Dendrimer compositions and methods for drug delivery to eye

Abstract

To provide dendrimer compositions and methods for drug delivery to the eye.SOLUTION: Dendrimer compositions and methods for the treatment of one or more inflammatory and / or angiogenic diseases and / or disorders of the eye include hydroxyl-terminated dendrimers complexed or conjugated with one or more active agents for the treatment or alleviation of one or more symptoms of the diseases of the eye, and / or for diagnosing the diseases and / or disorders of the eye. The dendrimers may include one or more ethylene diamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated generation-4, 5, 6, 7, 8, 9, or 10 dendrimers. The active agents may be VEGFR tyrosine kinase inhibitors including sunitinib or analogues thereof. Preferably, the compositions are suitable for administration via a systemic route to target activated microglia / macrophages in retina / choroid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit 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, which are incorporated by reference in their entireties.

[0002] FIELD OF THE INVENTION The present invention is generally in the field of drug delivery, and in particular, methods for selectively delivering drugs to activated immune cells within the eye and surrounding tissues. [Background technology]

[0003] Background of the Invention The development of neuroinflammatory changes within the retina is a significant factor in the pathogenesis of numerous retinal disorders, including glaucoma, diabetic retinopathy, and age-related macular degeneration. Abnormal immune responses resulting from physiological alterations in microglia, the primary resident innate immune cells within the retina, are thought to drive aspects of disease progression, including neurodegeneration and pathological neovascularization (Karlstetter et al., 2015; Silverman and Wong, 2018). Microglia become activated due to complex interactions between various retinal cell types and diverse pathological pathways. After activation, microglial cells lose their branched processes, proliferate, and rapidly migrate to the damaged area, resolving tissue damage. However, the persistent presence of tissue stress stimulates microglia to become hyperreactive, resulting in the 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 that affect vision. Retinal microglial activation occurs in mouse models of ischemia / reperfusion injury (I / R), as it occurs in ocular inflammatory diseases including glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, and branch vein occlusion (BVO). Retinal vascular occlusion, whether due to elevated intraocular pressure in I / R models or thrombus in BVO, causes a decrease in intraocular blood flow, resulting in retinal ischemia. This triggers neuronal death and initiates further microglial activation.

[0004] Enhanced production of pro-inflammatory and angiogenic factors induces the formation and growth of new blood vessels from the choroid into the subretinal space, mimicking features of exudative AMD in a laser-induced CNV mouse model (Lambert V, et al., Nat. Protoc. 8, 2197-2211 (2013)). Several conditions, such as ischemia, hypoxia, or inflammation, can promote neovascularization. Pathological intraocular neovascularization, particularly in the retina and choroid, can result in significant visual impairment. Diabetic retinopathy, neovascular age-related macular degeneration (AMD), retinopathy of prematurity, and retinal vascular occlusion are the leading causes of angiogenesis-related blindness.

[0005] Exudative (wet) AMD is characterized by serous or hemorrhagic separation of 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 (cacoid protrusions from capillary walls), intraretinal hemorrhage, and cotton-wool spots (nerve fiber layer infarctions). As the disease progresses, retinal vessels gradually close, resulting in retinal ischemia and other symptoms, including venous abnormalities (beads, loops), intraretinal microvascular abnormalities, and increasing retinal hemorrhage and exudation. Nonproliferative DR is graded as mild, moderate, severe, or very severe, depending on the presence and severity of these lesions. More advanced stages of DR involve the formation of new blood vessels induced by retinal ischemia, which spread from the papilla (neovascularization of the optic nerve, NVD) or from elsewhere in the retina (neovascularization elsewhere, NVE). New vessels extending into the vitreous can cause vitreous hemorrhage and tractional retinal detachment associated with the associated retractile fibrous tissue. To date, the only treatment conclusively demonstrated to provide long-term benefit for DR is focal laser photocoagulation. The standard treatment for patients with AMD is intravitreal injection of anti-VEGF drugs into the eye to slow disease progression, and some studies have shown that anti-VEGF therapy may be useful in diabetic macular edema (DME). However, there are currently no systemic treatments available for ischemic retinopathy or AMD. This treatment, due to its retention in microglia and ability to be delivered systemically, would require less frequent injections, avoiding the frequent intraocular injections required by current anti-VEGF therapies. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lambert V, et al., Nat. Protoc. 8,2197-2211(2013) Summary of the Invention [Means for solving the problem]

[0007] It is therefore an object of the present invention to provide compositions and methods for the effective treatment of one or more inflammatory and / or neovascular diseases of the eye, particularly DME, DR, and AMD.

[0008] Another object of the present invention is to provide compositions and methods for targeted delivery of one or more active agents to affected tissues / cells within the eye via systemic administration with increased efficacy and reduced side effects.

[0009] A further object is to provide compositions and methods for the targeted delivery of one or more active agents to activated microglia associated with one or more inflammatory and / or neovascular diseases of the eye.

[0010] An object is also to provide compositions and methods that are effective in inhibiting or reducing pro-inflammatory and / or angiogenic factors associated with one or more inflammatory and / or neovascular diseases of the eye.

[0011] Summary of the Invention Compositions and methods have been developed for selectively delivering one or more therapeutic, prophylactic, and / or diagnostic agents to treat and / or diagnose one or more diseases and / or disorders of the eye. The compositions deliver one or more therapeutic, prophylactic, and / or diagnostic agents to selectively activate microglial cells so that diseased tissues / cells of the eye are treated and / or diagnosed.

[0012] The composition comprises a hydroxyl-terminated dendrimer complexed, covalently conjugated, or intramolecularly dispersed or encapsulated with one or more receptor tyrosine kinase inhibitors in an amount effective to reduce the number or activity of activated microglia and macrophages in the retina and / or choroid of a subject in need thereof. In some embodiments, the receptor tyrosine kinase inhibitor is a vascular endothelial growth factor receptor inhibitor, such as sunitinib, sorafenib, pazopanib, vandetanib, axitinib, cediranib, vatalanib, dasatinib, nintedanib, motesanib, and analogs thereof. Preferably, the receptor tyrosine kinase inhibitor is sunitinib or an analog thereof. In some embodiments, the diagnostic agent is a dye, such as a fluorescent dye, a near-infrared dye, a SPECT contrast agent, a PET contrast agent, and a radioisotope. Preferably, the diagnostic agent is the fluorescent dye indocyanine green (ICG).

[0013] In some embodiments, the dendrimer is a generation 4, 5, 6, 7, 8, 9, or 10 PAMAM dendrimer. In some embodiments, one or more therapeutic, prophylactic, and / or diagnostic agents are covalently conjugated to the dendrimer.

[0014] In some embodiments, the one or more therapeutic, prophylactic, and / or diagnostic agents are present in a concentration by weight of the agent relative to the dendrimer conjugate of between 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.

[0015] In some embodiments, one or more spacers or linkers between the dendrimer and the drug are added to provide a releasable (or cleavable) or non-releasable (or non-cleavable) form of the dendrimer-drug conjugate in vivo. In some embodiments, the linkage is via a suitable spacer that provides an ester bond between the drug and the dendrimer. In some embodiments, the linkage is via a suitable spacer that provides an ether bond between the drug and the dendrimer. In some embodiments, the linkage is via a suitable spacer that provides an amide bond between the drug and the dendrimer. In preferred embodiments, the one or more spacers / linkers between the dendrimer and the drug are adjusted to achieve the desired effective release kinetics in vivo.

[0016] The compositions are suitable for treating and / or diagnosing one or more inflammatory and / or neovascular diseases of the eye, such as age-related macular degeneration (AMD), retinitis pigmentosa, optic neuritis, uveitis, retinal detachment, temporal arteritis, retinal ischemia, atherosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, diabetic retinopathy, macular edema, retinal neovascularization, and choroidal neovascularization.

[0017] Methods of making the dendrimer compositions are provided. Dosage forms and pharmaceutical formulations containing an effective amount of the dendrimer compositions for administration to a subject in need thereof are also provided.

[0018] Methods for treating and / or diagnosing one or more ocular diseases and / or disorders are described, including administering an effective amount of a composition to a subject in need thereof. The method is effective for treating and / or diagnosing one or more ocular diseases and / or disorders, including age-related macular degeneration (AMD), retinitis pigmentosa, optic neuritis, uveitis, retinal detachment, temporal arteritis, retinal ischemia, atherosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, diabetic retinopathy, macular edema, retinal neovascularization, and choroidal neovascularization. In particular, the method is effective for treating and / or diagnosing one or more ocular diseases and / or disorders associated with activated microglia in tissues within and surrounding the eye. Typically, the composition is administered in an amount effective to target activated microglia, retinal pigment epithelial (RPE) cells, and / or choroidal neovascularization (CNV) lesions and / or to alleviate one or more symptoms of one or more ocular diseases and / or disorders.

[0019] Also provided is a method for administering compositions and pharmaceutical preparations.Typically, compositions and pharmaceutical preparations are administered via one or more systemic routes daily, weekly, biweekly, monthly, bimonthly, or less frequently.In some embodiments, compositions and pharmaceutical preparations are administered via one or more systemic routes once every four weeks or less frequently.In a preferred embodiment, compositions and pharmaceutical preparations are administered via intravenous, subcutaneous, or oral routes. [Brief explanation of the drawings]

[0020] [Figure 1-1] 1A and 1B are schemes showing the chemical reaction steps for synthesizing dendrimer-sunitinib conjugates. Sunitinib is conjugated to the dendrimer via a hydroxymethyl bond (FIG. 1A) and an amide bond (FIG. 1B), respectively. [Figure 1-2]1A and 1B are schemes showing the chemical reaction steps for synthesizing dendrimer-sunitinib conjugates. Sunitinib is conjugated to the dendrimer via a hydroxymethyl bond (FIG. 1A) and an amide bond (FIG. 1B), respectively.

[0021] [Figure 2-1] Figures 2A and 2B are bar graphs showing the levels of isolectin (Figure 2A) and IBA-1 (Figure 2B) analyzed by optical coherence tomography with ICG imaging at 4 or 24 hours after D-ICG administration, and show the signal area (μm2) at 1, 3, 7, and 14 days after laser for 4 hours (4h) and 24 hours (24h), respectively. [Figure 2-2] Figure 2C is a bar graph showing corrected total lesion fluorescence over 28 days following single systemic dendrimer-indocyanine green (D-ICG) administration 24 hours after laser injury localized to choroidal neovascularization (CNV) lesions in C57BL / 6 mice.

[0022] [Figure 3] Figure 3 is a bar graph showing the mean area of ​​choroidal neovascularization (CNV) (mm2) in eyes of mice treated with vehicle, aflibercept, low-dose (D-CSA low) or high-dose (D-CSA high) cleavable sunitinib analog (D-NSA), low-dose (D-NSA low) or high-dose (D-NSA high) non-cleavable sunitinib analog (D-NSA), and free sunitinib administered 24 hours after laser-induced rupture of Bruch's membrane in the eyes of C57BL / 6 mice (n=8 / group for all except D-NSA high, n=6). P values ​​are shown compared to vehicle control.

[0023] [Figure 4-1]Figure 4A is a bar graph showing the mean area (mm) of choroidal neovascularization (CNV) in eyes of mice treated with free sunitinib, cleavable sunitinib analog (D-CSA), non-cleavable sunitinib analog (D-NSA), and aflibercept administered 24 hours after laser-induced rupture of Bruch's membrane in the eyes of C57BL / 6 mice (n=8 / group) on days 7 and 14 after treatment, respectively. [Figure 4-2] FIG. 4B is a line graph showing the plasma concentrations (μg / ml) of dendrimer-sunitinib analogue conjugates over a period of 0 to 72 hours.

[0024] [Figure 5] FIG. 5 is a reaction scheme illustrating one synthetic strategy for N,N-didecethylsunitinib azide bearing an amide bond.

[0025] [Figure 6] 6A and 6B are schemes showing the chemical reaction steps for synthesizing an exemplary dendrimer-sunitinib conjugate via initial synthesis of a dendrimer-hexynoic acid conjugate (FIG. 6A) prior to synthesizing a dendrimer-didecetyl-sunitinib amide conjugate (FIG. 6B). A G4 PAMAM dendrimer is used as the exemplary dendrimer.

[0026] [Figure 7] FIG. 7 is a line graph showing the in vitro release profile (linker loss with AVT-4517 % w / w) of D-didecethylsunitinib conjugate (D-4517) pH 7.4 and pH 5.5 over 15 days by esterase at 37° C., which mimics plasma and intracellular conditions, respectively.

[0027] [Figure 8] FIG. 8 is a line graph showing plasma concentrations in μg / mL over time in female C57 / B16 mice injected IP with 5 or 50 mg / kg D-4517.

[0028] [Figure 9] Figures 9A and 9B are line graphs showing plasma concentrations in μg / mL over time (0-24 hours) for male and female groups of Sprague-Dawley rats, respectively, that received daily IP injections of 12 mg / kg D-4517 and daily oral doses of 30 mg / kg sunitinib (40.21 mg / kg sunitinib malate) on day 1 (Figure 9A) and day 14 (Figure 9B).

[0029] [Figure 10] FIG. 10 is a bar graph showing the mean area (μm) of choroidal neovascularization (CNV) in the eyes of mice treated with vehicle, aflibercept (40 μg), and three dose levels of D-didecethylsunitinib conjugate (D-4517) administered as a single subcutaneous dose of 2, 10, and 50 mg / kg, 24 hours after laser-induced rupture of Bruch's membrane in the eyes of C57BL / 6 mice, on day 14 after treatment.

[0030] [Figure 11] FIG. 11 is a scheme showing the synthesis of an exemplary dendrimer-conjugate (D-4517.2) in which N,N-didecethylsunitinib is conjugated to a dendrimer via an ether bond for enhanced in vivo stability.

[0031] [Figure 12] FIG. 12 is a schematic diagram showing the chemical structure of compound D-4517.2.

[0032] [Figure 13] FIG. 13 is a bar graph showing the weight percentage drug release (0.0%-0.50%) of D-didecethylsunitinib conjugate D-4517.2 in human, mouse, and rat plasma conditions at time points of 4, 24, and 48 hours, respectively.

[0033] [Figure 14]FIG. 14 is a synthetic scheme for dendrimer-N-acetyl-L-cysteine ​​methyl ester conjugates (dendrimer-NAC-carboxymethylated conjugates). DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description of the Invention I. Definition The terms "active agent" or "biologically active agent" are used interchangeably to refer to a chemical or biological compound that induces a desired pharmacological and / or physiological effect, which may be prophylactic, therapeutic, or diagnostic. These may be nucleic acids, nucleic acid analogs, small molecules having a molecular weight of less than 2 kDa, more typically less than 1 kDa, peptidomimetics, proteins or peptides, carbohydrates or sugars, lipids, or surfactants, or combinations thereof. The term also encompasses pharmaceutically acceptable, pharmacologically active derivatives of the active agent, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, and analogs.

[0035] The term "pharmaceutically acceptable salt" is art-recognized and includes relatively non-toxic, inorganic and organic acid addition salts of compounds. Examples of pharmaceutically acceptable salts include those derived from mineral acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Examples of inorganic bases suitable for forming salts include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc. Salts may also be formed with suitable organic bases, including those that are sufficiently non-toxic and strong to form such salts. By way of example, classes of such organic bases may include mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines, such as mono-, di-, and triethanolamine; amino acids, such as arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; and N-benzylphenethylamine.

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

[0037] The term "diagnostic agent" refers to an active agent that can be administered to localize, pinpoint, or define a pathological process. The diagnostic agent can label target cells to allow subsequent detection or imaging of these labeled target cells. In some embodiments, the diagnostic agent can target / bind to activated microglia, activated macrophages, and / or RPE cells via a dendrimer or suitable delivery vehicle.

