Tri-branched N-acetylgalactosamine modified hydroxyl polyamidoamine dendrimers and methods of use thereof

Abstract

It has been established that dendrimers conjugated to or complexed with the carbohydrate triantennary N-acetylgalactosamine (triantennary β-GalNAc) selectively accumulate in hepatocytes and selectively deliver therapeutic, prophylactic, or diagnostic agents to the liver. Compositions of dendrimers complexed with triantennary β-GalNAc and one or more agents, and methods of use thereof, have been developed for preventing, treating, or diagnosing liver injury, liver disease, or liver damage in subjects in need thereof. The compositions are particularly suitable for treating and / or ameliorating one or more symptoms of nonalcoholic fatty liver disease (NAFLD) and liver cancer with reduced toxicity.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 943,705, filed December 4, 2019, U.S. Provisional Application No. 63 / 067,155, filed August 18, 2020, U.S. Provisional Application No. 63 / 068,109, filed October 1, 2020, and U.S. Provisional Application No. 63 / 108,186, filed October 30, 2020, which are incorporated by reference in their entireties.

[0002] FIELD OF THE INVENTION The present invention is generally in the field of drug delivery, and in particular, methods for delivering drugs conjugated to dendrimers that selectively target sites or regions of the liver. [Background technology]

[0003] Background of the Invention Liver diseases, such as liver infections, cirrhosis, drug-induced liver failure, and hepatocellular carcinoma, continue to pose significant health challenges worldwide. The prevalence of liver disease is increasing worldwide, with an estimated 844 million people worldwide suffering from chronic liver disease, and approximately 2 million people die from liver damage each year.

[0004] Nonalcoholic fatty liver disease (NAFLD), also known as nonalcoholic steatohepatitis (NASH), is currently the most common liver disorder and is expected to become the leading indication for liver transplantation by 2030. NAFLD / NASH can lead to cirrhosis (scarring) or liver cancer and is associated with significant morbidity and mortality. The estimated global prevalence of NAFLD ranges from 6.3% to 33% in the general population, with a median prevalence of 20%. The median prevalence of NAFLD is generally higher in developed countries.

[0005] Despite these staggering numbers, current treatment options for many liver diseases are limited and lack the efficacy needed to treat advanced and severe cases. Although considerable progress has been made in understanding the epidemiology, natural history, and pathogenesis of NAFLD / NASH, effective treatments remain lacking, and evidence-based clinical guideline options for patient management are limited. Pharmacological treatment of patients with NAFLD is still evolving, with no single agent proven to be clearly effective in specifically altering the course of the disease.

[0006] Hepatocytes are the most abundant type of liver cell, constituting >80% of the liver biomass, and are primarily involved in most liver disorders, such as hepatocellular carcinoma, drug-induced liver failure, hepatitis, and non-alcoholic steatohepatitis. Effectively delivering drugs to diseased liver cells is a challenge. When injected, most drugs accumulate in the liver but tend to be eliminated through macrophages and Kupffer cells rather than hepatocytes, making it difficult to selectively and effectively target hepatocytes. Summary of the Invention [Means for solving the problem]

[0007] It is therefore an object of the present invention to provide compositions and methods for selectively reducing or preventing one or more symptoms of liver disease and / or liver damage.

[0008] It is also an object of the present invention to provide compositions, and methods of making and using them, that reduce or prevent pathological processes associated with the development and progression of liver disease and / or liver damage.

[0009] Yet another object of the present invention is to provide compositions and methods for selectively targeting active agents to hepatocytes at sites in need thereof.

[0010] Summary of the Invention Dendrimers complexed or conjugated with triantennary N-acetylgalactosamine (GalNAc) have been established to selectively deliver active agents to hepatocytes in vivo. In some embodiments, the dendrimer is covalently conjugated to the triantennary N-acetylgalactosamine (GalNAc) via an ester, ether, or amide bond, optionally with one or more linkers.

[0011] Provided are compositions and methods for treating or preventing one or more symptoms of liver disease and / or liver damage in a subject in need thereof.Typically, the method for treating liver disease in a subject comprises administering to the subject a formulation comprising a tri-antennary GalNAc-modified dendrimer that is complexed with, covalently conjugated to, or intermolecularly dispersed or encapsulated in one or more therapeutic or preventive agents.The formulation is typically administered in an amount effective to treat, alleviate, or prevent one or more symptoms of liver disease and / or liver damage in the recipient.Exemplary liver diseases and / or liver damage that can be treated include non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), drug-induced liver failure, hepatitis, liver fibrosis, cirrhosis, hepatocellular carcinoma, or a combination thereof.In some embodiments, the dendrimer is a hydroxyl-terminated dendrimer. In some embodiments, the dendrimer is a fourth, fifth, sixth, seventh, or eighth generation poly(amidoamine) (PAMAM) dendrimer.

[0012] This method is used to selectively deliver one or more active agents, such as therapeutic agents, preventive agents, and / or diagnostic agents, to recipient liver cells.Exemplary therapeutic agents that can be delivered include angiotensin II receptor blockers, farnesoid X receptor agonists, death receptor 5 agonists, sodium-glucose cotransporter type 2 (SGLT2) inhibitors, lysophosphatidic acid (LPA)1 receptor antagonists, endothelin-A receptor antagonists, PPARδ agonists, AT1 receptor antagonists, CCR5 / CCR2 antagonists, antifibrotic agents, anti-inflammatory agents, and / or antioxidants.In some embodiments, the angiotensin II receptor blockers are telmisartan, or telmisartan amide derivatives, or telmisartan ester derivatives.In some embodiments, the FXR agonists are chenodeoxycholic acid, or chenodeoxycholic acid amide derivatives, or chenodeoxycholic acid ester derivatives. Exemplary SGLT2 inhibitors that can be delivered include phlorizin, T-1095, canagliflozin, dapagliflozin, ipragliflozin, tofogliflozin, empagliflozin, luseogliflozin, ertugliflozin, remogliflozin etabonate, or derivatives thereof. In some embodiments, the PPARδ agonist is GW0742, GW0742-amide derivatives, and GW0742-ester derivatives. In some embodiments, the antioxidant is vitamin E or a derivative thereof.

[0013] Typically, the method delivers the active agent to the subject in an amount effective to achieve the desired physiological response in the subject.For example, in some embodiments, the method delivers the active agent to the subject in an amount effective to reduce the serum level of one or more of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), gamma-glutamyltransferase (GGT), total cholesterol (TC), low-density lipoprotein (LDP), fasting blood glucose, or a combination thereof in the subject.In some embodiments, the method delivers the active agent to the subject in an amount effective to reduce one or more of steatosis, inflammation, ballooning, fibrosis, cirrhosis, or a combination thereof in the subject. In some embodiments, the method delivers an active agent to a subject in an amount effective to reduce lobular inflammation in the liver; reduce the amount or presence of one or more pro-inflammatory cells, chemokines, and / or cytokines in the liver; or reduce one or more pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1α.

[0014] In some embodiments, the therapeutic, prophylactic, and / or diagnostic agent delivered to hepatocytes comprises a STING agonist, a CSF1R inhibitor, a PARP inhibitor, a VEGFR tyrosine kinase inhibitor, an EGFR tyrosine kinase inhibitor, a MEK inhibitor, a glutaminase inhibitor, a TIE II antagonist, a CXCR2 inhibitor, a CD73 inhibitor, an arginase inhibitor, a PI3K inhibitor, a TLR4 agonist, a TLR7 agonist, an SHP2 inhibitor, a chemotherapeutic agent, and a cytotoxic agent. In some embodiments, the STING agonist is the cyclic dinucleotide GMP-AMP or DMXAA. In some embodiments, the CSF1R inhibitor is selected from the group consisting of PLX3397, PLX108-01, ARRY-382, PLX7486, BLZ945, JNJ-40346527, and GW2580. In some embodiments, the PARP inhibitor is selected from the group consisting of olaparib, veliparib, niraparib, and rucaparib. In some embodiments, the VEGFR tyrosine kinase inhibitor is selected from the group consisting of sunitinib or its derivative or analog, sorafenib, pazopanib, vandetanib, axitinib, cediranib, vatalanib, dasatinib, nintedanib, and motesanib. In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD325901, PD035901, PD032901, and TAK-733. In some embodiments, the glutaminase inhibitor is selected from the group consisting of bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-L-norleucine (DON), azaserine, acivicin, and CB-839. In some embodiments, the CXCR2 inhibitor is navarixin, SB225002, or SB332235. In some embodiments, the CD73 inhibitor is APCP, quercetin, or tenofovir, or a derivative or analog thereof. In some embodiments, the arginase inhibitor is a derivative or analog of 2-(S)-amino-6-bromohexanoic acid.In some embodiments, the PI3K inhibitor is selected from the group consisting of alpelisib, ceravelisib, pilaralisib, WX-037, dactolisib, prexasertib, voxtalisib, PX-866, ZSTK474, buparlisib, pictilisib, and copanlisib. In some embodiments, the immunomodulatory agent is an SHP2 inhibitor. In some embodiments, the cytotoxic agent is auristatin E or mertansine. In some embodiments, the chemotherapeutic agent is amsacrine, bleomycin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxin, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, innotecan, leucovorin, daunorubicin, lomustine The active agent is selected from the group consisting of methadone, mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, taxol, trichostatin A and its derivatives, trastuzumab, cetuximab, rituximab, and bevacizumab. In a preferred embodiment, the method delivers an active agent to a subject in an amount effective to reduce tumor size in the subject and / or effective to enhance a tumor-specific cytotoxic T cell response.

[0015] In some embodiments, the therapeutic, prophylactic, and / or diagnostic agent is covalently conjugated to the dendrimer via a linkage selected from the group consisting of an ether, an ester, and an amide linkage, optionally via a linker or spacer moiety. In preferred embodiments, the therapeutic, prophylactic, and / or diagnostic agent is covalently conjugated to the dendrimer via an amide or ether linkage, optionally via a linker or spacer moiety.

[0016] In some embodiments, the preparation is formulated for intravenous, subcutaneous, or intramuscular administration to a subject, or for intestinal administration.In some embodiments, the preparation is administered before, together with, after, or alternating with one or more additional therapies or procedures.Exemplary additional procedures include administering one or more therapeutic, prophylactic, and / or diagnostic agents to prevent or treat one or more symptoms of liver injury-related diseases or conditions, such as infection, sepsis, diabetic complications, hypertension, obesity, high blood pressure, heart failure, kidney disease, and cancer.

[0017] Pharmaceutical formulations of tri-antennary GalNAc-modified dendrimers complexed with, covalently conjugated to, intermolecularly dispersed within, or encapsulated in, one or more therapeutic or prophylactic agents are also described. Kits containing tri-antennary GalNAc-modified dendrimers complexed with, covalently conjugated to, intermolecularly dispersed within, or encapsulated in, one or more therapeutic or prophylactic agents are also provided.

[0018] Also described are methods for making tri-antennary GalNAc-modified dendrimers and methods for making tri-antennary GalNAc-modified dendrimers that are complexed with, covalently conjugated to, inter-molecularly dispersed within, or encapsulated within, one or more therapeutic or prophylactic agents. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows a scheme for the synthesis of β-GalNAc-triantennary βEG3-azide (AB3 building block). The reagents and conditions are as follows: (i) scandium trifluoromethanesulfonate, DCE, 3 h, 80 °C; (ii) propargyl bromide, toluene, sodium hydroxide, water, TBAB; (iii) pyridine, thionyl chloride, chloroform, 65 °C, 2 h; (iv) tetrabutylammonium hydrogen sulfate, 50% NaOH, 16 h, room temperature; (v) CuSO4·5H2O, sodium ascorbate, THF, water, 10 h; (vi) DMF, tetrabutylammonium iodide, NaN3, 80 °C, 5 h; and (vii) sodium methoxide, anhydrous methanol, 30 °C, 3 h.

[0020] [Figure 2] Figure 2 shows the synthesis scheme for the dendrimer triantennary β-GalNAc-CY5. The reagents and conditions are as follows: (i) EDC, DMAP, DMF, room temperature, 24 hours; (ii) CuSO4·5H2O, sodium ascorbate, DMF, water, 8 hours; (iii) CuSO4·5H2O, sodium ascorbate, DMF, water, 8 hours.

[0021] [Figure 3] Figure 3 shows a scheme for the synthesis of telmisartan ester conjugates with dendrimer-triantennary β-GlcNAc. The reagents and conditions are as follows: (i) DCC, DMAP, DCM, room temperature, 4 hours; (ii) EDC, DMAP, DMF, room temperature, 24 hours; (iii) CuSO₄·5H₂O, sodium ascorbate, DMF, water, 8 hours; (iv) CuSO₄·5H₂O, sodium ascorbate, DMF, water, 8 hours.

[0022] [Figure 4]Figure 4 shows a scheme for the synthesis of telmisartanamide conjugates with dendrimer-triantennary β-GlcNAc. The reagents and conditions are as follows: (i) HATU, DIPEA, DCM, room temperature, 4 hours; (ii) EDC, DMAP, DMF, room temperature, 24 hours; (iii) CuSO₄·5H₂O, sodium ascorbate, DMF, water, 8 hours; (iv) CuSO₄·5H₂O, sodium ascorbate, DMF, water, 8 hours. [Figure 5] FIG. 5 is a bar graph showing the in vitro drug release from dendrimer-telmisartan ester conjugates over 18 days in plasma (pH 7.4, PBS) and intracellular conditions (pH 5.5, esterase).

[0023] [Figure 6] FIG. 6 is a bar graph showing in vitro drug release from dendrimer-telmisartanamide conjugates in plasma (pH 7.4, PBS) and intracellular conditions (pH 5.5, esterase) over 18 days.

[0024] [Figure 7] FIG. 7 is a bar graph showing in vitro drug release from dendrimer-telmisartanamide conjugates in human, mouse, and rat plasma at 37° C. for 48 hours.

[0025] [Figure 8] Figure 8 shows a scheme for the synthesis of a dendrimer-triantennary β-GlcNAc-azide-obeticholic acid conjugate. The reagents and conditions are as follows: (i) EDC, DMAP, DCM, room temperature, 4 hours; (ii) CuSO4-5H2O, sodium ascorbate, DMF, water, 8 hours; (iii) CuSO4-5H2O, sodium ascorbate, DMF, water, 8 hours.

[0026] [Figure 9-1]Figures 9A-9C are plots showing body weight in grams (Figure 9A), liver weight in grams (Figure 9B), and liver weight to body weight ratio (Figure 9C) of normal and nonalcoholic steatohepatitis (NASH) mice treated with vehicle, free telmisartan, obeticholic acid (OCA), high-dose dendrimer-triantennary β-GlcNAc-azide-telmisartan amide conjugate (D-Tel High), low-dose D-Tel (D-Tel Low), high-dose dendrimer-triantennary β-GlcNAc-azide-telmisartan ester conjugate (D-TelB High), high-dose dendrimer-triantennary β-GlcNAc-azide-obeticholic acid ester conjugate (D-OCA High), and low-dose D-OCA (D-OCA Low) at sacrifice at 9 weeks of age. Compared to vehicle, *p<0.05; ***p<0.001; ****p<0.0001. [Figure 9-2] Figures 9A-9C are plots showing body weight in grams (Figure 9A), liver weight in grams (Figure 9B), and liver weight to body weight ratio (Figure 9C) of normal and nonalcoholic steatohepatitis (NASH) mice treated with vehicle, free telmisartan, obeticholic acid (OCA), high-dose dendrimer-triantennary β-GlcNAc-azide-telmisartan amide conjugate (D-Tel High), low-dose D-Tel (D-Tel Low), high-dose dendrimer-triantennary β-GlcNAc-azide-telmisartan ester conjugate (D-TelB High), high-dose dendrimer-triantennary β-GlcNAc-azide-obeticholic acid ester conjugate (D-OCA High), and low-dose D-OCA (D-OCA Low) at sacrifice at 9 weeks of age. Compared to vehicle, *p<0.05; ***p<0.001; ****p<0.0001.

[0027] [Figure 10-1]Figures 10A and 10B are plots showing serum aminotransferase (ALT) levels (Figure 10A) and liver triglyceride levels (Figure 10B) of normal and NASH mice treated with vehicle, free telmisartan, OCA, D-Tel high, D-Tel low, D-TelB high, D-OCA high, and D-OCA low at 9 weeks of age. *p<0.05; **p<0.01; ****p<0.0001 compared to vehicle. [Figure 10-2] Figures 10A and 10B are plots showing serum aminotransferase (ALT) levels (Figure 10A) and liver triglyceride levels (Figure 10B) of normal and NASH mice treated with vehicle, free telmisartan, OCA, D-Tel high, D-Tel low, D-TelB high, D-OCA high, and D-OCA low at 9 weeks of age. *p<0.05; **p<0.01; ****p<0.0001 compared to vehicle.

[0028] [Figure 11-1] Figures 11A-11D are plots showing the nonalcoholic fatty liver disease (NAFLD) activity score (Figure 11A), steatosis score (Figure 11B), inflammation score (Figure 11C), and ballooning score (Figure 11D) in the livers of normal and NASH mice treated with vehicle, free telmisartan, OCA, D-Tel high, D-Tel low, D-TelB high, D-OCA high, and D-OCA low at 9 weeks of age. *p<0.05; **p<0.01; ****p<0.0001 compared to vehicle. [Figure 11-2]Figures 11A-11D are plots showing the nonalcoholic fatty liver disease (NAFLD) activity score (Figure 11A), steatosis score (Figure 11B), inflammation score (Figure 11C), and ballooning score (Figure 11D) in the livers of normal and NASH mice treated with vehicle, free telmisartan, OCA, D-Tel high, D-Tel low, D-TelB high, D-OCA high, and D-OCA low at 9 weeks of age. *p<0.05; **p<0.01; ****p<0.0001 compared to vehicle. [Figure 11-3] Figures 11A-11D are plots showing the nonalcoholic fatty liver disease (NAFLD) activity score (Figure 11A), steatosis score (Figure 11B), inflammation score (Figure 11C), and ballooning score (Figure 11D) in the livers of normal and NASH mice treated with vehicle, free telmisartan, OCA, D-Tel high, D-Tel low, D-TelB high, D-OCA high, and D-OCA low at 9 weeks of age. *p<0.05; **p<0.01; ****p<0.0001 compared to vehicle.

[0029] [Figure 12] Figure 12 is a plot showing Sirius Red-positive areas in the livers of normal and NASH mice treated with vehicle, free telmisartan, OCA, D-Tel high, D-Tel low, D-TelB high, D-OCA high, and D-OCA low at 9 weeks of age. *p<0.05; **p<0.01; ****p<0.0001 compared to vehicle.

[0030] [Figure 13-1] FIG. 13A is a synthetic scheme of dendrimers conjugated to two different classes of active agents, R1 and R2; FIG. 13B shows exemplary R1 groups including capecitabine and gemcitabine and their analogs, and FIG. 13C shows exemplary R2 groups, such as TIE II inhibitors and their analogs. [Figure 13-2]FIG. 13A is a synthetic scheme of dendrimers conjugated to two different classes of active agents, R1 and R2; FIG. 13B shows exemplary R1 groups including capecitabine and gemcitabine and their analogs, and FIG. 13C shows exemplary R2 groups, such as TIE II inhibitors and their analogs. [Figure 13-3] FIG. 13A is a synthetic scheme of dendrimers conjugated to two different classes of active agents, R1 and R2; FIG. 13B shows exemplary R1 groups including capecitabine and gemcitabine and their analogs, and FIG. 13C shows exemplary R2 groups, such as TIE II inhibitors and their analogs.

[0031] [Figure 14-1] 14A and 14B are synthetic schemes for dendrimers conjugated to two exemplary TLR4 agonists. [Figure 14-2] 14A and 14B are synthetic schemes for dendrimers conjugated to two exemplary TLR4 agonists.

[0032] [Figure 15] FIG. 15 is a synthetic scheme for dendrimers conjugated to exemplary CSF1R inhibitors.

[0033] [Figure 16] FIG. 16 is a synthetic scheme for dendrimer-N-acetyl-L-cysteine methyl ester conjugates.

[0034] [Figure 17-1] 17A and 17B are schemes showing the chemical reaction steps for the synthesis of a dendrimer-GW2580 ether conjugate (FIG. 17A) and a dendrimer-GW2580 ester conjugate (FIG. 17B), respectively. [Figure 17-2]17A and 17B are schemes showing the chemical reaction steps for the synthesis of a dendrimer-GW2580 ether conjugate (FIG. 17A) and a dendrimer-GW2580 ester conjugate (FIG. 17B), respectively. DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description of the Invention I. Definition The terms "active agent" or "bioactive agent" refer to a therapeutic, prophylactic, or diagnostic agent and are used interchangeably to refer to a chemical or biological compound that induces a desired pharmacological and / or physiological effect, which may be a prophylactic, therapeutic, or diagnostic agent. These may be nucleic acids, nucleic acid analogs, small molecules having a molecular weight of less than 2 kDa, more typically less than 1 kDa, peptidomimetics, proteins, or peptides, carbohydrates, or sugars, lipids, or surfactants, or combinations thereof. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the active agent, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, and analogs.

[0036] The term "therapeutic agent" refers to an active agent that can be administered to treat one or more symptoms of a disease or disorder.

[0037] The term "diagnostic agent" refers to an active agent that can be administered to localize, pinpoint, and define a pathological process. Diagnostic agents are capable of labeling target cells, thereby allowing for subsequent detection or imaging of these labeled target cells. The term "prophylactic agent" refers to an active agent that can be administered to prevent disease or to prevent a particular condition or symptom.

[0038] The term "prodrug" refers to a pharmacological substance (drug) that is administered to a subject in an inactive form (or a significantly less active form). Upon administration, the prodrug is metabolized in vivo by an enzyme or chemical reaction, or a combination of both, to a compound with the desired pharmacological activity. Prodrugs can be prepared by replacing appropriate functional groups present in the above compounds with "promoieties," for example, as described in H. Bundgaar, Design of Prodrugs (1985). For a further discussion of prodrugs, see, for example, Rautio, J. et al. Nature Reviews Drug Discovery. 7:255-270 (2008).

[0039] The terms "immune," "immunological," or "immune" response refer to the development in a recipient patient of a beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretory products) response to an immunogen. Such a response can be an active response induced by administration of the immunogen, or a passive response induced by administration of antibodies or primed T cells. Cellular immune responses are elicited by the presentation of polypeptide epitopes in association with class I or class II MHC molecules and result in antigen-specific CD4 + Helper T cells and / or CD8 + Activates cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglial cells, eosinophils, or other components of innate immunity. The presence of a cellular immune response can be determined by proliferation assays (CD4 + The relative contribution of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be discerned by isolating antibodies and T cells separately from immunized syngeneic animals and measuring the protective or therapeutic effect in a second subject.

[0040] The term "immunomodulatory agent" or "immunotherapeutic agent" refers to an active agent that can be administered to regulate, enhance, reduce, prolong, diminish, or otherwise alter one or more factors of the innate or adaptive immune response in a recipient. Generally, immunomodulatory agents can modulate the immune microenvironment for a desired immune response by targeting one or more immune cells or cell types at the target site, and thus are not necessarily specific for any cancer type. For example, blocking one molecule on immune cells, programmed cell death protein 1 (PD-1), has resulted in antitumor activity. In some embodiments, immunomodulatory agents are specifically delivered to inhibit or reduce suppressive immune cells, such as tumor-associated macrophages, for an enhanced antitumor response at the tumor site.

[0041] The term "immunosuppressive cells" refers to immune cells that promote tumor growth, angiogenesis, invasion, metastasis, resistance to therapy, or a combination thereof. Exemplary immunosuppressive cells include cancer-associated fibroblasts, myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), mesenchymal stromal cells (MSCs) and TIE2-expressing monocytes, and tumor-associated macrophages (TAMs).

[0042] The term "pro-inflammatory cell" refers to an immune cell that promotes pro-inflammatory activity, secretion of pro-inflammatory cytokines such as IL-12, IFN-γ, and TNF-α, or a combination thereof. Exemplary pro-inflammatory cells include pro-inflammatory M1 macrophages, or classically activated macrophages (CAMs).

[0043] The terms "pharmaceutically acceptable" or "biocompatible" refer to compositions, polymers, and other materials and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material, involved in the transfer or transport of any subject composition from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other components of the subject composition and not harmful to the patient. The term "pharmaceutically acceptable salt" is art-recognized and includes relatively non-toxic inorganic and organic acid addition salts of a compound. Examples of pharmaceutically acceptable salts include salts derived from inorganic acids, such as hydrochloric acid and sulfuric acid, and organic acids, such as ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of suitable inorganic bases for salt formation include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc. Salts can also be formed with suitable organic bases, including bases that are non-toxic and strong enough to form such salts. For illustrative purposes, classes of such organic bases may include mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines, such as mono-, di-, and triethanolamine; amino acids, such as arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; and N-benzylphenethylamine.

[0044] The term "therapeutically effective amount" refers to an amount of a therapeutic agent that, when incorporated into and / or onto a dendrimer, produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. The effective amount may vary depending on factors such as the disease or condition being treated, the particular targeted construct being administered, the subject's size, or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation. In some embodiments, the term "effective amount" refers to an amount of a prophylactic or therapeutic agent that reduces or attenuates the risk of developing a liver disease / disorder or reduces or attenuates one or more symptoms of a liver disease / disorder, e.g., reduces inflammation in the liver. Additional desired results also include reducing and / or inhibiting serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), and total cholesterol (TC), fat accumulation or steatosis, inflammation, ballooning, fibrosis, long-term morbidity, and mortality. In the case of cancer or tumor, an effective amount of drug can have the effect of reducing the number of cancer cells; reducing tumor size; inhibiting the infiltration of cancer cells into peripheral organs; inhibiting tumor metastasis; inhibiting tumor growth; and / or alleviating one or more of the symptoms associated with the disorder.An effective amount can be administered in one or more doses.

