Dendrimer compositions and methods for delivering drugs to the eye

Abstract

Dendrimer compositions and methods for treating one or more ocular inflammatory and / or angiogenic diseases and / or conditions, including hydroxyl-terminated dendrimers complexed or conjugated to one or more active agents for treating or reducing one or more symptoms of an ocular disease, and / or for diagnosing an ocular disease and / or condition. The dendrimers can include one or more ethylenediamine core poly(amidoamine) (PAMAM) hydroxyl-terminated generation 4, 5, 6, 7, 8, 9, or 10 dendrimers. The active agent can be a VEGFR tyrosine kinase inhibitor, including sunitinib or an analog thereof. Preferably, the compositions are suitable for administration via a systemic route to target activated microglia / macrophages in the retina / choroid.

Description

[0001] Cross-references to related applications

[0002] This application claims the entire contents of U.S. Provisional Application No. 62 / 943,724, filed December 4, 2019; U.S. Provisional Application No. 63 / 021,023, filed May 6, 2020; and U.S. Provisional Application No. 63 / 108,234, filed October 30, 2020, all of which are incorporated herein by reference. Invention Field

[0003] This invention relates generally to the field of drug delivery, and more specifically to a method for selectively delivering drugs to activate immune cells in the intraocular and peripheral tissues. Background of the Invention

[0005] Neuroinflammatory changes in the retina are a crucial factor in the pathogenesis of various retinal diseases, including glaucoma, diabetic retinopathy, and age-related macular degeneration. Microglia are the main resident innate immune cells in the retina, and their physiological changes leading to abnormal immune responses are considered to drive various aspects of disease progression, including neuronal degeneration and pathological angiogenesis (Karlstetter et al., 2015; Silverman and Wong, 2018). Microglia are activated due to complex interactions between different retinal cell types and pathological pathways. Upon activation, microglia lose their ramified protrusions, proliferate, and rapidly migrate to the damaged area, resolving tissue damage. However, persistent tissue stress can induce microglia overreaction, leading to excessive production of pro-inflammatory mediators that favor retinal degenerative changes. A chronic pro-inflammatory environment is a hallmark of retinal degenerative diseases and neurological disorders affecting vision. Activation of retinal microglia also occurs in mouse models of ischemia / reperfusion injury (I / R), such as in inflammatory eye diseases including glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, and branch vein occlusion (BVO). Retinal vascular occlusion (whether due to high intraocular pressure in the I / R model or BVO thrombosis) leads to reduced intraocular blood flow, resulting in retinal ischemia. This causes neuronal death, triggering further activation of microglia.

[0006] Increased production of pro-inflammatory and angiogenic factors induces the formation and growth of new blood vessels from the choroid into the subretinal space, similar to the characteristics of exudative AMD in laser-induced CNV mouse models (Lambert V, et al., Nat. Protoc. 8, 2197-2211 (2013)). Various conditions, such as ischemia, hypoxia, or inflammation, can promote neovascularization. Pathological ocular angiogenesis, particularly retinal and choroidal angiogenesis, can lead to significant visual impairment. Diabetic retinopathy, neovascular age-related macular degeneration (AMD), retinopathy of prematurity, and retinal vascular occlusion are the main causes of angiogenesis-related vision loss.

[0007] Exudative (wet) AMD is characterized by serous or hemorrhagic detachments in the retinal pigment epithelium or neurosensory layer. Patients may develop choroidal neovascularization (CNV), manifested as fluid accumulation, hemorrhage, and / or lipid exudation. The earliest stages of diabetic retinopathy (DR) are characterized by retinal vascular abnormalities, including microaneurysms (cystic eversion of capillary walls), intraretinal hemorrhage, and cotton wool spots (neurofibrillar infarction). As the disease progresses, the gradual closure of retinal vessels leads to retinal ischemia, resulting in signs including venous abnormalities (beading, ringing), intraretinal microvascular abnormalities, and increased retinal hemorrhage and exudation. Based on the presence and extent of the above lesions, nonproliferative DR is classified as mild, moderate, severe, and very severe. Later stages of DR involve the formation of new vessels induced by retinal ischemia, with new vessels spreading from the optic disc (optic disc neovascularization, NVD) or from other parts of the retina (other retinal neovascularization, NVE). New vessels extending into the vitreous can lead to vitreous hemorrhage and tractional retinal detachment of contractile fibrous tissue.

[0008] To date, the only treatment definitively proven to have long-term benefit for DR is focal laser photocoagulation. The standard treatment for AMD patients is intravitreal injection of anti-VEGF to slow disease progression, and studies have shown that anti-VEGF therapy may be effective for diabetic macular edema (DME). However, there are currently no systemic treatment options for ischemic retinopathy or AMD. Systemic treatment options would reduce injection frequency due to retention in microglia and the ability to be administered systemically, thus avoiding the frequent intravitreal injections required for current anti-VEGF therapies.

[0009] Therefore, one object of the present invention is to provide compositions and methods for effectively treating one or more ocular inflammatory and / or angiogenic diseases, particularly DME, DR and AMD.

[0010] Another object of the present invention is to provide compositions and methods for targeted delivery of one or more active agents to diseased tissues / cells in the eye via systemic administration, which have increased efficacy and reduced side effects.

[0011] Another object of the present invention is to provide compositions and methods for targeted delivery of one or more active agents to activated microglia associated with one or more ocular inflammatory and / or angiogenic diseases.

[0012] Another object of the present invention is to provide compositions and methods for effectively inhibiting or reducing pro-inflammatory and / or angiogenic factors associated with one or more ocular inflammatory and / or angiogenic diseases. Invention Overview

[0014] Compositions and methods have been developed for selectively delivering one or more therapeutic, preventative, and / or diagnostic agents to treat and / or diagnose one or more ocular diseases and / or conditions. The compositions selectively deliver one or more therapeutic, preventative, and / or diagnostic agents to activated microglia to treat and / or diagnose diseased tissues / cells of the eye.

[0015] The composition comprises a hydroxyl-terminated dendritic macromolecule complexed, covalently conjugated, or intramolecularly dispersed or encapsulated with one or more receptor tyrosine kinase inhibitors, wherein the amount of said one or more receptor tyrosine kinase inhibitors effectively reduces the number or activity of activated microglia and macrophages in the retina and / or choroid of a subject in need. In some embodiments, the receptor tyrosine kinase inhibitor is an inhibitor of vascular endothelial growth factor receptor, such as sunitinib, sorafenib, pazopanib, vandetanib, axitinib, sidedinibub, vatalanib, dasatinib, nintedanib, motesanib, and analogues thereof. Preferably, the receptor tyrosine kinase inhibitor is sunitinib or an analogue thereof. In some embodiments, the diagnostic agent is a dye, such as a fluorescent dye, a near-infrared dye, a SPECT imaging agent, a PET imaging agent, and a radioisotope. Preferably, the diagnostic agent is the fluorescent dye indocyanine green (ICG).

[0016] In some embodiments, the dendritic macromolecule is a 4th, 5th, 6th, 7th, 8th, 9th, or 10th generation PAMAM dendritic macromolecule. In some embodiments, one or more therapeutic, prophylactic, and / or diagnostic agents are covalently conjugated to the dendritic macromolecule.

[0017] In some embodiments, the drug concentration of one or more therapeutic, preventive and / or diagnostic agents in the dendritic macromolecular conjugate is from about 0.01% by weight (w / w) to about 30% w / w, from about 1% w / w to about 25% w / w, from about 5% w / w to about 20% w / w, and from about 10% w / w to about 15% w / w.

[0018] In some embodiments, one or more spacer groups or linkers are incorporated between the dendritic macromolecule and the drug to provide a releasable (or cleavable) or non-releasable (or non-cleavable) form of the dendritic macromolecule-drug complex in vivo. In some embodiments, linkage occurs by providing a suitable spacer group with an ester bond between the reagent and the dendritic macromolecule. In some embodiments, linkage occurs by providing a suitable spacer group with an ether bond between the reagent and the dendritic macromolecule. In some embodiments, linkage occurs by providing a suitable spacer group with an amide bond between the drug and the dendritic macromolecule. In preferred embodiments, the spacer / linker group between one or more dendritic macromolecules and the drug is tailored to achieve desired and efficient release kinetics in vivo.

[0019] The composition is suitable for the treatment and / or diagnosis of one or more inflammatory and / or angiogenic eye diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, optic neuritis, uveitis, retinal detachment, temporal arteritis, retinal ischemia, arteriosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, diabetic retinopathy, macular edema, retinal neovascularization, and choroidal neovascularization.

[0020] A method for preparing a dendritic macromolecular composition is provided. Dosage forms and pharmaceutical preparations comprising an effective amount of the dendritic macromolecular composition for administration to subjects in need are also provided.

[0021] A method is described for treating and / or diagnosing one or more ocular diseases and / or conditions by administering an effective amount of the composition to a subject in need. The method is effective in treating and / or diagnosing one or more ocular diseases and / or conditions, including age-related macular degeneration (AMD), retinitis pigmentosa, optic neuritis, uveitis, retinal detachment, temporal arteritis, retinal ischemia, arteriosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, diabetic retinopathy, macular edema, retinal neovascularization, and choroidal neovascularization. In particular, the method is effective in treating and / or diagnosing one or more ocular diseases and / or conditions associated with activated microglia in the intraocular and peripheral tissues. Typically, the composition is administered in an amount that effectively targets damage to activated microglia, retinal pigment epithelium (RPE) cells, and / or choroidal neovascularization (CNV), and / or alleviates one or more symptoms of one or more ocular diseases and / or conditions.

[0022] Methods for administering the compositions and pharmaceutical preparations are also provided. Typically, the compositions and pharmaceutical preparations are administered via one or more systemic routes at frequencies of daily, weekly, bi-weekly, monthly, bi-monthly, or less. In some embodiments, the compositions and pharmaceutical preparations are administered via one or more systemic routes at frequencies of once every four weeks or less. In preferred embodiments, the compositions and pharmaceutical preparations are administered via intravenous, subcutaneous, or oral routes. Attached Figure Description

[0023] Figure 1A and 1B This is a schematic diagram showing the chemical reaction steps for synthesizing the dendritic macromolecule-sunitinib conjugate. Sunitinib is linked by hydroxymethyl bonds ( Figure 1A ) and amide linkage ( Figure 1B It is associated with dendritic macromolecules.

[0024] Figure 2A and 2B To display isolectin levels ( Figure 2A ) and IBA-1( Figure 2B The horizontal bar graphs show the signal area (μm) at 4 hours (4h) and 24 hours (24h) on days 1, 3, 7, and 14 after laser irradiation. 2 The samples were analyzed 4 or 24 hours after D-ICG irradiation using optical coherence tomography combined with ICG imaging. Figure 2C This is a bar graph showing the corrected total lesion fluorescence during the 28-day period following a single systemic administration of dendritic macromolecule-indocyanine green (D-ICG) 24 hours after local laser injury to choroidal neovascularization (CNV) lesions in C57BL / 6 mice.

[0025] Figure 3 This shows the average area (CNV) of neovascularization in the choroid of mice eyes (mm²). 2 The bar graph represents the results of treatments in mice that received the following: a vector, aflibercept, low-dose (low D-CSA) or high-dose (high D-CSA) cleavable sunitinib analog (D-CSA), low-dose (low D-NSA) or high-dose (high D-NSA) non-cleavable sunitinib analog (D-NSA), and free sunitinib administered 24 hours after laser-induced Bruch membrane rupture in the eyes of C57BL / 6 mice (n=8 / group except for the high D-NSA group, where n=6). p-values ​​are expressed as a comparison with the vector control.

[0026] Figure 4A This is a bar graph showing the mean area (CNV) (mm*) of choroidal neovascularization in the eyes of mice treated with free sunitinib, cleavable sunitinib analog (D-CSA), non-cleavable sunitinib analog (D-NSA), and aflibercept, administered on days 7 and 14 post-treatment, 24 hours after laser-induced Bruch's membrane rupture in the eyes of C57BL / 6 mice (n=8 / group). Figure 4B It is a line graph showing the plasma concentration (μg / ml) of dendritic macromolecular sunitinib analog conjugates over time from 0 to 72 hours.

[0027] Figure 5 This is a reaction scheme that demonstrates a synthetic strategy for N,N-didesethylsunitinib azide with amide linkages.

[0028] Figure 6A and 6B This demonstrates the steps involved in synthesizing dendritic macromolecules-dideethyl-sunitinamide-conjugates ( Figure 6B First, dendritic macromolecular hexynic acid-conjugates were synthesized. Figure 6A This diagram illustrates the chemical reaction steps for synthesizing an exemplary dendritic macromolecule-sunitinib conjugate. G4 PAMAM dendritic macromolecules are used as an example dendritic macromolecule.

[0029] Figure 7 This is a line graph showing the in vitro release profile (loss of linker group with AVT-4517 % w / w) of D-didesethylsunitinib conjugate (D-4517) over 15 days at 37°C, using esterase, to simulate plasma and intracellular conditions, respectively.

[0030] Figure 8This is a line graph showing the change in plasma concentration (μg / mL) over time in female C57 / B16 mice injected with IP at 5 or 50 mg / kg D-4517.

[0031] Figure 9A and 9B On the first day ( Figure 9A ) and day 14 ( Figure 9B Line graph showing the change in plasma concentration (μg / mL) over time (0–24 hours) in male and female Sprague-Dawley rats that received daily IP injections of 12 mg / kg D-4517 and daily oral administration of 30 mg / kg sunitinib (40.21 mg / kg sunitinib malate).

[0032] Figure 10 To show the average area (CNV) of choroidal neovascularization in mouse eyes (μm) 2 The bar graph shows the results of laser-induced Bruch membrane rupture in the eyes of C57BL / 6 mice on day 14 post-treatment, 24 hours after the mice were treated with aflibercept (40 μg) at three dose levels of D-didesethylsunitinib conjugate (D-4517) administered subcutaneously at single doses of 2, 10, and 50 mg / kg.

[0033] Figure 11 An exemplary synthetic scheme for a dendritic macromolecule-conjugate (D-4517.2) is shown, wherein N,N-didesethylsunitinib is conjugated with a dendritic macromolecule having an ether linkage to enhance in vivo stability.

[0034] Figure 12 This is a schematic diagram showing the chemical structure of compound D-4517.2.

[0035] Figure 13 The bar chart shows the percentage of drug release (0.0%–0.50%) of D-diethylsunitinib conjugate D-4517.2 at each time point (4, 24, and 48 hours) under human, mouse, and rat plasma conditions.

[0036] Figure 14 It is a synthetic scheme for dendritic macromolecule-N-acetyl-L-cysteine ​​methyl ester conjugate (dendritic macromolecule-NAC-carboxymethyl conjugate). Invention Details

[0038] I. Definition

[0039] The terms "active agent" or "bioactive agent" are interchangeable therapeutic, preventative, or diagnostic agents, referring to a chemical or biological compound that induces a desired pharmacological and / or physiological effect, which may be preventative, therapeutic, or diagnostic. These can be nucleic acids, nucleic acid analogs, small molecules with a molecular weight less than 2 kDa, more typically less than 1 kDa, peptide mimics, proteins or peptides, carbohydrates or sugars, lipids or surfactants, or combinations thereof. The term also includes pharmaceutically acceptable pharmacologically active derivatives of the active agent, including but not limited to salts, esters, amides, prodrugs, active metabolites, and analogs.

[0040] The term "pharmaceutically acceptable salt" is recognized in the art to include relatively non-toxic inorganic and organic acid addition salts of compounds. Examples of pharmaceutically acceptable salts include salts derived from inorganic acids, such as hydrochloric acid and sulfuric acid, and salts derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of suitable inorganic bases for forming salts include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc. Salts can also form with suitable organic bases, including non-toxic organic bases that are strong enough to form such salts. For illustrative purposes, such organic bases can include monoalkylamines, dialkylamines, and trialkylamines, such as methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines, such as mono-, di-, and triethanolamine; amino acids, such as arginine and lysine; guanidine; N-methylglucosamine; N-methylglucosamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenylethylamine;

[0041] The term "therapeutic agent" refers to an active agent that can be administered to treat one or more symptoms of a disease or condition.

[0042] The term "diagnostic agent" refers to an active reagent that can be administered to reveal, identify, and elucidate the localization of a pathological process. Diagnostic agents can label target cells, thereby allowing subsequent detection or imaging of these labeled target cells. In some embodiments, diagnostic agents can target / bind to activated microglia, activated macrophages, and / or RPE cells via dendritic macromolecules or suitable delivery mediators.

[0043] The term "preventive agent" refers to an active agent that can be administered to prevent disease or certain conditions.

