Poly(ethylene glycol)-based dendrimer-like hyperbranched polymer, its manufacturing method and applications

Abstract

In certain embodiments, the present invention relates to dendrimer-like superbranched polymers for a variety of uses, such as medical or biopharmaceutical applications, or non-medical or industrial applications, including antibody purification, cosmetic applications, catalytic applications, electronic equipment, agriculture, food, filtration, and other applications. In embodiments, the present invention relates to a superbranched polymer for drug delivery, which comprises polyethylene glycol (PEG) units and at least one activator conjugated to the superbranched polymer. Methods for synthesizing, purifying, and characterizing such superbranched polymers, as well as methods for treating medical conditions such as eye diseases, are further provided.

Description

[Technical Field]

[0001] In certain embodiments, the present invention relates to dendrimer-like superbranched polymers for a variety of uses, such as medical or biopharmaceutical applications, or non-medical or industrial applications, including applications in antibody purification, cosmetics, catalysts, electronics, agriculture, food, filtration, and other applications. In medical applications, the superbranched molecules are conjugated with activators such as drugs, peptides, or proteins. Furthermore, in certain embodiments, the present invention relates to a superbranched polymer for drug delivery comprising polyethylene glycol (PEG) units and at least one activator conjugated to the superbranched polymer. In even more specific embodiments, the present invention relates to a method for synthesizing, purifying, and characterizing such dendrimer-like superbranched polymers. The present invention also relates, in certain embodiments, to a method for treating medical conditions, such as the treatment of eye diseases. [Background technology]

[0002] Controlled delivery and stabilization of therapeutic drugs have become a major research area in recent years. Controlled delivery improves treatment, facilitates administration, and leads to improved efficacy, better compliance, reduced side effects, and overall improved treatment outcomes.

[0003] The eye is a unique organ, possessing both perfection and complexity, and in many ways a microcosm of the body. Its easy accessibility allows for direct delivery of drugs / genes, maximizing therapeutic efficacy and minimizing side effects. Therefore, it presents a prime opportunity for nanomedicine. Developing appropriate delivery systems that can sustainably deliver therapeutic drugs to target tissues is a critical challenge for nanotechnology. Current delivery systems for anterior segment diseases, including punctal plugs, microparticle and nanoparticle encapsulation, microneedle systems, iontophoresis, and different types of intravitreal implants, represent cutting-edge tools for achieving sustained and controlled drug release within the eye.

[0004] Dendrimers and hyperbranched polymers have attracted the attention of scientists in the fields of drug and gene delivery over the past two decades due to their versatility, complexity, and highly branched properties. Dendrimers are tree-like, highly symmetrical, monodisperse, branched nanostructured polymers with repeatable components that have clearly defined size, adjustable structure, and potentially favorable biodistribution. Due to their unique biological properties, such as high drug loading, lipid bilayer interactions, targeting ability, plasma residence time, filtration, intracellular transport, biodistribution, transfection, and excellent colloidal and biological stability, dendrimers have been widely studied as a novel platform for bioactive substance delivery. For drug delivery, many dendrimers have been studied, including polymer dendrimers such as polyamidoamine (PAMAM), poly(propyleneimine) (PPI), polyesters, polyethers, poly-L-lysine, triazine, melamine, poly(glycerol-co-succinate), poly(glycerol), and poly[2,2-bis(hydroxymethyl)propionic acid]dendrimers, as well as other types of dendrimers such as peptides, liquid crystal-forming dendrimers, and carbosilanes. (For an overview, see, for example, “Dendrimer as nanocarrier for drug delivery” Prashant Kesharwani, Keerti Jain, Narendra Kumar Jain, Progress in Polymer Science 39(2014)268-307, and “Dendrimer-based drug delivery systems: history, challenges, and latest developments” Juan Wang, Boxuan Li, Li Qiu, Xin Qiao and Hu Yang, Journal of Biological Sciences.) See Engineering (2022) 16:18).

[0005] Among the various applications of dendrimers for drug delivery, those related to the treatment and management of ophthalmic diseases are particularly interesting. Ophthalmic drug therapy has several significant drawbacks, including frequent administration, poor osmosis, and / or rapid elimination. Using dendrimers as a strategy to overcome the limitations of conventional treatments for ophthalmic diseases represents a promising advance in this field, and the dendrimer-based approach to ophthalmic safety is intended to reflect the most advanced science to date. Several ophthalmic applications of dendrimers and dendrimer delivery systems are known. See “Dendrimer as nanocarrier for drug delivery” Prashant Kesharwani, Keerti Jain, Narendra Kumar Jain, Progress in Polymer Science 39(2014)268-307. However, most of these applications are still in the early stages of research, and to date, only a few commercially available products for the treatment of ophthalmic diseases using dendrimer delivery are known.

[0006] Therefore, there is a need to provide strategies for optimizing drug delivery and site-specific targeting. Furthermore, there is a need to provide delivery systems for sustained-release drugs that can extend the half-life of activators and improve their efficacy and bioavailability. In the delivery of biomolecules such as peptides and proteins, it is even more necessary to improve the binding activity of receptor-binding biomolecules and extend their half-lives by providing the possibility of multivalent delivery.

[0007] Furthermore, dendrimers can have a variety of applications in non-medical fields or industrial uses, such as antibody purification, cosmetics, catalysts, electronic devices, agriculture, food, filtration, energy storage, and building materials.

[0008] All references cited herein are incorporated in their entirety by reference for the purposes of this document. [Overview of the project]

[0009] Therefore, an objective of certain embodiments of the present invention is to provide a dendrimer platform based on polyethylene glycol components that is highly variable and flexible, and easy to synthesize, so that it can be adapted to a variety of uses and applications.

[0010] A further object of certain embodiments of the present invention is to provide a system with optimized drug delivery and site-specific targeting, particularly for treating ocular conditions.

[0011] Another object of certain embodiments of the present invention is to provide a delivery system for sustained-release drug delivery that can extend the half-life of the activator and improve the efficacy, binding activity, and bioavailability of the activator.

[0012] Another object of certain embodiments of the present invention is to provide a biodegradable drug delivery system in which the rate of degradation and the rate of activator release can be adjusted using a wide range of different biodegradable molecular groups, including hydrolyzable groups and bonds incorporated into the molecular structure of the drug delivery system. In certain embodiments, the biodegradable drug delivery system needs to be completely reabsorbed and degraded from local tissues and ultimately into initial building blocks that are readily excreted from the body.

[0013] Another object of certain embodiments of the present invention is to provide a biodegradable drug delivery system that enhances the binding affinity and / or binding activity of activators, such as peptides and proteins, to biological targets.

[0014] A further object and aspect of a particular embodiment of the present invention is to provide a method for treating a patient's disease / condition.

[0015] A further object of certain embodiments of the present invention is to provide dendrimers for non-medical applications or industrial uses such as antibody purification, cosmetic applications, catalytic applications, electronic equipment, agriculture, food, filtration, energy storage, building materials, coatings, adhesives, water purification, oil recovery, fragrance release, papermaking, environmental sensing and release systems, membranes, fibers, printing inks, surface chemical applications, thickeners, detergents, rheological modifiers, scaffolding, or applications in 3D printing.

[0016] The above objectives are solved by the present invention as described in the independent claims. Advantageous modifications are described in the dependent claims.

[0017] Some aspects of the present disclosure relate to a hyperbranched polymer comprising a core unit having at least three linkage sites c, and a plurality of polymer arms linked to the core unit at the linkage sites c, wherein at least one of the polymer arms is linked to a dendritic repeating unit (DCRU) by a hydrolyzable linkage, and the dendritic repeating unit (DCRU) comprises a branched unit linked to at least two polymer arms, each containing an end group or linked to a subsequent dendritic repeating unit which can be relinked to a further dendritic repeating unit, the polymer arms of the outermost dendritic repeating unit each containing an end group, the polymer arms are made of polyethylene glycol (PEG) units, and at least one of the outermost polymer arms is optionally conjugated with at least one activator. In some embodiments, at least 10%, preferably about 20-100%, of the chemical bonds of the linkages can be cleaved by hydrolysis. The bonds that can be cleaved by hydrolysis are preferably ester bonds. In certain embodiments, the ester bonds are introduced using a linker derived from an organic diacid.

[0018] In some embodiments, the building blocks or fragments of the superbranched polymer obtained / can be obtained after all hydrolyzable bonds of linkage are cleaved have an average molecular weight (Mn) of less than 50,000 Daltons. The activator may be covalently or noncovalently bonded to the superbranched polymer. In certain embodiments, the activator is covalently conjugated to the superbranched polymer.

[0019] In certain embodiments, the hyperbranched polymer is a G0-generation dendrimer-like hyperbranched polymer, where the surface end groups of the branched polymer are end groups of polymer arms linked to the core unit without further linkage to DCRUs. In other embodiments, the hyperbranched polymer is a high-generation Gx dendrimer-like hyperbranched polymer, where x is an integer from 1 to 10, defining the number of continuously linked dendritic repeating units in the hyperbranched polymer. Each polymer arm of the outermost dendritic repeating unit contains an end group, and at least one activator is conjugated to at least one of the outermost polymer arms.

[0020] In certain embodiments of this disclosure, the core unit and branched units of the hyperbranched polymer are the same or different and independent of each other, having 3-10, 4-8, 4-6, or 4 linkage sites c. In certain embodiments, the core unit and branched units are the same or different and each is derived from a polyol having at least three hydroxyl groups. In certain embodiments, the polyol is selected from the group consisting of glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomalt, lactitol, maltitol, mannitol, or sorbitol.

[0021] In certain embodiments of this disclosure, the polymer arms in the hyperbranched polymer include PEG units having an average molecular weight (Mw) in the range of about 1,000 to about 80,000 daltons, or about 10,000 to about 60,000 daltons, or about 15,000 to about 50,000 daltons. In certain embodiments, the average molecular weight of the polymer arm PEG units bound to the core is the same as or different from the average molecular weight of the polymer arms in the dendritic repeating unit. The average molecular weight of the polymer arm PEG units bound to the core may be higher or lower than the average molecular weight of the polymer arms in the dendritic repeating unit. For high-generation Gx hyperbranched polymers (x is an integer from 2 to 10), the average molecular weight of the polymer arm PEG units may decrease or increase from the innermost polymer arm to the outermost polymer arm.

[0022] In certain embodiments of this disclosure, terminal groups bonded to the outermost polymer arm are grafted directly to the end of the polymer arm or via a bifunctional linker containing hydrolyzable bonds such as carboxyl groups, dicarboxyl groups, carboxamide groups, dicarbboxamide groups, functionalized aliphatic groups, heteroaliphatic groups, or aromatic or heteroaromatic groups. In further embodiments, terminal groups bonded to the outermost polymer arm are electrophiles such as active ester groups (such as succinimidyl esters and succinimidyl carbonates), nitrophenyl carbonates, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, alkynes, azides, norbornene, epoxides, mesylates, tosylates, tresyl, cyanurates, orthopyridyl disulfide, or halogens, amines such as primary amines, hydroxyls, alcohols, thiols, These functional groups include nucleophiles such as azide anions and carboxyl groups, functional groups for click chemistry, functional groups for cyclization such as 1,3-dipolar cycloaddition, [3+2] cycloaddition such as alkene-nitrone cycloaddition or alkyne-nitrone cycloaddition, and [4+2] cycloaddition, functional groups for thiol-ene reactions, functional groups for hetero-Diels-Alder cycloaddition, functional groups for nucleophilic ring-opening, functional groups for non-aldol-type carbonyl reactions, functional groups for addition reactions to carbon-carbon multiple bonds, polymerizable vinyl groups, or a combination thereof.

[0023] In certain embodiments of this disclosure, the terminal groups bonded to the outermost polymer arms are linker-space functional groups selected from succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), succinimidyl azelate (SAZ), and succinimidyl glutaramide (SGA).

[0024] In certain embodiments of this disclosure, the terminal groups bonded to the outermost polymer arms are alkyne compounds such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]-nonine (BCN); or norbornene or trans-cyclooctene (TCO); or functional groups selected from azide, 3,4-dihydroxyphenylacetic acid (DHPA), or tetrazine (Tz).

[0025] In certain embodiments of the present disclosure, the connections between the core unit and the polymer arms linked to the first dendritic repeating unit, and / or the connections between successive dendritic repeating units, are formed by click chemistry.

[0026] In certain embodiments of this disclosure, the coupling is formed by click chemistry, which is formed by reacting a polymer arm functionalized with an alkyne, cycloalkyne, or strained or terminal alkene moiety with a polymer arm functionalized with an azide or tetrazine moiety in a SPAAC or IEDDA type click chemistry coupling reaction. In certain embodiments, the alkyne moiety is a dibenzocyclooctyne moiety.

[0027] In certain embodiments of the present disclosure, connections between a core unit and a polymer arm connected to a first dendritic repeating unit, and / or connections between successive dendritic repeating units, are formed between a polymer arm connected to a core unit and a polymer arm connected to a branching unit of a dendritic repeating unit, and / or between a polymer arm of a dendritic repeating unit and a polymer arm of a successive dendritic repeating unit.

[0028] In certain embodiments of this disclosure, the activator conjugated to at least one terminal group located on the surface of the hyperbranched polymer is selected from the group consisting of therapeutic activators or diagnostic activators.

[0029] In certain embodiments of this disclosure, the activator conjugated to at least one of the terminal groups located on the surface of the dendrimer is a steroid, a nonsteroidal anti-inflammatory drug (NSAID) (such as diclofenac, ibuprofen, meclofename, mefanamic A, sarsalate, sulindac, tolmetin, ketoprofen, diflunisal, piroxicam, naproxen, etodolac, flurbiprofen, fenoprofen C, indomethacin, celecoxib, ketorolac, nepafenac, etc.), an intraocular pressure lowering agent, an antibiotic such as ciprofloxacin, analgesics such as bupivacaine, calcium channel blockers such as nifedipine, cell cycle inhibitors such as simvastatin, a protein such as insulin, etc. Small molecule hydrophilic drugs, small molecule hydrophobic drugs, hydrophilic peptides and protein drugs (such as insulin, single-chain antibody fragments, Fab fragments, IgG antibodies, fusion antibodies, etc.), aptamers, in particular bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDS), steroids, antibiotics, analgesics, calcium channel blockers, cell cycle inhibitors, chemotherapeutic agents, antivirals, anesthetics, hormones, anticancer drugs, antitumor drugs, viruses, gene delivery viruses such as AAV, protein conjugates (such as nanobodies, afibodies, ankyrin, DARPin, etc.), or any combination thereof.

[0030] In certain aspects of this disclosure, the activators covalently or non-covalently conjugated to at least one of the terminal groups located on the surface of a hyperbranched polymer are peptides selected from the group consisting of compstatin, APL-1, Fc-III-4C, Beov (brolucizumab), Zimura (abacincaptado pegol), pegcetacoplan, abisipal pegol, lamparizumab, Fovista, listeganib, AXT107, elamipretide, THR149, ALM201, VGB3, and largazole.

[0031] In certain embodiments of this disclosure, the activator is covalently bonded to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% of the terminal groups located on the surface of the hyperbranched polymer.

[0032] In certain embodiments of this disclosure, the dendritic repeating unit is represented by formula (i), [ka] In the formula, A is a connection to a polymer arm connected to the core unit, or A is a connection to B of a preceding dendritic repeating unit represented by formula (i), and L A is a linker, m is either 0 or 1, n is an integer between 20 and 2000, o is an integer between 20 and 2000, n and o may be different or the same, X is a branching unit such as a polyol derivative unit, L B is a linker, p is either 0 or 1, B is a linkage to A of a continuous dendritic repeating unit, or a linkage to an activator, L A and L B The elements may be different or the same, m and p may be different or the same, y is an integer from 2 to 9, y = c-1, c is the linking site c of the branched unit X, and the repeating dendritic structures in the hyperbranched polymer may be different or the same.

[0033] In a more specific embodiment, A comprises a functional group formed by click chemistry such as triazole or dihydropyrazine, and / or linker L A and / or L B This includes diacid and / or acid diamide groups, succinic acid, glutarate, adipate, azelaic acid, or carboxyl and / or carboxamide groups such as glutaramide. In certain embodiments, the linker L A and / or L B This includes the structure represented by formula (ii), [Chemical formula] In the formula, U 1 and U 2 are independently NH or O, may be the same or different, and t is an integer from 0 to 10. The linker L A and / or L B further contains a polyethylene glycol unit between the bond to B and the carboxyl group, carboxamide group or the structure of formula (ii).

[0034] In certain embodiments of this disclosure, the present invention provides a method for producing the superbranched polymer described herein by divergent synthesis, the method comprising the steps of (a) providing a core unit having at least three linkage sites c, and polymer arms linked to the core unit and having functional groups suitable for click chemistry at the ends of a plurality of polymer arms, and (b) providing one polymer arm containing a functional group suitable for forming a linkage with a corresponding functional group (e.g., azide, alkyne, alkene, or tetrazine) of a polymer arm linked to the core by click chemistry, and at least two containing functional groups that do not react by click chemistry. The process includes the steps of: (c) providing a dendritic repeating unit precursor containing polymer arms; (d) forming a link between the polymer arms linked to the core and the polymer arms of the dendritic repeating unit precursor by click chemistry; (e) optionally converting functional groups of at least two polymer arms containing non-reactive functional groups in click chemistry to functional groups suitable for click chemistry; and (f) conjugating an activator containing functional groups to the outermost polymer arm by reacting it with the functional groups of the outermost polymer arm, thereby forming a hyperbranched polymer-activator conjugate. In the case of high-generation Gx hyperbranched polymers (where x is an integer from 2 to 10), step (d) may be essential, and before conjugating the activator in step (f), further consecutive dendritic repeating unit precursors are linked by click chemistry to the click-chemistry-suitable functional groups obtained in step (d) to form a hyperbranched polymer.

[0035] In a particular embodiment of the method, the dendritic repeating unit precursor in step (c) is represented by formula (iii), [ka] In the formula, C contains a functional group suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines), D contains a functional group unsuitable for click chemistry (such as succinimidyl), and L A, m, n, X, o, L B p and y are as defined above, and the repeating units of the dendritic structure may be the same or different.

[0036] In further embodiments of this method, the present invention relates to a method for producing the superbranched polymer described herein by convergent synthesis, comprising the steps of (I) providing a dendritic repeating unit precursor comprising one polymer arm comprising a functional group suitable for forming click chemistry links (such as an azide, alkyne, alkene, or tetrazine) and at least two polymer arms comprising a functional group that does not react in click chemistry, and (II) conjugating at least one of the at least two polymer arms comprising a functional group that does not react in click chemistry with an activator comprising a functional group, and (I (II) Providing a core unit having at least three linkage sites c, and polymer arms linked to the core unit, each polymer arm having a functional group suitable for click chemistry (e.g., azide, alkyne, alkene, or tetrazine) at its end; (IV) Forming a hyperbranched polymer-activator conjugate by forming a click chemistry linkage between the polymer arms linked to the core provided in step III) and polymer arms containing functional groups suitable for forming click chemistry links of the activator conjugate dendritic repeating unit precursor obtained in step II). In embodiments of this method, the dendritic repeating unit precursor in step I) is represented by formula (iii) above.

[0037] In the case of a high-generation Gx hyperbranched polymer (where x is an integer from 2 to 10), the activator-conjugated dendritic repeating unit precursor obtained in step II) can be linked by click chemistry to an inverse dendritic repeating unit precursor comprising one polymer arm containing a functional group that does not react by click chemistry and at least two polymer arms containing functional groups suitable for click chemistry (such as azides, alkynes, alkenes, or tetrazines). The functional group of the polymer arm that does not react by click chemistry is then converted to a functional group suitable for click chemistry before linking to a further inverse dendritic repeating unit precursor, or before forming a link with the polymer arm linked to the core by click chemistry in step IV), thereby forming a high-generation hyperbranched polymer. In one embodiment, by performing steps I) and II) on an activator-conjugated dendritic repeating unit precursor, a dendritic repeating unit precursor having different activators conjugated to polymer arms can be obtained. By using a mixture of the obtained activator-conjugated dendritic repeating unit precursors in step IV), a superbranched polymer-activator conjugate having different activators in different regions of the surface of the superbranched polymer is formed.

[0038] In a further embodiment, the present invention relates to a superbranched polymer described herein for use as a pharmaceutical agent. In a further embodiment, the present invention relates to a therapeutic method comprising treating a patient's disease or condition using a superbranched polymer of an embodiment of the present invention. In that embodiment, the superbranched polymer is used to treat eye diseases such as any posterior segment eye disease that affects the vascular system and integrity of the retina, macula, and choroid and leads to visual impairment, visual loss, or blindness, specifically eye diseases such as age-related macular degeneration (AMD), cystoid macular edema (CME), diabetic macular edema (DME), posterior uveitis, and diabetic retinopathy, as well as posterior segment eye conditions resulting from age, trauma, or surgical intervention.

[0039] In a further embodiment, superbranched polymers are used for retinal neovascularization, choroidal neovascularization, exudative AMD, atrophic AMD, retinal vein occlusion, diabetic macular edema, retinal degeneration, anterior chamber hemorrhage, presbyopia, corneal transplant rejection, retinoblastoma, melanoma, miosis, mydriasis, glaucoma, conjunctivitis, intraocular infection, choroidal neovascularization (CNV), intraocular tumors, retinal neuritis, inflammation, autoimmune uveitis, uveitis, proliferative vitreoretinopathy, and corneal degeneration, acute and chronic macular neuroretinopathy. Central serous chorioretinopathy, macular edema, acute multifocal placoid pigment epitheliopathy, Behçet's disease, birdshot chorioretinopathy, posterior uveitis, posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusion, central retinal vein occlusion, disseminated intravascular coagulation, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal artery microaneurysms, Koert's disease, parafoveal telangiectasia, unilateral retinal retinoretinopathy Membrane vein occlusion, papillary phlebitis, carotid artery disease (CAD), dendritic vasculitis, sickle cell retinopathy, vascular streaks, familial exudative vitreoretinopathy, Eels' disease, proliferative vitreoretinopathy, diabetic retinopathy, retinal diseases with tumors, congenital retinal pigment epithelial hypertrophy (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, complex hamartoma of the retina and retinal pigment epithelium, retinoblastoma, fundus vascular proliferative neoplasm, retinal astrocytoma, intraocular lymphoma, myopic retinal malignancy It is used to treat eye diseases selected from the group consisting of sexually transmitted diseases, acute retinitis pigmentosa, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal cancer, retinitis pigmentosa, Leber congenital amaurosis, choroidal agenesis, X-linked retinitis pigmentosa, Best vitiligo macular degeneration, X-linked retinoschisis, CNGA3 color blindness, CNGB3 color blindness, LHON, Stargardt disease, Usher syndrome, Norrie disease, Baldett-Beedl syndrome, and red-green color blindness.