[0045] The terms "inhibit" or "reduce" mean to decrease or diminish activity and amount. This can be complete or partial inhibition or reduction of activity or amount. Inhibition or reduction can be compared to a control or standard level. Inhibition can be 5, 10, 25, 50, 75, 80, 85, 90, 95, 99, or 100%, or any integer therebetween. For example, a dendrimer composition comprising one or more agents can inhibit or reduce serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), and total cholesterol (TC), fat accumulation or steatosis, inflammation, ballooning, fibrosis, long-term morbidity, and mortality in diseased livers by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% from a subject not administered the dendrimer composition or untreated or prior to treatment. In some embodiments, inhibition and reduction are compared at the mRNA, protein, cell, tissue, and organ level, for example, inhibition and reduction of tumor growth or tumor size / volume.

[0046] The term "treat" or "prevent" refers to improving, reducing, or otherwise stopping the occurrence or progression of a disease, disorder, or condition in an animal that may have a predisposition to the disease, disorder, and / or condition but has not yet been diagnosed with it; inhibiting a disease, disorder, or condition, e.g., preventing its progression; and alleviating a disease, disorder, or condition, e.g., causing regression of the disease, disorder, and / or condition. Treating a disease or condition includes improving at least one symptom of a particular disease or condition, even if the underlying pathophysiology is not affected, for example, treating pain in a subject by administering an analgesic, even if such an agent does not treat the cause of the pain. Desirable effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a liver disease / disorder are alleviated or eliminated, including, but not limited to, reducing and / or inhibiting elevations of transaminases, including alanine transaminase (ALT) and aspartate transaminase (AST), reducing the proliferation of cancerous cells in the case of liver cancer, increasing the quality of life of a subject suffering from the disease, reducing the dose of other medications required to treat the disease, slowing the progression of the disease, and / or prolonging the survival of the individual.

[0047] The phrase "enhancing T cell function" refers to inducing, causing, or stimulating T cells to have sustained or amplified biological function, or regenerating or reactivating exhausted or inactivated T cells. Examples of enhancing T cell function include: increased secretion of granzyme B and / or IFN-γ from CD8+ T cells, increased proliferation, and increased antigen responsiveness (e.g., elimination of viruses, pathogens, or tumors) compared to such levels before the intervention. In one embodiment, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. Formats for measuring this enhancement are known to those skilled in the art.

[0048] The term "tumor immunity" refers to the process by which tumors evade immune recognition and elimination. Thus, from a therapeutic perspective, tumor immunity is "treated" when such evasion is weakened and tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor elimination. The term "immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors can be immunogenic, but enhancing the immunogenicity of tumors helps the immune response eliminate tumor cells.

[0049] The term "biodegradable" refers to a material that breaks down or decays under physiological conditions into smaller units or chemical species that can be metabolized, eliminated, or excreted by the body. The degradation time of a material is a function of the material's composition and morphology.

[0050] The term "dendrimer" includes, but is not limited to, a molecular structure having an inner core, an inner layer (or "generation") of repeating units regularly attached to the inner core, and an outer surface of terminal groups attached to the outermost generation.

[0051] The term "functionalize" means to modify a compound or molecule in a manner that results in the attachment of a functional group or moiety. For example, a molecule may be functionalized by the introduction of a molecule that makes it a strong nucleophile or a strong electrophile.

[0052] The term "targeting moiety" refers to a moiety that localizes to or away from a specific location. The moiety can be, for example, a protein, a nucleic acid, a nucleic acid analog, a carbohydrate, or a small molecule. The entity can be, for example, a therapeutic compound, for example, a small molecule, or a diagnostic entity, for example, a detectable label. The location can be a tissue, a specific cell type or cell activation state, or a subcellular compartment. In some embodiments, the targeting moiety directs the localization of the active agent.

[0053] The term "extended residence time" refers to an increase in the time required for an agent to be cleared from a patient's body, or an organ or tissue of the patient, compared to a standard for comparison, e.g., a comparable agent not conjugated to a delivery vehicle, e.g., a dendrimer. In certain embodiments, "extended residence time" refers to an agent that is cleared with a half-life that is 10%, 20%, 50%, or 75% longer than a standard for comparison, e.g., a comparable agent not conjugated to a delivery vehicle, e.g., a dendrimer. In certain embodiments, "extended residence time" refers to an agent that is cleared with a half-life that is 2, 5, 10, 20, 50, 100, 200, or 10,000 times longer than a standard for comparison, e.g., a comparable agent that does not have a dendrimer that specifically targets a particular cell type associated with sites of inflammation and / or tumor regions.

[0054] The terms "incorporated" and "encapsulated" refer to incorporating, formulating, or otherwise including an active ingredient in and / or on a composition. For example, an active agent or other material may be incorporated (by covalent, ionic, or other bonding interactions), physically mixed with, or encapsulated within the dendrimer structure, including one or more surface functional groups of such a dendrimer.

[0055] The term "neutral surface charge" of a particle refers to an electrokinetic potential (zeta potential) of the particle being 0 mV. In some embodiments, the term "near-neutral surface charge" refers to a zeta potential of approximately 0 mV, e.g., -10 mV to 10 mV, -5 mV to 5 mV, preferably -1 mV to 1 mV. II. Composition

[0056] It has been established that dendrimers conjugated to or complexed with the carbohydrate triantennary N-acetylgalactosamine (GalNAc) selectively accumulate in hepatocytes and prevent and / or treat liver disease.

[0057] Compositions of carbohydrate-complexed dendrimers have been developed that are suitable for delivering one or more active agents, particularly one or more active agents for preventing, treating, or diagnosing liver injury, liver disease, or liver damage in a subject in need thereof. The compositions are particularly suitable for treating and / or ameliorating one or more symptoms of nonalcoholic fatty liver disease (NAFLD) and / or hepatocellular carcinoma (HCC).

[0058] Compositions of dendrimer-carbohydrate conjugates containing one or more prophylactic, therapeutic, and / or diagnostic agents encapsulated in, associated with, and / or conjugated to the dendrimer are provided. Typically, the one or more active agents are encapsulated in, associated with, and / or conjugated to the dendrimer conjugate at concentrations of about 0.01% w / w to about 30% w / w, about 1% w / w to about 25% w / w, about 5% w / w to about 20% w / w, and about 10% w / w to about 15% w / w. Preferably, the active agent is covalently conjugated to the dendrimer via one or more linkages, such as disulfides, esters, ethers, thioesters, carbamates, carbonates, hydrazines, and amides, optionally via one or more spacers. In some embodiments, the spacer is an active agent, such as telmisartan. Exemplary active agents include angiotensin II receptor blockers, FXR agonists, PPARδ agonists, antioxidants, anti-inflammatory agents, chemotherapeutic agents, anti-fibrotic agents, and anti-infective agents.

[0059] The presence of additional agents can affect the zeta potential or surface charge of the dendrimer. In one embodiment, the zeta potential of the dendrimer-carbohydrate conjugate is between -100 mV and 100 mV, between -50 mV and 50 mV, between -25 mV and 25 mV, between -20 mV and 20 mV, between -10 mV and 10 mV, between -10 mV and 5 mV, between -5 mV and 5 mV, or between -2 mV and 2 mV. In a preferred embodiment, the surface charge is neutral or near-neutral. The above ranges include all ranges between -100 mV and 100 mV.

[0060] A. Dendrimer Dendrimers are three-dimensional, highly branched, monodisperse, spherical, polyvalent macromolecules containing a high density of surface end groups (Tomalia, D.A., et al., Biochemical Society Transactions, 35, 61 (2007); and Sharma, A., et al., ACS Macro Letters, 3, 1079 (2014)). Due to their unique structural and physical features, dendrimers are useful as nanocarriers for various biomedical applications, including targeted drug / gene delivery, imaging, and diagnostics (Sharma, A., et al., RSC Advances, 4, 19242 (2014); Caminade, A.-M., et al., Journal of Materials Chemistry B, 2, 4055 (2014); Esfand, R., et al., Drug Discovery Today, 6, 427 (2001); and Kannan, RM, et al., Journal of Internal Medicine, 276, 579 (2014)).

[0061] Recent studies have shown that the surface groups of dendrimers significantly affect their biodistribution (Nance, E., et al., Biomaterials, 101, 96 (2016)). Hydroxyl-terminated fourth-generation PAMAM dendrimers (approximately 4 nm in size) without any targeting ligands crossed the dysfunctional BBB significantly more (>20-fold) in rabbit cerebral palsy (CP) models after systemic administration compared with healthy controls, selectively targeting activated microglia and astrocytes (Lesniak, W.G., et al., Mol Pharm, 10 (2013)).

[0062] The term "dendrimer" includes, but is not limited to, a molecular structure having an inner core, layers (or "generations") of repeating units regularly attached to and extending from the inner core, each layer having one or more branch points, and an exterior surface of terminal groups attached to the outermost generation. In some embodiments, the dendrimer has a regular dendrimer or "starburst" molecular structure.

[0063] Typically, dendrimers have diameters between about 1 nm and about 50 nm, more typically between about 1 nm and about 20 nm, between about 1 nm and about 10 nm, or between about 1 nm and about 5 nm. In some embodiments, the diameter is between about 1 nm and about 2 nm. Conjugates are generally in the same size range, but larger proteins, such as antibodies, can increase the size by 5 to 15 nm. Typically, agents are encapsulated at agent to dendrimer ratios of 1:1 to 4:1 for larger generation dendrimers. In preferred embodiments, the dendrimers have a diameter effective for targeting and prolonged retention in hepatocytes.

[0064] In some embodiments, the dendrimer has a molecular weight between about 500 daltons and about 100,000 daltons, preferably between about 500 daltons and about 50,000 daltons, and most preferably between about 1,000 daltons and about 20,000 daltons.

[0065] Suitable dendrimer scaffolds that can be used include poly(amidoamine), also known as PAMAM or STARBURST™ dendrimers; polypropylamine (POPAM), polyethyleneimine, polylysine, polyester, iptycene, aliphatic poly(ether), and / or aromatic polyether dendrimers. The dendrimers can have carboxyl, amine, and / or hydroxyl termini. In preferred embodiments, the dendrimers are hydroxyl terminated. Each dendrimer in a dendrimer complex can be of similar or different chemical nature to the other dendrimers (e.g., a first dendrimer can comprise a PAMAM dendrimer and a second dendrimer can be a POPAM dendrimer).

[0066] The term "PAMAM dendrimer" refers to a poly(amidoamine) dendrimer that may contain different cores with amidoamine building blocks and may have carboxyl, amine, and hydroxyl termini of any generation, including, but not limited to, a first-generation PAMAM dendrimer, a second-generation PAMAM dendrimer, a third-generation PAMAM dendrimer, a fourth-generation PAMAM dendrimer, a fifth-generation PAMAM dendrimer, a sixth-generation PAMAM dendrimer, a seventh-generation PAMAM dendrimer, an eighth-generation PAMAM dendrimer, a ninth-generation PAMAM dendrimer, or a tenth-generation PAMAM dendrimer. In preferred embodiments, the dendrimer is soluble in the formulation and is a fourth-, fifth-, or sixth-generation ("G") dendrimer (i.e., a G4-G6 dendrimer), and / or a G4-G10 dendrimer, a G6-G10 dendrimer, or a G2-G10 dendrimer. The dendrimer may have hydroxyl groups attached to its functional surface groups. In preferred embodiments, the dendrimer is a 4th, 5th, 6th, 7th, or 8th generation hydroxyl-terminated polyamidoamine dendrimer.

[0067] Methods for producing dendrimers are known to those skilled in the art and generally involve a two-step, iterative reaction sequence that produces concentric shells (generations) of dendritic β-alanine units around a central starting core (e.g., an ethylenediamine core). Each subsequent growth step represents a new "generation" of polymer with a larger molecular diameter, doubling the number of reactive surface sites and approximately doubling the molecular weight of the previous generation. Suitable dendrimer scaffolds for use are commercially available in a variety of generations. Preferably, the dendrimer composition is based on a dendrimer scaffold of generations 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Such scaffolds have 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, and 4096 reactive sites, respectively. Thus, dendrimeric compounds based on these scaffold structures can have a corresponding maximum number of combined targeting moieties, such as tri-antennary N-acetylgalactosamine (GalNAc), and active agents, either directly or indirectly through a linker.

[0068] In some embodiments, the dendrimer comprises multiple hydroxyl groups. Some exemplary high-density hydroxyl-group-containing dendrimers include commercially available polyester dendritic polymers, such as hyperbranched 2,2-bis(hydroxyl-methyl)propionic acid polyester polymers (e.g., hyperbranched bis-MPA polyester-64-hydroxyl, 4th generation), and dendritic polyglycerols.

[0069] In some embodiments, the high-density hydroxyl-containing dendrimer is an oligoethylene glycol (OEG)-like dendrimer. For example, second-generation OEG dendrimers (D2-OH-60) can be synthesized using highly efficient and robust atom-economic chemical reactions, such as Cu(I)-catalyzed alkyne-azide click chemistry and photocatalytic thiol-ene click chemistry. High-density polyol dendrimers of very low generation with minimal reaction steps can be achieved by using orthogonal hypermonomer and hypercore strategies, as described, for example, in WO2019094952. In some embodiments, the dendrimer backbone has non-cleavable polyether bonds throughout the structure to avoid in vivo dendrimer collapse and enable elimination of such dendrimers from the body as a single entity (non-biodegradable).

[0070] In some embodiments, the dendrimers specifically target particular tissue regions and / or cell types, such as hepatocytes, tumor-associated macrophages (TAMs) in liver tumors / cancers, or pro-inflammatory macrophages involved in autoimmune diseases of the liver.

[0071] In a preferred embodiment, the dendrimer has multiple hydroxyl (-OH) groups at the termini of the dendrimer. The preferred surface density of hydroxyl (-OH) groups is at least 1 OH group / nm 2 (number of hydroxyl surface groups / nm 2 For example, in some embodiments, the surface density of hydroxyl groups is greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10; preferably at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 surface hydroxyl groups / nm 2 In a further embodiment, the surface density of hydroxyl (—OH) groups is from about 1 to about 50, preferably from 5 to 20, hydroxyl surface groups / nm 2 (number of hydroxyl surface groups / nm 2(surface area at 1000 nm) and has a molecular weight between about 500 Da and about 10 kDa. In some embodiments, the percentage of free, i.e., non-conjugated, hydroxyl groups among the total surface groups (conjugated and non-conjugated) on the dendrimer is greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, and / or less than 100%.

[0072] In some embodiments, dendrimers may have a fraction of their hydroxyl groups exposed on the exterior surface and another fraction in the interior core of the dendrimer. In preferred embodiments, dendrimers have at least one OH group / nm 3 (Number of hydroxyl groups / nm 3 For example, in some embodiments, the volume density of hydroxyl groups is 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, greater than 15, greater than 20, greater than 25, greater than 30, greater than 35, greater than 40, greater than 45, and greater than 50 hydroxyl groups / nm 3 In some embodiments, the volume density of hydroxyl groups is from about 4 to about 50 hydroxyl groups / nm 3 between about 5 and about 30 groups / nm 3 between about 10 and about 20 hydroxyl groups / nm 3 It is between.

[0073] B. Dendrimers modified with triantennary N-acetylgalactosamine (GalNAc) It has been established that dendrimers conjugated to or complexed with the carbohydrate triantennary N-acetylgalactosamine (GalNAc) selectively accumulate in hepatocytes. Described are compositions of dendrimers modified by the addition of triantennary N-acetylgalactosamine (GalNAc) to the dendrimer surface.

[0074] The asialoglycoprotein receptor (ASGPR), abundantly expressed on hepatocytes, can selectively recognize galactose and N-acetylgalactosamine (GalNAc) through its carbohydrate recognition domain (CRD), which tightly binds to the receptor. Efficient binding of carbohydrate moieties to the ASGPR receptor allows their selective internalization within hepatocytes via receptor-mediated endocytosis. The low pH in endosomes disrupts the tetravalent calcium chelation between the sugar ligand and the ASGPR receptor, thereby releasing the ligand into the hepatocyte. Once the ligand is released, the receptor complex recycles, allowing large amounts of ligand to be internalized into hepatocytes without saturation effects. Binding of GalNAc to the ASGPR occurs on the sinusoidal surface of hepatocytes, but hepatocytes contain approximately 500,000 ASGPR receptors per cell, of which approximately 5%–10% are present on the cell surface at any given time. Previous studies have shown that ligand binding to ASGPR depends on the sugar type (GalNAc > Gal) and the number of sugars (4 = 3 > 2 > 1). The X-ray crystal structure of the extracellular domain of ASGPR revealed a shallow carbohydrate-binding pocket, which explains the need for multivalency. Therefore, multivalent binding has been explored, and the binding affinity of trivalent and tetravalent carbohydrate constructs to ASGPR is 100–1000 times stronger than that of monovalent ligands due to the glycocluster effect.

[0075] Biantennary and triantennary GalNAc ligands conjugated to siRNA demonstrated significantly higher levels of GalNAc-siRNA in the liver of C57BL / 6 mice after subcutaneous administration, with 94% of the GalNAc-siRNA localized to hepatocytes. Furthermore, these siRNA conjugates mediated efficient gene silencing. Further studies reported that antisense oligonucleotides (ASOs) linked to triantennary GalNAc were up to 10-fold more potent than the parent ASOs in mouse models.

[0076] Carbohydrate-protein interactions play an important role in biological processes, such as receptor-mediated endocytosis, and have been applied to cell recognition studies and biomedical material design. Carbohydrate-terminated dendrimers (glycodendrimers) are endowed with enhanced binding affinity for their cognate receptors, enabling them to interact with specific cell types with avidity and selectivity for targeted drug delivery. The introduction of carbohydrate moieties into drug delivery platforms also provides biocompatibility and increases the water solubility of dendrimer conjugates.

[0077] Triantennary GalNAc provides effective multivalent binding to ASGPR in hepatocytes. Thus, in a preferred embodiment, dendrimers are modified with one or more triantennary GalNAc groups at one or more surface terminal groups (e.g., -OH).

[0078] Triantennary GalNAc modification of a dendrimer results in a set of three GalNAc at each surface end group. In some embodiments, three β-GalNAc molecules are grafted to the building block via one or more linkers to produce an AB3 building block (i.e., a triantennary GalNAc dendron) suitable for conjugation with a surface functional group of a dendrimer.

[0079] In one embodiment, three β-GalNAc molecules are grafted to a propargylated pentaerythritol building block via one or more linkers to yield an AB3 building block suitable for conjugation with surface functional groups of a dendrimer, as shown below. [ka]

[0080] In some embodiments, conjugation of the triantennary GalNAc through one or more surface groups occurs via about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or 30% of the total available surface functional groups, preferably hydroxyl groups, of the dendrimer prior to conjugation. In other embodiments, conjugation of the triantennary β-GalNAc occurs on less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the total available surface functional groups of the dendrimer prior to conjugation. In a preferred embodiment, the dendrimer is conjugated to an effective amount of triantennary β-GalNAc for binding to and / or targeting ASGPR on hepatocytes, and is also conjugated to an effective amount of an active agent for treating, preventing, and / or imaging liver disease or disorder.

[0081] C. Dendrimer Complex Dendrimers modified with triantennary GalNAc (dendrimer-triantennary GalNAc) can contain one or more therapeutic or prophylactic agents complexed with the dendrimer, covalently conjugated thereto, or dispersed or encapsulated therein. Conjugation of one or more agents to the dendrimer component of the dendrimer-triantennary GalNAc complex can occur before, simultaneously with, or after conjugation of the dendrimer with triantennary GalNAc. Compositions and methods for conjugating agents to dendrimers are known in the art and are described in detail in U.S. Patent Application Publication Nos. 2011 / 0034422, 2012 / 0003155, and 2013 / 0136697.

[0082] In some embodiments, one or more active agents are covalently attached to the dendrimer component of the dendrimer-triantennary GalNAc. In some embodiments, the active agent is functionalized for conjugation with the dendrimer, optionally via one or more spacers or linking moieties. The functionalized active agent and / or linking moiety are designed to have a desired release rate of the active agent from the dendrimer-triantennary GalNAc in vivo. The functionalized active agent and / or linking moiety can be designed to be cleaved by hydrolysis, enzymes, or a combination thereof to provide sustained release of the active agent in vivo. If a cleavable form is desired, both the composition of the linking moiety and its point of attachment to the active agent are selected so that cleavage of the linking moiety releases either the active agent or a suitable prodrug thereof. In some embodiments, the functionalized active agent and / or linking moiety are designed to be cleaved at a minimal or insignificant rate in vivo. The composition of the linking moiety can also be selected based on the desired release rate of the active agent. In a preferred embodiment, the one or more active agents are functionalized to be non-cleavable or hardly cleavable from the dendrimer-triantennary GalNAc in vivo, e.g., via one or more amide or ether linkages, optionally with one or more spacers / linkers.

[0083] In some embodiments, the binding between the active agent and the dendrimer occurs via one or more of disulfide, ester, ether, thioester, carbamate, carbonate, hydrazine, or amide linkages. In preferred embodiments, the binding occurs via a suitable spacer that provides an ester or amide bond between the active agent and the dendrimer depending on the desired release kinetics of the active agent. In some cases, an ester bond is introduced for the release of the active agent. In other cases, an amide bond is introduced for the non-release of the active agent.

[0084] The linking moiety generally comprises one or more organic functional groups. Examples of suitable organic functional groups include secondary amide (-CONH-), tertiary amide (-CONR-), sulfonamide (-S(O)2-NR-), secondary carbamate (-OCONH-; -NHCOO-), tertiary carbamate (-OCONR-; -NRCOO-), carbonate (-OC(O)-O-), urea (-NHCONH-; -NRCONH-; -NHCONR-, -NRCONR-), carbinol (-CHOH-, -CROH-), disulfide group, hydrazone, hydrazide, ether (-O-), and ester (-COO-, -CHOC-, CHROC-), where R is an alkyl group, an aryl group, or a heterocyclic group. In general, the identity of the one or more organic functional groups in the linking moiety can be selected with respect to the desired release rate of the active agent. In addition, one or more organic functional groups can be selected to facilitate covalent binding of an active agent to the dendrimer. In a preferred embodiment, binding can occur via a suitable spacer that provides a disulfide bridge between the active agent and the dendrimer. The dendrimer-triantennary GalNAc conjugate can rapidly release the active agent in vivo by thiol exchange under reducing conditions found in the body.

[0085] In certain embodiments, the linking moiety comprises one or more of the above organic functional groups in combination with a spacer group. The spacer group can be composed of any collection of atoms, including oligomeric and polymeric chains; however, the total number of atoms in the spacer group is preferably between 3 and 200 atoms, more preferably between 3 and 150 atoms, more preferably between 3 and 100 atoms, and most preferably between 3 and 50 atoms. Examples of suitable spacer groups include alkyl groups, heteroalkyl groups, alkylaryl groups, oligo- and polyethylene glycol chains, and oligo- and poly(amino acid) chains. Variations in the spacer group provide additional control over the release of the active agent in vivo. In embodiments in which the linking moiety comprises a spacer group, one or more organic functional groups are typically used to connect the spacer group to both the active agent and the dendrimer.

[0086] Useful reactions and strategies for covalently linking active agents to dendrimers are known in the art. See, for example, March, "Advanced Organic Chemistry," 5th Edition, 2001, Wiley-Interscience Publication, New York) and Hermanson, "Bioconjugate Techniques," 1996, Elsevier Academic Press, USA. The appropriate method for covalently linking a given active agent can be selected based on the desired linking moiety and the overall structure of the agent and dendrimer relative to functional group compatibility, protecting group strategy, and the presence of labile bonds.

[0087] Optimal drug loading necessarily depends on many factors, including the choice of drug, the structure and size of the dendrimer, and the tissue to be treated. In some embodiments, one or more active agents are encapsulated, associated, and / or conjugated to the dendrimer-triantennary GalNAc complex, preferably through one or more surface groups of the dendrimer, at a concentration of about 0.01% to about 45% by weight, preferably about 0.1% to about 30% by weight, about 0.1% to about 20% by weight, about 0.1% to about 10% by weight, about 1% to about 10% by weight, about 1% to about 5% by weight, about 3% to about 20% by weight, and about 3% to about 10% by weight. However, optimal drug loading for any given drug, dendrimer, and target site can be identified by routine methods, such as those described.

[0088] In some embodiments, conjugation of the dendrimer with the active agent occurs prior to conjugation of the dendrimer with the triantennary GalNAc. In some embodiments, conjugation of the active agent and / or linker with the dendrimer occurs through one or more surface and / or internal groups. Thus, in some embodiments, conjugation of the active agent / linker occurs through about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the total available surface functional groups, preferably hydroxyl groups, of the dendrimer prior to conjugation. In other embodiments, conjugation of the active agent / linker occurs on less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the total available surface functional groups of the dendrimer prior to conjugation and / or modification with the triantennary β-GalNAc. In a preferred embodiment, the dendrimer complex retains an effective amount of surface functional groups for modification with triantennary β-GalNAc for targeting to hepatocytes, yet is conjugated to an effective amount of an active agent for treating, preventing, and / or imaging a disease or disorder. D. Coupling Agents and Spacers

[0089] Dendrimer complexes can be formed with therapeutic active agents or compounds conjugated or bound to the dendrimer. Optionally, the active agent is conjugated to the dendrimer via one or more spacer / linkers through different linkages, such as disulfide, ester, carbonate, carbamate, thioester, hydrazine, hydrazide, and amide linkages. The one or more spacer / linkers between the dendrimer and the active agent can be designed to provide a releasable or non-releasable form of the dendrimer-active complex in vivo. In some embodiments, the conjugation occurs through a suitable spacer that provides an ester bond between the active agent and the dendrimer. In some embodiments, the conjugation occurs through a suitable spacer that provides an amide or ether bond between the active agent and the dendrimer. In preferred embodiments, the one or more spacer / linkers between the dendrimer and the active agent are added to achieve desired and effective release kinetics in vivo.