[0038] The term "prophylactic agent" refers to an active agent that can be administered to prevent disease or to prevent a particular condition.

[0039] The phrases "pharmaceutically acceptable" or "biocompatible" refer to compositions, polymers, and other materials, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material, involved in carrying or transporting any subject composition from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not injurious to the patient.

[0040] The term "therapeutically effective amount" refers to an amount of a therapeutic agent that, when incorporated into and / or onto a dendrimer, exerts some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. The effective amount may vary depending on factors such as the disease or condition being treated, the specific target structure to which it is administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound without undue experimentation. In some embodiments, the term "effective amount" refers to an amount of a therapeutic or prophylactic agent that reduces or diminishes one or more symptoms of an ocular disease or disorder, such as reducing or inhibiting one or more proinflammatory cytokines and / or cells associated with affected tissues / cells of the eye, thereby reducing inflammation. In the case of retinal and / or choroidal neovascularization, an effective amount of a drug may be effective in reducing retinal and / or choroidal neovascularization; inhibiting, to some extent, the growth / proliferation of vascular endothelial cells; and / or alleviating, to some extent, one or more symptoms associated with the disorder. An effective amount can be administered in one or more administrations.

[0041] The terms "inhibit" or "reduce" in the context of inhibition refer to a decrease or decrease in activity or amount. This can be a complete inhibition or reduction or a partial inhibition or reduction of activity or amount. Inhibition or reduction can be compared to a control or standard level. Inhibition can be 5, 10, 25, 50, 75, 80, 85, 90, 95, 99, or 100%. For example, a dendrimer composition containing one or more therapeutic agents 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% from the activity and / or amount of the same cells in comparable diseased tissue of a subject that has not received or been treated with the dendrimer composition (i.e., unconjugated active agent). In some embodiments, inhibition and reduction are compared at the mRNA, protein, cell, tissue, and organ levels. For example, inhibition and / or reduction of pro-inflammatory cytokines (e.g., TNF-α, interleukin-1β (IL-β), or interferon-γ (IFN-γ)) secreted by activated microglia and macrophages in the diseased retina and / or choroid.

[0042] The terms "treating" a disease, disorder, or condition, or "preventing" such a condition from occurring in an animal believed to be predisposed to the disease, disorder, and / or condition but not yet diagnosed with the condition, include: inhibiting the disease, disorder, or condition, e.g., preventing its progression; and alleviating the disease, disorder, or condition, e.g., causing regression of the disease, disorder, and / or condition. Treating a disease or condition includes ameliorating at least one symptom of a particular disease or condition even when the underlying pathophysiology is unaffected, such as treating pain in a subject by administering an analgesic even if the analgesic does not treat the cause of the pain. Desirable effects of treatment include slowing the rate of disease progression, remission or alleviation of the disease state, and remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with an ocular disease or disorder are reduced or eliminated, including, but not limited to, a reduction in the proliferation of pro-inflammatory cells, a decrease in symptoms resulting from the disease, an enhancement or restoration of visual field, a decrease in the degree and rate of vision loss, an increase in the quality of life of an individual suffering from the disease, a reduction in the dose of other medications required to treat the disease, a delay in the progression of the disease, and / or an increase in the survival of the individual.

[0043] The term "biodegradable" refers to a material that will break down or erode under physiological conditions into smaller units or chemical species that can be metabolized, eliminated, or excreted by a subject. The degradation time is a function of composition and form.

[0044] The term "dendrimer" includes, but is not limited to, a molecular architecture having an inner core, inner layers (or "generations") of repeating units regularly attached to the inner core, and an outer surface of terminal groups attached to the outermost generation.

[0045] The term "functionalization" means modifying a compound or molecule in a manner that results in the attachment of a functional group or moiety. For example, a molecule may be functionalized by the introduction of a molecule that makes it a strong nucleophile or a strong electrophile.

[0046] The term "targeting moiety" refers to a moiety that localizes to or leaves a specific location. The moiety may be, for example, a protein, a nucleic acid, a nucleic acid analog, a carbohydrate, or a small molecule. The entity may be, for example, a therapeutic compound such as a small molecule, or a diagnostic entity such as a detectable label. The location may be a tissue, a particular cell type or cell activation state, or a subcellular compartment. In some embodiments, the targeting moiety directs the localization of an active agent.

[0047] The term "extended residence time" refers to an increase in the time required for a drug to clear from a patient's body or from the patient's organs or tissues. In certain embodiments, "extended residence time" refers to a drug that clears with a half-life that is 10%, 20%, 50%, or 75% longer than a comparison standard, such as a comparable drug that is not conjugated to a delivery vehicle such as a dendrimer. In certain embodiments, "extended residence time" refers to a drug that clears with a half-life that is 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 times longer than a comparison standard, such as a comparable drug that is not conjugated to a dendrimer that specifically targets a particular cell type associated with a tumor.

[0048] The terms "incorporated" and "encapsulated" refer to incorporating, formulating, or otherwise including an active agent within and / or on the surface of a composition that allows for release, such as sustained release, of such agent in a desired application. The active agent or other material can be incorporated into a dendrimer, including by including one or more surface functional groups of such a dendrimer (by covalent, ionic, or other bonding interactions), physical mixing, entrapment of the agent within the dendrimer structure, encapsulation within the dendrimer structure, etc. II. Composition

[0049] Dendrimer complexes suitable for delivering one or more active agents, particularly one or more active agents that prevent, treat, or diagnose one or more diseases or disorders of the eye.

[0050] The dendrimer complex composition comprises one or more prophylactic, therapeutic, and / or diagnostic agents encapsulated, associated with, and / or conjugated within the dendrimer complex at a weight concentration of about 0.01% weight / 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 prophylactic, therapeutic, and / or diagnostic agent is covalently conjugated to the dendrimer via one or more bonds selected from disulfide, ester, ether, thioester, carbamate, carbonate, hydrazine, and amide, optionally via one or more spacers. Preferably, the hydroxyl group of a hydroxyl-terminated dendrimer is covalently conjugated to one or more active agents via at least one ether bond, optionally via one or more linkers / spacers. In a preferred embodiment, the surface groups of hydroxyl-terminated dendrimers are modified via an etherification reaction prior to conjugation with one or more linkers and active agents. When one or more linkers are present between the dendrimer and the active agent, the covalent bond between the dendrimer surface group and the linker is an ether bond. In another embodiment, at generation 3.5 of the dendrimer, alkyne functionality is introduced using a polyethyl glycol (PEG) linker with an amine at one end and a hexyne at the other to generate a generation 4 bifunctional dendrimer. An exemplary bifunctional dendrimer is shown as Compound 1 in Figure 11, with seven alkyne arms and 57 hydroxyl groups on the surface.

[0051] In some embodiments, the spacer is a prophylactic, therapeutic, and / or diagnostic agent, such as sunitinib. Exemplary active agents include anti-angiogenic agents, anti-inflammatory agents, and anti-infective agents.

[0052] The presence of additional agents may affect the zeta potential or surface charge of the particles. In one embodiment, the zeta potential of the dendrimer 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 nearly neutral. The above ranges include all values ​​between -100 mV and 100 mV. A. Dendrimer

[0053] Dendrimers are three-dimensional, highly branched, monodisperse, spherical, and multivalent macromolecules with a high density of surface end groups (Tomalia, D.A., et al., Biochemical Society Transactions, 35, 61 (2007); and Sharma, A., et al., ACS MacroLetters, 3, 1079 (2014)). Due to their unique structure and physical characteristics, dendrimers are useful as nanocarriers in various biomedical applications, including targeted drug / gene delivery, imaging, and diagnostics (Sharma, A., et al., RSC Advances, 4, 19242 (2014); Caminade, A.-M., et al., Journal of Materials Chemistry B, 2, 4055 (2014); Esfand, R., et al., Drug Discovery Today, 6, 427 (2001); and Kannan, RM, et al., Journal of Internal Medicine, 276, 579 (2014)).

[0054] Dendrimer surface groups have a significant effect on their biodistribution (Nance, E., et al., Biomaterials, 101, 96 (2016)). Hydroxyl-terminated fourth-generation PAMAM dendrimers (approximately 4 nm in size) without any targeting ligands crossed the compromised BBB significantly more (>20-fold) in a rabbit model of cerebral palsy (CP) after systemic administration compared to healthy controls, selectively targeting activated microglia and astrocytes (Lesniak, W.G., et al., Mol Pharm, 10 (2013)).

[0055] The term "dendrimer" includes a molecular architecture having an inner core and layers (or "generations") of repeating units attached to and extending from the inner core, each layer having one or more branch points, with terminal groups attached to the outermost generation. In some embodiments, dendrimers have a regular dendrimer or "starburst" molecular structure.

[0056] Typically, dendrimers have diameters 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 in the same size range, although larger proteins such as antibodies can increase in size by 5-15 nm. Drugs are typically encapsulated at a drug to dendrimer ratio of between 1:1 and 4:1 for larger generation dendrimers. In preferred embodiments, the dendrimers have an effective diameter to penetrate ocular tissues and be retained within target cells for extended periods of time.

[0057] In some embodiments, the dendrimer has a molecular weight between about 500 and about 100,000 daltons, preferably between about 500 and about 50,000 daltons, and most preferably between about 1,000 and about 20,000 daltons.

[0058] Suitable dendrimer scaffolds that can be used include poly(amidoamine), also known as PAMAM or STARBURST™ dendrimers; polypropylamine (POPAM), polyethyleneimine, polylysine, polyester, iptycene, aliphatic poly(ether), and / or aromatic polyether dendrimers. The dendrimers can have carboxylic acid, amine, and / or hydroxyl termini. In preferred embodiments, the dendrimers are hydroxyl terminated. Each dendrimer in a dendrimer complex can be the same or can be of similar or different chemical nature to the other dendrimers (e.g., a first dendrimer can comprise a PAMAM dendrimer, while a second dendrimer can be a POPAM dendrimer).

[0059] The term "PAMAM dendrimer" refers to a poly(amidoamine) dendrimer that may contain different cores with amidoamine building blocks and can have carboxylic acid, amine, and hydroxyl termini of any generation, including, but not limited to, 1st generation PAMAM dendrimers, 2nd generation PAMAM dendrimers, 3rd generation PAMAM dendrimers, 4th generation PAMAM dendrimers, 5th generation PAMAM dendrimers, 6th generation PAMAM dendrimers, 7th generation PAMAM dendrimers, 8th generation PAMAM dendrimers, 9th generation PAMAM dendrimers, or 10th generation PAMAM dendrimers. In preferred embodiments, the dendrimers are soluble in formulations and are 4th, 5th, or 6th generation ("G") dendrimers. In preferred embodiments, the dendrimers have multiple hydroxyl groups attached to their functional surface groups.

[0060] Methods for producing dendrimers are known to those skilled in the art and generally involve a two-step iterative reaction sequence that produces concentric shells (generations) of dendritic β-alanine units around a central initiator core (e.g., an ethylenediamine core). Each subsequent growth step represents a new "generation" of polymer with a larger molecular diameter, twice the number of reactive surface sites, and approximately twice the molecular weight of the preceding generation. Suitable dendrimer scaffolds are commercially available in various generations. Preferably, dendrimer compounds are based on 0-, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-generation dendrimer scaffolds. Such scaffolds have 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, and 4096 reactive sites, respectively. Dendrimer compounds based on these scaffolds therefore have a corresponding number of targeting moiety-modulator combinations.

[0061] In some embodiments, the dendrimer comprises multiple hydroxyl groups. Some exemplary high-density hydroxyl-group-containing dendrimers include commercially available polyester dendritic polymers, such as hyperbranched 2,2-bis(hydroxyl-methyl)propionate polymers (e.g., hyperbranched bis-MPA polyester-64-hydroxyl, 4th generation), and dendritic polyglycerols.

[0062] In some embodiments, the dense hydroxyl-containing dendrimer is an oligoethylene glycol (OEG)-like dendrimer. For example, second-generation OEG dendrimers (D2-OH-60) can be synthesized using highly efficient, robust, and atom-economical chemical reactions, such as Cu(I)-catalyzed alkyne-azide click and photocatalyzed thiol-ene click chemistry. Highly dense polyol dendrimers at very low generations with minimal reaction steps can be achieved by using orthogonal hypermonomer and hypercore strategies, as described, for example, in WO2019094952. In some embodiments, the dendrimer backbone has non-cleavable polyether bonds throughout the structure to avoid dendrimer breakdown in vivo and enable elimination of such dendrimers as a single entity (non-biodegradable) from the body.

[0063] In some embodiments, dendrimers can specifically target particular tissue regions and / or cell types, preferably activated microglia and macrophages associated with one or more ocular diseases, hi preferred embodiments, dendrimers can specifically target particular tissue regions and / or cell types without the addition of a targeting moiety.

[0064] In a preferred embodiment, the dendrimer has a plurality of hydroxyl (-OH) groups on the surface of the dendrimer. The preferred surface density of hydroxyl (-OH) groups is at least 1 OH group / nm 2 (number of hydroxyl surface groups / nm 2 For example, in some embodiments, the surface density of hydroxyl groups is greater than 2, 3, 4, 5, 6, 7, 8, 9, 10; preferably at least 10, 15, 20, 25, 30, 35, 40, 45, 50, or 50 surface groups / 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 / nm2 The hydroxyl-terminated dendrimer has a surface area in units of 0.05 mm and a molecular weight between about 500 Da and about 10 kDa. In preferred embodiments, the percentage of free, i.e., unconjugated, hydroxyl groups among all surface groups (conjugated and unconjugated) on the dendrimer is greater than 70%, 75%, 80%, 85%, 90%, 95%, and / or less than 100%. For fourth-generation PAMAM dendrimers, the preferred number of free, i.e., unconjugated, hydroxyl groups is greater than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63 out of a total of 64 surface termini / groups. In further embodiments, the hydroxyl-terminated dendrimer has a number of free hydroxyl groups effective for selectively targeting activated microglia, activated microphages, and / or RPE cells associated with one or more diseases and / or disorders of the eye.

[0065] In some embodiments, dendrimers may have some of the hydroxyl groups exposed on the exterior surface, with other groups in the interior core of the dendrimer. In preferred embodiments, the dendrimer has at least 1 OH group / nm 3 (Number of hydroxyl groups / nm 3 For example, in some embodiments, the volume density of hydroxyl groups is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10, 15, 20, 25, 30, 35, 40, 45, and 50 hydroxyl groups / nm 3 In some embodiments, the volume density of hydroxyl groups is from about 4 to about 50 hydroxyl groups / nm 3 between about 5 and about 30 hydroxyl groups / nm 3 more preferably between about 10 and about 20 hydroxyl groups / nm 3 It is between. B. Coupling Agents and Spacers

[0066] Dendrimer complexes can be formed with therapeutically active agents or compounds conjugated or bound to dendrimers, dendritic polymers, or hyperbranched polymers. If desired, the active agent is conjugated to the dendrimer via one or more spacer / linkers via various linkages, such as disulfide, ester, carbonate, carbamate, thioester, hydrazine, hydrazide, and amide bonds. The one or more spacer / linkers between the dendrimer and the agent can be designed to provide a releasable (or cleavable) or non-releasable (or non-cleavable) form of the dendrimer-active complex in vivo. In some embodiments, conjugation occurs via a suitable spacer that provides an ester bond between the agent and the dendrimer. In some embodiments, conjugation occurs via a suitable spacer that provides an amide bond between the agent and the dendrimer. In preferred embodiments, one or more spacer / linkers between the dendrimer and the agent are added to achieve desired and effective release kinetics in vivo. In further embodiments, the conjugation of the dendrimer and / or linker does not significantly affect the activity of the active agent, e.g., in the case of a VEGFR TKR inhibitor, the VEGFR TKR inhibitor retains its binding affinity for one or more VEGFR TKRs after conjugation to a dendrimer at a level comparable to that of the unconjugated VEGFR TKR inhibitor.