[0044] The phrase "pharmaceutically acceptable" or "biocompatible" refers to compositions, polymers, and other materials and / or dosage forms that, to the extent of reasonable medical judgment, are suitable for use in contact with tissues of humans and animals without excessive toxicity, irritation, allergic reactions, or other problems or complications, and that are proportionate to a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, solvent, or encapsulating material, relating to the delivery or transfer of any test composition from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable," meaning it is compatible with the other components of the test composition and harmless to the patient.

[0045] The term "therapeutic effective amount" refers to the amount of a therapeutic agent that, when bound to and / or onto a dendritic macromolecule, produces some desired effect over a reasonable benefit / risk ratio applicable to any medical treatment. Effective amounts can vary depending on factors such as the disease or condition being treated, the specific target construct being administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art can determine the effective amount of a particular compound empirically without extensive experimentation. In some embodiments, the term "effective amount" refers to the amount of a therapeutic or preventative agent to reduce or alleviate symptoms of one or more ocular diseases or conditions, for example, by reducing inflammation through reducing or inhibiting one or more pro-inflammatory cytokines and / or cells associated with diseased tissue / cells in the eye. In the case of retinal and / or choroidal neovascularization, an effective amount of the drug may have the effect of reducing retinal and / or choroidal neovascularization; inhibiting vascular endothelial cell growth / proliferation to some extent; and / or alleviating one or more symptoms associated with the condition to some extent. Effective amounts may be administered once or multiple times.

[0046] In the context of inhibition, the term "inhibition" or "reduction" refers to a reduction or decrease in activity and quantity. This can be a complete inhibition or reduction of activity or quantity, or a partial inhibition or reduction.

[0047] Inhibition or reduction can be compared to control or standard levels. Inhibition can be 5, 10, 25, 50, 75, 80, 85, 90, 95, 99, or 100%. For example, a dendritic macromolecular composition containing one or more therapeutic agents can inhibit or reduce the activity and / or number of activated microglia and macrophages in the diseased retina and / or choroid of a subject by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% compared to the activity and / or number of the same cells in the equivalent diseased tissue of a subject who has not received or been treated with the dendritic macromolecular composition (i.e., without conjugated active agent). In some embodiments, inhibition and reduction are compared at the mRNA, protein, cellular, tissue, and organ levels. For example, inhibition and / or reduction of pro-inflammatory cytokines (such as TNF-α, interleukin-1β (IL-1β), or interferon-γ (IFN-γ)) secreted by activated microglia and macrophages in the diseased retina and / or choroid.

[0048] The terms "treatment" or "prevention" may refer to a disease, disorder, or condition occurring in animals susceptible to it but not yet diagnosed with it; to suppress a disease, disorder, or condition, such as by inhibiting its progression; and to alleviate said disease, disorder, or condition, such as by causing its remission. Treating a disease or condition includes improving at least one symptom of the specific disease or condition, even if the underlying pathophysiology is unaffected, for example, treating a subject's pain by administering an analgesic, even if the agent does not treat the cause of the pain. The expected effects of treatment include slowing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with an eye disease or disorder are reduced or eliminated, including but not limited to reducing the proliferation of pro-inflammatory cells, reducing symptoms caused by said disease, enhancing or restoring vision, reducing the degree and rate of vision loss, improving the quality of life of a person with said disease, reducing the dosage of other medications required to treat said disease, slowing the progression of said disease, and / or prolonging the individual's survival.

[0049] The term "biodegradable" refers to materials that, under physiological conditions, degrade or decay into smaller units or chemical substances that can be metabolized, eliminated, or excreted by the subject. Degradation time is a function of composition and form.

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

[0051] The term "functionalization" refers to modifying a compound or molecule in a way that results in the connection of functional groups or parts thereof. For example, a molecule can be functionalized by introducing molecules that make it a strong nucleophile or a strong electrophile.

[0052] The term "targeting portion" specifies a portion located at or away from a particular region. This portion can be, for example, a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule. The entity can be, for example, a therapeutic compound such as a small molecule, or a diagnostic entity such as a detectable marker. The region can be a tissue, a specific cell type or state of cell activation, or a subcellular compartment. In some embodiments, the targeting portion guides the localization of the active agent.

[0053] The term "extended residence time" refers to an increased time required for a drug to be cleared from a patient or their organs or tissues. In some embodiments, "extended residence time" means that the cleared drug is cleared with a half-life that is 10%, 20%, 50%, or 75% longer than a comparative standard (e.g., a comparable agent not conjugated with a delivery medium such as a dendritic macromolecule). In some embodiments, "extended residence time" means that the drug is cleared with a half-life that is 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, or 10000 times longer than a comparative standard (e.g., a comparable agent that does not specifically target dendritic macromolecules associated with specific cell types of tumors).

[0054] The terms "incorporated" and "encapsulated" refer to the incorporation, formulation, or other inclusion of an active agent into and / or onto a composition that allows the active agent to be released (e.g., sustained release) in a desired application. Active agents or other materials can be incorporated into dendritic macromolecules, including incorporation into one or more surface functional groups of such dendritic macromolecules (through covalent, ionic, or other binding interactions), physical mixing, encapsulating the reagent within a dendritic structure, or encapsulating it within a dendritic structure.

[0055] II. Composition

[0056] Dendritic macromolecular complexes suitable for delivering one or more active agents, particularly one or more active agents for the prevention, treatment or diagnosis of one or more eye diseases or disorders.

[0057] The composition of the dendritic macromolecular complex includes one or more preventative, therapeutic, and / or diagnostic agents encapsulated, associated, and / or conjugated within the dendritic macromolecular complex at weight concentrations of about 0.01% by weight (w / w) to about 30% w / w, about 1% w / w to about 25% w / w, about 5% w / w to about 20% w / w, and about 10% w / w to about 15% w / w. In some embodiments, the preventative, therapeutic, and / or diagnostic agents are covalently conjugated to the dendritic macromolecular complex via one or more linking bonds selected from disulfides, esters, ethers, thioesters, carbamates, carbonates, hydrazines, and amides, optionally via one or more spacer groups. Preferably, the hydroxyl groups of the hydroxyl-terminated dendritic macromolecular complex are covalently conjugated to one or more active agents via at least one ether linking bond, optionally via one or more linker / spacer groups. In a preferred embodiment, the surface groups of the hydroxyl-terminated dendritic macromolecular complex are modified by an etherification reaction prior to conjugation with one or more linker groups and active agents. When one or more linkers exist between the dendritic macromolecule and the surfactant, the covalent bond between the surface groups of the dendritic macromolecule and the linker is an ether bond. In other embodiments, at generation 3.5 of the dendritic macromolecule, an alkyne functional group is introduced using a polyethylene glycol (PEG) linker, which has an amine at one end and an alkyne at the other end, to generate a generation 4 bifunctional dendritic macromolecule. Exemplary bifunctional dendritic macromolecules in... Figure 11 It is shown as compound 1, which has 7 alkyne arms and 57 hydroxyl groups on its surface.

[0058] In some embodiments, the spacer group is a prophylactic agent, therapeutic agent, and / or diagnostic agent, such as sunitinib. Exemplary active agents include anti-angiogenic agents, anti-inflammatory drugs, and anti-infective agents.

[0059] The presence of additional reagents can affect the zeta potential or the surface charge of the particles. In one embodiment, the zeta potential of the dendritic macromolecules is between -100 mV and 100 mV, between -50 mV and 50 mV, between -25 mV and 25 mV, between -20 mV and 20 mV, between -10 mV and 10 mV, between -10 mV and 5 mV, between -5 mV and 5 mV, or between -2 mV and 2 mV. In a preferred embodiment, the surface charge is neutral or near neutral. The above ranges encompass all values ​​from -100 mV to 100 mV.

[0060] A. Dendritic macromolecules

[0061] Dendritic macromolecules are three-dimensional, hyperbranched, monodisperse, spherical, and multivalent macromolecules with high-density surface end groups (Tomalia, DA, et al., Biochemical Society Transactions, 35, 61 (2007); and Sharma, A., et al., ACS Macro Letters, 3, 1079 (2014)). Due to their unique structural and physical characteristics, dendritic macromolecules can be used as nanocarriers for a variety of biomedical applications, including targeted drug / gene delivery, imaging, and diagnostics (Sharma, A., et al., RSC Advances, 4, 19242 (2014); Caminade, A.-M., et al., Journal of Materials Chemistry B, 2, 4055 (2014); Esfand, R., et al., Drug Discovery Today, 6, 427 (2001); and Kannan, RM, et al., Journal of Internal Medicine, 276, 579 (2014)).

[0062] Dendritic macromolecules have significant effects on their biodistribution (Nance, E., et al., Biomaterials, 101, 96 (2016)). Compared with healthy controls, in a rabbit model of cerebral palsy (CP), hydroxyl-terminated fourth-generation PAMAM dendritic macromolecules (~4 nm in size) without any targeting ligands showed significantly more cross-linking with damaged BBB (>20-fold) and selectively targeted activated microglia and astrocytes when systemically administered, (Lesniak, WG, et al., MolPharm, 10 (2013)).

[0063] The term "dendritic macromolecule" refers to a molecular structure having a core and layers (or "generations") of repeating units connected to and extending from the core, each layer having one or more branching points, and an outer surface with terminal groups attached to the outermost layer. In some embodiments, dendritic macromolecules have a regular dendritic or "starburst" molecular structure.

[0064] Typically, dendritic macromolecules have a diameter of about 1 nm to about 50 nm, more preferably 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 between about 1 nm and about 2 nm. Conjugates are generally within the same size range, although large proteins such as antibodies may increase the size by 5-15 nm. Generally, for larger generations of dendritic macromolecules, the agent-to-dendritic macromolecule encapsulation ratio is 1:1 to 4:1. In a preferred embodiment, the diameter of the dendritic macromolecule allows for effective penetration of ocular tissue and prolonged retention in target cells.

[0065] In some embodiments, the molecular weight of the dendritic macromolecule is from about 500 Daltons to about 100,000 Daltons, preferably from about 500 Daltons to about 50,000 Daltons, and most preferably from about 1,000 Daltons to about 20,000 Daltons.

[0066] Suitable dendritic macromolecular scaffolds that can be used include poly(amidoamine), also known as PAMAM, or STARBURST. TM Dendritic macromolecules; polypropyleneamine (POPAM), polyethyleneimine, polylysine, polyester, iptycene, aliphatic poly(ether), and / or aromatic polyether dendritic macromolecules. The dendritic macromolecules may have carboxyl, amino, and / or hydroxyl terminals. In a preferred embodiment, the dendritic macromolecules have hydroxyl terminals. Each dendritic macromolecule in the dendritic macromolecule complex may have the same, similar, or different chemical properties as other dendritic macromolecules (e.g., the first dendritic macromolecule may include a PAMAM dendritic macromolecule, while the second dendritic macromolecule may be a POPAM dendritic macromolecule).

[0067] The term "PAMAM dendrimer" refers to a poly(amidoamine) dendrimer that may contain different cores, has an amidoamine structural module, and may have any generation of carboxylic acid, amine, and hydroxyl terminals, including but not limited to first-generation, second-generation, third-generation, fourth-generation, fifth-generation, sixth-generation, seventh-generation, eighth-generation, ninth-generation, or tenth-generation PAMAM dendrimers. In a preferred embodiment, the dendrimer is soluble in the formulation and is a fourth, fifth, or sixth-generation ("G") dendrimer. In a preferred embodiment, the dendrimer has multiple hydroxyl groups attached to its functional surface groups.

[0068] Methods for preparing dendritic macromolecules are known to those skilled in the art and typically involve a two-step iterative reaction sequence that generates concentric shells (generations) of dendritic β-alanine units around a central initiator core (e.g., an ethylenediamine core). Each subsequent growth step represents a new “generation” polymer with a larger molecular diameter, twice the number of reactive surface sites, and approximately twice the molecular weight of the previous generation. Suitable dendritic macromolecular scaffolds are available in multigenerational products. Preferably, the dendritic compounds are based on generation 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 dendritic scaffolds. These scaffolds have 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, and 4096 reactive sites, respectively. Thus, dendritic compounds based on these scaffolds have a corresponding number of combined targeting moieties and modulators.

[0069] In some embodiments, the dendritic macromolecule includes multiple hydroxyl groups. Some exemplary high-density hydroxyl-containing dendritic macromolecules include commercially available polyester dendritic polymers, such as hyperbranched 2,2-bis(hydroxy-methyl)propionic acid polyester polymers (e.g., hyperbranched bis-MPA polyester-64-hydroxy, fourth generation), and dendritic polyglycerol.

[0070] In some embodiments, the high-density hydroxyl-containing dendritic macromolecule is an oligoethylene glycol (OEG)-like dendritic macromolecule. For example, second-generation OEG dendritic macromolecules (D2-OH-60) can be synthesized using efficient, robust, and atom-economical chemical reactions, such as Cu(I)-catalyzed alkyne-azide click chemistry and photocatalyzed thiol-ene click chemistry. Very low-generation, highly dense polyol dendritic macromolecules with minimal reaction steps can be achieved using orthogonal supermonomers and supernuclear strategies, as described, for example, in WO2019094952. In some embodiments, the dendritic macromolecule backbone has non-breakable polyether bonds throughout the structure to prevent in vivo disintegration of the dendritic macromolecule and allow for the elimination from the body as a single entity of such a dendritic macromolecule (non-biodegradable).

[0071] In some embodiments, the dendritic macromolecule can specifically target specific tissue regions and / or cell types, preferably activated microglia and macrophages associated with one or more ocular diseases. In a preferred embodiment, the dendritic macromolecule can specifically target specific tissue regions and / or cell types without adding a targeting portion.

[0072] In a preferred embodiment, the dendritic macromolecule has multiple hydroxyl (-OH) groups on its surface. The preferred surface density of the hydroxyl (-OH) groups is at least 1 OH group / nm. 2(Number of hydroxyl surface groups / surface area in nm). For example, in some embodiments, the surface density of hydroxyl groups is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10; preferably at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more surface groups / surface area, in nm. 2 In a further embodiment, the surface density of hydroxyl (-OH) groups is between about 1 and about 50, preferably 5-20 OH groups / nm. 2 (Number of hydroxyl surface groups / in nm) 2 The surface area is measured in units of [surface area], while the molecular weight is between approximately 500 Daltons and approximately 10,000 Daltons. In a preferred embodiment, the percentage of free (i.e., unconjugated) hydroxyl groups in the total surface groups (conjugated and unconjugated) on the dendritic macromolecule is greater than 70%, 75%, 80%, 85%, 90%, 95%, and / or less than 100%. In the case of the 4th generation PAMAM dendritic macromolecule, the preferred number of free (i.e., unconjugated) hydroxyl groups in a total of 64 surface ends / groups is greater than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63. In a further embodiment, the hydroxyl-terminated dendritic macromolecule has an effective number of free hydroxyl groups for selectively targeting activated microglia, activated microphages, and / or RPE cells associated with one or more ocular diseases and / or conditions.

[0073] In some embodiments, the dendritic macromolecule may have a portion of hydroxyl groups exposed on its outer surface, while other hydroxyl groups are located in the core of the dendritic macromolecule. In a preferred embodiment, the volume density of hydroxyl (-OH) groups in the dendritic macromolecule is at least 1 OH group / nm. 3 (Number of hydroxyl groups / in nm) 3 (in units of volume). For example, in some embodiments, the volume density of hydroxyl groups is 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10, 15, 20, 25, 30, 35, 40, 45, and 50 hydroxyl groups / nm. 3 The volume is measured in units of hydroxyl groups. In some embodiments, the volume density of hydroxyl groups is approximately 4 to approximately 50 hydroxyl groups / nm. 3 Preferably about 5 to about 30 hydroxyl groups / nm 3 More preferably about 10 to about 20 hydroxyl groups / nm 3 .

[0074] B. Coupling agents and spacers

[0075] Dendritic macromolecular complexes can be formed from therapeutic agents or compounds conjugated or linked to dendritic macromolecules, dendritic polymers, or hyperbranched polymers. Optionally, the active agent is conjugated to the dendritic macromolecule via one or more spacer / linker groups, through various linkages such as disulfide, ester, carbonate, carbamate, thioester, hydrazine, acylhydrazine, and amide linkages. One or more spacer / linker groups between the dendritic macromolecule and the drug can be designed to provide a releaseable (or cleavable) or non-releaseable (or non-cleavable) form of the dendritic macromolecule-active complex in vivo. In some embodiments, the linkage occurs by providing a suitable spacer group with an ester bond between the agent and the dendritic macromolecule. In some embodiments, the linkage occurs by providing a suitable spacer group with an amide bond between the drug and the dendritic macromolecule. In a preferred embodiment, one or more spacer / linker groups are incorporated between the dendritic macromolecule and the drug to achieve desired and efficient release kinetics in vivo. In a further embodiment, the conjugation of the dendritic macromolecule and / or linker group does not significantly affect the activity of the active agent. For example, with regard to VEGFR TKR inhibitors, after conjugation with dendritic macromolecules, their binding affinity to one or more VEGFR TKR inhibitors remains at a level comparable to that of unconjugated VEGFR TKR inhibitors.

