Dendrimer composition for the treatment of atherosclerosis, obesity, and metabolic syndrome

Abstract

A dendrimer-drug composition has been developed to simultaneously and independently address atherosclerosis, obesity, and related metabolic disorders by delivering the drug in a targeted manner, targeting not only reactive inflammatory cells / macrophages within plaques but also reactive inflammatory cells / macrophages within adipose tissue. The dendrimer is preferably a glucose dendrimer, a hydroxyl-terminated PAMAM dendrimer, or a sugar-modified dendrimer, most preferably a glucose dendrimer. The drug is preferably a PPAR-α agonist; a PPAR-γ agonist; a dual PPAR agonist (e.g., a PPAR-α / γ agonist); a GLP-1 receptor agonist (e.g., semaglutide); metformin; an SGLT2 agonist, a GIP-1 receptor agonist, or an antagonist; a dual GLP-1 / GIP-1 receptor agonist (e.g., tyrzepatide); a mitochondrial uncoupling agent (e.g., niclosamide); or a combination thereof. These compositions have the ability to overcome the side effects of current drugs for obesity and heart disease and open a new avenue for resolving these disorders through selective cell targeting.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims the benefit and priority to U.S. Provisional Application No. 63 / 611,586 filed December 18, 2023, the entirety of which is incorporated by reference into this application.

[0003] Statement regarding federal government-sponsored research

[0004] The present invention was made with government support under Project Nos. NS113140 and AG063831, supported by the National Institutes of Health. The government holds certain rights to the present invention.

[0005] Field of invention

[0006] The disclosed invention generally relates to a dendrimer-conjugated compound for the treatment of obesity, atherosclerosis and / or metabolic syndrome, in particular, in the field of therapeutic agents that induce weight loss, and a method of using the same. Background Technology

[0007] Atherosclerosis is the leading cause of death worldwide, as it is a major cause of fatal cardiovascular diseases and events, including heart attacks, strokes, and limb ischemia leading to amputation. The focus of therapy remains on lifestyle changes, including smoking cessation, diet, and exercise, to mitigate risk factors, lower cholesterol, and manage obesity and type 2 diabetes. While these approaches are effective in slowing the progression of atherosclerosis, patient compliance remains low, and the incidence of life-threatening limb-threatening sequelae remains high. Due to the absence of plaque treatment and regression therapies, surgical intervention is the only option, and patients often progress from angioplasty and stent placement to the need for bypass surgery. Particularly in the lower extremities, bypass grafts exhibit high failure rates, and the need for re-intervention increases not only the risk of death but also the emotional, social, and financial burdens associated with this massive global issue. Therefore, there is an urgent unmet need to develop plaque regression therapies that can be rapidly applied in clinical practice.

[0008] Obesity is a common, serious, and costly chronic disease affecting adults and children in the United States that is on the rise. Obesity can cause type 2 diabetes, heart disease, and some cancers. A weight higher than what is considered healthy for a given height is described as overweight or obese. The Body Mass Index (BMI) is a screening tool for overweight and obesity. A BMI of 25.0 to <30 falls within the overweight range. A BMI of 30.0 or higher falls within the obesity range. Obesity is commonly subdivided into the following categories: Class 1: BMI 30 to <35; Class 2: BMI 35 to <40; Class 3: BMI 40 or higher. Class 3 obesity is sometimes classified as "severe" obesity.

[0009] Metabolic syndrome is a group of conditions that collectively raises the risk of coronary heart disease, diabetes, stroke, and other serious health problems. Metabolic syndrome is also referred to as insulin resistance syndrome. Metabolic syndrome is common in the United States; approximately one in three adults has metabolic syndrome. It is often diagnosed when an individual has abdominal obesity or an "apple-shaped" body. Excess fat in the abdominal area is a greater risk factor for heart disease than excess fat in other parts of the body. If blood pressure rises and remains high for a long period, it can damage the heart and blood vessels. High blood pressure can also cause plaque to accumulate in the arteries. Plaque can lead to cardiovascular diseases such as heart attacks or strokes.

[0010] High blood sugar levels can also damage blood vessels and increase the risk of blood clots. Blood clots can cause cardiovascular disease. High blood triglyceride levels can raise LDL cholesterol levels, thereby increasing the risk of heart disease.

[0011] HDL cholesterol helps remove LDL cholesterol from blood vessels, which can reduce the risk of plaque accumulation in the blood vessels.

[0012] Effective treatment options for all of these indications are limited. Atherosclerosis is the leading cause of death worldwide, as it is a major cause of fatal cardiovascular diseases and events, including heart attacks, strokes, and limb ischemia leading to amputation. Due to the absence of plaque treatment and regressive therapies, surgical intervention is the only option, and patients often progress from angioplasty and stent placement to the need for bypass surgery. Therapeutic approaches capable of penetrating plaque to target specific cells and deliver drugs into them will present a major opportunity to address the unmet needs in this field.

[0013] Macrophages are not only involved in plaque proliferation but are also found in adipose tissue in obesity. In normal individuals, macrophages account for 10% of the cells in white adipose tissue, but this increases to up to 50% in obesity, leading to a chronic low-grade inflammatory state associated with comorbidities of obesity such as insulin resistance, type 2 diabetes, atherosclerosis, and related cardiovascular diseases. Inflammation resulting in increased cytokine production from adipose tissue macrophages can exacerbate metabolic syndromes such as type 2 diabetes and obesity due to increased lipid deposition, glucose intolerance, and insulin resistance (Liang, W., Qi, Y., Yi, H., Mao, C., Meng, Q., Wang, H., & Zheng, C. (2022). Frontiers in Immunology , 13 , 908749; https: / / doi.org / 10.3389 / fimmu.2022.908749).

[0014] Targeting inflammation in obesity by specifically targeting adipocytes provides a unique mechanism for treating obesity and related comorbidities as well as metabolic syndrome.

[0015] Therefore, the object of the present invention is to provide a safer and / or more effective composition for the treatment of atherosclerosis, obesity, and other metabolic diseases.

[0016] Atherosclerosis is often associated with obesity. Atherosclerosis and obesity share commonalities in terms of the role of inflammatory cells. Targeting inflammation in obesity provides a unique mechanism for treating obesity and its associated comorbidities as well as metabolic syndrome. A dendrimer-drug composition has been developed that simultaneously and independently addresses atherosclerosis, obesity, and related metabolic disorders by delivering drugs in a targeted manner through the targeting of reactive inflammatory cells / macrophages within plaques as well as reactive inflammatory cells / macrophages within adipose tissue.

[0017] The dendrimer is preferably a glucose dendrimer, a hydroxyl-terminated PAMAM dendrimer, or a sugar-modified dendrimer, most preferably a glucose dendrimer. Preferred glucose dendrimers include G1, G2, and G3 glucose dendrimers, and preferred PAMAM dendrimers include G3, G4, G5, and G6 hydroxyl-terminated PAMAM dendrimers. The density and loading of the formulation on the dendrimer, the mode of attachment of the formulation to the dendrimer, the size and chemical composition of the dendrimer, as well as the size and composition of the linker to the formulation (if present), govern the rate, selectivity, and activity of the formulation to the delivery site (where the formulation binds to the receptor). In one preferred embodiment, a cleavable linker binds the formulation to the dendrimer. In the most preferred embodiment, the cleavable linker is an ester. In some forms, the linker contains a triazole moiety. In some forms, the formulation (e.g., drug) has a loading amount of about 2 weight percent to about 35 weight percent of the composition. In some forms, the formulation (e.g., drug) has a loading amount of about 2 weight percent to about 35 weight percent of the dendrimer-formulation conjugate (e.g., dendrimer-drug conjugate).

[0018] The dendrimer-drug compositions of small molecule drugs include PPAR-α / γ agonists such as tesaglitajar; metformin; semaglutide (Ozempic) ® Glucagon-like peptide-1 (GLP-1) receptor agonists such as ); canagliflozin (INVOKANA) ®Sodium-glucose co-transporter 2 (SGLT2) agonists, GIP-1 receptor agonists, or antagonists such as ); dual GLP-1 / GIP-1 receptor agonists (e.g., tyrzepatide); mitochondrial uncouplers (e.g., niclosamide); or combinations thereof. The efficacy of the exemplary dendrimer conjugate dendrimer-tesaglitazar is ApoE - / - It was verified in a mouse model.

[0019] Typically, the route of administration—oral, intranasal, or applied to other mucosal surfaces—affects the rate and dosage of the dendrimer conjugate into the brain. Dendrimers increase brain absorption, solubility, target binding, and PK, and help the drug to be localized to the correct compartment. In some embodiments, dendrimers are used to retain the therapeutic agent in the peripheral circulation rather than the central circulation. Brief explanation of the drawing

[0020] Fig. 1 is a protocol for feeding a high-fat diet ("HFD") to mice. Figures 2a~2d % change in body weight for HFD mice treated with free Tesa compared to dendrimer-conjugated tesaglitazar (Tesa) (D-Tesa) Fig. 2a ), body weight (g)( Fig. 2b ), fat amount %( Fig. 2c ) and lean body mass %( Fig. 2d It is the graph of ). Figures 3a–3c This illustrates that D-Tesa improves markers of atherosclerosis: PWV improves with D-Tesa treatment but not with free tesaglitasar ( Fig. 3a )(n=8~10 male mice per group); **** p<0.0001, by two-way ANOVA. Systolic blood pressure ( Fig. 3b ) did not change throughout the experiment. Body weight( Fig. 3c) showed an increase due to HFD administration but decreased after D-Tesa treatment (n=8–9 male mice per group). Fig. 3c During the treatment, untreated mice did not lose weight, but D-Tesa treated mice showed a steady decrease. Fig. 4 This is a graph of the percentage of lesion area comparing D-Tesa-treated and untreated HFD mice. Figures 5a~5d is Ach capacity ( Figs. 5a, 5c ) or SNP dose( Figs. 5c, 5d Relaxation (% PE max) according to D-Tesa dose for ) Figs. 5a, 5c ) and log EC 50 ( Figs. 5b, 5d This is a graph of acetylcholine ( Ai ) and sodium nitroprusside ( Bi It was induced by an increase in the concentration of ). Ach( Aii ) and SNP( Bii -LogEC50 for ) is calculated from the dose-response curve. (n=4 per group; * p<0.05, ** p<0.01, based on one-way ANOVA). Figures 6a and 6b This is a graph showing that D-Tesa treatment leads to an increase in PPAR-α / γ. The aortic lysis extract of D-Tesa-treated HFD-ingested ApoE mice showed PPAR-α ( Fig. 6a ) and PPAR-γ( Fig. 6b ) shows the increase of everyone. Figures 7a–7f This proves the efficacy of D-Tesa and that free Tesa does not show side effects. VCO2( Fig. 7a ) and VO2( Fig. 7b ) increased in D-Tesa-treated mice compared to vehicle and glass Tesa-treated mice, resulting in increased energy consumption ( Fig. 7c It leads to an increase in ). More food intake ( Fig. 7dDespite this, the body weight of D-Tesa-treated mice was significantly lower than that of vehicle-treated mice after 3 weeks ( Fig. 7e Edema, measured by the degree of foot edema which is a common side effect of tesaglitazar, does not occur in D-Tesa-treated mice but is observed in free tesaglitazar-treated mice ( Fig. 7f ). Figs. 8a–8i This is representative data showing that D-Tesa upregulates ABCA1 through PPAR-α / γ in vitro. Figures 9a–9d This is representative data showing that PO D-Cy5 administration is localized to plaque and adipose tissue macrophages. Figures 10a~10m This is representative data showing that D-Tesa stops the progression of aortic plaque by improving cholesterol efflux. Figures 11a–11n This is representative data showing that D-Tesa improves aortic vascular stiffness. Figs. 12a~12l This is representative data showing that D-Tesa stops endothelial dysfunction caused by the progression of atherosclerosis. Figs. 13a–13n This is representative data showing that the delivery of D-Tesa to white adipose tissue (WAT) leads to a reduction in inflammation and browning of adipocytes. Figures 14a–14f This is representative data showing that the dendrimer conjugation of tessaglitazar avoids the renal side effects of systemic activation. Figures 15a~15m The physiological benefits of D-Tesa therapy include LDLr with intact apoE-mediated cholesterol efflux. - / - This is representative data showing that it is reproduced in. Figures 16a–16d This is representative data showing that intraperitoneal (IP) administration of D-Cy5 leads to localization into plaque and adipose tissue macrophages. Figures 17a–17f D-Tesa is female apoE - / - This is representative data showing that it avoids the side effects of weight gain and kidney function impairment in mice. Figures 18a–18lD-ApoE treated with D-Tesa - / - This is representative data showing that mice exhibit improvement in their inflammatory profiles. Fig. 19 This shows the structure of GD-semaglutide, an exemplary glucose dendrimer (GD) GLP-1 agonist conjugate. Fig. 20 This is a collection of micrographs showing that glucose dendrimers (GD) target mitochondria in adipocytes (differentiated 3T3L1 cells), while hydroxyl-terminated poly(amidoamine) (HD) exhibits lower uptake: ( top ) Difference in dendrimer mitochondrial uptake after 1 hour; ( bottom Difference in dendrimer mitochondrial uptake after 24 hours. Fig. 21 This is a synthetic reaction scheme for an exemplary esterase-degradable niclosamide click conjugate (niclosamide-PEG-N3). Fig. 22a This is a representative H-NMR trace of GD-niclosamide, an exemplary glucose dendrimer conjugated to a mitochondrial uncoupling agent. Fig. 22b This is a representative HPLC trace showing a newly formed conjugate at 335 nm. Fig. 23 This is the synthesis scheme for a dendrimer-tyrzepatide conjugate, which is an exemplary dendrimer-GIP1 conjugate. Specific details for implementing the invention

[0021] I. Definition

[0022] The terms “active agent” or “biologically active agent” are used interchangeably to refer to chemical or biological compounds that induce a desired pharmacological and / or physiological effect, which may be prophylactic, therapeutic, or diagnostic. These may be nucleic acids, nucleic acid analogs, small molecules having a molecular weight of less than 2 kD, more typically less than 1 kD, peptidomimetic compounds, proteins or peptides, carbohydrates or sugars, lipids, or combinations thereof. These terms also include pharmaceutically acceptable pharmacologically active derivatives of a formulation, including but not limited to salts, esters, amides, prodrugs, active metabolites, and analogs. An analog is a chemically modified active compound derived from a parent. The term “therapeutic agent” refers to a formulation that may be administered to treat one or more symptoms of a disease or disorder. The term "diagnostic agent" generally refers to a substance that can be administered to reveal the localization of a pathological process and to accurately locate and define it. Diagnostic agents can label target cells, enabling the subsequent detection or imaging of these labeled target cells.

[0023] The term “therapeutically effective amount” refers to an amount of therapeutic agent that, when incorporated into and / or on a dendrimer, produces a 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 specific targeted agent administered, the size of the subject, or the severity of the disease or condition. A person skilled in the art can determine the effective amount of a specific compound empirically without requiring excessive experimentation. In some embodiments, the term “effective amount” refers to an amount of therapeutic or prophylactic agent that reduces or alleviates the symptoms of one or more diseases.

[0024] In the context of inhibition, the terms "inhibit" or "reduce" mean a decrease or decline in activity or quantity. This may be a complete inhibition or reduction of activity or quantity, or a partial inhibition or reduction. Inhibition or reduction may be compared to a control or standard level. Inhibition may be 5, 10, 25, 50, 75, 80, 85, 90, 95, 99, or 100%. For example, a dendrimer composition containing one or more inhibitors may inhibit or reduce the activity and / or quantity of diseased neurons by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% from the activity and / or quantity of the same cells in an equivalent tissue of a subject that has not been administered or treated with the dendrimer composition. In some embodiments, inhibition and reduction are compared at the level of mRNA, proteins, cells, tissues, and organs. For example, inhibition and reduction of the rate of neuronal loss, the rate of brain weight reduction, or the rate of hippocampal volume reduction compared to an untreated control subject.

[0025] The terms “treating” or “preventing” mean improving, reducing, or halting the onset or progression of a disease, disorder, or condition in animals that may be susceptible to such disease, disorder, and / or condition but have not yet been diagnosed with it; suppressing the disease, disorder, or condition, e.g., delaying its progression; and alleviating the 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 of the symptoms of a specific disease or condition even if the underlying pathophysiology is not affected, e.g., treating the subject’s pain by administering analgesics even if the analgesics do not treat the cause of the pain. Desirable therapeutic effects include a reduction in the rate of disease progression, improvement or alleviation of the disease state, and remission or improved prognosis. For example, if one or more symptoms associated with depression are alleviated or eliminated (including, but not limited to, reducing levels of anxiety, agitation, or restlessness; improving feelings of sadness, tearfulness, emptiness, or hopelessness; increasing the quality of life of those suffering from the disease; reducing the dosage of other medications needed to treat the disease; and delaying the progression of the disease), the individual is successfully "treated."

[0026] The phrases “pharmaceuticalally acceptable” or “biocompatible” refer to compositions, polymers, and other materials and / or dosage forms that are suitable for use in contact with human and animal tissues and meet a reasonable benefit-risk ratio within the scope of reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications. The phrase “pharmaceuticalally acceptable carrier” refers to pharmaceutically acceptable materials, compositions, or vehicles, e.g., liquid or solid fillers, diluents, solvents, or encapsulating materials, involved in transporting or carrying any of the subject compositions from one organ or part of the body to another. Each carrier must be “acceptable” in the sense that it is compatible with other components of the subject composition and does not cause harm to the patient.

[0027] The term "biodegradable" generally refers to materials that decompose or erode under physiological conditions into smaller units or chemical species that can be metabolized, removed, or excreted within the body. Decomposition time is a function of composition and morphology.

[0028] 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 this initiator core, and an outer surface of terminals attached to the outermost generation.

[0029] The term "functionalize" refers to modifying a compound or molecule in a way that results in the attachment of functional groups or moiety. For example, a molecule can be functionalized by the introduction of a molecule that makes the molecule a strong nucleophile or a strong electrophile.

[0030] The term "targeting moiety" refers to a moiety that is localized to or away from a specific location. The moiety can be, for example, proteins, nucleic acids, nucleic acid analogs, carbohydrates, or small molecules. The location can be a tissue, a specific cell type, a subcellular compartment, or a molecule such as a receptor.

[0031] The term “prolonged residence time” refers to an increase in the time required for the agent to be removed from the patient’s body, or from the patient’s organs or tissues. In certain embodiments, “prolonged residence time” refers to the agent being removed with a half-life 10%, 20%, 50%, or 75% longer than a comparative standard such as a similar agent without conjugation to a delivery vehicle such as a dendrimer. In certain embodiments, “prolonged residence time” refers to the agent being removed with a half-life 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, or 10000 times longer than a comparative standard such as a similar agent without a dendrimer specifically targeting a specific cell type.

[0032] The terms “incorporated” and “encapsulated” refer to incorporating, formulating, or otherwise including such formulations within and / or on top of a composition that enables the release of the formulation, e.g., sustained release, in the intended application. The formulation or other material may be incorporated into the dendrimer by binding to one or more surface functional groups of the dendrimer (by covalent, ionic, or other bonding interactions) and / or physically mixing, wrapping the formulation within the dendrimer structure and / or encapsulating the formulation within the dendrimer structure.

[0033] "Hydroxyl-terminated" in relation to dendrimers refers to dendrimers having hydroxyl groups on their surface. These hydroxyl groups are not attached to the ends of the dendrimers through sugar moiety (e.g., sugar moiety).

[0034] "Sugar-terminated" in relation to dendrimers refers to dendrimers that contain sugar moiety (e.g., sugar moiety) on the surface rather than the core.

[0035] "Sugar-based" in relation to dendrimers refers to a dendrimer that includes a core, or a sugar moiety (e.g., sugar moiety) on the core and surface.