[0040] In certain embodiments, the superbranched polymer is formulated for direct injection into the patient's treatment site, for example, by parenteral administration, intratumoral injection, or intraocular injection such as intravitreous, anterior chamber, subconjunctival, retrobulbar, sub-Tenon's capsule, subretinal, or choroidal injection. The superbranched polymer can be administered by direct injection, orally, incorporated into a gel, or incorporated into an implant.

[0041] definition The terms “hyperbranched polymer” or “hyperbranched polymer,” or simply “branched polymer” or “branched polymer,” are used interchangeably herein to refer to dendrimer-like branched polymers or polymers having a dendrimer-like dendritic structure. The term “dendrimer” is used herein as a synonym. However, while dendrimers are monodisperse, highly symmetrical molecules with strictly defined compositions, the hyperbranched polymers of the present invention are polydisperse molecules, as they contain polyethylene glycol arms or units that have some degree of polydispersity, similar to most synthetic polymer structures. Although the polydispersity of PEG chains and precursor molecules containing them may be low, this suggests that the dendrimer-like hyperbranched polymers described herein also possess polydispersity.

[0042] Polydispersion is represented by the polydispersity index D, where D = M w / M n This is the result. Here, M w This is the weight-average molar mass, and M n This is the number-average molar mass measured by gel permeation chromatography. For most polyethylene glycol (PEG) materials, the polydispersity index is a parameter specified in the product specifications by the manufacturer as an indicator of the uniformity and quality of the material. The polydispersity of PEG multi-arm precursors may be less than 1.3, less than 1.2, or less than 1.1.

[0043] The term "biodegradable" refers to a material or object (such as the superbranched polymers according to the present invention) that decomposes in vivo, i.e., when placed in the body of a human or animal, or in vitro, when immersed in an aqueous solution under physiological conditions such as pH 7.2-7.4 at 37°C. In the context of the present invention, as disclosed in detail below herein, superbranched polymers, when administered or accumulated in the body of a human or animal, slowly biodegrade and are excreted over time. In certain embodiments, biodegradation occurs at least partially by ester hydrolysis in the aqueous environment within the body. Biodegradation may occur by hydrolysis or enzymatic cleavage of covalent or conjugation bonds within linker groups and / or polymer arms. Superbranched polymers decompose slowly and are excreted via physiological pathways. In certain embodiments, the superbranched polymers of the present invention are biodegradable and stable over long periods (e.g., about 1 month, 3 months, or 6 months). In certain embodiments, the hyperbranched polymer does not biodegrade until, for example, an activator or at least a significant amount thereof (e.g., at least 50%, at least 75%, or at least 90%) has been released therefrom.

[0044] In this specification, the terms “precursor,” “component,” or “building block” refer to molecules or compounds that react with each other, are linked together via covalent bonds, and form a hyperbranched polymer.

[0045] The portions of precursor molecules still present within the final dendrimer-like superbranched polymer are also referred to herein as “units” or “polymer arms.” Thus, “units” or “polymer arms” belong to the main building blocks or components of the polymer superbranched polymer. For example, a superbranched polymer suitable for use in the present invention may include, in addition to core units and branching units, identical or different polyethylene glycol units or arms, as further disclosed herein.

[0046] As used herein, the term “core unit” refers to the central structural unit of a hyperbranched polymer from which polymer arms or dendritic repeating units (DCRUs) or dendrons are derived. The core unit has at least three linkage sites c (or valences), each of which is linked, i.e., covalently bonded, to a polymer arm or dendritic repeating unit. For example, in the case of a generation G0 multi-armed PEG branched polymer, the core unit can be derived from a polyol compound in which each hydroxyl group is poly(ethoxylated).

[0047] An exemplary core unit structure having three connection points, where connection point c is indicated by OH, is shown below. [ka]

[0048] As used herein, the terms “branching unit” or “branching point” refer to a constituent unit within a dendritic repeating unit having at least three linkage sites c' (or valences), to which a polymer arm or another dendritic repeating unit is linked. A branching unit may have the same or different chemical structure as the core unit.

[0049] As used herein, the term “dendritic repeating unit” (DCRU), also known as “dendron,” refers to a constituent repeating unit with a branch point and polymer arms extending therefrom, where c’≧3 is the number of connecting points. This may be continuously connected to a total of c polymer arms extending from a core unit and / or other DCRUs to form a dendrimer-like hyperbranched structure.

[0050] As used herein, the term “end group” refers to a constituent unit located at the end of a polymer arm or DCRU, such as a functional group. In a hyperbranched polymer, end groups on the outermost surface of the hyperbranched polymer can be used to conjugate or bond activator molecules to the hyperbranched polymer. End groups may consist of linkers having hydrolyzable groups linked to the end functional group.

[0051] The term "generation" (abbreviated as "G") refers to a set of dendritic repeat units that are located the same number of dendritic repeat units away from the free valence of a dendron.

[0052] As used herein, the term “dendron” refers to a portion of a hyperbranched polymer having only one free valence, comprising only DCRUs and terminal groups, where each pathway from the free valence to any terminal group contains the same number of constituent repeating units.

[0053] As used herein, the term “conjugate” includes covalent or non-covalent bonds between an activator and a hyperbranched polymer. Conjugation may also include non-covalent bonds to terminal groups of a hyperbranched polymer that have affinity for the activator molecule, and may be a means of linking an activator molecule to a hyperbranched polymer.

[0054] As used herein, the term “release” (and accordingly, terms such as “released,” “release,” etc.) refers to the chemical separation and provision of an activator from the superbranched polymer of the present invention to the surrounding environment. The released activator may or may not still be bound to molecular fragments of the superbranched polymer. The surrounding environment may be an in vitro or in vivo environment, as described herein. In certain embodiments, the surrounding environment is vitreous fluid and / or ocular tissue such as the retina and choroid. The bond between the API and the superbranched polymer may be covalent. In this case, the API can be separated from the superbranched polymer by chemical reactions such as hydrolysis of the bond formed by the linker group. Furthermore, by employing multiple hydrolyzable bond chemistrys, the API can be released from the same or mixed superbranched polymer at multiple rates to achieve a desired release profile. Furthermore, the superbranched polymer may be functionalized with end groups that bond non-covalently to the API, and the API can be released according to the bond affinity kinetics of the end group-API pairs of the superbranched polymer. Multiple non-covalent end group-API pairs can be employed to achieve a desired release profile.

[0055] The term "100% release of the activator" should be interpreted as 95% to 100%. This controlled release method is achieved by several parameters that are characteristics of the drug delivery system, as disclosed herein. Each of these characteristics of the drug delivery system can be involved in controlled release, either individually or in combination with each other.

[0056] In the context of this invention, the term “sustained release” is intended to characterize products such as biodegradable hyperbranched polymers that are formulated to make the activator available over a long period of time, thereby enabling a reduction in the frequency of administration compared to immediate-release formulations (e.g., solutions of the activator applied topically to the eye, i.e., eye drops). Other terms that may be used interchangeably with “sustained release” herein include “sustained release” or “controlled release.” In the sense of this invention, the term “sustained release” includes constant release of the activator, decreasing release of the activator, increasing release of the activator, and any combination thereof (e.g., decreasing release of the activator after constant release of the activator). In the sense of this invention, the terms “decreased” or “decrease” refer to a decrease in activator release over time. Specifically, the term “sustained release” refers to the release of an activator from a hyperbranched polymer or a drug delivery system containing a hyperbranched polymer in a predetermined manner, in contrast to immediate release such as bolus injection. In certain embodiments, controlled release refers to the amount of activator released over the total number of days required for 100% release of the activator in an aqueous solution under in vitro physiological conditions such as pH 7.2-7.4 and 37°C.

[0057] As used herein, the term “longer period” means any period of time that a person skilled in the art would consider to be an extended treatment of a disease, in particular a period of at least about one week, or at least about one month, for example, up to about 12 months, or any intermediate period such as about 1 to about 6 months, about 2 to about 4 months, about 2 to about 3 months, or about 3 to about 4 months, or any other period otherwise disclosed herein.

[0058] "Zero-order" release, or "substantially zero-order" release, or "near zero-order" release, is defined as a relatively straight line in a graph of the proportion of released activator versus time. In certain embodiments of the present invention, substantially zero-order release is defined as an amount of activator released that is within 20% of the elapsed time.

[0059] The terms “API,” “Active(pharmaceutical) ingredient,” “Active(pharmaceutical) agent,” “Active(pharmaceutical) ingredient,” “(active) therapeutic agent,” “active drug,” and “drug” are used interchangeably herein and refer to substances used in a finished medicine product (FPP) that are intended to impart pharmacological activity or have a direct effect on the diagnosis, cure, alleviation, treatment, or prevention of a disease or on the restoration, correction, or modification of a patient’s physiological function, and substances used in the preparation of such a finished medicine product.

[0060] The activators used in accordance with the present invention may be activators for the treatment and / or prevention of disease or disorder, or diagnostic agents such as markers. In embodiments of the present invention, the activator is a low-water-solubility activator (i.e., having water solubility of less than about 1000 μg / mL or less than about 100 μg / mL). In other embodiments of the present invention, the activator is a high-water-solubility activator (i.e., having water solubility of about 1000 μg / mL or more, or even more than about 10 μg / mL). This definition does not depend on drugs approved by government agencies.

[0061] For the purposes of the present invention, the activator can be used in all possible forms, including free acids, free bases, polymorphs or any pharmaceutically acceptable salts, anhydrides, hydrates, cocrystals, or other solvates or derivatives (such as prodrugs or conjugates). The activator may need to be functionalized in order to be conjugated to a hyperbranched polymer, unless it already has functional groups suitable for conjugation. In this description or claims, whenever the activator is mentioned without further specification, even if not explicitly stated, it refers to the activator in any such polymorph, pharmaceutically acceptable salt, anhydride, or solvate (including hydrate) form. With respect to the activator, preferred solid forms include, without limitation, pure substance forms in any physical form known to those skilled in the art.

[0062] As used herein, the term “therapeutically effective” refers to the amount of activator required to produce the desired therapeutic outcome after administration. For example, in the context of the present invention, one desired therapeutic outcome is a reduction in symptoms associated with dry eye (DED), as measured by in vivo tests known to those skilled in the art, such as an increase in Schirmer tear film score, a decrease in staining values ​​measured by conjunctival lysamine green staining or corneal fluorescein staining, a decrease in the score for dry eye severity and / or dry eye frequency on a visual analog scale (VAS), a decrease in the standard patient-assessed score for ocular surface disease index and / or dry eye, and a decrease in best corrected visual acuity. In one embodiment, "therapeutic effectiveness" refers to the amount of activator in a sustained-release intratubular implant that, in terms of therapeutic effect, can achieve a tear film concentration equivalent to a cyclosporine concentration of 0.236 μg / mL (which is considered necessary for immunomodulation (Tang-Liu and Acheampong, Clin. Pharmacokinet. 44(3), pp. 247-261)), and once this tear film concentration is achieved, can be maintained over a long period, particularly substantially throughout the entire remaining period of implantation.

[0063] In this specification, the term “patient” includes both human and animal patients. Therefore, the biodegradable drug delivery system according to the present invention is suitable for human or veterinary medical use. Generally, “subject” refers to the individual (human or animal) to whom the drug delivery system according to the present invention is administered. “Patient” refers to a subject requiring treatment due to a specific physiological or pathological condition. “Patient” is not necessarily diagnosed with a specific physiological or pathological condition prior to receiving the drug delivery system.

[0064] The molecular weights of the hyperbranched polymers, polymer precursors, polymer units, arms, etc., used for the purposes of the present invention and disclosed herein can be determined by analytical methods known in the art. For example, the molecular weight of polyethylene glycol can be determined by any method known in the art, including gel electrophoresis, e.g., SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), gel permeation chromatography (GPC) including GPC or dynamic light scattering (DLS) with a static light scattering detector (SLS), liquid chromatography (LC), and mass spectrometry (e.g., matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) spectroscopy or electrospray ionization (ESI) mass spectrometry). The molecular weights of polymers containing polyethylene glycol precursors disclosed herein are average molecular weights (based on the molecular weight distribution of the polymer) and can therefore be represented by various average values, including weight-average molecular weight (Mw) and number-average molecular weight (Mn). For multi-arm PEG precursors used in some aspects of the present invention, the molecular weights expressed herein are the number-average molecular weight (Mn) measured by gel permeation chromatography according to standard methods known in the art, using suitable molecular weight standards such as polyethylene glycol or polystyrene standards. Typically, materials, particularly multi-arm precursors, are purchased with specific molecular weights and polydispersity defined by the vendor. Suitable PEG precursors are available from numerous suppliers, such as Jenkem Technology and Xiamen SinoPeg Biotech Co. Ltd.

[0065] As used herein, the term "Day 1" refers to the point in time immediately following "Day 0." Therefore, whenever "Day 1" is used, it always refers to the period of one day or approximately 24 hours that has already elapsed since the administration of the drug delivery system.

[0066] As used herein, the term “approximately” in relation to a measured quantity refers to the normal variation in that measured quantity that can be expected by a person skilled in the art when performing a measurement and exercising a level of care appropriate to the purpose of the measurement and the precision of the measuring instrument.

[0067] The term "at least about" in relation to a measured quantity refers to the normal variation in the measured quantity and amounts higher than the measured quantity that a person skilled in the art would expect when performing a measurement and exercising a level of care appropriate to the purpose of the measurement and the precision of the measuring instrument.

[0068] As used herein, the term “mean” refers to the central or representative value within a set of data (points), calculated by dividing the sum of the data (points) in the set by that number (i.e., the mean of the dataset).

[0069] As used herein, unless otherwise explicitly indicated by the context, the singular forms "a," "an," and "the" refer to multiple objects.

[0070] In this specification, the term "and / or" as used in expressions such as "A and / or B" is intended to include both "A and B" and "A or B".

[0071] Open terms such as "include," "including," "contain," and "containing" all mean "comprising." These unrestricted transition clauses are used to introduce an unrestricted list of elements, method steps, etc., that do not exclude additional elements or method steps not listed.

[0072] Where used herein in conjunction with a specific value or number, the term “maximum” means including that respective value or number.

[0073] The terms "from A to B," "of A to B," and "to A to B" are used interchangeably in this specification and all refer to the range from A to B, including the upper and lower limits of A and B.

[0074] Throughout this disclosure, various aspects of the invention are presented in range form. It should be understood that the range form is for convenience and brevity only and should not be interpreted as an inflexible limitation on the scope of the invention. Therefore, the range description should be considered to specifically disclose not only the individual numbers within that range, but also all possible subranges. For example, a range description such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range. A stated range of numbers includes the number defining the range and each integer within the defined range.

[0075] As used herein, the abbreviation "PBS" means phosphate-buffered saline.

[0076] As used herein, the abbreviation "PEG" refers to polyethylene glycol. [Brief explanation of the drawing]

[0077] [Figure 1] The following schematic diagrams illustrate different synthesis methods a to c for dendrimers or hyperbranched polymers. [Figure 2] The generations of dendrimers or superbranched polymers are schematically shown. [Figure 3] a) A schematic diagram of the formation of a PEG superbranched polymer by DBCO-azide coupling is shown, and b) A schematic 3D model of an 8-arm PEG core with 8 4-arm PEG branched repeating units conjugated with a peptide is shown. [Figure 4] The structures of the peptide compstatin, APL-1(Mod2), and APL-1(Mod3) are shown. [Figure 5] Examples 6 and 7 show the purification apparatus using a) dialysis and b) SEC column filtration. [Figure 6] This figure shows the UHPLC analysis of the ultrabranched polymer-compstatin conjugate purified by dialysis in Example 6. [Figure 7] This figure shows the UHPLC analysis of the ultrabranched polymer-compstatin conjugate purified by SEC column filtration in Example 7. [Figure 8a] This is a UHPLC graph of standard curves for Compstatin samples (12.5, 25, 50, 100, 200 μg / mL) with calibration curves. [Figure 8b] This is a UHPLC graph of the 4-arm G0 hyperbranched polymer-compstatin conjugate from Example 1. [Figure 9] Based on Example 8, a graph comparing the substitution rates of different linker PEGs containing compstatin and compstatin lysine is shown. [Figure 10] The graph shows the optimization conditions for compstatin conjugation in Example 8. [Figure 11a] The SPR results for Example 9 are shown, and for C3 binding, the KD values ​​for four compstatin samples from different vendors are shown in a1-4. [Figure 11b] The SPR results for Example 9 are shown, and the equilibrium analysis for the C3 bond is presented. [Figure 12a] The SPR results for Example 9 are shown, and for C3b binding, the KD values ​​for four compstatin samples from different vendors are shown in a1-4. [Figure 12b] The SPR results for Example 9 are shown, and the equilibrium analysis for C3b bonds is presented. [Figure 13] The SPR results for Example 9 are shown, with the C3 and C3b bonds represented as a) APL-1 (amine acetylated), b) APL-1 (amine acetate), and c) APL-1 (lysine-terminated). [Figure 14] The SPR results for IgG binding are shown, with a-c being Fc-III 4C and d being Fc-III. [Figure 15]This shows a comparison of SPRs between free compstatin and polyvalent compstatin. [Figure 16] The SPR results for a) 8a-40k-PEG-[(4a-2kPEG-(compstatin)3]8, b) 4a-40kPEG-SGA-(compstatin)4, c) 4a-40kPEG-SS-(compstatin)4, and d) SS-compstatin (hydrolyzed) are shown compared to free compstatin. [Figure 17] This graph plots the dissociation constant (KD) of the hyperbranched polymer-compstatin conjugate against the corresponding number of peptide substitutions. [Figure 18] This is a diagram of the Alternative Pathway (AP) hemolysis assay. [Figure 19] This shows the IC50 results in the AP hemolysis test for hyperbranched polymer-compstatin conjugates. [Figure 20] This calibration curve shows the relationship between the hydrodynamic radius Rh and half-life T1 / 2, measured in the vitreous fluid of New Zealand white rabbits, to predict the sustained release of dendrimer drug conjugates. [Figure 21a] This shows the degradation effect when the temperature is varied between 35°C and 39°C under constant pH 7.4 conditions. [Figure 21b] This shows the degradation effect when the temperature is varied between 35°C and 39°C under constant pH 7.4 conditions. [Figure 21c] This shows the degradation effect when the temperature is varied between 35°C and 39°C under constant pH 7.4 conditions. [Figure 22a] This shows the degradation effect when the pH is varied from 7.0 to 8.5 at a constant temperature of 37°C. [Figure 22b] This shows the degradation effect when the pH is varied from 7.0 to 8.5 at a constant temperature of 37°C. [Figure 22c] This shows the degradation effect when the pH is varied from 7.0 to 8.5 at a constant temperature of 37°C. [Modes for carrying out the invention]

[0078] In a particular embodiment, the present invention relates to a hyperbranched polymer (dendrimer) comprising a polyethylene glycol polymer unit and an activator covalently bonded or conjugated to at least one of the outermost arms of the hyperbranched polymer. The conjugation includes covalent and non-covalent bonding to terminal groups of the peptide hyperbranched polymer, such as those having affinity for the activator molecule, and can also be a means of linking the activator to the hyperbranched polymer.

[0079] Dendrimers are monodisperse polymers with multiple reactive end groups on their surface. Dendrimers are often compared to dendritic structures, i.e., branched molecular structures that offer a wide variety of possible end groups and exceptional structural control. Elements are added to the dendrimer structure through a series of chemical reactions, constructing a spherical structure branched from a starting atom or core unit. The central core unit has at least two or at least three reactive functional groups, and the repeating branching is organized into a series of "radial concentric layers" called "generations." Superbranched polymers can have the same molecular structure as dendrimers even if they are not monodisperse, as they can be constructed using polydisperse precursors or units.

[0080] The inventors have found that the strategies for optimizing drug delivery and site-specific targeting using dendrimer-like hyperbranched polymers described herein offer several advantages for drug delivery, and that the advantages of dendrimers can be utilized even for hyperbranched polymers that have a structure similar to dendrimers but are not monodisperse molecules. For example, dendrimer-like hyperbranched polymers can provide various terminal functions that can be used to modulate the hydrophobicity / hydrophilicity of hyperbranched polymers used as carriers of activators, or they can be used as conjugation precursors to target molecules to enhance the interaction between APIs and hyperbranched polymers, for example, by improving the binding activity of polyvalent binding to receptors and / or conjugate biomolecules such as peptides and proteins.

[0081] Multiple surface groups on a hyperbranched polymer allow for the immobilization of more APIs in a desired binding manner, enabling controlled release under different degradation conditions or kinetics. Hyperbranched polymer-drug conjugates can improve the stability and solubility of delivered therapeutic agents, reduce systemic effects compared to free drugs, and enhance efficacy at the target site. Furthermore, hyperbranched polymers may possess symmetrical structures that provide numerous intramolecular cavities for capturing unbound API molecules. Additionally, the large external hydration radius, particularly in PEG-based dendrimer structures, extends the half-life of dendrimer-drug conjugates in vivo, such as in the vitreous humor, allowing for the control and regulation of sustained release of activators.