[0076] The term "spacer group" includes parts and compositions used to link a therapeutic active agent to a dendritic macromolecule. A spacer group can be a single chemical entity or two or more chemical entities linked together to bridge the dendritic macromolecule and the active agent. Spacer groups can include any small chemical entity, peptide, or polymer having mercapto, thiopyridine, succinimide, maleimide, vinyl sulfone, or carbonate end groups.

[0077] The spacer group can be selected from a class of compounds terminated with a mercapto, thiopyridine, succinimide, maleimide, vinyl sulfone, or carbonate group. The spacer group can include thiopyridine-terminated compounds, such as dithiodipyridine, N-succinimide-3-(2-pyridyldithio)-propionate (SPDP), succinimide-6-(3-[2-pyridyldithio]-propionamido)hexanoate LC-SPDP, or sulfonyl LC-SPDP. The spacer group may also include peptides, wherein the peptides are linear or cyclic peptides substantially having a thiol group, such as glutathione, homocysteine, cysteine ​​and its derivatives, arg-gly-asp-cys (RGDC), cyclic (Arg-G1y-Asp-d-Phe-Cys)(c(RGDfc)), cyclic (Arg-G1y-Asp-D-Tyr-Cys), and cyclic (Arg-Ala-Asp-d-Tyr-Cys). In some embodiments, the spacer group includes thiolic acid derivatives such as 3-mercaptopropionic acid, thioacetic acid, 4-mercaptobutyric acid, thiolan-2-one, 6-mercaptohexanoic acid, 5-mercaptovalerate, and other thiol derivatives such as 2-mercaptoethanol and 2-mercaptoethylamine. In some embodiments, the spacer group includes thiosalicylic acid and its derivatives, (4-succinimide-oxycarbonyl-methyl-α-2-pyridylthio)toluene, and (3-[2-pyridyldithio]propionylhydrazine). In some embodiments, the spacer group includes maleimide-terminated compounds, wherein the spacer group comprises a polymer or small chemical entity, such as bismaleimide diethylene glycol and bismaleimide triethylene glycol, bismaleimide ethane and bismaleimide hexane. In some embodiments, the spacer group includes vinyl sulfones, such as 1,6-hexane-bisvinyl sulfone. In some embodiments, the spacer group includes thioglycosides, such as thioglucose. In other embodiments, the spacer group includes reduced proteins, such as bovine serum albumin and human serum albumin, and any thiol-terminated compound capable of forming disulfide bonds. In a particular embodiment, the spacer group includes polyethylene glycol having maleimide, succinimide, and thiol-terminated groups.

[0078] The therapeutic active agent, imaging agent, and / or targeting moiety can be covalently linked, intramolecularly dispersed, or encapsulated. The dendritic macromolecule is preferably a first-generation (G1), G2, G3, G4, G5, G6, G7, G8, G9, or G10 PAMAM dendritic macromolecule having carboxyl, hydroxyl, or amine terminal groups. In a preferred embodiment, the dendritic macromolecule is linked to the active agent via a spacer group terminated with an ether or amide bond.

[0079] In some implementations, the non-released form of the dendritic macromolecule / active agent complex provides enhanced therapeutic efficacy compared to the releaseable or cleavable form of the same dendritic macromolecule / active agent complex.

[0080] Therefore, in some embodiments, one or more surfactants are conjugated to a dendritic macromolecule via spacer groups, said spacer groups being connected to the dendritic macromolecule in a non-releasable manner (e.g., via ether or amide bonds). In some embodiments, one or more surfactants are connected to the spacer groups in a non-releasable manner, for example, via ether or amide bonds. Therefore, in some embodiments, one or more surfactants are connected to the dendritic macromolecule via spacer groups connected to the dendritic macromolecule, and are connected to the surfactant in a non-releasable manner. In an exemplary embodiment, one or more surfactants are connected to the dendritic macromolecule via spacer groups, said spacer groups being connected to the dendritic macromolecule and the surfactant via amide and / or ether bonds. An exemplary spacer group is polyethylene glycol (PEG).

[0081] 1. Dendritic macromolecules are conjugated with surfactants via ether linkages.

[0082] In some embodiments, the composition comprises a hydroxyl-terminated dendritic macromolecule conjugated to an active agent via an ether linkage, optionally having one or more linker / spacer groups.

[0083] In a preferred embodiment, the covalent bonds (if conjugated without any connecting portion) between the surface groups and linkers of the dendritic macromolecule or between the dendritic macromolecule and the active agent are stable under in vivo conditions, i.e., minimally cleavable when administered to a subject and / or completely eliminated from the body. For example, in a preferred embodiment, less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or less than 0.1% of the total dendritic macromolecule complex containing the active agent cleaves within 24, 48, or 72 hours after in vivo administration. In one embodiment, the covalent bond is an ether bond. In a further preferred embodiment, the covalent bonds between the surface groups and linkers of the dendritic macromolecule, or between the dendritic macromolecule and the active agent (if without any connecting portion), are not hydrolyzable or enzymatically cleavable bonds, such as ester bonds.

[0084] In some embodiments, one or more hydroxyl groups of the hydroxyl-terminated dendritic macromolecule are conjugated with one or more linking moieties and one or more active agents via one or more ether bonds, as shown in formula (I).

[0085]

[0086] Where D is a G2 to G10 poly(amidoamine) (PAMAM) dendritic macromolecule; L is one or more linking moies or spacers; X is an active agent or its analogue; n is an integer from 1 to 100; m is an integer from 16 to 4096; and

[0087] Y is a linker selected from secondary amides (-CONH-), tertiary amides (-CONR-), sulfonamides (-S(O)2-NR-), secondary carbamates (-OCONH-; -NHCOO-), tertiary carbamates (-OCONR-; -NRCOO-), carbonates (-OC(O)-O-), ureas (-NHCONH-; -NRCONH-; -NHCONR-, -NRCONR-), methanol (-CHOH-, -CROH-), dithiols, hydrazones, hydrazides, and ethers (-O-), wherein R is an alkyl, aryl, or heterocyclic group. Preferably, Y is a bond or linkage that is minimally cleavable in vivo.

[0088] In some implementations, X is an inhibitor of vascular endothelial growth factor receptor (VEGFR) and / or TIE2 receptor tyrosine kinase.

[0089] In a preferred embodiment, Y is a secondary amide (-CONH-).

[0090] In one embodiment, D is a fourth-generation PAMAM dendritic macromolecule; L is one or more linking or spacer moieties; X is sunitinib or an analogue thereof; n is about 5-15; m is an integer about 49-59; where n+m=64.

[0091] In another embodiment, D is a fourth-generation PAMAM dendritic macromolecule; L is one or more linking or spacer moieties; X is N,N-didesethylsunitinib; Y is a secondary amide (-CONH-); n is about 5-15; m is an integer about 49-59; where n+m=64.

[0092] In one specific implementation, Formula I has the following structure (also known as D-4517.2):

[0093] Structure I: Chemical structure of D-4517.2

[0094]

[0095] C. Treatment, prevention, and diagnostic agents

[0096] The advantage of dendritic macromolecules is that multiple therapeutics, preventatives, and / or diagnostics can be delivered using the same dendritic macromolecule. In some embodiments, one or more types of active agents are encapsulated, compounded, or conjugated to the dendritic macromolecule. In another embodiment, the dendritic macromolecule is covalently linked to at least one detectable moiety, the amount of which is effective for detecting one or more diseased or damaged cells / tissues in a subject. In specific embodiments, the dendritic macromolecule composition has multiple agents compounded or conjugated with the dendritic macromolecule, such as immunotherapeutic agents, antiepileptic agents, steroids that reduce swelling, antibiotics, anti-angiogenic agents, and / or diagnostics. In some embodiments, the dendritic macromolecule is compounded or conjugated with two or more different classes of active agents to provide simultaneous delivery at the target site with different or independent release kinetics. For example, sunitinib and anti-inflammatory agents can both be conjugated to the same dendritic macromolecule for delivery to target cells / tissues. In another embodiment, dendritic macromolecule complexes, each carrying different types of active agents, are simultaneously administered for combination therapy. In some implementations, there are one or more therapeutic agents that target the underlying cause of a disease or condition, and one or more therapeutic agents that relieve one or more symptoms of the disease or condition.

[0097] Suitable active agents include therapeutic agents, diagnostic agents, and / or preventative agents. The agent can be a biomolecule, such as an enzyme, protein, peptide, or nucleic acid, or a small molecule agent (e.g., molecular weight less than 2000 Daltons, preferably less than 1500 Daltons, more preferably 300-700 Daltons), including organic, inorganic, and organometallic agents. The agent can be encapsulated within a dendritic macromolecule, dispersed within a dendritic macromolecule, and / or covalently or non-covalently bound to the surface of the dendritic macromolecule. Exemplary therapeutic agents include anti-inflammatory drugs, anti-angiogenic agents, antioxidants, vasodilators, neuroactive agents, neuroprotective agents, and anti-infective agents. In some embodiments, the dendritic macromolecule is linked to the target moiety, imaging agent, and / or therapeutic agent via a linker or spacer ending in a disulfide bond, ester bond, ether bond, thioester bond, carbamate bond, carbonate bond, hydrazine bond, or amide bond.

[0098] Dendritic macromolecules can be used to deliver one or more additional active agents, particularly one or more active agents to prevent or treat symptoms of one or more eye diseases. Exemplary therapeutic agents administered with dendritic macromolecules include tyrosine kinase inhibitors, such as VEGFR tyrosine kinase inhibitors. In a preferred embodiment, the drug is a small molecule tyrosine kinase inhibitor.

[0099] Representative anti-angiogenic drugs include, but are not limited to, vascular endothelial growth factor (VEGF) antibodies, such as bevacizumab. and rhuFAb V2 (ranibizumab, ), and other anti-VEGF compounds, including aflibercept. (pegaptanim sodium, anti-VEGF aptamer or EYE001) (Eyetech Pharmaceuticals); pigment epithelium-derived factor (PEDF); COX-2 inhibitors, such as celecoxib and rofecoxib Interferon-alpha; Interleukin-12 (IL-12); Thalidomide and its derivatives such as lenalidomide Squalamine; endostatin; angiostatin; ribozyme inhibitors, such as (Sirna Therpeutics); Multifunctional anti-angiogenic agents, such as (AE-941) (Aeterna Laboratories, Quebec City, Canada); receptor tyrosine kinase (RTK) inhibitors, such as sunitinib Tyrosine kinase inhibitors, such as sorafenib and erlotinib Antibodies targeting the epidermal growth factor receptor, such as panitumumab cetuximab And other anti-angiogenic agents known in the art.

[0100] Other active agents suitable for anti-angiogenic therapy include those targeting the platelet-derived growth factor family, epidermal growth factor family, fibroblast growth factor family, transforming growth factor-β superfamily (TGF-β1, activin, follicle-stabilizing hormone, and bone morphogenetic protein), angiopoietin-like family, galactagogue family, integrin superfamily, as well as active agents targeting pigment epithelial-derived factor, hepatocyte growth factor, angiopoietin, endothelin, hypoxia-inducible factor, insulin-like growth factor, cytokines, matrix metalloproteinases and their inhibitors, and glycosylated proteins.

[0101] Tyrosine kinase inhibitors

[0102] In some implementations, the dendritic macromolecule is complexed or conjugated with one or more tyrosine kinase inhibitors.

[0103] Tyrosine kinases are important cell signaling proteins with various biological activities, including cell proliferation and migration. Angiogenesis involves multiple kinases, including receptor tyrosine kinases such as vascular endothelial growth factor receptor (VEGFR). Clinically developed anti-angiogenic tyrosine kinase inhibitors primarily target VEGFR-1, -2, -3, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), PDGFR-β, KIT, fms-associated tyrosine kinase 3 (FLT3), colony-stimulating factor-1 receptor (CSF-1R), Raf, and RET.

[0104] VEGFR inhibitors

[0105] In some implementations, dendritic macromolecules are complexed or conjugated with one or more VEGFR tyrosine kinase inhibitors. The VEGFR family includes three associated receptor tyrosine kinases, known as VEGFR-1, -2, and -3, which mediate the angiogenesis of VEGF ligands (Hicklin DJ, Ellis LM. J Clin Oncol. (2005), 23(5):1011-27). The VEGF family encoded in the mammalian genome includes five members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). VEGF is an important stimulator of endothelial cell proliferation and migration. Increased expression of the angiogenic factor VEGF-A promotes three common age-related eye diseases—"wet" and "dry" age-related macular degeneration and degeneration and cataracts in animal models (Marneros AG, EMBO Molecular Medicine, 2016; 8(3):208). Therefore, in some embodiments, the dendritic macromolecule is conjugated with one or more active agents that effectively reduce the amount and / or activity of one or more VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PIGF).

[0106] The most well-known angiogenesis inhibitors target the vascular endothelial growth factor signaling pathway, such as the monoclonal antibody bevacizumab (…). Genentech / Roche) and two kinase inhibitors, sunitinib (SU11248), Pfizer) and sorafenib (BAY43-9006, Bevacizumab was the first clinically approved angiogenesis inhibitor, initially used to treat colorectal cancer and more recently for breast and lung cancer. Another clinically available anti-VEGF drug is piperatanib sodium, an aptamer for neovascular AMD. Unlike bevacizumab, which binds to all VEGF isoforms, piperatanib targets only VEGF165, the isoform responsible for pathological ocular neovascularization. In some embodiments, the dendritic macromolecule is conjugated to one or more VEGF inhibitors, including bevacizumab and piperatanib sodium.

[0107] Small molecule tyrosine kinase inhibitors sunitinib and sorafenib target the VEGF receptor (VEGFR), primarily VEGFR-2. Both drugs have shown benefit in patients with renal cell carcinoma (Motzer Motzer RJ, Bukowski RM, J Clin Oneal. (2006); 24(35):5601-8). Sunitinib is a potent angiogenesis inhibitor; rabbit corneal neovascularization models have shown that topical sunitinib is almost three times more effective than bevacizumab (Pérez-Santonja JJ et al., Am J Ophthalmol. 2010 Oct; 150(4):519-528). Sorafenib inhibits Raf serine kinase. Sildenafil is an oral tyrosine kinase inhibitor of the VEGF receptor (VEGFR).

[0108] In some implementations, the dendritic macromolecule is conjugated to one or more VEGF receptor inhibitors, including sunitinib (SU11248). Sorafenib (BAY439006) ), Pazopanib (GW786034); ), Vandetanil (ZD 6474; Axitinib (AG013736), Sildenafil (AZD2171); Vatalanib (PTK787; ZK222584), dasatinib, nintedanib, and motracene (AMG706). In a preferred embodiment, the vascular endothelial growth factor receptor inhibitor can be functionalized with one or more spacer / linker groups, for example, with ether, ester, or amide linkages, to facilitate conjugation to dendritic macromolecules and / or for desired release kinetics. For example, sunitinib can be modified to have ester or amide linkages. Figure 1A and 1B ). Figure 1A (via hydroxymethyl linkage) and Figure 1B(Through amide linkages) illustrates an exemplary conjugation of a VEGF receptor inhibitor (e.g., sunitinib) to a dendritic macromolecule. In a preferred embodiment, the conjugation of the dendritic macromolecule and / or one or more linkers does not significantly affect the activity of the active agent. In a further preferred embodiment, the VEGF receptor inhibitor is conjugated to a dendritic macromolecule, with or without spacer groups, in a manner that minimizes the reduction in inhibition (e.g., less than 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, and 100-fold). For example, in the case of sunitinib, after conjugation to a dendritic macromolecule, its binding affinity to one or more VEGFR TKR proteins remains at a level comparable to that of unconjugated sunitinib.

[0109] Other VEGF receptor inhibitors with functional spacer groups / connecting bonds are shown below.

[0110] Structure II ab: Chemical structure of sorafenib analogues

[0111]

[0112] Structure III ad: Chemical structure of nintedanib analog 1

[0113]

[0114] Structure IV: Chemical structure of Orantinib analogues

[0115]

[0116] In some implementations, a dendritic macromolecular complex comprising one or more VEGF receptor inhibitors is administered in doses that effectively reduce or inhibit endothelial cell angiogenesis and / or vascular endothelial cell proliferation, reduce retinal and / or choroidal angiogenesis, and / or alleviate one or more symptoms associated with an eye disease or condition.