[0036] An "analog" with respect to a given compound refers to another compound that is structurally similar to, functionally similar to, or both of the specified compound. Structural similarity may be determined using any criterion known in the art, such as the Tanimoto coefficient, which provides a quantitative measure of similarity between two compounds based on molecular descriptors. Preferably, the molecular descriptors are 2D characteristics such as fingerprints, phase indices, and maximum common substructures, or 3D characteristics such as overall shape and molecular field. The Tanimoto coefficient ranges from 0 to 1 for different molecular pairs and identical molecular pairs, respectively. If a compound has a Tanimoto coefficient of 0.5 to 1.0, preferably 0.7 to 1.0, and most preferably 0.85 to 1.0 with respect to the specified compound, it may be considered an analog of the specified compound. If a compound produces the same pharmacological effect, physiological effect, or both as the specified compound, it is functionally similar to the specified compound. "Analogous" may also refer to modifications including, but not limited to, hydrolysis, reduction, or oxidation products of a compound. Hydrolysis, reduction, and oxidation reactions are known in the art.

[0037] Immune Targeting in Obesity and Crosstalk Between Obesity and Atherosclerosis

[0038] Macrophages play a crucial role in plaque formation in atherosclerosis and in adipose tissue in obesity. Adipose tissue macrophages can account for up to 40–50% of all cells in white adipose tissue, and obesity leads to a state of low-grade inflammation associated with various comorbidities such as atherosclerosis, cardiovascular disease, and type 2 diabetes. Inflammation of adipose tissue macrophages results in increased cytokine production and signaling, subsequently leading to metabolic syndrome characterized by lipid deposition, glucose toxicity / glucose intolerance, and insulin resistance. Targeting inflammation in obesity provides a unique mechanism for treating obesity and its associated comorbidities, as well as metabolic syndrome.

[0039] Previous studies have definitively established the major roles of both innate and adaptive immunity in the development and progression of atherosclerosis. The innate immune response in atherosclerosis is initiated by chronic inflammation, which is a characteristic of the disease. In particular, chronic inflammation at branching points and other oscillatory shear sites induces endothelial cell activation and the expression of adhesion molecules E-selectin and VCAM-1, thereby attracting dendritic cells and monocytes and stimulating the differentiation of monocytes into macrophages through the local production of macrophage colony-stimulating factor (mCSF). Subsequent cholesterol uptake by macrophages leads to the formation of foam cells, a characteristic of atherosclerosis. Pro-inflammatory M1 macrophages further contribute to the exacerbation of local inflammation, inducing EC activation, promoting VSMC migration and proliferation, and causing a vicious cycle of inflammation and cellular disturbance that leads to plaque growth. Macrophages and dendritic cells in plaques act as antigen-presenting cells and activate adaptive immune responses. Th1 cells or pro-inflammatory cytokines, such as TNF-α and IFN-γ, promote atherogenic inflammation and increase plaque instability, which can lead to comorbidities such as heart attack and stroke. Due to the significant roles played by both the innate and adaptive immune systems in plaque formation and progression, targeting the inflammatory system is a promising strategy in the treatment of atherosclerosis.

[0040] Targeting of the immune system in atherosclerosis: Previous therapies focused on IL-β binding (the CANTOS study using the IL1β antibody canakinumab showed some improvement in cardiovascular events). Treatment with the IL1 receptor antagonist Anakinra (MRC-ILA-HEART study, showed short-term benefits but not long-term benefits) and antibodies targeting IL6 receptors or ligands (tocilizumab and ziltivekimab) showed a reduction in inflammation (long-term ZEUS study is ongoing); the use of TNF-α inhibitors appears to be associated with a reduction in CVD; small molecule anti-inflammatory agents such as colchicine showed improvement in CV events; CXCL1 neutralizing antibodies, MLN1202 (a highly specific humanized monoclonal antibody preventing CCL2 binding), and the CXCR4 antagonist Plerixafor; Immuno-checkpoint inhibitors targeting CD28, CD80, CD86, CTLA-4, and PD-1, or CD70, CD27, and CD137 (TNF superfamily); examples of these include abatacept, a CTLA4-Ig construct that blocks CD80 / CD86 from interacting with CD28 to activate T cells, or TRAF-STOP therapy using SMI 6860766 or 6877002. Other targets include IL2 administration (LILACS and IVORY studies) and rituximab (anti-CD20 antibody) targeting B cells. Other small molecules that reduce MACE are as follows: statins, GLP1 agonists, e.g., semaglutide and analogs (e.g., Ozembic ® ), Lp-PLA2 inhibitors, e.g., darapladib, sPLA2 inhibitors, e.g., verespladib, 5-LO inhibitors, VIA-2291 (atrelutone); methotrexate, PPAR-α / γ agonists, e.g., tesaglitasar, metformin all showed some degree of efficacy. However, the trials did not show sufficient efficacy, or the drugs exhibited debilitating side effects that prevented further progression.

[0041] These debilitating side effects can be prevented by using hydroxyl and glucose dendrimers to specifically deliver these drugs to macrophages and / or neurons.

[0042] Immune Targeting in Obesity and Confusion Between Obesity and Atherosclerosis

[0043] Macrophages play a crucial role in plaque formation in atherosclerosis and in adipose tissue in obesity. Adipose tissue macrophages can account for up to 40–50% of all cells in white adipose tissue, and obesity leads to a state of low-grade inflammation associated with various comorbidities such as atherosclerosis, cardiovascular disease, and type 2 diabetes. Inflammation of adipose tissue macrophages results in increased cytokine production and signaling, subsequently leading to metabolic syndrome characterized by lipid deposition, glucose toxicity / glucose intolerance, and insulin resistance. Targeting inflammation in obesity provides a unique mechanism for treating obesity and its associated comorbidities, as well as metabolic syndrome.

[0044] II. Composition

[0045] A. Dendrimer

[0046] Dendrimers are three-dimensional, hyperbranched, monodisperse, spherical, and multivalent macromolecules containing surface end groups (Reference [Tomalia, DA, et al ., Biochemical Society Transactions , 35, 61 (2007)]; and [Sharma, A., et al ., ACS Macro Letters , 3, 1079 (2014)]).

[0047] The term “dendrimer” includes, but is not limited to, a molecular structure having an inner core (“G0”) and layers of repeating units (or “generations”) attached to and extending from the inner core (each layer having one or more branching points), and an outer surface of terminals attached to the outermost generation. In some embodiments, the dendrimer has a regular dendrimer molecular structure, and in other cases, may have a hyperbranched structure with irregular branching lengths.

[0048] Generally, the dendrimers have a diameter of about 1 nm to about 60 nm, more preferably about 1 nm to about 50 nm, about 1 nm to about 40 nm, about 1 nm to about 30 nm, about 1 nm to about 20 nm, about 1 nm to about 10 nm, or about 1 nm to about 5 nm. In some embodiments, the diameter is about 1 nm to about 2 nm. The preferred size of the dendrimers for passing through the blood-brain barrier ("BBB") is less than 5 nm, whereas those for failing to pass through the BBB and remaining in peripheral blood circulation are greater than 5 nm. In some embodiments, the dendrimers have a diameter effective for penetrating the BBB for the delivery of an agent conjugated thereto and remaining near or inside target neurons and / or glial cells. In some embodiments, the dendrimer has a diameter effective for penetrating the BBB for the delivery of the formulation conjugated thereto and for internalizing into target neurons and / or glial cells, e.g., neurons, oligodendrocytes, astrocytes, microglia, and glial support cells. In some embodiments, the dendrimer has a diameter effective for penetrating a barrier interface, such as the blood nerve barrier ("BNB"), for the delivery of the formulation conjugated thereto and for internalizing into neurons and glial cells of the peripheral nervous system, e.g., neurons, Schwann cells, satellite cells, and glial support cells. In some embodiments, the dendrimer has a diameter effective for remaining in the peripheral circulation for the delivery of the formulation conjugated thereto to target cells of the peripheral nervous system.

[0049] In some embodiments, the dendrimers have a molecular weight of about 500 daltons to about 100,000 daltons, about 500 daltons to about 50,000 daltons, or about 1,000 daltons to about 20,000 daltons. A dendrimer size of <30,000 Da is preferred for transport across the blood-brain barrier (BBB), and a size of >50,000 Da is preferred for periphery confinement.

[0050] In some embodiments, the dendrimer has a hypercore (e.g., dipentaerythritol) and one or more monosaccharide branching units. In some embodiments, the monosaccharide branching units are bonded to the core or preceding layer of the monomer through a linker such as a polyethylene glycol chain. In a preferred embodiment, the hypercore is dipentaerythritol, and the monosaccharide branching units are glucose-based branching units as shown in Structures II–IV. In the most preferred embodiment, the dendrimer is made entirely of glucose building blocks. PAMAM dendrimers modified by sugar may also be effective, but dendrimers made of sugar, in particular glucose, are most preferred. Particularly preferred glucose dendrimers are G1 to G3 glucose dendrimers, e.g., G1, G2, and / or G3 glucose dendrimers.

[0051] Suitable dendrimer scaffolds for use in conjugates include, but are not limited to, poly(amidoamines) also known as PAMAM or STARBURST dendrimers; polypropylamine (POPAM), polyethyleneimine, polylysine, polyester, yptisene, aliphatic poly(ether), aromatic polyether dendrimers, dendrimers of sugars (e.g., glucose, galactose, mannose, fructose, etc.), and copolymers thereof, e.g., copolymers of sugars and alkylene glycols (e.g., dendrimers formed by glucose and ethylene glycol building blocks). The dendrimers may have multiple surface functional groups such as carboxyl, amine, hydroxyl, and / or acetamide. The terms "surface functional group" and "terminal group" are used interchangeably in this application. In some embodiments, the dendrimer has surface hydroxyl groups. In some embodiments, one or more of these surface functional groups are further modified with other molecules, for example, sugars (e.g., glucose, galactose, mannose, fructose, etc.) and / or polyalkylene glycols, for example, polyethylene glycol, so as to have sugar molecules and / or polyalkylene glycols as terminal moiety / molecules. Preferred PAMAM dendrimers include hydroxyl-terminated PAMAM dendrimers, particularly G3 to G6 hydroxyl-terminated PAMAM dendrimers, for example, G3, G4, G5, and G6 hydroxyl-terminated PAMAM dendrimers. The dendrimer may be any generation, including but not limited to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth generations. In some embodiments, the dendrimer is a PAMAM dendrimer used as a platform and is modified with functional groups to increase the number of surface hydroxyl groups.

[0052] In some embodiments, the dendrimer-activator conjugate is localized to the peripheral circulation and can specifically target specific tissue regions and / or cell types, such as peripheral neurons and macrophages, using higher-generation dendrimers (e.g., 4th, 5th, or 6th generation PAMAM dendrimers, 2nd, 3rd, or higher-generation glucose-based dendrimers). Additionally, or alternatively, the dendrimer-activator conjugate may be localized to the peripheral circulation by appropriate functionalization of the dendrimer (e.g., PEGylation).

[0053] In some embodiments, the dendrimer may use a specific generation of dendrimer, for example, a second generation (G2), G3, G4, and G5 PAMAM dendrimer and / or a glucose dendrimer, to specifically target specific tissue regions and / or cell types of the central nervous system (CNS), peripheral nervous system (PNS), and / or eye, for example, neurons and glial cells of the CNS and / or PNS. In the most preferred embodiment, the dendrimer is made entirely of glucose building blocks. Although PAMAM dendrimers modified by sugars may also be effective, dendrimers made of sugars, particularly glucose, are most preferred.

[0054] Monosaccharide-based dendrimers

[0055] In some embodiments, the branching unit comprises a monosaccharide. In some embodiments, the monosaccharide branching unit is attached to the core or previous layer of the monomer through a linker such as a polyethylene glycol chain. In some embodiments, the monosaccharide branching unit is a glucose-based branching unit. In some embodiments, the branching unit may include PEG and / or alkyl chain linkers between different dendrimer generations. For example, the glucose layer is connected via a PEG linker and a triazole ring. In some embodiments, the branching unit is identical for each generation of dendrimer generated from the core. Thus, for example, the branching unit is a glucose-based branching unit for generating a first-generation dendrimer, a second-generation dendrimer, and a third-generation dendrimer.

[0056] In some embodiments, the dendrimer has a hypercore (e.g., dipentaerythritol) and one or more monosaccharide branching units. In some embodiments, the hypercore is dipentaerythritol, and the monosaccharide branching unit is a glucose-based branching unit. In further embodiments, the spacer molecule is also alkyl (CH2) n - It can be a hydrocarbon-like unit.

[0057] In some embodiments, a dendrimer synthesized using a glucose building block, having a surface made mostly of glucose moiety, enables specific targeting of cells including damaged neurons, ganglion cells, and other neuronal cells of the brain, eye, and / or peripheral nervous system. In some embodiments, the glucose-based dendrimer selectively targets or is enriched within target neurons and / or glial cells. In some embodiments, the glucose-based dendrimer selectively targets or is enriched on the surface of target neurons and / or glial cells. In some embodiments, the glucose-based dendrimer selectively targets or is enriched within target neuronal cells, and selectively targets or is enriched on the surface of target neurons and / or glial cells. In some embodiments, the glucose-based dendrimer selectively targets or is enriched within and / or on the surface of damaged and / or diseased or hyperactive neurons and / or glial cells.

[0058] In some cases, the dendrimers comprise an effective number of sugar molecules and terminal groups, e.g., glucose and / or hydroxyl groups, to target one or more neurons and / or glial cells of the CNS, PNS, and / or eye. The terminal hydroxyl groups of these dendrimers may be part of a terminal glucose molecule, an extra hydroxyl group that is not part of a glucose molecule, or a combination thereof. In some embodiments, all terminal hydroxyl groups are part of a terminal glucose molecule. In some embodiments, the number of sugar molecules on the dendrimer ends is determined by the generation number.

[0059] In some embodiments, the dendrimer is prepared from glucose and oligoethylene glycol building blocks. Exemplary glucose dendrimers are shown in structures V and VII.

[0060] Some exemplary glucose dendrimers include a first-generation glucose dendrimer having 24 hydroxyl (-OH) terminal groups, a second-generation glucose dendrimer having 96 hydroxyl (-OH) terminal groups, a third-generation glucose dendrimer having 396 hydroxyl (-OH) terminal groups, and a fourth-generation glucose dendrimer having 1584 hydroxyl (-OH) terminal groups. For example, the glucose dendrimer is a second-generation glucose-based dendrimer having 24 glucose molecules in the periphery and 6 embedded glucose molecules in the backbone bonded together with PEG segments.

[0061] A dendrimer composition, referred to as "glucose dendrimers," has been developed that can selectively accumulate inside neurons, particularly in the nuclei of damaged or overactivated neurons. These dendrimers can also accumulate at high levels inside activated microglia. However, compared to hydroxyl dendrimers, which accumulate primarily in microglia, these dendrimers primarily migrate to neurons. Glucose dendrimers are described in "Dendrimer compositions for targeted delivery of therapeutic agents to neurons" in U.S. Patent Application No. US 63 / 327,610, filed April 5, 2022, by inventors Kannan Rangaramanujam, Rishi Sharma, Anjali Sharma, Sujatha Kannan, Nirnath Sah, Mira Sachdeva, and Siva P. Kambhampati of Johns Hopkins University.

[0062] The glucose dendrimer comprises (a) a central core, (b) one or more branched units (wherein the branched units are monosaccharide glucose-based branched units, optionally, a linker is conjugated thereto), and optionally (c) one or more therapeutic, prophylactic, and / or diagnostic agents. Generally, one or more branched units are conjugated to the central core, and the surface groups of the dendrimer are monosaccharide glucose molecules. In some embodiments, the central core is dipentaerythritol or a hexapropagylated derivative thereof. In some embodiments, the branched units are conjugated to the central core via a linker such as a hydrocarbon or an oligoethylene glycol chain. In one preferred embodiment, the branched units are β-D-glucopyranoside tetraethylene glycol azide having the following structure

[0063]

[0064] Or it is a peracetylated derivative thereof.

[0065] In some embodiments, the glucose dendrimer is a first-generation, second-generation, third-generation, fourth-generation, fifth-generation, or sixth-generation dendrimer. In one embodiment, the dendrimer is a first-generation dendrimer having the following structure:

[0066]

[0067] In a preferred embodiment, the dendrimer is a second-generation dendrimer having the following structure:

[0068]

[0069] In some embodiments, one or more therapeutic agents, prophylactic agents and / or diagnostic agents are encapsulated, bound, and / or conjugated within the dendrimer at a concentration of about 0.01 wt% to about 30 wt%, preferably about 1 wt% to about 20 wt%, more preferably about 5 wt% to about 20 wt%. In some embodiments, the dendrimer is conjugated to a small molecule, an antibody or its antigen-binding fragment, a nucleic acid, or a polypeptide. In some embodiments, the therapeutic agent conjugated to the dendrimer is an anti-inflammatory agent, an antioxidant, or an immunomodulator. In other embodiments, the dendrimer is conjugated to one or more diagnostic agents, such as a fluorescent dye, a near-infrared dye, a SPECT imaging agent, a PET imaging agent, and a radioisotope.

[0070] In some embodiments, the dendrimer and the therapeutic agent, prophylactic agent, or diagnostic agent(s) are joined through one or more linkers or coupling agents, such as one or more hydrocarbons or oligoethylene glycol chains. Exemplary bonds are disulfide, ester, ether, thioester, and amide bonds.

[0071] PAMAM Dendrimer

[0072] The term "PAMAM dendrimer" refers to a poly(amidoamine) dendrimer that may have carboxyl, amine, acetamide, and / or hydroxyl terminal groups of any generation, including but not limited to first-generation PAMAM dendrimers, second-generation PAMAM dendrimers, third-generation PAMAM dendrimers, fourth-generation PAMAM dendrimers, fifth-generation PAMAM dendrimers, sixth-generation PAMAM dendrimers, seventh-generation PAMAM dendrimers, eighth-generation PAMAM dendrimers, ninth-generation PAMAM dendrimers, or tenth-generation PAMAM dendrimers, and may include different cores together with an amidoamine building block. In some embodiments, the dendrimer is a fourth, fifth, or sixth-generation ("G") dendrimer. In some embodiments, the PAMAM dendrimer has hydroxyl-terminal groups.

[0073] Generally, the overall structure of a dendrimer can be divided into an internal core moiety followed by radially attached branching units (i.e., generations) (which are further decorated with chemical functional groups having desired terminal groups on the outer surface of the dendrimer).

[0074] In some embodiments, the dendrimers are in the form of nanoparticles, as described in detail in U.S. Publication Nos. US 2011 / 0034422, US 2012 / 0003155, and US 2013 / 0136697. For example, the molecular weight of the dendrimers may vary to produce polymeric nanoparticles that form particles having characteristics such as drug release rates optimized for specific applications. In general, conjugation to dendrimers can further improve the safety and efficacy of these formulations. For example, dendrimer conjugation can alter specific receptor activity and / or modify biodistribution. For example, the use of higher-generation dendrimers and / or dendrimers having a molecular weight greater than 24k Da can confine these formulations to the peripheral nervous system, thereby preventing their psychoactive effects.

[0075] In some embodiments, different dendrimer variants, including but not limited to dendrons and tectodendrimers, may be used as a delivery vehicle for bonding and delivering one or more activators. A dendron is a dendritic wedge comprising one type of functional group (functional group, f=1) in the core and another type of functional group (f=8, 16, 32, etc.) in the periphery. A tectodendrimer generally consists of a central dendrimer and a plurality of dendrimers attached to the periphery.

[0076] 1. Core

[0077] In some embodiments, the dendrimer is manufactured using a method of assembling the dendrimer from a polyfunctional core and expanding it outward by a series of reactions. The polyfunctional core moiety enables the stepwise addition of branched units (i.e., generations) around the core.