[0082] Furthermore, biodegradable synthetic dendrimers have the advantage of incorporating controllably degradable functional groups, such as hydrolyzable or enzymatically cleavable bonds. Upon degradation, fragments with small molecular weights and low hydration radii are produced, generating different half-lives that determine whether they are excreted from the body. The incorporated degradable groups can be used to adjust and control the release rate of activators associated with the dendrimer.

[0083] Superbranched polymers In certain embodiments of the present invention, a hyperbranched polymer is provided, formed from a plurality of building blocks of a dendritic structure (excluding activators), e.g., a core unit, polymer arms, branching units, linkers and / or extenders, and dendritic repeating units. In one embodiment, a hyperbranched polymer is provided, comprising a core unit having at least three linking sites c, and a plurality of polymer arms linked to the core unit at the linking sites c, each polymer arm comprising an end group, or comprising a branching unit linked to at least two polymer arms each comprising an end group, or linked to the next dendritic repeating unit, which is linked to a dendritic repeating unit that can be linked again to further dendritic repeating units, each polymer arm of the outermost dendritic repeating unit comprising an end group, and the polymer arms comprising or composed of linear polyethylene glycol (PEG) units, and at least one activator is conjugated to at least one of the end groups located on the outermost polymer arms of the hyperbranched polymer. The hyperbranched polymer is biodegradable in an aqueous environment by comprising chemical bonds that can be cleaved by hydrolysis.

[0084] In embodiments, the superbranched molecule is formed from building blocks linked at least partially by hydrolyzable bonds, or from links positioned such that complete hydrolysis of all hydrolyzable bonds in the polymer yields hydrolyzable fragments, each having a molecular weight of less than 40 kDa. This can be achieved by selecting suitable building blocks or precursors having a molecular weight of less than 40 kDa and linking them via hydrolyzable, typically acid-unstable chemical bonds, such as ester or amide bonds, as further described herein. For example, by including a diacid linker linking constituent repeating units, each having a molecular weight of less than 40 kDa, hydrolysis can yield hydrolyzable fragments, each satisfying the desired molecular weight limit. When using non-hydrolyzable bonds, such as bonds formed by some click chemistry reaction, such as alkyne-azide coupling, these bonds must be located between building blocks that satisfy the molecular weight limit of hydrolyzable fragments of less than 40 kDa.

[0085] In certain embodiments of the present invention, the overall molecular size and number of surface groups of the superbranched polymer gradually increase with the addition of a continuous layer of monomers called generations. Biodegradable superbranched polymers can be synthesized by divergent synthesis, convergent synthesis, or a combination of both. See Figure 1. In the divergent method, monomers or so-called dendritic repeating units (DCRUs) are added in a repeating sequence, starting with a continuous increase in the number of branches from a multivalent core to the surface molecule. The molecular size and number of surface groups gradually increase with the addition of a continuous layer of monomers called generations. The convergent method involves the synthesis of the superbranched polymer from the surface to the core, resulting in the formation of conical wedge units or dendrons, which are bonded to the multivalent core in the final step.

[0086] The hyperbranched polymers of certain embodiments of the present invention include, as major building units, a core unit, a plurality of branched units which may optionally be derived from polyols, a plurality of polymer arms which may include polyethylene (PEG) units, and optionally hydrolyzable linker groups, connecting groups between building units, terminal groups and conjugate activators such as peptides. All of these components or building units are further described below herein.

[0087] The links formed between different polymer arms in a hyperbranched polymer may include hydrolyzable bonds, which are formed by introducing appropriate linker groups between the PEG arms and the functional groups that link different units to form the hyperbranched polymer. These linkers that form hydrolyzable bonds promote biodegradation in aquatic environments, such as within the human or animal body. Hydrolyzable chemical bonds may also be acid-unstable to promote cleavage in more acidic environments, such as those found in tumors at the cellular level.

[0088] Hydrolyzable chemical bonds may include bonds or linkages selected from the group consisting of amines, amides, urethanes, esters, anhydrides, ethers, acetals, ketals, nitriles, isonitriles, isothiocyanates, or imine bonds, and combinations thereof. These bonds are typically formed during the synthesis of hyperbranched polymers by condensation reactions or click chemistry of appropriately functionalized precursors. In certain preferred embodiments, the hydrolyzable bond is an ester bond, such as an ester bond formed using diacid linkers such as succinic acid, glutaric acid, adipic acid, and higher homologues.

[0089] An exemplary structure of a superbranched polymer in a specific embodiment of the present invention is shown below. [ka]

[0090] In this formula for a generation G1 hyperbranched polymer, X represents a core unit or a branched unit derived from a polyol such as glycerol, each of which is linked to three polyethylene glycol arms, and the branched unit is linked to a polyethylene glycol arm of the central core unit via a linker group Y by one of its polyethylene glycol arms. This linker Y includes hydrolyzable bonds such as ester bonds or amide bonds, as further defined herein. n represents the number of polyethylene glycol repeating units in the polymer arms.

[0091] The building blocks forming the hyperbranched polymer include a core unit, polymer arms (e.g., arms made of polyethylene glycol (PEG)), a bifunctional linking group or linker, a bifunctional extender, a dendritic repeating unit containing branched units, and functional terminal groups. In embodiments of the present invention, the building blocks have an average molecular weight (Mn) of less than 50,000 daltons, for example, less than 45,000 daltons, less than 40,000 daltons, less than 35,000 daltons, or less than 30,000 daltons.

[0092] Core unit The core unit is the center of a hyperbranched polymer from which polymer arms, dendritic repeating units (DCRUs), or dendrons are derived. The core unit has at least three linkage sites c (or valences), each to which a polymer arm or dendritic repeating unit is linked, i.e., covalently bonded. The polymer arms may be linked to the core unit by hydrolyzable bonds, preferably by non-hydrolyzable bonds such as ether bonds.

[0093] In certain embodiments, the core unit has 3-10, 4-8, 4-6, or 4 linkage sites c. The core unit can be derived from a molecule or chemical structure having a number of c functional groups to which polymer arms are bound. For example, in certain embodiments, the core unit is derived from a polyol having at least 3 hydroxyl groups, or 4, 5, 6, 7, 8, 9, or 10 hydroxyl groups.

[0094] In such embodiments, the polyol can be selected from the group consisting of glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomalt, lactitol, maltitol, mannitol, or sorbitol. In certain embodiments, the core unit derived from the polyol is ethoxylated at each of its hydroxyl groups to form a multi-arm precursor having arms which are polymer PEG arms end-capped with terminal or functional groups.

[0095] An exemplary core unit structure having three connecting parts may be represented by the following formula, where connecting part c is denoted by OH. [ka]

[0096] Therefore, the core unit of the multi-arm precursor that can be used to form the hyperbranched polymer of a particular embodiment of the present invention is a structure suitable for providing a desired number of arms of the precursor. For example, in the case of a 4-arm precursor, the core unit may be a pentaerythritol or ethylenediamine structure, while in the case of an 8-arm precursor, the core unit may be a hexaglycerol structure. In a particular embodiment of the present invention, the core unit is pegged by polyethylene glycol arms at linking sites c, as shown in the structure below. The linking sites at the ends are again shown as OH groups and can be linked to linkers, functional groups, or other DCRUs. [ka]

[0097] The terminal linking sites or terminal groups are again shown as OH groups, and by linking linkers, functional groups, extenders, or other DCRUs, further generations of hyperbranched polymers can be formed.

[0098] In embodiments of the present invention, building blocks comprising multi-arm PEG precursors derived from ethoxylated polyol core units, as further defined below, are used, and these multi-arm PEG precursor building blocks have an average molecular weight (Mn) of less than 50,000 daltons, for example less than 45,000 daltons, less than 40,000 daltons, less than 35,000 daltons, or less than 30,000 daltons.

[0099] generation Similar to dendrimers, the arrangement of the constituent repeating units in the superbranched polymer of the present invention can be specified by generation. In certain embodiments, the superbranched polymer may be a G0 superbranched polymer, or a G1-G10 superbranched polymer such as a G1, G2, G3, G4, or G5 superbranched polymer, and is generally a Gx superbranched polymer, where x is an integer from 1 to 10. The abbreviation G represents the generation, and its number represents the total number of dendritic repeating units that are linked to each other in a continuous line.

[0100] As shown in Figure 2, the number of branches and outermost terminal groups increases with each generation.

[0101] In exemplary embodiments, the superbranched polymer is a G0 branched polymer, where the end groups located on the surface of the superbranched polymer are the end groups of polymer arms linked to a core unit. The G0 branched polymer is also called a multi-armed PEG molecule, in which an activator is covalently bonded to at least one of its arms.

[0102] In another exemplary embodiment, the hyperbranched polymer is a high-generation Gx hyperbranched polymer, where x is an integer between 1 and 10, defining the number of continuously linked dendritic repeating units in the hyperbranched polymer, and each polymer arm of the outermost dendritic repeating unit contains a terminal group, and at least one activator is conjugated to at least one of the outermost polymer arms.

[0103] Exemplary embodiments of the present invention include G1-G10 hyperbranched polymers such as G1-G8, G1-G6, or G1-G4 (G1, G2, G3, or G4, etc.).

[0104] Different DCRUs, such as the same DCRU, or DCRUs with different molecular weights (due to different PEG arm lengths) or different numbers of arms, can be used to form different generations in a superbranched polymer. Furthermore, to control the degradation rate at different junctions within the superbranched polymer, for example, different generations of DCRUs within the superbranched polymer can be linked to each other using the same linkers and functional groups, or using different linkers and functional groups.

[0105] Branching unit The branching unit can be selected from the same chemical substances as the core unit described above. Similar to the core unit, the branching unit is a branched chemical structure that includes branching points and multiple linkage sites.

[0106] The core unit is located at the center of a superbranched polymer and there is only one of them, while branching units exist within the dendritic repeating units (DCRUs) of the superbranched polymer. A G0 superbranched polymer contains a core unit but no branching units. A higher-generation superbranched polymer, known as Gx, contains multiple branching units. The branching units in a superbranched polymer may have the same chemical structure as the core unit, or they may be different. For example, the core unit of a G1 superbranched polymer may be derived from pentaerythritol with linkage site c=4, and the superbranched polymer may contain four DCRUs, each with a branching unit derived from glycerol with linkage site c'=3, so the entire G1 superbranched polymer contains eight terminal (outermost) groups. If the four DCRUs also have branching units derived from pentaerythritol, similar to the core unit, the superbranched polymer will have a total of 12 outermost terminal groups.

[0107] Polymer Arm In certain embodiments, the polymer arms of a hyperbranched polymer consist of polyethylene glycol (PEG) polymer units. In generation G0 branched polymers, the polymer arms are linked to a core unit, for example, via ether bonds, and have terminal groups located on the surface of the branched polymer. An activator is covalently bonded to at least some of these terminal groups. In higher-generation Gx hyperbranched polymers, the polymer arms are also present in continuous, linked dendritic repeating units.

[0108] Therefore, in some embodiments, the polymer arms contained in the hyperbranched polymer consist of or include at least one polyethylene glycol unit. Polyethylene glycol (PEG, also called polyethylene oxide) refers to a polymer having repeating groups (CH2CH2O)n (where n is at least 3).

[0109] Therefore, polymer arms having polyethylene glycol have at least three of these repeating groups linearly linked to each other. PEG polymer arms can be terminated with terminal groups such as nucleophiles or electrophiles, dibenzocyclooctin (or other strained alkynes), strained alkenes, tetrazines, or azides, and can be used for conjugation with activators or linkage with DCRU precursors to construct next-generation hyperbranched polymers.

[0110] The polymer arm may have PEG units having an average molecular weight (Mw) in the range of approximately 1,000 to 100,000 daltons, or approximately 10,000 to 60,000 daltons, or approximately 15,000 to 50,000 daltons.

[0111] In certain embodiments, the average molecular weight (Mn) of the PEG units of the polymer arms bonded to the core unit may be the same as or different from the polymer arms in the dendritic repeating unit. For example, the average molecular weight of the PEG units of the polymer arms bonded to the core may be higher or lower than that of the polymer arms in the dendritic repeating unit. In one embodiment, for a high-generation Gx biodegradable superbranched polymer (x is an integer from 2 to 10), the average molecular weight of the polymer arm PEG units may decrease or increase from the innermost polymer arm to the outermost polymer arm. For example, the polymer arms bonded to the core unit may have a large molecular weight, and the DCRU may have a smaller molecular weight, or vice versa. The molecular weight of the polymer arms may change with the DCRU generation. As an example, a G2 superbranched polymer with 24 outermost conjugation sites may be constructed from a 4-arm 40k PEG core bonded to 4 4-arm 20k PEG DCRUs, and further bonded to 12 3-arm 30k PEGs. In this context, K refers to kilodaltons (kDa), so 4-arm 40k PEG has four polymer PEG arms and a total molecular weight of 40 kDa.

[0112] Dendritic repeating units A dendritic repeating unit (DCRU) is a substructure in a hyperbranched polymer of higher generation Gx as defined herein, having c'≧3 linkage sites and including branching points and polymer arms derived therefrom. It may be continuously linked to a total of c' polymer arms extending from a core unit and / or other DCRUs to form a dendritic dendrimer structure.

[0113] In certain embodiments, the dendritic repeating units in a dendrimer can be represented by general formula (i). [ka]

[0114] In this equation (i), A is a connection to a polymer arm connected to the core unit, or A is a connection to B of the preceding dendritic repeating unit represented by equation (i), and L A is the linker base, m is either 0 or 1, indicating whether a linker is present or absent, n is an integer between 3 and 2000, or between 20 and 2000, o is an integer between 3 and 2000, or between 20 and 2000, n and o may be different or the same, X is the branching unit, L B is a linker group, p is either 0 or 1, meaning whether a linker is present or absent, B is a terminal group located on the surface of a hyperbranched polymer, or a linkage of a continuous dendritic repeating unit to A, or a linkage to an activator, L A and L B A and B may be different or the same, m and p may be different or the same, y is an integer from 2 to 9, y = c'-1, and c' is the linkage site c' of branched unit X. In certain embodiments of the superbranched polymer containing the DCRU of formula (i), the linkage between A and B may include a functional group formed by click chemistry, such as triazole or dihydropyrazine.

[0115] The branched unit can be derived from polyols such as glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomalt, lactitol, maltitol, mannitol, or sorbitol. In formula (i) above, the branched unit is ethoxylated at all of its linkage sites c, and optionally a linker group L can be linked via an ether bond. A A is connected to A via a single PEG polymer arm, and optionally a linker group L B The DCRUs are connected to y PEG polymer arms, each connected to B via the DCRUs. Different DCRUs can be used in the hyperbranched polymer, such as the same DCRU, or DCRUs with different molecular weights (due to differences in PEG arm length) or different numbers of arms.

[0116] Linker group By incorporating linker groups that are susceptible to hydrolysis into dendritic structural units, biodegradation of hyperbranched polymers becomes possible under physiological conditions. High molecular weight hyperbranched polymer conjugates are broken down into small, low molecular weight constituent units, which are then excreted from the body through normal physiological pathways.

[0117] In certain embodiments of the superbranched polymer containing DCRU of formula (i), the linker group L A and / or L B The linkers comprise dicarboxyl groups and / or carboxamide groups of varying chain lengths, or combinations thereof, which can be derived from diacid groups such as succinic acid, glutaric acid, adipic acid, azelaic acid, or acid diamide groups such as glutaramide. These groups are linked to PEG polymer arms A and / or B via ester or amide bonds, which are hydrolyzable at different rates under physiological conditions in vivo, depending on the length of the acid chain. In certain embodiments, the linkers form ester bonds and are derived from diacids.

[0118] In a particular embodiment, the linker L A and / or L B This includes the structure represented by formula (ii), [ka] In the formula, U 1 and U 2 , are independently NH or O, and may be the same or different, and t is an integer from 0 to 10. For example, in succinate linker, U 1 and U 2 Both are oxygen, and t is 2. Regarding the linker end group, the linker of formula (ii) contains a terminal functional group at one of its ends. In the succinate linker of the above example, it reacts with N-hydroxysuccinimidyl to produce a succinimidyl succinate linker end group, which can be used to conjugate an amine-functionalizing activator to a hyperbranched polymer having this linker end group on the outermost polymer arm. In certain embodiments, linker L A and / or L B It further includes polyethylene glycol units between the bond to B and the carboxyl group, carboxamide group, or structure of formula (ii).

[0119] The linker of formula (ii) introduces hydrolyzable bonds to a superbranched polymer, which can be used to adjust the degradation rate of the superbranched polymer and / or the release rate of activators conjugated from the superbranched polymer. For example, the biodegradation / hydrolysis rate of ester bonds in these linkers decreases from succinate ester (C4) to azelaic acid ester (C9). In embodiments of the present invention, this can be used to control the degradation rate of a superbranched polymer and / or the release of activators conjugated to the superbranched polymer via these linkers. For example, succinimidyl succinate (SS) degrades in a few days, while succinimidyl glutarate (SG) degrades in a few weeks.

[0120] Linker base L A and L BThe other end may be connected to a terminal group such as an ester, for example, a succinimidyl (NHS) group formed by esterification of a linkeric acid group with N-hydroxysuccinimide, as will be further explained below, or a click chemistry functional group such as DBCO or an azide.

[0121] extender By incorporating bifunctional extender units as additional building blocks, in addition to or instead of linkers, the length of polymer arms between arms connected to, for example, core and branch units can be extended, providing greater flexibility and / or more hydrolytic cleavage points for the dendrimer. Such extenders are typically linear bifunctional polymer chains, such as linear PEG extenders.

[0122] In certain embodiments, the hyperbranched polymer comprises at least one extender unit comprising or consisting of polyethylene glycol (PEG) units, the extender unit being linear and bifunctional, and linked to polymer arms of a dendritic repeating unit, or to polymer arms linked to a core unit and then to either the terminal groups or polymer arms of the next dendritic repeating unit.

[0123] In this embodiment, the extender unit comprises at least one linker, which may be located at either or both ends of the extender unit, and is a bifunctional linker comprising a hydrolyzable bond containing a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, a heteroaliphatic, or aromatic or heteroaromatic group.

[0124] By including an extender, the hydration radius of the dendrimer can be increased, thereby extending the in vivo half-life of the hyperbranched polymer.

[0125] PEG and DCRU precursors for G0 branched polymers The core elements of the superbranched polymer in a particular embodiment of the present invention may include one or more multi-arm PEG precursors having 2 to 10 arms, 4 to 8 arms, or 4, 5, 6, 7, or 8 arms. Note that since the multi-arm precursor includes a core, for example, a 2-arm PEG precursor differs from a simple linear PEG due to the presence of the core structure. The PEG precursors used in the superbranched polymer may have different numbers of arms or the same number of arms. In a particular embodiment, the PEG precursors used in the superbranched polymer of the present invention have 3, 4, and / or 8 arms. In a particular embodiment, PEG precursors with a combination of 4 arms and 3 arms, or a combination of 4 arms and 8 arms, and any combination thereof are used. For example, by combining an 8-arm core unit with 8 4-arm DCRU precursors and then linking these again with 24 3-arm precursors, a superbranched polymer having 48 conjugation sites on the outermost arms is obtained. In another exemplary embodiment, a 4-arm core unit is combined with four 3-arm precursors, which are then linked again with eight 4-arm precursors to obtain a hyperbranched polymer having 24 conjugation sites in the outermost arms. Multi-arm PEG precursors for G0 branched polymers and DCRUs in embodiments of the present invention are commercially available, for example, from JenKem Technology, SinoPEG, or Sigma-Aldrich, and optionally contain various functional end groups for further derivatization.

[0126] In certain embodiments of the present invention, polyethylene glycol units used as core building blocks or DCRU precursors have an average molecular weight in the range of about 1,000 to about 80,000 daltons, or about 10,000 to about 60,000 daltons, or about 15,000 to about 50,000 daltons. In some embodiments, polyethylene glycol units have an average molecular weight in the range of about 10,000 to about 40,000 daltons, or about 20,000 daltons. PEG precursors with the same average molecular weight may be used, or PEG precursors with different average molecular weights may be combined with each other. The average molecular weight of the PEG precursors used in the present invention is given as the number-average molecular weight (Mn), which in certain embodiments can be determined by gel permeation chromatography against a polystyrene standard according to a standardized method.

[0127] In a 4-arm PEG, each arm may have an average arm length (or molecular weight) obtained by dividing the total molecular weight of PEG by 4. Therefore, one precursor usable in the present invention, the 4a20kPEG precursor, has four arms, each with an average molecular weight of approximately 5000 daltons (+ / -500), bound to a pentaerythritol core unit. Therefore, the 8a20kPEG precursor, which can be used in addition to the 4a20kPEG precursor in the present invention, has eight arms, each with an average molecular weight of 2500 daltons (+ / -250), bound to a tripentaerythritol or hexaglycerol core unit.

[0128] Generally, when referring to polymer precursors with a specific average molecular weight, such as 15kPEG precursors, the indicated average molecular weight (e.g., Mn of 15,000 or 20,000, respectively) refers to the polymer unit portion of the precursor before the addition of end groups (in this specification, "20k" means 20,000 daltons (+ / - 2,000 Da), and "15k" means 15,000 daltons (+ / - 1,500 Da)—the same abbreviations are used herein for PEG or other polymer precursors of other average molecular weights). In certain embodiments, the Mn of the polymer unit portion of the precursor is determined by gel permeation chromatography against a polystyrene standard according to a standardized method. The degree of substitution by end groups disclosed herein refers to the degree of substitution after functionalization of the end groups. 1 This may be determined by 1H-NMR.

[0129] In certain embodiments, a precursor suitable for use in the formation of DCRU is generally represented by formula (iii), [ka] In the formula, C contains a functional group suitable for click chemistry (such as an alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (such as succinimidyl), and L A is the linker base, m is either 0 or 1, indicating whether a linker is present or absent, n is an integer between 3 and 2000, or between 20 and 2000, o is an integer between 3 and 2000, or between 20 and 2000, n and o may be different or the same, X is the branching unit, L B is a linker group, p is either 0 or 1, and indicates whether a linker is present or absent, L A and L B m and p may be different or the same, y is an integer between 2 and 9, y = c'-1, and c' is the connection point c' of branching unit X.