[0117] TIE II antagonists

[0118] In some embodiments, the dendritic macromolecule is conjugated or conjugated with one or more TIE II inhibitors. Angiopoietin-1 receptor, also known as CD202B (differentiation cluster 202B) and TIE II, is a human protein encoded by the TEK gene. TIE2 is an angiopoietin receptor. Angiopoietin is a protein growth factor required for angiogenesis (vascularization) and supports tumor growth and development. Therefore, in some embodiments, the dendritic macromolecule is conjugated with one or more TIE II antagonists.

[0119] In some embodiments, the active agent is an inhibitor of TIE II receptor tyrosine kinase. Examples of VEGFR / TIE II inhibitors include CEP-11981 and rebastinib. TIE II antagonists can be functionalized, for example with ether, ester, ethyl, or amide linkages, to facilitate conjugation to dendritic macromolecules and / or for desired release kinetics. The chemical structure of an exemplary TIE II antagonist is shown in Structure XXI. The dissociation constant K for the TIE II inhibitory effect of the free TIE II antagonist (structure V) is also shown. d The dissociation constant K of the TIE II inhibitory effect of a dendritic macromolecular conjugated TIE II antagonist (structure XXI) is approximately 8.8 nm. d The wavelength is approximately 25 nm. Therefore, in a preferred embodiment, the TIE II antagonist is conjugated to a dendritic macromolecule with or without spacer groups in a manner that minimizes the reduction in TIE II inhibition (e.g., less than 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, and 100 times). In a preferred embodiment, the active agent is an inhibitor of vascular endothelial growth factor receptor (VEGFR) and TIE II receptor tyrosine kinase.

[0120] Structure V: TIE II antagonist 1

[0121]

[0122] anti-inflammatory agents

[0123] In some embodiments, one or more active agents associated with or complexed with dendritic macromolecules are one or more anti-inflammatory agents. Anti-inflammatory agents can reduce inflammation and include steroid and non-steroidal drugs. Suitable steroid active agents include glucocorticoids, progestins, mineralocorticoids, and corticosteroids. In some embodiments, one or more active agents are one or more corticosteroids.

[0124] Exemplary anti-inflammatory agents include triamcinolone, fluocinolone, methylprednisolone, prednisolone, dexamethasone, clotiprednisolone, fluocinolone, ibuprofen, aspirin, and naproxen. Exemplary immunomodulatory agents include cyclosporine, tacrolimus, and rapamycin. Examples of nonsteroidal anti-inflammatory drugs (NSAIDs) include mefenamic acid, aspirin, diflunisal, salicylsalicylic acid, ibuprofen, naproxen, fenprofen, ketoprofen, dextroketoprofen, flurbiprofen, oxaprazin, loxoprofen, indomethacin, sulindac, etodoxacin, ketoroxylic acid, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, itoxicam, meclofenac, flufenamic acid, tolmfenac, elecoxib, rofecoxib, vardicoxib, parecoxib, romecoxib, etodoxacin, firocoxib, sulphonanilides, nimesulide, niflufenic acid, and ricoxone. In preferred embodiments, the active agent is triamcinolone, prednisolone, dexamethasone, or an analogue thereof. Exemplary analogues of triamcinolone, prednisolone, and dexamethasone are shown below.

[0125]

[0126] In some embodiments, the active agent is N-acetyl-L-cysteine ​​or a derivative or analogue thereof, or a prodrug. In a preferred embodiment, N-acetyl-L-cysteine ​​is conjugated to a hydroxyl-terminated PAMAM dendrimer via a non-cleavable linker bond to minimize the release of free N-acetyl-cysteine ​​after in vivo administration. Exemplary synthetic routes for non-release (or non-cleavable) forms of dendrimer / N-acetyl-cysteine ​​complexes are as follows: Figure 14 As shown. In one embodiment, the dendritic macromolecular complex is a dendritic macromolecular-NAC-carboxymethylated conjugate, such as... Figure 14 As shown, the non-released form of the dendritic macromolecule / N-acetyl-cysteine ​​complex (e.g., N-acetyl-L-cysteine ​​conjugated to a hydroxyl-terminated PAMAM dendritic macromolecule via an ester linker) provides enhanced therapeutic efficacy compared to the releasable or cleavable forms of the dendritic macromolecule / N-acetyl-cysteine ​​complex.

[0127] In some embodiments, one or more active agents are polysialic acid (e.g., low molecular weight polysialic acid (polySia avDP20) with an average degree of polymerization of 20), transporter ligands (e.g., diazepam binding inhibitors (DBI)), interferon-β (IFN-β), and minocycline.

[0128] In some cases, one or more active agents are anti-infective drugs. Exemplary anti-infective agents include antiviral agents, antibacterial agents, antiparasitic agents, and antifungal agents. Exemplary antibiotics include moxifloxacin, ciprofloxacin, erythromycin, levofloxacin, cefazolin, vancomycin, tigecycline, gentamicin, tobramycin, ceftazidime, ofloxacin, and gatifloxacin; antifungal drugs include amphotericin B, voriconazole, and natamycin.

[0129] Diagnostic reagents

[0130] Dendritic macromolecular nanoparticles may include diagnostic agents for determining the location of drug delivery particles. These drugs may also be used for prevention. In some embodiments, the dendritic macromolecules are conjugated with one or more diagnostic agents, including indocyanine green, fluorescein (e.g., fluorescein isocyanate), boron-dipyrrole methylene, rhodamine, and Bengal rose red. In a preferred embodiment, the diagnostic agent is indocyanine green as shown below:

[0131] Structure VII: Chemical structure of indocyanine green

[0132]

[0133] Other examples of diagnostic reagents include paramagnetic molecules, fluorescent compounds, magnetic molecules and radionuclides, X-ray imaging reagents, and contrast agents. Other suitable examples of contrast agents include radiation-impermeable gases or gas-emitting compounds. Dendritic macromolecular complexes may also include reagents for determining the location of the drug delivery composition. Reagents used for this purpose include fluorescent tags, radionuclides, and contrast agents.

[0134] Exemplary diagnostic agents include dyes, such as fluorescent dyes and near-infrared dyes, SPECT imaging agents, PET imaging agents, and radioisotopes. Representative dyes include carbocyanine dyes, indole carbocyanine dyes, oxocarbocyanine dyes, thüicarbocyanine and cyanine, polymethine dyes, coumarin dyes, rhodamine dyes, xanthracene dyes, fluorescein dyes, boron dipyrrolemethane dyes (BODIPY dyes), Cy5 dyes, Cy5.5 dyes, Cy7 dyes, VivoTag-680 dyes, VivoTag-S680 dyes, VivoTag-S750 dyes, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, DyLight647, HiLyteFluor 647, HiLyte Fluor 680, HiLyte Fluor 750, and IRDye. 800CW, IRDye 800RS, IRDye700DX, ADS780WS, ADS830WS and ADS832WS.

[0135] Examples of SPECT or PET imaging agents include chelating agents such as diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diamine dithiol, activated mercaptoacetyl-glycyl-glycine (MAG3), and hydrazinotinamide (HYNIC).

[0136] Exemplary isotopes include Tc-94m, Tc-99m, In-111, Ga-67, Ga-68, Gd3+, Y-86, Y-90, Lu-177, Re-186, Re-188, Cu-64, Cu-67, Co-55, Co-57, F-18, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, and Dy-166.

[0137] In a preferred embodiment, the dendritic macromolecular complex comprises one or more radioactive isotopes suitable for positron emission tomography (PET) imaging. Exemplary positron-emitting radioactive isotopes include carbon-11 (… 11 C), Copper-64 ( 64 Cu), nitrogen-13 ( 13 N), Oxygen-15 ( 15 O), gallium-68( 68 Ga) and fluorine-18 ( 18F), for example, 2-deoxy-2- 18 F-fluoro-β-D-glucose (F-fluoro-β-D-glucose) 18 F-FDG).

[0138] In a preferred embodiment, one or more diagnostic agents may be functionalized with one or more spacer / linker groups, for example with ether, ester or amide linkages, to facilitate conjugation to dendritic macromolecules and / or for desired release kinetics.

[0139] In a further embodiment, the single dendritic macromolecular complex composition can simultaneously treat and / or diagnose diseases or conditions in one or more sites in the body.

[0140] III. Pharmaceutical Preparations

[0141] Pharmaceutical compositions comprising one or more dendritic macromolecular complexes can be formulated in a conventional manner using one or more physiologically acceptable carriers, said carriers comprising excipients and adjuvants that facilitate the processing of the active compound into a pharmaceutically usable formulation. The appropriate formulation depends on the chosen route of administration. Pharmaceutical formulations contain one or more dendritic macromolecular complexes in combination with one or more pharmaceutically acceptable excipients. Representative excipients include solvents, diluents, pH adjusters, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tension agents, stabilizers, and combinations thereof. Suitable pharmaceutically acceptable excipients are preferably selected from materials generally considered to be safe (GRAS) and capable of being administered to an individual without causing undesirable biological side effects or undesirable interactions.

[0142] In a preferred embodiment, the composition is formulated for parenteral delivery to the eye. In some embodiments, the composition is formulated for subcutaneous or intravitreal injection. Typically, the composition is formulated in sterile saline or a buffer solution for injection into the tissue or cells to be treated. The composition may be lyophilized and stored in disposable vials for immediate rehydration prior to use. Other methods for rehydration and administration are known to those skilled in the art.

[0143] Remington's Pharmaceutical Sciences, 20th edition, Lippincott Williams & Wilkins, Baltimore, MD, 2000, page 704, provides suitable formulations and examples of ophthalmic drugs administered in pharmaceutically acceptable salt forms, including timolol maleate, brimonidine tartrate, and diclofenac sodium.

[0144] For ease of administration and uniform dosage, the composition is preferably formulated in unit dose form. The phrase "unit dose form" refers to a physically discrete unit of the conjugate suitable for the patient to be treated. However, it should be understood that the total single dose of the composition will be determined by the attending physician within reasonable medical judgment. The therapeutically effective dose can be initially estimated in cell culture experiments or animal models (typically mice, rabbits, dogs, or pigs). This animal model is also used to obtain the desired concentration range and route of administration. Such information should then be used to determine the effective dose and route of administration to humans. The therapeutic efficacy and toxicity of the conjugate can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals, such as ED50 (the dose that is therapeutically effective in 50% of the population) and LD50 (the dose that is lethal to 50% of the population). The dose ratio of toxicity to therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions exhibiting a large therapeutic index are preferred. Data obtained from cell culture experiments and animal studies can be used to determine the dose range for human use.

[0145] Pharmaceutical compositions formulated for administration via parenteral (intramuscular, intraperitoneal, intravenous, or subcutaneous) and enteral routes are described. In a preferred embodiment, the composition is administered systemically. In one embodiment, the composition is administered subcutaneously. In another embodiment, the composition is administered orally.

[0146] A. Parenteral administration

[0147] The phrases “parenteral administration” and “via parenteral administration” are recognized terms in the art that encompass administration routes other than enteral and local administration, such as injection, and include, but are not limited to, intravenous, intramuscular, intrapleural, intravascular, intracardiac, intraarterial, intrasheath, intracapsular, intracardiac, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, spinal, and intrasternal injections and infusions. In some embodiments, the dendritic macromolecule is administered parenterally, for example via subdural, intravenous, intrasheath, intravenous, intraarterial, intraamniotic, intraperitoneal, or subcutaneous routes. In a preferred embodiment, the dendritic macromolecule composition is administered via subcutaneous injection.

[0148] For liquid formulations, pharmaceutically acceptable carriers can be, for example, aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Parenteral carriers include, for example, sodium chloride solutions, Ringer's glucose, glucose and sodium chloride, lactated Ringer's reagent, and fixed oils. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcohol / aqueous solutions, cyclodextrins, emulsions, or suspensions, including saline and buffer media. Dendritic macromolecules can also be administered in emulsion form, such as water-in-oil. Examples of oils are petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, cod liver oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineral oil. Fatty acids suitable for parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0149] Preparations suitable for parenteral administration may include antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Intravenous excipients may include liquid and nutritional supplements, and electrolyte supplements (such as Ringer's glucose-based supplements). Generally, water, saline, aqueous glucose solutions, and related sugar solutions, as well as glycols such as propylene glycol or polyethylene glycol, are preferred liquid carriers, especially for injectable solutions.

[0150] Injectable drug carriers are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, JBLippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pp. 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 15th edition, pp. 622-630 (2009)).

[0151] B. Enteral administration

[0152] In some embodiments, the composition is formulated for oral administration. The carrier or diluent may be a solid carrier, such as a capsule or tablet, or a diluent for solid dosage forms, a liquid carrier, or a diluent for liquid dosage forms, or a mixture thereof.

[0153] For liquid formulations, pharmaceutically acceptable carriers can be, for example, aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcohol / aqueous solutions, cyclodextrins, emulsions, or suspensions, including saline solutions and buffer media.

[0154] Examples of oils are petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, cod liver oil, sesame oil, cottonseed oil, corn oil, petrolatum, and mineral oil. Suitable fatty acids for parenteral preparations include, for example, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0155] Excipients include, for example, sodium chloride solution, Ringer's glucose, glucose and sodium chloride, lactated Ringer's reagent, and fixative oil. Formulations include, for example, aqueous and non-aqueous isotonic sterile injectable solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Carriers may include, for example, liquid and nutritional supplements, electrolyte supplements, such as those based on Ringer's glucose. Generally, water, saline, aqueous glucose solutions, and related sugar solutions are preferred liquid carriers. They can also be formulated with proteins, fats, carbohydrates, and other ingredients of infant formula.

[0156] In a preferred embodiment, the composition is formulated for oral administration. The oral formulation may be in the form of chewing gum, gum strips, tablets, capsules, or lozenges. Encapsulating materials used to prepare enteric-coated oral formulations include cellulose acetate, polyphthalate acetate, hydroxypropyl methylcellulose phthalate, and methacrylate copolymers. Solid oral formulations such as capsules or tablets are preferred. Elixirs and syrups are also well-known oral formulations.

[0157] IV. Production Method

[0158] A. Methods for preparing dendritic macromolecules

[0159] Dendritic macromolecules can be prepared through a variety of chemical reaction steps. They are typically synthesized using methods that allow control over their structure at each stage of construction. Dendritic structures are synthesized primarily through two distinct pathways: divergent or convergent.

[0160] In some embodiments, a divergent approach is used to prepare dendritic macromolecules, wherein the dendritic macromolecules are assembled from a multifunctional core that extends outward through a series of reactions (typically the Michael reaction). This strategy involves coupling monomer molecules with reactive and protective groups to the multifunctional core moiety, thereby progressively increasing generations around the core, followed by removal of the protective groups. For example, the PAMAM-NH2 dendritic macromolecule is synthesized first by coupling an N-(2-aminoethyl)acrylamide monomer to an ammonia core.

[0161] In other embodiments, a convergent process is used to prepare dendritic macromolecules, wherein the dendritic macromolecules are constructed from small molecules that terminate at the surface of the spheres and the reaction proceeds inward, building inward and eventually attaching to the core.

[0162] There are many other synthetic routes for the preparation of dendritic macromolecules, such as orthogonal method, accelerated method, two-stage convergence method or supernuclear method, supermonomer method or branched monomer method, double exponential method; orthogonal coupling method or two-step method, two-monomer method, AB2-CD2 method.

[0163] In some embodiments, the core, one or more branching units, one or more linker / spacer groups, and / or one or more surface groups of the dendritic macromolecule can be modified by click chemistry to allow conjugation with additional functional groups (branching units, linker / spacer groups, surface groups, etc.), monomers, and / or surfactants, using one or more copper-assisted azide-alkyne cycloadditions (CuAAC), Diels-Alder reactions, thiol-ene and thiol-alkyne reactions, and azide-alkyne reactions (Arseneault M et al., Molecules. 2015 May 20; 20(5):9263-94). In some embodiments, pre-prepared dendrites are clicked onto a high-density hydroxyl polymer. "Click chemistry" refers to the coupling of, for example, two distinct parts (e.g., a core group and a branching unit; or a branching unit and a surface group) through a 1,3-dipolar addition reaction between an alkyne portion (or its equivalent) on the surface of the first part and an azide portion (e.g., present on a triazine composition) or its equivalent (or any active end group, such as a primary amine end group, a hydroxyl end group, a carboxylic acid end group, a thiol end group, etc.) on the second part.

[0164] In some embodiments, the synthesis of dendritic macromolecules responds to one or more reactions selected from thiol-ene click reactions, thiol-alkyne click reactions, CuAAC, Diels-Alder click reactions, azide-alkyne click reactions, Michael addition, epoxide ring opening, esterification, silane chemistry, and combinations thereof.