[0078] Exemplary chemical structures suitable for core moiety are dipentaerythritol, pentaerythritol, 2-(aminomethyl)-2-(hydroxymethyl)propane-1,3-diol, 2-ethyl-2-(hydroxymethyl)propane-1,3-diol, 3,3',3'',3'''-silanetetrayltetrakis(propane-1-thiol), 3,3-divinylpenta-1,4-diene, 3,3',3''-nitrilotripropionic acid, 3,3',3''-nitrilotris(N-(2-aminoethyl)propanamide), 3,3',3'',3'''-(ethane-1,2-diylbis(azanetriyl))tetrapropanamide, 3-(carboxymethyl)-3-hydroxypentanediic acid, 2,2'-((2,2-bis((2-hydroxyethoxy)methyl)propane-1,3-diyl)bis(oxy))bis(ethanol-1-ol), tetrakis(3-(trichlorosilyl)propyl)silane, 1-thioglycerol, 2,2,4,4,6,6-hexachloro-1,3,5,215,415,615-triazatrifinine, 3-(hydroxymethyl)-5,5-dimethylhexane-2,4-diol, 4,4',4''-(ethanol-1,1,1-triyl)triphenol, 2,4,6-trichloro-1,3,5-triazine, 5-(hydroxymethyl)benzene-1,2,3-triol, 5-(hydroxymethyl)benzene-1,3-diol, It comprises 1,3,5-tris(dimethyl(vinyl)silyl)benzene, a carbosiloxane core, nitrilotrimethanol, ethylenediamine, propane-1,3-diamine, butan-1,4-diamine, 2,2',2''-nitrilotris(ethanol-1-ol), alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, cucurbituril, benzene-1,2,3,4,5,6-hexathiol, monosaccharides, disaccharides, trisaccharides, oligosaccharides, or their azide-, alkyne-modified moiety. In some embodiments, the core moiety is chitosan. Thus, azide-modified chitosan or alkyne-modified chitosan is suitable for conjugation to branched units using click chemistry.

[0079] In some embodiments, the core moiety is ethylenediamine or tetra(ethylene oxide). In some embodiments, the core moiety is dipentaerythritol. An exemplary chemical structure suitable for use as a core moiety is as follows: Table 1 It appears in.

[0080] [Table 1]

[0081] Structural description of various building blocks (core, branch unit, surface functional group, monomer) for the synthesis of dendrimers

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] 2. Branch unit

[0089] Exemplary chemical structures suitable for branching units include monosaccharides. In some embodiments, the monosaccharide branching unit is attached to the core or preceding layer of the monomer via a linker such as a polyethylene glycol chain. In a preferred embodiment, the monosaccharide branching unit is a glucose-based branching unit. Exemplary glucose-based branching units are shown in Structures II-IV. Since these are spacer molecules, they are also alkyl (CH2) n - It can be a hydrocarbon-like unit.

[0090] The branching unit is a PEG or alkyl chain linker between different dendrimer generations, for example, the glucose layer is connected through a PEG linker and a triazole ring.

[0091] In a preferred embodiment, the branching unit is the same for each generation of dendrimer generated from the core. Therefore, in one embodiment, the branching unit is a glucose-based branching unit for generating a first generation dendrimer as shown in structures V to VII.

[0092] In some embodiments, the branch unit is a hyper-monomer, i.e., AB n It is a building block. Exemplary hyper-monomers include the AB4, AB5, AB6, AB7, and AB8 building blocks. The hyper-monomer strategy dramatically increases the number of available terminal groups. An exemplary AB4 hyper-monomer is peracetylated β-D-glucopyranoside tetraethylene glycol azide as shown in Structure III.

[0093] Table 1The chemical structures listed are also suitable as building blocks for forming the branched units of dendrimers. For example, the branched units of the dendrimer are dipentaerythritol, pentaerythritol, 2-(aminomethyl)-2-(hydroxymethyl)propane-1,3-diol, 2-ethyl-2-(hydroxymethyl)propane-1,3-diol, 3,3',3'',3'''-silanetetrayltetrakis(propane-1-thiol), 3,3-divinylpenta-1,4-diene, 3,3',3''-nitrilotripropionic acid, 3,3',3''-nitrilotris(N-(2-aminoethyl)propanamide), 3,3',3'',3'''-(ethane-1,2-diylbis(azanetriyl))tetrapropanamide, 3-(carboxymethyl)-3-hydroxypentanediic acid, 2,2'-((2,2-bis((2-hydroxyethoxy)methyl)propane-1,3-diyl)bis(oxy))bis(ethanol-1-ol), tetrakis(3-(trichlorosilyl)propyl)silane, 1-thioglycerol, 2,2,4,4,6,6-hexachloro-1,3,5,215,415,615-triazatrifinine, 3-(hydroxymethyl)-5,5-dimethylhexane-2,4-diol, 4,4',4''-(ethanol-1,1,1-triyl)triphenol, 2,4,6-trichloro-1,3,5-triazine, 5-(hydroxymethyl)benzene-1,2,3-triol, 5-(hydroxymethyl)benzene-1,3-diol, It is formed by 1,3,5-tris(dimethyl(vinyl)silyl)benzene, carbosiloxane core, nitrilotrimethanol, ethylenediamine, propane-1,3-diamine, butan-1,4-diamine, 2,2',2''-nitrilotris(ethanol-1-ol), alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, cucurbituril, benzene-1,2,3,4,5,6-hexathiol, monosaccharides, disaccharides, trisaccharides, oligosaccharides or their azide-, alkyne-modified moiety, or a combination thereof.

[0094] Other examples of chemical structures suitable for forming the branched units of the dendrimer disclosed in this application include, but are not limited to, sugar moiety, e.g., glucose, galactose, mannose, and fructose, and alkylene glycols, e.g., ethylene glycol, and combinations thereof. In some embodiments, the branched unit is chitosan. Thus, azide-modified chitosan or alkyne-modified chitosan is suitable for conjugation to a core moiety or to the same or different branched units using click chemistry. In some embodiments, the branched unit is methyl acrylate or ethylenediamine, or a combination thereof. In some embodiments, the branched unit is polyethylene glycerol linear or branched. In some embodiments, the branched unit is a copolymer of alkylene glycol (e.g., ethylene glycol) and a sugar moiety, e.g., glucose, galactose, mannose, and / or fructose.

[0095] 3. Surface functional groups

[0096] The surface functional groups / molecules of the dendrimer are not limited to primary amine terminals, hydroxyl terminals, carboxylic acid terminals, acetamide terminals, sugar molecules, oligo- or poly-alkylene glycols and / or thiol terminals. In some embodiments, the desired terminal functional group may be added via one of the bonding methods to the core and branch units.

[0097] In some embodiments, the surface functional group is a hydroxyl group, for example, a hydroxyl group of a PAMAM dendrimer, a hydroxyl group of a second-generation PEG dendrimer as shown in Structure I, or a hydroxyl group of the terminal glucose of a dendrimer prepared from a glucose-based branched unit as shown in Structures V and VII. In some embodiments, the desired surface functional group may be modified or added through one of the bonding methods to the core and branched unit. Exemplary surface functional groups include hydroxyl terminal groups, amine terminal groups, carboxylic acid terminal groups, acetamide terminal groups and thiol terminal groups, and combinations thereof.

[0098] In some embodiments, the dendrimer may specifically target specific tissue regions and / or cell types, such as cells and tissues of the central nervous system (CNS), peripheral nervous system (PNS), and / or the eye. In some embodiments, the dendrimer specifically targets neurons and / or glial cells of the CNS. In some embodiments, the dendrimer specifically targets neurons and / or glial cells of the PNS. In some embodiments, the glucose dendrimers are of the first generation (G1), G2, G3, G4, and G5.

[0099] In some embodiments, the dendrimer comprises an effective number of terminal glucose and / or hydroxyl groups to target one or more neurons and / or glial cells of the CNS, PNS and / or eye.

[0100] Glucose dendrimers are preferred. In some embodiments, the dendrimers are prepared from glucose and oligoethylene glycol building blocks. An exemplary first-generation glucose dendrimer is shown in Structure VI, and a second-generation glucose dendrimer is shown in Structure VIII.

[0101] In some embodiments, the dendrimer has a plurality of surface functional groups (also referred to as surface functional groups or peripheral functional groups in this application), such as hydroxyl (-OH) groups, amine groups, acetamide groups, and / or carboxyl groups, on the periphery of the dendrimer. In some embodiments, the surface density of these peripheral functional groups is at least one group / nm 2 (Number of surface functional groups / Surface area (nm) 2 )) is. For example, in some embodiments, the surface density of surface functional groups such as hydroxyl groups is greater than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 OH groups / nm 2 , for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 OH groups / nm 2 is. In some embodiments, the bulk density of surface functional groups, such as hydroxyl groups, is about 1 to about 50 groups / nm. 3 , about 5 to about 30 units / nm 3 , or about 10 to about 20 units / nm 3 is. In additional embodiments, the surface density of surface functional groups such as hydroxyl groups is about 1 to about 50, preferably 5 to 20 groups / nm. 2 (Number of surface functional groups / Surface area (nm) 2 Each surface functional moiety has a molecular weight of about 100 Da to about 10 kDa, preferably about 100 Da to 1000 Da.

[0102] In some embodiments, the amount of surface functional groups of the dendrimer, for example, any one of those described above, for example, hydroxyl groups, is in the range of at least 40%, at least 50%, more than 40%, more than 50%, or more than 40% to 100%.

[0103] In some embodiments, one or more surface functional groups on the periphery of the dendrimer, for example, any one of those described above, may be further modified by conjugating with one or more carbohydrate molecules and / or one or more polyalkylene glycols, for example, polyethylene glycol. In these embodiments, the surface density of the terminal carbohydrate moiety / molecule and / or polyalkylene glycol may be any of the ranges described above for the hydroxyl groups. Hydroxyl-terminated PAMAM dendrimers, PAMAM dendrimers modified on the surface with sugar moiety (where >10% of the surface groups are modified by sugar, in particular, glucose), and glucose dendrimers (where the dendrimer is prepared from glucose building blocks) are preferred. For delivery to the brain, a composition having a total molecular weight of <30,000 Da is preferred. For confinement primarily to the peripheral circulation, a composition having a total molecular weight of >50,000 Da is preferred. When a dendrimer is formed of or contains a sugar moiety / molecule at its ends, e.g., glucose, the terminal hydroxyl groups of these dendrimers may be part of the terminal sugar moiety / molecule, extra hydroxyl groups that are not part of the sugar moiety / molecule, or a combination thereof. In some embodiments, all terminal hydroxyl groups are part of the terminal sugar moiety / molecule.

[0104] a. Hydroxyl-terminated dendrimer

[0105] In some embodiments, the dendrimer comprises a plurality of hydroxyl groups. Some exemplary high-density hydroxyl group-containing dendrimers comprise commercially available polyester resinous polymers, e.g., hyperbranched 2,2-bis(hydroxyl-methyl)propionic acid polyester polymers (e.g., hyperbranched bis-MPA polyester-64-hydroxyl, 4th generation), and resinous polyglycerols. In some embodiments, the hydroxyl-terminated dendrimer comprises hydroxyl-terminated PAMAM dendrimers, particularly G3 to G6 hydroxyl-terminated PAMAM dendrimers, e.g., G3, G4, G5, and G6 hydroxyl-terminated PAMAM dendrimers.

[0106] In some embodiments, the high-density hydroxyl group-containing dendrimers are oligoethylene glycol (OEG)-like dendrimers. For example, second-generation OEG dendrimers (D2-OH-60) as shown in Structure I can be synthesized using highly efficient, powerful, and atomic-economical chemical reactions such as Cu(I)-catalyzed alkyne-azide click and photocatalyzed thiol-en click chemistry. Very low-generation high-density polyol dendrimers with minimal reaction steps can be achieved using orthogonal hyper-monomer and hypercore strategies, for example, as described in International Publication WO 2019 / 094952. In some embodiments, the dendrimer backbone has uncleavable polyether bonds throughout the structure, thereby avoiding the degradation of the dendrimers in vivo and enabling the removal of these dendrimers from the body as a single entity (non-biodegradable).

[0107]

[0108] Structure I. 2nd Generation (G2) Oligoethylene Glycol-like Dendrimer

[0109] In some embodiments, the dendrimer has a plurality of hydroxyl (-OH) groups on the periphery of the dendrimer. In some embodiments, the surface density of the hydroxyl (-OH) groups is at least one OH group / nm 2 (Number of surface hydroxyl groups / Surface area (nm) 2 )) is. For example, in some embodiments, the surface density of hydroxyl groups is greater than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 OH groups / nm 2 , for example, nm 2 At least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 per. In some embodiments, the bulk density of hydroxyl groups is about 1 to about 50 groups / nm 3 , about 5 to about 30 units / nm 3 , or about 10 to about 20 units / nm 3 is. In a further embodiment, the surface density of hydroxyl (-OH) groups is about 1 to about 50, or 5 to 20 OH groups / nm, having a molecular weight of about 100 Da to about 10 kDa, preferably about 100 Da to 1000 Da. 2 (Number of surface hydroxyl groups / Surface area (nm) 2 )) is. In some embodiments, the amount of surface hydroxyl groups of the dendrimer is preferably greater than 35%, at least 40%, at least 50%, greater than 40%, greater than 50%, or greater than 40% to 100%. In some embodiments, some hydroxyl groups of the dendrimer may be exposed on the outer surface and the remainder may be in the inner core of the dendrimer.

[0110] In some embodiments, the dendrimer specifically targets a specific tissue region and / or cell type after administration into the body. In some embodiments, the dendrimer specifically targets a specific tissue region and / or cell type without a targeting moiety. In some embodiments, the dendrimer comprises an effective number of hydroxyl groups to target CNS cells and / or PNS cells, such as microglia, astrocytes and / or neurons, associated with diseases, disorders, or damage to the central or peripheral nervous system. In some embodiments, the dendrimer specifically targets a specific tissue region and / or cell type without a targeting moiety, and the activator conjugated thereto binds directly to receptors on the surface and / or inside the target neurons and / or glial cells.

[0111] In some embodiments, the dendrimers can specifically target specific tissue regions and / or cell types, preferably cells and tissues of the central nervous system (CNS) and / or the eye. In some embodiments, the dendrimers specifically target neurons of the CNS and the eye. Unmodified PAMAM dendrimers having hydroxyl terminals do not accumulate in neurons of the brain and / or retinal ganglion cells (RGCs) of the eye as much as these glucose dendrimers. Glucose dendrimers having terminal glucose monosaccharides and high-density hydroxyl functional groups effectively target neurons in a generation-dependent manner. The examples demonstrate efficacy with the second generation (G2), and G3 and G4 should also be effective. G5 and above are more difficult to use.

[0112] In a preferred embodiment, the dendrimer comprises an effective number of terminal glucose and / or hydroxyl groups to target one or more neurons in the CNS or eye. The hydroxyl groups on the surface of the dendrimer are part of the glucose molecule. There are no extra hydroxyl groups added to the glucose molecule on the surface. The number of sugar molecules on the surface is determined by the generation number. All generations are expected to target neurons.

[0113] In some embodiments, the dendrimers are prepared from glucose and oligoethylene glycol building blocks. Exemplary glucose dendrimers, for example, first-generation dendrimers as shown in structures IV–VI and second-generation dendrimers as shown in FIGS. 1a and 1b, are shown in the examples. Some exemplary glucose dendrimers include a first-generation glucose dendrimer having 24 hydroxyl (-OH) terminal groups, a second-generation glucose dendrimer having 96 hydroxyl (-OH) terminal groups, a third-generation glucose dendrimer having 396 hydroxyl (-OH) terminal groups, and a fourth-generation glucose dendrimer having 1,584 hydroxyl (-OH) terminal groups. In one preferred embodiment, the glucose dendrimer is a second-generation glucose-based dendrimer having 24 glucose molecules in the periphery and 6 embedded glucose molecules in the backbone bonded together with PEG segments.

[0114] b. Carbohydrate-modified dendrimer

[0115] In some embodiments, the dendrimer comprises one or more carbohydrate molecules at its ends. These terminal carbohydrate molecules may be prepared by conjugating one or more surface functional groups of the dendrimer, such as amine groups, carboxyl groups, or hydroxyl groups, to one or more carbohydrate molecules. In a preferred embodiment, the dendrimer prior to carbohydrate conjugation is a hydroxyl-terminated dendrimer, for example, a hydroxyl-terminated PAMAM dendrimer, and one or more of the hydroxyl groups are conjugated to one or more carbohydrate molecules.

[0116] In some embodiments, hydroxyl-terminated dendrimers modified with surface glucose molecules selectively target central and / or peripheral nerves and / or glial cells in vitro and in vivo; or selectively accumulate on the surface and / or inside these target nerve cells, glial cells and / or macrophages, so that activator(s) conjugated thereto bind to one or more receptors on / inside the target nerves and / or glial cells.

[0117] In some embodiments, the carbohydrate moiety used to modify one or more surface functional groups of the dendrimer is a monosaccharide. Exemplary monosaccharides suitable for modifying the dendrimer include glucose, glucosamine, galactose, mannose, fructose, dehydroascorbic acid, urate, and myo-inositol. In some embodiments, the dendrimer is conjugated to glucose and comprises glucose as a terminal moiety / molecule. In some embodiments, the hydroxyl-terminated dendrimer is modified to one or more glucose moietys for the dendrimer ("D-Glu"). In some embodiments, the dendrimer is conjugated to galactose. In some embodiments, the dendrimer is conjugated to mannose. In some embodiments, the dendrimer is conjugated to fructose. In some embodiments, the dendrimer is conjugated to one or more monosaccharides other than glucose, e.g., galactose, mannose, and / or fructose. For example, the carbohydrate moiety is an oligosaccharide that is terminaled with one or more monosaccharides, including glucose, glucosamine, mannose, and fructose, and exposes these sugar moiety on the surface for bonding.

[0118] Glucose dendrimers or glucose-modified dendrimers are used to obtain selective uptake by target cells. A drug conjugated to a dendrimer binds to a receptor or other site of action. In a preferred embodiment, the dendrimer or a dendrimer modified with a functional group has an affinity for one or more of the formulation receptors, e.g., CB1 receptors, CB2 receptors, and CB3 receptors, and is conjugated to one or more drugs suitable for binding thereto. In some embodiments, the dendrimer is a G-protein coupled receptor, e.g., GPR55, GPR18, GPR3, GPR6, GPR12, GPR40, GPR43, GPR41, GPR120, GPR23, GPR92, GPR84, GPR119, or GPR35; an adenosine receptor, e.g., adenosine A3; a muscarinic acetylcholine receptor, e.g., M1 and M4; It is conjugated to one or more carbohydrate moietyes that have affinity for and are suitable for binding to one or more non-receptor receptors, such as serotonin receptors, e.g., 5-HT1A, 5-HT2A; opioid receptors, e.g., μ- and δ-opioid receptors; and tachykinin NK2 receptors. In some embodiments, the dendrimer is conjugated to one or more carbohydrate moietyes that have affinity for and are suitable for transporting through one or more of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, GLUT7, GLUT8, GLUT9, GLUT10, GLUT11, GLUT12, GLUT13, and GLUT14, or is prepared as a sugar moiety. In further embodiments, the dendrimer is conjugated to one or more glucose and / or glucosamine moietyes. In another embodiment, the dendrimer is conjugated to one or more oligosaccharides terminated by glucose and / or glucosamine moiety, that is, the glucose and / or glucosamine moiety is exposed on the surface of the dendrimer conjugate suitable for binding to one or more of GLUT, formulation receptors and / or non-formulation receptors.