[0130] In certain embodiments, useful PEG precursors for forming DCRUs of hyperbranched polymers are NHS dicarboxylic acid ester-terminated multi-armed PEG precursors derived from commercially available multi-armed PEG compounds, such as the four-armed structure derived from pentaerythritol of formula (iv). [ka]

[0131] A PEG precursor useful for forming DCRU in a particular embodiment can be represented by the following formula (v): [ka] In the formula, n is determined by the molecular weight of each PEG arm, m is an integer from 0 to 10, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and x is the number of arms (thus, for example, 2, 4, 8, etc., see above). When m is 1, each arm is terminated with a succinimidyl succinate (SS) end group; when m is 2, each arm is terminated with a succinimidyl glutarate (SG) group; when m is 3, each arm is terminated with a succinimidyl adipate (SAP) group; and when m is 6, each arm is terminated with a succinimidyl azelate (SAZ) group. Using these specific electrophilic end groups, a multi-arm PEG unit may be abbreviated in the form, for example, 4a20kPEG-SAP, which refers to a 4-arm PEG having succinimidyl adipate end groups and a molecular weight of 20000 Da. In the above formula, R is a suitable core structure for providing the desired number of arms. As shown in the above formula, in the case of a 4-arm PEG unit and precursor, R may be a pentaerythritol structure, while in the case of an 8-arm PEG unit and precursor, R may be a hexaglycerol structure.

[0132] In certain embodiments, the PEG precursor used is 4a20kPEG-SG or 4a20kPEG-SAP.

[0133] Precursors having nucleophilic end groups instead of electrophilic end groups can also be used. In certain embodiments, the nucleophilic end group used as the hyperbranched polymer PEG precursor is an amine (denoted as "NH2") end group. Thiol (-SH) end groups or other nucleophilic end groups can also be used.

[0134] In certain embodiments, 4-arm PEG with an average molecular weight of approximately 20,000 daltons and 4-arm PEG with an average molecular weight of approximately 40,000 daltons can be used to form the hyperbranched polymer according to the present invention.

[0135] Functional groups for linking the building blocks of hyperbranched polymers To synthesize a hyperbranched polymer, the polymer arms or precursors have pairs of functional groups that react with each other, i.e., a first functional group on a first polymer arm or precursor can react with a second functional group on a second polymer arm or precursor on a different DCRU precursor.

[0136] In one embodiment, a first multi-arm precursor comprising a core unit and PEG arms connected thereto comprises a first functional group, and a second multi-arm precursor DCRU comprises one second functional group that can react with the first functional group, but all other terminal groups of the second DCRU precursor do not react with the first functional group, and these functional groups are located at the ends of the arms of the precursor or DCRU. The first and second functional groups may be grafted directly onto the ends of the arms or via a linker, preferably a hydrolyzable linker as defined elsewhere herein. The functional groups can react with each other to form covalent bonds, for example, in click chemistry reactions or electrophilic nucleophilic reactions, and are configured to participate in other chemical crosslinking reactions as described below.

[0137] In certain embodiments of the present invention, the first and second functional groups are selected from electrophiles and nucleophiles, functional groups for click chemistry, functional groups for cycloaddition, particularly 1,3-dipolar cycloaddition, hetero-Diels-Alder cycloaddition, functional groups for nucleophilic ring-opening, functional groups for non-aldol-type carbonyl reactions, functional groups for addition reactions to carbon-carbon multiple bonds, polymerizable vinyl groups, or combinations thereof. Those skilled in the art will know that a particular pair of functional groups can be classified into several of these groups. For example, in click chemistry, an azide reacting with dibenzocyclooctyne can also be considered an electrophilic nucleophilic reaction pair.

[0138] In certain embodiments of the hyperbranched polymers of the present invention, the linkage between different parts of the hyperbranched polymer, such as the core unit and polymer arms linked to the DCRU, is formed by click chemistry reactions such as strain-enhanced alkyne-azide cycloaddition (SPAAC), also known as Cu-free click reaction, or reverse electron-required Diels-Alder coupling (IEDDA) type click chemistry coupling reactions. An overview of such types of reactions is provided by reference in H.Colb; M.G.Finn; K.B.Sharpless (2001). “Click Chemistry: Diverse Chemical Function from a Few Good Reactions”, Angewandte Chemie International Edition, 40(11):2004-2021).

[0139] Other suitable click chemistry reactions for linking the constituent units of hyperbranched polymers in a particular embodiment include aldehyde / ketone condensation, cyanobenzothiazole condensation, strain-accelerated oxidation-controlled cyclooctin-1,2-quinone cycloaddition (SPOCQ), 1,3-dipolar cycloaddition, alkene-nitrone cycloaddition or alkyne-nitrone cycloaddition, [3+2] cycloaddition, [4+2] cycloaddition, and hetero-Diels-Alder reactions.

[0140] SPAAC requires cyclic alkynes such as dibenzylcyclooctyne (DBCO) and bicyclo[6.1.0]nonine (BCN) to react with aliphatic azides. This strained chemical reaction allows the reaction to proceed efficiently without the need for the copper catalyst required for copper(I) catalyzed azide-alkyne click chemistry (CuAAC). Similarly, in IEDDA, ​​no catalyst is required for the reaction of norbornene and tetrazine. Therefore, the advantages of SPAAC and IEDDA compared to electrophilic nucleophilic reactions such as CuAAC and NHS-NH2 are that they do not require a catalyst and do not produce byproducts after the reaction is complete.

[0141] The coupling reaction of SPAAC and IEDDA is a bioorthogonal reaction that yields selective and quantitative yields under mild conditions without interfering with the intrinsic biochemical processes in vivo. These click chemistry reactions utilize reagent pairs such as cyclooctyne and azide, which react mutually exclusive and efficiently while being inert to naturally occurring functional groups.

[0142] Scheme A [ka] R1 and R2 are either the same or different residues.

[0143] This reaction is suitable for forming the hyperbranched polymers of the embodiments of the present invention from the corresponding functionalization precursors and DCRUs described herein. Among the many known cyclooctins, dibenzocyclooctin (DBCO) compounds constitute a class of reagents with a moderately fast reaction rate and good stability in aqueous buffer. Within the physiological temperature and pH range, the DBCO group does not react with amine or hydroxyl groups naturally present in many biomolecules, or with amine or hydroxyl groups present as different functional groups in some hyperbranched polymers. Furthermore, the reaction between the DBCO group and the azide group is very fast and yields high yields.

[0144] The advantages of DBCO-based SPAACs include, for example, their biocompatibility, as they do not require cytotoxic copper catalysts, which can leave undesirable traces on hyperbranched polymers. Another advantage is the use of mild reaction conditions; DCRU coupling and activator conjugation can be performed under physiological conditions in aqueous buffers or common organic solvents. Furthermore, since azide groups react with DBCO only in the presence of amines, hydroxyls, thiols, acid groups, and other protein functional groups, the DBCO and azide moieties are stable over long periods and possess high selectivity and specificity. These reactions also form stable triazoles at high reaction rates and in quantitative yields, leaving no byproducts. Similar advantages can be obtained with IEDDA coupling reactions and other types of catalyst-free click chemistry reactions described herein.

[0145] In exemplary embodiments, a hyperbranched polymer containing a hydrolyzable linker derived from a diacid can be formed using the following precursors for click chemistry: [ka] And, [ka] Either one of the following, [ka] And, [ka] Either one of the following, [ka] And, [ka] Either one of the following, [ka] And, [ka] either (wherein t is m, and n and m are defined similarly in this specification with respect to formula (v) above).

[0146] In addition to the 4-arm PEG, other core / branch unit bonds described herein may also be used. In other embodiments, the precursor may also include hydrolyzable bonds, such as carboxamide bonds instead of ester bonds, or ester and amide bonds in the SGA linker units described herein.

[0147] In certain embodiments, the linkage in a superbranched polymer can be selectively formed using a DCRU precursor containing one functional group for click chemistry bond formation, while the other terminal functional groups of the DCRU remain inactive and can subsequently be used in other subsequent reactions such as the growth or conjugation of the superbranched polymer. In other embodiments, the linkage in a superbranched polymer can be selectively formed using an electrophilic-nucleophilic precursor or other functional group that does not react in click chemistry, while the other terminal functional groups of the DCRU, containing the functional group for click chemistry bond formation, remain inactive and can subsequently be used in other subsequent reactions such as the growth or conjugation with a click chemistry reaction of the superbranched polymer.

[0148] Functional group pairs for click chemistry may include selected functional groups for cycloaddition, particularly 1,3-dipolar cycloaddition, [3+2] cycloaddition such as alkene-nitrone cycloaddition or alkyne-nitrone cycloaddition, [4+2] cycloaddition, hetero-Diels-Alder cycloaddition, functional groups for thiol-ene reactions, functional groups for nucleophilic ring-opening, functional groups for non-aldol-type carbonyl reactions, functional groups for addition reactions to carbon-carbon multiple bonds, and functional groups for Michael-type addition.

[0149] For example, the first functional group is an alkyne compound such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]-nonine (BCN), or norbornene or trans-cyclooctene (TCO), and the second functional group is an azide, 3,4-dihydroxyphenylacetic acid (DHPA), or tetrazine (Tz). In these embodiments, the DBCO, BCN, norbornene, TCO, azide, DHPA, and Tz functional groups may be grafted to the ends of the multi-arm precursor via hydrolyzable linkers such as acidic groups, diacidic groups, amide groups, functionalized aliphatic, heteroaliphatic, or aromatic or heteroaromatic groups, or may be directly linked to the PEG.

[0150] In another embodiment, the first and second functional groups are selected for [3+2] cycloaddition reactions such as alkene-nitrone cycloaddition or alkyne-nitrone cycloaddition. In a further embodiment, the first and second functional groups are selected for [4+2] cycloaddition reactions, particularly hetero-Diels-Alder reactions, where the first functional group is an aldehyde or imine compound and the second functional group is a 1,3-diene compound, an unsaturated carbonyl compound, or a nitrosoalkene compound. In a further embodiment, the first and second functional groups are selected for nucleophilic ring-opening, where the first functional group is selected from epoxides, thiirane, aziridine, or lactams and the second functional group is the nucleophile described above. In another embodiment, the first and second functional groups are selected for a non-aldol type carbonyl reaction, the first functional group being an aldehyde or ketone compound, and the second functional group being a primary amine, hydrazide, acylhydrazide, or aminooxy compound, forming an imine, amide, isourea, hydrazone, acylhydrazone, or oxime bond.

[0151] Conjugation of activators The attachment or conjugation of activators to the outermost polymer arms of a hyperbranched polymer can also be carried out by click chemistry, as described above for linking the building blocks of the hyperbranched polymer, or by electrophilic and nucleophilic reactions and other types of coupling reactions as described herein.

[0152] Therefore, in one embodiment, the first functional group on the outermost polymer arm of the hyperbranched polymer may be a nucleophile, the second functional group on the activator may be an electrophile, or vice versa, and the reaction between the first and second functional groups is an electrophilic nucleophilic reaction that forms a covalent bond.

[0153] The nucleophile may be selected from one of the amines, such as a primary amine, hydroxyl, thiol, carboxyl, or hydrazide group. In certain embodiments, one of the functional groups comprises a nucleophile such as a primary amine.

[0154] Electrophiles that can be used in embodiments of the present invention may be selected from active ester groups such as succinimidyl esters, succinimidyl carbonates, nitrophenyl carbonates, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, norbornene, epoxides, mesylates, tosylates, tresyl, cyanurates, orthopyridyl disulfide, or halogens. These electrophiles contain functional groups that participate in electrophilic nucleophilic reactions and preferably further contain reactive groups that form linkers to PEGs containing hydrolyzable groups or bonds, such as glutaric acid esters. For example, in one embodiment of the present invention, the succinimidyl ester may contain reactive groups such as succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), succinimidyl azelate (SAZ), or succinimidyl glutaramide. The electrophilic nucleophilic reaction for linking multi-armed PEG precursors is described, for example, in US2002 / 0042473A1, which is incorporated by reference.

[0155] The activator can be appropriately derivatized with a functional group as described above, unless it already possesses a functional group suitable for linking with the hyperbranched polymer. For example, a peptide having a primary amino group may be conjugated to a hyperbranched polymer having an activated ester group on its surface via an electrophilic nucleophilic reaction.

[0156] In certain embodiments, activators, particularly peptides, may be conjugated to hyperbranched polymers via click chemistry reactions. In these embodiments, an activator or peptide having a primary amino group at its terminus is first reacted with a DBCO-NHS compound or an azide-NHS compound to produce an activator or peptide functionalized with a DBCO or azide group suitable for reacting with the corresponding functional group on the terminus of the hyperbranched polymer, thereby obtaining a conjugate with high reproducibility.

[0157] Suitable reaction products for click chemistry functionalization of activators or peptides having a primary amino group at the terminal include, for example, N-hydroxysuccinimidyl azidoacetate (NHS-azido), N-hydroxysuccinimidyl azidobutyrate or other azidic acid-NHS esters, and dibenzocyclooctin-N-hydroxysuccinimidyl (DBCO-NHS) of various acid chain lengths. Both azido-NHS esters and DBCO-NHS esters can be used in combination with acids of different chain lengths (e.g., those already described as linkers herein) to alter the biodegradation rate and the release of the activator from the hyperbranched polymer. Such click chemistry reagents are commercially available from vendors such as Sigma-Aldrich or Thermo Fisher Scientific and others.

[0158] In certain embodiments, an activator or peptide having a thiol functional group such as a cysteinethiol group may be conjugated to a hyperbranched polymer via a maleimide-thiol click chemistry reaction according to the following reaction scheme.

[0159] Scheme B [ka] R1 is the terminal of a hyperbranched polymer, and R2 is a peptide or activator. The thiol-maleimide reaction is a thiol-Michael addition reaction that produces a thio-succinimide bond. This reaction is rapid and chemoselective to thiols at pH 6.5 to pH 7.5.

[0160] For example, a maleimide-functionalized end of a superbranched polymer can be used to conjugate a peptide or activator via a maleimide-thiol reaction. In another embodiment, a DBCO or azide-functionalized end of a superbranched polymer can be subjected to maleimide end-functionalization by reacting it with a click chemistry linker having an azide or DBCO functional group and a maleimide group at the other end, and then used for conjugation with a thiol group of a peptide or activator. Suitable DBCO-maleimide or azide-maleimide linkers may optionally be extended with a PEG moiety and are commercially available from Sigma-Aldrich, TCI, Thermo Fisher, and others.

[0161] Examples include compounds such as DBCO-maleimide, DBCO-PEG3-maleimide, DBCO-PEG4-maleimide, and azide-PEG3-maleimide, which have the following exemplary structures: [ka]

[0162] In certain embodiments, the activator is conjugated to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% of the outermost polymer arms. The average substitution rate of the activator conjugated to the surface end groups of the hyperbranched polymer may be measured by UHPLC, as further described herein.

[0163] Activating agent: The activator in the biodegradable microparticles of the embodiments of the present invention may be a therapeutically active agent, a diagnostically active agent, or a combination thereof. It may be a single activator or a plurality of activators.

[0164] In some embodiments, the hyperbranched polymer contains two or more different activators in different dendrons or regions on the surface of the hyperbranched polymer. The two or more activators may each be bonded to the same or different hydrolyzable groups to control the release of the activators at different rates. Furthermore, the activators may be bonded to the dendrimer with or without hydrolyzable linkers or arm / extensioners, or a combination thereof, to control the release of the activators at different rates.

[0165] In certain embodiments, the activator conjugated to at least one of the outermost polymer arms of the hyperbranched polymer is a peptide selected from the group consisting of Compstatin, APL-1, Fc-III-4C, Beov (brolucizumab), Zimra (abacincaptado pegol), Pegcetacoplan, Abisipal pegol, Lamparizumab, Fovista, Listeganib, AXT107, Elamipretide, THR149, ALM201, VGB3, and Largazole.

[0166] Therapeutically active drugs include steroids, nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, meclofename, mefanamic A, sarsalate, sulindac, tolmetin, ketoprofen, diflunisal, piroxicam, naproxen, etodolac, flurbiprofen, fenoprofen C, indomethacin, celecoxib, ketorolac, and nepafenac, intraocular pressure lowerers, antibiotics such as ciprofloxacin, analgesics such as bupivacaine, calcium channel blockers such as nifedipine, cell cycle inhibitors such as simvastatin, proteins such as insulin, small molecule hydrophilic drugs including carboxylates and amine salts, and small molecules These may include hydrophobic drugs, hydrophilic peptides and protein drugs (e.g., insulin, single-chain antibody fragments, Fab fragments, IgG antibodies, fusion antibodies, etc.), aptamers, in particular bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDS), steroids, antibiotics, analgesics, calcium channel blockers, cell cycle inhibitors, chemotherapy drugs, antiviral drugs, anesthetics, hormones, anticancer drugs, antitumor drugs, viruses, viruses for gene delivery such as AAV, protein conjugates such as nanobodies, aphibodies, ankyrin, DARPin, etc., or any combination thereof.

[0167] In some embodiments, the steroid may be a corticosteroid, which may include hydrocortisone, loteprednol, cortisol, cortisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, aldosterone, or fludrocortisone.

[0168] In some embodiments, the NSAID may include diclofenac (e.g., diclofenac sodium), flurbiprofen (e.g., flurbiprofen sodium), ketrolac (e.g., ketrolactromethamine), bromfenac, or nepafenac.

[0169] In some embodiments, the IOP-lowering agent and / or glaucoma treatment agent is a prostaglandin analog (e.g., bimatoprost, latanoprost, travoprost, or latanoprostenbunod), a Rho kinase inhibitor (e.g., netaludil), an adrenergic agonist (epinephrine or dipivefrin), or a beta-adrenergic antagonist also known as a beta-blocker (e.g., timolol, levobunolol, metipranolol, carteo This may include alpha-2 adrenergic agonists (e.g., apraclonidine, brimonidine, or brimonidine tartrate), carbonic anhydrase inhibitors (e.g., brinzolamide, dichlorfenamide, metazolamide, acetazolamide, or dorzolamide), pilocarpine, ecothiophate, demercarium, physostigmine, and / or isofluorophate.

[0170] In some embodiments, the antiinfective agent may include ciprofloxacin, tobramycin, erythromycin, ofloxacin, gentamicin, fluoroquinolone antibiotics, antibiotics including moxifloxacin and / or gatifloxacin, antiviral agents including ganciclovir, idoxuridine, vidarabine and / or trifluridine, and / or antifungal agents including amphotericin B, natamycin, voriconazole, fluconazole, miconazole, clotrimazole, ketoconazole, posaconazole, echinocandin, caspofungin and / or micafungin.

[0171] In some embodiments, the antimetabolite may include methotrexate, mycophenolic acid, or azathioprine. In some embodiments, the antifibrotic agent may include mitomycin C or 5-fluorouracil.

[0172] In some embodiments, the angiogenesis inhibitor may include anti-VEGF agents (e.g., aflibercept, ranibizumab, bevacizumab), PDGF-B inhibitors (e.g., Fovista®), complement antagonists (e.g., eculizumab), tyrosine kinase inhibitors (e.g., sunitinib, axitinib), and / or integrin antagonists (e.g., natalizumab and vedolizumab).

[0173] In some embodiments, nanobodies can be conjugated to hyperbranched polymers. Nanobodies are described, for example, Yang et al. (2020), Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics, Front. Oncol. 10:1182, which is incorporated herein by reference in its entirety. Nanobodies are, 68 GaNOTA-Anti-HER2-VHH1, 68 GaNOTA-Anti-HER2-VHH1, 99m Tc-NM-02, 131 I-SGMIB-Anti-HER2-VHH1, 68 GaNOTA-Anti-MMR-VHH2, 99m The following may be selected from Tc-Anti-PD-L1, L-DOS47 + doxorubicin, L-DOS47 + cisplatin / vinorelbine, KN035 + trastuzumab / docetaxel, KN035, KN044, TC-210 T cells, CD19 / CD20 bispecific CAR T cells, BCMA CAR T cells, or TAS266 nanobodies.

[0174] In some embodiments, non-immunoglobulin affinity proteins, such as affibody molecules, can be conjugated to hyperbranched polymers. Affibody molecules are described, for example, in Stahl et al., Affibody Molecules in Biotechnological and Medical Applications, Trends in Biotechnology 2017, 35(8) pp. 691-712, the full text of which is incorporated herein by reference.

[0175] In some embodiments, binding proteins such as ankyrin and DARPin can be conjugated to hyperbranched polymers. Ankyrin and DARPin are described, for example, in the overview by Caputi et al., Current Opinion in Pharmacology 2020, 51:93-101, which is incorporated herein by reference in full. Ankyrin and DARPin may be selected from MP0250, a tripspecific DARPin drug candidate capable of binding to VEGF-A and hepatocyte growth factor (HGF), as well as one molecule of MP0250 capable of binding to two molecules of human serum albumin (HSA), avisipalpegol (MP0112 or AGN-150998), brolucizumab, ranibizumab, or aflibercept.

[0176] In some embodiments, the cytoprotective agent may include ebselen, sulforaphane, ortiplasm, or dimethyl fumarate. In some embodiments, the neuroprotective agent may include ursodiol, memantine, or acetylcysteine. In some embodiments, the anesthetic may include lidocaine, propalacaine, or bupivacaine.

[0177] In some embodiments, the activator may be dexamethasone, ketorolac, diclofenac, vancomycin, moxifloxacin, gatifloxin, becifloxacin, travoprost, 5-fluorouracil, methotrexate, mitomycin C, prednisolone, bevacizumab (Avastin®), ranibizumab (Lucentis®), sunitinib, pegaptanib (Macugen®), timolol, latanoprost, brimonidine, nepafenac, bromfenac, triamcinolone, difluprednate, fluocinolide, aflibercept, or a combination thereof. In some embodiments, the agent may be dexamethasone, ketorolac, diclofenac, moxifloxacin, travoprost, 5-fluorouracil, or methotrexate.