[0067] The term "spacer" includes moieties and compositions used to attach therapeutically active agents to dendrimers. A spacer can be a single chemical entity or two or more chemical entities linked together to bridge the dendrimer and the active agent. Spacers can include any small chemical entity, peptide, or polymer with sulfhydryl, thiopyridine, succinimidyl, maleimide, vinyl sulfone, and carbonate termini.

[0068] The spacer can be selected from the classes of compounds terminated with sulfhydryl, thiopyridine, succinimidyl, maleimide, vinyl sulfone, and carbonate groups. The spacer can include a thiopyridine-terminated compound, such as dithiodipyridine, N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate LC-SPDP, or sulfo-LC-SPDP. The spacer can also comprise a peptide, which is linear or cyclic, essentially containing a sulfhydryl group, such as glutathione, homocysteine, cysteine ​​and its derivatives, arg-gly-asp-cys (RGDC), cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDfC)), cyclo(Arg-Gly-Asp-d-Tyr-Cys), and cyclo(Arg-Ala-Asp-d-Tyr-Cys). In some embodiments, the spacer comprises a mercapto acid derivative, such as 3-mercaptopropionic acid, mercaptoacetic acid, 4-mercaptobutyric acid, thiolan-2-one, 6-mercaptohexanoic acid, 5-mercaptovaleric acid, and other mercapto derivatives, such as 2-mercaptoethanol and 2-mercaptoethylamine. In some embodiments, the spacer comprises thiosalicylic acid and its derivatives, (4-succinimidyloxycarbonyl-methyl-alpha-2-pyridylthio)toluene, (3-[2-pyridylthio]propionylhydrazide. In some embodiments, the spacer comprises a maleimide terminus, which comprises a polymer or a small chemical entity, such as bis-maleimidodiethylene glycol and bis-maleimidotriethylene glycol, bis-maleimidoethane, and bismaleimidohexane. In some embodiments, the spacer comprises a vinyl sulfone, such as 1,6-hexane-bis-vinyl sulfone. In some embodiments, the spacer comprises a thioglycoside, such as thioglucose. In other embodiments, the spacer comprises reduced proteins, such as bovine serum albumin and human serum albumin, any thiol-terminated compound capable of forming a disulfide bond.In certain embodiments, the spacer comprises maleimide, succinimidyl, and thiol-terminated polyethylene glycol.

[0069] The therapeutically active agent, imaging agent, and / or targeting moiety can be covalently bound or dispersed or encapsulated intramolecularly. The dendrimer is preferably a 1st generation (G1), G2, G3, G4, G5, G6, G7, G8, G9, or G10 PAMAM dendrimer with carboxylic acid, hydroxyl, or amine terminus. In a preferred embodiment, the dendrimer is linked to the active agent via a spacer terminating in an ether or amide bond.

[0070] In some embodiments, the non-releasable form of a dendrimer / active agent conjugate provides enhanced therapeutic efficacy compared to the releasable or cleavable form of the same dendrimer / active agent conjugate. Thus, in some embodiments, one or more active agents are conjugated to the dendrimer via a spacer that is non-releasably linked to the dendrimer, for example, by an ether or amide bond. In some embodiments, one or more active agents are non-releasably linked to the spacer, for example, by an ether or amide bond. Thus, in some embodiments, one or more active agents are linked to the dendrimer via a spacer that is non-releasably linked to the dendrimer and to the active agent. In an exemplary embodiment, one or more active agents are linked to the dendrimer via a spacer that is linked to the dendrimer and to the active agent via an amide and / or ether bond. An exemplary spacer is polyethylene glycol (PEG). 1. Dendrimer conjugation to active agents via ether bonds

[0071] In some embodiments, the composition comprises a hydroxyl-terminated dendrimer conjugated via an ether bond to an active agent, optionally by one or more linkers / spacers.

[0072] In a preferred embodiment, the covalent bond between the surface group of the dendrimer and the linker, or between the dendrimer and the active agent (when conjugated without any linking moiety), is stable under in vivo conditions, i.e., minimally cleavable when administered to a subject and / or excreted intact 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 all dendrimer complexes have active agents cleaved 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 bond between the surface group of the dendrimer and the linker, or between the dendrimer and the active agent (when conjugated without any linking moiety), is not a hydrolytically or enzymatically cleavable bond, such as an ester bond.

[0073] In some embodiments, one or more hydroxyl groups of the hydroxyl-terminated dendrimer are conjugated to one or more linking moieties and one or more active agents via one or more ether linkages, as shown in formula (I) below: [ka] where D is a G2 to G10 poly(amidoamine) (PAMAM) dendrimer; L is one or more linking moieties or spacers; X is an active agent or analog thereof; n is an integer from 1 to 100; m is an integer from 16 to 4096; Y is a linker selected from secondary amide (-CONH-), tertiary amide (-CONR-), sulfonamide (-S(O)-NR-), secondary carbamate (-OCONH-; -NHCOO-), tertiary carbamate (-OCONR-; -NRCOO-), carbonate (-OC(O)-O-), urea (-NHCONH-; -NRCONH-; -NHCONR-, -NRCONR-), carbinol (-CHOH-, -CROH-), disulfide group, hydrazone, hydrazide, and ether (-O-), where R is an alkyl group, an aryl group, or a heterocyclic group. Preferably, Y is a bond or linkage that is minimally cleavable in vivo.

[0074] In some embodiments, X is an inhibitor of vascular endothelial growth factor receptor (VEGFR) and / or TIE2 receptor tyrosine kinase.

[0075] In a preferred embodiment, Y is a secondary amide (-CONH-).

[0076] In one embodiment, D is a fourth generation PAMAM dendrimer; L is one or more linking or spacer moieties; X is sunitinib, or an analog thereof; n is about 5 to 15; m is an integer between about 49 and 59; and n+m=64.

[0077] In another embodiment, D is a fourth generation PAMAM dendrimer; L is one or more linking or spacer moieties; X is N,N-didecethylsunitinib; Y is a secondary amide (-CONH-); n is about 5 to 15; m is an integer between about 49 and 59; and n+m=64.

[0078] In certain embodiments, Formula I has the following structure (also referred to as D-4517.2): Structure I: Chemical structure of D-4517.2 [ka] C. Therapeutic, Prophylactic, and Diagnostic Agents

[0079] Dendrimers have the advantage that multiple therapeutic, prophylactic, and / or diagnostic agents can be delivered with the same dendrimer. In some embodiments, one or more types of active agents are encapsulated, complexed, or conjugated to the dendrimer. In other embodiments, the dendrimer is covalently attached to at least one detectable moiety in an amount effective to detect one or more diseased or damaged cells / tissues in a subject. In certain embodiments, the dendrimer composition has multiple agents complexed or conjugated to the dendrimer, such as immunotherapeutic agents, anticonvulsants, steroids that reduce swelling, antibiotics, antiangiogenic agents, and / or diagnostic agents. In some embodiments, the dendrimer is complexed or conjugated to two or more different classes of active agents, resulting in simultaneous delivery with different or independent release kinetics at the target site. For example, sunitinib and an anti-inflammatory agent can be conjugated to the same dendrimer for delivery to target cells / tissues. In further embodiments, dendrimer complexes, each carrying a different class of active agent, are administered simultaneously for combination treatment: in some embodiments, one or more therapeutic agents that target the underlying cause of a disease or condition, and one or more therapeutic agents that alleviate one or more symptoms of a disease or condition.

[0080] Suitable active agents include therapeutic, diagnostic, and / or prophylactic agents. Agents can include biomolecules such as enzymes, proteins, polypeptides, or nucleic acids, or small molecule agents (e.g., molecular weights less than 2000 Daltons, preferably less than 1500 Daltons, and more preferably 300-700 Daltons), including organic, inorganic, and organometallic agents. Agents can be encapsulated within the dendrimer, dispersed within the dendrimer, and / or associated with the dendrimer surface, either covalently or noncovalently. Exemplary therapeutic agents include anti-inflammatory agents, anti-angiogenic agents, antioxidants, vasodilators, neuroactive agents, neuroprotective agents, and anti-infective agents. In some embodiments, dendrimers are linked to targeting moieties, imaging agents, and / or therapeutic agents via linkers or spacers terminating in disulfide, ester, ether, thioester, carbamate, carbonate, hydrazine, or amide bonds.

[0081] Dendrimers can be used to deliver one or more additional active agents, particularly one or more active agents that prevent or treat one or more symptoms of ocular disease. Exemplary therapeutic agents that can be administered with dendrimers include tyrosine kinase inhibitors, such as VEGFR tyrosine kinase inhibitors. In a preferred embodiment, the agent is a small molecule tyrosine kinase inhibitor.

[0082] Representative anti-angiogenic agents include, but are not limited to, antibodies against vascular endothelial growth factor (VEGF), such as bevacizumab (AVASTIN®) and rhuFAb V2 (ranibizumab, LUCENTIS®), as well as other anti-VEGF compounds including aflibercept (EYLEA®); MACUGEN® (pegaptanim sodium, anti-VEGF aptamer or EYE001) (Eyetech Pharmaceuticals; pigment epithelium-derived factor (PEDF); COX-2 inhibitors, such as celecoxib (CELEBREX®) and rofecoxib (VIOXX®); interferon alpha; interleukin-12 (IL-12); thalidomide (THALOMID®) and its derivatives, such as lenalidomide (REVLIMID®); squalamine; endostatin; angiostatin; ribozyme inhibitors, such as ANGIOZYME® (Sirna Therapeutics); multifunctional antiangiogenic agents, such as NEOVASTAT® (AE-941) (Aeterna Laboratories, Quebec) City, Canada); receptor tyrosine kinase (RTK) inhibitors, such as sunitinib (SUTENT®); tyrosine kinase inhibitors, such as sorafenib (Nexavar®) and erlotinib (Tarceva®); antibodies against the epidermal growth factor receptor, such as panitumumab (VECTIBIX®) and cetuximab (ERBITUX®), and other anti-angiogenic agents known in the art.

[0083] Other active agents suitable for antiangiogenic therapy include those targeting members of the platelet-derived growth factor family, epidermal growth factor family, fibroblast growth factor family, transforming growth factor-β superfamily (TGF-β1, activin, follistatin, and bone morphogenetic protein), angiopoietin-like family, galectin family, integrin superfamily, as well as pigment epithelium-derived factor, hepatocyte growth factor, angiopoietin, endothelin, hypoxia-inducible factor, insulin-like growth factor, cytokines, matrix metalloproteinases and their inhibitors, and glycosylated proteins. tyrosine kinase inhibitors

[0084] In some embodiments, the dendrimer is complexed or conjugated to one or more tyrosine kinase inhibitors.

[0085] Tyrosine kinases are important cell signaling proteins with diverse biological activities, including cell proliferation and migration. Numerous kinases, including receptor tyrosine kinases such as vascular epidermal growth factor receptor (VEGFR), are involved in angiogenesis. Antiangiogenic tyrosine kinase inhibitors in clinical development primarily target VEGFR-1, -2, -3, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), PDGFR-β, KIT, fms-related tyrosine kinase 3 (FLT3), colony-stimulating factor-1 receptor (CSF-1R), Raf, and RET. VEGFR inhibitors

[0086] In some embodiments, the dendrimer is complexed or conjugated to one or more VEGFR tyrosine kinase inhibitors. The VEGFR family includes three related receptor tyrosine kinases known as VEGFR-1, -2, and -3, which mediate the angiogenic effects of VEGF ligands (Hicklin DJ, Ellis LM. J Clin Oncol. (2005), 23(5):1011-27). The VEGF family encoded by mammalian genomes is VEGF, which contains five members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). VEGF is a key stimulator of endothelial cell proliferation and migration. Increased expression of the angiogenic factor VEGF-A also promotes three common age-related eye conditions—the "wet" and "dry" forms of age-related macular degeneration—as well as cataracts in animal models (Marneros AG, EMBO Molecular Medicine, 2016; 8 (3): 208). Thus, in some embodiments, the dendrimer is conjugated to one or more active agents effective to reduce the amount and / or activity of one or more of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF).

[0087] The most prominent angiogenesis inhibitors target the vascular endothelial growth factor signaling pathway, such as the monoclonal antibody bevacizumab (AVASTIN®, Genentech / Roche) and two kinase inhibitors, sunitinib (SU11248, SUTENT®, Pfizer) and sorafenib (BAY43-9006, NEXAVAR®, Bayer). Bevacizumab was the first angiogenesis inhibitor to be clinically approved, initially for the treatment of colorectal cancer and more recently for the treatment of breast and lung cancer. Another clinically available anti-VEGF agent is pegaptanib sodium, an aptamer for neovascular AMD. Unlike bevacizumab, which binds to all VEGF isoforms, pegaptanib targets only VEGF165, the isoform responsible for pathological ocular neovascularization. In some embodiments, the dendrimer is conjugated to one or more VEGF inhibitors, including bevacizumab and pegaptanib sodium.

[0088] The small molecule tyrosine kinase inhibitors sunitinib and sorafenib target VEGF receptors (VEGFRs), primarily VEGFR-2. Both drugs have been shown to benefit patients with renal cell carcinoma (Motzer RJ, Bukowski RM, J Clin Oncol. (2006); 24(35):5601-8). Sunitinib is a potent inhibitor of angiogenesis and has been shown to inhibit corneal neovascularization in rabbit models. Studies suggest that topical sunitinib is nearly three times more effective than bevacizumab (Perez-Santonja JJ et al., Am JOphthalmol. 2010 Oct;150(4):519-528). Sorafenib inhibits Raf serine kinase. Cediranib is an oral tyrosine kinase inhibitor of VEGF receptors (VEGFRs).

[0089] In some embodiments, the dendrimer is conjugated to one or more VEGF receptor inhibitors, including sunitinib (SU11248; SUTENT®), sorafenib (BAY439006; NEXAVAR®), pazopanib (GW786034; VOTRIENT®), vandetanib (ZD6474; ZACTIMA®), axitinib (AG013736), cediranib (AZD2171; RECENTIN®), vatalanib (PTK787; ZK222584), dasatinib, nintedanib, and motesanib (AMG706). In preferred embodiments, the VEGF receptor inhibitor can be functionalized with one or more spacers / linkers, e.g., with ether, ester, or amide bonds, e.g., to facilitate conjugation to the dendrimer and / or for desired release kinetics. For example, sunitinib can be modified to sunitinib with an ester bond or an amide bond (FIGS. 1A and 1B). Exemplary conjugation of a VEGF receptor inhibitor, such as sunitinib, to a dendrimer is shown in FIG. 1A (via a hydroxymethyl bond) and FIG. 1B (via an amide bond). In preferred embodiments, the conjugation of the dendrimer and / or one or more linkers does not significantly affect the activity of the active agent. In further preferred embodiments, the VEGF receptor inhibitor is conjugated to the dendrimer with or without a spacer such that the reduction in inhibition is minimized, for example, by less than 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, and 100-fold. For example, sunitinib retains its binding affinity to one or more of the VEGFR TKRs after conjugation to a dendrimer at a level comparable to that of unconjugated sunitinib.