[0119] In some embodiments, the dendrimer has multiple carbohydrate moieties / molecules, such as monosaccharides, e.g., glucose, at the periphery of the dendrimer, or has sugar building blocks for the dendrimer. In some embodiments, the surface density of the carbohydrate molecules, such as monosaccharides, e.g., glucose, is at least 1 carbohydrate molecule / nm 2 (Number of surface carbohydrate groups / Surface area (nm) 2 )) is. In some embodiments, the surface density of the carbohydrate molecule is greater than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 OH groups / nm 2 , for example, nm 2 It is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 per. For example, the surface density of a carbohydrate molecule is nm 2 It is more than 10 per. In some embodiments, the bulk density of surface carbohydrate molecules is about 1 to about 50 units / nm 3 , about 5 to about 30 units / nm 3 , or about 10 to about 20 units / nm 3 is. In additional embodiments, the surface density of the carbohydrate molecule is nm 2 About 1 to about 50, about 5 to about 20 per unit (number of surface carbohydrate molecules / surface area (nm) 2 Each carbohydrate moiety has a molecular weight of about 100 Da to about 1000 Da. In these embodiments, that is, when one or more surface functional groups of the dendrimer are modified to introduce one or more sugar moiety / molecules at the ends, the terminal hydroxyl group may be part of the terminal sugar moiety / molecule, or an extra hydroxyl group that is not modified into the sugar moiety / molecule and is not part of the sugar moiety / molecule, or a combination thereof.

[0120] In some embodiments, carbohydrate molecules such as monosaccharides, e.g., glucose, are present in an amount of about 1% to 40% by weight of the total weight of the glycosylated dendrimer, e.g., about 2% to 20% by weight, about 5% to 15% by weight, or 9% to 12% by weight of the total weight of the glycosylated dendrimer. For example, in some embodiments, the carbohydrate moiety is present in an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total weight of the glycosylated dendrimer after conjugation. In some embodiments, conjugation of carbohydrate molecules through one or more surface functional groups occurs through about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% of the total available surface functional groups of the dendrimer prior to conjugation, preferably hydroxyl groups. In other embodiments, conjugation of carbohydrate molecules occurs at less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, or less than 40% of the total available surface functional groups of the dendrimer prior to conjugation.

[0121] c. Polyalkylene glycol modified dendrimer

[0122] In some embodiments, the dendrimer comprises one or more polyalkylene glycols at the ends. These terminal polyalkylene glycols may be prepared by conjugating one or more of the surface functional groups of the dendrimer, for example, hydroxyl groups, to a polyalkylene glycol, for example, PEG. In some embodiments, the dendrimer prior to conjugation is a hydroxyl-terminated dendrimer, for example, a hydroxyl-terminated PAMAM dendrimer, and at least some of the surface hydroxyl groups are conjugated to PEG.

[0123] In some embodiments, the dendrimer has multiple polyalkylene glycols, such as PEG, at the periphery of the dendrimer. In some embodiments, the surface density of the polyalkylene glycol, such as PEG, is at least one polyalkylene glycol / nm 2 (Number of surface polyalkylene glycols / Surface area (nm) 2 )) is. In some embodiments, the surface density of the polyalkylene glycol is nm 2 More than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 per, for example, at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50. For example, the surface density of polyalkylene glycol is nm 2 It is greater than 10 per unit. In some embodiments, the bulk density of the surface polyalkylene glycol is about 1 to about 50 groups / nm 3 , about 5 to about 30 units / nm 3 , or about 10 to about 20 units / nm 3 is. In additional embodiments, the surface density of a polyalkylene glycol such as PEG is nm, having a molecular weight of about 100 Da to about 10 kDa. 2 About 1 to about 50 per, about 5 to about 20 (number of surface polyalkylene glycols / surface area (nm) 2 ))am.

[0124] In some embodiments, polyalkylene glycol molecules such as PEG may be present in an amount of about 1% to 40% by weight of the total weight of the pegylated dendrimer, for example, about 2% to 20% by weight, about 5% to 15% by weight, or 9% to 12% by weight of the total weight of the pegylated dendrimer. For example, in some embodiments, polyalkylene glycol molecules such as PEG are present in an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the total weight of the pegylated dendrimer after conjugation.

[0125] In some embodiments, the conjugation of a polyalkylene glycol molecule, such as PEG, through one or more surface functional groups of the dendrimer occurs through about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% of the total available surface functional groups of the dendrimer prior to conjugation, preferably hydroxyl groups. In other embodiments, the conjugation of a polyalkylene glycol molecule, such as PEG, occurs at less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, or less than 40% of the total available surface functional groups of the dendrimer prior to conjugation.

[0126] B. Treatment

[0127] Previous studies have definitively established the major roles of both innate and adaptive immunity in the development and progression of atherosclerosis. The innate immune response in atherosclerosis is initiated by chronic inflammation, which is a characteristic of the disease. In particular, chronic inflammation at branching points and other oscillatory shear sites induces endothelial cell activation and the expression of adhesion molecules E-selectin and VCAM-1, which attract dendritic cells and monocytes. Furthermore, it stimulates the differentiation of monocytes into macrophages through the local production of macrophage colony-stimulating factor (mCSF). Subsequent cholesterol uptake by macrophages leads to the formation of foam cells, a characteristic of atherosclerosis. Pro-inflammatory M1 macrophages further contribute to the exacerbation of local inflammation, inducing EC activation, promoting the migration and proliferation of VSMCs, and triggering a vicious cycle of inflammation and cellular disturbance that leads to plaque growth. Macrophages and dendritic cells within the plaque act as antigen-presenting cells and activate the adaptive immune response. Th1 or inflammatory cytokines, such as TNF-α and IFN-γ, promote atherogenic inflammation and increase plaque instability, which can lead to comorbidities such as heart attack and stroke. Due to the significant roles played by both the innate and adaptive immune systems in plaque formation and progression, targeting the inflammatory system is a promising strategy in the treatment of atherosclerosis.

[0128] Targeting of the immune system in atherosclerosis: Previous therapies focused on IL-β binding (the CANTOS study using the IL1β antibody canakinumab showed some improvement in cardiovascular events). Treatment with the IL1 receptor antagonist Anakinra (MRC-ILA-HEART study, showed short-term benefits but not long-term benefits) and antibodies targeting IL6 receptors or ligands (tocilizumab and ziltivekimab) showed a reduction in inflammation (long-term ZEUS study is ongoing); the use of TNF-α inhibitors appears to be associated with a reduction in CVD; small molecule anti-inflammatory agents such as colchicine showed improvement in CV events; CXCL1 neutralizing antibodies, MLN1202 (a highly specific humanized monoclonal antibody preventing CCL2 binding), and the CXCR4 antagonist Plerixafor; Immuno-checkpoint inhibitors targeting CD28, CD80, CD86, CTLA-4, and PD-1, or CD70, CD27, and CD137 (TNF superfamily); examples of these include abatacept, a CTLA4-Ig construct that blocks CD80 / CD86 from interacting with CD28 to activate T cells, or TRAF-STOP therapy using SMI 6860766 or 6877002. Other targets include IL2 administration (LILACS and IVORY studies) and rituximab (anti-CD20 antibody) targeting B cells. Other small molecules that reduce MACE are as follows: statins, GLP1 agonists, e.g., semaglutide and analogs (e.g., Ozembic ® ), Lp-PLA2 inhibitors, e.g., darapladib, sPLA2 inhibitors, e.g., verespladib, 5-LO inhibitors, VIA-2291 (atrelutone); methotrexate, PPAR-α / γ agonists, e.g., tesaglitasar, metformin all showed some degree of efficacy. However, the trials did not show sufficient efficacy, or the drugs exhibited debilitating side effects that prevented further progression.

[0129] PPAR-α / γ agonists [tesaglitajar], metformin, glucagon-like peptide-1 (GLP-1) receptor agonists [e.g., semaglutide (Ozempic)] ® )], sodium-glucose co-transporter 2 (SGLT2) agonists [e.g., canagliflozin (Invokana ® A dendrimer-drug composition of a small molecule drug containing )] is described.

[0130] These debilitating side effects can be prevented by using hydroxyl and glucose dendrimers to specifically deliver these drugs to macrophages and / or neurons.

[0131] C. Dendrimer-Formulation Conjugate

[0132] A dendrimer-activator conjugate may be formed from a formulation and / or formulation derivative that is covalently or non-covalently attached to a dendrimer, a dendritic polymer, or a hyperbranched polymer. Methods for conjugating one or more activators to a dendrimer are known, for example, as described in U.S. Applications No. US 2011 / 0034422, US 2012 / 0003155, and US 2013 / 0136697. While a covalent conjugate is preferred, an ionic complex (cation-anion complex) may also be used.

[0133] In some embodiments, one or more activators are covalently bonded to one or more terminal groups of the dendrimer, such as terminal hydroxyl groups. In some embodiments, the dendrimer conjugate comprises one or more activators bonded to the dendrimer through one or more spacers. The spacers between the dendrimer and the activators may be designed to provide a releaseable or non-releaseable form of the dendrimer conjugate in vivo. For example, the spacers may be cleavable or may include cleavable chemical bonds, for example, by exposure to the intracellular compartment of a target neuron and / or glial cell, or when binding to receptors on or inside the surface of a target neuron and / or glial cell in vivo. Examples of cleavable bonds that can be used as spacers in a dendrimer-activator conjugate include esterase-sensitive ester bonds, glutathione-sensitive disulfide bonds, phosphatase-sensitive phosphodiester bonds, oligopeptides such as lysosome-releasing triglycil peptide linkers, acid-cleavable hydrazine bonds, etc. In some embodiments, the spacer between the dendrimer and the activator may provide desirable and effective release kinetics in vivo. In some embodiments, the spacer between the dendrimer and the activator may be non-degradable or may include non-degradable chemical bonds such as amide, ether, and aminoalkyl bonds.

[0134] Generally, the spacer between the dendrimer and the activator has a length sufficient for the activator attached thereto to reach and bind to a target receptor on the surface and / or inside the target cell. For example, the spacer between the dendrimer and the activator has a length ranging from 50 Da to 2000 Da depending on the desired release kinetics and the desired receptor binding flexibility. The length of the spacer may vary depending on the location of the target receptor (e.g., cell surface, cytoplasm, or intercellular compartment of the cell) and / or the receptor density when located on the cell surface.

[0135] The dendrimers may be 2nd, 3rd, 4th, 5th, 6th, and up to 10th generation. In some embodiments, the dendrimers are conjugated to one or more activators through spacers comprising cleavable (ester, disulfide, phosphodiester, triglycyl peptide, and hydrazine) or non-cleavable (amide, ether, and aminoalkyl) bonds.

[0136] The density of activators covalently or non-covalently attached to the dendrimer can be adjusted based on the specific agent or agent derivative being delivered, the target receptor, the target neuron and / or glial cell, the location of the target neuron and / or glial cell, etc. For example, multiple activators attached to the dendrimer are present in the periphery of the dendrimer, and the surface density of the activators is at least one activator / nm 2 (Number of conjugated active agents / Surface area (nm) 2 )), preferably 3 to 10. For example, in some embodiments, the surface density of the active agent is nm 2 More than 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 per, e.g., at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50. In some embodiments, the bulk density of the active agent is about 1 to about 50 groups / nm. 3 (groups / nm 3 ), about 5 to about 30 units / nm 3 , or about 10 to about 20 units / nm 3 am.

[0137] Typically, the dendrimer-activator conjugate has a hydrodynamic volume in the nanometer range. For example, in some embodiments, a glucose dendrimer-activator conjugate comprising one or more formulations or formulation derivatives conjugated to a dendrimer has a diameter of about 2 nm to about 100 nm, or greater than 100 nm, up to 500 nm, depending on the generation of the dendrimer, the chemical composition, and the amount of activator conjugated thereto. In some embodiments, the dendrimer-activator conjugate comprising one or more formulations or formulation derivatives conjugated to a dendrimer has a diameter effective for penetrating brain tissue and remaining on and / or inside the target neuron and / or glial cell for a sufficient time for the activator to bind to targeted receptors on and / or inside the target neuron and / or glial cell. In some embodiments, a dendrimer-activator conjugate comprising one or more agents or agent derivatives conjugated to a dendrimer is maintained in peripheral circulation and has an effective diameter to remain on the surface and / or inside of a target neuron and / or glial cell for a sufficient time for the activator to bind to a targeted receptor on the surface and / or inside of the target neuron and / or glial cell.

[0138] The dendrimer-activator conjugate may be neutral, positively charged, or negatively charged. In some embodiments, the dendrimer-activator conjugate is neutral. The presence of a formulation or formulation derivative may affect the surface charge of the dendrimer-activator conjugate. In some embodiments, the surface charge of the dendrimer conjugate to the formulation or formulation derivative is -100 mV to 100 mV, -50 mV to 50 mV, -25 mV to 25 mV, -20 mV to 20 mV, -10 mV to 10 mV, -10 mV to 5 mV, -5 mV to 5 mV, or -2 mV to 2 mV. The above ranges include all values ​​from -100 mV to 100 mV. In a preferred embodiment, the surface charge of the dendrimer-activator conjugate is neutral or nearly neutral, i.e., about -10 mV to about 10 mV.

[0139] An exemplary dendrimer-activator conjugate is represented by Chemical Formula I. The dendrimer of the exemplary conjugate comprises surface hydroxyl groups, wherein one or more of the surface hydroxyl groups are conjugated to one or more activators through one or more spacers as shown in Chemical Formula I below.

[0140] [Chemical Formula I]

[0141]

[0142] Here, D may be a 1st to 10th generation or 2nd to 10th generation dendrimer, for example, any of those described above, for example, PAMAM (e.g., hydroxyl-terminated PAMAM dendrimer) or a glucose-based dendrimer; and each L may be any suitable chemical moiety suitable for providing customized drug release and receptor binding, preferably comprising a triazole moiety; Y is a bond, or a 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, phosphodiester group ( The bond may be selected from ), hydrazino group, hydrazone, hydrazide, ester (-C(O)-O-), ether (-O-) and oligopeptide (e.g., triglycyl peptide), wherein R is an alkyl group, an aryl group, or a heterocyclic group; each X may be a formulation or a derivative of a formulation, wherein the functional group of X (e.g., an amino group including a primary, secondary, or tertiary amino group; a carboxyl group; or a hydroxyl group) forms part of the bond Y; n may be an integer from 1 to 100; and m may be an integer from 16 to 4096. The dendrimer may be a PAMAM or glucose dendrimer that is 100% hydroxyl. m and n depend on the size of the dendrimer D, and n should be such that the weight percentage of the drug in the total conjugate is 5 to 20 percent. This range is also suitable for binding and internalization.

[0143] The oxygen atom represented by Formula I is derived from a surface functional group of the dendrimer, e.g., a surface hydroxyl group, where the surface hydroxyl group may or may not be part of a terminal sugar moiety / molecule (e.g., glucose). Although not exemplified in Formula I, one or more hydroxyl groups of the dendrimer not conjugated to the activator may be modified with one or more carbohydrates and / or polyalkylene glycols, e.g., PEG.

[0144] The formulation of Formula I and / or formulation derivative X may bind to a target receptor on the surface of a target cell or inside a target cell when administered to a subject requiring it. In some embodiments, when the formulation and / or formulation derivative X bind to the target receptor, the formulation X remains attached to the dendrimer. In these embodiments, after binding, the formulation X may be released from the dendrimer or remain attached to the dendrimer as an intact dendrimer-activator conjugate. In some embodiments, the formulation and / or formulation derivative X is released from the dendrimer at a location very close to the target receptor and then binds to the target receptor.

[0145] In some embodiments, each L may be represented as -A'-L1-B'-L2-, where A' may be a carbonyl (-C(O)-) or a bond (single, double, and triple bonds, e.g., including a single bond), and B' may be a bond (single, double, and triple bonds, e.g., including a single bond), an amide, an ester, an ether, a thiol, a dithiol, an aryl, a heteroaryl, a polyaryl, a heteropolyaryl, or a heterocyclic compound; L1 and L2 may independently be bonded, alkylene, heteroalkylene, aryl, aralkyl, ether, polyether, thiol, dithiol, thiol ether, polythioether, oligopeptide, polypeptide, oligo(alkylene glycol), or polyalkylene glycol, or L1 and L2 may independently be composed of a combination of these groups, for example, a combination of alkylene and polyether, a combination of alkylene and thiol or dithiol, a combination of alkylene and oligopeptide, a combination of alkylene, polyether, and thiol or dithiol, or a combination of polyether and thiol or dithiol. In some forms, L1-B'-L2- together form a chemical moiety selected from -alkylene-triazole-di(alkylene glycol)-, -di(alkylene glycol)-triazole-alkylene-, -alkylene-triazole-oligo(alkylene glycol)-, -oligo(alkylene glycol)-triazole-alkylene-, -alkylene-triazole-poly(alkylene glycol)-, -poly(alkylene glycol)-triazole-alkylene-, -alkylene-triazole-ether-, -alkylene-triazole-alkylene-, -alkylene-amide-alkylene- and combinations thereof.

[0146] In some embodiments, B' may be a bond (single, double, and triple bonds, e.g., including a single bond), an amide group, or a heterocyclic group, e.g., a triazole group.

[0147] In some embodiments, L1 is a bond; an alkylene, e.g., C1-C 10Alkylene, C1-C8 alkylene, C1-C6 alkylene, C1-C5 alkylene, C1-C4 alkylene or C1-C3 alkylene; or oligo- or poly-(alkylene glycol), e.g.,

[0148]

[0149] It can be,

[0150] Here, p is an integer of 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

[0151] In some embodiments, L2 is a bond; an alkylene, e.g., C1-C 10 Alkylene, C1-C8 alkylene, C1-C6 alkylene, C1-C5 alkylene, C1-C4 alkylene or C1-C3 alkylene; oligo- or poly-(alkylene glycol), e.g.,

[0152]

[0153] (wherein p is an integer of 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2); an oligo- or poly-peptide, e.g., a triglycyl peptide; a thiol; or a dithiol; or L2 may be composed of a combination of two or more of alkylene, oligo- or poly-(alkylene glycol), oligo- or poly-peptide, thiol, and dithiol. For example, L2 is

[0154]

[0155] It is displayed as,

[0156] Here, p, q, r, s, t and u are each integers of 0 to 10, 0 to 8, 0 to 6, 0 to 5, 0 to 4, 0 to 3, or 0 to 2, e.g., 0, 1 or 2; and G' is a thiol, dithiol, oligo-peptide, e.g., triglycyl peptide, or polypeptide.

[0157] In some embodiments, Y is a link that is minimally cleavable in vivo. In some embodiments, Y is a link that is cleavable in vivo. In some embodiments, Y is an amide (-CONH-), ester (-C(O)-O-), ether (-O-), phosphodiester, or disulfide group.

[0158] In some embodiments, L and Y are both single bonds, and D is directly connected to X (an activator or an analog thereof) through an ether bond.

[0159] In some embodiments, D is a 2nd generation PAMAM dendrimer, a 3rd generation PAMAM dendrimer, a 4th generation PAMAM dendrimer, a 5th generation PAMAM dendrimer, a 6th generation PAMAM dendrimer, a 1st generation glucose dendrimer, a 2nd generation glucose dendrimer, a 3rd generation glucose dendrimer, a 4th generation glucose dendrimer, a 5th generation glucose dendrimer, or a 6th generation glucose dendrimer.

[0160] More specific exemplary dendrimer-activator conjugates are shown in the following examples. In one preferred embodiment, the conjugate is formed from a hydroxyl dendrimer and a glucose dendrimer-drug conjugate of PPAR-α, PPAR-γ, and PPAR-α / γ agonists, including tesaglitazar, metformin, and other drugs of this class.