[0090] The spacer can be either a single chemical entity or two or more chemical entities that are linked together to bridge the dendrimer and the active agent. Spacers can include sulfhydryl, thiopyridine, succinimidyl, maleimide, vinyl sulfone, and any small chemical entity, peptide, or polymer with a carbonate terminus.

[0091] The spacer can be selected from the classes of compounds terminating in sulfhydryl, thiopyridine, succinimidyl, maleimide, vinyl sulfone, and carbonate groups. The spacer can include a thiopyridine-terminated compound, such as dithiodipyridine, N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate LC-SPDP, or sulfo-LC-SPDP. Spacers can also include peptides that are linear or cyclic in nature with sulfhydryl groups, such as glutathione, homocysteine, cysteine and its derivatives, arg-gly-asp-cys (RGDC), cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDfC)), cyclo(Arg-Gly-Asp-d-Tyr-Cys), and cyclo(Arg-Ala-Asp-d-Tyr-Cys). In some embodiments, the spacer comprises a mercapto acid derivative, such as 3-mercaptopropionic acid, mercaptoacetic acid, 4-mercaptobutyric acid, thiolan-2-one, 6-mercaptohexanoic acid, 5-mercaptovaleric acid, and other mercapto derivatives, such as 2-mercaptoethanol and 2-mercaptoethylamine. In some embodiments, the spacer comprises thiosalicylic acid and its derivatives, (4-succinimidyloxycarbonyl-methyl-alpha-2-pyridylthio)toluene, (3-[2-pyridylthio]propionylhydrazide. In some embodiments, the spacer comprises a maleimide terminus, and the spacer comprises a polymer or a small chemical entity, such as bis-maleimidodiethylene glycol and bis-maleimidotriethylene glycol, bis-maleimidoethane, bismaleimidohexane. In some embodiments, the spacer comprises a vinyl sulfone, such as 1,6-hexane-bis-vinyl sulfone. In some embodiments, the spacer comprises a thioglycoside, such as thioglucose. In other embodiments, the spacer comprises reduced proteins, such as bovine serum albumin and human serum albumin, any thiol-terminated compound capable of forming a disulfide bond.In certain embodiments, the spacer comprises maleimide, succinimidyl, and thiol-terminated polyethylene glycol.

[0092] The active agent and / or targeting moiety can be either covalently bound to the dendrimer, or dispersed or encapsulated within the molecule.The dendrimer is preferably a fourth, fifth, sixth, seventh, eighth, ninth, or tenth generation PAMAM dendrimer with a hydroxyl terminus.In a preferred embodiment, the dendrimer is linked to the active agent via a spacer that terminates with a disulfide, ester, ether, or amide bond.

[0093] In some embodiments, the non-releasable form of a dendrimer / active agent conjugate provides enhanced therapeutic efficacy compared to the releasable form of the same dendrimer / active agent conjugate. Thus, in some embodiments, one or more active agent(s) are conjugated to the dendrimer in a non-releasable form via a spacer that is linked to the dendrimer via, for example, an ether or amide bond. In some embodiments, one or more active agent(s) are linked to the spacer in a non-releasable form, for example, an ether or amide bond. Thus, in some embodiments, one or more active agent(s) are linked to the dendrimer via a spacer that is linked to the dendrimer and the active agent(s) in a non-releasable form. In an exemplary embodiment, one or more active agent(s) are linked to the dendrimer via a spacer that is linked to the dendrimer and the active agent(s) via an amide and / or ether bond. An exemplary spacer is polyethylene glycol (PEG). 1. Conjugation of dendrimers with active agents via ether linkages

[0094] In some embodiments, compositions are described that include a hydroxyl-terminated triantennary GalNAc-modified dendrimer conjugated to an active agent via an ether linkage, optionally by one or more linkers / spacers.

[0095] In preferred embodiments, the covalent bonds between the surface groups of the dendrimer and the linker, or between the dendrimer and the active agent (when conjugated without any linking moiety), are stable under in vivo conditions, i.e., are hardly cleaved when administered to a subject and / or are excreted intact from the body. For example, in preferred embodiments, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, or less than 0.1% of the total dendrimer conjugates cleave the active agent within 24 hours, 48 hours, or 72 hours after administration in vivo. In one embodiment, these covalent bonds are ether bonds. In a further preferred embodiment, the covalent bonds between the surface groups of the dendrimer and the linker, or between the dendrimer and the active agent (when conjugated without any linking moiety), are not hydrolytically cleavable or enzymatically cleavable bonds, such as ester bonds.

[0096] In some embodiments, one or more hydroxyl groups of a hydroxyl-terminated dendrimer conjugate with one or more linking moieties and one or more active agents via one or more ether linkages is shown below in formula (I): [ka] During the ceremony, D is a 2nd to 10th generation poly(amidoamine) (PAMAM) dendrimer; L is one or more linking moieties or spacers; X is an active agent, or a derivative, analog, or prodrug thereof; n is an integer from 1 to 100; and m is an integer from 16 to 4096; and Y is a linker selected from secondary amide (-CONH-), tertiary amide (-CONR-), sulfonamide (-S(O)-NR-), secondary carbamate (-OCONH-; -NHCOO-), tertiary carbamate (-OCONR-; -NRCOO-), carbonate (-OC(O)-O-), urea (-NHCONH-; -NRCONH-; -NHCONR-, -NRCONR-), carbinol (-CHOH-, -CROH-), disulfide group, hydrazone, hydrazide, and ether (-O-), where R is an alkyl group, an aryl group, or a heterocyclic group. Preferably, Y is a bond or linkage that is not cleavable in vivo.

[0097] In a preferred embodiment, Y is a secondary amide (-CONH-).

[0098] In one embodiment, D is a fourth or sixth generation hydroxyl-terminated PAMAM dendrimer; L is one or more linking or spacer moieties; X is an angiotensin II receptor blocker, a farnesoid X receptor agonist, a death receptor 5 agonist, a sodium-glucose cotransporter type 2 inhibitor, a lysophosphatidic acid 1 receptor antagonist, an endothelin-A receptor antagonist, a PPARδ agonist, an AT1 receptor antagonist, a CCR5 / CCR2 antagonist, an antifibrotic agent, an anti-inflammatory agent, and / or an antioxidant or a derivative, analog, or prodrug thereof; n is about 5 to 15; m is an integer between about 49 and 59; and n+m=64.

[0099] In one embodiment, Y is a secondary amide (-CONH-). E. Therapeutic, Prophylactic, and Diagnostic Agents

[0100] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more therapeutic, prophylactic, and diagnostic agents. The agent(s) contained in the dendrimer complex to be delivered can be a protein or peptide, a sugar or carbohydrate, a nucleic acid or oligonucleotide, a lipid, a small molecule (e.g., molecular weight less than 2500 daltons, preferably less than 2000 daltons, more preferably less than 1500 daltons, more preferably less than 300-700 daltons), or a combination thereof. The nucleic acid can be an oligonucleotide encoding a protein, such as a DNA expression cassette or mRNA. Exemplary oligonucleotides include siRNA, microRNA, DNA, and RNA. In some embodiments, the active agent is a therapeutic antibody.

[0101] Dendrimers have the advantage that multiple therapeutic, prophylactic, and / or diagnostic agents can be delivered via the same dendrimer. In some embodiments, one or more types of active agents can be encapsulated, complexed, or conjugated to the dendrimer. In certain embodiments, the dendrimer is complexed or conjugated to two or more different classes of active agents, providing simultaneous delivery with different or independent release kinetics at the target site. For example, one dendrimer can be covalently linked to one or more PPARδ agonists and one or more angiotensin II receptor blockers. In another embodiment, the dendrimer is covalently linked to at least one detectable moiety and at least one class of active agent. In a further embodiment, dendrimer complexes each containing a different class of active agent are administered simultaneously for combined treatment. In one embodiment, the dendrimer composition has multiple agents complexed or conjugated to the dendrimer, such as chemotherapeutic agents, immunotherapeutic agents, antifibrotic agents, steroids that reduce swelling, antibiotics, antiangiogenic agents, and / or diagnostic agents.

[0102] The selective targeting of triantennary GalNAc-modified dendrimers allows for the administration of weaker active agents to achieve the same therapeutic effect compared to the same active agent not conjugated to a dendrimer or compared to the same active agent conjugated to a dendrimer not modified with triantennary β-GalNAc, thus reducing the dose-related cytotoxicity and / or other side effects associated with the active agent. Dendrimers can also increase the solubility of one or more therapeutic, prophylactic, and / or diagnostic agents to be delivered. For example, telmisartan is a very hydrophobic drug, but when conjugated to a dendrimer, it becomes highly water-soluble, with a water solubility of approximately 60 mg / ml.

[0103] The active agent may also be a pharmaceutically acceptable prodrug of any of the compounds described below. A prodrug is a compound that, when metabolized in vivo, is converted into a compound with the desired pharmacological activity. Prodrugs can be prepared by replacing appropriate functional groups present in the above compounds with "promoieties," as described, for example, in H. Bundgaar, Design of Prodrugs (1985). Examples of prodrugs include ester, ether, or amide derivatives of the above compounds, polyethylene glycol derivatives of the above compounds, N-acylamine derivatives, dihydropyridine pyridine derivatives, amino-containing derivatives conjugated to polypeptides, 2-hydroxybenzamide derivatives, carbamate derivatives, N-oxide derivatives that are biologically reduced to active amines, and N-Mannich base derivatives. For further discussion of prodrugs, see, for example, Rautio, J. et al. Nature Reviews Drug Discovery. 7:255-270 (2008).

[0104] The active agent includes a therapeutic agent that has been shown to be effective for treating and preventing one or more liver diseases or disorders. Exemplary therapeutic agents include angiotensin II receptor blockers, farnesoid X receptor (FXR) agonists, sodium-glucose cotransporter type 2 (SGLT2) inhibitors, apoptosis signal-regulating kinase 1 (ASK-1) inhibitors, pyridinone derivatives, FGF-21 analogs, FGF-19 analogs, lysophosphatidic acid (LPA) 1 receptor antagonists, endothelin-A receptor antagonists, PPARα / δ agonists, AT1 receptor antagonists, CCR5 / CCR2 inhibitors, death receptor 5 (DR5) activators, anti-fibrotic agents, anti-inflammatory agents, and / or antioxidants. In some embodiments, the dendrimer-triantennary GalNAc is complexed or conjugated to one or more angiotensin II receptor blockers, FXR agonists, SGLT2 inhibitors, ASK-1 inhibitors, pyridinone derivatives, FGF-21 analogs, FGF-19 analogs, LPA1 receptor antagonists, endothelin-A receptor antagonists, PPARα / δ agonists, AT1 receptor antagonists, CCR5 / CCR2 antagonists, activators of DR5, anti-fibrotic agents, anti-inflammatory agents, antioxidants, or combinations thereof.

[0105] Peroxisome proliferator-activated receptor delta (PPARδ) is a member of the nuclear receptor family and is emerging as an important metabolic regulator with pleiotropic effects in various tissues, including adipose tissue, skeletal muscle, and liver. PPARδ agonists protect hepatocytes from cell death by reducing ROS production in hepatocytes, resulting in less liver fibrosis. Exemplary PPARδ agonists have been previously described. In some embodiments, the PPARδ agonist is an indanyl acetic acid derivative with a 4-thiazolyl-phenoxy tail group, as described in Rudolph J et al., J. Med. Chem. 2007, 50, 5, 984-1000 (2007). Exemplary PPARδ agonists have been previously described, for example, by Ham J et al., Eur J Med Chem. 53:190-202 (2012). Thus, in some embodiments, the tri-antennary β-GalNAc-modified dendrimer is complexed, covalently conjugated, or inter-molecularly dispersed or encapsulated with one or more PPARδ agonists, hi one embodiment, the tri-antennary β-GalNAc-modified dendrimer is complexed, covalently conjugated, or inter-molecularly dispersed or encapsulated with one or more PPARδ agonists, e.g., GW0742, GW501516, elafibranor, or derivatives, analogs, or prodrugs thereof.

[0106] Sodium-glucose cotransporter type 2 (SGLT2) inhibitors are hypoglycemic agents that promote weight loss, lower serum uric acid levels, and improve glycemic control. The beneficial effects of SGLT2 inhibitors on hepatic steatosis have been reported by Scheen AJ. Diabetes Metab. 2019;45(3):213-223; Omori et al., Metab.Clin. Exp. 2019 Jul 11. Exemplary SGLT2 inhibitors include phlorizin, T-1095, canagliflozin, dapagliflozin, ipragliflozin, tofogliflozin, empagliflozin, luseogliflozin, ertugliflozin, and remogliflozin etabonate. Thus, in some embodiments, the triantennary β-GalNAc-modified dendrimer is complexed with, covalently conjugated to, or inter-molecularly dispersed or encapsulated with one or more SGLT2 inhibitors.

[0107] Lysophosphatidic acid (LPA) is a lipid mediator produced primarily by activated platelets through the hydrolysis of lysophosphatidylcholine by autotaxin (ATX). LPA is a bioactive lipid involved in several functions, including proliferation, apoptosis, migration, and cancer cell invasion. LPA and the LPA1 receptor (LPA1R) are elevated in many inflammatory conditions, including pulmonary fibrosis, liver fibrosis, and systemic sclerosis. LPA exerts various physiological effects on its receptors in parenchymal cells, and LPA1R antagonists have shown antifibrotic effects in liver fibrosis, pulmonary fibrosis, and scleroderma models. Exemplary LPA1 receptor antagonists include BMS-986202, BMS-986020, VPC12249, AM966, AM095, Ki16425, and Ki16198. Thus, in some embodiments, the triantennary β-GalNAc-modified dendrimer is complexed with, covalently conjugated to, or inter-molecularly dispersed or encapsulated with one or more LPA1 receptor antagonists, such as BMS-986202, BMS-986020, VPC12249, AM966, AM095, Ki16425, Ki16198, or derivatives or analogs or prodrugs thereof.

[0108] The antifibrotic effect of ambrisentan, an endothelin-A receptor antagonist, has been demonstrated in a mouse nonalcoholic steatohepatitis model (World J Hepatol. 2016 Aug 8; 8(22): 933-941). Exemplary endothelin receptor antagonists include sitaxsentan, ambrisentan, macitentan, and zibotentan. Thus, in some embodiments, the triantennary β-GalNAc-modified dendrimer is complexed with, covalently conjugated to, or inter-molecularly dispersed or encapsulated with one or more endothelin receptor antagonists, such as sitaxsentan, ambrisentan, macitentan, and zibotentan, or derivatives, analogs, or prodrugs thereof.

[0109] Since oxidative stress is implicated in the pathogenesis of NAFLD, the role of antioxidants, such as vitamin E, which are known to react with reactive oxygen species (ROS), is to block the chain propagation of free radical reactions in a wide range of oxidative stress situations. In one embodiment, the active agent is vitamin E or a derivative or analog or prodrug thereof.

[0110] Typically, the active agent(s) are functionalized, e.g., by an ether, ester, or amide linkage, optionally with one or more spacers / linkers, to facilitate conjugation to the dendrimer and / or for desired release kinetics. In preferred embodiments, the active agent(s) are functionalized, e.g., via an ether or amide, optionally with one or more spacers / linkers, to be non-cleavable or nearly non-cleavable from the dendrimer in vivo.

[0111] In preferred embodiments, the active agent or agents delivered via the tri-antennary GalNAc-modified dendrimer are released from the dendrimer conjugate for at least 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days, preferably at least 1 week, 2 weeks, or 3 weeks, and more preferably at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration to a mammalian subject in an amount effective to be therapeutically effective in the target cell, tissue, or region.

[0112] 1. Angiotensin II receptor blockers (ARBs) In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more angiotensin II receptor blockers (ARBs). The renin-angiotensin pathway in hepatic stellate cells induces reactive oxygen species and accelerates liver fibrosis. In response to persistent liver injury, the renin-angiotensin system (RAS) locally accelerates inflammation, tissue repair, and fibrinogenesis by producing angiotensin II (Ang II), a vasoconstrictor agonist involved in the pathogenesis of liver fibrosis. The RAS is described as a single cascade in which renin converts angiotensinogen to angiotensin I (Ang I), which is then converted to angiotensin II (Ang II) by angiotensin-converting enzyme (ACE). Ang II mediates biological responses through two G protein-coupled receptors: Ang II receptor type 1 (AT1) and Ang II receptor type 2 (AT2). However, the fibrinogenic effects of Ang II are primarily mediated by the angiotensin receptor AT1.

[0113] Among the emerging treatment approaches for NAFLD is the antihypertensive agent telmisartan, which has positive effects on liver, lipid, and glucose metabolism, particularly through its action on the renin-angiotensin system, by blocking the ACE / AngII / AT1 axis and by increasing ACE2 / Ang(1-7) / Mas axis activation.

[0114] Thus, in some embodiments, the triantennary β-GalNAc-modified dendrimers are complexed with, covalently conjugated to, or inter-molecularly dispersed or encapsulated with one or more angiotensin II receptor blockers to treat, alleviate, or prevent one or more liver diseases or disorders, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, drug-induced liver failure, hepatitis, liver fibrosis, cirrhosis, or a combination thereof.

[0115] In some embodiments, the angiotensin II receptor blocker is optionally functionalized with one or more spacers / linkers, e.g., via ether, ester, or amide linkages, to facilitate conjugation with the dendrimer and / or for desired release kinetics. In preferred embodiments, the angiotensin II receptor blocker is functionalized to be non-cleavable or substantially non-cleavable from the dendrimer in vivo, e.g., via ether or amide, optionally via one or more spacers / linkers. In preferred embodiments, the angiotensin II receptor blocker or a derivative, analog, or prodrug thereof is conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry, optionally via one or more spacers / linkers, e.g., polyethylene glycol (PEG).

[0116] In one embodiment, the tri-branched β-GalNAc-modified dendrimer is complexed with, covalently conjugated to, or intermolecularly dispersed or encapsulated with telmisartan or its derivatives, analogs, or prodrugs, or its pharmacologically active salts. In some embodiments, telmisartan is functionalized, for example, by ether, ester, or amide linkages, optionally with one or more spacers / linkers, for example, polyethylene glycol (PEG). Exemplary conjugations of telmisartan with tri-branched β-GalNAc-modified hydroxyl-terminated PAMAM dendrimers as dendrimer-telmisartan ester conjugates and dendrimer-telmisartan amide conjugates are shown in Figure 3 and Figure 4, respectively.

[0117] 2. Farnesoid X receptor (FXR) agonists In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more agonists of the farnesoid X receptor (FXR). The farnesoid X receptor (FXR) is a major regulator of bile acid homeostasis through transcriptional regulation of genes involved in bile acid synthesis and membrane transport. Impaired bile acid outflow due to cholangiopathies leads to chronic cholestasis, resulting in abnormally elevated hepatic and systemic bile acid levels. Obeticholic acid (OCA) is a potent and selective FXR agonist that is 100-fold more potent than its endogenous ligand, chenodeoxycholic acid (CDCA).

[0118] In some embodiments, the triantennary β-GalNAc-modified dendrimers are complexed with, covalently conjugated to, or inter-molecularly dispersed or encapsulated with one or more FXR agonists, such as obeticholic acid and GW4064, for treating, alleviating, or preventing one or more liver diseases or disorders, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, drug-induced liver failure, hepatitis, liver fibrosis, cirrhosis, or a combination thereof.

[0119] In some embodiments, the FXR agonist is functionalized, for example, by an ether, ester, or amide linkage, as appropriate with one or more spacers / linkers, to facilitate conjugation to the dendrimer and / or for desired release kinetics. In preferred embodiments, the FXR agonist is functionalized to be non-cleavable or substantially non-cleavable from the dendrimer in vivo, for example, via an ether or amide, as appropriate with one or more spacers / linkers.

[0120] In one embodiment, the triantennary β-GalNAc-modified dendrimer is complexed with, covalently conjugated to, or inter-molecularly dispersed or encapsulated with obeticholic acid or its derivatives, analogs, or prodrugs, or pharmacologically active salts thereof. In some embodiments, the obeticholic acid is functionalized, for example, by ether, ester, or amide linkages, optionally with one or more spacers / linkers, such as polyethylene glycol (PEG). An exemplary conjugation of obeticholic acid with a triantennary β-GalNAc-modified hydroxyl-terminated PAMAM dendrimer as a dendrimer-obeticholic acid ester conjugate is shown in Figure 8.

[0121] 3. Death receptor 5 (DR5) agonists In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated to one or more agonists of death receptor 5 (DR5), also known as TRAIL receptor 2 (TRAIL-R2) and tumor necrosis factor receptor superfamily member 10B (TNFRSF10B), a cell surface receptor of the TNF receptor superfamily that binds tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

[0122] In some embodiments, the composition comprises one or more DR5 agonists for treating, alleviating, or preventing one or more liver diseases or disorders, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, drug-induced liver failure, hepatitis, liver fibrosis, cirrhosis, or a combination thereof.The DR5 agonist specifically binds to cells expressing DR5, induces the apoptosis cascade, and results in a statistically significant increase in cell death (i.e., apoptosis) when measured in at least one DR5 agonist-sensitive cell line (including but not limited to human colon cancer cell line Colo205 or human lung cancer cell line H2122).The DR5 agonist can be an antibody, apo2L / TRAIL, avimer, Fc-peptide fusion protein (e.g., peptibody), or small molecule DR5 agonist. In some embodiments, the DR5 agonist is an avimer (e.g., Nature Biotechnology 23:1556-1561 (2005)), or a human TRAIL ligand (e.g., U.S. Patent Nos. 6,284,236; and 6,998,116) DR5 agonist (e.g., U.S. Patent Application Publication Nos. 2012 / 0070432 and 2006 / 0275838). In some embodiments, the composition comprises recombinant soluble TRAIL.

[0123] In some embodiments, the composition comprises one or more DR5 agonistic antibodies.Exemplary DR5 agonistic antibodies are described by Lee H et al., Biomacromolecules 2016 17 (9), 3085-3093; Yada A et al., Ann. Oncol. 2008, 19, 1060-1067; Ichikawa, K. et al., Nat. Med. 2001, 7,954-960; Camidge, D. R et al., Clin. Cancer Res. 2010, 16, 1256-1263; Graves, J. D et al., Cancer Cell 2014, 26, 177-189.

[0124] In some embodiments, the composition comprises one or more DR5 oligomeric peptides and antibody agonists, such as those described in Li B et al., J Mol Biol 2006 Aug 18;361(3):522-36, the "anti-hDR5 peptides" and "anti-DR5 antibodies" of which are incorporated herein.

[0125] In some embodiments, the composition comprises one or more disulfide bond disrupting agents, such as those described in WangM et al., Cell Death Discovery (2019) 5:153; Ferreira, RB et al. Oncotarget8, 28971-28989 (2017); Law, ME et al. Breast Cancer Res. 18, 80 (2016); Ferreira, RB et al. Oncotarget 6, 10445-10459 (2015). In one embodiment, the composition comprises tcyDTDO, as shown below. Structure I: Chemical structure of tcyDTDO [ka]

[0126] Some small molecules that directly target DR5 to initiate apoptosis have been previously described (WangG et al., Nat Chem Biol. 2013 Feb;9(2):84-9). Thus, in some embodiments, the composition comprises one or more small molecules that directly target DR5. Some exemplary small molecules that directly target DR5 are listed below. Structure II: Chemical structure of a small molecule that directly targets DR5 [ka] 4. Anti-inflammatory

[0127] In some embodiments, the triantennary β-GalNAc-modified dendrimers are complexed with, covalently conjugated to, or inter-molecularly dispersed or encapsulated with one or more anti-inflammatory agents for treating, alleviating, or preventing one or more liver diseases or disorders, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, drug-induced liver failure, hepatitis, liver fibrosis, cirrhosis, or a combination thereof. Anti-inflammatory agents reduce inflammation and include steroidal and non-steroidal drugs.

[0128] Exemplary anti-inflammatory agents include triamcinolone acetonide, fluocinolone acetonide, dexamethasone, prednisolone, prednisone, methylprednisolone, hydrocortisone acetate, cortisone, diflucortolone, difluprednate, flucinonide, alclometasone, difluprednate, triamcinolone diacetate, betamethasone, betamethasone valerate, beclomethasone, and salts and prodrugs thereof. Glucocorticoid steroidal anti-inflammatory agents include prednisone, dexamethasone, and corticosteroids such as fluocinolone acetonide and methylprednisolone.

[0129] Examples of nonsteroidal drugs can be classified as NSAIDS and COX-2 inhibitors, including ibufenac, acetylsalicylic acid, benoxaprofen, naproxen, alminoproxen, bucloxic acid, ibuprofen, celecoxib, carprofen, etodolac, flufenamic acid, flurbiprofen, indomethacin, isoxepac, ketoprofen, mefenamic acid, oxaprozen, oxpinac, parecoxib, phenylbutazone, piroxicam, sulindac, suprofen, tiaprofenic acid, tolmetin, tramadol, valdecoxib salts, and prodrugs thereof.