[0178] In alternative embodiments, activators that can be used in the dendrimers and methods of the present invention include, but are not limited to, immunosuppressants, complement inhibitors (e.g., C5 inhibitors such as eculizumab or abasincapta dopegol), anti-inflammatory agents such as steroids, steroidal and nonsteroidal anti-inflammatory agents (e.g., COX1 or COX2 inhibitors), antiviral agents, antibiotics, antiglaucoma agents, anti-VEGF agents, analgesics, tyrosine kinase inhibitors, integrin inhibitors, IL-6 inhibitors, reactive aldehyde species (RASP) inhibitors, nitric oxide donating PgA, antihistamines, mast cell stabilizers, Rh kinase inhibitors, plasma kallikrein inhibitors, BCL-2 inhibitors, semaphorin antagonists, HtRA1 inhibitors, IGF-1R inhibitors, VEGF combination agents (multispecific anti-angiogenic agents), and combinations thereof.

[0179] Immunosuppressants include, but are not limited to, cyclosporine, mTOR inhibitors (e.g., rapamycin, tacrolimus, temsirolimus, sirolimus, everolimus, KU-0063794, WYE-354, AZD8055, metformin, or Torin-2), cyclophosphamide, atoposide, thiotepa, methotrexate, azathioprine, mercaptopurine, interferon, infliximab, etanercept, mycophenolate mofetil, 15-deoxyspargarin, thalidomide, glatiramer, leflunomide, vincristine, cytarabine, pharmaceutically acceptable salts thereof, and combinations thereof.

[0180] Nonsteroidal anti-inflammatory compounds (NSAIDs) include inhibitors of cyclooxygenase (COX) enzymes, such as cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) isozymes. A common class of NSAIDs includes salicylates, propionic acid derivatives, acetic acid derivatives, enolic acid derivatives, and anthranilic acid derivatives. Examples of nonsteroidal anti-inflammatory compounds include acetylsalicylic acid, diflunisal, salsalate, ibuprofen, dexiibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tolmetine, sulindac, etodolac, ketrolac, diclofenac, aceclofenac, nabumetone, piroxicam, tenoxicam, loroxicam, phenylbutazone, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, pharmaceutically acceptable salts thereof, and combinations thereof.

[0181] The anti-inflammatory agents that can be used in the dendrimers and methods of the present invention may include agents that target inflammatory cytokines such as TNFα, IL-1, IL-4, IL-5, or IL-17, or CD20. Such agents may include etanercept, infliximab, adalimumab, daclizumab, rituximab, tocilizumab, certolizumab pegol, golimumab, pharmaceutically acceptable salts thereof, and combinations thereof.

[0182] Analgesics that can be used in the dendrimer and method of the present invention include acetaminophen, acetaminosarol, aminochlortenoxazine, acetylsalicylic acid 2-amino-4-picolinic acid, acetylsalicylsalicylic acid, anilelysine, benoxaprofen, benzylmorphine, 5-bromosalicylic acid, busetin, buprenorphine, butorphanol, capsaicin, syncophene, silamdol, clometacin, clonixin, codeine, desomorphine, dezosine, dihydrocodeine, dihydromorphine, dimefeptanol, dipylocetyl, eptazosine, etoxazene, ethylmorphine, eugenol, This includes phloxtaphenine, phosphosal, graphenine, hydrocodone, hydromorphone, hydroxypethidine, ibufenac, p-lactophenetide, levorphanol, meptazinol, metazosin, methopone, morphine, nalbuffine, nicomorphine, norlevorphanol, normorphine, oxycodone, oxymorphone, pentazocine, phenazosin, phenocol, phenoperidine, phenylbutazone, phenylsalicylic acid, phenyllamidol, salicin, salicylamide, thiorphan, tramadol, diaselein, actarit, pharmaceutically acceptable salts thereof, and combinations thereof.

[0183] Antibiotics that can be used in the dendrimers and methods of the present invention include aminoglycosides, penicillins, cephalosporins, fluoroquinolones, macrolides, and combinations thereof. Aminoglycosides may include tobramycin, kanamycin A, amikacin, dibekacin, gentamicin, shisomycin, netylmycin, neomycin B, neomycin C, neomycin E, streptomycin, paramomycin, pharmaceutically acceptable salts thereof, and combinations thereof. Penicillins may include amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, pivampicillin, pivmecillinum, ticarcillin, pharmaceutically acceptable salts thereof, and combinations thereof. Cephalosporins include cefatril, cefadroxil, cephalexin, cephaloglysin, cephalonium, cephaloridine, cephalothin, cefapillin, cefatoridine, cefazflur, cefazedone, cefazolin, cefradin, ceffloxazine, ceftezol, cefaclor, cefamandol, cefmetazole, cefonisid, cefotetan, cefoxitin, cefprodil, cefuroxime, cefzonam, cefcapene, cefdaroxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefozidime, cefotaxime, cefpimisole, cefpodoxime, This may include cefteram, ceftibutene, ceftiofur, cefthiolen, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclizine, cefepime, ceffluprenum, cefoselis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftarolin, cefaclomedin, cephaloram, cephaparol, cefcanel, cefedrol, cefenpidone, cefetrizole, cefibitril, cefmethylene, cefmepidium, cefobesin, cefoxazole, cefrotyl, cefsmid, cefrastim, ceftioxide, pharmaceutically acceptable salts thereof, and combinations thereof.Fluoroquinolones may include ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, ofloxacin, norfloxacin, pharmaceutically acceptable salts thereof, and combinations thereof. Macrolides may include azithromycin, erythromycin, clarithromycin, dilithromycin, oxythromycin, telithromycin, pharmaceutically acceptable salts thereof, and combinations thereof.

[0184] The dendrimers and antiviral agents that can be used in the present invention include nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, fusion inhibitors, integrase inhibitors, nucleoside analogs, protease inhibitors, and reverse transcriptase inhibitors. Examples of antiviral drugs include abacavir, acyclovir, adefovir, amantadine, amprenavir, amprigen, arbidol, atazanavir, boceprevir, cidofovir, darunavir, delavirdin, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, phosphonet, ganciclovir, ivacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, interferon, lamivudine, and lopinavir. This includes, but is not limited to, roviride, maraviloc, moloxidine, methisazone, nelfinavir, nevirapine, nexavir, oseltamivir, pegylated interferon alfa-2a, penciclovir, peramivir, preconalil, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, pyramiding saquinavir, stabudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir, valganciclovir, bicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine, pharmaceutically acceptable salts thereof, and combinations thereof.

[0185] Steroidal anti-inflammatory agents that can be used in the dendrimers and methods of the present invention include dexamethasone, budesonide, triamcinolone, hydrocortisone, fluocinolone, loteprednol, prednisolone, mometasone, fluticasone, rimexolone, fluorometholone, beclomethasone, flunisolide, their pharmaceutically acceptable salts, and combinations thereof.

[0186] Agents for glaucoma that can be used in the dendrimers and methods of the present invention include beta blockers such as atenolol, propranolol, metipranolol, betaxolol, carteolol, levobetaxolol, levobunolol, timolol, their pharmaceutically acceptable salts, and combinations thereof, adrenergic agents or sympathomimetic agents such as epinephrine, dipivefrin, clonidine, apraclonidine, brimonidine, their pharmaceutically acceptable salts, and combinations thereof, parasympathetic stimulants or cholinergic agents such as pilocarpine, carbachol, phospholine iodide, physostigmine, their pharmaceutically acceptable salts, and combinations thereof, carbonic anhydrase inhibitors including topical or systemic agents such as acetazolamide, brinzolamide, dorzolamide, methazolamide, ethoxzolamide, dichlorphenamide, their pharmaceutically acceptable salts and combinations thereof, mydriatic cycloplegic agents such as atropine, cyclopentolate, succinylcholine, homatropine, phenylephrine, scopolamine, tropicamide, their pharmaceutically acceptable salts and combinations thereof, prostaglandins such as prostaglandin F2 alpha, anti-prostaglandins, prostaglandin precursors, or prostaglandin analog agents such as bimatoprost, latanoprost, travoprost, unoprostone, tafluprost, their pharmaceutically acceptable salts and combinations thereof.

[0187] Agents for anti-VEGF that can be used in the dendrimers and methods of the present invention include bevacizumab, pegaptanib, ranibizumab, brolucizumab, conbercept, aflibercept, their pharmaceutically acceptable salts, and combinations thereof.

[0188] The tyrosine kinase inhibitors that can be used in the dendrimers and methods of the present invention include deuclavacitinib, axitinib, avapritinib, capmatinib, pegimatinib, ripretinib, serpercatinib, selumetinib, tucatinib, entrectinib, erdaftinib, fedratinib, pexidartinib, upadacatinib, zanubrutinib, and ba Licitinib, binimetinib, dacomitinib, fostamatinib, gilteritinib, lalotrectinib, lorlatinib, acalabrutinib, brigatinib, midostaurin, neratinib, alectinib, cobimetinib, lenvatinib, osimertinib, ceritinib, nintedanib, afatinib, ibrutinib, trametinib, bosutinib, cabo Zantinib, ponatinib, regorafenib, tofacitinib, crizotinib, ruxolitinib, vandetanib, pazopanib, lapatinib, nilotinib, dasatinib, sunitinib (borolanib), sorafenib, erlotinib, gefitinib, imatinib, afatinib, bosutinib, cabozantinib, cejiranib, ceritinib, crizotinib, This includes dabrafenib, dasatinib, erlotinib, everolimus, gefitinib, imatinib, restaurtinib, nilotinib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib.In another embodiment, the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor, such as A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD 2076, KB SRC 4, KX2361, KX2-391, MLR 1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN 004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL 201, WH-4-023, XL 228, artenusine, bosutinib, damnacanthal, dasatinib, harbimycin A, indirubin, neratinib, lavendastine A, peritinib, piceatanol, salakatinib, SrcI1, foretinib, motesanib, tivozanib, LY2457546, MGCD-265, MGCD-510, tivantinib, AMG458, JNJ-3887, EMD1214063, BMS794833, PHI1665752, SGX-523, INCB280, their pharmaceutically acceptable salts and combinations thereof, but not limited thereto.

[0189] Examples of complement pathway modulators that can be used in the dendrimers and methods of the present invention include those targeting C1 / C1Q, C3, C3 convertase, C5, C5 convertase, C5a, C5aR, C6, C7, C8, C9, CD59, factor B, factor D, factor H, factor P, or combinations thereof. Specific drugs include Synrize, Berinart, Luconest, Stimulimab, Pegcetacoplan (GA), Eclidiumab, Labuirizumab, Abacopan, Pozerimab, Nomacopan, Zircopan, Viroberimab, Clobarimab, Abasin Capted Pegol, Semdisilan, BDB-001, Tesidorumab, Avdralimab, MOR210, ALXN1720, Danicopan, and Bemircopan. This may include ACH-5228, ACH-5548, BCX-9330, AMY-101, ANX005, ANX007, nalsoprimab, iptacopan, CLG561, GT103, ARGX-117, ALXN1820, NGM621, lamparizumab, NGM621, IONIS-FB-Lrx, GEM103, CLG561, pharmaceutically acceptable salts thereof, and combinations thereof.

[0190] Integrin inhibitors that can be used in the dendrimers and methods of the present invention include rifitegrast, vedolizumab, natalizumab, efalizumab, tyrofiban, eptifivatide, absiximab, IDL-2965, PLN-74809, PLN-1474, PN-943, 7HP349, MORF-057, OS2966, OTT166, AXT-107, JSM-6427, listiganib, THR-687(D / ced), pharmaceutically acceptable salts thereof, and combinations thereof.

[0191] Antihistamines that can be used in the dendrimers and methods of the present invention include loradatin, hydroxyzine, diphenhydramine, chlorpheniramine, brompheniramine, cyproheptadine, terfenadine, clemastine, triprolidine, carbinoxamine, diphenylpyraline, phenyndamine, azatadine, triperenamine, dexchlorpheniramine, dexbrompheniramine, methidilazine, trimprazine, doxylamine, pheniramine, pyriramine, chlorcyclidine, tondylamine, pharmaceutically acceptable salts thereof, and combinations thereof.

[0192] The IL-6 inhibitors that can be used in the dendrimers and methods of the present invention include sarilumab, tocilizumab, RG6179, pharmaceutically acceptable salts thereof, and combinations thereof.

[0193] HtrA1 inhibitors that can be used in the dendrimers and methods of the present invention include IC-500, FHTR2163, RG6147, pharmaceutically acceptable salts thereof, and combinations thereof.

[0194] RASP inhibitors that can be used in the dendrimer and method of the present invention include reproxalap and pharmaceutically acceptable salts thereof.

[0195] Rho kinase inhibitors that can be used in the dendrimers and methods of the present invention include netarducyl, lipasudil, HA-1077, Y-27632, H-1152P, INS-115644, Y-39983, SB772077BS, LX71D1, AR-12286, AMA-0076, AR-13533, pharmaceutically acceptable salts thereof, and combinations thereof.

[0196] Plasma kallikrein inhibitors that can be used in the dendrimers and methods of the present invention include ecalantide, lanadermab, velotralstat, ATN-249, KVD900, KVD824, THR-149, pharmaceutically acceptable salts thereof, and combinations thereof.

[0197] The nitric oxide-donating PgA that can be used in the dendrimers and methods of the present invention includes latanoprostenbunod, NCX470, NCX125, pharmaceutically acceptable salts thereof, and combinations thereof.

[0198] Mast cell stabilizers that can be used in the dendrimers and methods of the present invention include rhodoxamide, nedocromil, pemirolast, cromolyn (e.g., cromolyn sodium), pharmaceutically acceptable salts thereof, and combinations thereof.

[0199] IGF-1R inhibitors that can be used in the dendrimers and methods of the present invention include teprotutumab, VRDN-001, VRDN-002, VRDN-003, ganitumab, figtumumab, MEDI-573, cyclotumumab, darotuzumab, lobatumumab, AVE1642, BIIB022, xentuzumab, istilazumab, lincitinib, picropodophyllin, BMS-754807, BMS-536924, and BMS. This includes -554417, GSK1838705A, GSK1904529A, NVP-AEW541, NVP-ADW742, GTx-134, AG1024, KW-2450, PL-2258, NVP-AEW541, NSM-18, AZD3463, AZD9362, B1I885578, B1893923, TT-100, XL-228, A-928605, their pharmaceutically acceptable salts, and combinations thereof.

[0200] TRPV1 antagonists that can be used in the dendrimers and methods of the present invention include Ashibatrep, V116517, for example, U.S. Patent Application No. 2004 / 0157849, U.S. Patent Application No. 2004 / 0209884, U.S. Patent Application No. 2005 / 0113576, International Patent Application No. WO 05 / 016890, U.S. Patent Application No. 2004 / 0254188, U.S. Patent Application No. 2005 / 0043351, International Patent Application No. WO 05 / 040121, U.S. Patent Application No. 2005 / 0085512, and Gomtsyan et al., 2005, J. Med. Condensed azalea compounds, heterocyclic compounds, and amide compounds described in Chem. 48:744-752, for example, condensed pyridine derivatives described in U.S. Patent Application No. 2004 / 0138454, for example, pyridylpiperazinyl urea described in Swanson et al., 2005, J. Med. Chem. 48:1857-1872 and U.S. Patent Application No. 2005 / 0049241, as well as AMG8163 (Bannon et al., 2005, 11th World Congress on Pain) and BCTC (Sun et al., 2003, Chem. Lett.13:3611-3616), 2-(piperazin-1-yl)-1H-benzimidazole, pyridadinylpiperazine, for example, urea derivatives described in U.S. Patent Application No. 2005 / 0107388, U.S. Patent Application No. 2005 / 0187291, and U.S. Patent Application No. 2005 / 0154230, as well as A-425619 (El Kouhen et al., 2005, J. Pharmacol. Exp.Ther. 314:400-409), SB-366791 (Gunthorpe et al., 2004, Neuropharmacology 46:133-149) and AMG 9810 (Gawa et al., 2005, J. Contains cinnamides (including Pharmacol.Exp.Ther.313:474-484).

[0201] In some embodiments, TRPV1 antagonists useful in the methods and compositions disclosed herein include, for example, capsazepine, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide (e.g., commercially available as AMG9810 from Tocris Bioscience, Bristol, United Kingdom), 4-tertiary butylcyclohexane (commercially available as SYMSITIVE 1609 from Symrise GmbH in Holzminden, Germany), and TRPV1 antagonists disclosed in U.S. Patents Nos. 8,815,930, 6,933,311, 7,767,705, U.S. Patent Application Publications 2010 / 0249203 and 2011 / 0104301, and International Application WO / 2008 / 013861.

[0202] In some embodiments, useful TRPV1 antagonists for the methods, compositions, and devices disclosed herein include AMG-517 and AMG-628 (Amgen Inc., Thousand Oaks, Calif.). Useful TRPV1 antagonists for this application are also described, for example, in International Patent Application WO2006065484, International Patent Application WO2003070247, U.S. Patent Application US2005080095, and International Patent Application WO2005007642. Additional TRPV1 antagonists useful for the methods, compositions and devices disclosed herein include the TRPV1 antagonists ABT-102, AMG8562, AMG9810, BCTC, SB366791, JNJ17203212, I-TTX, JYL-1421, A-425619, N-[4-[6-[4(trifluoromethyl)phenyl)pyrimidine-4-yloxy]benzothiazole-2-yl]acetamide (also known as AL-49975 or AMG-517), (R)-N-(4-(6-(4-(1-(4-fluorophenyl)ethyl)piperazin-1-yl)pyrimidine-4-yloxy)benzo[d]thiazole-2-yl)acetamide (also known as AL-49976 or AMG-628), pharmaceutically acceptable salts thereof, and combinations thereof.

[0203] Other TRPV1 antagonists useful for the methods, compositions, and devices disclosed herein have low inhibitory activity against CYP3A4, for example, 1-(2-(3,3-dimethylbutyl)-4-(trifluoromethyl)benzyl)-3-(1-methyl-1H-indazole-4-yl)urea, methyl2,2-dimethyl-4-(2-((3-(1-methyl-1H-indazole-4-yl)ureido)methyl)-5-(trifluoromethyl)phenyl)butanoic acid, 1-(2-(4-hydroxy-3,3-dimethylbutyl)-4-(trifluoromethyl)benzyl)-3-(1-methyl-1H-indazole-4-yl)urea, 2,2-dimethyl-4-(2-((3-(1- Methyl-1H-indazole-4-yl)ureido(methyl)-5-trifluoromethyl)phenyl)butanoic acid, 1-[4-chloro-3-(3,3-dimethylbutyl)benzyl]-3-(1-methyl-1H-indazole-4-yl)urea, 1-(2-isobutyl-4-(trifluoromethyl)benzyl)-3-(1-methyl-1H-indazole-4-yl)urea, 1-(2-isopropyl-4-(trifluoromethyl)benzyl)-3-(1-methyl-1H-indazole-4-yl)urea, 1-(4-chloro-3-isopropylbenzyl)-3-(1-methyl-1H-indazole-4-yl)urea, pharmaceutically acceptable salts thereof, and combinations thereof.

[0204] TrkA antagonists that can be used in the dendrimers and methods of the present invention include VM902A, larotrectinib, entrectinib, ceritrectinib (LOXO-195, BAY 2731954), repotrectinib (TPX-0005), pharmaceutically acceptable salts thereof, and combinations thereof.

[0205] To achieve the objectives of the present invention, the activators include all forms, including free acids, free bases, polymorphs, pharmaceutically acceptable salts, anhydrides, hydrates, other solvates, stereoisomers, crystalline forms, cocrystals, prodrugs, conjugates (e.g., pegylated compounds), complexes, and mixtures thereof.

[0206] Diagnostically active agents may be, for example, contrast agents, markers, or visualization agents. Generally, diagnostic agents may be substances used to examine the body to detect impairments in the normal functioning of the body. In some cases, diagnostic agents may be agents with a functional purpose, such as being used to detect deformities, diseases, and pathophysiological aspects of the eye. For example, diagnostic agents may be important and effective diagnostic aids such as dyes to aid in the visualization of ocular tissue (e.g., dark quenchers such as fluorescein dye, indocyanine green, trypan blue, cyanine dyes, azo dyes, acridine, fluoron, oxazine, phenanthridine, naphthalimide, rhodamine, benzopyrone, perylene, benzantrone, and prabenzoxanthrone). Diagnostic agents may include paramagnetic molecules, fluorescent compounds, magnetic molecules, radionuclides, X-ray imaging agents, and / or contrast agents. In some embodiments, the diagnostic agent may include radiopharmaceuticals, contrast agents used in imaging techniques, allergen extracts, activated carbon, various test strips (e.g., cholesterol, ethanol, and glucose), pregnancy tests, breath tests with urea 13C, and various stains / markers. In some embodiments, the labeling portion is a fluorescent dye or dark quencher selected from the group consisting of coumarin, cyanine dyes, azo dyes, acridine, fluorone, oxazine, phenanthridine, naphthalimide, rhodamine, benzopyrone, perylene, benzantrone, and benzoxanthrone. In certain non-limiting embodiments, the fluorescent dye is a compound selected from the group consisting of coumarin, fluorescein, cyanine 3 (Cy3), cyanine 5 (Cy5), cyanine 7 (Cy7), Alexa dye, bodipy derivative, (E)-2-(4-(phenyldiazenyl)phenoxy)acetic acid, 3-(3′,3′-dimethyl-6-nitrospiro[chromen-2,2′-indoline]-1′-yl)propanoate (spiropyran), 3,5-dihydroxybenzoate, and (E)-2-(4-(phenyldiazenyl)phenoxy)acetic acid, or a combination thereof, or a residue thereof.

[0207] In certain embodiments of the present invention, the active agent can be further dispersed, embedded, or encapsulated within the voids of the hyperbranched polymer. In certain embodiments thereof, the active agent can be in particulate form.

[0208] Synthesis Several methods for producing hyperbranched polymers are known to those skilled in the art, and these methods can be mainly applied and suitably adapted to the embodiments of the present invention.