[0161] In another preferred embodiment, the hydroxyl dendrimer and glucose dendrimer-drug conjugate of a GLP-1 agonist is semaglutide (Ozempic ® , Rybelsus (RYBELSUS) ®), dulaglutide (Trulicity ® ), exenatide extended release, exenatide (Byetta ® ), liraglutide (Victoza) ® , Saxenda ® )(daily) and lixisenatide(adlyxin) ® Includes ).

[0162] D. Excipients

[0163] "Pharmaceuticalally acceptable" means a material that is biologically unacceptable or otherwise undesirable, that is, the material may be administered to a subject together with a selected compound without causing any undesirable biological effects or interacting in a harmful manner with any of the other components of a pharmaceutical composition containing it.

[0164] Any of the compounds having Formula I may be used therapeutically in combination with a pharmaceutically acceptable carrier. The compounds described in this application may be conveniently formulated into pharmaceutical compositions consisting of one or more compounds together with a pharmaceutically acceptable carrier. For example, literature [ Remington's Pharmaceutical Sciences [Refer to the latest edition, by EW Martin Mack Pub. Co., Easton, PA], which discloses a conventional method for preparing typical carriers and pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the compounds described in this application. These are typically standard carriers for the administration of compositions to humans. In one embodiment, solutions such as sterile water, saline solution, and a buffer solution of physiological pH, including human and non-human. Other compounds will be administered according to standard procedures used by a person skilled in the art.

[0165] The pharmaceutical composition described in this application may include, but is not limited to, a carrier, a thickener, a diluent, a buffer, a preservative, a surfactant, etc. in addition to the selected molecule.

[0166] Injectable preparations can be prepared in conventional forms such as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in a liquid prior to injection, or emulsions (Reference [Park et al., J Control Release , 342:53-65 (2022)]; [Nkanga, et al., Advanced Drug Delivery Reviews , 167:19-46, (2020)]; [Sheikh, et al., Asian Journal of Pharmaceutics , 10(4):S465-S471 (2016)]; [Rhee et al., Pharmaceutical Technology Drug Delivery [ , p.S6 (2010)]). Exemplary approaches for parenteral administration include the use of slow-release or sustained-release systems that allow for the maintenance of a constant dosage. For example, see U.S. Patent US 9,700,630, International Publication WO 2006 / 125620 and Korean Patent KR 101898816.

[0167] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions that may also contain buffers, diluents, and other suitable additives. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, e.g., olive oil, and injectable organic esters, e.g., ethyl oleate. Aqueous carriers include saline solutions and buffer media, e.g., water, alcoholic solutions / aqueous solutions, emulsions, or suspensions. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluids and nutritional supplements, electrolyte supplements (e.g., those based on Ringer's dextrose), etc. Preservatives and other additives, e.g., antimicrobial agents, antioxidants, chelating agents, inert gases, etc., may also be present.

[0168] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc. may be required or preferred.

[0169] A composition for oral administration may include a powder or granules, a suspension or solution in water or a non-aqueous medium, a capsule, a sachet, or a tablet. Thickeners, flavoring agents, diluents, emulsifiers, dispersion aids, or binders may be preferred.

[0170] III. Treatment Methods

[0171] Dendrimer-drug conjugates are administered orally, by injection, or into the mucosa to treat atherosclerosis through the action of drugs when delivered to macrophages and foam macrophages within plaques.

[0172] Dendrimer-drug conjugates are administered orally, by injection, or by mucosal delivery to treat obesity by delivery to adipose tissue macrophages (typically M2 type) and brain microglia / macrophages in the hypothalamus (where arcuate neurons are responsible for appetite).

[0173] A preferred embodiment is semaglutide (Ozempic) for atherosclerosis, obesity, or related metabolic syndrome. ® , Ribelsus ® ), dulaglutide (Trulicity ® ), exenatide extended release, exenatide (Byetta ® ), liraglutide (Victoza ® , Saxenda ® )(daily), lixisenatide(adlixin ® It includes hydroxyl dendrimers and glucose dendrimers-drug conjugates of GLP-1 agonists, including ).

[0174] Therapeutic approaches for metabolic syndrome ("MetS") are diverse and may target lipoproteins, blood pressure, or anthropometric indicators. Peroxisome proliferator-activated receptors ("PPARs") are involved in the metabolic regulation of lipid and lipoprotein levels, namely triglycerides ("TG"), blood glucose, and abdominal obesity. PPARs can be classified into α, β / δ, and γ subtypes. PPAR-α agonists (primarily fibrates and their variants, e.g., pemafibrate) and omega-3 fatty acids lower triglycerides. They primarily affect TG catabolism; in particular, fibrates increase levels of high-density lipoprotein cholesterol ("HDL-C"). PPAR-γ agonists (primarily glitazones) exhibit less activity toward TG but are potent glucose-lowering agents. Newer PPAR-α / δ agonists, for example, elafibranor, are designed as monotherapy drugs that achieve TG-lowering and HDL-C-elevating effects in addition to the insulin sensitivity-improving and antihyperglycemic effects of glitazones. They also show potential for the treatment of non-alcoholic fatty liver disease (NAFLD), which is closely associated with MetS.

[0175] PPARα agonists, including fibrates, and PPARγ agonists, such as thiazolidinediones, are used to treat hypertriglyceridemia and type 2 diabetes, respectively. PPARδ activation promotes mitochondrial and energy metabolism. In one embodiment, a PPAR-α / γ agonist ("PPAR") is a transcription factor that regulates genes involved in cell proliferation as well as lipid and carbohydrate metabolism. PPAR-α regulates the expression of genes involved in lipid and lipoprotein metabolism. PPAR-γ regulates fatty acid storage and increases the expression of proteins involved in lipid and glucose metabolism. 6 Both PPAR-α and PPAR-γ agonists inhibit macrophage foam cell formation. 14 Macrophage peroxisome proliferator-activated receptors (PPARs) are involved in both atherosclerosis and obesity.

[0176] Tesaglitazar is a dual PPAR-α / γ agonist with anti-inflammatory and anti-atherogenic properties. Previous studies have shown that tesaglitazar significantly improved lipid profiles in diabetic and non-diabetic patients with insulin resistance. In mice, tesaglitazar demonstrated benefits including reductions in plasma cholesterol and atherosclerotic plaques, as well as improvements in inflammatory markers. However, toxicity issues occurred during Phase 3 clinical trials. Patients receiving tesaglitazar experienced peripheral edema, weight gain, impaired renal function, dose-dependent hemoglobin reduction, myocardial ischemia, and worsening of congestive heart failure, leading to the termination of the clinical trial. Clinical development was discontinued due to off-target side effects, including fibrosarcoma formation, increased cardiovascular risk, and weight gain. Specific macrophage localization should reduce or prevent tesaglitazar-related side effects. Other side effects include anemia, leukopenia, edema, and increased cardiovascular risk. Weight gain is mentioned in some studies. Other glitasares (dual PPAR agonists) were associated with myocardial dysfunction and more serious cardiovascular events (8 to 9 others were tested but failed). Toxicity was observed with the highest dose of tesaglitasar 4.1 mg / kg daily PO for 12 to 24 weeks. Specific macrophage localization using dendrimers should reduce or prevent tesaglitasar-related side effects.

[0177] In a preferred embodiment, the conjugate is formed from hydroxyl dendrimers and glucose dendrimers-drug conjugates of PPAR-α, PPAR-γ, and PPAR-α / γ agonists, including tesaglitazar, metformin, and other drugs of this class, and is used to treat atherosclerosis through action on plaque macrophages and foam macrophages. Other specific exemplary dendrimer-activator conjugates are semaglutide (Ozempic ® , Ribelsus ® ), dulaglutide (Trulicity ®), exenatide extended release, exenatide (Byetta ® ), liraglutide (Victoza ® , Saxenda ® )(daily) and lixisenatide(adlixin ® It includes hydroxyl dendrimers and glucose dendrimers-drug conjugates of GLP-1 agonists, including ).

[0178] The conjugate should be useful for treating obesity using the composition through action on adipose tissue lipid macrophages (typically type M2) and brain microglia / macrophages in the hypothalamus.

[0179] The term "preventing" as used in this application refers to preventing the physical manifestation of abnormalities associated with a disease or pathological condition by administering a compound before the onset of clinical symptoms of the disease or pathological condition.

[0180] The terms "treatment" and "treating" refer to the medical management of a subject intended to cure, improve, stabilize, or prevent a disease, pathological condition, or disorder. These terms include active treatment, that is, treatment specifically aimed at improving a disease, pathological condition, or disorder, and also include causal treatment, that is, treatment aimed at eliminating the cause of the related disease, pathological condition, or disorder. Furthermore, these terms include palliative treatment, that is, treatment designed for the alleviation of symptoms rather than the cure of the disease, pathological condition, or disorder; preventive treatment, that is, treatment aimed at minimizing or partially or completely suppressing the onset of the related disease, pathological condition, or disorder; and supportive treatment, that is, treatment used to complement another specific therapy aimed at the improvement of the related disease, pathological condition, or disorder. Treatment is understood to be intended to cure, improve, stabilize, or prevent a disease, pathological condition, or disorder, but not to actually result in a cure, improvement, stabilization, or prevention. The effect of the treatment may be measured or evaluated as described in this application and as known in the art, appropriate to the relevant disease, pathological condition, or disorder. Such measurement and evaluation may be performed in qualitative and / or quantitative aspects. Accordingly, for example, the characteristics or features of the disease, pathological condition, or disorder and / or the symptoms of the disease, pathological condition, or disorder may be reduced by any effect or any amount.

[0181] The term "effective amount" of a compound as provided in this application refers to a sufficient amount of the compound to provide the desired result while being non-toxic. As noted below, the exact required amount will vary from subject to subject depending on the species, age and overall condition of the subject, the severity of the disease being treated, the specific compound used, the method of administration, etc. Therefore, it is impossible to specify the exact "effective amount." However, an appropriate effective amount can be determined by a person skilled in the art using only routine experiments.

[0182] The dosage or amount of the compound described in this application is sufficient to produce the desired effect in the method of delivery. The dosage must not be so large as to cause harmful side effects, such as unwanted cross-reactions or anaphylactic reactions. Generally, the dosage will vary depending on the subject's age, condition, gender, and severity of the disease, and this may be determined by a person skilled in the art. The dosage may be adjusted by a private physician based on the clinical condition of the subject. The dosage, administration schedule, and route of administration may vary.

[0183] The efficacy of administering a specific dose of a compound or composition according to the method described in this application may be determined by evaluating specific aspects of the medical history, signs, symptoms, and objective laboratory tests known to be useful in assessing the condition of a subject requiring treatment for atherosclerosis, obesity, metabolic syndrome, or symptoms thereof. These signs, symptoms, and objective laboratory tests will vary depending on the disease or condition being treated or prevented, as is known to any clinician treating such patients or to researchers conducting experiments in the relevant field. For example, a specific therapeutic regimen will be considered effective if, based on comparison with an appropriate control group and / or knowledge regarding the normal progression of the disease in a general population or specific individuals, (1) the subject's physical condition appears to be improved, (2) the progression of the disease or condition appears to be stabilized, slowed, or reversed, or (3) the need for other drugs to treat the disease or condition is reduced or eliminated.

[0184] The compounds and pharmaceutical compositions described in this application may be administered to a subject in various ways depending on whether topical or systemic treatment is preferred and the site to be treated. Accordingly, for example, the pharmaceutical compositions described in this application may be administered to a subject via vaginal, rectal, nasal, oral, by inhalation, or parenterally, e.g., intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intra-arterial, intra-lymphatic, intravenous, intradural, and intratracheal routes. Parenteral administration (where used) generally features injection and includes intravenous (IV), subcutaneous (SC), intramuscular (IM), epidural, and intra-articular injections, as well as surgical insertion of a depot into an organ or tissue of interest (see [Bittner, et al., BioDrugs ., 32:425-440 (2018)]; [Lee et al., J. Pharm. Investig ., 49: 459-476 (2019)]; [Chaudhary et al., Crit. Rev. Ther. Drug Carrier System ., 36:137-181 (2019)]).

[0185] The disclosed composition and method may be further understood through the following paragraphs:

[0186] 1. A composition comprising a hydroxyl dendrimer covalently conjugated to at least one drug, wherein the drug comprises a PPAR-α agonist; a PPAR-γ agonist; a dual PPAR agonist (e.g., a PPAR-α / γ agonist); a GLP-1 receptor agonist (e.g., semaglutide); metformin; an SGLT2 agonist, a GIP-1 receptor agonist, or an antagonist; a dual GLP-1 / GIP-1 receptor agonist (e.g., tyrzepatide); a mitochondrial uncoupling agent (e.g., niclosamide); or a combination thereof.

[0187] 2. A composition comprising a glucose dendrimer covalently conjugated to at least one drug, wherein the drug comprises a PPAR-α agonist; a PPAR-γ agonist; a dual PPAR agonist (e.g., a PPAR-α / γ agonist); a GLP-1 receptor agonist (e.g., semaglutide); metformin; an SGLT2 agonist, a GIP-1 receptor agonist, or an antagonist; a dual GLP-1 / GIP-1 receptor agonist (e.g., tyrzepatide); a mitochondrial uncoupling agent (e.g., niclosamide); a cannabinoid 1 receptor (CB1R) antagonist; or a combination thereof.

[0188] 3. A composition wherein, in any one of the preceding paragraphs, the drug comprises a PPAR-α agonist selected from the group consisting of tesaglitazar, fenofibrate, WY-14643, oleoyethanolamide, GW4148, GW 9578, GW2148, or combinations thereof; a PPAR-γ agonist; or a dual PPAR agonist (e.g., a PPAR-α / γ agonist).

[0189] 4. In any of the preceding paragraphs, the drug is semaglutide (e.g., Ozempic ® or Rebelsus ® ); dulaglutide (e.g., trulicity ® ); Exenatide extended release, exenatide (e.g., Byetta ® ) and analogs and derivatives thereof; liraglutide (e.g., Victoza ® , Saxenda ® )(daily); lixisenatide (e.g., adlixin ® A composition comprising a GLP-1 receptor agonist selected from the group consisting of ); and other incretin mimics. Compounds of the incretin mimic class include, but are not limited to, exenatide (e.g., Byetta, Bydureon), liraglutide (e.g., Victoza), sitagliptin (e.g., Januvia, Janumet, Janumet XR, Juvisync), saxagliptin (e.g., Onglyza, Kombiglyze XR), alogliptin (e.g., Nesina, Kazano, Oseni), albiglutide (e.g., Tanzeum), and linagliptin (e.g., Tradjenta, Jentadueto).

[0190] 5. A composition wherein, in any one of the preceding paragraphs, the drug is a GIP-1 receptor agonist or antagonist selected from the group consisting of SKL-14959, MA-38472-B1, or a combination thereof.

[0191] 6. A composition wherein, in any one of the preceding paragraphs, the drug is a dual GLP-1 / GIP-1 receptor agonist selected from the group consisting of tircepate, NNC0090-2746, NN9709, CT-388, retatrutide, or a combination thereof.

[0192] 7. A composition in which, in any one of the preceding paragraphs, the drug is metformin.

[0193] 8. In any of the preceding paragraphs, the drug is canagliflozin (e.g., Invokana ® ), Bexagliflozin (e.g., Brenzavvy) ® ), dapagliflozin (e.g., Farxiga) ® ), empagliflozin (e.g., Jardiance) ® ), Ertugliflozin (Steglatro) ® A composition comprising an SGLT2 agonist selected from a group consisting of ) or a combination thereof.

[0194] 9. A composition wherein, in any one of the preceding paragraphs, the drug comprises a mitochondrial uncoupling agent selected from the group consisting of niclosamide, BAM15, carbonyl cyanide 4-(trifluoro-methoxy)phenylhydrazone (FCCP), carbonyl cyanide-3-chlorophenylhydrazone (CCCP), dinitrophenol, analogs of these molecules, or combinations thereof.

[0195] 10. A composition wherein, in any one of the preceding paragraphs, the drug comprises a CB1R antagonist selected from the group consisting of AJ5012, JD5037, 3,4-diarylpirazoline, 1,5-diarylpyrrol-3-carboxamide, methylsulfonamide azetidine, 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-piperidino-1H-pyrazole-3-carboxamide (rimonabant) or a combination thereof.

[0196] 11. A composition wherein, in any one of the preceding paragraphs, the covalent bonding comprises a covalent bond between a modified or unmodified surface group or an internal group of the hydroxyl dendrimer or glucose dendrimer, wherein the covalent bond comprises an amide, ester, disulfide, ether, or phosphate bond.

[0197] 12. A composition in which, in any one of the preceding paragraphs, the covalent bonding comprises a covalent bond capable of being cleaved at a pH selected from neutral, acidic, or basic.

[0198] 13. A composition in which, in any one of the preceding paragraphs, the hydroxyl dendrimer or glucose dendrimer is based on a 1st to 7th generation polyamidoamine (PAMAM) dendrimer.

[0199] 14. A composition in which, in any one of the preceding paragraphs, the dendrimer is a glucose dendrimer, preferably a first-generation, second-generation, or third-generation glucose dendrimer.

[0200] 15. A composition according to any one of the preceding paragraphs, wherein the dendrimer is based on a 2nd to 7th generation PAMAM dendrimer modified with a sugar moiety, wherein the sugar moiety is selected from the group consisting of glucose, galactose, mannose, and fructose.

[0201] 16. A composition in which, in any one of the preceding paragraphs, the hydroxyl dendrimer or glucose dendrimer has at least a 10-fold increase in drug solubility compared to the free form of the drug, or the hydroxyl dendrimer, glucose dendrimer, or both increase the duration of effect compared to the free form of the drug.

[0202] 17. A composition wherein, in any one of the preceding paragraphs, the hydroxyl dendrimer is based on a higher generation dendrimer, preferably a 3rd, 4th, 5th, or 6th generation PAMAM dendrimer (e.g., a hydroxyl-terminated PAMAM dendrimer), or the glucose dendrimer is a 1st, 2nd, 3rd, or higher generation glucose dendrimer or is functionalized with poly(ethylene glycol), wherein the conjugate is limited to peripheral circulation.

[0203] 18. A composition in which, in any one of the preceding paragraphs, the drug has an amount of about 2% by weight to about 35% by weight.

[0204] 19. A composition comprising, in any one of the preceding paragraphs, a pharmaceutically acceptable excipient.

[0205] 20. A dosage form comprising the composition of any one of the preceding paragraphs for the treatment of atherosclerosis, obesity, diabetes, metabolic syndrome, blood sugar control, blood pressure reduction, lipid disorders, fatty liver disease, kidney disease treatment / prevention, or symptomatic treatment thereof in an individual.

[0206] 21. A formulation for the treatment of an obese individual in any of the preceding paragraphs, wherein the reduction in fat mass is greater than the reduction in lean body mass.

[0207] 22. A formulation for the treatment of an obese individual in any of the preceding paragraphs, wherein weight loss is achieved without significant loss of appetite.

[0208] 23. A formulation for the treatment of an obese individual in any of the preceding paragraphs, wherein the treatment is to convert white fat cells into brown fat cells.

[0209] 24. A method for treating one or more symptoms of atherosclerosis, abnormal blood glucose, hypotension, lipid disease, fatty liver disease, kidney disease, obesity or related metabolic disorders and combinations thereof, wherein the method comprises the step of administering a composition of any one of the preceding paragraphs or a formulation of any one of the preceding paragraphs to an individual requiring treatment thereof.

[0210] 25. A method for treating a disease or disorder, wherein the method comprises the step of using a composition of any one of the preceding paragraphs or a formulation of any one of the preceding paragraphs in combination with an existing treatment.

[0211] 26. A method for treating a disease or disorder, wherein the method comprises the step of preventing the recurrence of said disease or disorder by using the composition of any one of the preceding paragraphs or the formulation of any one of the preceding paragraphs after the termination or reduction of said treatment.