[0165] Any existing dendritic macromolecule platform can be used to prepare dendritic macromolecules with desired functionality, i.e., high-density surface hydroxyl groups by conjugating highly hydroxyl-containing moieties, such as 1-thioglycerol or pentylenetetrol. Exemplary dendritic platforms such as polyamide-amine (PAMAM), polypropyleneimine (PPI), poly-L-lysine, melamine, polyether hydroxylamine (PEHAM), polyesteramine (PEA), and polyglycerol can be synthesized and developed.

[0166] Furthermore, suitable dendritic macromolecules can be prepared by combining two or more dendrites. A dendrite is a wedge-shaped cross-section of a dendritic macromolecule with reactive focal functional groups. Many dendritic scaffolds are commercially available. They are of generations 1, 2, 3, 4, 5, and 6, with 2, 4, 8, 16, 32, and 64 reactive groups, respectively. In some embodiments, one type of surfactant is linked to one type of dendrite, while a different type of surfactant is linked to another type of dendrite. The two dendrites are then linked together to form a dendritic macromolecule. These two dendritic macromolecules can be linked by click chemistry, i.e., a 1,3-dipolar cycloaddition reaction between the azide moiety on one dendritic macromolecule and the alkyne moiety on the other dendritic macromolecule, forming a triazole linker.

[0167] Exemplary methods for preparing dendritic macromolecules are described in detail in International Patent Publications WO2009 / 046446, WO2015168347, WO2016025745, WO2016025741, WO2019094952 and U.S. Patent No. 8,889,101.

[0168] B. Dendritic macromolecular complexes

[0169] Dendritic macromolecular complexes can be formed from therapeutic agents or compounds conjugated or linked to dendritic macromolecules, dendritic polymers, or hyperbranched polymers. Techniques for conjugating one or more active agents to dendritic macromolecules are known in the art and are described in detail in U.S. Publications US 2011 / 0034422, US 2012 / 0003155, and US 2013 / 0136697.

[0170] In some embodiments, one or more active agents are covalently linked to a dendritic macromolecule. In some embodiments, the active agent is linked to the dendritic macromolecule via a linker designed to cleave in vivo. The linker can be designed to be hydrolyzable, enzymatically cleavable, or a combination thereof, thereby providing sustained release of the active drug in vivo. The composition of the linker and its connection point with the active agent are selected such that cleavage of the linker releases the active agent or a suitable prodrug. The composition of the linker can also be selected based on the desired release rate of the active agent.

[0171] In some embodiments, the linkage occurs via one or more of disulfide, ester, ether, thioester, carbamate, carbonate, hydrazine, or amide linkages. In preferred embodiments, the linkage occurs by providing an appropriate spacer group between the drug and the dendritic macromolecule, depending on the desired release kinetics of the active pharmaceutical ingredient. In some cases, an ester bond is introduced for a cleavable form of the active pharmaceutical ingredient. In other cases, an amide bond is introduced for a non-cleavable form of the active agent. Exemplary synthetic routes are described in Examples 4 and 5 to demonstrate the introduction of a non-cleavable linkage between the active agent and the dendritic macromolecule.

[0172] The linker typically includes one or more organic functional groups. Examples of suitable organic functional groups include secondary amides (-CONH-), tertiary amides (-CONR-), sulfonamides (-S(O)2-NR-), secondary carbamates (-OCONH-; -NHCOO-), tertiary carbamates (OCONR-; -NRCOO-), carbonates (-OC(O)-O-), ureas (-NHCONH-; -NRCONH-; -NHCONR-, -NRCONR-), methanol (-CHOH-, -CROH-), dithiols, hydrazones, hydrazides, ethers (-O-), and esters (-COO-, -CH2O2C-, CHRO2C-), where R is an alkyl, aryl, or heterocyclic group. Generally, the characteristics of the one or more organic functional groups within the linker can be selected based on the desired release rate of the surfactant. Furthermore, one or more organic functional groups can be selected to facilitate covalent bonding of the surfactant to the dendritic macromolecule. In a preferred embodiment, the connection can occur via a suitable spacer group that provides a disulfide bridge between the drug and the dendritic macromolecule. Under reducing conditions found in vivo, the dendritic macromolecular complex is able to rapidly release the drug in vivo via a thiol exchange reaction.

[0173] In some embodiments, the linking portion includes one or more of the aforementioned organic functional groups in combination with a spacer group. The spacer group can consist of any combination of atoms, including oligomeric and polymeric chains; however, the total number of atoms in the spacer group is preferably 3-200 atoms, more preferably 3-150 atoms, even more preferably 3-100 atoms, and most preferably 3-50 atoms. Examples of suitable spacer groups include alkyl, heteroalkyl, alkylaryl, oligo- and polyethylene glycol chains, and oligo- and poly(amino acid) chains. Variations in the spacer group provide additional control over the release of anti-inflammatory agents in vivo. In embodiments where the linking portion includes a spacer group, one or more organic functional groups are typically used to link the spacer group to the anti-inflammatory agent and the dendritic macromolecule.

[0174] Reactions and strategies for covalently linking surfactants to dendritic macromolecules are known in the art. See, for example, March, “Advanced Organic Chemistry,” 5th edition, 2001 (Wiley-Interscience Publication, New York) and Hermanson, “Bioconjugate Techniques,” 1996 (Elsevier Academic Press, USA). The choice of a suitable method for the covalent linking of a given surfactant can take into account the desired linker portion and the structure of the surfactant and dendritic macromolecule as a whole, as it involves functional group compatibility, protecting group strategies, and the presence of unstable bonds.

[0175] The optimal drug loading necessarily depends on many factors, including the choice of drug, the structure and size of the dendritic macromolecule, and the tissue to be treated. In some embodiments, one or more active drugs are encapsulated, associated, and / or conjugated to the dendritic macromolecule at concentrations of about 0.01% to about 45%, preferably about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 10%, about 1% to about 10%, about 1% to about 5%, about 3% to about 20% by weight, and about 3% to about 10% by weight. However, the optimal drug loading for any given drug, dendritic macromolecule, and target site can be determined by conventional methods (as described above).

[0176] In some embodiments, the conjugation of the active agent and / or linker occurs via one or more surface and / or internal groups. Thus, in some embodiments, the active agent / linker conjugation occurs prior to conjugation on about 1%, 2%, 3%, 4%, or 5% of the total available surface functional groups (preferably hydroxyl groups) of the dendritic macromolecule. In other embodiments, the active agent / linker conjugation occurs on less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, or less than 75% of the total available surface functional groups of the dendritic macromolecule prior to conjugation. In a preferred embodiment, the dendritic macromolecular complex retains an effective amount of surface functional groups for targeting specific cell types while being conjugated with an effective amount of an active agent for treating, preventing, and / or imaging diseases or conditions.

[0177] 1. Dendritic macromolecules bind to surfactants via ether linkages.

[0178] A method has been developed to incorporate one or more active agents into hydroxyl-terminated dendritic macromolecules via ether linkages, optionally through one or more linker / spacer groups.

[0179] In some embodiments, the surface or end groups of a hydroxyl-terminated dendritic macromolecule are modified by an etherification reaction prior to conjugation with one or more linker / spacer groups and one or more radionuclides. Etherification refers to the dehydration of an alcohol to form an ether. In some embodiments, one or more hydroxyl groups of the hydroxyl-terminated dendritic macromolecule undergo an etherification reaction prior to conjugation with one or more linker moieties and one or more active agents.

[0180] In some embodiments, ether linkages are introduced into the surface groups of the hydroxyl-terminated PAMAM dendrimer by reacting it with propargyl bromide in the presence of 2% sodium hydroxide solution in DMSO. In another embodiment, the etherification of the fourth-generation hydroxyl-terminated PAMAM dendrimer PAMAM-G4-OH is carried out using allyl bromide, anhydrous cesium carbonate, and tetrabutylammonium iodide in DMF.

[0181] In other embodiments, for the 3.5 generation dendrimer, a polyethylene glycol (PEG) linker is used to introduce an alkyne functional group, with an amine at one end and an alkyne at the other, to generate a 4th generation bifunctional dendrimer, i.e., having hydroxyl and ether linkages ready for further conjugation. Exemplary bifunctional dendrimers in... Figure 11 It is shown as compound 1, which has 7 alkyne arms and 57 hydroxyl groups on its surface.

[0182] V. Usage Instructions

[0183] Dendritic macromolecular complex compositions are generally suitable for treating one or more ocular-related diseases or conditions, particularly inflammatory and / or angiogenic diseases of the eye. This invention describes dendritic macromolecular compositions and methods thereof for targeted delivery of one or more active agents to diseased tissues / cells in the eye via systemic administration, with increased efficacy and reduced side effects, preferably by selectively targeting affected cells / tissues including activated microglia and activated macrophages, retinal pigment epithelial (RPE) cells, and / or choroidal neovascularization (CNV) lesions. Preferably, the dendritic macromolecular compositions and targeted delivery methods produce minimal dendritic macromolecules in non-damaged areas of the optic nerve or CNS. Methods for treating posterior ocular diseases are also described. In some embodiments, dendritic macromolecular complexes are used to treat exudative age-related macular degeneration (AMD). The methods generally involve administering an effective amount of the composition, comprising dendritic macromolecules and one or more active agents, to a subject in need.

[0184] Methods are provided for reducing and / or inhibiting the number or activity of activated microglia and macrophages in the retina and / or choroid of the eye of a subject in need of treatment. In some embodiments, administration is performed using an effective amount of a composition comprising a hydroxyl-terminated dendritic macromolecule compounded, covalently conjugated, or intramolecularly dispersed or encapsulated with one or more therapeutic agents to reduce and / or inhibit the number or activity of activated microglia and macrophages in the retina and / or choroid of the eye in need of treatment. In some embodiments, the composition is administered at a dose and via a route that inhibits or reduces the activation of microglia in the retina. In other embodiments, the composition may inhibit or reduce the phagocytic activity of microglia. In other embodiments, the composition comprising one or more receptor tyrosine kinase inhibitors may inhibit or reduce the activity and / or amount of activated microglia and macrophages in the diseased retina and / or choroid of a subject by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% of the activity and / or amount of the same cells in equivalent diseased tissue of a subject who has not received or has not been treated with a dendritic macromolecular composition (e.g., an unconjugated active agent).

[0185] Methods for reducing and / or inhibiting the expression and / or activity of VEGF and / or VEGFR in activated microglia, activated macrophages, and / or retinal pigment epithelium (RPE) cells in the diseased retina and / or choroid are also described. In some embodiments, the composition is administered via a systemic route, such as intravenous injection, subcutaneous injection, or oral administration. In preferred embodiments, the composition is not administered intravitreal or subchoroidally, which could cause direct damage and / or inflammation to the eye. Methods for reducing and / or inhibiting one or more pro-inflammatory cytokines secreted by activated microglia and macrophages in the diseased retina and / or choroid are also described. In some embodiments, treatment with an effective amount of the composition results in a reduction of the expression of one or more pro-inflammatory cytokines (e.g., TNF-α, interleukin-1β (IL-1β), or interferon-γ (IFN-γ)) secreted by activated microglia and macrophages in the diseased retina and / or choroid by approximately 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% compared to the expression in equivalent diseased tissue of subjects who did not receive treatment with the dendritic macromolecular composition or who did not receive treatment with the dendritic macromolecular composition.

[0186] Methods for reducing and / or inhibiting one or more pro-oxidative properties of activated microglia and macrophages in the diseased retina and / or choroid are also described. In some embodiments, treatment with an effective amount of the composition results in a reduction of oxidative stress in activated microglia and macrophages in the diseased retina and / or choroid, for example, by reducing nitric oxide (NO) production or inducible nitric oxide synthase (iNOS) activation (e.g., NOS2 expression), by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% compared to those in equivalent diseased tissues of subjects who have not received or have not been treated with the dendritic macromolecular composition (e.g., unconjugated active agent).

[0187] Methods for reducing and / or inhibiting abnormal vascular permeability and leakage and / or neovascularization in the eyes of subjects with this need are also described. In some embodiments, treatment with an effective amount of the composition results in a reduction of vascular leakage and / or neovascularization.

[0188] A. Treatment Plan

[0189] 1. Dosage and effective dose

[0190] Dosage and administration regimens depend on the severity and location of the condition or lesion and / or the method of administration, and are known to those skilled in the art. Therapeutic amounts of dendritic macromolecular compositions used to treat one or more ocular diseases are generally sufficient to treat, suppress, or alleviate one or more ocular-related symptoms.

[0191] In some in vivo methods, a therapeutically effective amount of a dendritic macromolecular complex is administered to a subject to reduce or inhibit ocular angiogenesis, particularly retinal and choroidal neovascularization. In some embodiments, an effective amount of the composition is used to reduce or inhibit endothelial cell angiogenesis and / or vascular endothelial cell proliferation.

[0192] A pharmaceutical composition is described comprising a therapeutically effective amount of a dendritic macromolecular composition and a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of a hydroxyl-terminated dendritic macromolecular conjugated to a VEGF receptor tyrosine kinase inhibitor. In some specific embodiments, the appropriate parenteral dose range is between about 0.1 mg / kg and about 200 mg / kg, including endpoints; about 0.5 mg / kg to about 100 mg / kg, including endpoints; about 1.0 mg / kg to about 40 mg / kg, including endpoints; and about 2.0 mg / kg to about 20 mg / kg, including endpoints. A higher initial dose may be given to load the drug in the patient and maximize uptake in diseased tissues (e.g., the eye). After the loading dose, the patient may receive a maintenance dose. The loading dose ranges from 10 to 100 mg / kg body weight, and the maintenance dose ranges from 0.1 to <10 mg / kg body weight. When administered enterally, the therapeutically required dose may be up to 10 times higher than the effective parenteral dose. Choose the optimal dose from the safety and efficacy results of each trial dose of each drug taken by the patient.

[0193] Dosage forms of pharmaceutical compositions comprising dendritic macromolecular compositions are also provided. A “dosage form” refers to the physical form of a dose of the therapeutic compound intended to be administered to a patient, such as a capsule or vial. As used herein, the term “unit dose” refers to the amount of the therapeutic compound administered to a patient in a single dose. In some embodiments, suitable unit doses are (assuming an average patient weight of 70 kg) from 5 mg / unit dose to about 14,000 mg / unit dose, including endpoints; between about 35 mg / unit dose and about 7,000 mg / unit dose, including endpoints; and between about 70 mg / unit dose and about 2,800 mg / unit dose, including endpoints; and between about 140 mg / unit dose and about 1,400 mg / unit dose, including endpoints.

[0194] The actual effective amount of dendritic macromolecular complexes can vary depending on a variety of factors, including the specific active pharmaceutical ingredient administered, the specific composition formulated, the route of administration, and the age, weight, condition, route of administration, and disease or disorder of the subject being treated.

[0195] Preferably, a dendritic macromolecular composition comprising one or more active agents (e.g., sunitinib) is delivered to cells in or around diseased or damaged tissue (e.g., microglia). For example, the amount of the dendritic macromolecular complex composition can effectively deliver one or more active agents to cells at or near the site of inflammation (particularly ocular inflammation). Thus, in some embodiments, the amount of the dendritic macromolecular complex composition comprising one or more active agents effectively improves inflammation in the subject. In a preferred embodiment, the effective amount of the dendritic macromolecular complex composition does not induce significant cytotoxicity in the subject's cells compared to untreated control subjects. Preferably, the amount of the dendritic macromolecular complex composition effectively prevents or reduces inflammation and / or further symptoms related to the disease or condition in the subject compared to untreated controls.

[0196] Generally, the timing and frequency of dosing will be adjusted to balance the efficacy of a given treatment or diagnostic regimen with the side effects of a given dosing system. Exemplary dosing frequencies include continuous infusion, single-dose administration, and multiple-dose administration, such as hourly, daily, weekly, monthly, or yearly administration.

[0197] In some embodiments, the dose is administered once, twice, or three times daily, or less frequently, such as every other day, two days, three days, four days, five days, or six days. In some embodiments, the dose is administered only once or twice weekly, every two weeks, every three weeks, or every four weeks. In some embodiments, the dose is administered once or twice monthly, every two months, every three months, every four months, every five months, or every six months, or less. In a preferred embodiment, the dose is administered once every four weeks or less frequently.

[0198] Those skilled in the art will understand that the dosing regimen can be of any length of time sufficient to treat the subject's condition. In some embodiments, the regimen includes one or more cycles of a treatment round, followed by a medication break (e.g., no medication). The treatment round can be, for example, the dosing rounds discussed above. Similarly, the medication break can be 1, 2, 3, 4, 5, 6, or 7 days; or 1, 2, 3, or 4 weeks; or 1, 2, 3, 4, 5, or 6 months.