[0090] Additional VEGF receptor inhibitors with functional spacers / linkages are shown below. Structure II ab: Chemical structures of sorafenib analogues [ka] Structure III a-d: Chemical structures of nintedanib analogue 1 [ka] Structure IV: Chemical structure of orantinib analogues [ka] Orantinib-amide-linker azide

[0091] In some embodiments, the dendrimer conjugates comprising one or more VEGF receptor inhibitors are administered in an amount effective to reduce or inhibit endothelial cell angiogenesis and / or vascular endothelial cell proliferation, reduce retinal and / or choroidal neovascularization, and / or alleviate one or more symptoms associated with an ocular disease or disorder. TIE II antagonists

[0092] In some embodiments, the dendrimer is complexed or conjugated with one or more inhibitors of TIE II. The angiopoietin-1 receptor, also known as CD202B (cluster of differentiation 202B) and TIE II, is a protein encoded by the TEK gene in humans. TIE2 is an angiopoietin receptor. Angiopoietins are protein growth factors required for the formation of blood vessels (angiogenesis), which supports tumor growth and development. Thus, in some embodiments, the dendrimer is conjugated to one or more TIE II antagonists.

[0093] In some embodiments, the active agent is an inhibitor of TIE II receptor tyrosine kinase. Exemplary inhibitors of VEGFR / TIE II include CEP-11981 and rebastinib. TIE II antagonists can be functionalized with, for example, ether, ester, ethyl, or amide bonds to facilitate conjugation with dendrimers and / or for desired release kinetics. The chemical structure of an exemplary TIE II antagonist is shown below as Structure XXI. The TIE II of the free TIE II antagonist (Structure V) II inhibition is determined by the dissociation constant, K d , approximately 8.8 nm, and the TIE II inhibition of the dendrimer-conjugated TIE II antagonist (structure XXI) has a dissociation constant, K d , about 25 nm. Thus, in preferred embodiments, the TIE II antagonist is conjugated to the dendrimer with or without a spacer to minimize, for example, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, and 100-fold reduction in TIE II inhibition. In preferred embodiments, the active agent is an inhibitor of vascular endothelial growth factor receptor (VEGFR) and TIE II receptor tyrosine kinase. Structure V: TIE II antagonist 1 [ka] anti-inflammatory agents

[0094] In some embodiments, the one or more active agents associated with or complexed to the dendrimer are one or more anti-inflammatory agents. Anti-inflammatory agents reduce inflammation and include steroidal and non-steroidal drugs. Suitable steroidal active agents include glucocorticoids, progestins, mineralocorticoids, and corticosteroids. In some embodiments, the one or more active agents are one or more corticosteroids.

[0095] Exemplary anti-inflammatory agents include triamcinolone acetonide, fluocinolone acetonide, methylprednisolone, prednisolone, dexamethasone, loteplendole, fluorometholone, ibuprofen, aspirin, and naproxen. Exemplary immunomodulatory agents include cyclosporine, tacrolimus, and rapamycin. Exemplary nonsteroidal anti-inflammatory drugs (NSAIDs) include mefenamic acid, aspirin, diflunisal, salsalate, ibuprofen, naproxen, fenoprofen, ketoprofen, deaketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, sulindac, etodolac, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, meclofenamic acid, flufenamic acid, tolfenamic acid, elecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, firocoxib, sulfonanilides, nimesulide, niflumic acid, and licofelone. In preferred embodiments, the active agent is triamcinolone acetonide, prednisolone, dexamethasone, or an analog thereof. Exemplary analogs of triamcinolone acetonide, prednisolone, and dexamethasone are shown below. Structure VI af: Chemical structures of analogues of triamcinolone acetonide, prednisolone, and dexamethasone [ka]

[0096] In some embodiments, the active agent is N-acetyl-L-cysteine, or a derivative, analog, or prodrug thereof. In a preferred embodiment, N-acetyl-L-cysteine ​​is conjugated to a hydroxyl-terminated PAMAM dendrimer via a non-cleavable bond, resulting in minimal release of free N-acetyl-cysteine ​​after in vivo administration. A synthetic route for an exemplary non-releasable (or non-cleavable) form of a dendrimer / N-acetyl-cysteine ​​conjugate is shown in FIG. 14. In one embodiment, the dendrimer conjugate is a dendrimer-NAC-carboxymethylated conjugate shown in FIG. 14. The non-releasable form of the dendrimer / N-acetyl-cysteine ​​conjugate provides enhanced therapeutic efficacy compared to a releasable or cleavable form of the dendrimer / N-acetyl-cysteine ​​conjugate, for example, N-acetyl-L-cysteine ​​conjugated to a hydroxyl-terminated PAMAM dendrimer via an ester bond.

[0097] In some embodiments, the one or more active agents are polysialic acid (e.g., low molecular weight polysia with an average degree of polymerization of 20 (polysiaavDP20)), translocator protein ligand (e.g., diazepam binding inhibitor (DBI)), interferon-β (IFN-β), and minocycline.

[0098] In some cases, one or more active agents are anti-infective agents. 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 agents: amphotericin, voriconazole, and natamycin. diagnostic agents

[0099] Dendrimer nanoparticles can contain diagnostic agents useful for determining the location of administered particles. These agents can also be used prophylactically. In some embodiments, the dendrimers are conjugated to one or more diagnostic agents, including indocyanine green, fluorescein (e.g., fluorescein isocyanate), boron-dipyrromethene, rhodamine, and rose bengal. In a preferred embodiment, the diagnostic agent is indocyanine green, as shown below: Structure VII: Chemical structure of indocyanine green [ka]

[0100] Additional examples of diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray contrast agents, and contrast media. Other examples of suitable contrast agents include radiopaque gases or gas-releasing compounds. The dendrimer complex can further include an agent useful for determining the location of the administered composition. Agents useful for this purpose include fluorescent tags, radionuclides, and contrast agents.

[0101] Exemplary diagnostic agents include dyes, such as fluorescent dyes and near-infrared dyes, SPECT imaging agents, PET imaging agents, and radioisotopes. Representative dyes include carbocyanines, indocarbocyanines, oxacarbocyanines, thiacarbocyanines, and merocyanines, polymethines, coumarins, rhodamines, xanthenes, fluoresceins, boron-dipyrromethanes (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IRDye 800CW ... These include the 800RS, IRDye 700DX, ADS780WS, ADS830WS, and ADS832WS.

[0102] Exemplary SPECT or PET imaging agents include chelating agents such as diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diaminedithiols, activated mercaptoacetyl-glycyl-glycyl-glycine (MAG3), and hydrazidonicotinamide (HYNIC).

[0103] 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.

[0104] In a preferred embodiment, the dendrimer conjugate comprises one or more radioisotopes suitable for positron emission tomography (PET) imaging. Exemplary positron-emitting radioisotopes include carbon-11 ( 11 C), copper-64( 64 Cu), nitrogen-13( 13 N), oxygen-15( 15 O), Gallium-68( 68 Ga), and fluorine-18( 18 F), for example, 2-deoxy-2- 18 F-fluoro-β-D-glucose ( 18 F-FDG).

[0105] In preferred embodiments, the one or more diagnostic agents are functionalized with one or more spacers / linkers, e.g., with ether, ester, or amide bonds, to facilitate conjugation to the dendrimer and / or to facilitate desired release kinetics.

[0106] In a further embodiment, a single dendrimer complex composition can simultaneously treat and / or diagnose a disease or condition at one or more locations in the body. III. Pharmaceutical Preparations

[0107] Pharmaceutical compositions containing one or more dendrimer conjugates may be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants that facilitate processing of the active compounds into pharmaceutically usable preparations. The appropriate formulation depends on the selected route of administration. Pharmaceutical formulations contain one or more dendrimer conjugates in combination with one or more pharmaceutically acceptable excipients. Representative excipients include solvents, diluents, pH adjusters, preservatives, antioxidants, suspending agents, wetting agents, viscosity adjusters, isotonicity agents, stabilizers, and combinations thereof. Suitable pharmaceutically acceptable excipients are preferably selected from materials generally recognized as safe (GRAS) and can be administered to individuals without causing undesired biological side effects or unwanted interactions.

[0108] 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 will be formulated with sterile saline or buffer solution for injection into the tissue or cell to be treated.The composition can be freeze-dried and stored in a single-use vial for rehydration immediately before use.Other means for rehydration and administration are known to those skilled in the art. Remington's Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704, provides suitable formulations, and examples of ophthalmic drugs administered in the form of pharmaceutically acceptable salts include timolol maleate, brimonidine tartrate, and sodium diclofenac.

[0109] The compositions are preferably formulated in unit dosage form for ease of administration and uniformity of dosage. The term "unit dosage form" refers to a physically discrete unit of the conjugate appropriate for the patient being treated. However, it will be understood that the total amount of a single administration of the composition will be determined by the attending physician within the scope of sound medical judgment. The therapeutically effective dose can be initially estimated in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. Animal models are also used to achieve a desired concentration range and route of administration. Such information should then be used to determine useful doses and routes of administration in humans. The therapeutic efficacy and toxicity of the conjugates can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, as the ED50 (the dose therapeutically effective in 50% of the population) and the LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for human use.

[0110] Pharmaceutical compositions formulated for parenteral administration (intramuscular, intraperitoneal, intravenous, or subcutaneous injection) and enteral administration routes are described. In a preferred embodiment, the compositions are administered systemically. In one embodiment, the compositions are administered via the subcutaneous route. In another embodiment, the compositions are administered orally. A. Parenteral Administration

[0111] The phrases "parenteral administration" and "administered parenterally" are art-recognized terms and include forms of administration other than enteral and topical administration, such as injection, including, but not limited to, intravenous, intramuscular, intrathoracic, intravascular, intrapericardial, intraarterial, intraspinal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. In some embodiments, the dendrimer is administered parenterally, for example, by subdural, intravenous, intraspinal, intraventricular, intraarterial, intraamniotic, intraperitoneal, or subcutaneous routes. In a preferred embodiment, the dendrimer composition is administered via subcutaneous injection.

[0112] For liquid formulations, pharmaceutically acceptable carriers can be, for example, aqueous or non-aqueous solutions, suspensions, emulsions, or oils. Parenteral vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions, or suspensions, including saline and buffered media. Dendrimers can also be administered in emulsions, such as water-in-oil. Examples of oils include those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish liver oil, sesame oil, cottonseed oil, corn oil, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include, for example, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0113] Formulations suitable for parenteral administration can include aqueous and non-aqueous sterile suspensions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose. In general, water, saline, aqueous dextrose, and related sugar solutions, as well as glycols, such as polypropylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

[0114] Injectable pharmaceutical carriers are well known to those skilled in the art (see, e.g., Pharmaceutics and Pharmacy Practice, JB Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHPH Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630 (2009)). B. Enteral Administration

[0115] In some embodiments, the composition is formulated to be administered enterally. The carrier or diluent may be a solid carrier such as a capsule or tablet, or a diluent for a solid formulation, a liquid carrier or diluent for a liquid formulation, or a mixture thereof.

[0116] For liquid preparations, pharmaceutically acceptable carriers can be, for example, aqueous or non-aqueous solutions, suspensions, emulsions, or oils.Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and injectable organic esters, such as ethyl oleate.Aqueous carriers include, for example, water, alcoholic / aqueous solutions, cyclodextrins, emulsions, or suspensions, including saline and buffered media.

[0117] Examples of oils are those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, fish liver oil, sesame oil, cottonseed oil, corn oil, olive, petrolatum and mineral oil.The fatty acid suitable for use in parenteral formulations includes, for example, oleic acid, stearic acid and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0118] Vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Formulations can include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain, for example, antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which can contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Vehicles can include, for example, fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. In general, water, saline, aqueous dextrose, and related sugar solutions are preferred liquid carriers. These can also be formulated with proteins, fats, sugars, and other components of infant formula.

[0119] In a preferred embodiment, the composition is formulated for oral administration.The oral formulation may take the form of chewing gum, gel strips, tablets, capsules, or lozenges.Encapsulating materials for preparing enteric-coated oral formulations include cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, and methacrylic acid ester copolymers.Solid oral formulations such as capsules or tablets are preferred.Elixirs and syrups are also well-known oral formulations. IV. Preparation Method A. Methods for Making Dendrimers

[0120] Dendrimers can be prepared via a variety of chemical reaction steps. Dendrimers are usually synthesized by methods that allow control of their structure at every stage of construction. Dendritic structures are mostly synthesized by two main different approaches: divergent or convergent.

[0121] In some embodiments, dendrimers are prepared using a divergent methodology in which dendrimers are assembled from an outwardly extending multifunctional core through a series of reactions, typically a Michael reaction. The strategy involves coupling monomer molecules bearing reactive and protecting groups with the multifunctional core moiety, resulting in stepwise generational additions around the core, followed by removal of the protecting groups. For example, PAMAM-NH2 dendrimers are first synthesized by coupling N-(2-aminoethyl)acrylamide monomers to an ammonia core.

[0122] In other embodiments, dendrimers are prepared using a convergent method, in which the dendrimer is built from small molecules that terminate on the surface of a sphere and the reaction proceeds to build inward, eventually attaching to the core.

[0123] Many other synthetic routes exist for the preparation of dendrimers, such as the orthogonal approach, the accelerated approach, the two-step convergent approach or hypercore approach, the hypermonomer approach or branched monomer approach, the double exponential approach; the orthogonal coupling approach or two-step approach, the two-monomer approach, the AB2-CD2 approach, etc.

[0124] In some embodiments, the dendrimer core, one or more branching units, one or more linkers / spacers, and / or one or more surface groups can be modified to conjugate to additional functional groups (such as branching units, linkers / spacers, surface groups), monomers, and / or active agents via click chemistry using one or more of copper-assisted azide-alkyne cycloaddition (CuAAC), Diels-Alder reaction, thiol-ene and thiol-yne reactions, and azide-alkyne reactions (ArseneaultM et al., Molecules. 2015 May 20;20(5):9263-94). Prefabricated dendrons are clicked onto the high-density hydroxyl polymer. "Click chemistry" involves the coupling of two different moieties (e.g., a core group and a branching unit; or a branching unit and a surface group) via a 1,3-dipolar cycloaddition reaction between, for example, an alkyne moiety (or its equivalent) on the surface of a first moiety and an azide moiety (e.g., present on a triazine composition) (or its equivalent) on a second moiety (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.).

[0125] In some embodiments, dendrimer synthesis relies on one or more reactions selected from thiol-ene click reaction, thiol-yne click reaction, CuAAC, Diels-Alder click reaction, azide-alkyne click reaction, Michael addition, epoxy ring-opening, esterification, silane chemistry, and combinations thereof.

[0126] Any existing dendritic platform can be used to create dendrimers of desired functionality, i.e., dendrimers with a high density of surface hydroxyl groups, by conjugating high hydroxyl-containing moieties such as 1-thio-glycerol or pentaerythritol. Exemplary dendritic platforms, such as polyamidoamine (PAMAM), poly(propyleneimine) (PPI), poly-L-lysine, melamine, poly(etherhydroxylamine) (PEHAM), poly(esteramine) (PEA), and polyglycerol, can be synthesized and investigated.

[0127] Even more suitable dendrons can be prepared by combining two or more dendrons. A dendron is a wedge-shaped section of a dendrimer that carries a reactive focal group. Many dendron scaffolds are commercially available. They come in generations 1, 2, 3, 4, 5, and 6, with 2, 4, 8, 16, 32, and 64 reactive groups, respectively. In one particular example, one type of activator is linked to one type of dendron, and a different type of activator is linked to another type of dendron. The two dendrons are then connected to form the dendrimer. The two dendrons can be linked via click chemistry, i.e., a 1,3-dipolar cycloaddition reaction between an azide moiety on one dendron and an alkyne moiety on another dendron to form a triazole linker.