[0212] 27. A method according to any one of the preceding paragraphs, wherein the formulation selectively delivers the hydroxyl dendrimer-drug conjugate or glucose dendrimer-drug conjugate to macrophages and foam macrophages within an atherosclerotic plaque.

[0213] 28. A method according to any one of the preceding paragraphs, wherein the formulation selectively delivers the hydroxyl dendrimer-drug conjugate or glucose dendrimer-drug conjugate to adipocytes in the adipose tissue of the individual and / or brain microglia / macrophages in the hypothalamus.

[0214] 29. A method wherein, in any one of the preceding paragraphs, the individual has type 2 diabetes, and the hydroxyl dendrimer-drug conjugate or glucose dendrimer-drug conjugate is administered in an amount effective for alleviating one or more symptoms of type 2 diabetes.

[0215] 30. A method wherein, in any one of the preceding paragraphs, the individual is an obese individual, and the hydroxyl dendrimer-drug conjugate or glucose dendrimer-drug conjugate is administered to the obese individual in an amount that causes weight loss.

[0216] 31. A method in which, in any one of the preceding paragraphs, the glucose dendrimer-drug conjugate is administered to an obese individual in an amount that causes weight loss.

[0217] 32. A method in which, in any one of the preceding paragraphs, the reduction in fat mass is higher than the reduction in lean body mass.

[0218] 33. A method in which, in any one of the preceding paragraphs, weight loss is achieved without notable loss of appetite.

[0219] 34. In any of the preceding paragraphs, the treatment is a method of converting white fat cells into brown fat cells.

[0220] 35. In any one of the preceding paragraphs, the obese individual is a human, method.

[0221] 36. A method in which, in any one of the preceding paragraphs, the hydroxyl dendrimer-drug conjugate or glucose dendrimer-drug conjugate does not cause toxic symptoms.

[0222] 37. A method in which, in any one of the preceding paragraphs, the formulation is administered via a route selected from the group consisting of mucosal administration, enteral administration or injection, intranasal, intravenous, oral, sublingual, subcutaneous, inhaled, transdermal, or intraperitoneal.

[0223] Examples

[0224] Example 1: PAMAM-OH dendrimers selectively deliver agents to macrophages within atherosclerotic plaques.

[0225] Materials and Methods

[0226] The feasibility of selective drug delivery to macrophages within atherosclerotic plaques was investigated using PAMAM-OH dendrimer-conjugated Cy5 (D-Cy5).

[0227] ApoE - / - By feeding mice a high-fat diet (HFD) for 4 months Fig. 1 As described in [figure], significant plaque accumulation was induced. Then, D-Cy5 (10 mg / kg; intraperitoneal injection) was administered to the mice. After 24 hours, the aorta was harvested, fixed, stained with CD68 (macrophages) and DAPI (nuclei), and imaged by confocal microscopy.

[0228] result

[0229] Control group and atherosclerosis ApoE - / - Representative confocal images obtained 24 hours after intraperitoneal injection of D-Cy5 into mice show that D-Cy5 was clearly internalized by macrophages within atherosclerotic plaques, as indicated by Cy5 signals accompanied by positive CD68 and nuclear staining, whereas healthy controls did not show D-Cy5 uptake, confirming that the PAMAM-OH dendrimer conjugated to the agent delivers the drug payload to macrophages within atherosclerotic plaques.

[0230] Example 2: PAMAM-OH-Tesa reduces arterial stiffness and body weight in atherosclerotic mice.

[0231] Atherosclerosis is associated with arterial stiffness, which can be evaluated by PWV (higher PWV = higher aortic stiffness), the in vivo standard for arterial stiffness in mice.

[0232] Materials and Methods

[0233] High-fat diet ("HFD") male ApoE - / - Mice exhibited a faster increase in PWV than expected in natural aging, indicating the progression of atherosclerosis.

[0234] After 4 months of HFD intake, mice were randomly assigned to two groups: 1) Treatment group: 1) HFD + D-Tesa (20 μg / kg orally twice a week) for 6 weeks, and 2) Untreated group: mice that continued to take HFD during the treatment period.

[0235] Plaque burden was evaluated using Oil Red O (ORO) staining in baseline (16-week HFD), untreated group (16-week HFD + 6-week HFD), and D-Tesa treated group (16-week HFD + 6-week HFD and treatment), and the percentage of plaque area / total area was determined as a marker of plaque burden using ImageJ.

[0236] Body composition analysis by qMRI. April-aged male ApoE - / - After administering HFD to mice, they were randomized to receive the following for 3 weeks: 1) vehicle (control), 2) D-Tesa (20 μg / kg, twice weekly), or 3) free Tesa (20 μg / kg, twice weekly). HFD was continued to be administered to the mice. qMRI evaluations of body weight, fat mass %, and lean body mass % were determined. (n=3–5 mice per group; * p<0.05, ** p<0.01, one-way ANOVA and Tukey post-hoc analysis)

[0237] result

[0238] Figures 2a~2d % change in body weight for HFD mice treated with free Tesa compared to dendrimer-conjugated tesaglitazar (Tesa) (D-Tesa) Fig. 2a ), body weight (g)( Fig. 2b ), fat amount %( Fig. 2c ) and lean body mass %( Fig. 2d This is a graph of ). D-Tesa reduces weight gain caused by a high-fat diet. D-Tesa treatment demonstrated that although body weight increases due to HFD administration, it decreases after D-Tesa treatment.

[0239] Figures 3a–3c This illustrates that D-Tesa improves markers of atherosclerosis.

[0240] While PWV improves with D-Tesa treatment ( Fig. 3a ), glass tessaglitazar did not show these effects. Body weight ( Fig. 3c ) showed an increase due to HFD administration but decreased after D-Tesa treatment (n=6–12 male mice per group). Fig. 3c During treatment, untreated mice did not lose body weight, whereas D-Tesa-treated mice showed a steady decrease in fat mass and an increase in lean body mass (n=8–9 male mice per group).

[0241] HFD resulted in the expected body weight gain. Although HFD was continued for the treated mice, D-Tesa treatment caused significant body weight loss, whereas the untreated mice continued to gain body weight.

[0242] D-Tesa induces body weight loss in male mice. One of the major side effects of tesaglitazar was weight gain. Surprisingly, mice administered D-Tesa were found to lose body weight despite continuing HFD intake without a decrease in the amount of food consumed. This indicates that the weight loss is not due to a decrease in food intake or appetite. While cationic dendrimers have been shown to induce weight loss resulting from non-specific accumulation in various tissues along with associated toxicity, the hydroxyl dendrimers used in this study are non-toxic and do not induce weight loss on their own. This is also reflected in the data showing similar body weight gain between mice administered empty dendrimers and mice supplied with only HFD. The results indicate that the weight loss in the HFD+D-Tesa group is caused by the action of tesaglitazar released into the cells of macrophages within adipose tissue.

[0243] Importantly, D-Tesa treatment reduces fat mass while increasing lean body mass, which is a highly desirable outcome for weight loss, diabetes, and heart medications. Typically, many drugs used for weight loss (e.g., semaglutide, ozempic) and diabetes have been shown to reduce both fat mass and lean body mass. While a reduction in fat mass is desirable for weight loss purposes, a reduction in lean body mass is undesirable, particularly in elderly patients. When patients discontinue these medications, fat mass may recover, but lean body mass does not, which can lead to health problems. In contrast, the selective reduction of fat mass and the increase in lean body mass demonstrated by D-Tesa in this application are quite surprising and unexpected. This may indicate a conversion of white adipose tissue to brown adipose tissue. Increased energy expenditure may also indicate an increase in brown adipose tissue.

[0244] Blood pressure (BP) was not affected by HFD and D-Tesa treatment, which confirms that changes in PWV are due to changes in aortic dynamics rather than simply an increase in BP.

[0245] Fig. 3a This shows that PWV (m / s) decreases at 40 weeks or more when treated with D-Tesa compared to untreated and free tesaglitazar. Systolic ( Fig. 3b ) Blood pressure did not change throughout the experiment. (n=6–16 male mice per group).

[0246] Fig. 4 This is a graph of the percentage of lesion area comparing D-Tesa-treated and untreated HFD mice.

[0247] Impairment of vascular reactivity is stopped by D-Tesa. Relaxation of the PE pre-constricted aorta from baseline, untreated, and D-Tesa treatments using acetylcholine ( Ai ) and sodium nitroprusside ( BiIt was induced by an increase in the concentration of ). Ach dose ( Figs. 5a, 5c ) or SNP dose( Figs. 5c, 5d Relaxation (% PE max) according to D-Tesa dose for ) Figs. 5a, 5c ) and log EC 50 ( Figs. 5b, 5d )of Figures 5a~5d Refer to the graph.

[0248] In summary, after 6 weeks, D-Tesa-treated mice Despite HFD intake It showed a lower PWV compared to untreated mice. D-Tesa treatment also slows plaque progression. Plaque deposition 6 weeks after baseline showed a significant increase in untreated mice ( Fig. 4 D-Tesa-treated mice exhibited a significantly lower plaque burden than untreated mice. This demonstrates that D-Tesa can be used to reduce plaque burden. The data prove that specifically targeting PPAR in macrophages is sufficient to improve plaque burden.

[0249] Furthermore, D-Tesa treatment improves the severity of aortic endothelial and smooth muscle cell (SMC) dysfunction. Endothelial dysfunction, characterized by a reduction in agonist-induced endothelial-dependent vasodilation, is a known feature of atherosclerosis. Targeting inflammatory cells with D-Tesa also improves endothelial dysfunction in atherosclerotic mice. Endothelial-dependent vasodilation of the aortic loop (pre-constricted with phenylephrine) is [induced by] acetylcholine (10 -9 ~10 -5Evaluation was performed using wire motor recording with M). Untreated mice exhibited greater endothelial dysfunction compared to baseline mice, as evidenced by lower -LogEC50. D-Tesa treatment showed a significantly increased -LogEC50 compared to the untreated group, similar to the baseline group, indicating that D-Tesa-treated mice preserved endothelial cell function even with continued HFD intake. The dose-response to sodium nitroprusside showed an increase in -LogEC50 in the untreated group compared to baseline, indicating SMC dysfunction. However, there was a significant improvement in -LogEC50 in the D-Tesa group compared to the untreated group, indicating that SMC function was preserved.

[0250] Example 3: D-Tesa upregulates PPAR-α and PPAR-γ in the aorta.

[0251] Materials and Methods

[0252] Aortic PPAR-α and PPAR-γ expression were evaluated by Western blotting. HFD supply ± D-Tesa-treated ApoE (- / -) The mouse thoracic descending aortic segment was homogenized, and PPAR-α and PPAR-γ were detected on separate blots along with GAPDH for normalization.

[0253] result

[0254] PPAR-α( Fig. 6a ) and PPAR-γ( Fig. 6b Protein expression is increased in D-Tesa-treated mice compared to untreated mice, which demonstrates successful D-Tesa delivery to the injury site and the release of functional tesaglitazar.

[0255] Example 4: D-Tesa reduces body weight in obese mice despite continued HFD, but not with free tesaglitazar.

[0256] Figures 7a–7f This proves the efficacy of D-Tesa and that free Tesa does not show side effects. VCO2( Fig. 7a ) and VO2( Fig. 7b ) increased in D-Tesa-treated mice compared to vehicle and glass Tesa-treated mice, resulting in increased energy consumption ( Fig. 7c It leads to an increase in ).

[0257] More food intake ( Fig. 7d Despite this, the body weight of D-Tesa-treated mice was significantly lower than that of vehicle-treated mice after 3 weeks ( Fig. 7e Edema, measured by the degree of foot edema which is a common side effect of tesaglitazar, does not occur in D-Tesa-treated mice but is observed in free tesaglitazar-treated mice ( Fig. 7f ).

[0258] Metabolic measurements performed on mice after treatment demonstrated that there was no decrease in food intake in D-Tesa-treated mice, and that an increase in HFD intake was actually observed. Fig. 7d ). More food intake ( Fig. 7d Despite this, the body weight of D-Tesa-treated mice was significantly lower than that of vehicle-treated mice after 3 weeks ( Fig. 7e ). Metabolic measurements also showed VCO2 in D-Tesa-treated mice compared to vehicle and glass Tesa-treated mice ( Fig. 7a ) and VO2( Fig. 7b Showing an increase in ) energy consumption ( Fig. 7c This led to an increase in ). This may be due to an increase in brown adipose tissue in D-Tesa-treated mice compared to white adipose tissue, which is generally associated with lower energy consumption in untreated and free Tesa-treated obese mice.

[0259] One of the notable side effects of free tesaglitazar treatment is peripheral edema. Mice treated with free tesaglitazar showed foot edema that did not appear in vehicle-treated mice. Fig. 7fIt showed a significant increase in thickness (mm). However, D-Tesa-treated mice did not show edema compared to vehicle-treated mice. Fig. 7f This demonstrated D-Tesa's ability to evade the side effects of free drugs through macrophage-specific targeting. No changes in activity were observed in D-Tesa-treated mice, indicating that body weight loss is not associated with negative effects.

[0260] Example 5: Localization of glucose dendrimers in plaque-associated macrophages

[0261] Macrophages in atherosclerotic plaques, adipose tissue macrophages, and microglia express increased glucose transporters (Glut) and exhibit increased localization of GD, which enables greater delivery of drugs to these cells. Glucose dendrimers are also localized in the hypothalamus of the brain (the region responsible for appetite and feeding) only in obese mice.

[0262] Example 6: Efficacy of Dendrimer-Tesaglitazar as a Potential Treatment for Atherosclerosis

[0263] Materials and Methods

[0264] Research Design

[0265] ApoE used in this study - / - Mice were purchased from Jackson Laboratory or produced in-house from mice also purchased from Jackson Laboratory. C57Bl / 6J mice were obtained from Jackson Laboratory and used as a healthy control group. Atherosclerosis was induced in mice aged 22–25 weeks by initiating a high-fat diet (Inotiv Teklad, catalog number TD.88137). Chow supply ApoE - / -Mice and healthy C57Bl / 6J mice were fed a regular chow diet throughout the entire study period. At the end of the study, the mice were anesthetized with 2% isoflurane. Foot thickness was measured using a digital caliper. Heparin was injected into the mice, and serum was collected via cardiac puncture. Euthanasia was completed by heart extraction under anesthesia.

[0266] All animal procedures and protocols used in this study were approved by the Animal Care and Use Committee (ACUC) of Johns Hopkins University. Mice were housed in the animal facility at Johns Hopkins University School of Medicine and provided with free access to food and water. The animal housing room was temperature-controlled with a 12-hour light / dark cycle.

[0267] Manufacturing and Characterization of D-Tesa

[0268] Tesaglitazar was conjugated to the surface of a fourth-generation hydroxyl-terminated polyamidoamine (PAMAM-OH) dendrimer as previously described. 20 Simply put, each dendrimer was synthesized to contain 10 tessaglitazar molecules attached via degradable ester bonds. Product formation was confirmed by mass spectrometry, and the purity of the synthesis was confirmed by HPLC.

[0269] In vitro THP-1 cell study

[0270] THP-1 monocyte cells were purchased from ATCC. Monocyte cells were placed in a 6-well plate at a ratio of 1 x 10⁶ per well. 6Cells were seeded. Transition from monocytes to macrophages was induced by incubating cells for 24 hours in RPMI 1640 medium supplemented with 10% FBS, 1% PSY (phytoene synthase-1), and phorbol 12-myristate 13-acetate (PMA). Subsequently, cells were incubated for 24 hours in serum-deficient DMEM / F12 (1:1) supplemented with 1% PSY. After incubating cells with oxLDL for 24 hours, they were incubated with free tesaglitasar, D-Tesa, for 24 hours or left untreated (control). Cells were lysed in MPER or subjected to nuclear fractionation using the NE-PER Nucleoplasm Extraction Kit (Thermo Fisher, Catalog No. 78833) according to the manufacturer's instructions.

[0271] D-Cy5 Localization Study

[0272] Dendrimer Cy5 (D-Cy5) was synthesized as previously described. 38-week-old ApoE that consumed HFD for 16 weeks - / - 10 mg / kg of PAMAM-OH D-Cy5 was administered to mice of the same age fed a standard diet and C57Blk / 6J mice fed a standard diet (ip). Upon sacrifice, the aorta was isolated, washed, and fixed in 10% formalin solution overnight. Aortic tissue was stained using DAPI and antibodies against CD68. Adipose tissue was isolated, fixed in 10% formalin solution for 30 minutes, and then cryopreserved in 30% sucrose overnight. Subsequently, the adipose tissue was embedded in an OCT and sectioned to a thickness of 20 μm using a Leica freeze sectioner. The adipose sections were stained with DAPI and TREM2. IF imaging was performed using a Leica SP8 confocal microscope.

[0273] Pulse wave transmission speed measurement

[0274] Mice were anesthetized with 2% isoflurane and placed on heated EKG pads (Indus Instruments) to monitor their heart rate. Heartbeats were measured along the thoracic and abdominal aorta using a 10 MHz Doppler. The pulse velocity was calculated by dividing the distance between two points by the time it took for the pulse wave to pass through the two points.

[0275] Blood pressure measurement

[0276] Prior to study enrollment, mice were subjected to a gradual acclimatization process for blood pressure measurement. First, the mice were secured to a blood pressure cone and placed under a heating lamp. Then, blood pressure was measured in conscious mice using a tail cuff (CODA Scientific non-invasive blood pressure measurement system).

[0277] ORO (Oil Red) staining and lesion analysis

[0278] The aorta was collected at the time of sacrifice and bisected longitudinally. Plaque deposition was measured using a commercially available ORO kit (abcam). Plaque was quantified along the ascending aorta, aortic arch, and descending aorta. The percentage of plaque area was measured using ImageJ.

[0279] Western blotting

[0280] Rapidly frozen tissue was ground into a powder and homogenized in RIPA buffer (Sigma-Aldrich) containing a protease inhibitor (Roche). The protein concentration of the tissue homogenates was quantified by performing the Bradford assay according to the manufacturer's instructions (BioRad). 4%–15% MP TGX gel (BioRad, Mini ®Protein samples were developed via SDS-PAGE on PROTEAN™ Precast Gel and transferred to nitrocellulose paper (BioRad Trans-Blot Turbo Transfer System). The blots were blocked for 1 hour in 3% skim milk powder in Tris-buffered saline with Tween 20 (TBST), followed by overnight incubation with a primary antibody diluted in 3% skim milk powder in TBST. After washing three times for 5 minutes each in TBST, the blots were incubated for 1 hour with a horseradish peroxidase (HRP) conjugated secondary antibody and imaged for chemiluminescence detection (Cytiva). The antibodies used were PPAR-α (Invitrogen, Catalog No. MA1-822), PPAR-γ (Invitrogen, Catalog No. MA5-14889), ABCA1 (Novus Biologicals, Catalog No. NB400-105), iNOS(Novus Biologicals, Catalog No. NB300-605), Arg1 (Novus Biologicals, catalog number NBP1-32731) and GAPDH (Novus Biologicals, catalog number NB300-221).

[0281] Wire Muscle Exercise Recording Method

[0282] In this study, aortic loops isolated from mice were applied to vascular reactivity studies as previously described. Briefly, a 2 mm aortic section was mounted on a wire muscle motion recorder (DMT), and 10 -6 Pre-constricted with M's phenylephrine. Endothelial-dependent vasodilation increased with acetylcholine concentration (10 -9 10 in M -5 It was determined according to (up to M). After pre-constricting blood vessels with phenylephrine first, the concentration increased (10 -910 in M -5 Endothelial cell-independent vasodilation was determined by incubating according to (up to M).