[0199] 2. Comparison

[0200] Treatment outcomes of dendritic macromolecular complex compositions comprising one or more active agents can be compared to controls. Suitable controls are known in the art and include, for example, untreated cells or untreated subjects. A typical control is a comparison of a subject's condition or symptoms before and after administration of the targeted drug. The condition or symptom can be a biochemical, molecular, physiological, or pathological reading. For example, the effect of the composition on a specific symptom, pharmacological, or physiological indicator can be compared to the condition of an untreated subject or a subject before treatment. In some embodiments, symptoms, pharmacological, or physiological indicators are measured in subjects before treatment and repeated once or multiple times after treatment begins. In some embodiments, the control is a reference level or an average value determined based on measurements of symptoms, pharmacological, or physiological indicators in one or more subjects (e.g., healthy subjects) who do not have the disease or condition to be treated. In some embodiments, the effect of treatment is compared to conventional treatments known in the art.

[0201] B. Subjects awaiting treatment

[0202] This composition and method are suitable for treating one or more eye diseases or conditions. The composition and method are suitable for relieving one or more symptoms associated with one or more eye diseases or conditions, such as discomfort, pain, dryness, excessive tearing, injury, infection, burns, and progressive vision loss.

[0203] In some embodiments, the eye disease to be treated is a posterior ocular disease, such as diabetic retinopathy, symptomatic vitreomacular adhesion / vitreous traction (sVMA / VMT), and wet (neovascular) or dry AMD (age-related macular degeneration). In some embodiments, the eye disease to be treated is one or more retinal and choroidal vascular diseases (e.g., AMD, retinopathy of prematurity, diabetic macular edema, retinal vein occlusion, retinopathy associated with chemotherapy toxicity, such as MEK retinopathy). In a preferred embodiment, the eye disease to be treated is age-related macular degeneration (AMD). Age-related macular degeneration (AMD) is a neurodegenerative, neuroinflammatory disease of the macular region that leads to central vision loss. The pathogenesis of age-related macular degeneration involves chronic neuroinflammation of the choroid (the vascular layer under the retina), retinal pigment epithelium (RPE), the cellular layer under the neurosensory retina, Bruch's membrane, and the neurosensory retina itself.

[0204] In other implementations, the eye disease to be treated is an inflammatory eye disease, i.e., an eye disease associated with inflammation of the ocular tissues, including, for example, AMD, retinitis pigmentosa, optic neuritis, sarcoma, retinal detachment, temporal arteritis, retinal ischemia, arteriosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, diabetic retinopathy, macular edema, Stargardt's disease (also known as Stargardt's macular dystrophy or juvenile macular degeneration), geographic atrophy, neuromyelitis optica, and also includes angiogenic diseases, including, for example, retinal neovascularization and choroidal neovascularization. Other conditions may also cause ocular inflammation and / or angiogenesis, such as infection, sickle cell disease, hypotension, etc.

[0205] Other examples of treatable eye diseases include amebic keratitis, fungal keratitis, bacterial keratitis, viral keratitis, onchorcercal keratitis, bacterial keratoconjunctivitis, viral keratoconjunctivitis, corneal dystrophy, Fuchs' endothelial dystrophy, meibomian gland dysfunction, anterior and posterior blepharitis, conjunctival hyperemia, conjunctival necrosis, scarring and fibrosis, punctate epithelial keratopathy, filamentous keratitis, corneal erosion, thinning, ulceration and perforation, and Sjögren's syndrome (Stevens-Johnson syndrome). Stevens-Johnson syndrome, autoimmune dry eye disease, environmental dry eye disease, corneal neovascularization, prevention and treatment of corneal transplant rejection, autoimmune uveitis, infectious uveitis, anterior uveitis, posterior uveitis (including toxoplasmosis), panuveitis, vitreous or retinal inflammatory diseases, prevention and treatment of endophthalmitis, macular edema, macular degeneration, age-related macular degeneration, proliferative and non-proliferative diabetic retinopathy, hypertensive retinopathy, retinal autoimmune diseases, primary and metastatic intraocular melanoma, other intraocular metastases, open-angle glaucoma, angle-closure glaucoma, pigmentary glaucoma and combinations thereof. Other conditions include corneal injury, burns or abrasions, cataracts, and related age-related degeneration of the eye or vision.

[0206] Dendritic macromolecular complexes can be administered in combination with one or more other therapeutically active agents known to treat the aforementioned conditions or diseases.

[0207] The invention will be further understood by referring to the following non-limiting embodiments. Example

[0208] Example 1: Targeted and sustained intracellular delivery to choroidal neovascularization lesions following a single systemic administration, as shown in the imaging.

[0209] method

[0210] The hydroxyl dendritic macromolecules (~14000 Da) are covalently bonded to the 2-3 indocyanine green (ICG) molecules (D-ICG) of each dendritic macromolecule via indestructible linkages. The hydroxyl dendritic macromolecules (~14000 Da) are also covalently bonded to the 2-3 tetramethylrhodamine (TRITC) molecules (D-TRITC) of each dendritic macromolecule via indestructible linkages.

[0211] Two studies were conducted in C57BL / 6 mice (n=5 / group) that received 100 μL of D-ICG or a mediator control via intravenous injection. In the first study, mice were administered D-ICG or a mediator control at 1, 3, 7, or 14 days after laser irradiation, and the eyes were analyzed by optical coherence tomography (OCT) combined with ICG imaging at 4 or 24 hours post-administration. Flat-mounts of the scleral choroid / retinal pigment epithelium (RPE) complex were stained with fluorescently labeled isolectin and IBA-1.

[0212] The second study evaluated the localization and persistence of dendritic macromolecular conjugates in CNV lesions. In this study, mice were intravenously injected with 100 μL D-ICG and 100 μL D-TRITC (1 hour after D-ICG) or a mediator irradiation 24 hours after laser irradiation. Mice were analyzed and sacrificed at 4, 7, 14, 21, and 28 days after administration (n=5 / group). For control, free ICG (1.23 mg / mL), 100 μL, IV administration (24 hours after laser irradiation), and analysis mice, sacrifice was performed at 2, 4, 7, and 14 days after administration (n=5 / group). Ocular analysis was performed using optical coherence tomography (OCT) combined with ICG imaging. Patches of the sclera-choroid / retinal pigment epithelium (RPE) complex were stained alone with fluorescently labeled IBA-1.

[0213] result

[0214] When the esterase-containing enzyme was evaluated at 37°C in PBS, pH 7.4, or citrate buffer (pH 5.5), no significant release of ICG or TRITC from dendritic macromolecules was observed under in vitro release conditions.

[0215] Following systemic administration in a laser-induced CNV mouse model, the ability of indocyanine green (ICG)-labeled hydroxyl dendritic macromolecules to target choroidal neovascularization (CNV) lesions and further penetrate macrophages and retinal pigment epithelium was evaluated.

[0216] Systemically administered D-ICG was selectively taken up by cells within CNV lesions within 24 hours of administration, while free ICG was nonspecifically distributed and typically cleared within hours. 24 hours after laser irradiation, reactive macrophages and microglia internalized dendritic macromolecular conjugates, with greater uptake observed in the early stages of CNV, consistent with efficacy studies (24 hours after laser irradiation). Co-localization with IBA-1 positive cells revealed dendritic macromolecular conjugates located in CNV lesion macrophages (data not shown). In the free ICG control group, free ICG was no longer present in the lesions during 7–14 days after laser irradiation. IBA-1 signal increased 24–48 hours after laser injury, while isolectin signal increased slightly later than 48 hours after laser injury. Figure 2A and 2B A single systemic D-ICG dose administered 24 hours after laser injury was confined to CNV lesions, and a significant D-ICG effect was still observed at the last time point, day 28. Figure 2C ).

[0217] Hydroxyl dendritic macromolecules are co-localized with reactive macrophages in the choroid, microglia / macrophages in the retina, and RPE cells at sites of inflammation / angiogenesis. D-ICG and D-TRITC appear to be intracellular and concentrated in the IBA-1 signaling region, consistent with previous studies, demonstrating the uptake of hydroxyl dendritic macromolecules in reactive microglia, macrophages, and RPE cells.

[0218] Hydroxy-dendritic macromolecules (D-ICG) selectively target CNV lesions after systemic administration and persist for at least 28 days post-administration, although they are systemically cleared within 48 hours. Therefore, D-ICG provides prolonged localization at CNV lesions, suitable for continuous and targeted therapy, such as monthly systemic (subcutaneous or oral) treatment of retinal diseases with minimal systemic exposure.

[0219] Example 2: Inhibition of choroidal angiogenesis in mice after systemic administration of targeted anti-VEGF therapy

[0220] method

[0221] A selectively inflammatory hydroxyl dendritic macromolecule (~14000 Da) was covalently conjugated to an analogue of sunitinib (an FDA-approved potent VEGF receptor tyrosine kinase inhibitor). A cleavable sunitinib analogue (D-CSA) was used. Figure 1A Compound 6) or non-cleavable sunitinib analogs (D-NSA) Figure 1BCompound 3) was used to prepare conjugates, and drug release was assessed at 37°C in PBS (pH 7.4) or citrate buffer (pH 5.5) containing esterase. Laser-induced Bruch's membrane rupture was performed in both eyes of C57BL / 6 mice (n = 8 / group) 24 hours prior to administration. Mice were intravenously injected (IV, 100 μL) with the mediator, D-CSA (equivalent to 5.25 (low) or 26.25 (high) mg / kg sunitinib), D-NSA (equivalent to 6.3 (low) or 15.75 (high) mg / kg sunitinib), or free sunitinib (32.5 mg / kg). As a positive control group, a group of mice received intravitreal administration of aflibercept. (IVT; 1 μL, 40 μg). Seven days after laser treatment, CNV area was measured by fluorescein angiography and scleral-choroid / RPE complex slices stained with isolectin IB4.

[0222] result

[0223] The efficacy of hydroxyl dendritic macromolecules covalently conjugated with sunitinib analogs was evaluated in a laser-induced choroidal neovascularization (CNV) mouse model.

[0224] D-CSA was prepared with 5 sunitinib analogs per dendritic macromolecule (10.5% w / w), and D-NSA was prepared with 7 sunitinib analogs per dendritic macromolecule (12.6% w / w). In vitro, D-CSA released ~65% sunitinib over 6 days at pH 5.5 (intracellular conditions) and ~15% sunitinib over 24 hours at pH 7.4 (plasma conditions). The release of sunitinib analogs from the D-NSA conjugate was minimal.

[0225] Compared with the mediator control, a statistically significant decrease in CNV area was observed at both IVT aflibercept and the IV dose levels of D-CSA and D-NSA (excluding free sunitinib) (even at doses 5-fold higher than low-dose D-CSA). Figure 3 ).

[0226] Binding affinity (Kd) to VEGFR2 was assessed using free sunitinib malate (0.13 nM), a sunitinib analog linked via a non-cleavable PEG linker (1 nM), and D-NSA (27 nM). Binding affinity data showed that D-NSA retained a higher binding affinity. Therefore, it has been demonstrated that the conjugation of sunitinib analogs to hydroxyl dendritic macromolecules maintains the nanomolar potency of VEGF RTK.

[0227] In a mouse model of laser-induced CNV, single-dose administration of D-CSA / D-NSA demonstrated efficacy comparable to intravitreal administration of aflibercept. The efficacy of non-cleavable sunitinib analogs in reducing CNV area suggests that release of sunitinib from dendritic macromolecules may not be necessary. Previous studies have shown that hydroxyl dendritic macromolecules and dendritic macromolecule drug conjugates persist in CNV lesions in mice and humans for >28 days and are completely cleared systemically within 24 hours, with no detectable hepatotoxicity or other off-target toxicities.

[0228] Example 3: Duration of therapeutic effect and systemic drug clearance method

[0229] The conjugate consists of a cleavable sunitinib analog (D-CSA). Figure 1A Compound 6) or non-cleavable sunitinib analogs (D-NSA) Figure 1B Compound 3) was prepared. Laser-induced Bruch's membrane rupture was performed in both eyes of C57BL / 6 mice (n=8 / group) 24 hours prior to administration. Mice were given a single intraperitoneal injection (IP, 100 μL) of the mediator, D-CSA (relative to 5.25 mg / kg sunitinib), D-NSA (relative to 6.3 mg / kg sunitinib), or free sunitinib (6.5 mg / kg). As a positive control group, one group of mice received intravitreal administration of aflibercept. Administration (IVT; 1 μL, 40 μg). CNV area was measured by fluorescein angiography and isolectin IB4-stained scleral-choroid / RPE complex planks at 7 and 14 days post-laser treatment. For plasma pharmacokinetics studies, plasma collection within 72 hours following IP administration was used to monitor the same Cy5-labeled dendritic macromolecules administered via a single IP injection.

[0230] result

[0231] The duration of treatment and clearance of hydroxyl dendritic macromolecules covalently conjugated with sunitinib analogs were evaluated in a laser-induced choroidal neovascularization mouse model (CNV).

[0232] Dendritic macromolecular conjugated sunitinib analogs D-CSA and D-NSA exhibited a durable response after a single intraperitoneal (IP) dose, with a reduction in CNV area on day 7 and a further reduction on day 14. A significant reduction in CNV area was observed with IVT aflibercept on day 7, but this reduction did not persist on day 14. Figure 4A ).

[0233] Both D-CSA and D-NSA in serum were cleared within 2 days after treatment. Figure 4BTherefore, the dendritic macromolecule-conjugated sunitinib analogs D-CSA and D-NSA have a prolonged local effect on CNV lesions, with lesion size continuing to decrease on day 14 post-treatment.

[0234] Example 4: Synthesis and characterization of N,N-didesethylsunitinamide azide

[0235] Examples 4 and 5 describe the design and synthesis of the dendritic macromolecule-didesethylsunitinib conjugate. Overexpression of vascular endothelial growth factor (VEGF) is associated with many angiogenesis-related diseases. Sunitinib, a receptor tyrosine kinase inhibitor, blocks VEGF receptors and exhibits excellent anti-angiogenic activity; it is FDA-approved for use in various types of cancer. Didesethylsunitinib is the active metabolite of sunitinib. Despite the excellent therapeutic value of sunitinib and its analogues, their associated toxicities have hindered their clinical development. The dendritic macromolecule-didesethylsunitinib conjugate aims to overcome the dose-related toxicity of sunitinib by linking it to a hydroxyl-terminated dendritic macromolecule. Figure 5 The synthetic scheme is outlined, using fourth-generation PAMAM as an example hydroxyl-terminated dendritic macromolecule.

[0236] Step 1: Synthesis of 5-fluoro-2,3-dihydro-1H-indol-2-one (Compound 2)

[0237] Triethylamine (6.12 mL, 1.2 equivalents) was added to a stirred mixture of 5-fluoro-2,3-dihydro-1H-indole-2,3-dione (6.0 g m, 1.0 equivalents) in n-butanol (10 V), followed by the addition of hydrazine hydrate (3.56 mL, 2.0 equivalents) at room temperature. The resulting solution was stirred at 100 °C for 16 h. The reaction progress was monitored by TLC (50% ethyl acetate in hexane). Once the reaction was determined to be complete, the reactants were evaporated to dryness under vacuum at 45 °C to give a dark brown solid. The solid was quenched with water (20 V), extracted with ethyl acetate (30 V), and the organic layer was washed with water. The organic layer was concentrated to dryness on a rotary evaporator. The crude product was purified by recrystallization from ethyl acetate to give a gray, fluffy solid (4.0 g, 72% yield). Figure 5 Compound 2 shown is composed of 1 Confirmed by H NMR, liquid chromatography, and mass spectrometry.

[0238] Step 2: Synthesis of 5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indole-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (Compound 4)

[0239] Pyrrolidine (4.42 mL, 2.0 equivalents) was added to a stirred solution of 5-fluoro-2,3-dihydro-1H-indol-2-one (compound 2) (4.0 gm, 1.0 equivalents) and 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (compound 3) (4.41 gm, 1.0 equivalents) in ethanol (10 V). The resulting solution was stirred at 80 °C for 3 hours. The reaction progress was monitored by TLC (10% methanol in DCM). After the reaction was deemed complete, the reactants were cooled to room temperature and 2 M HCl solution was added to adjust the pH to 3. A brownish-red precipitate formed and was filtered. The obtained solid was washed with ethanol (20 V), then with hexane (30 V), and filtered to give a reddish-orange solid (6.6 g, 82% yield). Figure 5 Compound 4 shown is obtained through 1 H NMR confirmed.