[0130] In preferred embodiments, the active agent is triamcinolone acetonide, prednisone, dexamethasone, or a derivative, analog, or prodrug thereof, or a pharmacologically active salt thereof. Exemplary analogs of triamcinolone acetonide, prednisone, and dexamethasone are shown below (Structure III). Structures III af: Chemical structures of analogues of triamcinolone acetonide, prednisone, and dexamethasone [ka]

[0131] In one embodiment, the anti-inflammatory agent is N-acetyl-L-cysteine. In a preferred embodiment, N-acetyl-L-cysteine is conjugated to a hydroxyl-terminated PAMAM dendrimer via a non-cleavable linkage such that little free N-acetyl-cysteine is released in vivo after administration. An exemplary synthetic route for a non-releasing (or non-cleaving) form of a dendrimer / N-acetyl-cysteine conjugate is shown in Figure 16. The non-releasing form of the dendrimer / N-acetyl-cysteine conjugate provides enhanced therapeutic efficacy compared to the releasing or cleaving form of the dendrimer / N-acetyl-cysteine conjugate.

[0132] Exemplary immunomodulatory agents include cyclosporine, tacrolimus, and rapamycin. In some embodiments, the anti-inflammatory agent is a biologic that blocks the action of one or more types of immune cells, such as T cells, or blocks proteins of the immune system, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-17-A, interleukin-12, and interleukin-23.

[0133] In some embodiments, the anti-inflammatory drug is a synthetic or natural anti-inflammatory protein. Specific antibodies can be added to immunosuppressive therapy to select immune components. In some embodiments, the anti-inflammatory drug is an anti-T cell antibody (e.g., antithymocyte globulin or antilymphocyte globulin), an anti-IL-2Rα receptor antibody (e.g., basiliximab or daclizumab), or an anti-CD20 antibody (e.g., rituximab).

[0134] Many inflammatory diseases can be associated with pathologically elevated signaling through toll-like receptor 4 (TLR4), the receptor for lipopolysaccharide (LPS). Thus, in some embodiments, the active agent is one or more TLR4 inhibitors.

[0135] 5. Anti-fibrotic agents In some embodiments, the triantennary β-GalNAc-modified dendrimer is complexed with, covalently conjugated to, or inter-molecularly dispersed or encapsulated with, one or more antifibrotic therapeutic agents for treating, alleviating, or preventing one or more liver diseases or disorders, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, drug-induced liver failure, hepatitis, liver fibrosis, cirrhosis, or a combination thereof. In some embodiments, one or more existing antifibrotic therapeutic agents shown to be effective for treating liver fibrosis are complexed with or conjugated to the triantennary β-GalNAc-modified dendrimer to enhance delivery to and accumulation within hepatocytes. For example, exemplary antifibrotic therapeutic agents include those discussed in Cohen-Naftaly M et al., Therap Adv Gastroenterol. 4(6): 391-417(2011) and Chang Y et al., J Clin Transl Hepatol. 28; 8(2): 222-229 (2020). In some embodiments, the antifibrotic therapeutic agent is an antioxidant, an anti-TNFα, a PPARγ agonist, an IFNα agonist, an angiotensin receptor blocker, an endothelin receptor antagonist, an anticoagulant, an FXR agonist, an antibody against connective tissue growth factor, insulin, pegylated interferon, or a combination thereof. In some embodiments, the antifibrotic therapeutic agent is pentoxifylline, tocopherol, pegylated interferon 2a, etanercept, recombinant IL-10, pioglitazone, vitamin E, Lovaza (fish oil), polyenylphosphatidylcholine, obeticholic acid, infliximab, pegylated IFN α-2b, ribavirin, Peg-IFN α-2b, glycyrrhizin, candesartan, losartan, irbesartan, ambrisentan, FG-3019 (human monoclonal antibody against connective tissue growth factor), warfarin, insulin, colchicine, or a combination thereof. 6. Immunomodulators for treating liver cancer

[0136] In some embodiments, the triantennary β-GalNAc-modified dendrimers are complexed with, covalently conjugated to, or inter-molecularly dispersed or encapsulated with one or more immunomodulatory agents. The terms "immunomodulatory agent" and "immunotherapeutic agent" refer to active agents that induce a specific effect on the recipient's immune system. Immunomodulation can include suppressing, reducing, enhancing, prolonging, or stimulating one or more physiological processes of the innate immune response or the adaptive immune response to an antigen compared to a control. Typically, immunomodulatory agents can modulate the immune microenvironment for a desired immune response (e.g., increasing anti-tumor activity or increasing anti-inflammatory activity at sites where it is needed in autoimmune diseases) by targeting one or more immune cells or cell types to the target site, but are not necessarily specific for any cancer type. In some embodiments, immunomodulatory agents are specifically delivered to kill, inhibit, or reduce the activity or amount of suppressive immune cells, such as tumor-associated macrophages, for an enhanced anti-tumor response at the tumor site.

[0137] Some exemplary immunomodulatory agents for use with triantennary β-GalNAc-modified dendrimers include interferon-stimulated gene (STING) agonists, colony-stimulating factor 1 receptor (CSF1R) inhibitors, poly(ADP-ribose) polymerase (PARP) inhibitors, VEGFR tyrosine kinase inhibitors, EGFR tyrosine kinase inhibitors, MEK inhibitors, glutaminase inhibitors, TIE II antagonists, CXCR2 inhibitors, CD73 inhibitors, arginase inhibitors, phosphatidylinositol-3-kinase (PI3K) inhibitors, Toll-like receptor 4 (TLR4) agonists, TLR7 agonists, and SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase-2) inhibitors. In a preferred embodiment, the triantennary β-GalNAc-modified dendrimer is complexed to, covalently conjugated to, or inter-molecularly dispersed or encapsulated with one or more of a STING agonist, a CSF1R inhibitor, a PARP inhibitor, a VEGFR tyrosine kinase inhibitor, an EGFR tyrosine kinase inhibitor, a MEK inhibitor, a glutaminase inhibitor, a TIE II antagonist, a CXCR2 inhibitor, a CD73 inhibitor, an arginase inhibitor, a PI3K inhibitor, a TLR4 agonist, a TLR7 agonist, an SHP2 inhibitor, or a combination thereof.

[0138] These dendrimer complexes are particularly suitable for targeting one or more suppressive immune cells in tumor regions of the liver and for reducing the number of cancer cells; reducing tumor size; inhibiting cancer cell infiltration into peripheral organs; inhibiting tumor metastasis; inhibiting tumor growth; and / or alleviating one or more symptoms associated with tumors / cancer. In some embodiments, dendrimers associated or conjugated to one or more immunomodulatory agents are used in combination with anti-tumor vaccines and / or adoptive cell therapy (ACT) as adjuvants, for example, to increase innate immune gene expression, infiltration and expansion of activated effector T cells, antigen dissemination, and durable immune responses.

[0139] In some embodiments, the immunomodulatory agent is any inhibitor that targets one or more of EGFR, ERBB2, VEGFR, Kit, PDGFR, ABL, SRC, mTOR, and combinations thereof. In some embodiments, the immunomodulatory agent is one or more inhibitors and analogs thereof, such as crizotinib, ceritinib, alectinib, brigatinib, bosutinib, dasatinib, imatinib, nilotinib, ponatinib, vemurafenib, dabrafenib, ibrutinib, palbociclib, ribociclib, cabozantinib, gefitinib, erlotinib, lapatinib, vandetanib, afatinib, osimertinib, ruxolitinib, tofacitinib, trametinib, axitinib, lenvatinib, nintedanib, pazopanib, regorafenib, sorafenib, sunitinib, vandetanib, bosutinib, dasatinib, dacomitinib, ponatinib, and combinations thereof. In some embodiments, the immunomodulatory agent is a tyrosine kinase inhibitor, such as a HER2 inhibitor or an EGFR tyrosine kinase inhibitor. Exemplary EGFR tyrosine kinase inhibitors include gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib.

[0140] The additional immunomodulatory agent can include one or more cytotoxic agents that are toxic to one or more immune cells, thus killing or inhibiting one or more types of suppressive immune cells. When selectively delivered to target immune cells, such as when conjugated to a dendrimer, these agents can selectively kill suppressive immune cells, thus altering the immunological microenvironment within and around the tumor. Cytotoxic immunomodulatory agents include auristatin E and mertansine. STING agonists

[0141] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more interferon-stimulated gene (STING) agonists. STING is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides (CDNs) and activates the TBK1 / IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce robust type I interferon and pro-inflammatory cytokine responses. STING is required for the induction of antitumor CD8 T responses in mouse cancer models. In the tumor microenvironment, T cells, endothelial cells, and fibroblasts stimulated ex vivo by STING agonists produce type I IFNs (Corrales, et al., Cell Rep (2015) 11(7):1018-30). In contrast, most studies have shown that tumor cells can inhibit STING pathway activation, possibly resulting in immune evasion during carcinogenesis (He, et al., Cancer Lett (2017) 402:203-12; Xia, et al., Cancer Res (2016)76(22):6747-59). Thus, in some embodiments, the dendrimer is associated with or conjugated to one or more STING agonists or analogs thereof. Exemplary STING agonists include cyclic dinucleotides such as 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) and DMXAA (also known as badimesan or ASA404). In one embodiment, a triantennary β-GalNAc-modified dendrimer is associated with or conjugated to DMXAA or a derivative, analog, or prodrug thereof. In a preferred embodiment, the complex or conjugate of triantennary β-GalNAc and DMXAA is effective to induce one or more of TNF-α, IP-10, IL-6, IFN-β, and RANTES at a target site.

[0142] In some embodiments, the STING agonist is functionalized with one or more spacers / linkers, such as ether, ester, or amide linkages, to facilitate conjugation with the dendrimer and / or for desired release kinetics. For example, DMXAA can be modified into a DMXAA analog, such as a DMXAA ester, DMXAA ether, or DMXAA amide. In a preferred embodiment, the STING agonist or its derivative, analog, or prodrug is conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry, optionally via one or more spacers / linkers, such as polyethylene glycol (PEG). An exemplary conjugation of a STING agonist, such as DMXAA, with a dendrimer, such as a fourth- or sixth-generation PAMAM dendrimer, is shown in Figure 1.

[0143] In preferred examples, dendrimer complexes comprising one or more STING agonists are administered in an amount effective to induce / enhance IFN-β production by tumor-infiltrating APCs (e.g., CD11c+CD11b− or CD11c+CD11b+ cells), induce / enhance one or more of TNF-α, IP-10, IL-6, IFN-β, and RANTES, inhibit tumor growth, reduce tumor size, increase long-term survival, improve response to immune checkpoint blockade, and / or induce immunological memory that protects against tumor re-challenge. Colony-stimulating factor 1 receptor (CSF1R) inhibitors

[0144] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more colony-stimulating factor 1 receptor (CSF1R) inhibitors. CSF1R belongs to the type III protein tyrosine kinase receptor family, and binding of CSF1 or its more recently identified ligand, IL-34, induces receptor homodimerization and subsequent activation of receptor signaling (Achkova D, Maher J. Biochem Soc Trans. (2016) 44:333-41). CSF1 receptor (CSF1R)-mediated signaling is crucial for the differentiation and survival of monocytic cell lines, and in particular macrophages (Stanley ER, Chitu V. Cold Spring Harb Perspect Biol (2014), 6(6)). Because the intratumoral presence of CSF1R+ macrophages correlates with poor survival in various tumor types (Pedersen MB, et al., Histopathology. (2014), 65:490-500; Zhang QW et al., PLoS One. (2012), 7:e50946), targeting CSF1R signaling in tumor-promoting TAMs represents an attractive strategy to eliminate or repolarize these cells. In addition to TAMs, CSF1R expression can be detected in other myeloid cells within the tumor microenvironment, such as dendritic cells, neutrophils, and myeloid-derived suppressor cells (MDSCs).

[0145] A variety of small molecules and monoclonal antibodies (mAbs) directed against CSF1R or its ligand, CSF1, are in clinical development as monotherapy and in combination with standard treatment modalities, such as chemotherapy and other cancer immunotherapy approaches. Among the small molecule class, pexidartinib (PLX3397), an oral tyrosine kinase inhibitor of CSF1R, cKIT, mutant fms-like tyrosine kinase 3 (FLT3), and platelet-derived growth factor receptor (PDGFR)-β, is the subject of the most extensive clinical development program as a monotherapy, with completed and ongoing trials in c-kit-mutant melanoma, prostate cancer, glioblastoma (GBM), classical Hodgkin lymphoma (cHL), neurofibromatosis, sarcoma, and leukemia. Additional CSF1R-targeting small molecules, including ARRY-382, PLX7486, BLZ945, and JNJ-40346527, are currently being tested in solid tumors and cHL. mAbs in clinical development include emactuzumab (RG7155), AMG820, IMC-CS4 (LY3022855), cabilalizumab, MCS110, and PD-0360324, the latter two being compounds that target the CSF1 ligand. The phrase "CSF1R inhibitor" is used as a general term for both receptor-targeted and ligand-targeted compounds.

[0146] Thus, in some embodiments, the triantennary β-GalNAc-modified dendrimer is associated with or conjugated to one or more agents for reducing or inhibiting the activity of the CSF1R signaling pathway, such as one or more CSF1R inhibitors or one or more compounds targeting the ligand CSF1. In some embodiments, the dendrimer is associated with or conjugated to one or more small molecule CSF1R inhibitors or analogs thereof. Exemplary small molecule CSF1R inhibitors are provided in Current Medicinal Chemistry, 2019, 26, 1-23. Exemplary CSF1R-targeting small molecules include pexidartinib (PLX3397, PLX108-01), ARRY-382, PLX7486, BLZ945, JNJ-40346527, and GW2580. The small molecule CSF1R inhibitor can be functionalized with one or more spacers / linkers, for example, by ether, ester, or amide linkages, as needed to facilitate conjugation to the dendrimer and / or for desired release kinetics. In a preferred embodiment, the small molecule CSF1R inhibitor, or a derivative, analog, or prodrug thereof, is conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry, as needed, via one or more spacers / linkers, for example, polyethylene glycol (PEG).

[0147] The chemical structures of exemplary CSF1R-targeting small molecules or analogs thereof suitable for conjugation to dendrimers are shown below. Structure IV: Chemical structure of CSF1R inhibitor 1 [ka] Structure V: Chemical structure of CSF1R inhibitor 2 [ka] Structure VI: Chemical structure of CSF1R inhibitor 3 [ka] Structure VII: Chemical structure of CSF1R inhibitor 4 [ka] Structure VIII: Chemical structure of CSF1R inhibitor 5 [ka] Structure IX: Chemical structure of CSF1R inhibitor 6 [ka] Structure X a-b: Chemical structures of a) CSF1R-E analogs and b) dendrimer-conjugated CSF1R-E [ka] Structure XI: Chemical structure of CSF1R-E analogue 1 [ka]

[0148] The binding affinity of CSF1R-E analog 1 (structure XI) is about 13 nm, and the binding affinity of dendrimer-conjugated CSF1R-E analog 1 (e.g., via alkyne-azide click chemistry) is about 200 nm. Thus, in preferred embodiments, the CSF1R inhibitor is conjugated to a dendrimer with or without a spacer to minimize reduction in binding affinity to CSF1R, e.g., by less than 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or 100-fold. Structure XII: Chemical structure of CSF1R inhibitor F [ka]

[0149] Exemplary CSF1R-targeting mAbs include emactuzumab (RG7155), AMG820, IMC-CS4 (LY3022855), and cabilalizumab. Exemplary mAbs target the ligands CSF1MCS110 and PD-0360324.

[0150] In a preferred embodiment, the dendrimer is conjugated to one or more CSF1R tyrosine kinase inhibitors, such as GW2580 (shown as Structure X). The CSF1R inhibitor can be functionalized with one or more spacers / linkers, such as ether, ester, or amide linkages, as needed to facilitate conjugation to the dendrimer and / or for desired release kinetics. For example, GW2580 can be modified into GW2580 analogs, including GW2580 ethers, GW2580 esters, and GW2580 amides. In a preferred embodiment, GW2580 or a derivative, analog, or prodrug thereof is conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry, as needed via one or more spacers / linkers, such as polyethylene glycol (PEG). Exemplary strategies for conjugating a CSF1R inhibitor, such as GW2580, to a dendrimer are shown in Figures 17A and 17B. Structure XIII: Chemical structure of GW2580 [ka]

[0151] In one embodiment, the dendrimer is conjugated to a CSF1R inhibitor or analog thereof having the following structure: Structure XIV: Chemical structure of AR004 [ka]

[0152] The synthetic route for the AR004-conjugated dendrimer is shown in FIG. Poly(ADP-ribose) polymerase (PARP) inhibitors

[0153] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated to one or more poly(ADP-ribose) polymerase (PARP) inhibitors. Poly(ADP-ribose) polymerase (PARP) is a family of 17 nuclear proteins characterized by a common catalytic site that transfers ADP-ribose groups onto specific acceptor proteins using NAD+ as a cofactor. Poly(ADP-ribose) polymerase (PARP) inhibitors

[0154] Olaparib (C 24 H 23 FN4O3) was the first PARP inhibitor introduced into clinical practice. Niraparib is a potent and selective inhibitor of PARP-1 and PARP-2. Rucaparib, as a single agent, is a potent PARP inhibitor approved by the FDA in December 2016 and by the EMA in May 2018 for the treatment of patients with gBRCAm or sBRCAm HGSOC who have relapsed after at least two lines of chemotherapy.

[0155] In some embodiments, the dendrimer comprises one or more PARP inhibitors, such as olaparib, niraparib, and rucaparib. The PARP inhibitor can be functionalized with one or more spacers / linkers, such as ether, ester, or amide linkages, to facilitate conjugation with the dendrimer and / or for desired release kinetics. In a preferred embodiment, the PARP inhibitor, or its derivative, analog, or prodrug, is conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry, optionally via one or more spacers / linkers, such as polyethylene glycol (PEG). VEGFR tyrosine kinase inhibitors

[0156] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more VEGFR tyrosine kinase inhibitors. Tyrosine kinases are important cell signaling proteins with diverse biological activities, including cell proliferation and migration. Several kinases, including receptor tyrosine kinases, such as vascular endothelial growth factor receptors (VEGFRs), are involved in angiogenesis. Antiangiogenic tyrosine kinase inhibitors currently under clinical development primarily target VEGFR-1, -2, -3, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), PDGFR-β, KIT, fms-related tyrosine kinase 3 (FLT3), colony-stimulating factor-1 receptor (CSF-1R), Raf, and RET.

[0157] The VEGFR family includes three related receptor tyrosine kinases known as VEGFR-1, -2, and -3, which mediate the angiogenic effects of VEGF ligands (Hicklin DJ, Ellis LM. J Clin Oncol. (2005), 23(5):1011-27). The VEGF family encoded in mammalian genomes includes five members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). VEGF is an important stimulator of endothelial cell proliferation and migration. VEGF-A (commonly referred to as VEGF) is a major mediator of tumor angiogenesis and transmits signals through VEGFR-2, the main VEGF signaling receptor (Kerbel RS, N Engl J Med. (2008), 358(19):2039-49).

[0158] The most notable angiogenesis inhibitors target the vascular endothelial growth factor (VEGF) signaling pathway, such as the monoclonal antibody bevacizumab (Avastin, Genentech / Roche) and the two kinase inhibitors sunitinib (SU11248, Sutent, Pfizer) and sorafenib (BAY43-9006, Nexavar, Bayer). Bevacizumab was the first angiogenesis inhibitor to receive clinical approval, initially for the treatment of colorectal cancer and more recently for the treatment of breast and lung cancer. The small-molecule tyrosine kinase inhibitors sunitinib and sorafenib target VEGF receptors (VEGFRs), primarily VEGFR-2, and have demonstrated clinical efficacy in a variety of cancer types. Both drugs have shown benefit in patients with renal cell carcinoma (Motzer RJ, Bukowski RM, J Clin Oncol. (2006); 24(35):5601-8). Additionally, sunitinib has been approved for the treatment of gastrointestinal stromal tumors (GISTs). Sorafenib also inhibits Raf serine kinase and has been approved for the treatment of hepatocellular carcinoma. Cediranib is an oral tyrosine kinase inhibitor of VEGF receptors (VEGFRs).

[0159] In some embodiments, the dendrimer is conjugated to one or more VEGF receptor inhibitors, including sunitinib (SU11248; SUTENT®), sorafenib (BAY439006; NEXAVAR®), pazopanib (GW786034; VOTRIENT®), vandetanib (ZD6474; ZACTIMA®), axitinib (AG013736), cediranib (AZD2171; RECENTIN®), vatalanib (PTK787; ZK222584), dasatinib, nintedanib, and motesanib (AMG706), or analogs thereof.

[0160] In some embodiments, the VEGF receptor inhibitor can be functionalized with one or more spacers / linkers, for example, ether, ester, or amide linkages, as needed, to facilitate conjugation with the dendrimer and / or for desired release kinetics. In a preferred embodiment, one or more VEGF receptor inhibitors, or their derivatives, analogs, or prodrugs, are conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry, as needed, via one or more spacers / linkers, for example, polyethylene glycol (PEG). For example, sunitinib can be modified to sunitinib with an ester linkage or an amide linkage (FIGS. 3A and 3B). Exemplary conjugations of a VEGF receptor inhibitor, such as sunitinib, with a dendrimer are shown in FIG. 3A (via a hydroxymethyl linkage) and 3B (via an amide linkage). In one embodiment, the sunitinib analog is N,N-didesethyl sunitinib.

[0161] Exemplary VEGF receptor inhibitor analogs with functional spacers / linkages are shown below in Structures XV, XVI, and XVII. Structure XV ab: Chemical structures of sorafenib analogues [ka] Structure XVI a-d: Chemical structures of nintedanib and analogues [ka] Structure XVII: Chemical structure of oratinib analogues [ka] MEK inhibitors

[0162] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more MEK inhibitors. The mitogen-activated protein kinase (MAPK) cascade is an important pathway for human cancer cell survival, dissemination, and resistance to drug therapy. The MAPK / ERK (extracellular signal-regulated kinase) pathway is a convergent signaling node that receives input from multiple stimuli, including internal metabolic stress and DNA damage pathways and altered protein concentrations, as well as input through signal transduction from external growth factors, cell-matrix interactions, and communication with other cells.

[0163] In some embodiments, the dendrimer is conjugated to one or more MEK inhibitors. Exemplary MEK inhibitors include refametinib, pimasertib, trametinib (GSK1120212), cobimetinib (or XL518), binimetinib (MEK162), selumetinib, CI-1040 (PD-184352), PD325901, PD035901, PD032901, and TAK-733, or analogs thereof. In preferred embodiments, the MEK inhibitor is optionally functionalized with one or more spacers / linkers, for example, by ether, ester, or amide linkages, to facilitate conjugation with the dendrimer and / or for desired release kinetics. In a preferred embodiment, the MEK inhibitor or its derivative, analog, or prodrug is conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry, optionally via one or more spacers / linkers, such as polyethylene glycol (PEG).For example, binimetinib can be modified into binimetinib ester, binimetinib ether, or binimetinib amide; trametinib can be modified into trametinib ether, trametinib ester, or trametinib amide; pimasertib can be modified into pimasertib ester and pimasertib ether. Exemplary MEK inhibitors and analogs thereof are shown below: binimetinib functionalized with a PEG linker and an azide group via an ester linkage (structure XVIII) and an ether linkage (structure XIX); a trametinib analog functionalized with a PEG linker and an azide group via an amide linkage (structure XX); and a pimasertib analog functionalized with a PEG linker and an azide group via an ester linkage (structure XXI). Structure XVIII: Chemical structure of binimetinib analogue 1 [ka] Structure XIX: Chemical structure of binimetinib analogue 2 [ka] Structure XX: Chemical structure of trametinib analogues [ka] Structure XXI: Chemical structure of pimasertib analogues [ka] glutaminase inhibitors

[0164] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more glutaminase inhibitors. Glutaminase (GLS), which is involved in the conversion of glutamine to glutamate, plays an essential role in the upregulation of cellular metabolism for tumor cell growth. Exemplary glutaminase inhibitors include bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethylsulfinide (BPTES), 6-diazo-5-oxo-L-norleucine (DON), azaserine, acivicin, and CB-839. In some embodiments, the glutaminase inhibitor is a BPTES analog, such as JHU-198, JHU-212, and JHU-329 (Thomas AG et al., Biochem Biophys Res Commun. (2014); 443(1): 32-36).

[0165] In some embodiments, the dendrimer is conjugated to one or more glutaminase inhibitors. Exemplary glutaminase inhibitors include BPTES, DON, azaserine, acivicin, CB-839, JHU-198, JHU-212, and JHU-329. The glutaminase inhibitor can be functionalized with one or more spacers / linkers, for example, via ether, ester, or amide linkages, to facilitate conjugation with the dendrimer and / or for desired release kinetics. In a preferred embodiment, the glutaminase inhibitor or its derivatives, analogs, or prodrugs are conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry, optionally via one or more spacers / linkers, for example, polyethylene glycol (PEG). In a preferred embodiment, the dendrimer is conjugated to CB-839 or its derivatives, analogs, or prodrugs, or a pharmacologically active salt thereof. CB-839 has the following structure: Structure XXII: Chemical structure of CB-839 [ka]

[0166] In some embodiments, the dendrimer is conjugated to a glutamine analog or the antagonist L-[αS,5S]-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin), or a derivative, analog, or prodrug thereof, or a pharmacologically active salt thereof. The chemical structure of acivicin is shown below in Structure XXIII. Structure XXIII: [ka]

[0167] Acivicin is currently undergoing clinical trials for the treatment of cancer. Dosages and formulations are known in the art, see, for example, Hidalgo, Clinical Cancer Research, 4(11): 2763-2770 (1998), U.S. Patent Nos. 3,856,807, 3,878,047, and 5,087,639. In one embodiment, a dendrimer is conjugated to acivicin. In a preferred embodiment, acivicin is functionalized, for example, by an ether, ester, N-alkyl, or amide linkage, optionally with one or more spacers / linkers, such as polyethylene glycol (PEG), before being conjugated to the dendrimer. TIE II antagonists

[0168] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more TIE II antagonists.The angiopoietin-1 receptor, also known as CD202B (CD classification 202B), is a protein encoded by the TEK gene in humans.Similarly, the angiopoietin receptor, also known as TIE2, is known as angiopoietin.Angiopoietin is a protein growth factor necessary for the formation of blood vessels (angiogenesis) that support tumor growth and development.Therefore, in some embodiments, the dendrimer is conjugated with one or more TIE II antagonists.