[0283] puller

[0284] The passive stiffness of the aortic ring was determined as previously reported. Simply put, the lumen diameter in ImageJ ( D ), wall thickness( t ) and blood vessel length( l To measure ), a 1.5 mm aortic ring was first imaged. Next, the ring was mounted on two parallel pins on a digital traction device (DMT) and pulled at a constant speed until fracture. During traction, data on the displacement and force applied by the ring in response to deformation were continuously recorded. Force ( F ) / Displacement( d Normalize the data into individual parameters of the side to obtain the data stress( S ) / Strain( λ Converted to ) data( S = F / 2 x t x l and λ= D / d ). By performing nonlinear regression, S=αe βxλ The stress-strain curve represented by the equation (where α and β are constants) was determined. The elastin-mediated incremental elastic modulus ( E inc At a strain of 1.5 for ), collagen-mediated E inc For , at a strain of 2.5, calculate the first derivative of the stress-strain curve to obtain the incremental elastic modulus ( E inc ) was produced.

[0285] Electrochemiluminescence multiple analysis method

[0286] Serum samples collected from mice were analyzed using the V-PLEX Inflammation Panel 1 Mouse Kit (Meso Scale Diagnostics, Catalog No. K15048D-1) according to the manufacturer's instructions. Multiple analysis was performed using 1:2 dilutions of serum, and each sample was run in duplicate. The Bradford assay (BioRad) was used to determine the protein concentration of each serum sample and to normalize the concentrations of each analyte.

[0287] Metabolism test

[0288] Quantitative MRI (qMRI) and indirect calorimetry experiments were performed in collaboration with the Johns Hopkins Center for Metabolism & Obesity Research.

[0289] For qMRI, the mouse was weighed before being guided into the insertion tube. The plunger was gently inserted into the tube to accurately position and secure the mouse for measurement. Data acquisition was started by inserting the insertion tube into the EchoMRI-100.

[0290] Indirect calorimetry was performed using 24 CLAMS indirect calorimeters (Columbus Instruments). Mice were housed individually and provided with HFD and water freely. Body weight and food intake were monitored daily. Data on oxygen consumed and carbon dioxide produced were collected by continuously supplying fresh indoor air, which was sampled periodically, to each cage.

[0291] Statistical analysis

[0292] Data are presented as mean ± standard error of the mean. The sample size (n) is reported for all data collections. Statistical analysis was performed in Prism 8. For normally distributed data, Student's t-test was used to compare the two means. For data with more than one mean, one-way ANOVA was used. Two-way ANOVA was used for group analysis. Statistical significance was considered to be p<0.05.

[0293] result

[0294] D-Tesa-mediated activation of PPAR-α / γ leads to an anti-inflammatory phenotype and increased cholesterol efflux in THP-1-derived macrophages.

[0295] The performance of D-Tesa was tested in vitro to determine whether the dendrimer formulation successfully delivers tesaglitazar to cells and activates PPAR-α / γ in atherosclerotic macrophages. To this end, THP-1 monocytes were stimulated with PMA to induce a macrophage phenotype and incubated with oxLDL to induce foam cell formation; subsequently, they were randomized to be administered D-Tesa, free tesaglitazar, or vehicle (DMSO) (Fig. 8a). Oil Red O staining showed a reduction in lipid deposition in D-Tesa-treated macrophages compared to the vehicle and free drugs (Fig. 8b).

[0296] A comparison of PPAR-α / γ nuclear translocations after treatment with free or D-Tesa revealed that the dendrimer conjugate could upregulate the protein expression of both PPAR-α and PPAR-γ on a larger scale than that of the free drug (Figs. 8c–8e). This upregulation was also associated with higher protein levels of ABCA1 (Figs. 8f and 8g), a well-established target gene for cholesterol efflux transporters and PPAR-γ. Furthermore, THP-1-derived macrophages treated with D-Tesa exhibited a phenotypic shift from pro-inflammatory to anti-inflammatory similar to that of macrophages treated with the free drug, which was evidenced by the loss of iNOS (Fig. 8h) and an increase in Arg1 (Fig. 8i). Thus, in vitro, D-Tesa demonstrated similar PPAR-α / γ activation while inducing superior cholesterol efflux and anti-inflammatory effects compared to the free drug.

[0297] PAMAM-OH dendrimers deliver payloads in vivo to WAT and resident macrophages within aortic plaques.

[0298] We investigated whether dendrimers could deliver payloads to cells within white adipose tissue (WAT) and plaques. To visualize drug delivery, atherosclerotic apoE - / - After administering cy5-labeled D-Tesa (D-Tesa-cy5) PO to mice, leukocytes, aorta, and WAT were stained with IF (immunofluorescence) (Fig. 9a). Smearing of the leukocyte soft layer revealed that the dendrimer conjugate was not absorbed by circulating leukocytes (Fig. 9b). Sectioned WAT showed that D-Tesa-cy5 was internalized by TREM2-positive macrophages (Fig. 9c). Similarly, the conjugate showed uptake in aortic macrophages, as evidenced by dendrimer co-localization with CD68-positive cells (Fig. 9d). This co-localization is related to atherosclerosis ApoE - / -It was observed only in mice and not in healthy age-matched wild-type control mice. Images taken with increasing depth into the plaque show that the dendrimers can penetrate deeply into the plaque, which is a major challenge in treating atherosclerosis (Figs. 16a–16d).

[0299] D-Tesah treatment in male and female apoE - / - Stops the progression of aortic plaque in mice by improving cholesterol efflux.

[0300] In vivo efficacy of D-Tesa in male and female apoE - / - The study was conducted on mice. Mice aged 22–25 weeks were fed a high-fat diet (HFD) for 12 weeks and then randomly assigned to one of four groups. 1) The first group, serving as the baseline, was sacrificed immediately after 16 weeks of HFD intake to provide pre-treatment physiological data; 2) D-Tesa (20 mg / kg PO twice weekly), 3) Free tesaglitasar (20 mg / kg PO twice weekly), and 4) Vehicle control group (PO DI twice weekly) mice continued to consume the high-fat diet (HFD) throughout the treatment period (Fig. 10a). Oil Red O staining (ORO) was performed on aortas isolated from baseline, vehicle-treated, and D-Tesa-treated mice (Fig. 10b). Vehicle-treated mice showed a significant increase in plaque deposition compared to baseline mice. However, atherosclerotic mice treated with D-Tesa had significantly lower plaque levels than vehicle-treated mice (Fig. 10c). Western blotting (Figs. 10d and 10i) revealed that this benefit was due to an increase in ABCA1 (Figs. 10e and 10j) as well as apoA (Figs. 10f and 10g), an apolipoprotein responsible for high-density lipoprotein-mediated cholesterol efflux. Additionally, there was a regulation of macrophages from an inflamed phenotype represented by iNOS (Figs. 10g and 10l) to an anti-inflammatory macrophage phenotype represented by Arg1 (Figs. 10h and 10m).

[0301] ApoE treated with D-Tesa - / - Mice exhibit active and passive aortic stiffness improvement.

[0302] During the administration and treatment period of the high-fat diet (HFD), the following parameters were monitored: body weight, blood pressure, and pulse wave velocity (PWV), the standard criterion for determining arterial stiffness. During the 16-week HFD administration, males and females demonstrated an increase in PWV, which is correlated with the development of atherosclerosis. Males showed a statistically significant increase during the first 8 weeks but decreased over the following 8 weeks. Females also demonstrated a significant increase in PWV during the first 8 weeks and another significant increase during the following 8 weeks, revealing sex differences in the progression of arterial stiffness in atherosclerosis. During the 6-week treatment period, male and female mice treated with D-Tesa demonstrated a significant decrease in PWV not observed in mice treated with free tesaglitasar (Figs. 11a and 11h). Throughout the entire study, neither male nor female mice demonstrated significant changes in blood pressure (Figs. 11b and 11i).

[0303] At the time of sacrifice, the aortic ring was subjected to an in vitro tensile test to generate stress-strain curves, and the passive vascular stiffness of the isolated aortic ring was determined (Figs. 11c and 11j). Vehicle-treated male and female ApoE - / - The mouse is baseline ApoE - / -It showed an increase in collagen-mediated stiffness compared to mice and age-matched feed control mice. In both sexes, D-Tesa treatment slightly improved elastin-mediated stiffness (Figs. 11d and 11k), but significantly reduced collagen-mediated stiffness compared to vehicle-treated mice (Figs. 11e and 11l). This reduction in passive vascular stiffness in both males and females corresponded to PWV data showing lower aortic stiffness in D-Tesa-treated mice. In males, an advantage in strain at rupture was also observed, exhibiting similar strain at rupture compared to vehicle-treated and free tesaglitazar-treated mice (Figs. 11f and 11g). This trend was not observed in females, suggesting a sex difference in the treatment.

[0304] D-Tesa stops the progression of endothelial dysfunction caused by the progression of atherosclerosis.

[0305] Vascular responsiveness was determined by determining endothelial-dependent vasodilation using acetylcholine (Ach) and endothelial-independent vasodilation using sodium nitroprusside (SNP) following phenylephrine preconstriction, and also by using the aortic loop. In males, endothelial-mediated vasodilation in the vehicle-treated group was observed to be significantly impaired 6 weeks from baseline (Fig. 12a). However, the significant positive effect of D-Tesa on the Ach (acetylcholine) dose-response was LogEC 50 (Fig. 12b) and maximum relaxation (Fig. 12c) were observed when compared to the vehicle, whereas they did not appear in mice treated with free tesaglitazar. In females, the same trend was observed (Fig. 12g). This is due to male apoE - / - Maximum relaxation in vehicle relative to baseline (Fig. 12g) and LogEC in females compared to mice 50This may be due to cardioprotection in females, as evidenced by the reduction in the difference in (Fig. 12h). Additionally, in both male (Fig. 12d–12f) and female (Fig. 12j–12l) atherosclerotic mice, SNP-mediated vasodilation remained constant, which suggests that the difference in each dose response is likely due to endothelial protection rather than differences in aortic smooth muscle cell function.

[0306] The selective delivery of tesaglitazar to macrophages within adipose tissue induces browning and causes weight loss.

[0307] D-Tesa treatment induced body weight loss that was not observed in mice treated with vehicle or free tesaglitazar (Fig. 13a). Body composition tests revealed that while body weight decreased, lean body mass, i.e., the proportion of body weight accounted for by muscle mass, increased (Fig. 13b). The decrease in total body weight was not due to appetite suppression, as D-Tesa-treated mice consumed significantly more than mice treated with vehicle and free drugs (Fig. 13c).

[0308] Western blot revealed that D-Tesa-treated mice exhibited anti-inflammatory effects on adipose tissue, as indicated by a decrease in iNOS and an increase in arg1 (Figs. 13d–13f). The dendrimer conjugate also induced an increase in uncoupling protein 1 (UCP-1), a marker for brown adipose tissue, to the same extent as free tesaglitazar (Fig. 13g). This browning causes phenotypic changes in adipocytes, leading to a reduction in size; D-Tesa treatment resulted in a significant reduction in adipocyte size compared to mice treated with vehicle and free tesaglitazar (Figs. 13h–13i). Metabolic tests were performed on male mice to investigate any benefits to body composition resulting from D-Tesa treatment. D-Tesa was observed to significantly increase the volumes of oxygen consumed (VO2) (Fig. 13j) and carbon dioxide produced (VCO2) (Fig. 13l) compared to mice treated with vehicle and free tesaglitazar. Since both parameters increased, the respiratory exchange ratio (RER) remained the same for all three groups (Fig. 13m). However, this revealed that D-Tesa-treated mice significantly increased the level of energy expenditure (EE) (Fig. 13n).

[0309] The macrophage-targeted delivery of tesaglitazar avoids side effects associated with free drug treatment.

[0310] Because D-Tesa utilizes macrophage-targeted delivery of free tesaglitazar, we tested whether dendrimer conjugation enables the avoidance of adverse outcomes observed in clinical trials. In addition to weight gain, another cited side effect of tesaglitazar was renal impairment leading to peripheral edema. Foot thickness was measured using digital calipers, and indeed, free tesaglitazar induced edema in all four feet. However, this same degree of edema was observed in D-Tesa-treated male ApoE- / - This was not observed in mice (Fig. 14a). Additionally, there was a slight decrease in hemoglobin associated with free tesaglitazare treatment that was not observed in D-Tesa (Fig. 14b). Western blot of the whole kidney (Fig. 14c) showed that while there was an increase in inducible nitric oxide synthase (iNOS) in free Tesa-treated mice (Fig. 14d) and no effect on arg1 (Fig. 14e), this was not observed in D-Tesa-treated mice. Similar levels of PPAR-α protein expression were present in both the free tesaglitazare and D-Tesa-treated groups (Fig. 14f), indicating that targeted PPAR activation does not confer the same renal toxicity as systemic activation. A similar trend was observed in female apoE - / - It was observed in mice (Figs. 17a–17f).

[0311] The physiological benefits of D-Tesa therapy include LDL with intact apoE-mediated cholesterol efflux. r- / - It is reproduced in mice.

[0312] ApoE - / - Since mice develop atherosclerosis through impaired cholesterol efflux, male and female LDLr with intact apoE-mediated cholesterol efflux - / - The experiment was repeated in mice. Male and female LDLr - / - All mice showed a significant decrease in the % change in body weight that was not observed with vehicle or free tesaglitazar treatment (Figs. 15a and 15h). Free tesaglitazar caused significant paw edema compared to vehicle-treated mice. However, this adverse effect was not observed with D-Tesa treatment (Figs. 15b and 15i). PWV is male atherosclerotic LDLr - / -In mice, it decreased after D-Tesa treatment, but not in females (Figs. 15c and 15j). Blood pressure remained constant throughout the experiment (Figs. 15d and 15j). After treatment, passive vascular stiffness did not change in males (Fig. 15e), but decreased in female mice (Fig. 15k). Acetylcholine-mediated vasoresponsiveness studies on isolated aorta showed that endothelial function in male and female LDLr treated with D-Tesa - / - It was shown to be superior in mice (Figs. 15f and 15l), which was highlighted by significantly higher maximum relaxation compared to vehicle and free tesaglitazar (Figs. 15g and 15m).

[0313] Ldlr KO mice are another widely used atherosclerosis model in which exposure to HFD induces plaque along with vascular stiffening and body weight gain. Data in Figures 15a–15m demonstrate the efficacy of D-Tesa treatment in the model, resulting in improved vascular responsiveness and body weight loss in both males and females. This indicates that the therapeutic benefits of D-Tesa are observed regardless of the model type inducing atherosclerosis and body weight gain.

[0314] Discussion

[0315] Obesity and atherosclerosis are chronic inflammatory diseases linked by the infiltration of macrophages into adipose tissue and arteries, respectively. Obesity accelerates atherosclerosis, and for every 1-point increase in BMI, the risk of atherosclerosis and coronary heart disease increases by 10%. 1 Atherosclerosis is a major cause of cardiovascular disease, which is the leading cause of death worldwide. 2 In addition, obesity increases the risk of premature death by 2 to 3 times in both men and women compared to individuals with a BMI of 23.5 to 24.9. 3

[0316] To date, there is no therapy that can cure atherosclerosis. Despite significant advancements in the diagnosis and treatment of atherosclerosis, the need for therapies that directly target plaque remains. Most recently, the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) demonstrated a reduction in systemic inflammation and cardiovascular events using a monoclonal antibody against interleukin-1β. 4 Ongoing research on atherosclerosis therapy involves the inactivation of cytokine release within plaques 5,6 and improvement of efferocytosis 7,8 This includes. Currently, the only treatment option to reduce the burden of severe plaque is surgical intervention. These options are limited to severe cases; while they alleviate the burden, they do not address the underlying cause of the disease. The immune system is emerging as a target for treating atherosclerosis and obesity because macrophages are the primary cell type driving disease progression. Macrophages residing in atherosclerotic plaques induce systemic inflammation and contribute to the accumulation of foam cells within the plaque.

[0317] Peroxisome proliferator-activated receptors (PPARs) are transcription factors of particular interest for treating chronic inflammation associated with obesity and atherosclerosis. The two isotypes of PPAR, PPAR-α and PPAR-γ, have been explored for their anti-atherosclerotic and anti-inflammatory functions. PPAR-α is involved in lipid and lipoprotein metabolism 4 It regulates the expression of genes involved in [it]. Activation of PPAR-α reduces cholesterol ester levels and lipoprotein accumulation, while also increasing fatty acid beta-oxidation. 9 PPAR-γ regulates fatty acid storage and increases the expression of proteins involved in lipid and glucose metabolism. 4In addition, PPAR-γ reduces monocyte infiltration into the vascular endothelium and decreases macrophage accumulation in plaques by inhibiting vascular smooth muscle cell migration and differentiation. 10 Both PPAR-α and PPAR-γ are expressed in macrophages and have well-established anti-inflammatory properties. PPAR-α and PPAR-γ have been shown to not only inhibit the acetylation of p65 NF-κB but also induce the deacetylation of p65 NF-κB, leading to the inactivation of NF-κB. 11 PPAR-γ activation demonstrated potent macrophage polarization toward an anti-inflammatory phenotype. PPAR-α and PPAR-γ have been reported to inhibit foam cell formation by regulating genes that improve reverse cholesterol transport. 5

[0318] Tesaglitazar is a PPAR agonist of particular interest because it is a dual PPAR-α / γ agonist. Previous studies have shown that tesaglitazar improved lipid profiles in diabetic and non-diabetic patients with insulin resistance. 11,14 In mice, this is plasma cholesterol 15 and reduction of atherosclerotic plaques and improvement of inflammation markers 16 It demonstrated benefits including [mention specific benefits]. However, toxicity issues occurred during Phase 3 clinical trials. Patients treated with tesaglitasar experienced peripheral edema, weight gain, and impaired renal function. 17 , dose-dependent hemoglobin decrease (anemia) 18 , worsening of myocardial ischemia and congestive heart failure 19 ...was experienced. Therefore, the trial was terminated due to a lack of benefit compared to the side effect profile resulting from off-target effects.

[0319] In this study, we hypothesized that targeting inflammatory macrophages in aortic plaques and white adipose tissue using dendrimer-conjugated formulations would achieve benefits for obesity and atherosclerosis while avoiding PPAR activation in healthy tissues. To leverage these dual PPAR-α / γ benefits and avoid harmful side effects, we tested tesaglitazar conjugated to fourth-generation hydroxyl-terminated polyamidoamine (PAMAM-OH) dendrimer nanoparticles (D-Tesa). These dendrimers are non-toxic and are not metabolized through the kidneys, as demonstrated in humans. Preferential uptake of PAMAM-OH dendrimers into activated macrophages in various preclinical animal species was revealed, leading to positive therapeutic outcomes. It was also found that D-Tesa possesses higher solubility than free drugs and achieves sustained intracellular release. 20

[0320] The influence of gender is an established variable in cardiometabolic disorders. Women show protection against cardiometabolic dysfunction until menopause, and it has been hypothesized that this is due to the benefits of estrogen. 21 For example, the risk of myocardial infarction, stroke, or other major adverse cardiovascular events is higher in men under 60 but higher in women over 60. 22 The study reports that the intima-media thickness, a parameter of atheroma formation, is higher in men than in women at age 35, but no such difference is reported at age 75. 23 In this study, we investigated the sex-specific effects on the development and treatment of atherosclerosis using male and female mice.