[0128] Exemplary methods for making dendrimers are detailed in International Patent Publication Nos. WO2009 / 046446, WO2015168347, WO2016025745, WO2016025741, WO2019094952, and U.S. Patent No. 8,889,101. B. Dendrimer Conjugates

[0129] Dendrimer conjugates can be formed with therapeutically active agents or compounds conjugated or attached to dendrimers, dendritic polymers, or hyperbranched polymers. Techniques for conjugating one or more active agents to dendrimers are known in the art and are described in detail in U.S. Published Application Nos. US2011 / 0034422, US2012 / 0003155, and US2013 / 0136697.

[0130] In some embodiments, one or more active agents are covalently bound to the dendrimer. In some embodiments, the active agent is attached to the dendrimer via a linking moiety designed to be cleaved in vivo. The linking moiety can be designed to be cleaved hydrolytically, enzymatically, or a combination thereof, to provide sustained release of the active agent in vivo. Both the composition of the linking moiety and its point of attachment to the active agent are selected so that cleavage of the linking moiety releases either the active agent or a suitable prodrug thereof. The composition of the linking moiety can also be selected in view of the desired release rate of the active agent.

[0131] In some embodiments, the bond is formed via one or more of a disulfide, ester, ether, thioester, carbamate, carbonate, hydrazine, or amide bond. In a preferred embodiment, the bond is formed via a suitable spacer that creates an ester or amide bond between the drug and the dendrimer, depending on the desired release kinetics of the active agent. In some cases, an ester bond is introduced for a cleavable form of the active agent. 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 bond between the active agent and the dendrimer.

[0132] The linking moiety generally comprises one or more organic functional groups. Examples of suitable organic functional groups include secondary amide (-CONH-), tertiary amide (-CONR-), sulfonamide (-S(O)2-NR-), secondary carbamate (-OCONH-; -NHCOO-), tertiary carbamate (-OCONR-; -NRCOO-), carbonate (-OC(O)-O-), urea (-NHCONH-; -NRCONH-; -NHCONR-, -NRCONR-), carbinol (-CHOH-, -CROH-), disulfide group, hydrazone, hydrazide, ether (-O-), and ester (-COO-, -CHOC-, CHROC-), where R is an alkyl group, an aryl group, or a heterocyclic group. Generally, the identity of the one or more organic functional groups in the linking moiety can be selected in view of the desired release rate of the active agent. Furthermore, one or more organic functional groups can be selected to facilitate covalent binding of an active agent to the dendrimer. In a preferred embodiment, binding can be achieved via a suitable spacer that creates a disulfide bridge between the agent and the dendrimer. The dendrimer conjugate can rapidly release the agent in vivo through a thiol exchange reaction under reducing conditions found in the body.

[0133] In certain embodiments, the linking moiety comprises one or more of the aforementioned organic functional groups in combination with a spacer group. The spacer group can be composed of any assembly of atoms, including oligomeric and polymeric chains; however, the total number of atoms in the spacer group is preferably between 3 and 200 atoms, more preferably between 3 and 150 atoms, more preferably between 3 and 100 atoms, and most preferably between 3 and 50 atoms. Examples of suitable spacer groups include alkyl groups, heteroalkyl groups, alkylaryl groups, oligo- and polyethylene glycol chains, and oligo- and poly(amino acid) chains. Variation in the spacer group provides additional control over the release of the anti-inflammatory agent in vivo. In embodiments in which the linking moiety comprises a spacer group, one or more organic functional groups will generally be used to connect the spacer group to both the anti-inflammatory agent and the dendrimer.

[0134] Reactions and strategies useful for covalently linking active agents to dendrimers are known in the art. See, for example, March, "Advanced Organic Chemistry," 5th Edition, 2001, Wiley-Interscience Publication, New York) and Hermanson, "Bioconjugate Techniques," 1996, Elsevier Academic Press, USA. The appropriate method for covalently linking a given active agent can be selected in view of the desired linking moiety and the overall structure of the active agent and dendrimer, as it involves functional group compatibility, protecting group strategies, and the presence of labile bonds.

[0135] The optimal drug loading will necessarily depend on many factors, including the choice of drug, the structure and size of the dendrimer, and the tissue to be treated. In some embodiments, one or more active agents are encapsulated, associated with, and / or conjugated to the dendrimer at a concentration 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, dendrimer, and targeting site can be determined by routine methods, such as those described.

[0136] In some embodiments, conjugation of the active agent and / or linker occurs via one or more surface and / or internal groups. Thus, in some embodiments, conjugation of the active agent / linker occurs via approximately 1%, 2%, 3%, 4%, or 5% of all available surface functional groups, preferably hydroxyl groups, of the dendrimer prior to conjugation. In other embodiments, conjugation of the active agent / linker occurs via less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of all available surface functional groups of the dendrimer prior to conjugation. In preferred embodiments, the dendrimer complex retains an effective amount of surface functional groups for targeting a specific cell type while being conjugated to an effective amount of an active agent for treating, preventing, and / or imaging a disease or disorder. 1. Conjugation of dendrimers with active agents via ether bonds

[0137] A method has been developed to incorporate one or more active agents onto hydroxyl-terminated dendrimers via ether bonds, optionally via one or more linkers / spacers.

[0138] In some embodiments, the surface or end groups of a hydroxyl-terminated dendrimer are modified via an etherification reaction prior to conjugation with one or more linkers / spacers and one or more radionuclides. Etherification involves the dehydration of an alcohol to form an ether. In some embodiments, one or more hydroxyl groups of a hydroxyl-terminated dendrimer undergo an etherification reaction prior to conjugation with one or more linking moieties and one or more active agents.

[0139] In some embodiments, ether linkages are introduced at the surface groups of hydroxyl PAMAM dendrimers by reaction with propargyl bromide in the presence of 2% sodium hydroxide solution in DMSO. In a further embodiment, the etherification reaction of the fourth generation hydroxyl-terminated PAMAM dendrimer PAMAM-G4-OH uses allyl bromide, anhydrous cesium carbonate, and tetrabutylammonium iodide in DMF.

[0140] In another embodiment, alkyne functionality is introduced into a generation 3.5 dendrimer using a polyethyl glycol (PEG) linker with an amine at one end and a hexyne at the other to produce a generation 4 bifunctional dendrimer, i.e., one containing hydroxyl groups and ether linkages in preparation for further conjugation. An exemplary bifunctional dendrimer is shown as compound 1 in Figure 11, which contains seven alkyne arms and 57 hydroxyl groups on the surface. V. How to use

[0141] Dendrimer conjugate compositions are generally suitable for treating one or more diseases or disorders related to the eye, particularly inflammatory and / or neovascular diseases of the eye. Dendrimer compositions and methods for targeted delivery of one or more active agents to diseased tissues / cells within the eye via systemic administration with increased efficacy and reduced side effects are described, preferably those that selectively target diseased cells / tissues, including activated microgels and activated macrophages, retinal pigment epithelial (RPE) cells, and / or choroidal neovascular (CNV) lesions. Preferably, the dendrimer compositions and methods for targeted delivery cause minimal dendrimer release in undamaged areas of the optic nerve or CNS. Methods for treating disorders of the fundus are also described. In some embodiments, dendrimer conjugates are used to treat the wet form of age-related macular degeneration (AMD). The methods typically involve administering to a subject in need thereof an effective amount of a composition comprising a dendrimer and one or more active agents.

[0142] Methods for reducing and / or inhibiting the number or activity of activated microglia and macrophages in the retina and / or choroid of an eye in need thereof are provided. In some embodiments, treatment using an effective amount of a composition comprising a hydroxyl-terminated dendrimer complexed, covalently conjugated, intramolecularly dispersed, or encapsulated with one or more therapeutic agents is administered to reduce and / or inhibit the number or activity of activated microglia and macrophages in the retina and / or choroid of an eye in need thereof. In some embodiments, the composition is administered in a dosage and by a route that inhibits or reduces microglial activation in the retina. In other embodiments, the composition can inhibit or reduce microglial phagocytic activity. In other embodiments, a composition comprising one or more receptor tyrosine kinase inhibitors can 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% compared to the activity and / or amount of the same cells in equivalent diseased tissue of a subject that has not received or been treated with the dendrimer composition (e.g., unconjugated active agent).

[0143] Also described are 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 epithelial (RPE) cells in the affected retina and / or choroid. In some embodiments, the compositions are administered systemically, such as intravenously, subcutaneously, or orally. In preferred embodiments, the compositions are not administered intravitreally or subchoroidally, which may cause direct damage and / or inflammation to the eye. Also described are methods for reducing and / or inhibiting one or more pro-inflammatory cytokines secreted by activated microglia and macrophages in the affected retina and / or choroid. In some embodiments, treatment with an effective amount of the composition results in about a 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99% reduction in 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 compared to that in comparable diseased tissues of subjects that have not received or been treated with the dendrimer composition (e.g., unconjugated active agent).

[0144] Also described are methods for reducing and / or inhibiting one or more pro-oxidant properties of activated microglia and macrophages in diseased retina and / or choroid. 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 by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99%, compared to that of comparable diseased tissue from a subject not receiving or treated with the dendrimer composition (e.g., unconjugated active agent), for example, by reducing nitric oxide (NO) production or inducible nitric oxide synthase (iNOS) activation (e.g., NOS2 expression).

[0145] Also described are methods for reducing and / or inhibiting abnormal vascular permeability and leakage and / or neovascularization in the eye of a subject in need thereof. In some embodiments, treatment with an effective amount of the composition results in a reduction in vascular leakage and / or neovascularization. A. Treatment Regimen 1. Dosage and Effective Amount

[0146] Dosage and administration regimens depend on the severity and location of the disorder or injury and / or the method of administration, and are known to those skilled in the art. A therapeutically effective amount of a dendrimer composition used to treat one or more ocular diseases is typically sufficient to treat, inhibit, or alleviate one or more symptoms associated with the eye.

[0147] In some in vivo procedures, the dendrimer conjugates are administered to a subject in a therapeutically effective amount to reduce or inhibit ocular neovascularization, 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.

[0148] Pharmaceutical compositions are described that include a therapeutically effective amount of a dendrimer composition and a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the pharmaceutical composition includes an effective amount of a hydroxyl-terminated dendrimer conjugated to a VEGF receptor tyrosine kinase inhibitor. In certain embodiments, dosage ranges suitable for parenteral use are between about 0.1 mg / kg and about 200 mg / kg, inclusive; between about 0.5 mg / kg and about 100 mg / kg, inclusive; between about 1.0 mg / kg and about 40 mg / kg, inclusive; and between about 2.0 mg / kg and about 20 mg / kg, inclusive. A higher dose may be given initially to load the patient with the drug and maximize uptake in affected tissues (e.g., the eye). After the loading dose, the patient may receive a maintenance dose. The loading dose may range from 10 to 100 mg / kg body weight, and the maintenance dose may range from 0.1 to <10 mg / kg body weight. When administered enterally, the dose required for treatment may be up to 10 times higher than the effective parenteral dose. The optimal dose is selected based on the safety and efficacy results of each dose tested for each drug in patients.

[0149] Dosage forms of pharmaceutical compositions comprising dendrimer compositions are also provided. "Dosage form" refers to the physical form of a dose of a therapeutic compound, such as a capsule or vial, intended for administration to a patient. As used herein, the term "dosage unit" refers to the amount of a therapeutic compound administered to a patient in a single dose. In some embodiments, dosage units suitable for use are between 5 mg / dosage unit and about 14,000 mg / dosage unit, inclusive; between about 35 mg / dosage unit and about 7,000 mg / dosage unit, inclusive; and between about 70 mg / dosage unit and about 2,800 mg / dosage unit, inclusive; and between about 140 mg / dosage unit and about 1,400 mg / dosage unit, inclusive (assuming an average patient weighs 70 kg).

[0150] The actual effective amount of the dendrimer conjugate can vary depending on factors including the particular active agent administered, the particular composition formulated, the mode of administration, and the age, weight, condition, and route of administration of the subject being treated, and the disease or disorder. Preferably, the dendrimer composition containing one or more active agents, such as sunitinib, is delivered to cells (e.g., microglia) within and surrounding diseased or damaged tissue. For example, the dendrimer conjugate composition can be in an amount effective to deliver one or more active agents to cells at or near a site of inflammation, particularly an inflammation site in the eye. Thus, in some embodiments, the dendrimer conjugate composition containing one or more active agents is in an amount effective to ameliorate inflammation in a subject. In preferred embodiments, an effective amount of the dendrimer conjugate composition does not induce significant cytotoxicity in cells of a subject compared to an untreated control subject. Preferably, the amount of the dendrimer conjugate composition is effective to prevent or reduce inflammation and / or other associated symptoms of a disease or disorder in a subject compared to an untreated control.

[0151] Generally, the timing and frequency of administration will be adjusted to balance the efficacy of a given treatment or diagnostic schedule with the side effects of a given delivery system. Exemplary dosing frequencies include continuous infusion, single and multiple administrations, e.g., hourly, daily, weekly, monthly, or yearly dosing.

[0152] In some embodiments, the dosage is administered to a human once, twice, or three times daily, or less frequently, for example, every other day, every second day, every third day, every fourth day, every fifth day, or every sixth day. In some embodiments, the dosage is administered only about once or twice every week, every two weeks, every three weeks, or every four weeks. In some embodiments, the dosage is administered about once or twice every month, every two months, every three months, every four months, every five months, or every six months, or less frequently. In a preferred embodiment, the dosage is administered once every four weeks or less frequently.

[0153] Those skilled in the art will understand that the administration regimen can be any length of time that is sufficient to treat the target disorder.In some embodiments, the regimen comprises one or more cycles of one round of treatment, followed by a drug-free day (for example, drug-free).The round of treatment can be, for example, the administration described above.Similarly, the drug-free day can be 1, 2, 3, 4, 5, 6, or 7 days; or 1, 2, 3, 4 weeks; or 1, 2, 3, 4, 5, or 6 months. 2. Control

[0154] The treatment results of a dendrimer conjugate composition containing one or more active agents can be compared to a control. 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 symptom before and after administration of a targeted agent. The condition or symptom can be a biochemical, molecular, physiological, or pathological readout. For example, the effect of the composition on a particular symptom, pharmacological, or physiological indicator can be compared to an untreated subject, or to the subject's condition before treatment. In some embodiments, the symptom, pharmacological, or physiological indicator is measured in the subject before treatment and again one or more times after treatment has begun. In some embodiments, the control is a reference level or average determined based on measurements of the symptom, pharmacological, or physiological indicator in one or more subjects (e.g., healthy subjects) who do not have the disease or condition being treated. In some embodiments, the effect of the treatment is compared to conventional treatments known in the art. B. Subject to Treatment

[0155] The compositions and methods are suitable for treating one or more diseases or disorders of the eye, and for alleviating one or more symptoms associated with one or more diseases or disorders of the eye, such as discomfort, pain, dryness, excessive tearing, damage, infection, burns, and gradual loss of vision.

[0156] In some embodiments, the ocular disorder to be treated is a fundus disease, such as diabetic eye disease, symptomatic vitreous adhesion / vitreous traction (sVMA / VMT), and wet (neovascular) or dry AMD (age-related macular degeneration). In some embodiments, the ocular disorder 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, e.g., MEK retinopathy). In a preferred embodiment, the ocular disorder to be treated is age-related macular degeneration (AMD). Age-related macular degeneration (AMD) is a neurodegenerative, neuroinflammatory disease of the macula that causes central vision loss. The pathogenesis of age-related macular degeneration involves chronic neuroinflammation of the choroid (subretinal vascular layer), retinal pigment epithelium (RPE), the neurosensory subretinal cell layer, Bruch's membrane, and the neurosensory retina itself.