[0169] The TIE II antagonist can be functionalized with one or more spacers / linkers, for example, by ether, ester, or amide linkages, as needed, to facilitate conjugation with, for example, a dendrimer, and / or for desired release kinetics. The chemical structure of an exemplary TIE II inhibitor is shown below in Structure XXIV. TIE II inhibition by the free TIE II antagonist is achieved with a dissociation constant K of about 8.8 nm. dand the TIE II inhibition of the dendrimer-conjugated TIE II antagonist (structure XXIV) has a dissociation constant K of approximately 25 nm. d Thus, in preferred embodiments, the TIE II antagonist is conjugated to the dendrimer with or without a spacer to minimize reduction in TIE II inhibition, e.g., less than 1-fold, less than 2-fold, less than 3-fold, less than 4-fold, less than 5-fold, less than 10-fold, less than 20-fold, less than 30-fold, less than 40-fold, less than 50-fold, and less than 100-fold. Structure XXIV: TIE II antagonist 1 [ka]

[0170] In some embodiments, dendrimers are complexed or conjugated with two or more different classes of active agents to provide simultaneous delivery with different or independent release kinetics at the target site. In one embodiment, a fourth- or sixth-generation PAMAM dendrimer is conjugated to a TIE II inhibitor and gemcitabine, or an analog thereof. In another embodiment, a fourth- or sixth-generation PAMAM dendrimer is conjugated to a TIE II inhibitor and capecitabine, or an analog thereof. Exemplary synthetic routes for dendrimers conjugated to two or more different classes of active agents are shown in Figures 13A-13C. CXCR2 inhibitors

[0171] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more CXCR2 inhibitors. CXCR2 is expressed by many tumor cells and has been implicated in chemotherapy resistance in various preclinical cancer models (Poeta VM et al., Front Immunol. 2019;10:379). In breast cancer cells, CXCR2 deletion resulted in a favorable response to paclitaxel. In melanoma models, the CXCR2 inhibitor navarixin showed synergistic effects with MEK inhibitors, while in ovarian tumor models, the CXCR2 inhibitor SB225002 improved the angiogenic effects of sorafenib. In human gastric cancer, reparixin, a CXCR1 and CXCR2 inhibitor, enhanced the efficacy of 5-fluorouracil.

[0172] Targeting CXCR2 also inhibits tumor growth by affecting myeloid cell infiltration. In pancreatic tumors, CXCR2 blockade prevented the accumulation of neutrophils that unleash T cell responses, inhibited metastatic dissemination, and resulted in improved responses to anti-PD-1. Interestingly, the combined response of CXCR2 and CCR2 inhibitors limited the complementary response of TAMs, increased antitumor immunity, and improved responses to FX. Finally, in a prostate cancer model, CXCR2 blockade with SB265610 reduced myeloid cell recruitment, enhanced docetaxel-induced senescence, and limited tumor growth.

[0173] Thus, in some embodiments, the dendrimer is associated with or conjugated to one or more CXCR2 inhibitors. Exemplary CXCR2 inhibitors include navarixin, SB225002, SB332235, SB265610, reparixin, and AZD5069. In a preferred embodiment, the dendrimer is conjugated to navarixin, SB225002, or SB332235, or a derivative, analog, or prodrug thereof, or a pharmacologically active salt thereof. The CXCR2 inhibitor can be functionalized, for example, by an ether, ester, N-alkyl, or amide linkage to facilitate conjugation with the dendrimer and / or for desired release kinetics. In some embodiments, the CXCR2 inhibitor is conjugated to the dendrimer via an N-alkyl linkage using click chemistry. CD73 inhibitors

[0174] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed with or conjugated to one or more CD73 inhibitors. CD73 converts extracellular adenosine monophosphate (AMP) into immunosuppressive adenosine, which blocks antitumor immune surveillance at the level of T cells and natural killer (NK) cells, dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). In cancer, upregulation of CD73 expression in tumor cells and tumor stromal cells leads to increased adenosine production, which inhibits the cytotoxicity, cytokine production, and proliferation of T cells and NK cells, and suppresses antigen-presenting cells (APCs); enhances regulatory T cell (Treg) proliferation and suppressive activity, and MDSC and macrophage M2 polarization. These changes enable tumor growth and disease progression.

[0175] Thus, in some embodiments, the dendrimer is conjugated to one or more CD73 inhibitors. Exemplary CD73 inhibitors include non-hydrolyzable AMP analogs, such as adenosine 5'-(α,β-methylene)diphosphate (APCP), flavonoid-based compounds, such as quercetin, and purine nucleotide analogs, such as tenofovir and sulfonic acid compounds. In preferred embodiments, the dendrimer is conjugated to one or more CD73 inhibitors, including APCP, quercetin, or tenofovir, or a derivative, analog, or prodrug thereof, or a pharmacologically active salt thereof. The CD73 inhibitor can be functionalized with one or more spacers / linkers, for example, by ether, ester, or amide linkages, as needed, to facilitate conjugation to the dendrimer and / or for desired release kinetics. In preferred embodiments, the CD73 inhibitor, or a derivative, analog, or prodrug thereof, is conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry.

[0176] In some embodiments, one or more CD73 inhibitors and / or derivatives or analogs thereof having the structures shown in Structures XXV a-i and XXVI a-c below are suitable for conjugation to a dendrimer. Structure XXV ai: Structures of CD73 inhibitors and their analogs [ka] Structure XXVI a-c: Structures of CD73 inhibitors and their analogues [ka] Arginase inhibitors

[0177] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more arginase inhibitors.The expression of the enzyme arginase 1 (Arg1) is a distinct characteristic of immunosuppressive myeloid cells, resulting in the depletion of L-arginine, a nutrient necessary for T cell and natural killer (NK) cell proliferation.Therefore, blocking Arg1 activity in cancer situations can shift the balance of L-arginine metabolism to favor lymphocyte proliferation.In fact, in mouse studies, injection of the arginase inhibitor nor-NOHA or genetic disruption of Arg1 in the myeloid compartment results in reduced tumor growth, indicating that Arg1 is pro-tumorigenic.

[0178] Thus, in some embodiments, the dendrimer is associated with or conjugated to one or more arginase inhibitors. In some embodiments, the one or more arginase inhibitors are boronic acid-based arginase inhibitors, such as derivatives of 2-(S)-amino-6-boronohexanoic acid (ABH) (Borek B et al., Bioorg Med Chem. 2020 Sep 15;28(18):115658), or derivatives, analogs, or prodrugs thereof, or pharmacologically active salts thereof. In a preferred embodiment, the dendrimer is conjugated to one or more arginase inhibitors, or derivatives, analogs, or prodrugs thereof, or pharmacologically active salts thereof. The arginase inhibitor can be functionalized with one or more spacers / linkers, for example, by ether, ester, amine, or amide linkages, as needed, to facilitate conjugation with the dendrimer and / or for desired release kinetics. In a preferred embodiment, the arginase inhibitor, or a derivative, analog, or prodrug thereof, is conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry.

[0179] In some embodiments, one or more arginase inhibitors and / or derivatives or analogs thereof having the structures shown in Structures XXVII a-g and Structures XXVIII a-h below are conjugated to a dendrimer. Structure XXVII ag: Structures of arginase inhibitors and their analogues [ka] Structure XXVIII a-h: Structures of arginase inhibitors and their analogues [ka] Phosphatidylinositol-3-kinase (PI3K) inhibitors

[0180] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more PI3K inhibitors. Dysregulation of PI3K / PTEN pathway components, resulting in hyperactive PI3K signaling, is frequently observed in various cancers and correlates with tumor growth and survival. Resistance to various anticancer treatments, including receptor tyrosine kinase (RTK) inhibitors and chemotherapeutic agents, is due to the absence or attenuation of downregulating signals along the PI3K / PTEN pathway. Macrophage PI3-kinase γ controls the critical switch between immune stimulation and suppression during inflammation and cancer. PI3Kγ signaling through Akt and mTor inhibits NFκB activation but stimulates C / EBPβ activation, thereby inducing a transcriptional program that promotes immune suppression during inflammation and tumor growth. In contrast, selective inactivation of macrophage PI3Kγ stimulates and prolongs NFκB activation, inhibits C / EBPβ activation, and thus promotes an immune-stimulatory transcriptional program that restores CD8+ T cell activation and cytotoxicity.

[0181] Thus, in some embodiments, the dendrimer is associated with or conjugated to one or more PI3K inhibitors. In preferred embodiments, the dendrimer is associated with or conjugated to one or more PI3K gamma inhibitors. Exemplary PI3K inhibitors include BYL719 (alpelisib), INK1117 (ceravelisib, MLN-1117, or TAK-117), XL147 (SAR245408), pilaralisib, WX-037, NVP-BEZ235 (dactolisib or BEZ235), LY3023414 (prexasertib), XL765 (voxtalisib or SAR245409), PX-866, ZSTK474, NVP-BKM120 (buparlisib), GDC-0941 (pictilisib), and BAY80-6946 (copanlisib). The PI3K inhibitor can be functionalized with one or more spacers / linkers, for example, via ether, ester, or amide linkages, as needed, to facilitate conjugation with the dendrimer and / or for desired release kinetics. In a preferred embodiment, the PI3K inhibitor, or its derivative, analog, or prodrug, is conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry, as needed, via one or more spacers / linkers, for example, polyethylene glycol (PEG). The chemical structures of exemplary PI3K inhibitors are shown below in Structure XXIX and Structure XXX. Structure XXIX a-k: Structures of PI3K inhibitors and their analogues [ka] Structures XXX af: Structures of PI3K inhibitors and their analogs [ka] Toll-like receptor 4 (TLR4) and TLR7 agonists

[0182] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more Toll-like receptor 4 (TLR4) agonists and / or Toll-like receptor 7 (TLR7) agonists. TLRs play a crucial role in activating immune responses. TLRs recognize conserved pathogen-associated molecular patterns (PAMPs) expressed on a wide range of microorganisms, as well as endogenous DAMPs released by stressed or dying cells.

[0183] In some embodiments, the dendrimer is associated with or conjugated to one or more TLR4 agonists. Exemplary TLR4 agonists include the synthetic toll-like receptor 4 agonists glucopyranosyl lipid A, Bacillus Calmette-Guerin (BCG), and monophosphoryl lipid A (MPLA). The TLR4 agonist can be functionalized with one or more spacers / linkers, for example, by ether, ester, or amide linkages, as needed to facilitate conjugation with the dendrimer and / or for desired release kinetics. In some embodiments, the dendrimer is a fourth-, fifth-, or sixth-generation hydroxyl-terminated PAMAM dendrimer. In a preferred embodiment, the TLR4 agonist, or a derivative, analog, or prodrug thereof, is conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry, optionally via one or more spacers / linkers, such as polyethylene glycol (PEG). Exemplary TLR4 agonists or analogs thereof are shown below. Structure XXXI ab: Structures of two TLR4 agonist analogs [ka]

[0184] Chemical synthesis routes for exemplary dendrimer-conjugated TLR4 agonists are shown in Figures 14A and 14B.

[0185] In some embodiments, the dendrimer is associated with or conjugated to one or more TLR7 agonists. Exemplary TLR7 agonists include imiquimod, resiquimod, gardiquimod, 852A, loxoribine, bropirimine, 3M-011, 3M-052, DSR-6434, DSR-29133, SC1, SZU-101, SM-276001, and SM-360320. In a preferred embodiment, the TLR agonist is resiquimod. The TLR7 agonist can be functionalized with one or more spacers / linkers, for example, by ether, ester, or amide linkage, as needed, to facilitate conjugation with the dendrimer and / or for desired release kinetics.

[0186] In some embodiments, dendrimers associated with or conjugated to one or more TLR4 or TLR7 agonists are used in combination with anti-tumor vaccines and / or adoptive cell therapy (ACT) as adjuvants, e.g., to increase innate immune gene expression, infiltration and expansion of activated effector T cells, antigen presentation, and durable immune responses. SHP2 inhibitors

[0187] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed or conjugated with one or more SHP2 inhibitors. SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase-2) is a non-receptor protein tyrosine phosphatase that removes tyrosine phosphorylation. Functionally, SHP2 acts as a key hub connecting several intracellular oncogenic signaling pathways, such as the Jak / STAT, PI3K / AKT, RAS / Raf / MAPK, and PD-1 / PD-L1 pathways. Mutations and / or overexpression of SHP2 are associated with genetic developmental diseases and cancer.

[0188] Thus, in some embodiments, the dendrimer is associated with or conjugated to one or more SHP2 inhibitors, or derivatives, analogs, or prodrugs thereof, or pharmacologically active salts thereof. Exemplary SHP2 inhibitors include inhibitors that target the catalytic site of SHP2 and inhibitors that target the allosteric site of SHP2, such as TNO155, RMC-4630, JAB-3068, JAB-3312, and RMC-4550. The SHP2 inhibitor can be functionalized with one or more spacers / linkers, for example, ether, ester, or amide linkages, as needed, to facilitate conjugation with the dendrimer and / or for desired release kinetics. In some embodiments, the dendrimer is a fourth-, fifth-, or sixth-generation hydroxyl-terminated PAMAM dendrimer. In a preferred embodiment, the SHP2 inhibitor, or a derivative, analog, or prodrug thereof, is conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry, optionally via one or more spacers / linkers, such as polyethylene glycol (PEG). Exemplary SHP2 inhibitors or analogs thereof are shown below. Structure XXXII ab: Structures of two SHP2 inhibitor analogues [ka]

[0189] Some exemplary immunomodulatory agents for use with dendrimers also include STING antagonists, JAK1 inhibitors, and anti-inflammatory agents. In preferred embodiments, dendrimers associated with or conjugated to one or more immunomodulatory agents, including STING antagonists, JAK1 inhibitors, and anti-inflammatory agents, are particularly suitable for targeting one or more pro-inflammatory immune cells.

[0190] 7. Additional agents for liver cancer In some embodiments, the triantennary β-GalNAc-modified dendrimers are complexed with, covalently conjugated to, or inter-molecularly dispersed or encapsulated with one or more additional therapeutic agents, including conventional cancer therapeutics, such as chemotherapeutics, cytokines, chemokines, and radiation therapy. Most chemotherapy drugs can be classified as alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. These drugs affect cell division or DNA synthesis and function in some way. Additional therapeutic agents include monoclonal antibodies and tyrosine kinase inhibitors, such as imatinib mesylate (GLEEVEC® or GLIVEC®), which directly target molecular abnormalities in certain types of cancer (chronic myeloid leukemia, gastrointestinal stromal tumors).

[0191] In some embodiments, the triantennary GalNAc-modified dendrimer is complexed with, covalently conjugated to, inter-molecularly dispersed within, or encapsulated within, one or more chemotherapeutic agents. Representative chemotherapeutic agents include amsacrine, bleomycin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxin, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, mechlorethamine, melphalan, mercaptopurine, mesna, and mesna. These include, but are not limited to, thotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, taxol, trichostatin A and its derivatives, trastuzumab (HERCEPTIN®), cetuximab, and rituximab (RITUXAN® or MABTHERA®), bevacizumab (AVASTIN®), and combinations thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabine, taurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2)5, and combinations thereof.

[0192] In one embodiment, the tri-antennary GalNAc-modified dendrimer is covalently conjugated to capecitabine via a spacer such as PEG, preferably via an ester, ether, or amide linkage.

[0193] In another embodiment, the tri-antennary GalNAc-modified dendrimer is covalently conjugated to gemcitabine via a spacer such as PEG, preferably via an ester, ether, or amide linkage.

[0194] In some embodiments, the active agent is a histone deacetylase (HDAC) inhibitor. In one embodiment, the active agent is vorinostat. In other embodiments, the active agent is a topoisomerase I and / or II inhibitor. In certain embodiments, the active agent is etoposide or camptothecin.

[0195] Additional anticancer agents include, but are not limited to, irinotecan, exemestane, octreotide, carmofur, clarithromycin, zinostatin, tamoxifen, tegafur, toremifene, doxifluridine, nimustine, vindesine, nedaplatin, pirarubicin, flutamide, fadrozole, prednisone, medroxyprogesterone, mitotane, mycophenolate mofetil, and mizoribine.

[0196] Representative anti-angiogenic agents include antibodies against vascular endothelial growth factor (VEGF), such as bevacizumab (AVASTIN®) and rhuFAb V2 (ranibizumab, LUCENTIS®), and other anti-VEGF compounds, including aflibercept (EYLEA®); MACUGEN® (pegaptanim sodium, an anti-VEGF aptamer, or EYE001) (Eyetech Pharmaceuticals; pigment epithelium-derived factor(s) (PEDF); COX-2 inhibitors, such as celecoxib (CELEBREX®) and rofecoxib (VIOXX®); interferon alpha; interleukin-12 (IL-12); thalidomide (THALOMID®) and its derivatives, such as lenalidomide (REVLIMID®); squalamine; endostatin; angiostatin; ribozyme inhibitors, such as ANGIOZYME® (Sirna Therapeutics); multifunctional antiangiogenic agents, such as NEOVASTAT® (AE-941) (Aeterna Laboratories, Quebec City, Canada); receptor tyrosine kinase (RTK) inhibitors, such as sunitinib (SUTENT®); tyrosine kinase inhibitors, such as sorafenib (Nexavar®) and erlotinib (Tarceva®); antibodies against epidermal growth factor receptors, such as panitumumab (VECTIBIX®) and cetuximab (ERBITUX®), and other anti-angiogenic agents known in the art.

[0197] In some instances, the active agent is an anti-infective agent. Exemplary anti-infective agents include antiviral agents, antibacterial agents, antiparasitic agents, and antifungal agents. Exemplary antibiotics include moxifloxacin, ciprofloxacin, erythromycin, levofloxacin, cefazolin, vancomycin, tigecycline, gentamicin, tobramycin, ceftazidime, ofloxacin, gatifloxacin; antifungal agents; amphotericin, voriconazole, and natamycin.

[0198] Any additional active compound can be functionalized with one or more spacers / linkers, for example, ether, ester, ethyl, or amide linkages, to facilitate conjugation to the dendrimer and / or for desired release kinetics. In a preferred embodiment, the active agent or its derivatives, analogs, or prodrugs are conjugated to the dendrimer via Cu(I)-catalyzed alkyne-azide click chemistry or thiol-ene click chemistry, optionally via one or more spacers / linkers, for example, polyethylene glycol (PEG). In some embodiments, the additional active agent is a chemotherapeutic agent or its derivatives, analogs, or prodrugs, or a pharmacologically active salt thereof. In one embodiment, the active agent complexed with or conjugated to the dendrimer is, for example, methotrexate, as shown in Structure XXXIII, or a derivative, analog, or prodrug thereof, or a pharmacologically active salt thereof. Structure XXXIII: Chemical Structures of Methotrexate Analogues [ka] 8. Agents for treating hypertension and other disorders

[0199] In some embodiments, the dendrimers are used to deliver one or more additional active agents, particularly one or more therapeutic, prophylactic, and / or diagnostic agents, to prevent or treat one or more symptoms of liver injury and / or related diseases or conditions, such as infection, sepsis, diabetic complications, hypertension, obesity, high blood pressure, heart failure, kidney disease, and cancer.

[0200] In some embodiments, other agents may be incorporated, such as chemotherapeutic agents, anti-angiogenic agents, and anti-excitotoxic agents, such as valproic acid, D-aminophosphonovaleric acid, D-aminophosphonoheptanoic acid, inhibitors of glutamate formation / release, such as anti-VEGF agents, including baclofen, NMDA receptor antagonists, ranibizumab, and aflibercept, and immunomodulatory agents, such as rapamycin.

[0201] Other therapeutic agents that may be delivered include the insulin sensitizer, pioglitazone.

[0202] In some embodiments, the active agent is an anti-infective agent. Exemplary anti-infective agents include antiviral agents, antibacterial agents, antiparasitic agents, and antifungal agents. 9. Diagnostic Agents

[0203] In some cases, the agent may comprise a diagnostic agent. Examples of diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray contrast agents, and contrast media. Other suitable contrast agents include radiopaque gases or gas-emitting compounds. The dendrimer complex can further comprise an agent useful for determining the location of the administered composition. Agents useful for this purpose include fluorescent tags, radionuclides, and contrast agents.

[0204] Exemplary diagnostic agents include dyes, fluorescent dyes, near-infrared dyes, SPECT contrast agents, PET contrast agents, and radioisotopes.

[0205] In a further embodiment, a single dendrimer complex composition can simultaneously treat and / or diagnose a disease or condition at one or more locations in the body. III. Pharmaceutical Preparations

[0206] Pharmaceutical compositions containing a dendrimer and one or more active agents, such as one or more angiotensin II receptor blockers, may be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants, that facilitate processing of the active compounds into pharmaceutically usable preparations. The formulation will depend on the selected route of administration. In preferred embodiments, the composition is formulated for parenteral delivery. In some embodiments, the composition is formulated for subcutaneous injection. Typically, the composition is formulated in a sterile saline or buffer solution for injection into the tissue or cells to be treated. The composition can be stored in a lyophilized state in single-use vials for rehydration immediately before use. Other means for rehydration and administration are known to those skilled in the art.

[0207] The pharmaceutical composition contains one or more dendrimers in combination with one or more pharmaceutically acceptable excipients.Representative excipients include solvents, diluents, pH adjusters, preservatives, antioxidants, suspending agents, wetting agents, viscosity adjusters, isotonicity agents, stabilizers, and combinations thereof.Suitable pharmaceutically acceptable excipients are preferably selected from materials that are generally recognized as safe (GRAS) and can be administered to individuals without causing undesired biological side effects or undesired interactions.See, for example, Remington's Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704.

[0208] The compositions are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. The phrase "unit dosage form" refers to a physically discrete unit of the conjugate appropriate for the patient being treated. However, it is understood that the total single dose of the composition will be determined by the attending physician within the scope of sound medical judgment. A therapeutically effective amount can be initially estimated in either cell culture assays or animal models, usually mice, rabbits, dogs, or pigs. Animal models are also used to achieve a desired concentration range and route of administration. Such information should then be useful for determining useful doses and routes for administration in humans. The therapeutic efficacy and toxicity of the conjugates can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal in 50% of the population). The dose ratio of toxic effects to therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions exhibiting large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.

[0209] Pharmaceutical compositions formulated for administration by parenteral administration (intramuscular, intraperitoneal, intravenous, or subcutaneous injection) and enteral routes of administration are described. A. Parenteral Administration

[0210] The phrases "parenteral administration" and "parenterally administered" are art-recognized terms and include modes of administration other than enteral and topical administration, such as injection, including, but not limited to, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Dendrimers can be administered parenterally, for example, by subdural, intravenous, intrathecal, intraventricular, intraarterial, intraamniotic, intraperitoneal, or subcutaneous routes. In a preferred embodiment, the dendrimer composition is administered by subcutaneous injection.

[0211] For liquid formulations, pharmaceutically acceptable carriers can be, for example, aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Parenteral vehicles (e.g., subcutaneous, intravenous, intraarterial, or intramuscular injection) include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, cyclodextrins, emulsions, or suspensions, including, for example, saline and buffered media. Dendrimers can also be administered in emulsions, such as water-in-oil emulsions. Examples of oils are oils of petroleum, animal, vegetable, or synthetic origin, petrolatum, and mineral oil. Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0212] Formulations suitable for parenteral administration may include aqueous and non-aqueous sterile suspensions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose. In general, water, saline, aqueous dextrose, and related sugar solutions, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

[0213] Injectable pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630 (2009)). B. Enteral Administration

[0214] The composition can be administered enterally. The carrier or diluent can be a solid carrier or diluent for a solid formulation, such as a capsule or tablet, a liquid carrier or diluent for a liquid formulation, or a combination thereof.

[0215] For liquid preparations, pharmaceutically acceptable carriers can be, for example, aqueous or non-aqueous solutions, suspensions, emulsions, or oils.Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and injectable organic esters, such as ethyl oleate.Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions, or suspensions, including saline and buffered media.

[0216] Examples of oils include oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, cod liver oil, sesame oil, cottonseed oil, corn oil, olive, petrolatum, and mineral oil. Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0217] Vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose, and sodium chloride, lactated Ringer's, and fixed oils. Formulations include aqueous and non-aqueous isotonic sterile injection solutions, which may contain, for example, antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Vehicles may include, for example, fluid and nutrient replenishers, electrolyte replenishers, and replenishers based on Ringer's dextrose. Generally, water, saline, aqueous dextrose, and related sugar solutions are preferred liquid carriers. They can also be formulated with proteins, fats, sugars, and other components of pediatric formulas.