[0240] Step 3: Synthesis of N-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrolo-3-yl)carbamate]ethyl}tert-butyl carbamate (Compound 6)

[0241] Triethylamine (6.08 mL, 2.0 equivalents), EDC.HCl (8.68 g, 2.1 equivalents), HOBT (3.94 g, 1.35 equivalents), and N-(2-aminoethyl)carbamate tert-butyl carboxylate (4.16 g, 1.2 equivalents) were added to a DMF solution of 5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (compound 4) at 0 °C (6.5 g, 1.0 equivalents). The reaction was stirred at room temperature for 16 minutes. The reaction mixture was diluted with water (20.0 V), stirred for 10 min, precipitated, and filtered to give a brown solid. The resulting solid was washed with ethyl acetate (15.0 V), then with hexane (15.0 V), filtered, and dried to give a brownish-orange solid, which was N-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrolo-3-yl)carbamate]ethyl} tert-butyl carbamate (compound 6) (7.5 g, 78% yield). Figure 5 Compound 6 shown is composed of 1 H NMR confirmed.

[0242] Step 4: Synthesis of N-(2-aminoethyl)-5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indole-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxamide (compound 7):

[0243] Trifluoroacetic acid (3.0 V) was added to a solution of N-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrolo-3-yl)carbamate]ethyl}carbamate (compound 6) (9.0 g, 1.0 equivalent) in DCM (10.0 V). The reaction was stirred at room temperature for 12 hours. The reactants were evaporated to dryness under vacuum at 45 °C to give a dark brown solid. The obtained solid was washed with diethyl ether (15.0 V), filtered, and dried to give an orange-yellow solid (6.0 g crude product). Figure 5 Compound 7 shown is obtained through 1 The results were confirmed by 1H NMR, liquid chromatography, and mass spectrometry.

[0244] Step 5: Synthesis of N-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrolo-3-yl)carbamoyl]ethyl}-3-[2-(2-propoxyethoxy)ethoxy]propionamide (compound 9):

[0245] To a solution of 3-[2-(2-propoxyethoxy)ethoxy]propionic acid (8) (5.95 g, 1.0 equivalent) in DMF (10.0 V), DIPEA (8.40 mL, 2.0 equivalent), EDC.HCl (6.90 g, 1.5 equivalent), HOBT (0.65 g, 0.2 equivalent), N-(2-aminoethyl)-5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrole-3-carboxamide (compound 7) (11.0 g, 1.0 equivalent) and DMAP (0.294 g, 0.1 equivalent) were added at 0-5 °C. The reaction mixture was stirred at room temperature for 3 h. The reaction progress was monitored by TCL (10% MeOH in DCM). The reaction mixture was diluted with water (20.0 V) and stirred for 10 min, a brown precipitate formed, and filtered. The resulting solid was purified by reversed-phase column chromatography to give N,N-dideethylsunitinamide azide as an orange solid (5.2 g, 37% yield). Compound 9 was derived from... 1 Confirmed by H NMR, liquid chromatography and mass spectrometry.

[0246] Example 5: Synthesis and characterization of dendritic macromolecule-didesethylsunitinib conjugate (D-4517)

[0247] method

[0248] Synthesis and characterization of intermediates and dendritic macromolecular conjugates:

[0249] Synthesis of dendritic macromolecular hexyne Figure 6ACompound 2):

[0250] Take a dry round-bottom flask (250 mL) and record its empty weight. Pour the required amount of G4-OH methanol solution into the round-bottom flask and evaporate at 60 °C for 2 hours. Transfer the flask to a high-vacuum assembly and apply high vacuum for 1 hour. Record the amount of G4-OH in the flask. After recording the weight of G4-OH, add 50-60 mL of anhydrous DMF to the flask and evaporate under reduced pressure to remove any trace amounts of methanol in the dendritic macromolecules that might affect the Steglich esterification efficiency. After the DMF evaporates, place the flask in a nitrogen atmosphere. Add anhydrous DMF (10 mL / g) to the flask, transfer the solution to an ultrasonic bath, and sonicate the reaction mixture until a clear solution is obtained. Dissolve 5-hexyneic acid in 2 mL of DMF and add it to the stirred solution. After 10 minutes, add EDC.HCl and DMAP to the stirred solution and stir the solution at room temperature for 48 hours. After completion, begin DMF dialysis in a 1 kDa molecular weight cutoff dialysis bag. Dialyze the solution in DMF for 8 hours, changing the DMF once. After 8 hours, add 30 mL of DI water to the solution in the bag and dialyze overnight. Dilute the reaction mixture with HPLC-grade water to a final volume of 300-350 mL. TFF was performed using a 3 kDa TFF cartridge in DI water. Perform 6-7 TFF cycles until the final retentate volume is approximately 100 mL, which was then lyophilized to obtain a viscous solid. The product yield was approximately 5.5 g (74%). Recordings were taken at 500 MHz in deuterated DMSO. 1 ¹H NMR was performed, with ~10 mg of the compound used for sample preparation. The loading of hexyneic acid was calculated using proton integration. The internal amide peak of the dendritic macromolecule in the range of δ 8.11–7.70 ppm served as a reference. The peak at δ 4.0 ppm corresponded to the ester-linked proton, and the peak at δ 1.6 ppm was from the CH₂ of the hexyneic acid. Proton integration indicated that each dendritic macromolecule linked 9–10 molecules of hexyneic acid. HPLC purity > 99%.

[0251] Table 1. Reagents used for the synthesis of dendritic macromolecular hexyneides ( Figure 6A Compound 2)

[0252] G4-OH 5-Hexyneic acid EDC.HCl DMAP MW 14279 112.13 191.7 122.17 quantity 10.5g 1.15g 3.52g 1.79g millimole: 0.73 10.3 18.4 14.7 equivalent 1 14 25 20

[0253] Synthesis of dendritic macromolecule-didesethylsunitinib conjugate ( Figure 6B Compound 3 in the compound:

[0254] Dendritic macromolecular hexyneide ( Figure 6ACompound 2) was placed in a 250 mL round-bottom flask. The compound was dissolved in 40 mL of anhydrous DMF by sonication. A sunitinib-azide solution dissolved in 20 mL of DMF was added to the reaction mixture, and the solution was stirred. Then, 10 mL of water was added to the reaction mixture to stop the precipitation of the copper salt. After stirring for 10 minutes, copper sulfate pentahydrate (dissolved in 3 mL of water) was added dropwise to the reaction flask. The stirred solution turned blue. After 5 minutes, sodium ascorbate (dissolved in 3 mL of water) was added dropwise to the reaction mixture, and the flask was transferred to an oil bath set to 40 °C. The reaction mixture was stirred and heated for 24 hours. After completion, the DMF was evaporated, and the reaction mixture was diluted with 300 mL of 10% DMAc aqueous solution. EDTA (500 μL, 0.5 M) solution was added to this solution to remove the copper salt by chelation. The reaction mixture was analyzed by TFF in water using a 3 kDa TFF cartridge. Eight to ten dialysate volumes were performed in a 10% DMAc aqueous solution, followed by five to six cycles in water as a buffer to remove trace amounts of solvent. The final retentate volume was approximately 150 mL, which was lyophilized to obtain a pale yellow solid. The product yield was 5.5 g. Results were recorded on a 500 MHz instrument in deuterated DMSO and deuterated water. 1 ¹H NMR, approximately 10 mg of the compound was used for sample preparation. 1 100 H NMR scans were performed. 1 ¹H NMR revealed the formation of the product, with 6-7 arms of the sunitinib molecule linked (Figure 4). Drug loading was calculated using proton integration, comparing the peaks corresponding to the dendritic macromolecule and the drug. The CH₂ peak at 1.8 ppm corresponds to hexynic acid, and the CH₂ peak at 4.0 ppm, linked to the ester, was locked as a reference peak from the dendritic macromolecule side. After triazole formation, [the following was observed]... 1 The drug molecule loading was calculated using new peaks in ¹H NMR, corresponding to a proton peak at δ 4.4 ppm for the CH₂ peak next to the triazole ring, two aromatic protons of sunitinib at 6.92–6.80 ppm, and two NH protons at 10.9–13.6 ppm. Following the click reaction, 1–4 triazoles were formed, and the characteristic proton peaks corresponding to triazoles appeared between δ 7.5–8.0 ppm, which were suppressed by the presence of internal amide protons. To confirm the linkage of sunitinib, peaks were recorded in D₂O. 1 ¹H NMR revealed the disappearance of the internal amide peak and the presence of a triazole peak at δ 7.7 ppm. HPLC purity > 99%.

[0255] Table 2. Reagents for the synthesis of dendritic macromolecular-didesethylsunitinib conjugate (D-4517)

[0256]

[0257] In vitro kinase binding assay protocol

[0258] Kinase-labeled T7 phage strains were prepared in *E. coli* host derived from strain BL21. *E. coli* were grown to the logarithmic phase, infected with T7 phage, and cultured at 32°C with shaking until lysis. The lysate was centrifuged and filtered to remove cell debris. The remaining kinase was produced in HEK-293 cells and subsequently labeled with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with a biotinylated small molecule ligand at room temperature for 30 minutes to generate affinity resin for kinase analysis. The ligand-linked beads were blocked with excess biotin and then sealed with blocking buffer. Washing with (Pierce), 1% BSA, 0.05% Tween 20, 1mM DTT) removes unbound ligands and reduces nonspecific binding. The binding reaction is performed by washing with 1x binding buffer (20%). The assay was performed using kinase-binding, ligand-linked affinity beads, and test compounds in 0.17x PBS, 0.05% Tween 20, and 6 mM DTT. Test compounds were prepared as 111X stock solutions in 100% DMSO. Kd was measured using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds used for Kd measurements were partitioned in 100% DMSO via acoustic transfer (non-contact partitioning). The compounds were then directly diluted to the assay to a final DMSO concentration of 0.9%. All reactions were performed in polypropylene 384-well plates. The final volume for each plate was 0.02 mL. The plates were incubated with shaking at room temperature for 1 hour, and the affinity beads were washed with washing buffer (1x PBS, 0.05% Tween 20). The beads were then resuspended in elution buffer (1xPBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated with shaking at room temperature for 30 minutes. The kinase concentration in the elution buffer was determined by qPCR.

[0259] Sample preparation:

[0260] Sunitinib malate, the sunitinib ester amide linker, and the D4-sunitinib conjugate were dissolved in an aqueous DMSO solution to form a solution with a free drug (sunitinib) concentration of 10 mM. Each sample solution was further diluted in DMSO to 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.123 μM, 41.2 nM, 13.7 nM, 4.57 nM, 1.52 nM, 0.508 nM, and 0.169 nM, respectively.

[0261] result

[0262] Synthesis and Characterization

[0263] The synthesis of dendritic macromolecule-dideethylsunitinib analogues is achieved through a three-step copper(I) catalyzed alkyne-azide click reaction. Figure 6A and 6B The process was achieved through a series of steps. The first step involved linking several hexyneic acid linkers via esterification, thereby functionalizing the alkyne surface and partially modifying the surface hydroxyl groups of the dendritic macromolecule. The second step involved introducing linkers onto di-deethylsunitinib with azide ends. The third step involved a two-part click reaction. To synthesize the dendritic macromolecule hexyneic acid, a received ethylenediamine-core hydroxyl polyamidoamine dendritic macromolecule (G4-OH, pharmaceutical grade >95% HPLC purity) was used. First, partial esterification of the OH-terminated dendritic macromolecule (compound 1) with 5-hexyneic acid was performed using EDC.HCl and DMAP in anhydrous N,N-dimethylformamide. Figure 6A To obtain compound 2. Using 1 The compound was confirmed by 1H NMR. The loading of the hexyne linker was calculated by proton integration by comparing the linker protons in the range of δ 8.11–7.70 ppm with the internal amide protons of the dendritic macromolecule. The peak at δ 4.0 ppm corresponds to the ester-linked proton, and the peak at δ 1.6 ppm refers to the CH2 from the hexyneic acid. Proton integration suggests that each dendritic macromolecule is linked to 8–10 alkyne molecules. The purity of the construct was evaluated by HPLC and found to be >99%. The linker 3-[2-(2-propoxyethoxy)ethoxy]propionic acid was linked to didesethylsunitinib using EDC.HCl, DMAP, DIPEA, and HOBt in anhydrous DMF, allowing the azide to participate in the click reaction. The drug is linked to the amide via the linker. The dendritic hexyne (2) and didesethylsunitinib PEG azide were subjected to the click reaction. Copper-catalyzed click reactions are among the most efficient chemical transformations, revolutionizing drug discovery and serving as an excellent tool for the binding of small or large molecules to macromolecules, polymers, and antibodies. They are renowned for their ease of operation, mild reaction conditions, compatibility with diverse functional groups, regioselectivity, and enhanced reaction rates, enabling the production of cleaner products in high yields. Click reactions were carried out using copper sulfate pentahydrate (II) and sodium ascorbate in the presence of DMF:H2O (1:1). The reaction was carried out overnight at room temperature, followed by purification via tangential flow filtration. The formation of product (3) was achieved through… 1 The H NMR spectrum confirmed the presence of this conjugate. 1The 1H NMR spectra clearly showed peaks corresponding to the dendritic macromolecule, the drug, and the linker groups attached to them, and the drug loading was calculated by comparing these peaks using the proton integration method. When the spectra were recorded in deuterated DMSO, the internal amide protons of the dendritic macromolecule were present between δ 8.5–7.5 ppm. These amide peaks served as reference standards for the remaining peaks. The -NH peaks of the drug appeared at δ 13.6 and 10.8 ppm. The drug contained four protons, and one triazole proton formed after the click reaction, which merged with the internal amide peaks, between δ 8.5–7.5 ppm. Additionally, two aromatic protons near the fluorine group in sunitinib appeared between δ 6.95–6.85 ppm. A sharp triazole peak at δ 7.7 ppm was observed when the NMR solvent was converted from deuterated DMSO to CD3OD; this is a characteristic peak of the click conversion. After the click reaction, the CH2 present next to the azide decreased and could be observed at δ 4.4 ppm. Comparison of proton NMR spectra of drug linkers, dendritic macromolecular intermediates, and final conjugates. 1 H NMR confirmation. The purity of the dendritic macromolecular drug conjugate, intermediate, and drug linker was assessed using HPLC. The final conjugate purity was >99% by HPLC analysis. In HPLC, the dendritic macromolecular G4-OH and the dendritic macromolecular hexynylated intermediate were visible in the 210 nm channel, and didesethylsunitinib was visible at 430 nm. The retention time of compound 2 was approximately 16.9 min, but once the hydrophobic drug molecule was linked to the dendritic macromolecular, the peak of the final conjugate shifted to the right, with a retention time of approximately 27 min, confirming the linkage between the hydrophobic drug and the dendritic macromolecular structure. Once the drug was linked to the dendritic macromolecular, corresponding peaks were observed in both the 210 nm (dendritic macromolecular absorption wavelength) and 430 nm (drug absorption wavelength) channels, further confirming product formation. The drug loading of the dendritic macromolecular conjugate was approximately 12.6% wt / wt, corresponding to 7 drug molecules linked to each dendritic macromolecular molecule.

[0264] Combining affinity:

[0265] The comparative kinase binding affinity of the D-didesethyl-sunitinib conjugate (compound D-4517), free sunitinib malate, and the sunitinib linker (AVT-4517) was evaluated, and the results are shown in Table 3. The binding affinity of free sunitinib was 0.13 nM. After PEG linker attachment, the binding affinity decreased by approximately 8-fold to 1.0 nM. The binding affinity of the conjugate was 27 nM. The results indicate that the conjugation of the drug to the surface of the dendritic macromolecule maintains the binding affinity of the drug to the RTK domain in the nanomolar range. This suggests that the conjugate itself is active and can bind to the receptor without releasing the drug. This study is the first to demonstrate that the conjugation of a small molecule inhibitor (300-400 Da) to a large dendritic macromolecule (14000 Da) can still maintain the nanomolar binding of the small molecule inhibitor.

[0266] Table 3: D-Deshydroxyethyl-sunitinib conjugate kinase (VGEFR2) binding assay:

[0267]

[0268] Stability studies in human and rat plasma:

[0269] Under physiological conditions, the in vitro stability of D-didesethylsunitinib (D-4517) in human and rat plasma was further evaluated. The results in Table 4 show that the conjugate D4517 was very stable, releasing 2% (w / w) in human plasma and 4% (w / w) in rat plasma after 48 hours.

[0270] Table 4: In vitro stability study of the percentage of drug release by weight of D-didesethylsunitinib (D-4517) in human and rat plasma at 37°C.