[0157] In other embodiments, the ocular disorder being treated is an ocular inflammatory disease, i.e., an ocular disease associated with inflammation of ocular tissues, including, for example, AMD, retinitis pigmentosa, optic neuritis, sarcoid, retinal detachment, temporal arteritis, retinal ischemia, atherosclerotic arteriopathy, hypertensive arteriopathy, retinal artery occlusion, retinal vein occlusion, diabetic retinopathy, macular edema, Stargardt disease (also known as Stargardt macular dystrophy or juvenile macular degeneration), geographic atrophy, neuromyelitis optica, and angiogenic diseases, including, for example, retinal neovascularization and choroidal neovascularization. Other conditions may result in inflammation and / or angiogenesis in the eye, such as infections, sickle cell disease, hypotension, etc.

[0158] Further examples of ocular disorders that may be treated include amoebic keratitis, fungal keratitis, bacterial keratitis, viral keratitis, onchocercal keratitis, bacterial keratoconjunctivitis, viral keratoconjunctivitis, corneal dystrophic diseases, Fuch's endothelial dystrophy, meibomian gland dysfunction, anterior and posterior blepharitis, conjunctival hyperemia, conjunctival necrosis, cicatricial scarring and fibrosis, punctate epithelial keratopathy, filamentous keratitis, corneal erosion, thinning, ulcers and perforations, Sjogren's syndrome, Stevens-Johnson syndrome, autoimmune dry eye disease, environmental dry eye disease. These include diseases such as corneal neovascularization, corneal transplant rejection, autoimmune uveitis, infectious uveitis, anterior uveitis, posterior uveitis (including toxoplasmosis), panuveitis, inflammatory diseases of the vitreous or retina, endophthalmitis, macular edema, macular degeneration, age-related macular degeneration, proliferative and non-proliferative diabetic retinopathy, hypertensive retinopathy, autoimmune diseases of the retina, primary and metastatic intraocular melanoma, other intraocular metastatic tumors, open-angle glaucoma, angle-closure glaucoma, pigmentary glaucoma, and combinations thereof. Other disorders include corneal injury, burns, or abrasions, cataracts, and associated age-related degeneration of the eye or visual field.

[0159] The dendrimer conjugates can be administered in combination with one or more additional therapeutically active agents known to be capable of treating the conditions or diseases discussed above.

[0160] The present invention will be further understood by reference to the following non-limiting examples. [Example]

[0161] Example 1 Targeted and sustained intracellular delivery to choroidal neovascular lesions after a single systemic administration demonstrated by imaging method Hydroxyl dendrimers (approximately 14,000 Da) were covalently conjugated via non-cleavable bonds to two to three indocyanine green (ICG) molecules (D-ICG) per dendrimer. Hydroxyl dendrimers (approximately 14,000 Da) were covalently conjugated via non-cleavable bonds to two to three tetramethylrhodamine (TRITC) molecules (D-TRITC) per dendrimer.

[0162] Two studies were performed in C57BL / 6 mice (n = 5 / group) administered 100 μL of D-ICG or vehicle control intravenously. In the first study, mice were administered D-ICG or vehicle control on days 1, 3, 7, or 14 after laser irradiation, and eyes were analyzed by optical coherence tomography (OCT) imaging of ICG at 4 or 24 hours after administration. Flat mounts of the sclera-choroid / retinal pigment epithelium (RPE) complex were stained with fluorescently labeled isolectin and IBA-1.

[0163] The second study evaluated the localization and persistence of the dendrimer conjugate in CNV lesions. Mice were intravenously administered 100 μL of D-ICG and 100 μL of D-TRITC (1 h after D-ICG) or vehicle control 24 h after laser. Mice were analyzed and sacrificed at 4, 7, 14, 21, and 28 days after administration (n = 5 per group). In the control group, 100 μL of free ICG (1.23 mg / mL) was administered IV 24 h after laser, and mice were analyzed and sacrificed at 2, 4, 7, and 14 days after administration (n = 5 per group). Eyes were analyzed by optical coherence tomography (OCT) with ICG imaging. Flat mounts of the sclera-choroid / retinal pigment epithelium (RPE) complex were stained with fluorescently labeled IBA-1 alone. result

[0164] No significant release of ICG or TRITC was observed from the dendrimers under in vitro release when assessed with esterase in PBS at pH 7.4 or citrate buffer at pH 5.5 at 37°C. The ability of indocyanine green (ICG)-labeled hydroxyl dendrimers was evaluated for targeting choroidal neovascular (CNV) lesions, as well as macrophages and within the retinal pigment epithelium, after systemic administration in a mouse model of laser-induced CNV.

[0165] Systemically administered D-ICG was selectively taken up by cells within CNV lesions within 24 hours after administration, whereas free ICG was distributed nonspecifically and typically disappeared within a few hours. Reactive macrophages and microglia endocytosed the dendrimer conjugate 24 hours after laser, and the lesions showed further uptake during the early stages of CNV, consistent with efficacy studies (24 hours after laser). The dendrimer conjugate localized within macrophages in CNV lesions, as indicated by colocalization with IBA-1-positive cells (data not shown). The free ICG control group demonstrated that free ICG was no longer present within the lesions between 7 and 14 days after laser. IBA-1 signal increased from 24 to 48 hours after laser injury, and isolectin signaling increased slightly at 48 hours after laser (Figures 1A and 2B). A single systemic D-ICG dose given 24 hours after laser injury localized to the CNV lesion, and significant D-ICG was still present at the last time point, i.e., 28 days (Fig. 2C).

[0166] Hydroxyl dendrimers colocalized with reactive macrophages in the choroid, microglia / macrophages in the retina, and RPE cells at sites of inflammation / neovascularization. D-ICG and D-TRITC appeared to be intracellular and focused in areas of IBA-1 signal, consistent with previous studies demonstrating uptake of hydroxyl dendrimers in reactive microglia, macrophages, and RPE cells. The hydroxyl dendrimer (D-ICG) selectively targeted CNV lesions after systemic administration and persisted for at least 28 days after administration, despite systemic clearance within 48 hours. Thus, the hydroxyl dendrimer provided long-term localization at CNV lesions, suitable for sustained and targeted therapy, e.g., once-monthly systemic (subcutaneous or oral) treatment for retinal diseases with minimal systemic exposure. Example 2 Suppression of murine choroidal neovascularization after systemic administration of targeted anti-VEGF therapy method

[0167] A hydroxyl dendrimer (approximately 14,000 Da) selectively targeting inflammation was covalently conjugated with an analog of sunitinib, a potent FDA-approved VFGF receptor tyrosine kinase inhibitor. Conjugates were prepared with either a cleavable sunitinib analog (D-CSA, compound 6 in Figure 1A) or a noncleavable sunitinib analog (D-NSA, compound 3 in Figure 1B), and drug release was assessed by esterase in PBS at pH 7.4 or citrate buffer at pH 5.5 at 37°C. Laser-induced rupture of Bruch's membrane was performed in both eyes of C57BL / 6 mice (n = 8 / group) 24 h before dose administration. Mice were intravenously administered (IV, 100 μL) vehicle, D-CSA (5.25 (low) or 26.25 (high) mg / kg sunitinib equivalent), D-NSA (6.3 (low) or 15.75 (high) mg / kg sunitinib equivalent), or free sunitinib (32.5 mg / kg). As a positive control, a cohort of mice received intravitreal administration (IVT; 1 μL, 40 μg) of aflibercept (EYLEA®). CNV area was measured 7 days after laser treatment by both fluorescein angiography and flatmounting of the sclera-choroid / RPE complex stained with isolectin IB4. result

[0168] The efficacy of hydroxyl dendrimers covalently conjugated to analogues of sunitinib was evaluated in a mouse model of laser-induced choroidal neovascularization (CNV).

[0169] D-CSA was prepared with five sunitinib analogs per dendrimer (10.5% w / w), and D-NSA was prepared with seven sunitinib analogs per dendrimer (12.6% w / w). Over a 6-day in vitro study, D-CSA released approximately 65% ​​of sunitinib at pH 5.5 (intracellular conditions) via esterases, and approximately 15% of sunitinib was released over 24 hours at pH 7.4 (plasma conditions). Release of sunitinib analogs from the D-NSA conjugates was minimal.

[0170] Statistically significant reductions in CNV area were observed at IV dose levels of both IVT aflibercept and D-CSA and D-NSA without free sunitinib (an additional 5-fold higher dose compared to low-dose D-CSA) compared to vehicle control (Figure 3).

[0171] The binding affinity (Kd) for VEGFR2 was evaluated for free sunitinib maleate (0.13 nM), a sunitinib analog conjugated via a non-cleavable PEG linker (1 nM), and D-NSA (27 nM). Binding affinity data showed that high binding affinity was maintained with D-NSA. Thus, conjugation of a sunitinib analog to a hydroxyl dendrimer demonstrated nanomolar potency for VEGF RTK.

[0172] A single dose of D-CSA / D-NSA administered to a mouse model of laser-induced CNV demonstrated efficacy equivalent to that of intravitreally administered aflibercept. The efficacy of non-cleavable sunitinib analogs in reducing CNV area suggests that sunitinib release from the dendrimer may not be required. Previous studies have shown that hydroxyl dendrimers and dendrimer-drug conjugates are retained in CNV lesions for >28 days and cleared intact from the system within 24 hours in mice and humans without detectable liver or other off-target toxicity. Example 3 Duration of efficacy and systemic drug clearance method

[0173] Conjugates were made with a cleavable sunitinib analog (D-CSA, compound 6 in Figure 1A) or a non-cleavable sunitinib analog (D-NSA, compound 3 in Figure 1B). Laser-induced rupture of Bruch's membrane was performed in both eyes of C57BL / 6 mice (n = 8 per group) 24 hours before dose administration. Mice received a single intraperitoneal injection (IP, 100 μL) of vehicle, D-CSA (5.25 mg / kg sunitinib equivalent), D-NSA (6.3 mg / kg sunitinib equivalent), or free sunitinib (6.5 mg / kg). As a positive control, a cohort of mice received aflibercept (EYLEA®) intravitreally (IVT; 1 μL, 40 μg). CNV area was measured 7 and 14 days after laser treatment by both fluorescein angiography and flat mounts of the sclera-choroid / RPE complex stained with isolectin IB4. In plasma pharmacokinetic studies, the same dendrimers labeled with Cy5 administered via a single IP injection were monitored by plasma collection up to 72 hours after IP administration. result

[0174] The therapeutic duration and clearance of hydroxyl dendrimers covalently conjugated with sunitinib analogues were evaluated in a mouse model of laser-induced choroidal neovascularization (CNV).

[0175] The dendrimer-conjugated sunitinib analogs, D-CSA and D-NSA, demonstrated durable responses from a single IP dose, with a reduction in CNV area at 7 days post-treatment and a further reduction in CNV area at 14 days post-treatment. A significant reduction in CNV area was observed with IVT aflibercept at 7 days post-treatment, but the reduction did not persist at 14 days post-treatment (Figure 4A).

[0176] Both D-CSA and D-NSA disappeared from serum within 2 days after treatment (Figure 4B). Thus, the dendrimer-conjugated sunitinib analogs D-CSA and D-NSA exerted long-term local efficacy against CNV lesions, with lesion size continuing to decrease 14 days after treatment. Example 4 Synthesis and characterization of N,N-didecethylsunitinib amide azide

[0177] The design and synthesis of a dendrimer-didecetylsunitinib conjugate are described in Examples 4 and 5. Overexpression of vascular endothelial growth factor (VEGF) has been implicated in several angiogenesis-related diseases. Sunitinib is a receptor tyrosine kinase inhibitor that blocks VEGF receptors and has excellent antiangiogenic activity. It has been approved by the FDA for use in various types of cancer. Dicetylsunitinib is the active metabolite of sunitinib. Despite the excellent therapeutic value of sunitinib and its analogs, their clinical development is hindered by associated toxicities. The dendrimer-didecetylsunitinib conjugate aims to overcome the dose-related toxicity of sunitinib by linking it to a hydroxyl-terminated dendrimer. The synthetic scheme is outlined in Figure 5, and fourth-generation PAMAM is used as an exemplary hydroxyl-terminated dendrimer.

[0178] Step 1: Synthesis of 5-fluoro-2,3-dihydro-1H-indol-2-one (compound 2) To a stirred solution of 5-fluoro-2,3-dihydro-1H-indole-2,3-dione (6.0 gm, 1.0 eq.) in n-butanol (10 V) was added triethylamine (6.12 mL, 1.2 eq.) followed by hydrazine hydrate (3.56 mL, 2.0 eq.) at room temperature. The resulting solution was stirred at 100° C. for 16 hours. The reaction progress was monitored by TLC (50% ethyl acetate in hexane). Once the reaction was deemed complete, the reaction product itself was evaporated to dryness under vacuum at 45° C. to give a dark brown solid. The resulting 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 fluffy gray solid (4.0 g, 72% yield). Compound 2, shown in FIG. 5, was prepared by: 1Confirmation was achieved by 1 H NMR, liquid chromatography, and mass spectroscopy.

[0179] Step 2: Synthesis 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) To a stirred solution of 5-fluoro-2,3-dihydro-1H-indol-2-one (compound 2) (4.0 gm, 1.0 eq.) and 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (compound 3) (4.41 gm, 1.0 eq.) in ethanol (10 V) was added pyrrolidine (4.42 mL, 2.0 eq.) at room temperature. The resulting solution was stirred at 80° C. for 3 hours. The progress of the reaction was monitored by TLC (10% methanol in DCM). Once the reaction was deemed complete, the reaction mixture was cooled to room temperature and 2 M HCl solution was added to pH=3. A brown precipitate formed and was filtered. The resulting solid was washed with ethanol (20 V), followed by hexane (30 V), and filtered to give a reddish-orange solid (6.6 g, 82% yield). Compound 4, shown in FIG. 5, was prepared by: 1 H This was confirmed by NMR.

[0180] Step 3: Synthesis of tert-butyl N-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrol-3-yl)formamido]ethyl}carbamate (Compound 6) To a 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) (6.5 g, 1.0 equiv.) in DMF was added triethylamine (6.08 mL, 2.0 equiv.), EDC.HCl (8.68 g, 2.1 equiv.), HOBT (3.94 g, 1.35 equiv.), and tert-butyl N-(2-aminoethyl)carbamate (4.16 g, 1.2 equiv.) at 0° C. The reaction was stirred at room temperature for 16 hours. The reaction mixture was diluted with water (20 The resulting solid was diluted with ethyl acetate (15.0 V), stirred for 10 minutes to allow precipitation, 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 as tert-butyl N-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrol-3-yl)formamido]ethyl}carbamate (Compound 6) (7.5 g, 78% yield). Compound 6, shown in Figure 5, was prepared by 1 Confirmed by 1 H NMR.

[0181] Step 4: Synthesis of 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): To a solution of tert-butyl N-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrol-3-yl)formamido]ethyl}carbamate (compound 6) (9.0 g, 1.0 equiv.) in DCM (10.0 V) was added trifluoroacetic acid (3.0 V) at 0-5° C. The reaction was stirred at room temperature for 12 hours. The reaction product itself was evaporated to dryness under vacuum at 45° C. to give a dark brown solid. The resulting solid was washed with diethyl ether (15.0 V), filtered, and dried to give an orange-yellow solid (6.0 g crude). Compound 7, shown in FIG. 5, was prepared by 1Confirmation was achieved by 1 H NMR, liquid chromatography, and mass spectroscopy.