[0218] In a preferred embodiment, the composition is formulated for oral administration.Oral formulations can be in the form of chewing gum, gel strips, tablets, capsules, or lozenges.The encapsulating material for preparing enteric-coated oral formulations includes cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, and methacrylic acid ester copolymer.Solid oral formulations, such as capsules or tablets, are preferred.Elixir and syrup are also well-known oral formulations. IV. Preparation Method A. Methods for Making Dendrimers

[0219] Dendrimers can be prepared via a variety of chemical reaction steps. Dendrimers are usually synthesized according to methods that allow control of their structure at every stage of construction. Most dendrimer structures are synthesized by two main different approaches: divergent or convergent approaches.

[0220] In some embodiments, dendrimers are prepared using a different method, where dendrimers are assembled from a multifunctional core and then extended outward through a series of reactions, typically a Michael reaction. The strategy involves coupling monomer molecules bearing reactive and protecting groups to the multifunctional core moiety, thereby gradually adding generations around the core, followed by removal of the protecting groups. For example, PAMAM-NH2 dendrimers are synthesized by first coupling N-(2-aminoethyl)acrylamide monomers to an ammonia core.

[0221] In other embodiments, dendrimers are prepared using convergent methods, where the dendrimer is built from small molecules that terminate at the surface of a sphere and the reaction proceeds to build inward, eventually reaching the core.

[0222] Many other synthetic routes exist for preparing dendrimers, such as the orthogonal approach, the accelerated approach, the double-stage convergent approach, or hypercore approach, the hypermonomer approach, or branched monomer approach, the double exponential approach; the orthogonal coupling approach, or two-step approach, the two-monomer approach, and the AB2-CD2 approach.

[0223] In some embodiments, the dendrimer core, one or more branching units, one or more linker / spacer, and / or one or more surface groups can be modified to enable conjugation with additional functional groups (branching units, linkers / spacers, surface groups, etc.), monomers, and / or active agents via click chemistry using one or more of copper-assisted azide-alkyne cycloaddition (CuAAC), Diels-Alder reactions, thiol-ene and thiol-yne reactions, and azide-alkyne reactions (Arseneault M et al., Molecules. 2015 May 20;20(5):9263-94). In some embodiments, prefabricated dendrons are click-reacted onto the high-density hydroxypolymer. "Click chemistry" involves, for example, coupling two different moieties (e.g., a core group and a branching unit; or a branching unit and a surface group) via a 1,3-dipolar cycloaddition reaction between an alkyne moiety (or its equivalent) on the surface of the first moiety and an azide moiety (e.g., present on a triazine composition or its equivalent) or any active end group, such as a primary amine end group, a hydroxyl end group, a carboxylic acid end group, a thiol end group, etc., on the second moiety.

[0224] In some embodiments, dendrimer synthesis relies on one or more reactions, such as thiol-ene click reaction, thiol-yne click reaction, CuAAC, Diels-Alder click reaction, azide-alkyne click reaction, Michael addition, epoxy ring-opening, esterification, silane chemistry, and combinations thereof.

[0225] Any existing dendritic platform can be used to create dendrimers with the desired functionality, i.e., a high density of surface hydroxyl groups, by conjugating high-hydroxyl-containing moieties, such as 1-thio-glycerol or pentaerythritol. Exemplary dendritic platforms can be synthesized and explored, such as polyamidoamine (PAMAM), poly(propyleneimine) (PPI), poly-L-lysine, melamine, poly(etherhydroxylamine) (PEHAM), poly(esteramine) (PEA), and polyglycerol.

[0226] Dendrimers can also be prepared by combining two or more dendrons. A dendron is a wedge-shaped segment of a dendrimer that contains a reactive central functional group. Many dendron scaffolds are commercially available. They are first-, second-, third-, fourth-, fifth-, and sixth-generation scaffolds, with 2, 4, 8, 16, 32, and 64 reactive groups, respectively. In certain embodiments, one type of activator is linked to one type of dendron, and a different type of activator is linked to another type of dendron. The two dendrons are then connected to form a dendrimer. The two dendrons can be linked via click chemistry, i.e., a 1,3-dipolar cycloaddition reaction between an azide moiety on one dendron and an alkyne moiety on another dendron, to form a triazole linker.

[0227] Exemplary methods for making dendrimers are described in detail in WO2009 / 046446, WO2015168347, WO2016025745, WO2016025741, WO2019094952, and U.S. Pat. No. 8,889,101. B. Conjugation of Triantennary N-Acetylgalactosamine (GalNAc) with Dendrimers

[0228] In some embodiments, β-GalNAc-tri-branched PEG3-azide is prepared as shown in Figure 1. In some embodiments, a tri-antennary building block is prepared in which three molecules of β-GalNAc-azide are grafted onto a propargylated pentaerythritol building block, optionally via a linker such as PEG, to produce an AB3-type orthogonal building block. In other embodiments, an AB4 monomer, such as pentaerythritol or a derivative thereof, is used as a core to conjugate three molecules of β-GalNAc. In some embodiments, the synthesis begins with a glycosylation reaction of β-D-GalNAc pentoacetate (e.g., compound 1 in Figure 1) with 2-[2-(2-azidoethoxy)ethoxy]ethan-1-ol (compound 2 in Figure 1), resulting in a peracetylated β-GalNAc-azide via a PEG spacer / linker (e.g., compound 3 in Figure 1). In some embodiments, pentaerythritol (compound 4) is selectively modified with three propargyl arms to produce tripropargyl pentaerythritol (e.g., compound 5 in Figure 1). In some embodiments, the remaining hydroxyl group on tripropargyl pentaerythritol is reacted with bis-chlorotetraethylene glycol (compound 7) to produce an intermediate compound, the AB3 building block (e.g., compound 8 in Figure 1). In some embodiments, peracetylated β-GalNAc-PEG3-azide is click-reacted with the AB3 building block (e.g., compound 8) using conventional CuAAC click reaction conditions to produce compound 9. In some embodiments, the success of the click reaction is determined by: 1 Confirmation is achieved by H NMR, HRMS, and HPLC. In some embodiments, the terminal chloride group of compound 9 is exchanged with an azide by nucleophilic substitution to yield compound 10. In some embodiments, the final step is transesterification to yield the deacetylated β-GalNAc-tri-antennary PEG3 azide (compound 11) building block, the GalNAc dendron.

[0229] In some embodiments, the β-GalNAc-tri-branched PEG3-azide is conjugated to a dendrimer as shown in Figure 2. In some embodiments, a fourth- or sixth-generation hydroxyl-terminated PAMAM dendrimer undergoes partial esterification with 5-hexynoic acid to yield a compound having two or more hexyne arms, preferably 5-20, or 10-15, or 12-14 hexyne arms, attached to the dendrimer. In some embodiments, one or more β-GalNAc-tri-branched PEG3-azides are conjugated to a dendrimer having hexyne arms attached thereto using a copper-catalyzed click (CuAAc) reaction to yield a β-GalNAc-tri-branched modified dendrimer. In a preferred embodiment, one or more hexyne arms conjugated to the dendrimer are for conjugation with a GalNAc dendron or β-GalNAc-tri-antennary PEG3-azide, and one or more hexyne arms conjugated to the dendrimer are for conjugation with a drug or imaging agent. In one embodiment, 5-6 hexyne arms are for conjugation with a GalNAc dendron or β-GalNAc-tri-antennary PEG3-azide, and 5-7 hexyne arms are for conjugation with a drug and / or imaging agent. Introduction of 5-6 dendron arms results in 15-18 GalNAc units in the final structure. V. How to use

[0230] A method for selectively delivering an active agent to hepatocytes is provided. It has been established that triantennary β-GalNAc-modified dendrimer compositions bind to the asialoglycoprotein receptor (ASGPR) on hepatocytes. Efficient binding to the ASGPR receptor directs selective internalization of the dendrimer-triantennary β-GalNAc within hepatocytes via receptor-mediated endocytosis. The low pH in endosomes within hepatocytes disrupts the interaction between the triantennary β-GalNAc ligand and the ASGPR receptor, causing release of the ligand into hepatocytes. A method for using a triantennary β-GalNAc-modified dendrimer composition for selective delivery, accumulation, and intracellular release of one or more active agents into hepatocytes is described. A. Methods of Treating Liver Injuries and Diseases

[0231] Methods of using dendrimer-triantennary GalNAc-modified compositions to treat or prevent one or more liver diseases or disorders in a subject are described.

[0232] Dendrimer-triantennary GalNAc compositions comprising one or more active agents for treating or preventing a liver disease or disorder can be administered to a subject to treat, prevent, and / or diagnose one or more symptoms of one or more liver disorders and / or diseases in the subject. The method may include the step of identifying and / or selecting a subject in need.

[0233] A method for treating or preventing one or more symptoms of one or more liver disorders and / or diseases comprises administering to a subject a dendrimer complexed, covalently conjugated, intercellularly dispersed, or encapsulated with one or more therapeutic or prophylactic agents in an amount effective to treat, alleviate, or prevent one or more symptoms of one or more liver disorders or diseases. In a preferred embodiment, a dendrimer composition, or a formulation thereof, comprising one or more antioxidants and / or angiotensin II type I receptor blockers is administered in an amount effective to treat or prevent one or more symptoms of one or more liver disorders and / or diseases, for example, to reduce lobular inflammation in the liver.

[0234] In one embodiment, a method for treating or preventing one or more liver disorders and / or diseases comprises administering to a subject a composition comprising a 4th, 5th, 6th, 7th, or 8th generation triantennary β-GalNAc-modified hydroxyl-terminated PAMAM dendrimer covalently conjugated to one or more angiotensin II type I receptor blockers, in an amount effective to treat or prevent one or more symptoms of the one or more liver disorders and / or diseases. 1. Liver disorders and diseases to be treated

[0235] In some embodiments, for treating, preventing, and / or diagnosing one or more liver disorders and / or diseases, a tri-antennary GalNAc-modified dendrimer complexed or conjugated to one or more active agents is administered to a subject to treat, prevent, and / or diagnose one or more symptoms of one or more liver disorders and / or diseases in the subject.

[0236] Dendrimer-triantennary β-GalNAc compositions are effective for treating or ameliorating one or more symptoms of liver disease or disorders, such as acute or chronic liver disease. Exemplary indications that can be treated include, but are not limited to, acute liver failure (acute hepatitis, fulminant hepatitis) due to neoplastic invasion, acute Budd-Chiari syndrome, heatstroke, mushroom ingestion, metabolic diseases such as Wilson's disease, or viral liver diseases caused by herpes simplex virus, cytomegalovirus, Epstein-Barr virus, parvovirus, hepatitis viruses (e.g., hepatitis A, hepatitis E, and hepatitis D+B virus infections), or drug-induced liver injury, including rifampicin-induced hepatotoxicity, acetaminophen-induced hepatotoxicity, illegal drug-induced toxicity, such as 3,4-methylenedioxy-N-methylamphetamine (MDMA, also known as ecstasy), or cocaine-induced toxicity, acute ischemic hepatocellular injury, or hypoxic hepatitis, or diseases resulting from traumatic liver injury. The methods can treat and prevent any hyperacute, acute, or subacute liver disease defined by the development of encephalopathy, coagulopathy, and jaundice in individuals with previously normal livers.

[0237] Symptoms and clinical manifestations of acute liver disease include jaundice and encephalopathy, as well as liver dysfunction (e.g., loss of metabolic function, decreased gluconeogenesis resulting in hypoglycemia, decreased lactate clearance resulting in lactic acidosis, decreased ammonia clearance resulting in hyperammonemia, and reduced synthetic capacity resulting in coagulation disorders). Acute liver disease and injury are often associated with multiple systemic symptoms, including immunoparesis, which contributes to a high risk of sepsis; systemic inflammatory responses with high energy consumption or catabolic rates; portal hypertension; renal dysfunction; myocardial injury; pancreatitis (especially acetaminophen-related disease); inadequate glucocorticoid production in the adrenal glands, which contributes to hypotension; and acute lung injury, which results in acute respiratory distress syndrome.

[0238] All of the described methods may also include a step of identifying and selecting a subject in need of treatment or who would benefit from administration with the composition. In some embodiments, the subject has been medically diagnosed with acute liver disease or injury by exhibiting clinical (e.g., physical) symptoms of the disease. In other embodiments, the subject has been medically diagnosed with subacute or chronic liver disease by exhibiting clinical (or physical) symptoms indicative of an increased risk or likelihood of developing acute liver disease. Thus, in some embodiments, a formulation of a dendrimer composition of the present disclosure is administered to the subject prior to clinical diagnosis of acute liver disease.

[0239] In preferred embodiments, the method treats or prevents non-alcoholic steatohepatitis, liver fibrosis associated with non-alcoholic steatohepatitis, primary biliary cholangitis. i. Non-alcoholic fatty liver disease (NAFLD)

[0240] In some embodiments, dendrimer-triantennary β-GalNAc compositions treat or alleviate one or more symptoms associated with nonalcoholic fatty liver disease (NAFLD). NAFLD represents a clinical-pathological spectrum of diseases primarily manifested as excessive accumulation of fat (steatosis) in hepatocytes. NAFLD encompasses the entire spectrum of disease, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which can lead to life-threatening cirrhosis and, in its most severe form, hepatocellular carcinoma. It is considered a hepatic manifestation of metabolic syndrome, with other pathologies including obesity, insulin resistance, hypertension, and hyperlipidemia. Histologically, NASH is characterized by signs of hepatic steatosis and intralobular inflammation with ballooning of hepatocytes. The estimated prevalence of NASH is much lower than that of NAFLD, ranging from 3-5%. It has been reported that 20% of NASH patients develop cirrhosis, and 30-40% of patients with NASH cirrhosis experience liver-related death.

[0241] In some embodiments, the dendrimer composition is administered in an amount effective to prevent conversion of NAFLD to NASH and to ameliorate the pathophysiology of the disease.

[0242] NAFLD is broadly classified into two phenotypes: nonalcoholic fatty liver disease (NAFL), characterized by isolated steatosis, and a more fulminant subtype, nonalcoholic steatohepatitis (NASH), characterized by cellular injury, infiltration of inflammatory cells, and hepatocyte ballooning that can progress to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). In some embodiments, the dendrimer composition is used in an amount effective to treat or ameliorate one or more symptoms of nonalcoholic steatohepatitis (NASH).

[0243] Methods for treating and / or preventing one or more symptoms of NAFLD or NASH typically involve administering to a subject in need thereof an effective amount of a composition comprising a tri-antennary β-GalNAc-modified hydroxyl-terminated PAMAM dendrimer and one or more agents to treat and / or alleviate one or more symptoms associated with NAFLD or NASH. In one embodiment, the dendrimer composition comprises a fourth-, fifth-, or sixth-generation tri-antennary β-GalNAc-modified hydroxyl-terminated PAMAM dendrimer covalently conjugated to one or more angiotensin II type I receptor blockers.

[0244] In some embodiments, the dendrimer-triantennary β-GalNAc composition is administered in an amount effective to inhibit or reduce serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), and total cholesterol (TC), fat accumulation or steatosis, inflammation, ballooning, fibrosis, long-term morbidity and mortality. ii. Liver cancer

[0245] In some embodiments, a dendrimer-triantennary β-GalNAc composition conjugated or complexed with one or more immunomodulatory agents, one or more chemotherapeutic agents, and / or additional therapeutic or diagnostic agents is administered to a subject with a proliferative disease, such as a benign or malignant tumor. In some embodiments, the subject being treated has been diagnosed with stage I, stage II, stage III, or stage IV cancer. The term cancer specifically refers to malignant tumors. In addition to uncontrolled growth, malignant tumors exhibit metastasis. In this process, small populations of cancer-like cells break away from the tumor, invade blood or lymphatic vessels, and travel to other tissues where they continue to proliferate. In this way, a primary tumor at one site can give rise to secondary tumors at another site.

[0246] In some embodiments, the dendrimer-triantennary β-GalNAc composition treats or alleviates one or more symptoms associated with liver cancer. In some embodiments, the subject has been medically diagnosed with liver cancer.

[0247] In some embodiments, the dendrimer-triantennary β-GalNAc compositions treat or alleviate one or more symptoms associated with hepatocellular carcinoma (HCC). The development of HCC results from an interaction between environmental and genetic factors. Liver cirrhosis, hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, excessive alcohol consumption, aflatoxin B1 intake, and nonalcoholic steatohepatitis (NASH) are important risk factors for the development of HCC.

[0248] Methods for treating and / or preventing one or more symptoms of liver cancer typically involve administering to a subject in need thereof an effective amount of a composition comprising a triantennary β-GalNAc-modified hydroxyl-terminated PAMAM dendrimer and one or more agents to treat and / or alleviate one or more symptoms associated with liver cancer or HCC. In one embodiment, a dendrimer composition comprising a fourth-, fifth-, or sixth-generation triantennary β-GalNAc-modified hydroxyl-terminated PAMAM dendrimer is complexed to, covalently conjugated to, or inter-molecularly dispersed or encapsulated with one or more of: a STING agonist, a CSF1R inhibitor, a PARP inhibitor, a VEGFR tyrosine kinase inhibitor, an EGFR tyrosine kinase inhibitor, a MEK inhibitor, a glutaminase inhibitor, a TIE II antagonist, a CXCR2 inhibitor, a CD73 inhibitor, an arginase inhibitor, a PI3K inhibitor, a TLR4 agonist, a TLR7 agonist, an SHP2 inhibitor, or a combination thereof.

[0249] In some embodiments, the dendrimer-triantennary β-GalNAc compositions are administered in an amount effective to reduce the number and / or proliferation of cancer cells, reduce tumor size, inhibit cancer cell invasion into peripheral organs, inhibit tumor metastasis, inhibit tumor growth, increase long-term survival, improve response to immune checkpoint blockade, and / or induce immunological memory that protects against tumor re-challenge. 2. Dosage and Effective Amount

[0250] Dosage and administration regimens depend on the severity and location of the injury or insult and / or the method of administration and can be determined by one skilled in the art. A therapeutically effective amount of a dendrimer composition used to treat liver injury and / or disease is typically sufficient to reduce or alleviate one or more symptoms of liver injury and / or disease.

[0251] Preferably, the active agent does not target or otherwise modulate the activity or amount of healthy cells not present in or associated with the diseased / damaged tissue, or targets or modulates them at a reduced level compared to cells associated with the diseased / damaged liver, thus reducing by-products and other side effects associated with the composition.

[0252] Pharmaceutical compositions are described that include a therapeutically effective amount of a dendrimer composition and a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the pharmaceutical composition includes an effective amount of a tri-branched GalNAc-modified hydroxyl-terminated PAMAM dendrimer conjugated to telmisartan. In some embodiments, suitable dosage ranges for use are between about 0.1 mg / kg and about 100 mg / kg, inclusive; between about 0.5 mg / kg and about 40 mg / kg, inclusive; between about 1.0 mg / kg and about 20 mg / kg, inclusive; and between about 2.0 mg / kg and about 10 mg / kg, inclusive.

[0253] Dosage forms of pharmaceutical compositions comprising dendrimer compositions are also provided. "Dosage form" refers to the physical form of a dose of a therapeutic compound, such as a capsule or vial, intended to be administered to a patient. The term "dosage unit" refers to the amount of a therapeutic compound administered to a patient in a single dose. In some embodiments, dosage units suitable for use (assuming an average adult patient weight of 70 kg) range from 5 mg / dosage unit to about 7000 mg / dosage unit, including those between about 35 mg / dosage unit and about 2800 mg / dosage unit; and those between about 70 mg / dosage unit and about 1400 mg / dosage unit; and those between about 140 mg / dosage unit and about 700 mg / dosage unit.

[0254] The actual effective amount of the dendrimer conjugate may vary depending on factors including the specific active agent administered, the specific composition formulated, the mode of administration, and the age, weight, condition, and route of administration and disease or disorder of the subject being treated. The subject is preferably a human. Generally, dosages are lower for intravenous injection or infusion compared to other systemic routes of administration, such as oral administration, and are based on body weight per patient compared to topical application, local, or regional administration based on the area to be treated.

[0255] Generally, the timing and frequency of administration is adjusted to balance the effectiveness of a given treatment or diagnostic schedule with the side effects of a given delivery system. Exemplary dosages include continuous infusion, single and multiple administrations, such as hourly, daily, weekly, monthly, or yearly administrations.

[0256] In some embodiments, the dosage is administered to a human once, twice, or three times daily, or less frequently, i.e., every 1, 2, 3, 4, 5, or 6 days. In some embodiments, the dosage is administered about once or twice per week, every 2 weeks, every 3 weeks, or every 4 weeks. In some embodiments, the dosage is administered about once or twice per month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or less frequently.

[0257] It is understood by those skilled in the art that the dosing regimen can be any period sufficient to treat the disorder in the subject.In some embodiments, the regimen comprises one or more cycles, where one round of treatment is followed by a rest period (for example, drug-free).The rest period can be 1, 2, 3, 4, 5, 6 or 7 days; or 1, 2, 3, 4 weeks, or 1, 2, 3, 4, 5 or 6 months. 3. Control

[0258] The effect of a dendrimer composition comprising one or more agents can be compared to a control or alternative treatment. Suitable controls are known in the art and include, for example, untreated or placebo-treated subjects. A typical control is a comparison of a subject's condition or symptom before and after administration of a targeted agent. The condition or symptom can be a biochemical, molecular, physiological, or pathological readout. For example, the effect of a composition on a particular symptom, pharmacological, or physiological indicator can be compared to an untreated subject, or to the subject's condition before treatment. In some embodiments, the symptom, pharmacological, or physiological indicator is measured in a subject before treatment and again one or more times after treatment has begun. In some embodiments, the control is a reference level or average value determined based on measurements of the symptom, pharmacological, or physiological indicator in one or more subjects (e.g., healthy subjects) who do not have the disease or condition being treated. In some embodiments, the effect of a treatment is compared to a conventional treatment known in the art. In some embodiments, the untreated control subject suffers from the same acute liver disease or condition as the treated subject. B. Combination Treatments and Procedures

[0259] The composition can be administered alone or in combination with one or more conventional therapies. In some embodiments, the conventional therapy includes one or more administrations of the composition in combination with one or more additional active agents. Combination therapy includes administering the active agents together in the same mixture or in separate mixtures. Thus, in some embodiments, the pharmaceutical composition includes two, three, or more active agents. Such formulations typically include an effective amount of an agent that targets the treatment site. The additional active agent(s) can have the same or different mechanisms of action. In some embodiments, the combination provides an additive effect for treating a liver condition. In some embodiments, the combination provides more than an additive effect for treating a disease or disorder.

[0260] The additional therapy or procedure may be administered simultaneously or sequentially with the administration of the dendrimer composition. In some embodiments, the additional therapy is administered during a drug cycle or during a drug holiday that is part of the composition dosing regimen. For example, in some embodiments, the additional therapy or procedure is surgery, radiation therapy, chemotherapy, liver transplant, stem cell transplant, or mesenchymal stem cells (MSCs).

[0261] Exemplary additional therapies or procedures include lifestyle modifications such as avoiding saturated fats, excessive sugary foods, soft drinks, fast food, and refined carbohydrates, and encouraging moderate exercise. Diabetic patients can be treated with lifestyle modifications and, if necessary, oral sulfonylurea-gliclazide, glimeperide, and / or insulin. Dyslipidemia can be managed with statins, and hypertension can be managed with antihypertensive agents.

[0262] In some embodiments, the compositions and methods are used before, with, after, or alternating with treatment with one or more additional therapies or procedures. Additional therapeutic agents include traditional cancer treatments, such as chemotherapy agents, cytokines, chemokines, and radiation therapy. Most chemotherapy drugs can be classified as alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. These drugs affect cell division or DNA synthesis and function in some way. Additional therapeutic agents include monoclonal antibodies and tyrosine kinase inhibitors, such as imatinib mesylate (GLEEVEC® or GLIVEC®), which directly target molecular abnormalities in certain types of cancer (chronic myeloid leukemia, gastrointestinal stromal tumors).

[0263] Representative chemotherapeutic agents include amsacrine, bleomycin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxin, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, mechlorethamine, melphalan, mercaptopurine, mesna, and methotrexate. , mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, taxol, trichostatin A and its derivatives, trastuzumab (HERCEPTIN®), cetuximab, and rituximab (RITUXAN® or MABTHERA®), bevacizumab (AVASTIN®), and combinations thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabine, staurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2)5, and combinations thereof.

[0264] In some embodiments, the compositions and methods are used prior to or in conjunction with immunotherapy to inhibit checkpoint proteins, such as components of the PD-1 / PD-L1 axis or the CD28-CTLA-4 axis, using one or more immune checkpoint modulators (e.g., PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists), adoptive T cell therapy, and / or cancer vaccines. Exemplary immune checkpoint modulators used in immunotherapy include pembrolizumab (anti-PD1 mAb), durvalumab (anti-PDL1 mAb), PDR001 (anti-PD1 mAb), atezolizumab (anti-PDL1 mAb), nivolumab (anti-PD1 mAb), tremelimumab (anti-CTLA4 mAb), avelumab (anti-PDL1 mAb), and RG7876 (CD40 agonist mAb).

[0265] Methods of adoptive T cell therapy are known in the art and are used in clinical practice. Generally, adoptive T cell therapy involves the isolation and ex vivo expansion of tumor-specific T cells to achieve a higher number of T cells than can be achieved by vaccination alone. The tumor-specific T cells are then infused into a patient with cancer in an attempt to equip the immune system with the ability to overcome residual tumors through T cells that can attack and kill the cancer. Several forms of adoptive T cell therapy can be used to treat cancer, including, but not limited to, culturing tumor-infiltrating lymphocytes or TILs; isolating and expanding a specific T cell or clone; and using T cells engineered to recognize and attack tumors. In some embodiments, T cells are collected directly from the patient's blood. Methods for in vitro priming and activating T cells for adaptive T cell therapy are known in the art. See, e.g., Wang, et al, Blood, 109(11):4865-4872 (2007) and Hervas-Stubbs, et al, J. Immunol., 189(7):3299-310 (2012).