[0209] In certain embodiments of the present invention, synthetic methods for producing dendrimers have been developed, among which divergent synthesis and convergent synthesis are the two most common synthetic methods used by chemists. In principle, these methods can also be used to synthesize the hyperbranched polymers of certain embodiments of the present invention. In the divergent method, monomers are added in repeated sequence, continuously increasing the number of branches from a polyvalent core to surface molecules. The size of the molecule and the number of surface groups gradually increase with the addition of successive layers of monomers called generations (see Figure 2). The convergent method involves the synthesis of hyperbranched polymers from the surface to the core, resulting in the formation of conical wedge-shaped units or dendrons, which are attached to the polyvalent core in the final step. Furthermore, in the embodiments of the present invention, a method combining the divergent method and the convergent method can also be employed. For example, as shown in Figure 1, in an embodiment of the synthetic method combining the divergent method and the convergent method, the first-generation DCRU is linked to the core unit, and the DCRUs of the second and subsequent generations are first linked to each other and then to the first-generation DRCU. In the embodiments of the present invention, any variation of the synthetic steps combining the divergent method and the convergent method can be used according to the specific hyperbranched polymer structure targeted.

[0210] In one embodiment of the present invention, a method for divergently synthesizing a hyperbranched polymer is provided, (a) providing a core unit having at least three linking sites c and a plurality of polymer arms linked to the core unit and having functional groups suitable for click chemistry at the ends of the polymer arms; (b) providing a dendritic structure repeating unit precursor, wherein the precursor is A polymer arm linked to a core, containing a functional group suitable for forming a linkage by click chemistry with the corresponding functional group (e.g., azide, alkyne, alkene, or tetrazine) of the polymer arm, The step of providing a dendritic repeating unit precursor comprising at least two polymer arms containing non-reactive functional groups in click chemistry, (c) The step of forming a link between the polymer arm connected to the core and the polymer arm of the dendritic structure repeating unit precursor by click chemistry, (d) Optionally, a step of converting functional groups of at least two polymer arms containing non-reactive functional groups in click chemistry to functional groups suitable for click chemistry, (e) The step of conjugating an activator containing functional groups to the outermost polymer arm by reacting it with the functional groups of the outermost polymer arm to form a hyperbranched polymer-activator conjugate.

[0211] In certain embodiments of high-generation Gx hyperbranched polymers (where x is an integer from 2 to 10), step (d) is essential. Before conjugating the activator in step (f), the functional groups suitable for click chemistry obtained in step (d) are further linked by click chemistry to successive dendritic repeating unit precursors, thereby forming a hyperbranched polymer.

[0212] For example, step d) is the NHS group [ka] This can be carried out by reacting a PEG arm having an SS (succinimidyl succinate), SG (succinimidyl glutarate), SAP (succinimidyl adipate), or SAZ (succinimidyl azelate) NHS-terminal group with a DBCO-amine click chemistry linker such as DS (dibenzocyclooctinamide succinate), DG (dibenzocyclooctinamide glutarate), DAP (dibenzocyclooctinamide adipate), or DAZ (dibenzocyclooctinamide azelate) group.

[0213] Similarly, the conversion of PEG-NHS-terminated PEG arms to azide-terminated PEG arms can be carried out by reacting the NHS group with an azide-amine click chemistry linker such as azide-PEG2-NH2. Such azide-amine click chemistry linkers are commercially available from several vendors and have the structures shown below. [ka] n defines the number of PEG repeating units.

[0214] In this synthesis method, the dendritic repeating unit precursor in step (c) can be represented by formula (iii), [ka] In the formula, C comprises a functional group suitable for click chemistry, such as an alkyne, alkene, azide, or tetrazine, D comprises a functional group that does not react in click chemistry, such as succinimidyl or a primary amine, and L A is the linker base, m is either 0 or 1, indicating whether a linker is present or absent, n is an integer between 3 and 2000, or between 20 and 2000, o is an integer between 3 and 2000, or between 20 and 2000, n and o may be different or the same, X is the branching unit, L Bis a linker group, p is either 0 or 1, meaning whether a linker is present or absent, B contains terminal groups located on the surface of the hyperbranched polymer, or bonds linked to A or the activator of a continuous dendritic repeating unit, L A and L B m and p may be different or the same, y is an integer between 2 and 9, y = c'-1, c' is the linkage site c' of the branched unit X, and the DCRU precursors used to synthesize the hyperbranched polymer may be the same or different.

[0215] Examples of precursors with four arms include 4-aPEG-NHS(3) azide(1) or 4-armed PEG-NHS(3) DBCO(1) compounds, and similar structures with or without hydrolyzable linker groups, such as those linked via ester bonds, amide bonds, or a combination of both. See, for example, the following structures. [ka] [ka] Or, the following exemplary precursor pairs, [ka] And, [ka] Either one of the following, [ka] And, [ka] Either one of the following, [ka] And, [ka] Either one of the following, [ka] And, [ka] either (wherein t is m, and n and m are defined similarly in this specification with respect to formula (v) above).

[0216] Referring to Figure 3, according to a particular embodiment, exemplary synthesis schemes for 4-4 arm PEG unit peptide conjugate G1 hyperbranched polymers (Figure 3a) and 8-4 arm PEG hyperbranched polymers are shown. In a general embodiment, a multi-arm PEG having terminal functional groups for click chemistry, such as DBCO or azide, can be used as the core of the hyperbranched polymer. Another branched PEG with functional groups then reacts with the core PEG via click chemistry. The branched PEG contains two types of functional groups, one of which, such as azide or DBCO, can bind to the core PEG for the growth of the hyperbranched polymer in the click chemistry reaction, while the remaining branched PEG (i.e., DCRU) is inert to the core PEG and can be used for the growth of the next generation of hyperbranched polymers or as a precursor for terminal bioconjugation (Figure 3a). Based on this general method, multiple generations of hyperbranched polymers can be synthesized to realize different numbers of terminal functional groups. Figure 3b shows the 3D structure of a hyperbranched polymer that starts with an 8-arm PEG core, conjugates with 8 4-arm PEG branches to realize 24 terminal groups on the surface, and is ultimately conjugated with up to 24 peptides.

[0217] As shown in the examples, two cyclic peptides, compstatin and APL-1, which are C3 binding inhibitors, as well as Fc-III-4C, an immunoglobulin G (IgG) binding peptide ligand, can be exemplary used as APIs conjugated with PEG hyperbranched polymers. The primary amine groups on the peptides can be used as nucleophiles and react with electrophilic NHS groups on the outermost polymer arms of the hyperbranched polymer.

[0218] Alternatively, the functional groups of the outermost polymer arm containing non-reactive functional groups in click chemistry can be converted to functional groups suitable for click chemistry, and then activators such as peptides having click chemistry-suitable functional groups (alkynes, alkenes, azides, tetrazines, etc.) can be functionalized to perform click chemistry conjugation of activators in the outermost polymer arm of a hyperbranched polymer.

[0219] Suitable ester groups on the outermost polymer PEG arms of the hyperbranched polymers of a particular embodiment, such as succinic acid (S-), glutaric acid (G-), adipic acid (AP-), and azelaic acid (AZ-), are hydrolyzable under physiological conditions and degraded under controlled pH conditions to release peptides in vivo. The controlled release and binding affinity of the peptide moiety can be characterized by ultra-high performance liquid chromatography (UHPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and surface plasmon resonance (SPR), as further described herein.

[0220] In an alternative embodiment, the convergent synthesis of the superbranched polymer of the present invention is provided. I) A step of providing a dendritic repeating unit precursor, wherein the precursor is A polymer arm containing a functional group suitable for forming a linkage by click chemistry (e.g., azide, alkyne, alkene, or tetrazine), A step of providing a dendritic repeating unit precursor comprising at least two polymer arms containing non-reactive functional groups in click chemistry, II) A step of conjugating an activator containing a functional group to at least one of at least two polymer arms containing a functional group that does not react in click chemistry of a dendritic repeating unit precursor, III) A step of providing a core unit having at least three connecting portions c, and a plurality of polymer arms connected to the core unit, each having a functional group suitable for click chemistry (e.g., azide, alkyne, alkene, or tetrazine) at the end of the polymer arm, IV) The step of forming a biodegradable hyperbranched polymer-activator conjugate by forming a click chemistry linkage between a polymer arm linked to the core provided in step III) and a polymer arm containing a functional group suitable for forming a click chemistry linkage of the activator conjugate dendritic repeating unit precursor obtained in step II).

[0221] In this method, the dendritic repeating unit precursor in step I) is represented by the above formula (iii).

[0222] In a specific embodiment of the high-generation Gx biodegradable superbranched polymer, x is an integer between 2 and 10, and the activator-conjugated dendritic repeating unit precursor obtained in step II) is linked by click chemistry to an inverse dendritic repeating unit precursor comprising one polymer arm containing a click-chemistry-inactive functional group and at least two polymer arms containing click-chemistry-suitable functional groups (such as azides, alkynes, alkenes, or tetrazines), and the click-chemistry-inactive functional group of the one polymer arm is subsequently converted to a click-chemistry-suitable functional group before linking to further inverse dendritic repeating unit precursors or before forming a click-chemistry link with the polymer arm linked to the core in step IV), thereby forming a high-generation biodegradable superbranched polymer.

[0223] In contrast to divergent synthesis methods, convergent synthesis methods enable the synthesis of superbranched polymers having two or more different activators in different dendrons or regions on the surface of the superbranched polymer. This allows for the clustering of multiple activators on the surface of the superbranched polymer. In a specific embodiment of the convergent synthesis method of the present invention, steps I) and II) can be performed on each activator-conjugated DCRU precursor to obtain dendritic repeating unit precursors having different activators conjugated to polymer arms, and a mixture of the obtained activator-conjugated DCRU precursors can be used in step IV) to form biodegradable superbranched polymer-activator conjugates having different activators in different regions on the surface of the superbranched polymer. Such clustered superbranched polymers may be used, for example, in combination therapy involving the administration of multiple activators.

[0224] In alternative embodiments of the divergent and convergent synthesis methods described above, these methods can also be carried out using reversed functional groups, i.e., using other reactions and functional groups to form links within the hyperbranched polymer and click chemistry functional groups for terminal conjugation. In such “reverse” embodiments of the described synthesis methods, links within the hyperbranched polymer can be selectively formed using electrophilic-nucleophilic precursors or other functional groups that do not react with click chemistry functional groups, while all other terminal functional groups of the DCRU that are not involved in linking with the core or previous DCRU contain functional groups for click chemistry bond formation and remain inactive in the bond formation reaction. These terminal click chemistry functional groups can then be used in other subsequent reactions, such as the growth of the hyperbranched polymer of conjugation by click chemistry reactions.

[0225] Therefore, in another embodiment of the present invention, a method for divergently synthesizing superbranched polymers is provided. (a) A core unit having at least three connecting portions c, and a plurality of polymer arms connected to the core unit, each having a functional group at the end of the polymer arm that does not react in click chemistry, (b) A step of providing a dendritic repeating unit precursor, wherein the precursor is In reactions other than click chemistry, one polymer arm contains a functional group suitable for forming a link with the corresponding functional group of the polymer arm linked to the core (e.g., electrophile or nucleophile, e.g., amine, NHS), A step of providing a dendritic repeating unit precursor comprising at least two polymer arms containing functional groups suitable for click chemistry, (c) A step of forming a link between the polymer arm linked to the core and the polymer arm of the dendritic structure repeating unit precursor by reacting functional groups that do not react in click chemistry, (d) Optionally, the step of converting functional groups of at least two polymer arms containing functional groups suitable for click chemistry to functional groups that do not react to click chemistry, (e) The step of conjugating an activator containing functional groups to the outermost polymer arm by reacting it with the functional groups of the outermost polymer arm to form a hyperbranched polymer-activator conjugate.

[0226] In certain embodiments of high-generation Gx hyperbranched polymers (where x is an integer from 2 to 10), step (d) is essential. Before conjugating the activator in step (f), a series of repeating dendritic structural unit precursors are further linked to the click-chemistry-inactive functional groups obtained in step (d) to form a hyperbranched polymer.

[0227] In this synthesis method, the dendritic repeating unit precursor in step (c) can be represented by the above formula (iii).

[0228] In a further alternative embodiment of the convergence method, the convergence synthesis of hyperbranched polymers of the present invention is provided. I. A step of providing a dendritic repeating unit precursor, wherein the precursor is A polymer arm containing a non-reactive functional group in click chemistry, A step of providing a dendritic repeating unit precursor comprising: at least two polymer arms containing functional groups (such as azides, alkynes, alkenes, or tetrazines) suitable for forming links by click chemistry; II. A step of conjugating an activator containing a functional group to at least one of at least two polymer arms containing a functional group suitable for click chemistry of a dendritic repeating unit precursor, III. A step of providing a core unit having at least three connecting portions c, and a plurality of polymer arms connected to the core unit, each having a functional group at the end of the polymer arm that does not react in click chemistry, IV. The step of forming a biodegradable hyperbranched polymer-activator conjugate by forming a linkage between a polymer arm linked to the core provided in step III) and a polymer arm containing a functional group that does not react in the click chemistry of the activator conjugate dendritic repeating unit precursor obtained in step II).

[0229] Exemplary reaction conditions for forming a dendrimer include reacting the core precursor and DCRU in a suitable solvent such as DMF at a relatively mild temperature of 10–50°C, e.g., 30–45°C, for several hours, e.g., overnight, up to 24 hours, or even 48 hours.

[0230] Purification and Characterization The ultrabranched polymer reaction mixture obtained by the synthesis method described herein can be purified, for example, by filtration, dialysis, SEC column filtration, centrifugation, or UHPLC.

[0231] In exemplary embodiments, the synthesis reaction mixture of a hyperbranched polymer conjugated with an activator such as a peptide may be diluted, filtered through, for example, a 0.45 μm mesh, then purified by, for example, ultracentrifugation using a 100 kDa membrane, followed by the addition of a glucose buffer and lyophilization to obtain the final product. For administration in therapeutic methods, the lyophilized product may optionally be reconstituted by adding a solvent containing further glucose buffer.

[0232] The glucose buffer may optionally be added before or after lyophilization to improve the solubility and stability of the dendrimer peptide or protein conjugate, for example, by preventing peptide precipitation. Since the PEG-based dendrimers of the embodiments of the present invention behave similarly to synthetic proteins, the stability and solubility of dendrimer conjugates other than peptides are also improved by the addition of the glucose buffer. Exemplary glucose buffer formulations for use in embodiments of the present invention may include solutions in which sugars such as trehalose, monophosphates, and diphosphates are dissolved in water at appropriate concentrations, for example, 3% (or 30 mg / mL) and pH approximately 6.4.

[0233] Dialysis is a common purification method that separates molecules in a solution based on differences in diffusion rates through a semipermeable membrane, such as a dialysis tube. To purify hyperbranched polymer reaction mixtures, which may contain molecules of different sizes, such as free peptides (e.g., approximately 1.5 kDa MW), free PEG / DCRU precursors (e.g., approximately 10-40 kDa MW), small hyperbranched polymer conjugates like G0 (e.g., approximately 20-50 kDa MW), and larger conjugates like high-generation Gx hyperbranched polymer conjugates (e.g., over approximately 50 kDa), the solution can be filled into a dialysis tube with a membrane of a specific pore size to define a cutoff, and then immersed in a large volume of solvent. Molecules smaller than the pore size elute from the tube into the solvent, while molecules larger than the pore size remain inside the tube. Dialysis tubes are commercially available from companies such as Spectra / Por® Float-A-Lyzer G2 Dialysis Devices and Spectrum® Laboratories, and several different molecular weight cutoffs are available to meet the requirements of specific separation tasks. Figure 5 a) shows the corresponding experimental setup for purification by dialysis.

[0234] By selecting appropriate molecular weight cutoff dialysis tubes and membranes, and optionally using a series of separation steps with different cutoff dialysis tubes, most impurities such as excess peptides, unreacted precursors, and small-sized intermediates can be removed from the product.

[0235] Another purification method applicable in certain embodiments of the present invention is size exclusion chromatography (SEC), for example, the use of an SEC column. For example, Zeba® Spin Desalting columns (manufactured by Thermo Fisher Scientific), designed for protein purification and removing salts and small impurities, can be used for the purification of hyperbranched polymer conjugates. For example, columns with different pore sizes, such as 7 kDa and 40 kDa, may be used. The purification mechanism is based on size exclusion chromatography, where small particles are trapped in the pores of the stationary phase material, and larger particles, such as the hyperbranched polymer conjugates of certain embodiments of the present invention, are eluted through the column and collected in a purified form. Figure 5 b) shows the corresponding experimental setup for purification by SEC column filtration.

[0236] The resulting purified product can be characterized by ultra-high-performance liquid chromatography (UHPLC), an efficient technique that enables more sensitive analysis due to its good chromatographic separation and resolution of the analyte. UHPLC offers advantages such as rapid analysis, high-resolution separation, reduced solvent and sample usage, and improved sensitivity and accuracy. Based on a calibration curve using free activators and free precursor units, and a comparison of the solutions before and after purification, the amount of the target product in the purified solution can be determined by peak area integration.

[0237] Purification by dialysis yields a product solution containing over 99% of the ultrabranched polymer-peptide conjugates, based on peak area integration by UHPLC (see Example 6). Purification by SEC column yields a product solution containing over 98% of the ultrabranched polymer-peptide conjugates, based on peak area integration by UHPLC (see Example 7). All of the purification methods described demonstrate highly efficient purification capabilities.

[0238] SDS-PAGE can be used to determine the molecular weight of the hyperbranched polymer-conjugate in certain embodiments of the present invention. SDS-PAGE is an analytical technique for separating substances based on molecular weight. When separating samples by electrophoresis under potential through a gel matrix, smaller compounds move faster due to less resistance from the gel matrix, while larger molecules move more slowly. Sodium dodecyl sulfate (SDS) is a surfactant that can detach macromolecules such as proteins and separate compounds based solely on molecular size, eliminating the influence of their structure and charge.

[0239] In an embodiment of the present invention, a storage-stable formulation is provided that can be reconstituted with a suitable solvent before use in treatment by freeze-drying a superbranched molecule.

[0240] Polyvalent shell binding To obtain biological efficacy, it is desirable that the biomolecules conjugated to the hyperbranched polymer of the present invention exhibit the same or similar affinity to the receptor. Furthermore, efficacy can be improved if the half-life of the receptor-binding biomolecule is extended by polyvalent binding. For example, in antigen binding, affinity is defined as the strength required for the interaction between the antigen-binding site of the antibody and the antigen epitope. Binding activity is the total strength required for the interaction between a polyvalent antibody and multiple antigen epitopes. This definition can also be applied to other biomolecules that bind to specific targets or receptor sites. Therefore, polyvalent binding leads to improved binding activity. The concept of polyvalency, the resulting concept of binding activity, and a model for quantifying binding activity are explained in Kitov et al., “On the Nature of the Multivalency Effect: A Thermodynamic Model”, JACS 2003, 125, 16271-16284, the full text of which is incorporated herein by reference.

[0241] Kitov describes the interaction between a series of dendrimer-conjugated polyvalent oligosaccharide ligands and Shiga-like toxins based on various polyvalent PANAM dendrimer structures. In particular, Kitov found that additional branching of polyvalent ligand dendrimers increases the probability of interaction with the receptor, even in cases where such interaction would not normally be expected. Furthermore, Kitov concludes that "when all binding sites need to be inhibited to achieve the desired effect, the proportion of uninhibited binding sites can be precisely controlled by selecting the appropriate number of branches in the assembly of the polyvalent inhibitor." Therefore, for example, in a polyvalent inhibitor system, while individual inhibitors may not specifically interact with the receptor, the inhibitor molecules conjugated to the additional branches provide a higher inhibitory effect, resulting in extended half-life, improved efficacy, and enhanced binding activity, even through the potential for polyvalent binding.

[0242] Khalili et al., “Fab-PEG-Fab as a Potential Antibody Mimetic”, Bioconjugate Chem. 2013, 24, 1870-1882 (the entire article is incorporated herein by reference) exemplifies divalent PEG conjugated to a protein-binding ligand and illustrates the improved binding activity due to divalentity.

[0243] Specific embodiments of the present invention utilize the concepts described below with reference to Examples 8 and 9, and Figures 18 and 19.

[0244] Embodiments of the present invention relate to methods for treating diseases using antibodies conjugated to dendrimers described herein. For example, the dendrimers of the embodiments of the present invention may be used to improve the pharmacokinetics of antibodies delivered as delivery targets conjugated to or attached to the hyperbranched polymers described herein. Suitable delivery targets are selected from, for example, anti-VEGF, aflibercept, falisimab, bevacizumab, anti-TNF-α, infliximab, etanercept, adalimumab, anti-IL-6R, sarilumab, anti-IL-6, siltuximab, anti-C5, rabirizumab, eculizumab, anti-CD20, ocrelizumab, rituximab, anti-IGF-1R, or teprotumumab. These antibodies, delivered conjugated to dendrimers, conjugate non-covalently to the antibody drug and exert a chaperone effect, extending the blood half-life upon intravenous administration or infusion into the vitreous fluid (IVT). The high molecular weight of the dendrimer-antibody conjugate prevents the bound antibody from being excreted from the bloodstream via the kidneys, and slows its diffusion from therapeutic target sites such as the vitreous humor.

[0245] The antibody maintains its function while bound to the dendrimer of the embodiment of the present invention. Sustained release from the dendrimer allows the antibody to be delivered to the target tissue without interference. The dendrimer can be designed to be broken down into lower molecular weight fragments, such as those less than 50,000 kDa, as described herein, and ultimately excreted by the kidney.

[0246] Furthermore, the nanoscale size of the dendrimer-antibody conjugates in embodiments of the present invention enables passive targeting of leaky vessels, such as tumors or choroidal neovascularization (CNVs), for enhanced vascular permeability and retention (EPR) effects. EPR allows for subcutaneous (SC) or intravenous (IV) administration routes by reducing off-target effects. This enables SC or IV delivery to CNV areas in the eye.