[0182] Step 5: Synthesis of N-{2-[(5-{[(3Z)-5-fluoro-2-oxo-2,3-dihydro-1H-indol-3-ylidene]methyl}-2,4-dimethyl-1H-pyrrol-3-yl)formamido]ethyl}-3-[2-(2-propoxyethoxy)ethoxy]propanamide (compound 9): To a solution of 3-[2-(2-propoxyethoxy)ethoxy]propanoic acid (8) (5.95 g, 1.0 equiv.) in DMF (10.0 mL), DIPEA (8.40 mL, 2.0 equiv.), EDC.HCl (6.90 g, 1.5 equiv.), HOBT (0.65 g, 0.2 equiv.), 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 equiv.), and DMAP (0.294 g, 0.1 equiv.) were added at 0-5 °C. The reaction was stirred at room temperature for 3 h. The progress of the reaction was monitored by TLC (10% MeOH in DCM). The reaction mixture was diluted with water (20.0 V) and stirred for 10 minutes to form a brown precipitate, which was filtered. The resulting solid was purified by reverse-phase column chromatography to give N,N-didecethylsunitinib amide azide as an orange solid (5.2 g, 37% yield). Compound 9 was obtained by 1 Confirmation was achieved by 1 H NMR, liquid chromatography, and mass spectroscopy.

[0183] Example 5 Synthesis and characterization of dendrimer-didecethylsunitinib conjugate (D-4517) method Synthesis and characterization of intermediates and dendrimer conjugates: Synthesis of dendrimer hexyne (compound 2 in Figure 6A): A dry round-bottom flask (250 mL) was taken and its weight was recorded. The desired amount of methanol solution of G4-OH was poured into the round-bottom flask and allowed to evaporate at 60 °C for 2 hours. The flask was then transferred to a high-vacuum assembly for 1 hour. The amount of G4-OH in the flask was recorded. Once the weight of G4-OH was recorded, 50–60 mL of anhydrous DMF was added to the flask and evaporated under reduced pressure to remove any traces of methanol present in the dendrimer, which could affect the efficiency of the Steglich esterification. After evaporation of the DMF, the flask was placed under a nitrogen atmosphere. Anhydrous DMF (10 mL / gram) was added to the flask, the solution was transferred to an ultrasonic bath, and the reaction mixture was sonicated until a clear solution was achieved. 5-Hexynoic acid was dissolved in 2 mL of DMF and added to the stirring solution. After 10 minutes, EDC.HCl and DMAP were added to the stirring solution, and the solution was left stirring at room temperature for 48 hours. Upon completion, DMF dialysis was initiated using a 1 kDa molecular weight cutoff dialysis bag. DMF dialysis was performed for 8 hours, with one change of DMF. After 8 hours, 30 mL of deionized water was added to the solution in the bag and dialyzed against water overnight. The reaction mixture was diluted with HPLC water to a final volume of 300-350 mL. TFF was performed in deionized water using a 3 kDa TFF cartridge. Six to seven TFF cycles were performed, and the final retentate volume was approximately 100 mL. This was lyophilized to obtain a viscous solid. The product yield was approximately 5.5 g (74%). 1 H NMR was recorded on a 500 MHz instrument in deuterated DMSO; approximately 10 mg of compound was used for sample preparation. The hexynoic acid loading was calculated by proton integration. The internal amide peak of the dendrimer between δ 8.11 and 7.70 ppm was used as the reference peak. The peak at δ 4.0 ppm corresponds to the ester bond proton, and the peak at δ 1.6 ppm is the CH2 from the hexynoic acid. Proton integration suggested the binding of 9–10 molecules of hexynoic acid per dendrimer. HPLC purity was >99%. [Table 1]

[0184] Synthesis of dendrimer-didecethylsunitinib conjugate (compound 3 in Figure 6B): Dendrimer hexyne (compound 2 in Figure 6A) was placed in a 250 mL round-bottom flask. The compound was dissolved in 40 mL of anhydrous DMF by sonication. The sunitinib-azide solution was added to the reaction mixture by dissolving it in 20 mL of DMF, and the solution was stirred. 10 mL of water was then added to the reaction mixture to quench the precipitation of copper salts in the reaction mixture. 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 reaction vial was transferred to an oil bath set at 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 in water. EDTA (500 microliters, 0.5 M) solution was added to this solution to remove the copper salts by chelation. TFF was performed on the reaction mixture in water using a 3 kDa TFF cartridge. Eight to ten diafiltration volumes were performed with 10% DMAc in water, followed by five to six cycles with water as buffer to remove traces of solvent. The final retentate volume was approximately 150 mL, which was lyophilized to yield a pale yellow solid. The product yield was 5.5 g. 1 H NMR was recorded on a 500 MHz instrument in deuterated DMSO and deuterated water, and approximately 10 mg of compound was used for sample preparation. 100 scans were performed. 1 H NMR was performed. 1 H NMR showed the formation of the product, with 6–7 arms of the sunitinib molecule attached (Figure 4). Drug loading was calculated by proton integration, where peaks correspond to the dendrimer and drug. The CH2 peak at 1.8 ppm corresponds to the hexynoic acid, and the ester-linked CH2 at 4.0 ppm is locked as a reference peak from the dendrimer side. After triazole formation, the protons correspond to the CH2 peak next to the triazole ring at δ 4.4 ppm. 1New peaks in H NMR, two aromatic protons from sunitinib between 6.92 and 6.80 ppm, and two NH protons at 10.9 and 13.6 ppm, were used to calculate the loading of the drug molecule. After the click reaction, there was formation of 1-4 triazole, and the signature proton peak corresponding to the triazole appears to be between δ 7.5 and 8.0 ppm, which is suppressed by the presence of internal amide protons. To confirm sunitinib binding, 1 H NMR was recorded in DO and observed the disappearance of the internal amide peak and the presence of a triazole peak at δ 7.7 ppm. HPLC purity was >99%. [Table 2]

[0185] Protocol for in vitro kinase binding assay Kinase-tagged T7 phage stocks were prepared in E. coli hosts derived from the BL21 strain. E. coli were grown to logarithmic phase, infected with T7 phage, and incubated with shaking at 32°C until lysis. The lysate was centrifuged and filtered to remove cellular 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 biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SEABLOCK® (Pierce), 1% BSA, 0.05% Tween® 20, 1 mM DTT) to remove unbound ligand and thereby reduce nonspecific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1x binding buffer (20% SEABLOCK®, 0.17x PBS, 0.05% Tween® 20, 6mM DTT). Test compounds were prepared as 111x stocks in 100% DMSO. Kd was determined using an 11-point, 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements were dispensed by acoustic transfer in 100% DMSO (non-contact dispensing). Compounds were then diluted directly into the assay to a final DMSO concentration of 0.9%. All reactions were performed in polypropylene 384-well plates. Each had a final volume of 0.02 ml. The assay plate was incubated at room temperature with shaking for 1 hour, and the affinity beads were washed with wash buffer (1x PBS, 0.05% Tween® 20). The beads were then resuspended in elution buffer (1x PBS, 0.05% Tween® 20, 0.5 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. Kinase concentrations in the eluates were measured by qPCR.

[0186] Sample preparation: Sunitinib maleate, sunitinib ester amide linker, and D4-sunitinib conjugate were dissolved in aqueous DMSO 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.

[0187] result Synthesis and Characterization: The synthesis of dendrimeric didecethylsunitinib analogs was achieved in three steps via copper(I)-catalyzed alkyne-azide click reaction (Figures 6A and 6B). In the first step, partial modification of the dendrimer surface hydroxyl groups was achieved by attaching several hexynoic acid linker arms via esterification to alkyne surface groups. The second step involved the introduction of linker arms onto the azide-terminated didecethylsunitinib. The third step involved the click reaction of both moieties. For the synthesis of dendrimeric hexynoic acid, the as-received ethylenediamine-core hydroxyl polyamidoamine dendrimer (G4-OH, Pharma grade >95% HPLC purity) was used. Partial esterification of the OH-terminated dendrimer (compound 1) with 5-hexynoic acid was first carried out using EDC.HCl and DMAP in anhydrous N,N-dimethylformamide (Figure 6A), yielding compound 2. 1The compound was confirmed using H NMR. The loading of the hexyne linker was calculated by proton integration by comparing the linker protons to the internal amide protons of the dendrimer between δ 8.11 and 7.70 ppm. The peak at δ 4.0 ppm, corresponding to the ester bond proton, and the peak at δ 1.6 ppm refer to CH2 from the hexynoic acid. Proton integration suggested the attachment of 8–10 molecules of alkyne linker per dendrimer. The purity of the construct was assessed using HPLC and found to be >99%. The linker 3-[2-(2-propoxyethoxy)ethoxy]propanoic acid was coupled to didecethylsunitinib using EDC.HCl, DMAP, DIPEA, and HOBt in anhydrous DMF, followed by precipitation of the azide via a click reaction. The drug was attached via the amide bond through the linker. A click reaction was performed using the dendrimer hexyne (2) and didecethylsunitinib PEG azide. The copper-catalyzed click reaction is one of the most efficient chemical transformations that has revolutionized the field of drug discovery and is an excellent tool for the conjugation of small or large molecules with macromolecules, polymers, and antibodies. Its ease of implementation, milder reaction conditions, compatibility with different functional groups, regioselectivity, and enhanced reaction rate have been found to lead to cleaner products in greater yields. Copper(II) sulfate pentahydrate and sodium ascorbate were used in the click reaction in the presence of DMF:HO (1:1). The reaction was carried out at room temperature overnight and then purified by tangential flow filtration. The formation of product (3) was confirmed by 1 The conjugate was confirmed by H NMR. 1The H NMR spectrum clearly shows peaks corresponding to the dendrimer, the drug, and the linker attached thereto. Drug loading was calculated by comparing these peaks with the aid of proton integration. The initial amide protons from the dendrimer are present between δ 8.5 and 7.5 ppm when the spectrum is recorded in deuterated DMSO. These amide peaks serve as the reference standard for the remaining peaks. The -NH peaks from the drug appear at δ 13.6 and 10.8 ppm. Four protons from the drug and one triazole proton formed after the click reaction integrated with the internal amide peak appear between δ 8.5 and 7.5 ppm. Additionally, two aromatic protons from sunitinib next to the fluorine group appear between δ 6.95 and 6.85 ppm. A sharp triazole peak at δ 7.7 ppm, a signature peak of the click transformation, is observed when the NMR solvent is switched from deuterated DMSO to CD3OD. After the click, the CH2 next to the azide is downshielded and can be observed at δ 4.4 ppm. Comparison of the proton NMR spectra of the drug linker, dendrimer intermediate, and final conjugate is shown below. 1 The purity of the dendrimer-drug conjugate, intermediates, and drug linker was assessed using HPLC. The final conjugate was >99% pure by HPLC. The dendrimer G4-OH and dendrimer hexyne intermediate were visible in the 210 nm channel, and didecethylsunitinib was visible at 430 nm in HPLC. The retention time of compound 2 is approximately 16.9 min, but upon binding of the hydrophobic drug molecule to the dendrimer, the peak of the final conjugate shifts to the right, confirming binding of the hydrophobic drug to the dendrimer construct at approximately 27 min. Upon binding of the drug to the dendrimer, corresponding peaks were observed in both the 210 nm (dendrimer absorption wavelength) and 430 nm (drug absorption wavelength) channels, further confirming product formation. The drug loading of the dendrimer conjugate was approximately 12.6% wt / wt, corresponding to seven molecules of drug bound per dendrimer molecule.

[0188] Binding affinity: The comparative kinase binding affinities of the D-didecetyl-sunitinib conjugate (compound D-4517), free sunitinib maleate, and sunitinib blinker (AVT-4517) were evaluated, and the results are presented in Table 3. The binding affinity of free sunitinib is 0.13 nM. After attachment of the PEG linker, the binding affinity decreased approximately 8-fold to 1.0 nM. The conjugate exhibited a binding affinity of 27 nM. The results demonstrate that conjugation of the drug to the dendrimer surface preserves the binding affinity of the drug to the RTK domain in the nanomolar range. This indicates that the conjugate itself is active and can bind to the receptor without drug release. This is the first time that conjugation of a small molecule inhibitor (300-400 Da) to a large dendrimer (14,000 Da) has been shown to still retain nanomolar binding of the small molecule inhibitor. [Table 3]

[0189] Stability studies in human and rat plasma: The in vitro stability of D-didecethylsunitinib (D-4517) in human and rat plasma was further evaluated under physiological conditions. The results presented in Table 4 suggest that the conjugate D4517 is highly stable, with a 2% (weight percentage) release in human plasma and a 4% (weight percentage) release in rat plasma after 48 hours. [Table 4]

[0190] In vitro drug release studies: In vitro drug release studies were performed at pH 7.4 and pH 5.5 with esterase at 37°C, which mimic plasma and intracellular conditions, respectively. Release studies were performed in duplicate. The results are presented in Figure 7. Under intracellular conditions, less than 2% by weight of the drug arm was released in 15 days. Under plasma conditions, approximately 2% was released in 24 hours, and approximately 4% was released in 15 days. The ester bond between the dendrimer and the linker resulted in the loss of the linker to AVT-4517 over time in D-4517. Under both conditions, the conjugate demonstrated good stability.

[0191] Example 6 In vivo pharmacokinetics of dendrimer-didecethylsunitinib conjugate (D-4517) D-4517 pharmacokinetics was evaluated in vivo in mice. Female C57 / B16 mice were IP injected with 5 or 50 mg / kg of D-4517, and blood samples were collected to determine plasma D-4517 concentrations. Peak plasma concentrations were observed at 0.5 hours, the first time point sampled. Exposure based on Cmax and AUC was dose-related and approximately dose-proportional. Final disappearance T 1 / 2 was approximately 1 hour after both dose levels. PK parameters estimated by non-compartmental methods are shown in Table 5 below, and mean plasma concentrations versus time are shown in Figure 8. [Table 5]

[0192] Toxicokinetic data were collected in rats. Sprague-Dawley rats were given daily IP injections of 12 mg / kg, or a single dose of 168 mg / kg D-4517, or a daily oral dose of 30 mg / kg sunitinib (40.21 mg / kg sunitinib maleate). Blood samples were collected to determine plasma concentrations of the drug. Noncompartmental toxicokinetic parameters were estimated.

[0193] Figures 9A and 9B show plasma concentration versus time profiles for the 12 mg / kg D-4517 and sunitinib groups on days 1 and 14. No significant gender differences were observed in D-4517 plasma concentrations, except for the 24-hour time point on day 1, where males had higher concentrations than females. D-4517 plasma concentrations declined more rapidly than sunitinib. Terminal half-lives could not be reliably estimated due to a lack of sufficient time points at the terminal stage. While sunitinib was measurable 24 hours after dosing, D-4517 was not measurable beyond 8 hours after dosing. Consequently, AUC estimates were higher for sunitinib compared with D-4517. Following a single 168 mg / kg D4517 dose, plasma concentrations were high at 1 hour after dosing, but only one animal had a measurable concentration at the next sampled time point, 24 hours.

[0194] Pharmacokinetic results indicate that doses of D-4517 resulted in comparable maximum concentrations with lower overall exposure compared to sunitinib. Separate rats were given sunitinib maleate orally for 14 days at a dose of 40.21 mg / kg. D-4517 was not associated with mortality or affected clinical observations, body weight, food consumption, or clinical pathology parameters (hematology, clinical chemistry, and urinalysis). D-4517-related macroscopic findings were limited to yellow discoloration of adipose tissue and mesentery in males and females at 12 mg / kg and / or 168 mg / kg, which correlated with subacute / chronic inflammation associated with intraperitoneal administration of the test article. Organ weight changes included a statistically significant decrease in spleen weight in males at 168 mg / kg; however, this observation had no microscopic correlation. Non-adverse microscopic findings included minimal focal pigmentation in the choroid of the eye and subacute / chronic inflammation in the abdominal fat / mesentery in males and females at 12 mg / kg and 168 mg / kg. Inflammation appeared to be secondary to the intraperitoneal injection of test article and was observed secondarily along the serosal surfaces of the stomach, liver, and spleen.