[0266] Histologically, adoptive T cell therapy strategies largely focus on the infusion of tumor antigen-specific cytotoxic T cells (CTLs), which can directly kill tumor cells. However, CD4+ helper T (Th) cells, such as Th1, Th2, Tfh, Treg, and Th17, can also be used. Th cells can activate antigen-specific effector cells and recruit cells of the innate immune system, such as macrophages and dendritic cells, to help antigen-presenting cells (APCs). Th cells primed by antigens can directly activate antigen-specific CTLs. As a result of APC activation, antigen-specific Th1 cells have been implicated as initiators of epitope or determinant expansion, which is the expansion of immunity to other antigens in tumors. The ability to induce epitope expansion can expand the immune response to many potential antigens in tumors and lead to more efficient tumor cell killing through its ability to initiate heterologous responses. In this way, adoptive T cell therapy can be used to stimulate endogenous immunity.

[0267] In some embodiments, the T cells express chimeric antigen receptors (CARs, CAR T cells, or CARTs). Artificial T cell receptors are engineered receptors that graft specific specificities onto immune effector cells. Typically, these receptors are used to graft the specificity of monoclonal antibodies onto T cells and can be engineered to target virtually any tumor-associated antigen. First-generation CARs typically have the intracellular domain from the CD3ζ chain, which is the primary transmitter of signals from endogenous TCRs. Second-generation CARs add intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to T cells, and third-generation CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further enhance efficacy.

[0268] In some embodiments, the compositions and methods are used prior to or in conjunction with a cancer vaccine, such as a dendritic cell cancer vaccine. Vaccination typically involves administering an antigen (e.g., a cancer antigen) to a subject with an adjuvant to induce therapeutic T cells in vivo. In some embodiments, the cancer vaccine is a dendritic cell cancer vaccine in which antigens delivered by dendritic cells are primed ex vivo to present the cancer antigen. Examples include PROVENGE® (sipuleucel-T), a dendritic cell-based vaccine for treating prostate cancer (Ledford, et al., Nature, 519, 17-18 (05 March 2015)). Such vaccines, as well as other compositions and methods for immunotherapy, are reviewed in Palucka, et al., Nature Reviews Cancer, 12, 265-277 (April 2012).

[0269] In some embodiments, the compositions and methods are used prior to or in conjunction with surgical resection of a tumor, for example, to prevent metastasis of the primary tumor. In some embodiments, the compositions and methods are used to enhance the body's own anti-tumor immune function.

[0270] In vivo efficacy studies of these triantennary GalNAc-modified dendrimers can be evaluated in a mouse model of non-alcoholic steatohepatitis, for example, the STAM™ model of non-alcoholic steatohepatitis (mouse). method

[0271] Pathogen-free, 14-day pregnant C57BL / 6 mice can be obtained from Japan SLC, Inc. (Japan). NASH can be established in male mice by subcutaneously injecting 200 μg of streptozotocin (STZ, Sigma, USA) once on day 2 of age and then feeding them ad libitum with a high-fat diet (CLEA Japan Inc., Japan) from 4 weeks of age onwards (day 28 ± 2 days).

[0272] NASH mice can be randomized based on their body weight at 6 weeks of age (day 42 ± 2 days) one day before treatment initiation into 8 groups of 8 mice and 2 groups of 4 mice. Litter-matched control mice (n=8) that did not undergo STZ priming can be fed a normal diet ad libitum and serve as control purposes. Animals will be euthanized before the end of the study if they show a weight loss of >25% within one week or a weight loss of >20% compared to the previous day. Animals will be euthanized before the end of the study if they show signs of moribundity, such as prone position. No samples will be collected from euthanized animals.

[0273] During the treatment period, individual body weights are measured daily.

[0274] Mice are monitored daily for survival, clinical signs, and behavior.

[0275] group Group 1 (normal): 8 normal mice are fed normal diet ad libitum without any treatment and are sacrificed at the age of 9 weeks. Group 2 (vehicle): Eight NASH mice are administered vehicle [saline] intraperitoneally at a volume of 10 mL / kg every other day from 6 to 9 weeks of age. Group 3 (telmisartan): Eight NASH mice are orally administered pure water supplemented with a 10 mg / kg dose of telmisartan once daily from 6 to 9 weeks of age. Group 4 (Obeticholic Acid, or "OCA"): Eight NASH mice are orally administered 1% methylcellulose supplemented with a 30 mg / kg dose of OCA once daily from 6 to 9 weeks of age. Group 5 (dendrimer-triantennary β-GlcNAc-azide-telmisartanamide conjugate, or "D-Tel" high): Eight NASH mice will receive intraperitoneal administration of vehicle supplemented with a 90 mg / kg dose of D-Tel every other day from 6 to 9 weeks of age. Group 6 (D-Tel low): Eight NASH mice will receive vehicle supplemented with an 18 mg / kg dose of D-Tel intraperitoneally every other day from 6 to 9 weeks of age. Group 7 (dendrimer-triantennary β-GlcNAc-azide-telmisartan ester conjugate or "D-TelB" high): Eight NASH mice are intraperitoneally administered vehicle supplemented with a 90 mg / kg dose of D-TelB every other day from 6 to 9 weeks of age. Group 8 (D-OCA high): Eight NASH mice are administered vehicle supplemented with a 315 mg / kg dose of D-OCA intraperitoneally every other day from 6 to 9 weeks of age. Group 9 (D-OCA low): Eight NASH mice are administered vehicle supplemented with a 63 mg / kg dose of D-OCA intraperitoneally every other day from 6 to 9 weeks of age. Group 10 (D-Cy5-6 wks): Four NASH mice receive a single intraperitoneal injection of vehicle supplemented with a 50 mg / kg dose of D-Cy5 at 6 weeks of age. Group 11 (D-Cy5-9 wks): Four NASH mice receive a single intraperitoneal injection of vehicle supplemented with a 50 mg / kg dose of D-Cy5 at 9 weeks of age.

[0276] Mice in groups 10 and 11 will be sacrificed 48 hours after administration at 6 and 9 weeks of age. Mice in groups 1-9 will be sacrificed at 9 weeks of age for later assays, and mice in groups 10 and 11 will be sacrificed at 6 and 9 weeks of age for later assays. Organ weight measurements: Measure individual liver weights, Calculate the liver weight to body weight ratio. Biochemical assays (groups 1-9): Non-fasting serum ALT levels were determined by FUJI DRI CHEM (Fujifilm, Japan). Liver triglycerides were quantified using a Triglyceride E Test Kit (FUJIFUILM Wako Pure Chemical Corporation, Japan). Histological analysis of liver sections (following routine methods) (Groups 1-9): HE staining and estimation of NAFLD activity score, Sirius red staining and estimation of percentage of fibrotic area,

[0277] Sample collection and fixation: After completion of the in-life portion of the study, the following samples will be collected for further analysis or transport:

[0278] Animals in groups 10-11 were anesthetized with isoflurane and perfused through the left ventricle with saline (followed by 4% neutral buffered formalin, NBF, pH 7.4) for 20-30 minutes. The animals were dissected and tissue samples (left and right kidneys, liver) were serially collected. The thickness of the samples was approximately less than 5 mm to ensure proper fixation. A flat surface was prepared for the area of interest. The samples were immediately placed in 4% NBF for fixation. The samples were fixed overnight at room temperature in 4% NBF.

[0279] After fixation, the samples are subjected to the following process.

[0280] Sample processing 1. Place tissue in PBS for 5 minutes three times; 2. Place tissue in 10% sucrose (in PBS) for 24 hours at 4°C; 3. Place tissue in 20% sucrose (in PBS) for 24 hours at 4°C; 4. Place tissue in 30% sucrose (in PBS) for 24 hours at 4°C; 5. Place tissue in 30% sucrose (in PBS):OCT (1:1) at 4°C for 24 hours.

[0281] Tissue embedding procedure Place the tissue and 30% sucrose:OCT (1:1–2) into the embedding module and adjust the orientation of the tissue; Place the module on a flat surface of dry ice and wait until it solidifies; Store the embedded models at −80°C; Section preparation The embedded model was placed in a ThermoHM550 microtome and 10 μm-thick sections were prepared in the axial direction; The slides are stored at -80°C until use. sample Freeze serum samples (groups 1-9), Freeze liver samples (groups 1-11). Freezing liver sections (groups 10-11) OCT-embedded liver blocks (groups 10–11), OCT-embedded kidney blocks (groups 10–11), Statistics Test (Groups 1-9) Statistical tests are performed using the Bonferroni multiple comparison test. A P value of <0.05 is considered statistically significant. V. Kit

[0282] The composition can be packaged in a kit.The kit can include a single dose or multiple doses of the composition comprising one or more active agents that are encapsulated in, associated with, or conjugated with a dendrimer, and instructions for administering the composition.Specifically, the instructions indicate that an effective amount of the composition is administered to an individual with a specific liver condition / disease as directed.The composition can be formulated as described above with reference to specific treatment methods, and can be packaged in any convenient format.

[0283] The present invention will be further understood with reference to the following non-limiting examples. [Example]

[0284] Example 1 Synthesis of β-GalNAc-triantennary PEG3-azide building blocks Triantennary Gal-NAc-based hydroxyl PAMAM dendrimers were evaluated for site-specific drug targeting and delivery to hepatocytes. Surface GalNAc sugars were shown to confer a multivalent binding effect on ASGPR, enabling the dendrimers to selectively target and internalize hepatocytes in vivo in the STAM model of nonalcoholic steatohepatitis.

[0285] Four different dendrimer-drug conjugates were synthesized and evaluated in this model: 1) D-GalNAc-Cy5 for targeting, 2) D-GalNAc-telmisartan ester (cleavable drug linker, angiotensin 2 receptor blocker), 3) D-GalNAc-telmisartan amide (non-cleavable drug linker), and D-obeticholic acid (cleavable drug linker). Accurate loading of combinations of targeting ligands, imaging dyes, and therapeutic agents has been successfully demonstrated. The dendrimers can be further engineered to attach diverse combinations of therapeutic molecules. These results indicate that GalNAc PAMAM dendrimers represent an effective platform for the treatment of liver disease. method

[0286] The synthesis scheme for β-GalNAc-tri-branched PEG3-azide (AB3 building block) is shown in Figure 1. Reagents and conditions: (i) scandium trifluoromethanesulfonate, DCE, 3 h, 80 °C; (ii) propargyl bromide, toluene, sodium hydroxide, water, TBAB; (iii) pyridine, thionyl chloride, chloroform, 65 °C, 2 h; (iv) tetrabutylammonium hydrogen sulfate, 50% NaOH, 16 h, room temperature; (v) CuSO4·5H2O, sodium ascorbate, THF, water, 10 h; (vi) DMF, tetrabutylammonium iodide, NaN3, 80 °C, 5 h; (vii) sodium methoxide, absolute ethanol, 30 °C, 3 h.

[0287] Three molecules of β-GalNAc-PEG3 azide were grafted onto a propargylated pentaerythritol building block to produce an AB3-type orthogonal building block. The synthesis began with the glycosylation reaction of β-D-GalNAc pentoacetate (1, Figure 1) with 2-[2-(2-azidoethoxy)ethoxy]ethan-1-ol (2) in the presence of scandium trifluoromethanesulfonate in dichloromethane, resulting in peracetylated β-GalNAc-PEG3 azide (3). Pentaerythritol 4 was selectively modified with three propargyl arms in the presence of sodium hydroxide and tetrabutylammonium bromide in DMSO according to literature methods, resulting in tripropargyl pentaerythritol (5). The single remaining hydroxyl group on compound (5) was reacted with bis-chlorotetraethylene glycol (7) using sodium hydroxide and TBAB in DMSO to give intermediate compound (8). During the next synthetic step, peracetylated β-GalNAc-PEG3-azide was clicked with the AB3 building block (8) using standard CuAAC click reaction conditions (copper(II) sulfonate pentahydrate and sodium ascorbate in THF:water) to give compound (9). A successful click reaction was confirmed by: 1 Confirm by 1 H NMR, HRMS, and HPLC. 1 In H NMR, a single sharp singlet of triazole was observed at δ 7.9 ppm. Other characteristic peaks were the acetic acid peak between δ 2.0 and 1.74 ppm, the GalNAc protons at δ 5.2 and 3.2 ppm, and the NH of GalNAc at δ 7.78 ppm. In the next synthetic step, the terminal chloride group of compound (9) was replaced by an azide by nucleophilic substitution in the presence of sodium azide and tetrabutylammonium iodide in DMF to give compound (10). The final step was transesterification using Zemplen conditions, where the reaction was carried out in methanol using sodium methoxide to give the deacetylated β-GalNAc-tri-branched PEG3 azide building block (11). result

[0288] Successful completion of the reaction is confirmed by 1 The synthesis was confirmed by H NMR, where the peak corresponding to the O-acetate completely disappeared and all sugar protons were shifted upfield. 1 Characterization using 1 H NMR, HPLC, and HRMS confirmed the desired compound. Example 2 Synthesis and characterization of fluorescently labeled hepatocyte-targeting dendrimer-triantennary β-GalNAc-CY5

[0289] After the synthesis of the targeting dendron (11), the synthesis of the dendrimer-triantennary β-GalNAc-CY5 was performed, and the selective hepatocyte targeting ability of this dendrimer was evaluated using confocal microscopy and fluorescence spectroscopy. method

[0290] Dendrimer synthesis began with the fourth-generation PAMAM hydroxyl-terminated dendrimer 12 (Figure 2), and partial esterification with 5-hexynoic acid 13 was achieved using Steglich esterification to yield compound 14. For this reaction, EDC-HCl was used as the coupling reagent along with 4-dimethylaminopyridine (DMAP) to attach 12 to 14 hexyne arms to the dendrimer. The structure of compound 14 is shown below. 1 Confirmed by H NMR, a peak for the esterified CH2 was observed at δ 4.0 ppm, and other multiplets corresponding to CH2 from the hexyne arms were observed at δ 1.7–1.6 ppm. To accurately quantify the loading of hexyne arms, the internal amide peaks of the dendrimer at δ 8.0–7.7 ppm were used as reference points, and the number of attached arms was calculated using proton integration. Once the hexyne arms were introduced onto the dendrimer surface, GalNAc dendron (3) (Figure 1) was coupled to dendrimer (14) using a copper-catalyzed click (CuAAc) reaction to yield dendrimer (15). 1H NMR confirmed that 5–6 arms of the GalNAc dendron (11) were attached to the dendrimer. Five to six arms of the GalNAc dendron were maintained for targeting, and five to seven arms of the hexyne were left untouched for conjugation to drugs or imaging agents. The introduction of five to six arms of the dendron resulted in 15–18 GalNAc units in the final structure.

[0291] For loading calculations, the proton integration method was used. 1 In H NMR, the internal amide protons plus 15 triazole protons from the dendron and 5–6 protons from the newly synthesized triazole were observed between δ 8.0 and 7.7 ppm. The NH peak from GalNAc was observed at δ 7.6 ppm, and an N-acetyl singlet corresponding to 45 protons was observed at δ 1.78 ppm. 1 When H NMR was recorded in DO, all NH-related signals disappeared due to exchange with water, and two distinct triazole peaks corresponding to 15 and 5 protons were clearly observed at δ 8.0 and 7.8 ppm. All GalNAc and dendrimer signals could be observed between δ 5.0 and 1.5 ppm. Once the targeting moiety was attached to the dendrimer, the next step was to attach a fluorescent tag to this dendrimer. The near-infrared dye CY5 was used as the fluorescent tag. To attach CY5 azide (16) to dendrimer (15), a CuAAc reaction was used to generate fluorescent-GalNAc dendrimer (17). result

[0292] The click reaction successfully produced CY5-labeled dendrimers with two to three CY5 molecules attached (a representative small molecule useful as a diagnostic agent and predictor of outcomes from small molecule drugs). The final dendrimers were 1The dendrimer was characterized by H NMR, and the CY5 loading was calculated using proton integration. The peak corresponding to CY5 appeared between δ 7.5 and 6.2 ppm. The entire synthetic sequence was followed by HPLC. The HPLC spectrum of G4-OH appeared at 6.1 min. The HPLC spectrum shifted to the hydrophobic side at 9.4 min when a hexyne arm was added to the dendrimer, and shifted again to the hydrophilic side at 7.7 min when a water-soluble triantennary GalNAc dendron was conjugated to the dendrimer. The addition of CY5 also shifted the peak to the right at 8.4 min. The final CY5-labeled dendrimer (17) was >98% pure as determined by HPLC. Example 3 Synthesis and characterization of dendrimer-GalNAc-telmisartan conjugates with enzyme-cleavable and non-cleavable linkers for the treatment of NASH

[0293] After the successful completion of the fluorescent dendrimers, the next goal was to synthesize dendrimers bearing targeting moieties and drugs for liver damage. NASH is a well-recognized global health problem that leads to diseases such as cirrhosis and hepatocellular carcinoma. Many different types of therapeutic molecules, such as PPAR gamma, antioxidants, and cytoprotective agents, have been used with limited or no success. method

[0294] Currently, there are no approved drugs for the treatment of NASH and other chronic liver diseases. Recently, telmisartan, an angiotensin receptor blocker (ARB) and partial agonist of peroxisome proliferator receptors, has shown promise in many animal models of NASH by increasing insulin sensitivity and inhibiting fat accumulation in the liver. Despite positive results, its clinical application has been hindered by dose-related toxicity and other side effects such as hypotension.

[0295] To overcome these challenges, highly specific targeted drug delivery nanoplatforms are needed to deliver drug loads to the desired organs or tissues. To this end, telmisartan was conjugated to dendrimer-GalNAc (15) using two different linkages: telmisartan ester (Figure 3) and telmisartan amide (Figure 4). To synthesize telmisartan-ester PEG4 azide (19), telmisartan was reacted with 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol (2) in the presence of DCC and DMAP in anhydrous DCM. Telmisartan ester-PEG4 azide was achieved in quantitative yield.

[0296] For the synthesis of dendrimer-triantennary β-GlcNAc-azido-telmisartan ester conjugates, the products were: 1 Confirmed using 1 H NMR and LCMS. 1 In H NMR, the aromatic protons (14) appear between δ 7.8 and 7.1 ppm, the benzyl CH2 appears at δ 5.6 ppm, the CH2 next to the CH3 appears at δ 1.85 ppm, and the triplet corresponding to the CH3 appears at δ 1.0 ppm. After confirming the successful formation of telmisartan azide, it was clicked with GalNAc dendrimer (15) having 6-7 arms of a hexyne linker using CuAAC click reaction. The click reaction successfully produced dendrimer-triantennary GalNAc(4-5)-telmisartan(7-8) (20). The product was confirmed by 1H NMR was performed again. The dendrimer internal amide peak, triazole peak, aromatic protons from telmisartan, and NH corresponding to GalNAc appear between δ 8.0 and 7.0 ppm. The benzyl CH2 appears at δ 5.6 ppm. Other important peaks to observe are the N-acetyl peak at δ 1.7 ppm and the CH3 peak at δ 0.9 ppm. Drug loading was calculated using proton integration. 7–9 molecules of telmisartan ester were confirmed to be conjugated to the dendrimer. The weight percent loading of telmisartan was approximately 14%, suggesting that 8 molecules of telmisartan were conjugated. Although telmisartan is a very hydrophobic drug, the conjugate is highly water-soluble, with a solubility of approximately 60 mg / mL. The purity of the final conjugate is >96%.

[0297] After successfully completing the synthesis of dendrimer telmisartan with enzyme-sensitive ester linkages, we synthesized a dendrimer-telmisartan amide conjugate, which should be more stable under physiological conditions (Figure 4). To achieve this, the linker azide to telmisartan was first introduced by coupling with 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-amine (21) using HATU and DIPEA to give telmisartan-PEG3-amide azide (22). The compound was 1 The azide-functionalized telmisartan was characterized using H NMR and LCMS. After the formation of the azide-functionalized telmisartan, it was conjugated to dendrimer-GalNAc (15) using CuAAc to give dendrimer-GalNAc(4-5)-telmisartanamide (6-7) (23).

[0298] Regarding the synthesis of dendrimer-triantennary β-GlcNAc-azide-telmisartanamide conjugate, product confirmation was performed. 1H NMR was performed. After the drug molecules were attached, all signature peaks from the drug were observed in the final compound. The dendrimer internal amide peak, triazole peak, aromatic protons from telmisartan, and NH corresponding to GalNAc appeared between δ 8.2 and 7.1 ppm. The benzyl CH2 appeared at δ 5.6 ppm. Other significant peaks were the N-acetyl peak at δ 1.8 ppm and the CH3 peak at δ 1.0 ppm. 1 When H NMR was recorded in DO, the internal amide peak corresponding to the dendrimer disappeared in exchange, and a singlet corresponding to the triazole appeared at δ 8.0 ppm. Drug loading was calculated using proton integration. result

[0299] Six molecules of telmisartan amide were confirmed to be conjugated to the dendrimer. The weight percent loading of telmisartan was approximately 11%, suggesting that six molecules of telmisartan were conjugated. The final conjugate was highly water-soluble, with a solubility of approximately 60-70 mg / ml.

[0300] The progress of the entire synthesis process was monitored by HPLC. The retention time of the GalNAc triantennary conjugate (15) is 7.8 min, but upon addition of the hydrophobic telmisartan azide, the retention time of the final conjugate (23) shifts to the hydrophobic side, reaching 10.4 min. The purity of the final conjugate is >95%. Example 4 Binding affinity of telmisartan and the telmisartan linker to the human angiotensin II AT1 receptor method

[0301] The binding affinities of telmisartan, telmisartan-ester-PEG4 azide, and telmisartan-PEG3-amide azide were evaluated using a human angiotensin II AT1 receptor (radioligand antagonist) binding assay (Table 1). Cell membrane homogenates (8 μg of protein) were incubated with 0.05 nM [125I][Sar1-Ile8]angiotensin II in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA, and 0.1% BSA at 37°C for 120 min in the absence or presence of test compounds. Nonspecific binding was determined in the presence of 10 μM angiotensin II. After incubation, the sample solution was rapidly filtered under vacuum through glass fiber filters (GF / B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). After drying, the filters were counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). result

[0302] Results are expressed as percent inhibition of control radioligand specific binding. Saralasin is used as a standard reference compound and is tested at several concentrations in each experiment to generate a competition curve from which its IC50 is calculated.

[0303] Sample preparation: Telmisartan, telmisartan ester linker, and telmisartan amide linker were dissolved in DMSO aqueous solution to form a solution with a free drug (telmisartan) concentration of 10 mM. Each sample solution was further diluted in DMSO to 10 μM, 3.33 μM, 1.1 μM, 0.37 μM, 0.123 μM, 41.2 nM, 13.7 nM, 4.57 nM, 1.52 nM, 0.508 nM, and 0.169 nM, respectively, for binding studies.

[0304] The binding affinities of the modified drug linkers remained in the nanomolar range (Table 1). Table 1. Binding affinities of telmisartan, telmisartan-ester-PEG4 azide, and telmisartan-PEG3-amide azide [Table 1] Example 5 Release studies of dendrimer-telmisartan-ester and dendrimer-telmisartan-amide method

[0305] The drug release profiles of both conjugates were evaluated in plasma (pH 7.4, PBS) and intracellular conditions (pH 5.5, esterase). result

[0306] The results show that under physiological plasma conditions, the dendrimer-ester-linked construct is highly stable, with only 14% drug release observed over 18 days, whereas under intracellular conditions, 94% of the drug is released over 18 days (Figure 5). However, release experiments with the dendrimer-telmisartan amide conjugate showed that under physiological plasma conditions, less than 2% of the drug was released over 18 days, and under intracellular conditions, less than 10% of the drug was released over 18 days (Figure 6). The amide-linked drug conjugate is more stable than the ester-linked dendrimer conjugate under physiological conditions.

[0307] The stability of the D-telmisartanamide conjugate was also evaluated in human, mouse, and rat plasma at 37° C., with only 3% of the drug released over a 2-day period (FIG. 7). Example 6 Synthesis and Characterization of Dendrimer-GalNAc-Obiticholic Acid Conjugates for the Treatment of NASH

[0308] Obeticholic acid, a highly potent drug, is a semisynthetic bile acid analogue and the most active physiological ligand of the farnesoid X receptor, which has shown promising results in NASH but has dose-related toxicity issues. To conjugate obeticholic acid to dendrimer-GalNAc-hexynoic acid (15), the carboxylic acid functional handle (24) of obeticholic acid was selectively esterified with a PEG-azide linker (25) using an EDC-DMAP coupling reaction (Figure 8). The obeticholic acid linker azide (26) was then attached to 1 The azide-terminated obeticholic acid was successfully conjugated to dendrimer-GalNAc-hexyne (15) using a click reaction to give dendrimer-GalNAc(4-5)-obeticholic acid conjugate (6-7) (27). The dendrimer and intermediates were characterized using H NMR, HRMS, and HPLC. 1 The dendrimer was fully characterized using H NMR (Figure 8) and HPLC. The drug loading of the dendrimer was calculated by proton integration using the dendrimer internal amide protons as reference peaks. The methyl peak at δ 0.6 ppm belongs to obeticholic acid, which helped calculate the exact drug loading. Six molecules of obeticholic acid are conjugated to the dendrimer, resulting in a drug loading of approximately 9.5%. The purity of the final conjugate is greater than 99%, and the solubility range is approximately 100 mg / mL. Example 7 In vivo efficacy study of triantennary Galnac-modified hydroxyl dendrimers in a murine non-alcoholic steatohepatitis model method Mice and NASH models

[0309] Pathogen-free, 14-day-old pregnant C57BL / 6 mice were obtained from Japan SLC, Inc. (Japan). NASH was established in male mice by a single subcutaneous injection of 200 μg of streptozotocin (STZ, Sigma, USA) on postnatal day 2 and by feeding them ad libitum with a high-fat diet (CLEA Japan Inc., Japan) from 4 weeks of age (day 28 ± 2 days). At 6 weeks of age (day 42 ± 2 days), the day before treatment began, NASH mice were randomized based on their body weight into eight groups of eight mice and two groups of four mice. Control mice (n = 8) from the same litter that did not undergo STZ priming were fed a normal diet ad libitum and served as controls (see below for details on group assignment). Animals were euthanized before the end of the study if they showed a >25% weight loss within 1 week or a >20% weight loss compared to the previous day. If animals showed signs of moribundity, such as prone position, they were euthanized before the end of the study. No samples were collected from euthanized animals. Individual body weights were measured daily during the treatment period. Mice were monitored daily for survival, clinical signs, and behavior.