[0247] Examples of diseases treatable with dendrimer antibody conjugates of specific embodiments include exudative AMD, cancer (e.g., anti-VEGF dendrimer conjugate, IVT or SC), RA, PsA, COPD (e.g., anti-TNF-α dendrimer conjugate, IV or SC administration), PNH, aHUS, MG, glomerular diseases, GA (e.g., dendrimer conjugate containing anti-C5, labirizumab, or eculizumab, IV, IVT, or SC administration), RA (e.g., dendrimer conjugate containing rifaximab, IV administration), or TED (e.g., dendrimer conjugate having anti-IGF-1R, IV or SC administration).

[0248] Further embodiments of the present invention relate to methods for treating diseases using peptides conjugated to the dendrimers described herein. For example, the dendrimers of the embodiments of the present invention can be used to improve the pharmacokinetics of peptides delivered as delivery targets conjugated to or attached to the hyperbranched polymers described herein. Suitable delivery targets can be selected from, for example, anti-C3, C3B, sifoble, GLP-1RA, liraglutide, Victoza, Saxenda, semaglutide, Ozempic, Rybelsus, Wegoby, exenatide, hormone therapy, HGH (somatotripin), insulin, estrogen, and the like.

[0249] Compared to larger proteins, peptides have the advantages of lower immunogenicity and superior stability. However, peptides have the disadvantage of rapid excretion and sometimes low solubility, limiting their therapeutic usefulness. In embodiments of the present invention, conjugation to dendrimers can be a successful strategy for peptide delivery in therapeutic treatments, such as dendrimer-Syfovre conjugates, to increase solubility and extend half-life. Syfovre also exhibits improved binding activity through divalent conjugation. Conjugation with dendrimers, as in embodiments of the present invention, surpasses simple PEG conjugation, resulting in higher molecular weight, longer half-life, and higher valency, i.e., higher binding activity. As the dendrimer gradually biodegrades into smaller fragments, these high molecular weight molecules are removed, preventing accumulation in the body.

[0250] Examples of diseases that can be treated with dendrimer peptide conjugates of specific embodiments include GA, PNH (anti-C3, C3B dendrimer conjugates, IVT, IV, or SC, etc.), type 2 diabetes, obesity (GLP-1RA dendrimer conjugate, etc.), and hormone deficiency syndromes (hormone dendrimer conjugates, inhalation, IV, or SC, etc.).

[0251] Further embodiments of the present invention relate to methods for treating diseases using aptamers conjugated to the dendrimers described herein. For example, the dendrimers of the embodiments of the present invention can be used to improve the pharmacokinetics of aptamers delivered as delivery targets conjugated to or attached to the hyperbranched polymers described herein. Suitable delivery targets are selected from, for example, anti-C5, Izervay, anti-VEGF165, Macugen, anti-CXCL12 / SDF-1, or NOX-A12.

[0252] Aptamers are similar to peptides in that they have low immunogenicity. However, their stability in vivo is a concern, which can be addressed by conjugation to dendrimers as described herein. Aptamers also have excellent water solubility. Conjugation to PEG is an effective strategy for extending the half-life of aptamers such as Macugen and Izervay. The dendrimer conjugation described herein goes beyond simple PEG conjugation, achieving higher molecular weight, longer half-life, and higher valency (higher binding activity). As the dendrimers gradually biodegrade into smaller fragments, these high molecular weight molecules are removed, preventing accumulation in the body.

[0253] Examples of diseases that can be treated with dendrimer-aptamer conjugates of specific embodiments include exudative AMD (e.g., with anti-VEGF165 dendrimer conjugates), PNH, aHUS, MG, glomerular diseases, GA (e.g., with anti-C5 or izervay dendrimer conjugates), CLL, and pancreatic cancer (e.g., with anti-CXCL12 / SDF-1 dendrimer conjugates).

[0254] To determine the biological efficacy of the hyperbranched polymer-conjugate of a particular embodiment of the present invention, a binding assay can be performed to analyze the binding affinity of the peptide conjugated to the hyperbranched polymer.

[0255] Complement activation is essential for the normal inflammatory response to foreign pathogens, but improper activation leads to tissue damage in many pathological conditions. Complement component C3 is a common factor in the activation of the classical, alternative, and lectin pathways of complement activation. Unregulated complement activation can lead to a variety of life-threatening or debilitating diseases.

[0256] Compstatin is a 13-base peptide (1e-Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys-Thr-NH2) cyclized by a disulfide bond, and is a novel and promising inhibitor of complement system activation. It was initially isolated from a phage display random peptide library screened against C3b.

[0257] APL-1 (le-Cys-Val-MeTrp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-NH2) has a structure similar to compstatin, and its sequence contains two different amino acids. According to literature reports, the dissociation constant K for C3 of APL-1 is D While K is 10 nM, compstatin's K D The concentration is 13 μM, and there is approximately a 100-fold difference in C3 binding affinity. The peptide sequences of compstatin and APL-1 are shown below.

[0258] Scheme C [ka] Fc-III-4C is an immunoglobulin G (IgG)-binding peptide ligand consisting of 15 residues, in which four cysteine ​​residues form two disulfide bonds to create a bicyclic structure. The proposed structure of the Fc-III-4C bicyclic peptide is shown below.

[0259] Scheme D [ka] The binding affinity of Fc-III-4C peptide to human IgG is 2.45 nM (K). D It was measured as ), which is higher than that of protein A / G (Pro-A / G) bound IgG. Importantly, the Fc-III-4C peptide has been reported to have high affinity for various IgGs of different species and is therefore also used as a peptide-based antibody affinity tag.

[0260] The three peptides mentioned above can be used to analyze the binding affinity of hyperbranched polymer conjugates composed of these peptides using a surface resonance plasmon instrument at Mosaic Biosciences, Inc., USA. Surface plasmon resonance (SPR) coupling analysis can be used to study intermolecular interactions. SPR is an optical technique that detects the interaction between two different molecules, one of which is mobile and the other fixed on a thin film. In this analysis, a C3 target is fixed to the surface of a thin film or chip, and a hyperbranched polymer-peptide conjugate solution is flowed over it. Differences in signals as the hyperbranched polymer-peptide conjugate associates / dissociates on the C3 target are monitored.

[0261] These assays show that the binding of free compstatin, APL-1, and Fc-III 4C to C3 and C3b is consistent with the dissociation constant K reported in the literature. D The selected assay demonstrates good agreement with the values, making it a reliable tool.

[0262] The same assay can be applied to test the binding affinity of hyperbranched polymer-peptide conjugate peptides by immobilizing C3 on an assay chip and flowing various concentrations of hyperbranched polymer-peptide conjugate solutions through it. When multiple peptides are polyvalently bound to a hyperbranched polymer, the hyperbranched polymer is expected to dissociate very slowly, while the free peptides are expected to dissociate rapidly. This measurement allows for a comparison of the dissociation rates of different generations of hyperbranched polymers.

[0263] This theoretical prediction has been confirmed by SPR measurements. When comparing the binding affinity of compstatin, APL-1, and Fc-III 4C superbranched polymer-conjugates in specific embodiments of the present invention using the same assay, a fast association rate is observed. During the dissociation time, the response from free compstatin decreases rapidly, and its rate is the same as the association rate, as expected. On the other hand, the superbranched polymer-peptide conjugate dissociates much more slowly. While we do not intend to be bound to any particular theory, this is thought to be caused by multiple interactions of the multiple peptides conjugated to the superbranched polymer, such as polyvalent bonds. This slow dissociation phase indicates that the polyvalent conjugated superbranched polymer-peptide is cooperatively bound to the C3 surface.

[0264] Similar experiments have shown that peptides hydrolyzed from hyperbranched polymers during biodegradation can exhibit nearly the same binding affinity as free peptides. The hydrolyzed peptides contain acid ester bonds (part of the linker on the hyperbranched polymer) formed by the degradation, and similar SPR signals indicate that these do not affect the C3 bond. Therefore, it is considered that ester bonds do not alter the physiological activity of the peptide.

[0265] Furthermore, quantitative K D In the analysis, the K of the hyperbranched polymer-conjugate D It can be seen that the value decreases as the amount of peptides substituted with hyperbranched polymers increases.

[0266] Referring to Figures 18 and 19, as described in Example 10, the IC50 (median inhibitory concentration) was measured by an alternative pathway (AP) hemolysis assay using four different compstatin-conjugated hyperbranched polymers of the present invention. The G1 PEG hyperbranched polymer compstatin showed improved IC50 compared to free compstatin, suggesting improved binding activity due to binding affinity. While we do not intend to be bound by any particular theory, it is thought that in high-generation hyperbranched polymer peptide conjugates, enabling multiple binding events or polyvalent binding contributes to receptor inhibition and improves efficiency. Furthermore, hyperbranched polymer conjugates with longer polymer arms conjugated to the peptide appear to show improved IC50 because they have greater flexibility to interact with the receptor compared to shorter polymer arms, which may result in steric repulsion issues. These results have been confirmed by a classical pathway (CP) hemolysis assay, which has shown that high-valence compstatin conjugates in certain embodiments of the present invention are more effective in inhibiting CP hemolysis compared to free compstatin alone. Furthermore, as shown in Example 10, the dendrimer binders such as APL-1 used in this example can maintain their stability and activity in vivo over a long period of time.

[0267] Release kinetics In certain embodiments, the hyperbranched polymers of the present invention can be used for sustained-release drug delivery. Generally, the in vivo half-life of a drug can be extended by conjugating a therapeutic agent to a hyperbranched polymer. The hyperbranched polymer structure can be adapted to regulate the release of the conjugated agent in several ways, thereby providing a hyperbranched polymer-based drug delivery system. For example, adjusting or appropriately selecting the precursor components and DCRUs that form the hyperbranched polymer, such as the length and molecular weight of the polymer arms, the type of linker used, and the links formed between the hyperbranched polymer portions and used for conjugation, affects the release of the agent.

[0268] Furthermore, the release of activators with multiple binding sites on a dendrimer can be slowed down by multiple bonding of the activator to the dendrimer functional end groups intramolecularly and / or intermolecularly, i.e., by linking two or more dendrimers via a single multiple-bonded activator. For example, multiple bonding to a dendrimer can be used to extend the half-life of the activator. This is because the bonds of multiple conjugations must be cleaved in order to completely release the activator from the dendrimer.

[0269] Dendrimers for drug delivery can be considered as large support or carrier media. Due to their high symmetry and regular spherical structure, dendrimers have a high hydration radius or hydrodynamic radius R of the bound activator. h It can be used to expand the dendrimer structure. In particular, the large hydrodynamic radius of PEG-based dendrimer structures can be used to further extend the half-life of dendrimer drug conjugates in vivo, e.g., in the vitreous humor, and to control and regulate the sustained release of activators. Estimated from the Stokes-Einstein equations,

number

[0270] Therefore, embodiments of the present invention utilize large-sized dendrimers to delay the release of activators in vivo by appropriately adjusting the overall size of the dendrimer drug conjugate. Hydrodynamic radius R hSince this can be easily determined, for example, by size exclusion chromatography (SEC), it is possible to predictably adjust the release rate or half-life of the activator bound to the dendrimer from the calibration information of the SEC measurement. Example 11 below illustrates a method for correlating dendrimer size with release rate.

[0271] Therefore, the biodegradable synthetic dendrimer of the embodiment of the present invention has a hydrodynamic radius R that decreases gradually during decomposition. h This offers the advantage of incorporating controlled degradable functional groups that generate smaller fragments with different half-lives that determine their mobility, and / or lead to their elimination from the body. For example, a first-generation (G1) dendrimer (e.g., 4a40k-PEG(SGA)-[4a20k-PEG(SG)-(Fc-III-4C)3]4) consisting of a 4-arm 40kDa PEG core and four 4-arm 20kDa dendrons conjugated with 12 peptides or proteins, each 1.7kDa, has a molecular weight of approximately 145kDa. When one, two, three, or all four dendrons are cleaved, the molecular weight decreases stepwise, producing fragments of approximately 115kDa, 90kDa, and 65kDa, ultimately leaving a 40k core and four dendrons, each approximately 25kDa, with each fragment having a different hydrodynamic radius and diffusion rate. In the same polymer, a 20 kDa linear PEG extender is incorporated between the core unit and the arms of the branched unit, and the molecular weight cascade contains a dendrimer of approximately 22 kDa and degradation fragments of approximately 175 kDa, 130 kDa, 85 kDa, 45 kDa, 40 kDa, 25, and 20 kDa, with these dendrimers having different, longer release rates and a wider distribution of fragment half-lives.

[0272] The multiple half-lives of the degradable dendrimer in the embodiment of the present invention are as follows: hThis is based on a first half-life, followed by another half-life based on degradable fragments (dendrons, dendron analogs, or dendron analogs with linear PEG extensions) formed by cleaving hydrolyzable bonds within the hyperbranched polymer structure. All of these multiple sequentially degrading species have different R2 levels. h These different types of R exhibit a series of substructures with different hydrodynamic radii, which decompose and separate from the initial dendrimer structure, resulting in different clearance rates and half-lives. h This correlates with different building blocks of different molecular weights, number of arms, linear PEG extensions, and / or linkers. By constructing hyperbranched dendrimer polymers from building blocks having different molecular weights and number of arms, using linear PEG extensions and bifunctional linkers of different lengths between hydrolyzable bonds, the dendrimers of embodiments of the present invention can be designed to degrade and remove fragments at multiple rates and half-lives to suit individual activators and / or therapeutic purposes / or dosage forms.

[0273] Specifically, to adjust and control the release rate of the active substance conjugated to or associated with the dendrimer, incorporated degradable linker groups (e.g., diacid derivative esters of linkers based on succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), succinimidyl azelate (SAZ), e.g., succinate diester (S), glutarate diester (G), adipate diester (AP), or azelate diester (AZ)) can be used. For example, in one embodiment, a dendron building block can be prepared using different degradable bonds (SS, SG, SAP, SAZ, etc.), and if the ester bonds are the same, a homogeneous dendrimer can be obtained in which all dendrons degrade at the same rate.

[0274] In another embodiment, the building blocks of the dendrons can be fabricated with different degradable bonds (S, G, AP, AZ, etc.), and different ester bonds result in heterogeneous dendrimers in which the dendrons degrade at different rates. For example, in one embodiment, dendrimers can be fabricated using S, G, AP, or AZ dendrons, or mixtures thereof, and the core structure can be clicked by click chemistry links.

[0275] In other embodiments, a specific release profile can be adjusted by blending a mixture of multiple homogeneous dendrimers, and the half-life clearance rate can be adjusted by dry or wet blending.

[0276] The PEG-based embodiments of the present invention may also be called nanodroplets because they have a large hydrodynamic radius even with a low solids content. For weight or solids content similar to the drug or peptide itself, the dendrimers have a much higher hydrodynamic radius (see Figure 20 and Example 11). Therefore, the diffusion rate is slower, and the in vivo half-life T of the conjugated activator is reduced. 1 / 2 It will be longer.

[0277] In certain embodiments, linkers of formula (ii) used in superbranched polymers introduce hydrolyzable bonds to the superbranched polymer, which can be used to adjust the degradation rate of the superbranched polymer and / or the release rate of activators conjugated from the superbranched polymer. For example, the biodegradation / hydrolysis rate of ester bonds in these linkers increases from succinate esters (C4) to azelaic acid esters (C9). The shorter the diacid linker chain length, the faster the hydrolysis rate of the ester bonds it forms. Therefore, the hydrolysis rate decreases in the order of SS > SG > SAP > SAZ > SGA ester bonds. In embodiments of the present invention, this can be used to control the degradation rate of superbranched polymers and / or the release of activators conjugated to the superbranched polymer via these linkers. For example, esters formed from succinimidyl succinate (SS) groups degrade in a few days, while esters from succinimidyl glutarate (SG) groups degrade in a few weeks. By using different linkers within the superbranched polymer, the rate of decomposition between the bonds of different generations of DCRUs within the superbranched polymer can be controlled, or the release of activators from the superbranched polymer can be controlled by conjugating them.

[0278] For linkages formed by click chemistry reactions, in embodiments of this disclosure, the hydrolysis of adjacent ester bonds can also be slowed by extending the spacer structure between the DBCO / azide functional group and the functional group to which it binds to the polymer arm, for example, by using alkylene chains or pegylation. The greater the distance between the DBCO / azide functional group and the next hydrolyzable ester group, the slower the hydrolysis of the ester. Degradation control can be enabled by using different linkers in the hyperbranched polymer and in the activator conjugation site. By using a short-chain linker group such as succinic acid for the DCRU linkage and a long-chain linker such as SAZ for the conjugation site, it is possible to degrade the ester group in the hyperbranched polymer first during degradation, followed by the DCRU-activator conjugation. Conversely, it is also possible to intentionally design the conjugation site to degrade first for activator release by the short-chain linker. This can be used to control the half-life of the activator and change the release rate.

[0279] Furthermore, depending on the chain length of the diacid linker, the hydrolysis of the ester bond is dependent on the pH and / or temperature of the environment. This can be used in certain embodiments to control the release of the activator, for example, site-directed release in specific tumor cells that have a higher pH than the surrounding cells.

[0280] In certain embodiments, the sustained-release drug-delivery hyperbranched polymers of the present invention are formulated to make the activator available over a long period of time, thereby enabling a reduction in the frequency of administration compared to immediate-release dosage forms (e.g., solutions of the activator applied topically to the eye (i.e., eye drops)). In certain embodiments, the release of the activator includes constant activator release, gradually decreasing activator release, and any combination thereof (e.g., gradually decreasing activator release after constant activator release). "Sustained release" can be measured in vitro in an aqueous solution under physiological conditions such as pH 7.2-7.4 and 37°C, and is considered to be the same as or substantially the same as when the hyperbranched polymer is administered in vivo to the subject.

[0281] In various embodiments of the present invention, the release of the activator follows zero-order release dynamics or substantially zero-order release dynamics, preferably without a "burst" of activator at the start of the period.

[0282] Embodiments of the present invention may provide, for example, the release of a therapeutically effective amount of the activator over a period of up to 1 year, up to 9 months, up to 6 months, up to 3 months, up to 1 month, or up to about 25 days after administration. Other embodiments of the present invention may provide the release of a therapeutically effective amount of the activator over a period of up to about 14 days, or up to about 21 days, or over a period of about 6 hours or more, or over a period of about 12 hours, or 24 hours or more, or over a period of about 48 hours or more, or over a period of about 72 hours or more, or over a period of about 7 days or more, or over a period of about 10 days or more after administration. The present invention intends to achieve all of the above shorter and longer periods in any combination of ranges.

[0283] Some aspects of the present disclosure relate to pharmaceutically acceptable superbranched polymers for controlled release of activators conjugated to the superbranched polymer, wherein the controlled release is characterized in that the amount of activator released on day 1 is 0 to 50% of the total activator, the daily release of activator from day 2 to the final release day is 0 to 50% of the total activator, and / or the number of days required for 100% release of the total activator is at least 2 days.

[0284] In one embodiment, controlled release of the activator is characterized by the amount of activator released on day 1 being 0-50% of the total activator, the daily release of activator from day 2 to the final release day being 0-50% of the total activator, and / or the number of days required for 100% release of the total activator being at least 2 days. In another embodiment, the amount of activator released on day 1 is 0-25%, 0-20%, 0-10%, 0-5%, or about 0% of the total activator, the daily release of activator from day 2 to the final release day being 0-50% of the total activator, and / or the number of days required for 100% release of the total activator being 3 to 30 days, 25 days or less, or 16 days or less.

[0285] Sustained-release drug delivery system and administration The hyperbranched polymers of certain embodiments of the present invention can be used for drug delivery to patients, for example, for ophthalmic drug delivery, because they have many advantages as carrier systems. The hyperbranched polymers can be used for drug delivery, gene delivery, antioxidant delivery, peptide delivery, biomedical imaging, and genetic testing in ophthalmology.

[0286] Hyperbranched polymers can be transported into and out of cells. Drug delivery using hyperbranched polymers can utilize various ophthalmic application routes, and their tunable properties, such as water solubility, permeability, bioavailability, and biocompatibility, can be broadly modified to meet the specific needs of various medical applications.

[0287] In certain embodiments, a sustained-release biodegradable drug delivery system comprising the hyperbranched polymer described herein is provided. In certain embodiments of the present invention, the hyperbranched polymer or a drug delivery system comprising the same may be formulated for direct or indirect administration via various routes, such as oral, parenteral, surgical insertion, or injection.

[0288] To formulate a drug delivery system, the hyperbranched polymer may be incorporated into a suitable carrier such as a solvent or solvent mixture, or into a hydrogel or organogel.

[0289] In certain embodiments, the hyperbranched polymer is formulated for direct injection into the patient's treatment site, for example, by parenteral administration or by intraocular injection, such as intratumoral injection, intravitreous, anterior chamber, subconjunctival, retrobulbar, sub-Tenon's capsule, subretinal, or suprachoroidal injection. It can be formulated for injection into the anterior chamber, vitreous humor, episclera, posterior sub-Tenon's space (inferior fornix), subconjunctival, intraocular, periocular, posterior, sub-Tenon's space, retina, subretinal, intracanaliculi, intravitreous, intrascleral, intrachoroidal, suprachoroidal, retina, subretinal, or the surface of the lens, cornea or conjunctiva, lacrimal puncta (canaliculi, superior / inferior canaliculi), palpebral fornix, superior / inferior palpebral fornix, sub-Tenon's space, choroid, suprachoroidal, Tenon, cornea, cancerous tissue, organs, prostate, breast, joint cavity, subdural, teeth, subcutaneous, carpal tunnel, perivascular, surgically formed spaces or injuries, cavities, and latent cavities.

[0290] In embodiments of the present invention, the drug delivery system is used to manufacture or form a medical implant, in which the superbranched polymer is embedded or dispersed within a hydrogel or organogel matrix.

[0291] Treatment method According to certain embodiments of the present invention, a hyperbranched polymer or a biodegradable drug delivery system comprising a hyperbranched polymer is configured to be used as a drug for the treatment of a patient's disease or medical condition.

