristics.

3. Equation (I) 【Chemistry 4】 (In the formula, m, n, and p represent integers between 0 and 100.) The compound according to any one of claims 1 to 2, represented by [the specified compound].

4. The compound according to claim 3, wherein m, n, and p are 1.

5. A compound according to any one of claims 1 to 4, comprising a drug that is a decomposing agent, stabilizer, inhibitor, modulator, or activator.

6. The compound according to claim 5, wherein the decomposition agent is PROTAC.

7. A compound according to any one of claims 1 to 4, comprising a drug that is a protein binder.

8. The aforementioned protein binder has a binding affinity K for proteins with a mass of less than 20.0 mM. D The compound according to claim 7, having the following characteristics.

9. The compound according to claim 7 or 8, wherein the protein is an extracellular protein, an intracellular protein, an intrinsic membrane protein, a peripheral membrane protein, a lipid-anchored protein, a globular protein, or a glycoprotein.

10. A compound according to any one of claims 1 to 4, comprising a probe which is a diagnostic agent.

11. The compound according to claim 10, wherein the diagnostic agent includes a detectable label.

12. The compound according to any one of claims 1 to 11, wherein the chemical arm is an atom, a drug, a probe, or part of a drug, binder, or probe.

13. The chemical arm has a binding affinity K to film components that mediate endocytosis less than 20.0 mM. D The compound according to claim 12, having the following characteristics.

14. The compound according to any one of claims 1 to 13, wherein the membrane component mediating endocytosis is a cell membrane lipid, a carbohydrate, or a protein.

15. The compound according to any one of claims 1 to 13, wherein the membrane component mediating the endocytosis is a glycolipid, glycoprotein, phospholipid, ceramide, and cholesterol.

16. The compound according to any one of claims 1 to 13, wherein the membrane component mediating the endocytosis is a glycolipid or a glycoprotein containing 2 to 100 linear or branched monosaccharide units.

17. The compound according to any one of claims 1 to 13, wherein the membrane component mediating the endocytosis is an endogenous membrane protein, a peripheral membrane protein, a lipid-anchored protein, a globular protein, or a glycoprotein.

18. A pharmaceutical composition comprising a compound according to any one of claims 1 to 17, and a pharmaceutically acceptable excipient, carrier, or diluent.

19. A nanostructure comprising a lipid bilayer, a membrane component that mediates endocytosis, and a liquid or cytoplasm encapsulated by a compound according to any one of claims 1 to 17.

20. Binding affinity K to membrane components that mediate endocytosis less than 20.0 mM D The nanostructure according to claim 19, having the above characteristics.

21. A method for preparing the nanostructure according to claim 19 or 20, comprising contacting a cell or vesicle containing an endocytosis-mediated membrane component with the compound according to any one of claims 1 to 17.

22. The method according to claim 21, wherein the compound is brought into contact in vivo with a cell or vesicle containing a membrane component that mediates endocytosis.

23. The method according to claim 21, wherein the compound is brought into contact in vitro or ex vivo with a cell or vesicle containing the membrane component that mediates the endocytosis.

24. A method for internalizing a compound into a cell, comprising contacting a cell containing a membrane component that mediates endocytosis with a compound according to any one of claims 1 to 17.

25. A method for internalizing a compound into a cell, comprising contacting a cell containing a membrane component that mediates endocytosis with a nanostructure according to any one of claims 19 to 20.

26. A method for isolating a compound from a nanostructure according to any one of claims 19 to 20, comprising dissolving the nanostructure and separating the compound from the dissolved substance.

27. A method for determining the qualitative or quantitative presence of a compound or nanostructure described in any one of claims 1 to 20 in cells, bodies, solutions, or media, comprising: centrifuging a sample containing cells, bodies, solutions, or media; and detecting the compound or nanostructure.

28. A method for isolating a compound or nanostructure according to any one of claims 1 to 20 from cells, bodies, solutions, or media, comprising centrifugation of a sample comprising cells, bodies, solutions, or media.

29. The method according to any one of claims 27 to 28, wherein the medium is a cell culture medium, a tissue culture medium, an organ culture medium, a body fluid, a tissue, or an organ.