[0310] Grouping Group 1 (normal): Eight normal mice were fed a normal diet ad libitum without any treatment and were sacrificed at the age of 9 weeks. Group 2 (vehicle): Eight NASH mice were intraperitoneally administered vehicle (saline) at a volume of 10 mL / kg every other day from 6 to 9 weeks of age. Group 3 (telmisartan): Eight NASH mice were orally administered pure water supplemented with a 10 mg / kg dose of telmisartan once daily from 6 to 9 weeks of age. Group 4 (Obeticholic Acid, or "OCA"): Eight NASH mice were orally administered 1% methylcellulose supplemented with a 30 mg / kg dose of OCA once daily from 6 to 9 weeks of age. Group 5 (dendrimer-triantennary β-GlcNAc-azide-telmisartanamide conjugate, or “D-Tel” high): Eight NASH mice received intraperitoneal administration of vehicle supplemented with a 90 mg / kg dose of D-Tel every other day from 6 to 9 weeks of age. Group 6 (D-Tel low): Eight NASH mice received intraperitoneal administration of vehicle supplemented with an 18 mg / kg dose of D-Tel every other day from 6 to 9 weeks of age. Group 7 (dendrimer-triantennary β-GlcNAc-azide-telmisartan ester conjugate or “D-TelB” high): Eight NASH mice were intraperitoneally administered vehicle supplemented with a 90 mg / kg dose of D-TelB every other day from 6 to 9 weeks of age. Group 8 (D-OCA high): Eight NASH mice were intraperitoneally administered vehicle supplemented with a 315 mg / kg dose of D-OCA (equivalent to approximately 30 mg / kg of dendrimer-conjugated OCA) every other day from 6 to 9 weeks of age. Group 9 (D-OCA low): Eight NASH mice were intraperitoneally administered vehicle supplemented with a 63 mg / kg dose of D-OCA (equivalent to approximately 6 mg / kg of dendrimer-conjugated OCA) every other day from 6 to 9 weeks of age. Group 10 (D-Cy5-6 wks): Four NASH mice received a single intraperitoneal injection of vehicle supplemented with a 50 mg / kg dose of D-Cy5 at 6 weeks of age. Group 11 (D-Cy5-9 wks): Four NASH mice received a single intraperitoneal injection of vehicle supplemented with a 50 mg / kg dose of D-Cy5 at 9 weeks of age.

[0311] Mice in groups 10 and 11 were sacrificed 48 hours after administration at 6 and 9 weeks of age. Mice in groups 1-9 were sacrificed at 9 weeks of age for later assays, and groups 10 and 11 were sacrificed at 6 and 9 weeks of age for later assays. Individual liver weights were measured, and liver weight-to-body weight ratios were calculated. Biochemical assays (groups 1-9):

[0312] Nonfasting serum ALT levels were quantified using FUJI DRI CHEM (Fujifilm, Japan). Liver triglycerides were quantified using a Triglyceride E Test Kit (FUJIFILM Wako Pure Chemical Corporation, Japan). Histological analysis of liver sections (groups 1-9):

[0313] HE staining and estimation of NAFLD activity score were performed by routine methods. Sirius red staining and estimation of percentage of fibrotic area were also calculated. Sample collection and fixation:

[0314] After the in-life portion of the study was completed, the following samples were collected for further analysis or transport: Animals in groups 10-11 were anesthetized with isoflurane and perfused through the left ventricle with saline (followed by 4% neutral buffered formalin, NBF, pH 7.4) for 20-30 minutes. The animals were dissected and tissue samples (left and right kidneys, liver) were collected serially. The thickness of the samples was approximately less than 5 mm to ensure proper fixation. A flat surface was trimmed to the area of interest. The samples were immediately placed in 4% NBF for fixation. The samples were fixed overnight at room temperature in 4% NBF. result

[0315] Individual body weights were measured throughout the measurement period. Untreated (vehicle) or treated experimental NASH mice maintained similar body weights throughout the study period. Normal mice, weighing approximately 26–27 grams throughout the experiment, were larger than all NASH mice, but NASH mice in all treatment groups showed similar body weights throughout the treatment period. At week 9, i.e., three weeks after the start of treatment, there was no significant difference in body weights between the different treatment groups (Figure 9A). All treatment groups, except for the group treated with free telmisartan, showed similar liver weights at week 9. NASH mice treated with free telmisartan had reduced liver weights compared to vehicle-treated NASH mice (Figure 9B). Figure 9C shows the liver weight to body weight ratios for all experimental groups.

[0316] Biochemical assays measuring nonfasting serum ALT and liver triglyceride levels were performed at 9 weeks of age (FIGS. 10A and 10B).

[0317] Histopathological analysis was performed on the livers of normal and NASH mice from all treatment groups sacrificed at 9 weeks of age. Nonalcoholic fatty liver disease (NAFLD) activity scores, steatosis scores, inflammation scores, and ballooning scores are shown in Figures 11A-11D. Sirius red staining was used to assess the degree of fibrosis in the livers of normal and NASH mice from all treatment groups (Figure 12).

[0318] Obeticholic acid (OCA) is a potent and selective farnesoid X receptor agonist (FXRa). Immunohistochemical analysis of liver tissue demonstrated that dendrimer-triantennary β-GlcNAc-azide-obeticholic acid ester conjugate (D-OCA) significantly reduced NAFLD scores, liver fibrosis, and hepatocellular ballooning compared with free OCA and vehicle controls (p<0.05, n=8). Low-dose D-OCA treatment showed a significant reduction in steatosis scores, while high-dose D-OCA treatment showed an improved reduction in fibrosis scores compared with the free OCA group. Biochemical analysis also suggested that treatment with hepatocyte-targeting hydroxyl dendrimer-conjugated obeticholic acid improved liver function.

[0319] In summary, we have established that hepatocyte-targeted hydroxyl dendrimer therapeutics enable selective targeting of FXRa to hepatocytes through asialoglycoprotein receptor (ASGP-R)-mediated uptake after systemic administration, enhancing drug efficacy and reducing dose- and site-specific toxicity. Selective targeting of FXRa to hepatocytes improves functional outcomes in NASH models. This targeted approach significantly reduces the systemic off-target toxicity observed with current FXRa compounds. Previous studies with hydroxyl-terminated dendrimers demonstrated sustained localization within target cells for up to one month. Overall, the hepatocyte-targeted hydroxyl dendrimer approach provides a platform for developing a broad range of drugs to treat liver disease.

[0320] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosed invention belongs. Publications cited herein and the material for which they are cited are expressly incorporated by reference.

[0321] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims. The present invention provides, for example, the following items. (Item 1) 1. A method of treating or preventing one or more symptoms of liver disease and / or liver damage in a subject in need thereof, comprising: administering to a subject a composition comprising a dendrimer covalently conjugated to a triantennary N-acetylgalactosamine (GalNAc) and complexed to, covalently conjugated to, inter-molecularly dispersed within, or encapsulated within, one or more therapeutic or prophylactic agents; The method, wherein the composition is administered in an amount effective to treat, alleviate, or prevent one or more symptoms of liver disease and / or liver damage. (Item 2) 2. The method of claim 1, wherein the dendrimer is covalently conjugated to a triantennary N-acetylgalactosamine (GalNAc) via an ester, ether, or amide bond, optionally with one or more linkers. (Item 3) 3. The method according to claim 1 or 2, wherein the dendrimer is a hydroxyl-terminated dendrimer. (Item 4) 4. The method of any one of items 1 to 3, wherein the dendrimer is a fourth, fifth, sixth, seventh, or eighth generation poly(amidoamine) dendrimer. (Item 5) 5. The method of any one of items 1 to 4, wherein the therapeutic agent is one or more agents selected from the group consisting of angiotensin II receptor blockers, farnesoid X receptor agonists, death receptor 5 agonists, sodium-glucose cotransporter type 2 inhibitors, lysophosphatidic acid 1 receptor antagonists, endothelin-A receptor antagonists, PPARδ agonists, AT1 receptor antagonists, CCR5 / CCR2 antagonists, anti-fibrotic agents, anti-inflammatory agents, antioxidants, STING agonists, CSF1R inhibitors, PARP inhibitors, VEGFR tyrosine kinase inhibitors, EGFR tyrosine kinase inhibitors, MEK inhibitors, glutaminase inhibitors, TIE II antagonists, CXCR2 inhibitors, CD73 inhibitors, arginase inhibitors, PI3K inhibitors, TLR4 agonists, TLR7 agonists, SHP2 inhibitors, chemotherapeutic agents, and combinations thereof. (Item 6) 6. The method of any one of items 1 to 5, wherein the agent is covalently conjugated to the dendrimer, optionally via a linker or spacer moiety. (Item 7) 7. The method of any one of items 1 to 6, wherein the linker or spacer moiety is attached to the dendrimer via a linkage selected from the group consisting of an ether, an ester, and an amide linkage. (Item 8) 8. The method of any one of items 1 to 7, wherein the linker or spacer moiety is attached to the dendrimer via an amide or ether linkage. (Item 9) 9. The method of any one of items 1 to 8, wherein the one or more liver diseases and / or liver disorders are selected from the group consisting of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, drug-induced liver failure, hepatitis, liver fibrosis, cirrhosis, hepatocellular carcinoma, or a combination thereof. (Item 10) 10. The method according to any one of items 5 to 9, wherein the angiotensin II receptor blocker is telmisartan, or a derivative, analogue or prodrug thereof, optionally a telmisartan-amide derivative or a telmisartan-ester derivative. (Item 11) 10. The method according to any one of items 5 to 9, wherein the FXR agonist is chenodeoxycholic acid, or a derivative, analogue or prodrug thereof, optionally a chenodeoxycholic acid-amide derivative or a chenodeoxycholic acid-ester derivative. (Item 12) 10. The method of any one of items 5 to 9, wherein the one or more SGLT2 inhibitors are selected from the group consisting of phlorizin, T-1095, canagliflozin, dapagliflozin, ipragliflozin, tofogliflozin, empagliflozin, luseogliflozin, ertugliflozin, and remogliflozin etabonate, or derivatives, analogs, or prodrugs thereof. (Item 13) 10. The method according to any one of items 5 to 9, wherein the PPARδ agonist is GW0742, or a derivative, analogue or prodrug thereof, optionally a GW0742-amide derivative or a GW0742-ester derivative. (Item 14) 10. The method according to any one of items 5 to 9, wherein the antioxidant is vitamin E, or a derivative, analog, or prodrug thereof. (Item 15) 15. The method of any one of items 1-14, wherein the composition is administered in an amount effective to reduce serum levels of one or more of alanine aminotransferase, aspartate aminotransferase, triglycerides, gamma-glutamyltransferase, total cholesterol, low-density lipoprotein, fasting blood glucose, or a combination thereof in the subject. (Item 16) 15. The method of any one of items 1-14, wherein the composition is administered in an amount effective to reduce one or more of steatosis, inflammation, ballooning, fibrosis, cirrhosis, or a combination thereof in the subject. (Item 17) 15. The method of any one of items 1 to 14, wherein the formulation is administered in an amount effective to reduce lobular inflammation in the liver of said subject. (Item 18) 15. The method of any one of items 1 to 14, wherein the composition is administered in an amount effective to reduce the amount or presence of one or more pro-inflammatory cells, chemokines, and / or cytokines in the liver of the subject. (Item 19) 19. The method of claim 18, wherein the composition is administered in an amount effective to reduce one or more pro-inflammatory cytokines in the subject, or wherein the pro-inflammatory cytokines are selected from the group consisting of TNF-α, IFN-γ, IL-6, IL-1β, IL-23, and IL-17. (Item 20) 6. The method of any one of items 1 to 5, wherein the therapeutic agent is one or more agents selected from the group consisting of a STING agonist, a CSF1R inhibitor, a PARP inhibitor, a VEGFR tyrosine kinase inhibitor, an EGFR tyrosine kinase inhibitor, a MEK inhibitor, a glutaminase inhibitor, a TIE II antagonist, a CXCR2 inhibitor, a CD73 inhibitor, an arginase inhibitor, a PI3K inhibitor, a TLR4 agonist, a TLR7 agonist, a SHP2 inhibitor, a cytotoxic agent, a chemotherapeutic agent, and combinations thereof. (Item 21) the STING agonist is the cyclic dinucleotide GMP-AMP or DMXAA; the CSF1R inhibitor is selected from the group consisting of PLX3397, PLX108-01, ARRY-382, PLX7486, BLZ945, JNJ-40346527, and GW2580; the PARP inhibitor is selected from the group consisting of olaparib, veliparib, niraparib, and rucaparib; the VEGFR tyrosine kinase inhibitor is selected from the group consisting of sunitinib or a derivative or analog thereof, sorafenib, pazopanib, vandetanib, axitinib, cediranib, vatalanib, dasatinib, nintedanib, and motesanib; the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD325901, PD035901, PD032901, and TAK-733; the glutaminase inhibitor is selected from the group consisting of bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-L-norleucine (DON), azaserine, acivicin, and CB-839; the CXCR2 inhibitor is navarixin, SB225002, or SB332235; the CD73 inhibitor is APCP, quercetin, or tenofovir, or a derivative or analog thereof; the arginase inhibitor is a derivative or analog of 2-(S)-amino-6-boronohexanoic acid; the PI3K inhibitor is selected from the group consisting of alpelisib, ceravelisib, pilalalisib, WX-037, dactolisib, prexasertib, voxtalisib, PX-866, ZSTK474, buparlisib, pictilisib, and copanlisib; the immunomodulatory agent is an SHP2 inhibitor, or the cytotoxic agent is auristatin E or mertansine; Item 20. The method according to item 20. (Item 22) The chemotherapeutic agent is selected from the group consisting of amsacrine, bleomycin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxin, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, daunorubicin, lomustine, mechlorethamine, melfala 21. The method of item 20, wherein the medicament is selected from the group consisting of benzodiazepine, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, taxol, trichostatin A and its derivatives, trastuzumab, cetuximab, rituximab, and bevacizumab. (Item 23) 23. The method of any of items 20 to 22, wherein the composition is administered in an amount effective to reduce tumor size in the subject and / or effective to enhance a tumor-specific cytotoxic T cell response. (Item 24) 24. The method according to any of items 1 to 23, wherein the composition is formulated as a pharmaceutically acceptable preparation for intravenous, subcutaneous, or intramuscular administration. (Item 25) 24. The method according to any one of items 1 to 23, wherein the composition is formulated as a pharmaceutically acceptable formulation for enteral administration. (Item 26) 25. The method of any one of items 1 to 24, wherein the composition is administered via an intravenous, subcutaneous, or intramuscular route. (Item 27) 26. The method of any one of items 1 to 23 or 25, wherein the composition is administered via enteral administration. (Item 28) 28. The method of any one of items 1 to 27, wherein the composition is administered to the subject before, along with, after, or alternating with treatment of the subject with one or more additional therapies or procedures. (Item 29) 29. The method of claim 28, wherein the one or more additional steps comprise administering one or more therapeutic, prophylactic, and / or diagnostic agents to prevent or treat one or more symptoms of a disease or condition related to liver injury in the subject, including infection, sepsis, diabetic complications, hypertension, obesity, high blood pressure, heart failure, kidney disease, and cancer. (Item 30) 30. A pharmaceutical composition for use in the method according to any one of items 1 to 29. (Item 31) 1. A method of making a dendrimer having one or more tri-antennary GalNAc molecules thereon, comprising: (a) Hypermonomer AB 3 preparing a step comprising carrying out propargylation of the hypermonomer with three reactive groups; (b) conjugating one azide group onto an N-acetylgalactosamine (GalNAc) molecule, preferably via a spacer, to produce a GalNAc-azide building block; (c) the hypermonomer AB from step (a). 3 and the GalNAc-azide building blocks from step (b) and performing copper(I)-catalyzed alkyne azide click chemistry to yield triantennary GalNAc; and (d) conjugating the triantennary GalNAc to a reactive end group of a dendrimer. A method comprising: (Item 32) Prior to step (d), the hypermonomer AB having no GalNAc is preferably separated via a spacer. 3 32. The method according to item 31, further comprising the step of introducing an azide group onto one reactive group of (Item 33) 33. The method of claim 31 or 32, further comprising the step of introducing a terminal alkyne onto the dendrimer by reacting one or more terminal functional groups of the dendrimer with a hexynoic acid. (Item 34) 34. The method of any one of items 31 to 33, wherein step (d) of conjugating a triantennary GalNAc onto one reactive group of the dendrimer is achieved via copper(I)-catalyzed alkyne azide click chemistry. (Item 35) 35. The method according to any one of items 31 to 34, wherein the dendrimer is a 4th, 5th, 6th, 7th, or 8th generation hydroxyl-terminated polyamidoamine dendrimer. (Item 36) 36. The method of any one of items 31 to 35, wherein the dendrimer is further complexed and / or conjugated to one or more therapeutic, prophylactic, and / or diagnostic agents.

Claims

1. 1. A composition for treating or preventing one or more symptoms of liver disease and / or liver damage in a subject in need thereof, comprising: the composition comprises a hydroxyl-terminated dendrimer covalently conjugated to a triantennary N-acetylgalactosamine (GalNAc) and covalently conjugated to one or more therapeutic agents; each of the one or more therapeutic agents is covalently conjugated via a linker or spacer moiety attached to the dendrimer via an ether or ester linkage; The composition is administered in an amount effective to treat, alleviate, or prevent one or more symptoms of said liver disease and / or liver damage.

2. 2. The composition of claim 1, wherein the dendrimer is covalently conjugated to the triantennary GalNAc via an ester, ether, or amide bond, optionally by one or more linkers.

3. 3. The composition of claim 1 or 2, wherein the dendrimer is a fourth, fifth, sixth, seventh, or eighth generation poly(amidoamine) dendrimer.

4. The one or more therapeutic agents may be selected from the group consisting of an angiotensin II receptor blocker, a farnesoid X receptor agonist, a death receptor 5 agonist, a sodium-glucose cotransporter type 2 (SGLT2) inhibitor, a lysophosphatidic acid 1 receptor antagonist, an endothelin-A receptor antagonist, a PPARδ agonist, an AT1 receptor antagonist, a CCR5 / CCR2 antagonist, an anti-fibrotic agent, an anti-inflammatory agent, an antioxidant, a STING agonist, a CSF1R inhibitor, a PARP inhibitor, a VEGFR tyrosine kinase inhibitor, an EGFR tyrosine kinase inhibitor, a MEK inhibitor, a glutaminase inhibitor, a TIE inhibitor, a vasopressin ... II antagonist, CXCR2 inhibitor, CD73 inhibitor, arginase inhibitor, PI3K inhibitor, TLR4 agonist, TLR7 agonist, SHP2 inhibitor, chemotherapeutic agent, and combinations thereof.

5. 5. The composition of any one of claims 1 to 4, wherein the liver disease and / or liver damage is selected from the group consisting of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, drug-induced liver failure, hepatitis, liver fibrosis, cirrhosis, hepatocellular carcinoma, or a combination thereof.

6. The composition of claim 4 or 5, wherein the angiotensin II receptor blocker is telmisartan, or a derivative, analogue or prodrug thereof, optionally a telmisartan-amide derivative or a telmisartan-ester derivative.

7. The composition of claim 4 or 5, wherein the FXR agonist is chenodeoxycholic acid, or a derivative, analogue or prodrug thereof, optionally a chenodeoxycholic acid-amide derivative or a chenodeoxycholic acid-ester derivative.

8. The composition of claim 4 or 5, wherein the SGLT2 inhibitor is selected from the group consisting of phlorizin, T-1095, canagliflozin, dapagliflozin, ipragliflozin, tofogliflozin, empagliflozin, luseogliflozin, ertugliflozin, and remogliflozin etabonate, or derivatives, analogs, or prodrugs thereof.

9. The composition of claim 4 or 5, wherein the PPARδ agonist is GW0742, or a derivative, analogue or prodrug thereof, optionally a GW0742-amide derivative or a GW0742-ester derivative.

10. 6. The composition of claim 4 or 5, wherein the antioxidant is vitamin E, or a derivative, analog, or prodrug thereof.

11. 11. The composition of any one of claims 1 to 10, wherein the composition is administered in an amount effective to reduce serum levels of one or more of alanine aminotransferase, aspartate aminotransferase, triglycerides, gamma-glutamyltransferase, total cholesterol, low-density lipoprotein, fasting blood glucose, or a combination thereof in the subject.

12. 11. The composition of any one of claims 1 to 10, wherein the composition is administered in an amount effective to reduce one or more of steatosis, inflammation, ballooning, fibrosis, cirrhosis, or a combination thereof in the subject.

13. A composition described in any one of claims 1 to 10, wherein the composition is administered in an amount effective to reduce lobular inflammation in the liver of the subject.

14. 11. The composition of any one of claims 1 to 10, wherein the composition is administered in an amount effective to reduce the amount or presence of one or more pro-inflammatory cells, chemokines, and / or cytokines in the liver of the subject.

15. 15. The composition of claim 14, wherein the composition is administered in an amount effective to reduce one or more pro-inflammatory cytokines in the subject, or wherein the pro-inflammatory cytokines are selected from the group consisting of TNF-α, IFN-γ, IL-6, IL-1β, IL-23, and IL-17.

16. 4. The composition of any one of claims 1-3, wherein the one or more therapeutic agents are selected from the group consisting of a STING agonist, a CSF1R inhibitor, a PARP inhibitor, a VEGFR tyrosine kinase inhibitor, an EGFR tyrosine kinase inhibitor, a MEK inhibitor, a glutaminase inhibitor, a TIE II antagonist, a CXCR2 inhibitor, a CD73 inhibitor, an arginase inhibitor, a PI3K inhibitor, a TLR4 agonist, a TLR7 agonist, an SHP2 inhibitor, a cytotoxic agent, a chemotherapeutic agent, and combinations thereof.

17. the STING agonist is the cyclic dinucleotide GMP-AMP or DMXAA; the CSF1R inhibitor is selected from the group consisting of PLX3397, PLX108-01, ARRY-382, PLX7486, BLZ945, JNJ-40346527, and GW2580; the PARP inhibitor is selected from the group consisting of olaparib, veliparib, niraparib, and rucaparib; the VEGFR tyrosine kinase inhibitor is selected from the group consisting of sunitinib or a derivative or analog thereof, sorafenib, pazopanib, vandetanib, axitinib, cediranib, vatalanib, dasatinib, nintedanib, and motesanib; the MEK inhibitor is selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, PD325901, PD035901, PD032901, and TAK-733; the glutaminase inhibitor is selected from the group consisting of bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-L-norleucine (DON), azaserine, acivicin, and CB-839; the CXCR2 inhibitor is navarixin, SB225002, or SB332235; the CD73 inhibitor is APCP, quercetin, or tenofovir, or a derivative or analog thereof; the arginase inhibitor is a derivative or analog of 2-(S)-amino-6-boronohexanoic acid; the PI3K inhibitor is selected from the group consisting of alpelisib, selavelisib, pilalalisib, WX-037, dactolisib, prexasertib, voxtalisib, PX-866, ZSTK474, buparlisib, pictilisib, and copanlisib; the immunomodulatory agent is an SHP2 inhibitor, or the cytotoxic agent is auristatin E or mertansine; 17. The composition of claim 16.

18. The chemotherapeutic agent is selected from the group consisting of amsacrine, bleomycin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxin, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, daunorubicin, lomustine, mechlorethamine, and melphalan. , mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, taxol, trichostatin A and its derivatives, trastuzumab, cetuximab, rituximab, and bevacizumab.

19. The composition of any of claims 16 to 18, wherein the composition is administered in an amount effective to reduce tumor size and / or enhance a tumor-specific cytotoxic T cell response in the subject.

20. The composition of any of claims 1 to 19, wherein the composition is formulated as a pharmaceutically acceptable preparation for intravenous, subcutaneous, or intramuscular administration.

21. The composition of any one of claims 1 to 19, wherein the composition is formulated as a pharmaceutically acceptable formulation for enteral administration.

22. The composition of any one of claims 1 to 21, wherein the composition is administered via an intravenous, subcutaneous, or intramuscular route.

23. The composition of any one of claims 1 to 19 or 21, wherein the composition is administered via enteral administration.

24. 24. The composition of any one of claims 1 to 23, wherein the composition is administered to the subject before, along with, after, or alternating with treatment of the subject with one or more additional therapies or procedures.

25. 25. The composition of claim 24, wherein the one or more additional procedures comprise administering one or more therapeutic, prophylactic, and / or diagnostic agents to prevent or treat one or more symptoms of a disease or condition related to liver injury in the subject, including infection, sepsis, diabetic complications, hypertension, obesity, high blood pressure, heart failure, renal disease, and cancer.

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