[0321] There are two commonly used mouse models for atherosclerosis research: the apolipoprotein E knockout mouse (ApoE - / - ) and low-density lipoprotein knockout mice (LDLr - / - ). Both models are on a C57Blk / 6 background. LDLr- / - Mice are deficient in LDL receptors, which causes a delay in the clearance of LDL and leads to an increase in plasma cholesterol levels. When fed a standard diet, these mice do not develop atherosclerosis, because they require a source of high cholesterol from a high-fat diet to promote atherosclerosis. LDLr - / - The lipid profile of is similar to that of humans with hypercholesterolemia. ApoE - / - Mice are deficient in apolipoprotein E, a protein necessary for mammalian lipid metabolism. ApoE - / - Mice induce lesions corresponding to human atheromas more closely. Since both models mimic the disease progression of atherosclerosis and obesity, the efficacy of D-Tesa was examined in both mouse models. This study demonstrated the ability of D-Tesa to deliver tesaglitazarr into atherosclerotic macrophages, address the causes of atherosclerosis, improve atherosclerosis, and reduce obesity. The in vitro mechanism of D-Tesa was confirmed in THP-1-derived macrophages transformed into foam cells using elevated concentrations of oxidized LDL. D-Tesa treatment showed upregulation of PPAR-α / γ, the cholesterol efflux transporter ABCA1, and the anti-inflammatory marker Arg1 to the same or higher scale as in cells treated with free tesaglitazarr. Dendrimer conjugation enables enhanced efficacy due to dendrimer-mediated macrophage targeting, improved solubility, and intracellular release. Conducting these studies on THP-1 cells also provides insight into the ability of drugs to be applied to humans, as these monocytes are of human origin.

[0322] The efficacy of the conjugate was evaluated in vivo. To obtain the best indications for application, this study was designed to replicate clinical profiles and therapeutic regimens with high compliance potential. ApoE in atherosclerosis - / -A model was used. Mice were fed a high-fat diet for 16 weeks starting from 22–25 weeks of age prior to the start of treatment. ApoE from HFD intake for 16 weeks - / - It is well established that mice develop potent plaque deposition accompanied by fibrous caps in the aorta. This age group was selected because it corresponds to middle age in humans, meaning it is the group that can derive the greatest benefit from this treatment in clinical practice.

[0323] Dendrimer localization studies demonstrate that hydroxyl-terminated PAMAM dendrimers can deliver payloads into arterial plaque deposits, specifically to a depth of approximately 90% within the atheroma. This is considered the first report of therapy delivered into atherosclerotic plaques. Because the payload can be delivered into plaque macrophages, an improvement in the inflammatory response was observed in both treated male and female atherosclerotic mice. Interestingly, free tesaglitazar was found to induce increases in the pro-inflammatory markers INF-γ and KC / GRO, which are likely due to off-target activation of other organs such as the kidney or adipose tissue. However, these increases were not observed in D-Tesa-treated mice due to macrophage-specific delivery. The first tested marker of atherosclerotic burden was active arterial stiffening via PWV. When mice began HFD intake, there was an increase in PWV corresponding to the duration of HFD intake. Sex-based differences were observed in the progression of arterial stiffness; males developed arterial stiffness rapidly but diminished after 8 weeks of HFD intake, whereas females exhibited this stiffness much more gradually due to cardioprotection. After D-Tesa treatment every 2 weeks for 6 weeks, both males and females were observed to show significantly lower PWVs, on a scale nearly identical to that before the initiation of HFD. Arterial stiffness is a major cause and consequence of harmful cardiovascular diseases that can lead to more serious complications, such as heart failure, due to the increased load placed on the heart caused by non-compliant blood vessels. In vitro tensile tests also revealed that passive vascular stiffness improved after D-Tesa treatment and was consistent with active vascular stiffness. Aorta isolated from D-Tesa-treated male mice could withstand higher stress at the same maximum strain, indicating that the aorta of D-Tesa-treated males is less brittle and can handle more strain at the same level of maximum strain before fracture.Throughout the experiment, blood pressure in both males and females remained constant despite the occurrence of atherosclerosis and weight gain, which is consistent with literature showing that obesity precedes hypertension. This also demonstrates that the difference in arterial stiffness is due to actual changes in the mechanical properties of the aorta rather than differences in distensional pressure caused by hypertension.

[0324] Vascular reactivity studies demonstrate that D-Tesa was able to halt endothelial dysfunction, another marker of cardiovascular health. Endothelial cells in the aorta of D-Tesa-treated mice were found to exhibit preservation of function not observed in mice treated with vehicle or free tesaglitasar, which is likely due to a reduction in the inflammatory profile generated by plaque macrophages located in close proximity to the endothelial cells.

[0325] This delivery method avoided the side effects associated with free tesaglitazar. Therefore, dendrimer conjugation is believed to provide the benefits of the free drug while avoiding dose-dependent side effects typically observed in clinical trials. Specifically, increased peripheral edema resulting from free drug treatment was observed in the form of increased foot edema. This edema was caused by PPAR activation in healthy kidney tissue, which does not occur in mice treated with D-Tesa. In addition to reducing plaque burden and addressing the underlying causes of atherosclerosis, D-Tesa-treated mice also demonstrated the benefit of body weight reduction in both obese atherosclerotic male and female mice. This body weight reduction was not due to appetite suppression, which distinguishes it from current therapies for glucagon-like peptide-1 (GLP-1) activation. The data show that this body weight reduction was due to macrophage-mediated browning of adipose tissue, as evidenced by the upregulation of UCP-1. This browning leads to improvements in metabolic parameters, such as increases in VO2, VCO2, and EE. Interestingly, iNOS, a marker for inflammatory macrophages, was also found to be significantly upregulated in the adipose tissue of free tesaglitazare-treated mice compared to vehicle-treated mice, suggesting an inflammatory effect of systemic activation in adipose tissue. This effect was not observed in D-Tesa-treated mice, contributing to the benefits of targeted therapy. Weight loss further contributed to the benefits of D-Tesa and future dendrimer-conjugated therapies for obesity. Weight gain is a comorbidity for conditions other than atherosclerosis and cardiovascular disease. These data demonstrate the potential for dendrimer technology to be used to suppress weight gain through macrophage-induced adipose tissue browning.

[0326] After just 6 weeks, these benefits were observed at a much lower dose (2 / 7) of free tesaglitazar used in previous studies, even though the mice maintained a high-fat diet. Previous studies using free tesaglitazar changed the mice's diet from HFD to a standard diet at the start of treatment. However, this design demonstrated that D-Tesa can achieve therapeutic effects even during the continuation of an atherogenic diet. The improved safety profile of D-Tesa compared to free tesaglitazar, along with the convenience of oral administration, presents a promising therapeutic agent that addresses the urgent need for treating atherosclerosis by targeting the immune system to resolve the root cause of plaque progression.

[0327] References

[0328] 1. Hu F. Obesity Epidemiology. Oxford University Press; 2008.

[0329] 2. Cardiovascular diseases (CVDs). who.int / news-room / fact-sheets / detail / cardiovascular-diseases-(cvds)

[0330] 3. Adams KF, et al., N Engl J Med. 2006;355:763-778.

[0331] 4. Ridker PM, et al . N Engl J Med . 2017;377:1119-1131.

[0332] 5. Fatkhullina AR, et al . Immunity . 2018;49:943-957 e949.

[0333] 6. Fatkhullina AR, et al .Biochemistry (Mosc). 2016;81:1358-1370.

[0334] 7. Flores AM, et al., Pro-efferocytic nanoparticles are specifically taken up by lesional macrophages and prevent atherosclerosis. Nat Nanotechnol . 2020;15:154-161.

[0335] 8. Jarr KU, et al., The pleiotropic benefits of statins include the ability to reduce CD47 and amplify the effect of pro-efferocytic therapies in atherosclerosis. Nat Cardiovasc Res. 2022;1:253-262.

[0336] 9. Rigamonti E, et al ., Regulation of macrophage functions by PPAR-alpha, PPAR-gamma, and LXRs in mice and men. Arterioscler Thromb Vasc Biol . 2008;28:1050-1059.

[0337] 10. Yin L, et al . , The Role of Peroxisome Proliferator-Activated Receptor Gamma and Atherosclerosis: Post-translational Modification and Selective Modulators. Front Physiol. 2022;13:826811.

[0338] 11. Korbecki J, et al. Self-regulation of the inflammatory response by peroxisome proliferator-activated receptors. Inflamm Res . 2019;68:443-458.

[0339] 12. Fagerberg B, et al . Tesaglitazar, a novel dual peroxisome proliferator-activated receptor alpha / gamma agonist, dose-dependently improves the metabolic abnormalities associated with insulin resistance in a non-diabetic population. Diabetologia . 2005;48:1716-1725.

[0340] 13. Schuster H, et al. Tesaglitazar, a dual peroxisome proliferator-activated receptor alpha / gamma agonist, improves apolipoprotein levels in non-diabetic subjects with insulin resistance. Atherosclerosis . 2008;197:355-362.

[0341] 14. Goldstein BJ, Rosenstock J, Anzalone D, Tou C, Ohman KP. Effect of tesaglitazar, a dual PPAR alpha / gamma agonist, on glucose and lipid abnormalities in patients with type 2 diabetes: a 12-week dose-ranging trial. Curr Med Res Opin. 2006;22:2575-2590.

[0342] 15. Zadelaar AS, et al . Dual PPARalpha / gamma agonist tesaglitazar reduces atherosclerosis in insulin-resistant and hypercholesterolemic ApoE*3Leiden mice. Arterioscler Thromb Vasc Biol . 2006;26:2560-2566.

[0343] 16. Chira EC, et al . Tesaglitazar, a dual peroxisome proliferator-activated receptor alpha / gamma agonist, reduces atherosclerosis in female low density lipoprotein receptor deficient mice. Atherosclerosis . 2007;195:100-109.

[0344] 17. Bays H, et al . A double-blind, randomised trial of tesaglitazar versus pioglitazone in patients with type 2 diabetes mellitus. Diab Vasc Dis Res . 2007;4:181-193.

[0345] 18. Insulin, other hypoglycemic drugs, and glucagon. Annual 31 ed.; 2009.

[0346] 19. Kalliora C, Drosatos K. The Glitazars Paradox: Cardiotoxicity of the Metabolically Beneficial Dual PPARalpha and PPARgamma Activation. J Cardiovasc Pharmacol. 2020;76:514-526.

[0347] 20. DeRidder L, Dendrimer-tesaglitazar conjugate induces a phenotype shift of microglia and enhances beta-amyloid phagocytosis. Nanoscale . 2021;13:939-952.

[0348] 21. Ryczkowska K, et al. . Menopause and women's cardiovascular health: is it really an obvious relationship? Arch Med Sci. 2023;19:458-466.

[0349] 22. Sturlaugsdottir R, et al . Prevalence and determinants of carotid plaque in the cross-sectional REFINE-Reykjavik study. BMJ Open . 2016;6:e012457.

[0350] 23. Sinning C, et al. Sex differences in early carotid atherosclerosis (from the community-based Gutenberg-Heart Study). Am J Cardiol. 2011;107:1841-1847.

Claims

Claim 1 A composition comprising a hydroxyl dendrimer covalently conjugated to at least one drug, wherein the drug comprises a PPAR-α agonist; a PPAR-γ agonist; a dual PPAR agonist (e.g., a PPAR-α / γ agonist); a GLP-1 receptor agonist (e.g., semaglutide); metformin; an SGLT2 agonist, a GIP-1 receptor agonist, or an antagonist; a dual GLP-1 / GIP-1 receptor agonist (e.g., tyrzepatide); a mitochondrial uncoupler (e.g., niclosamide); or a combination thereof. Claim 2 A composition comprising a glucose dendrimer covalently conjugated to at least one drug, wherein the drug comprises a PPAR-α agonist; a PPAR-γ agonist; a dual PPAR agonist (e.g., a PPAR-α / γ agonist); a GLP-1 receptor agonist (e.g., semaglutide); metformin; an SGLT2 agonist, a GIP-1 receptor agonist or antagonist; a dual GLP-1 / GIP-1 receptor agonist (e.g., tyrzepatide); a mitochondrial uncoupling agent (e.g., niclosamide); a cannabinoid 1 receptor (CB1R) antagonist; or a combination thereof. Claim 3 A composition according to claim 1 or 2, wherein the drug comprises a PPAR-α agonist selected from the group consisting of tesaglitazar, fenofibrate, WY-14643, oleoyethanolamide, GW4148, GW 9578, GW2148 or combinations thereof; a PPAR-γ agonist; or a dual PPAR agonist (e.g., a PPAR-α / γ agonist). Claim 4 In claim 1 or 2, the drug is semaglutide (e.g., Ozempic) ® or Rybelsus ® ); dulaglutide (e.g., Trulicity) ® ); Exenatide extended release, exenatide (e.g., Byetta) ® ) and analogs and derivatives thereof; liraglutide (Victoza) ® , Saxenda ® )(Daily); Lixisenatide(Adlyxin) ® A composition comprising a GLP-1 receptor agonist selected from the group consisting of ); and other incretin mimics. Claim 5 A composition according to claim 1 or 2, wherein the drug is a GIP-1 receptor agonist or antagonist selected from the group consisting of SKL-14959, MA-38472-B1 or a combination thereof. Claim 6 A composition according to claim 1 or 2, wherein the drug is a dual GLP-1 / GIP-1 receptor agonist selected from the group consisting of tyrzepatide, NNC0090-2746, NN9709, CT-388, retatrutide, or a combination thereof. Claim 7 A composition according to claim 1 or 2, wherein the drug is metformin. Claim 8 In paragraph 1 or 2, the drug is canagliflozin (e.g., Invokana) ® ), Bexagliflozin (e.g., Brenzavvy) ® ), dapagliflozin (e.g., Farxiga) ® ), empagliflozin (e.g., Jardiance) ® ), Ertugliflozin (Steglatro) ® A composition comprising an SGLT2 agonist selected from the group consisting of ) or a combination thereof. Claim 9 A composition according to claim 1 or 2, wherein the drug comprises a mitochondrial uncoupling agent selected from the group consisting of niclosamide, BAM15, carbonyl cyanide 4-(trifluoro-methoxy)phenylhydrazone (FCCP), carbonyl cyanide-3-chlorophenylhydrazone (CCCP), dinitrophenol, analogs of these molecules, or combinations thereof. Claim 10 A composition according to claim 1 or 2, wherein the drug comprises a CB1R antagonist selected from the group consisting of AJ5012, JD5037, 3,4-diarylpirazoline, 1,5-diarylpyrrol-3-carboxamide, methylsulfonamide azetidine, 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-piperidino-1H-pyrazole-3-carboxamide (rimonabant), or combinations thereof. Claim 11 A composition according to any one of claims 1 to 10, wherein the covalent bonding comprises a covalent bond between a modified or unmodified surface group or an internal group of the hydroxyl dendrimer or glucose dendrimer, and the covalent bond comprises an amide, ester, disulfide, ether, or phosphate bond. Claim 12 A composition according to any one of claims 1 to 11, wherein the covalent bonding comprises a covalent bond capable of being cleaved at a pH selected from neutral, acidic, or basic. Claim 13 A composition according to any one of claims 1 to 12, wherein the hydroxyl dendrimer or glucose dendrimer is based on a 1st to 7th generation polyamidoamine (PAMAM) dendrimer. Claim 14 A composition according to any one of claims 2 to 13, wherein the dendrimer is a glucose dendrimer, preferably a first-generation, second-generation, or third-generation glucose dendrimer. Claim 15 A composition according to any one of claims 1 to 13, wherein the dendrimer is based on a 2nd to 7th generation PAMAM dendrimer modified with a sugar moiety, wherein the sugar moiety is selected from the group consisting of glucose, galactose, mannose, and fructose. Claim 16 A composition according to any one of claims 1 to 15, wherein the hydroxyl dendrimer or glucose dendrimer has at least a 10-fold increase in drug solubility compared to the free form of the drug; or the hydroxyl dendrimer, glucose dendrimer, or both increase the duration of effect compared to the free form of the drug. Claim 17 A composition according to any one of claims 1 to 16, wherein the hydroxyl dendrimer is based on a higher generation dendrimer, preferably a 3rd, 4th, 5th, or 6th generation PAMAM dendrimer (e.g., a hydroxyl-terminated PAMAM dendrimer); or the glucose dendrimer is a 1st, 2nd, 3rd, or higher generation glucose dendrimer or is functionalized with poly(ethylene glycol), wherein the conjugate is limited to peripheral circulation. Claim 18 A composition according to any one of claims 1 to 17, wherein the drug has a loading of about 2% by weight to about 35% by weight. Claim 19 A composition comprising, in any one of claims 1 to 18, a pharmaceutically acceptable excipient. Claim 20 A preparation formulated in a dosage for the treatment of atherosclerosis, obesity, diabetes, metabolic syndrome, blood sugar control, blood pressure reduction, lipid disorders, fatty liver disease, kidney disease treatment / prevention, or symptomatic treatment thereof, comprising a composition of any one of claims 1 to 19. Claim 21 In paragraph 20, the above-mentioned preparation is for the treatment of an obese individual, and the treatment of the said obese individual is such that the reduction in fat mass is greater than the reduction in lean body mass. Claim 22 In paragraph 20 or 21, the said preparation is for the treatment of an obese individual, and said treatment of the obese individual is such that weight loss is achieved without a notable decrease in appetite. Claim 23 A preparation according to any one of claims 20 to 22, wherein the preparation is for the treatment of an obese individual, and the treatment of the obese individual is to convert white fat cells into brown fat cells. Claim 24 A method for treating one or more symptoms of atherosclerosis, abnormal blood glucose, hypotension, lipid disease, fatty liver disease, kidney disease, obesity or related metabolic disorders and combinations thereof, wherein the method comprises the step of administering a composition of any one of claims 1 to 19 or a preparation of any one of claims 20 to 23 to an individual requiring treatment thereof. Claim 25 A method for treating a disease or disorder, wherein the method comprises the step of using a composition of any one of claims 1 to 19 or a preparation of any one of claims 20 to 23 in combination with an existing treatment. Claim 26 A method for treating a disease or disorder, wherein the method comprises the step of preventing the recurrence of said disease or disorder by using a composition of any one of claims 1 to 19 or a preparation of any one of claims 20 to 23 after the termination or reduction of existing treatment. Claim 27 A method according to any one of claims 24 to 26, wherein the preparation selectively delivers the hydroxyl dendrimer-drug conjugate or glucose dendrimer-drug conjugate to macrophages and foam macrophages within an atherosclerotic plaque. Claim 28 A method according to any one of claims 24 to 27, wherein the formulation selectively delivers the hydroxyl dendrimer-drug conjugate or glucose dendrimer-drug conjugate to adipocytes in the adipose tissue of the individual and / or brain microglia / macrophages in the hypothalamus. Claim 29 A method according to any one of claims 24 to 28, wherein the individual has type 2 diabetes, and the hydroxyl dendrimer-drug conjugate or glucose dendrimer-drug conjugate is administered in an amount effective for alleviating one or more symptoms of type 2 diabetes. Claim 30 A method according to any one of claims 24 to 29, wherein the individual is an obese individual, and the hydroxyl dendrimer-drug conjugate or glucose dendrimer-drug conjugate is administered to the obese individual in an amount that causes weight loss. Claim 31 A method according to claim 30, wherein the glucose dendrimer-drug conjugate is administered to an obese individual in an amount that causes weight loss. Claim 32 In paragraph 30 or 31, the above method is a method in which the reduction in fat mass is greater than the reduction in lean body mass. Claim 33 A method according to any one of paragraphs 30 to 32, wherein the method enables weight loss to be achieved without a notable decrease in appetite. Claim 34 A method according to any one of paragraphs 30 to 33, wherein the treatment converts white fat cells into brown fat cells. Claim 35 A method in which, in any one of paragraphs 30 to 34, the obese individual is a human. Claim 36 A method according to any one of claims 24 to 35, wherein the hydroxyl dendrimer-drug conjugate or glucose dendrimer-drug conjugate does not cause toxic symptoms. Claim 37 A method according to any one of claims 24 to 36, wherein the preparation is administered via a route selected from the group consisting of mucosal administration, enteral administration or injection, nasal, intravenous, oral, sublingual, subcutaneous, inhaled, transdermal, or peritoneal.

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