30. A method for treating a subject, comprising administering a compound or nanostructure described in any one of claims 1 to 20 to a subject in need thereof.

31. The method according to claim 30, wherein the effective amount of the compound or nanostructure for treating the target is less than the effective amount of a chemical arm-less agent or probe for treating the target.

32. The method according to claim 30, wherein the effective amount of the compound or nanostructure for treating the target is equal to the effective amount of the chemical arm-less agent or probe for treating the target.

33. The method according to claim 30, wherein the effective amount of the compound or nanostructure for treating the target is greater than the effective amount of the chemical arm-less agent or probe for treating the target.

34. A method for identifying an endocytosis agent, comprising contacting a compound with first cells and second cells, wherein the presence of endocytosis-mediated membrane components in the second cells is modulated compared to that in the first cells, and determining that the compound is an endocytosis agent by comparing the activity of the compound in contact with the first cells with the activity of the compound in contact with the second cells.

35. The method according to claim 34, wherein the presence of the membrane component mediating endocytosis in the second cell is less than in the first cell.

36. The method according to claim 35, wherein the presence of the membrane component mediating endocytosis in the second cell is reduced by gene editing, knockdown, or silencing.

37. The method according to claim 34, wherein the presence of the membrane component mediating endocytosis in the second cell is greater than in the first cell.

38. The method according to claim 37, wherein the presence of the endocytosis-mediated membrane component in the second cell is increased by gene editing or introduction of DNA or RNA encoding the endocytosis-mediated membrane component.

39. The method according to any one of claims 34 to 38, wherein the compound is brought into contact in vivo with the first cell and the second cell.

40. The method according to any one of claims 34 to 38, wherein the compound is brought into contact with the first cell and the second cell in vitro or ex vivo.

41. The aforementioned compound has a binding affinity K to endocytosis-mediated membrane components of less than 20.0 mM. D The method according to any one of claims 34 to 40, further comprising determining that it has

42. The method according to any one of claims 34 to 41, further comprising administering a compound or nanostructure to a subject requiring it.

43. A method for identifying membrane components that mediate endocytosis, comprising contacting a cell with a compound or nanostructure described in any one of claims 1 to 29, wherein the compound includes a detectable label, and the membrane components that mediate endocytosis within the cell can be identified by determining the interaction between the membrane components that mediate endocytosis and the detectable label.

44. A method for identifying endocytosis-mediated membrane components, comprising comparing the sensitivity of a first cell to a second cell to treatment with a compound or nanostructure described in any one of claims 1 to 20, and comparing the genomic expression or protein abundance of the membrane component in the first cell and the second cell, wherein the endocytosis-mediated membrane component is identified by an increase in the sensitivity of the first cell or the second cell to the compound or nanostructure.

45. The method according to claim 44, wherein a first cell having a greater abundance of membrane components is more sensitive than a second cell having a less abundance of membrane components or no membrane components at all.

46. The method according to claim 44, wherein a first cell with higher expression of the membrane component is more sensitive than a second cell with lower or no expression of the membrane component.

47. The endocytosis-mediated membrane component has a binding affinity K to the compound or nanostructure according to any one of claims 1 to 25, which is less than 20.0 mM. D The method according to any one of claims 44 to 46, further comprising determining that it has

48. The method according to 44-47, wherein the sensitivity of the first and second cells is determined by evaluating the changes in biological processes when the first and second cells are brought into contact with the compound or nanostructure.

49. A method for selecting a target for treatment with a compound or nanostructure according to any one of claims 1 to 20, which has binding affinity to an endocytosis-mediating membrane component, comprising: determining the qualitative or quantitative presence of the endocytosis-mediating membrane component in a sample obtained from a potential target; and, if the endocytosis-mediating membrane component is present in the sample, administering the compound or nanostructure to the potential target.

50. The method according to claim 49, wherein the administration route or effective amount of the compound or nanostructure is determined from the qualitative or quantitative presence of endocytosis-mediated membrane components in the sample.

51. The method according to any one of claims 49 to 50, wherein the compound or nanostructure has a binding affinity for the endocytosis-mediating membrane component of less than 20 mM.

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