[0195] Overall, D-4571 was well tolerated following single or repeated IP dosing. These observations contrast with sunitinib maleate, which was associated with mortality as well as a variety of clinical and pathological changes in female rats.

[0196] Example 7 A single subcutaneous administration study of dendrimer-didecethylsunitinib conjugate (D-4517) To evaluate the preferred administration route in humans, a single subcutaneous dose study of D-4517 was performed in a mouse model of laser-induced CNV. Control mice (n = 8 / group) received an intravitreal injection of either vehicle or aflibercept (40 μg) one day after laser treatment. Three dose levels of D-4517 (2, 10, and 50 mg / kg; n = 8 / group) were administered as a single subcutaneous dose one day after laser treatment. After 14 days, mice were sacrificed, and flat mounts of the sclera-choroid / RPE complex were stained with DAPI and isolectin IB4. CNV area was measured using fluorescence microscopy and imaging software. As shown in Figure 10, all three doses of D-4517 given as a single subcutaneous dose significantly reduced CNV lesion area. The response in D-4517-treated animals was more consistent than that observed in aflibercept-treated animals. This study demonstrates that significant efficacy was observed from subcutaneously administered D-4517 in the CNV model.

[0197] Example 8 Conjugation of didecethylsunitinib via a non-cleavable ether bond on dendrimers method Synthesis of dendrimer conjugates via non-cleavable ether bonds on dendrimers

[0198] The synthesis began with the construction of a bifunctional dendrimer. At generation 3.5 of the dendrimer, seven alkyne functional groups were introduced using a polyethyl glycol (PEG) linker with an amine at one end and a hexyne at the other, resulting in a generation 4 bifunctional dendrimer (compound 1 in Figure 11) with seven alkyne arms and 57 hydroxyl groups on the surface. The structure of the dendrimer was confirmed by 1H NMR spectroscopy. A clickable didecethylsunitinib analog (compound 2 in Figure 11, also known as AVT-4517) containing didecethylsunitinib, three ethylene glycol (PEG) spacers, and a terminal azide was synthesized and engaged in a click reaction with alkyne groups on the surface of the dendrimer. The active agent, compound 2, is prepared using a five-step synthesis shown in Figure 5.

[0199] VT-4517 (compound 2 in Figure 11) was finally reacted with a bifunctional dendrimer bearing hexyne groups (compound 1 in Figure 11) via copper(I)-catalyzed alkyne-azide click chemistry to yield D-4517.2 (compound 3 in Figure 11), whose complete structure is shown in Figure 12. After conjugation of this analog with the dendrimer, D-4517.2 was purified by tangential flow filtration (TFF) to remove all impurities and refined into the final formulation. D-4517.2 conjugate 1 H-NMR analysis

[0200] The formation of product D-4517.2 was confirmed by 1H NMR. The 1H NMR spectrum of the conjugate clearly showed peaks corresponding to the dendrimer, drug, and the linker attached thereto. Drug loading was calculated by comparing these peaks with the aid of proton integration. Internal amide protons from the dendrimer are present between δ 8.5 and 7.5 ppm when the spectrum was recorded in deuterated DMSO. These amide peaks serve as the reference standard for the remaining peaks. -NH peaks from the drug appear at δ 13.6 and 10.8 ppm. Four protons from the drug and one triazole proton formed after the click reaction were integrated with the internal amide peak, resulting in a peak between δ 8.5 and 7.5 ppm. Additionally, two aromatic protons from sunitinib next to the fluorine group appear at δ 6.95 and 6.85 ppm. A sharp triazole peak at δ 7.7 ppm, a signature peak for click transformation, is observed when the NMR solvent is switched from deuterated DMSO to CD3OD. After the click, the CH2 next to the azide is downshielded and can be observed at δ 4.4 ppm. NMR was also used to quantify the number of drug molecules conjugated to the hydroxyl dendrimer. Drug loading was calculated by proton integration by comparing the drug protons with the internal amide protons of the dendrimer. HPLC analysis to assess the purity of D-4517.2

[0201] The purity of the dendrimer-drug conjugate, intermediates, and drug linker was assessed using HPLC. The final conjugate was >99% pure by HPLC. The dendrimer G4-OH and dendrimer hexyne intermediate were visible in the 210 nm channel, and didecethylsunitinib was visible at 430 nm by HPLC. The retention time of compound 2 was approximately 16.9 min, but upon binding of the hydrophobic drug molecule to the dendrimer, the peak of the final conjugate shifted to the right, reaching approximately 27 min, confirming the attachment of the hydrophobic drug to the dendrimer construct. Upon binding of the drug to the dendrimer, the corresponding peak could be observed in both the 210 nm (dendrimer absorption wavelength) and 430 nm (drug absorption wavelength) channels, further confirming product formation. The drug loading of the dendrimer conjugate was approximately 12.6% wt / wt, corresponding to seven molecules of drug attached per dendrimer molecule.

[0202] Size and Zeta Potential 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 dendrimer in deionized water (18.2 Ω) to produce a final solution of 0.5 mg / mL. The solution was then filtered through a 0.2 μm syringe filter (Pall Corporation, 0.2 μm HT Tuffryn membrane) directly into a cell (UV-transparent disposable cuvette, dimensions: 12.5 × 12.5 × 45 mm). For zeta potential measurements, samples were prepared at a concentration of 0.2 mg / mL in 10 mM NaCl using the procedure described above. A Malvern Zetasizer Nanoseries 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).

[0203] Size Exclusion Chromatography Multi-Angle Laser Scattering (SEC-MALS) The molar mass of D-4517.2 will be determined by size exclusion chromatography multi-angle laser scattering (SEC-MALS).

[0204] result D-4517 has nanomolar affinity for VEGFR2 and does not require the release of the active drug, AVT-4517. To further increase the stability of the conjugate under physiological conditions and further reduce drug release from the conjugate, as observed in D-4517 buffer and plasma stability studies, the cleavable ester bond on the dendrimer surface was replaced with a non-cleavable bond, as demonstrated in the structure of D-4517.2 ( FIG. 12 ). The structure of D-4517.2 lacks a cleavable bond.

[0205] D-4517.2 is a covalent conjugate of a fourth-generation hydroxyl-terminated PAMAM dendrimer, consisting of an ethylenediamine (EDA) core, amidoamine repeating units [CH2CH2CONHCH2CH2N], and 64 hydroxyl end groups (chemical formula: C 622 H1 184 N 186 O 188 ), where a didecethylsunitinib analog (AVT-4517) was conjugated to the dendrimer by a highly efficient click chemistry approach. The hydroxyl, fourth-generation, PAMAM dendrimers are monodisperse and produced with high compositional purity (>95%). In the preparation of D-4517.2, seven of the 64 hydroxyl groups on the dendrimer were modified to conjugate AVT-4517 (approximately 12.6% of the total mass).

[0206] Stability studies in human, mouse, and rat plasma The in vitro stability of dendrimer didecetylsunitinib conjugates, D-4517 and D4517.2, in human, mouse, and rat plasma was evaluated under physiological conditions. The results are presented in Figure 13. Compared to D4517 (2% (weight percentage) released in human plasma and 4% (weight percentage) released in rat plasma), the plasma stability of D4517.2 is significantly improved. At 48 hours, less than 0.5% of the drug (by weight) was released from the dendrimer-drug conjugate in all three plasma species.

[0207] binding affinity The comparative kinase binding affinities of D-4517 and D-4517.2 were evaluated and the results are presented in Table 6. [Table 6]

[0208] The IC50 results for D-4517.2 were lower than D-4517 for all assays tested, indicating stronger binding between D4517.2 and the tyrosine kinase receptor.

[0209] 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 belongs. Publications cited herein and the references for which they are cited are specifically incorporated by reference.

[0210] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims. The present invention provides, for example, the following items. (Item 1) a composition comprising a hydroxyl-terminated dendrimer complexed, covalently conjugated, intramolecularly dispersed or encapsulated with one or more receptor tyrosine kinase inhibitors; one or more pharmaceutically acceptable excipients suitable for systemic administration; A pharmaceutical formulation comprising the composition is in an amount effective to treat or alleviate one or more symptoms associated with one or more diseases and / or disorders of the eye; Pharmaceutical preparations. (Item 2) 10. The formulation of claim 1, wherein the dendrimer is complexed, covalently conjugated, intramolecularly dispersed or encapsulated with one or more additional therapeutic, prophylactic and / or diagnostic agents. (Item 3) 3. The formulation according to item 1 or 2, wherein the receptor tyrosine kinase inhibitor is a receptor tyrosine kinase inhibitor of vascular endothelial growth factor receptor (VEGFR). (Item 4) 4. The formulation of any one of items 1 to 3, wherein the VEGFR inhibitor is selected from the group consisting of sunitinib, sorafenib, pazopanib, vandetanib, axitinib, cediranib, vatalanib, dasatinib, nintedanib, motesanib, and analogs thereof. (Item 5) 5. The formulation of any one of items 1 to 4, wherein the VEGFR inhibitor is sunitinib or an analog thereof. (Item 6) The formulation according to any one of items 2 to 5, wherein the diagnostic agent is selected from the group consisting of a dye, a fluorescent dye, a near-infrared dye, a SPECT contrast agent, a PET contrast agent, and a radioisotope. 7. The formulation of item 6, wherein the fluorescent dye is selected from the group consisting of indocyanine green, fluorescein isoiocinate, boron-dipyrromethene, rhodamine, rose bengal, and combinations thereof. (Item 8) 8. The formulation of item 7, wherein the diagnostic agent is indocyanine green. (Item 9) 9. The formulation of any one of items 1 to 8, wherein the dendrimer is a 4th, 5th, 6th, 7th, 8th, 9th, or 10th generation poly(amidoamine) (PAMAM) hydroxyl-terminated dendrimer. (Item 10) 10. The formulation of any one of items 1 to 9, wherein the one or more receptor tyrosine kinase inhibitors are covalently conjugated to the dendrimer. (Item 11) 11. The formulation of any one of items 1 to 10, wherein the one or more receptor tyrosine kinase inhibitors are in a concentration by weight of the dendrimer conjugate of between about 0.01% w / w and about 30% w / w, between about 1% w / w and about 25% w / w, between about 5% w / w and about 20% w / w, and between about 10% w / w and about 15% w / w. (Item 12) 12. The formulation according to any one of items 1 to 11, wherein the one or more diseases and / or disorders of the eye are inflammatory and / or neovascular diseases of the eye. (Item 13) 13. The formulation of any one of items 1 to 12, wherein the one or more diseases and / or disorders of the eye are selected from the group consisting of age-related macular degeneration (AMD), retinitis pigmentosa, optic neuritis, uveitis, retinal detachment, temporal arteritis, retinal ischemia, atherosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, diabetic retinopathy, macular edema, retinal neovascularization, and choroidal neovascularization. (Item 14) 14. A method for treating and / or diagnosing said one or more diseases and / or disorders of the eye, comprising administering to a subject in need thereof the formulation of any one of items 1 to 13. (Item 15) 15. The method of item 14, wherein the one or more diseases and / or disorders of the eye are inflammatory and / or neovascular diseases of the eye. (Item 16) Item 16. The method of item 14 or item 15, wherein the one or more diseases and / or disorders of the eye are selected from the group consisting of age-related macular degeneration (AMD), retinitis pigmentosa, optic neuritis, uveitis, retinal detachment, temporal arteritis, retinal ischemia, atherosclerotic retinopathy, hypertensive retinopathy, retinal artery occlusion, retinal vein occlusion, diabetic retinopathy, macular edema, retinal neovascularization, and choroidal neovascularization. (Item 17) 17. The method of any one of items 14 to 16, wherein the one or more diseases and / or disorders of the eye are diseases and / or disorders associated with activated microglia, activated macrophages, and / or retinal pigment epithelial (RPE) cells of the eye. (Item 18) 18. The method of any one of items 14 to 17, wherein the composition is in an amount effective to target the activated microglia, activated macrophages, and / or RPE cells in the one or more diseases and / or disorders of the eye. (Item 19) 19. The method of any one of items 14 to 18, wherein the composition is in an amount effective to reduce the number and / or activity of the activated microglia and / or activated macrophages in the eye. (Item 20) 21. The method of any one of items 14 to 19, wherein the composition is administered in an amount effective to alleviate one or more symptoms of the one or more diseases and / or disorders of the eye. 21. The method of any one of items 14 to 20, wherein the composition is administered via systemic administration. (Item 22) 22. The method of any one of items 14 to 21, wherein the composition is administered intravenously, subcutaneously, or orally. (Item 23) 23. The method of any one of items 14 to 22, wherein the composition is administered to the subject for a period selected from the group consisting of daily, weekly, biweekly, monthly, and bimonthly. (Item 24) Formula (I): [ka] wherein D is a poly(amidoamine) (PAMAM) dendrimer selected from generation 2, 3, 4, 5, 6, 7, 8, 9, and 10; L is one or more linking moieties or spacers; X is an active agent, or a derivative, analog, or prodrug thereof; n is an integer from 1 to 100, m is an integer from 16 to 4096, and Y is a linker selected from secondary amide (-CONH-), tertiary amide (-CONR-), sulfonamide (-S(O)2-NR-), secondary carbamate (-OCONH-; -NHCOO-), tertiary carbamate (-OCONR-; -NRCOO-), carbonate (-OC(O)-O-), urea (-NHCONH-; -NRCONH-; -NHCONR-, -NRCONR-), carbinol (-CHOH-, -CROH-), disulfide group, hydrazone, hydrazide, and ether (-O-), and R is an alkyl group, an aryl group, or a heterocyclic group. Dendrimer. (Item 25) D is a poly(amidoamine) (PAMAM) dendrimer selected from generations 4, 5, 6, 7, and 8; L is one or more linking or spacer moieties; X is an inhibitor of vascular endothelial growth factor receptor (VEGFR) or an inhibitor of TIE2 receptor tyrosine kinase; n is an integer from 1 to 100, m is an integer from 16 to 4096; Y is a minimally cleavable bond or linkage in vivo. 25. The dendrimer according to item 24. (Item 26) D is a G4 PAMAM dendrimer; L is one or more linking or spacer moieties; X is sunitinib or a derivative, analogue or prodrug thereof; Y is a secondary amide (—CONH—), n is an integer from 5 to 15, m is an integer from 49 to 59; n+m=64, 25. The dendrimer according to item 24. (Item 27) D is a G4 PAMAM dendrimer; L is polyethylene glycol with a triazole linker; X is N,N-didecethylsunitinib, or a derivative, analog, or prodrug thereof; Y is a secondary amide (—CONH—), n is an integer from 5 to 15, m is an integer from 49 to 59; n+m=64, 25. The dendrimer according to item 24. (Item 28) Formula I has the following structure: [ka] 25. The dendrimer according to item 24, having the formula: (Item 29) Formula I has the following structure: [ka] 29. The dendrimer according to item 28, having the formula: (Item 30) 30. The dendrimer according to any of items 24 to 29, wherein the dendrimer has a diameter of about 1 nm to about 20 nm. (Item 31) 31. The dendrimer according to any of items 24 to 30, wherein the dendrimer has a diameter of about 2 nm to about 10 nm. (Item 32) 32. The dendrimer according to any of items 24 to 31, wherein the dendrimer has a surface charge of 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 (inclusive). (Item 33) 33. The dendrimer according to any of items 24 to 32, wherein from about 0.1% to about 40% of the total surface groups of the dendrimer are covalently bonded to an active agent or analog thereof.

Claims

[Claim 1] The invention described in the present specification.

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