[0271]

[0272] In vitro drug release studies:

[0273] In vitro drug release studies were conducted at pH 7.4 and pH 5.5, containing esterase, at 37°C to simulate plasma and intracellular conditions, respectively. Release studies were performed in duplicate. Results are as follows: Figure 7 As shown. Under intracellular conditions, less than 2% by weight of the drug arm was released within 15 days. Under plasma conditions, ~2% was released within 24 hours and ~4% within 15 days. Ester bonds between the dendritic macromolecule and the linker lead to the loss of the AVT-4517 linker in D-4517 over time. The conjugate exhibited good stability under both conditions.

[0274] Example 6: In vivo pharmacokinetics of dendritic macromolecule-didesethylsunitinib conjugate (D-4517)

[0275] Pharmacokinetics of D-4517 were evaluated in mice. Female C57 / Bl6 mice were injected intraperitoneally with 5 or 50 mg / kg D-4517 and blood samples were collected to determine plasma D-4517 concentrations. Peak plasma concentrations were observed at the first time point (0.5 h) after sampling. Exposure based on Cmax and AUC was dose-related and approximately dose-proportional.

[0276] The terminal elimination time T is approximately 1 hour after two dose levels. 1 / 2 The PK parameters estimated using the noncompartmental method are shown in Table 5 below, and the relationship between mean plasma concentration and time is shown in [Table 5]. Figure 8 .

[0277] Table 5: Plasma D-4517 concentration in mice after IP injection.

[0278]

[0279] Toxicokinetic data were collected in rats. Sprague-Dawley rats received daily intraperitoneal injections of 12 mg / kg or a single dose of 168 mg / kg D-4517 orally daily of 30 mg / kg sunitinib (40.21 mg / kg sunitinib malate). Blood samples were collected, and plasma drug concentrations were determined. Non-compartmental toxicokinetic parameters were estimated.

[0280] Figure 9A and 9B The relationship between plasma concentrations and time on days 1 and 14 was shown in the 12 mg / kg D-4517 and sunitinib groups. There appeared to be no significant sex difference in D-4517 plasma concentrations, but for sunitinib, concentrations were higher in males than in females, except at the 24-hour time point on day 1. The rate of decline in D-4517 plasma concentrations was faster than that of sunitinib. The terminal half-life could not be reliably estimated due to insufficient time points in the terminal phase. Sunitinib was measurable 24 hours after administration, but D-4517 was not measurable 8 hours after administration. Therefore, the estimated AUC for sunitinib was higher than that for D-4517. Following a single dose of 168 mg / kg D4517, plasma concentrations were high 1 hour post-administration, but only one animal reached measurable concentrations at the next sampling time point (24 hours).

[0281] Pharmacodynamic results showed that the maximum concentrations produced by D-4517 were comparable to those produced by sunitinib, with lower total exposure. Rats were orally administered sunitinib malate for 14 days at a dose of 40.21 mg / kg. D-4517 was not associated with mortality and had no effect on clinical outcomes, body weight, food consumption, or clinicopathological parameters (hematology, clinical chemistry, and urinalysis). Gross necropsy findings associated with D-4517 were limited to yellowing of the adipose tissue and mesentery in male and female rats at the 12 mg / kg and / or 168 mg / kg dose groups, which was associated with subacute / chronic inflammation associated with intraperitoneal administration of the test substance. In males at the 168 mg / kg dose group, organ weight changes included a statistically significant decrease in spleen weight, although this observation was not microscopically relevant. Non-adverse microscopic findings in males and females at the 12 mg / kg and 168 mg / kg dose groups included microfocal pigmentation in the ocular choroid and subacute / chronic inflammation in the abdominal adipose tissue / mesentery. Inflammation may occur secondary to intraperitoneal injection of the test substance, and secondary inflammation was observed along the serosa of the stomach, liver, and spleen.

[0282] Overall, D-4571 was well tolerated after single or repeated intraperitoneal (IP) administration. These observations contrast with those of sunitinib malate, which, in addition to causing death in female rats, led to a variety of clinical and pathological changes.

[0283] Example 7: Single subcutaneous administration study of dendritic macromolecule-didesethylsunitinib conjugate (D-4517)

[0284] To evaluate the preferred route of administration in humans, a single subcutaneous administration study of D-4517 was conducted in a laser-induced CNV mouse model. One day after laser treatment, control mice (n=8 / group) received an intravitreal injection of either the carrier or aflibercept (40 μg). Three dose levels of D-4517 (2, 10, and 50 mg / kg; n=8 / group) were administered subcutaneously as a single dose one day after laser treatment. Fourteen days later, mice were sacrificed, and scleral-choroid / RPE complex slices were stained with DAPI and isolectin IB4. CNV area was measured using fluorescence microscopy and imaging software. Figure 10 As shown, all three doses of D-4517, administered as a single subcutaneous dose, significantly reduced the area of ​​CNV lesions. The responses in the D-4517 treatment group were more consistent than those observed in the aflibercept treatment group. This study demonstrates the significant efficacy of subcutaneous administration of D-4517 in a CNV model.

[0285] Example 8: Di-deethylsunitinib conjugation via non-cleavable ether linkages on a dendritic macromolecule

[0286] method

[0287] Dendritic macromolecular conjugates were synthesized via indestructible ether linkages on dendritic macromolecules.

[0288] Synthesis began with the construction of a bifunctional dendritic macromolecule. In the 3.5th generation of the dendritic macromolecule, seven alkyne functional groups were introduced using a polyethylene glycol (PEG) linker, which has an amine at one end and an alkyne group at the other, to generate a fourth-generation bifunctional dendritic macromolecule with seven alkyne arms and 57 hydroxyl groups on its surface. Figure 11 Compound 1 in the compound). l 1H NMR spectroscopy confirmed the structure of the dendritic macromolecules.

[0289] Clickable di-desethylsunitinib analogues ( Figure 11 Compound 2, also known as AVT-4517 (comprising di-deethylsunitinib, three ethylene glycol (PEG3) spacers, and a terminal azide), was synthesized to participate in a click reaction with alkyne groups on the surface of a dendritic macromolecule. Figure 5 The five-step synthetic method shown is used to prepare active pharmaceutical compound 2.

[0290] Click chemistry of alkynes-azides catalyzed by copper(I), AVT-4517 Figure 11 Compound 2) ultimately reacts with a bifunctional dendritic macromolecule with a hexyne group ( Figure 11 The reaction of compound 1) yields a product with... Figure 12 The complete structure shown is D-4517.2 ( Figure 11 Compound 3 in the literature. After conjugating the analogue with a dendritic macromolecule, D-4517.2 was purified by tangential flow filtration (TFF) to remove any impurities and purify it into the final formulation.

[0291] D-4517.2 conjugate 1 H-NMR analysis

[0292] The formation of product D-4517.2 is due to... l H NMR confirmed the conjugate. l¹H NMR spectra clearly showed peaks corresponding to the dendritic macromolecule, the drug, and the linker groups attached to them, and the drug loading was calculated by comparing these peaks using the proton integration method. When the spectra were recorded in deuterated DMSO, the internal amide protons of the dendritic macromolecule were located between δ 8.5–7.5 ppm. These amide peaks served as reference standards for the remaining peaks. The -NH peaks of the drug appeared at δ 13.6 and 10.8 ppm. The drug contained four protons; a triazole proton formed after the click reaction merged with the internal amide peak, located between δ 8.5–7.5 ppm. Additionally, two aromatic protons near the fluorine group in sunitinib were located between δ 6.95–6.85 ppm. A sharp triazole peak at δ 7.7 ppm was observed when the NMR solvent was converted from deuterated DMSO to CD3OD; this is a characteristic peak of the click conversion. After the click reaction, the CH₂ present next to the azide was down-shielded and could be observed at δ 4.4 ppm. NMR can also be used to quantify the number of drug molecules conjugated to hydroxyl dendritic macromolecules. Drug loading is calculated using the proton integration method by comparing amide protons within the dendritic macromolecule with drug protons.

[0293] HPLC analysis used to evaluate the purity of D-4517.2

[0294] The purity of the dendritic macromolecular drug conjugate, intermediates, and drug linkers was evaluated using HPLC. The final conjugate purity was >99% by HPLC analysis. In HPLC, the dendritic macromolecular G4-OH and the dendritic macromolecular hexyne intermediate were visible in the 210 nm channel, and didesethylsunitinib was visible at 430 nm. The retention time of compound 2 was approximately 16.9 min, but once the hydrophobic drug molecule was linked to the dendritic macromolecular structure, the peak of the final conjugate shifted to the right, with a retention time of approximately 27 min, confirming the linkage between the hydrophobic drug and the dendritic structure. Once the drug was linked, corresponding peaks were observed in the 210 nm (dendritic absorption wavelength) and 430 nm (drug absorption wavelength) channels, further confirming product formation. The drug loading of the dendritic macromolecular conjugate was approximately 12.6% wt / wt, corresponding to 7 drug molecules linked to each dendritic macromolecular molecule.

[0295] Size and ζ potential

[0296] The size and zeta potential distribution of D-4517.2 were determined using a Zetasizer Nano ZS instrument. For size measurements, samples were prepared by dissolving the dendritic macromolecules in deionized water (18.2 Ω) to a final concentration of 0.5 mg / mL. The solution was then filtered directly into the cells through a 0.2 μm syringe filter (Pall Corporation, 0.2 μm HT Tuffryn membrane) (UV transmission disposable cuvette, size: 12.5 x 12.5 x 45 mm). For zeta potential measurements, samples were prepared in 10 mM NaCl at a concentration of 0.2 mg / mL using the procedure described above. A Malvern Zetasizer Nanosizer disposable folded capillary cell was used for the measurements. The size of D-4517 was 5.5 ± 0.5 nm, and the zeta potential was slightly positive (+5.4 ± 0.4 mV).

[0297] Size exclusion chromatography with multi-angle laser scattering (SEC-MALS)

[0298] The molar mass of D-4517.2 will be determined by size exclusion chromatography with multi-angle laser scattering (SEC-MALS).

[0299] result

[0300] D-4517 exhibits nanomolar affinity for VEGFR2, eliminating the need for release of the active drug AVT-4517. To further enhance the stability of the conjugate under physiological conditions and further reduce drug release from the conjugate observed in D-4517 buffer and plasma stability studies, the cleavable ester linkages on the dendritic macromolecule surface were replaced with non-cleavable linkages, as shown in the structure of D-4517.2. Figure 12 The structure of D-4517.2 does not contain any breakable bonds.

[0301] D-4517.2 is a covalent conjugate of a fourth-generation hydroxyl-terminated PAMAM dendrimer containing an ethylenediamine (EDA) core, an amide amine repeating unit [CH2CH2CONHCH2CH2N], and 64 hydroxyl terminal groups (chemical formula: C). 622 H 1184 N 186 O 188 This product contains a di-deethylsunitinib analog (AVT-4517) conjugated to a dendritic macromolecule via an efficient click chemistry method. The hydroxyl-based fourth-generation PAMAM dendritic macromolecule is monodisperse and produced with high compositional purity (>95%). To prepare D-4517.2, 7 of the 64 hydroxyl groups on the dendritic macromolecule were modified to link AVT-4517 (approximately ~12.6% of the total mass).

[0302] Stability studies in human, mouse and rat plasma

[0303] The in vitro stability of the dendritic macromolecule didesethylsunitinib conjugates D-4517 and D-4517.2 in human, mouse, and rat plasma was evaluated under physiological conditions. Results are shown below. Figure 13 Compared to D4517 (2% (w / w) release in human plasma and 4% (w / w) release in rat plasma), D4517.2 exhibited significantly improved plasma stability. At 48 hours, less than 0.5% (w / w) of the drug was released from the dendritic macromolecular drug conjugate in all three plasma groups.

[0304] Affinity

[0305] The kinase binding affinity of D-4517 and D-4517.2 was evaluated, and the results are shown in Table 6.

[0306] Table 6. Dendritic macromolecular didesethylsunitinib conjugates, D-4517 and D-4517.2, and their binding analysis.

[0307] Compound Name Gene symbol Modified genes Kd(nM) D4517 VEGFR2 ＝ 27 D4517 VEGFR1 ＝ 1100 D4517 CSF1R ＝ 82 D4517 KIT ＝ 3.4 D4517 PDGFRA ＝ 16 D4517 PDGFRB ＝ 11 D-4517.2 CSF1R ＝ 41 D-4517.2 VEGFR1 ＝ 890 D-4517.2 KIT ＝ 3 D-4517.2 PDGFRA ＝ 11 D-4517.2 PDGFRB ＝ 7.5 D-4517.2 VEGFR2 ＝ 14

[0308] In all experimental analyses, the IC50 result of D-4517.2 was lower than that of D-4517, indicating that D4517.2 binds more strongly to the tyrosine kinase receptor.

[0309] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention pertains. Publications cited herein and the material cited herein are specifically incorporated herein by reference.

[0310] Those skilled in the art will recognize, or be able to determine, using only conventional experiments, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

Claims

1. Pharmaceutical preparations, including Dendritic macromolecules conjugated to receptor tyrosine kinase inhibitors, wherein the dendritic macromolecules are selected from: or ， in: D is a poly(amidoamine) (PAMAM) dendritic macromolecule selected from the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th and 10th generations; n is an integer from 1 to 100; m is an integer from 16 to 4096; and One or more pharmaceutically acceptable excipients suitable for systemic administration. The dosage of the preparation is effective in treating or alleviating one or more symptoms associated with one or more eye diseases and / or conditions.

2. The formulation according to claim 1: in: D is a poly(amidoamine) (PAMAM) dendritic macromolecule selected from the 4th, 5th, 6th, 7th and 8th generations.

3. The formulation according to claim 1, wherein: D is a G4 PAMAM dendritic macromolecule; n is an integer from 5 to 15; m is an integer from 49 to 59; and n+m=64.

4. The formulation according to claim 1, wherein the dendritic macromolecule has the following structure: ;or 。 5. The formulation according to claim 4, wherein the dendritic macromolecule has the following structure: 。 6. The formulation according to any one of claims 1-5, wherein the dendritic macromolecule has a diameter of 1 nm to 20 nm.

7. The formulation according to any one of claims 1-6, wherein the dendritic macromolecule has a diameter of 2 nm to 10 nm.

8. The formulation according to any one of claims 1-7, wherein the surface charge of the dendritic macromolecule is between -20 mV and 20 mV, including the endpoints.

9. The formulation according to any one of claims 1-7, wherein the surface charge of the dendritic macromolecule is between -10 mV and 10 mV, including the endpoints.

10. The formulation according to any one of claims 1-7, wherein the surface charge of the dendritic polymer is between -10 mV and 5 mV, including the endpoints.

11. The formulation according to any one of claims 1-7, wherein the surface charge of the dendritic polymer is between -5 mV and 5 mV, including the endpoints.

12. The formulation according to any one of claims 1-7, wherein the surface charge of the dendritic polymer is between -2 mV and 2 mV, including the endpoints.

13. The formulation according to any one of claims 1-12, wherein 0.1% to 40% of the total surface groups of the dendritic macromolecule are covalently linked to the active agent or the like.

14. Use of the pharmaceutical preparation of any one of claims 1-13 in the preparation of a medicament for treating an eye disease or condition in a subject in need.

15. The use according to claim 14, wherein the eye disease or condition is an inflammatory and / or angiogenic disease of the eye.

16. The use according to claim 14, wherein the eye disease or condition is selected from age-related macular degeneration (AMD), retinitis pigmentosa, optic neuritis, uveitis, retinal detachment, temporal arteritis, retinal ischemia, arteriosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, diabetic retinopathy, macular edema, retinal neovascularization, and choroidal neovascularization.

17. The use according to claim 14, wherein the eye disease or condition is wet age-related macular degeneration (AMD).

18. The use according to claim 14, wherein the eye disease or condition is diabetic macular edema (DME).

19. The use according to claim 14, wherein the eye disease or condition is associated with activated microglia, activated macrophages and / or retinal pigment epithelial (RPE) cells in the eye.

20. The use according to claim 19, wherein the formulation is administered to the subject in an amount that effectively targets activated microglia, activated macrophages, and / or RPE cells in the eye.

21. The use according to claim 19, wherein the formulation is administered to the subject in an amount that effectively reduces the number and / or activity of activated microglia and / or activated macrophages in the eye.

22. The use according to claim 14, wherein the formulation is administered to the subject in an amount that effectively relieves one or more symptoms of the eye disease or condition.

23. The use according to any one of claims 20-22, wherein the formulation is administered systemically to the subject.

24. The use according to any one of claims 20-22, wherein the formulation is administered to the subject intravenously, subcutaneously or orally.

25. The use according to any one of claims 20-24, wherein the formulation is administered to the subject at time intervals selected from daily, weekly, bi-weekly, monthly, and bi-monthly.

Images