[0292] In one embodiment, a method for treating a patient's disease or condition involves administering a hyperbranched polymer to the patient to release an activator over a long period of time.

[0293] A therapeutic method according to one embodiment of the present invention includes ophthalmic treatment. In such treatment, a superbranched polymer is used to release an active agent in the eye over a long period of time. In this embodiment, the disease or condition to be treated is an eye disease, any posterior segment eye disease that affects the vascular system and integrity of the retina, macula, and choroid and leads to impaired vision, vision loss, or blindness, particularly posterior segment eye conditions resulting from age, trauma, or surgical intervention, such as age-related macular degeneration (AMD), cystoid macular edema (CME), diabetic macular edema (DME), posterior uveitis, and diabetic retinopathy.

[0294] This treatment approach also includes the treatment of glaucoma, elevated intraocular pressure, anterior chamber hemorrhage, presbyopia, cataracts, retinal vein occlusion, inflammation, miosis, mydriasis, conjunctivitis, intraocular infections, choroidal neovascularization (CNV), intraocular tumors, and retinal neuritis.

[0295] Eye diseases include retinal neovascularization, choroidal neovascularization, exudative AMD, atrophic AMD, retinal vein occlusion, diabetic macular edema, retinal degeneration, corneal transplant rejection, retinoblastoma, melanoma, glaucoma, autoimmune uveitis, uveitis, proliferative vitreoretinopathy, and corneal degeneration, acute and chronic macular neuroretinopathy, central serous chorioretinopathy, macular edema, acute multifocal placoid pigment epitheliopathy, Behçet's disease, and bird's disease. Schott's chorioretinopathy, posterior uveitis, posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusion, central retinal vein occlusion, disseminated intravascular coagulation, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal artery microaneurysms, Cort's disease, parafoveal telangiectasia, unilateral retinal vein occlusion, papillary phlebitis, carotid artery disease (CAD), tree Iceberg vasculitis, sickle cell retinopathy, vascular streaks, familial exudative vitreoretinopathy, Eels' disease, proliferative vitreoretinopathy, diabetic retinopathy, retinal diseases with tumors, congenital retinal pigment epithelial hypertrophy (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, complex hamartoma of the retina and retinal pigment epithelium, retinoblastoma, fundus vascular proliferative neoplasm, retinal astrocytoma, intraocular lymphoma, myopic retinal degeneration, acute It may be one of the following: retinitis pigmentosa, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal cancer, retinitis pigmentosa, Leber congenital amaurosis, choroidal agenesis, X-linked retinitis pigmentosa, Best vitiligo macular degeneration, X-linked retinoschisis, CNGA3 color blindness, CNGB3 color blindness, LHON, Stargardt disease, Usher syndrome, Norrie disease, Baldett-Beedl syndrome, and red-green color blindness.

[0296] The methods described in this section may include administering ultrabranched molecular weight polymers in combination with other medications, also known as combination therapy.

[0297] In one embodiment, the combination therapy involves administering the superbranched polymer in combination with one or more additional agents on the same day or on different days. In one embodiment, the additional agents to be administered in the combination therapy may be in liquid form or contained in an oral dosage form. Thus, the additional agents may be any small molecule, large molecule, protein, nanoparticle, or any other activator described herein. In another embodiment, a superbranched polymer conjugated with multiple activators, such as those obtained by the convergent synthesis described above, may be used in combination therapy with the administration of multiple activators. Using the superbranched polymer of a particular embodiment, different agents can be conjugated to different regions on the surface of the superbranched polymer.

[0298] Therapeutic methods involving the administration of a superbranched polymer may include injection into the vitreous, anterior chamber, subconjunctival, retrobulbar, subtenon's capsule, subretinal, or choroidal regions. The method of administration may be local or oral.

[0299] The active or additional agents to be administered in combination therapy may be diagnostic agents. Diagnostic agents may be substances used to examine the body to detect impairments in normal bodily functions, as described above. In some cases, diagnostic agents may be agents with a functional purpose, such as being used to detect eye deformities, diseases, and pathophysiological aspects.

[0300] Exemplary embodiments of medical procedures using the drug conjugate dendrimer of the present invention are summarized in Table A below.

[0301] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] Industrial and non-medical applications In embodiments of the present invention, the hyperbranched polymer / dendrimer can also be used for non-medical or industrial applications. In certain embodiments, the dendrimer does not contain hydrolyzable bonds. In other embodiments, the dendrimer may contain hydrolyzable bonds as described herein.

[0302] Table B below outlines and illustrates the non-medical and industrial applications of the dendrimers of the embodiments of the present invention.

[0303] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0304] Further exemplary uses and applications are shown in Table C.

[0305] [Table 3] [Examples]

[0306] The following examples are included to illustrate specific aspects and embodiments of the invention as described in the claims. However, those skilled in the art should understand that the following description is illustrative and should not be construed as limiting the invention in any way.

[0307] Materials and abbreviations used in the examples: Degradable bifunctional 4-arm PEG-(NHS)3-(azide)1 (10kK, 20k, and 40kDa), and 4-arm PEG-DBCO (10k, 20k, and 40kDa) were purchased from XIAMEN SINOPEG BIOTECH Co. Ltd. All NHS-terminated PEGs were purchased from JenKem Technology USA.

[0308] Compstatin (ICVVQDWGHHRCT, disulfide cross-linked: Cys2-Cys12, TFA and acetate forms) was purchased from MedChemExpress. APL-1 (ICV{L-1-Me-Trp}QDWGAHRCT, disulfide cross-linked: Cys2-Cys12, TFA and acetate forms) and Fc-III 4C (CDCAWHLGELVWCTC, disulfide cross-linked: Cys1-Cys15, Cys3-Cys13, TFA and acetate forms) were purchased from Alan Scientific. The structures are shown in Figure 4.

[0309] The solvents and other reagents, including methanol, acetonitrile, PBS buffer, and triethylamine, were purchased from VWR.

[0310] Example 1 Branched synthesis of branched polymer G0 peptide conjugates

[0311] Scheme 1 [ka]

[0312] The peptide was weighed and dissolved in anhydrous methanol. 4-arm PEG-SS-NHS (molecular weight = 40 kDa) in a molar ratio of 1:6 to the peptide was slowly added to the peptide (compstatin) solution with vigorous stirring. A small amount of triethylamine (5-10 μL) was added dropwise to the reaction mixture. The reaction was carried out at room temperature for 1-4 hours. The final product was recovered and purified by dialyzing in methanol for 24 hours using a 10 kDa molecular weight cutoff tube. The purified ultrabranched polymer G0-peptide conjugate was recovered from the dialyzing tube, and the solvent was removed using a rotary evaporator. The dried powder was stored at -20°C for characterization. The composition is shown in Table 1.

[0313] [Table 4]

[0314] Example 2 Branched synthesis of ultrabranched polymer G1-peptide conjugates

[0315] Scheme 2 [ka]

[0316] Terminal functional group transformation of 4-arm PEG-DBCO: A certain amount of 4-arm PEG-NHS (MW=40kDa) was dissolved in anhydrous methanol. DBCOamine was weighed in a molar ratio of 1:1 to 4-arm PEG-NHS and dissolved in anhydrous acetonitrile. This DBCOamine solution was added dropwise to the 4-arm PEG-NHS solution with vigorous stirring. The reaction was carried out at room temperature for 1-4 hours, and the solvent was removed using a rotary evaporator. The dried crude product was used in the next step of synthesis. The composition is shown in Step 1 of Table 2.

[0317] Formation of hyperbranched polymers of 4-arm PEG-[4-arm PEG-(NHS)3]4(G1): A certain amount of 4-arm PEG-DBCO was dissolved in a 1:1 mixture of anhydrous methanol and acetonitrile. 4-arm PEG-(N3)1(NHS)3 (MW=20kDa) was weighed in a 1:1 molar ratio to 4-arm PEG-DBCO and dissolved in anhydrous methanol. This 4-arm PEG-(N3)1(NHS)3 solution was slowly added to the 4-arm PEG-DBCO solution with vigorous stirring. The reaction was carried out at room temperature for 1-4 hours, and the solvent was removed using a rotary evaporator. The dried crude product can be further purified by dialysis if necessary. The composition is shown in Step 2 of Table 2.

[0318] Peptide conjugation of hyperbranched polymer G1-peptide conjugates A certain amount of peptide was weighed and dissolved in anhydrous methanol. 4-arm PEG-[4-arm PEG-(NHS)3]4(G1) was slowly added to the peptide solution in a molar ratio of 1:18 with vigorous stirring. A small amount of triethylamine (5-10 μL) was added dropwise to the reaction mixture. The reaction was carried out at room temperature for 1-4 hours. The final product was recovered and purified by dialyzing in methanol for 24 hours using a 40 kDa molecular weight cutoff tube. The purified ultrabranched polymer G1-peptide conjugate was recovered from the dialyzing tube, and the solvent was removed using a rotary evaporator. The dried powder was stored at -20°C for characterization. The composition is shown in step 3 of Table 2.

[0319] [Table 5-1] [Table 5-2]

[0320] Example 3 Branched synthesis of ultrabranched polymer G2-peptide conjugates

[0321] Scheme 3 [ka]

[0322] Terminal functional group transformation of 4-arm PEG-[4-arm PEG(DBCO)3]4 A certain amount of 4-arm PEG-[4-arm PEG-(NHS)3]4(G1) obtained in Step 2 of Example 2 was dissolved in anhydrous methanol. DBCO-amine in a molar ratio of 1:1 to 4-arm PEG-[4-arm PEG-(NHS)3]4(G1) was weighed and dissolved in anhydrous acetonitrile. This DBCO-amine solution was added dropwise to the 4-arm PEG-[4-arm PEG-(NHS)3]4(G1) solution with vigorous stirring. The reaction was carried out at room temperature for 1 to 4 hours, and the solvent was removed using a rotary evaporator. The dried crude product was used in the synthesis of the next step. The composition is shown in Step 1 of Table 3.

[0323] Formation of a hyperbranched polymer of 4-arm PEG-{4-arm PEG-[4-arm PEG(NHS)3]3}4 A certain amount of 4-arm PEG-[4-arm PEG(DBCO)3]4 was dissolved in a 1:1 mixture of anhydrous methanol and acetonitrile. 4-arm PEG-(N3)1(NHS)3 (MW=10kDa) and 4-arm PEG-[4-arm PEG(DBCO)3]4 were weighed to a molar ratio of 1:1 and dissolved in anhydrous methanol. The 4-arm PEG-(N3)1(NHS)3 solution was slowly added to the 4-arm PEG-[4-arm PEG(DBCO)3]4 solution with vigorous stirring. The reaction was carried out at room temperature for 1-4 hours, and the solvent was removed using a rotary evaporator. The dried crude product can be further purified by dialysis if necessary. The composition is shown in Step 2 of Table 3.

[0324] Peptide conjugation of hyperbranched polymer G2-peptide conjugates A certain amount of peptide was weighed and dissolved in anhydrous methanol. 4-arm PEG-{4-arm PEG-[4-arm PEG(NHS)3]3}4(G2) was slowly added to the peptide solution in a molar ratio of 1:18 with vigorous stirring. A small amount of triethylamine (5-10 μL) was added dropwise to the reaction mixture. The reaction was carried out at room temperature for 1-4 hours. The final product was recovered and purified by dialyzing in methanol for 24 hours using a 40 kDa molecular weight cutoff tube. The purified ultrabranched polymer G2-peptide conjugate was recovered from the dialyzing tube, and the solvent was removed using a rotary evaporator. The dried powder was stored at -20°C for characterization. The composition is shown in step 3 of Table 3.

[0325] [Table 6-1] [Table 6-2]

[0326] Example 4 Convergent synthesis of hyperbranched polymer G1-peptide conjugates

[0327] Scheme 4 [ka]

[0328] Peptide conjugation of 4-arm PEG-(N3)1(peptide)3 A certain amount of peptide was weighed and dissolved in anhydrous methanol. 4-arm PEG-(N3)1(NHS)3 (MW=20kDa) in a molar ratio of 1:4.5 to the peptide was slowly added to the peptide solution with vigorous stirring. A small amount of triethylamine (5-10 μL) was added dropwise to the reaction mixture. The reaction was carried out at room temperature for 1-4 hours. The solvent was removed using a rotary evaporator. The dried crude product was used in the next step of synthesis. The composition is shown in Table 4, Step 1.

[0329] G1-peptide conjugate hyperbranched polymer formation A certain amount of 4-arm PEG-(N3)1(peptide)3 was dissolved in anhydrous methanol. 4-arm PEG-DBCO (MW=40kDa) in a molar ratio of 1:1 to PEG-(N3)1(peptide)3 was weighed and dissolved in anhydrous methanol. This 4-arm PEG-(N3)1(peptide)3 solution was slowly added to the 4-arm PEG-DBCO solution with vigorous stirring. The reaction was carried out at room temperature for 1–4 hours. The conjugates were collected in methanol for 24 hours in a dialysis tube with a molecular weight cutoff of 40kDa and purified. The solvent was removed using a rotary evaporator, and the dried powder was stored at -20°C for characterization. The composition is shown in Step 2 of Table 4.

[0330] [Table 7]

[0331] Example 5 Peptide conversion and G0 conjugation of hyperbranched polymers

[0332] Scheme 5 [ka] Route 1 Peptide-DBCO conversion: Step 1. A certain amount of peptide (compstatin, MW=1.5kDa) was dissolved in 1 mL of mixed solvent (anhydrous methanol:acetonitrile = 1:1). DBCO-NHS was weighed and dissolved in 1 mL of mixed solvent (anhydrous methanol:acetonitrile = 1:1). The peptide solution was added dropwise to the DBCO-NHS solution with vigorous stirring. The reaction was carried out at room temperature for 1-4 hours. The dried crude product was used in the synthesis of the next step. The composition is shown in Table 5 for Step 1.

[0333] Dendrimer formation of 8-arm PEG-(peptide)8: Step 2. A certain amount of 8-arm 20k PEG-azide was dissolved in 1 mL of a mixed solvent (anhydrous methanol:acetonitrile = 1:1) and slowly added to the peptide-DBCO solution with vigorous stirring. The reaction was carried out at room temperature for 1 to 4 hours, and the solvent was removed using a rotary evaporator. The dried crude product can be further purified by dialysis if necessary. The composition is shown in Table 5 for Step 2.

[0334] [Table 8]

[0335] Route 2 Peptide-Azide Conversion: Step 1. A certain amount of peptide (compstatin, MW=1.5kDa) was dissolved in 1 mL of mixed solvent (anhydrous methanol:acetonitrile = 1:1). Azide-NHS was weighed and dissolved in 1 mL of mixed solvent (anhydrous methanol:acetonitrile = 1:1). The peptide solution was added dropwise to the azide-NHS solution with vigorous stirring. The reaction was carried out at room temperature for 1-4 hours. The dried crude product was used in the synthesis of the next step. The composition is shown in Table 6 for Step 1.

[0336] Dendrimer formation of 8-arm PEG-(peptide)8: Step 2. A certain amount of 8-arm 20k PEG-DBCO was dissolved in 1 mL of a mixed solvent (anhydrous methanol:acetonitrile = 1:1) and slowly added to the peptide-azide solution with vigorous stirring. The reaction was carried out at room temperature for 1 to 4 hours, and the solvent was removed using a rotary evaporator. The dried crude product can be further purified by dialysis if necessary. The composition is shown in Table 6 for Step 2.

[0337] [Table 9]

[0338] Example 6 Purification of hyperbranched polymers and peptide conjugates by dialysis method The products obtained in Examples 1-5 were dissolved in methanol to a concentration of 10 mg / mL or higher. This solution (1-5 mL) was packed into dialysis tubes with specific molecular weight cutoffs (Spectra / Por® Float-A-Lyzer G2 Dialysis Devices, Spectrum® Laboratories). Next, the tubes were placed in a beaker of 500 mL of methanol at room temperature for 24 hours, and the low molecular weight components diffused from the tubes were separated. See Figure 5a). After processing, the products were removed from the tubes, collected in glass vials, and characterized.

[0339] [Table 10]

[0340] The products were characterized by UHPLC. Figure 6 shows the UHPLC analysis of the 4-arm 40k PEG-[4-arm 20k PEG-SG-(compstatin)3]4 conjugate of Example 2 G1, purified by dialyzing in methanol through a molecular weight cutoff membrane of 8-10 kDa, primarily to remove low molecular weight peptides (MW approximately 1.5 kDa). The blue line represents the sample before purification, and the green line represents the sample after purification. Based on peak area integration, the peak with a retention time of approximately 4 minutes, attributed to free compstatin, was reduced from 45% to less than 1% in the sample. On the other hand, the starting material in the mixture, 4-arm 40k PEG-SG-(DBCO)4 (retention time approximately 9 minutes, MW approximately 40 kDa), had a molecular weight greater than 8-10 kDa and remained in the tube. In addition, the hyperbranched polymer conjugate (MW approximately 120 kDa) also remained in the tube, causing the peak to shift slightly to the left from approximately 8.5 minutes to 9 minutes. The separation of the hyperbranched polymer conjugate from the PEG-DBCO precursor can be achieved by using a different dialysis tube with a higher molecular weight cutoff.

[0341] [Table 11]

[0342] These data demonstrate that dialysis can very efficiently purify hyperbranched polymer-peptide conjugates to a purity of over 99%.

[0343] Example 7 Purification of ultrabranched polymers and peptide conjugates by SEC column filtration The SEC column (Zeba® Spin Desalting column) was opened and washed twice with 1 mL of methanol. The products obtained in Examples 1-5 were dissolved in methanol to a concentration of 10 mg / mL or higher (1-2 mL), and this solution was packed into the column so that the solution would flow through the column by gravity. See Figure 5b). The eluate was collected in a glass vial and characterized.

[0344] [Table 12]

[0345] Figure 7 shows the UHPLC analysis results of the 4-arm 40k PEG-SS-compstatin conjugate of G0 from Example 1 before (black line) and after (blue line) purification. Based on peak area integration, the free compstatin content decreased from 38.8% to 1.6%, demonstrating highly efficient purification capabilities.

[0346] [Table 13]

[0347] These data demonstrate that SEC column filtration can very efficiently purify ultrabranched polymer-peptide conjugates with a purity of over 98%.

[0348] Example 8 Purity and substitution rate by UHPLC Ultra-high-performance liquid chromatography (UHPLC) is an efficient technique that enables more sensitive analysis due to its superior chromatographic separation and resolution of analytes. It offers advantages such as high-speed analysis, high-resolution separation, reduced solvent and sample usage, and improved sensitivity and accuracy. A Waters XBridge BEH300 C18 column (3.5 μm, 2.1 × 100 mm, PN1860036080) was used with mobile phase A (0.1% trifluoroacetic acid in water) and mobile phase B (0.1% trifluoroacetic acid in acetonitrile) to characterize the hyperbranched polymers and hyperbranched polymer-peptide conjugates of Examples 1-5.

[0349] For UHPLC analysis, peptide powders were dissolved in PBS:methanol = 9:1 at concentrations of 12.5, 25, 50, 100, and 200 μg / mL and injected into the UHPLC system. The peak area of ​​each sample was integrated and used as a baseline for calculating peptide concentrations (see Figure 8a). The inset shows the standard curve of peptide concentration against peak integrated area.

[0350] The dried G0 PEG-hyperbranched polymer-peptide was dissolved in PBS:methanol = 9:1 at a concentration of 1 mg / mL and analyzed by UHPLC. Figure 8b shows a typical UHPLC graph of the 4-arm PEG-hyperbranched polymer-compstatin conjugate of G0 from Example 1. The peak at retention time 22 minutes is from free compstatin, and the peak centered at 42 minutes is from the 4-arm PEG-hyperbranched polymer-compstatin conjugate. Using the compstatin standard curve, the concentration of each component in the product was calculated, and by dividing the number of moles of the hyperbranched polymer-conjugate peptide by the number of moles of PEG, the amount of peptide substitution could be estimated according to the following formula.

number

[0351] This method provides a practical way to compare and optimize reaction methods. Compstatin peptide samples in different salt forms—trifluoroacetic acid (TFA), acetate, and lysine—were selected from different vendors, Ambeed, Genscript, and MCE, to react with the same 4-arm 40k PEG-SGA-NHS under the same reaction conditions. Table 11 shows the conjugation results. Overall, the substitution rate of each peptide on the G0 4-arm PEG was approximately 80%, with a variability of ±10% from one another.

[0352] [Table 14] The substitution of compstatin and compstatin lysine in different PEGs, such as 4-arm 40k PEG-SGA and 4-arm 40k PEG-SS, was investigated using the same analytical method. These reactions were carried out under the same conditions, with only the PEG changed and each compound in molar ratio. Each reaction was repeated three times, and as shown in Figure 9, these reactions showed good reproducibility, with an average of approximately 3 peptide substitutions in the 4-arm PEGs.

[0353] Other reaction conditions, such as reaction time, solvent, and catalyst use, were also investigated, and the results are shown in Figure 10. The results show that reacting in methanol with triethylamine as a catalyst is the optimal condition for achieving the highest peptide substitution rate, but the reaction time has little effect on the change in the number of substitutions.

[0354] Table 12 below lists several superbranched polymers from G0 to G2 synthesized by convergent or divergent methods. The highest molecular weight superbranched polymer is the G2 superbranched polymer, with approximately 240 kDa and a structure containing approximately 36 terminal functional groups. In most conjugation results, the peptide substitution rate exceeded 50%, indicating that these methods exhibit good reproducibility.

[0355] [Table 15-1] [Table 15-2]

[0356] The same synthesis method was applied to the conjugation of hyperbranched polymers using APL-1 and Fc-III 4C, and the substitution results are shown in Tables 13 and 14. The substitution rates of APL-1 and Fc-III 4C are lower than those of compstatin.

[0357] [Table 16]

[0358] [Table 17]

[0359] Example 9 Binding assay To study molecular interactions, surface plasmon resonance (SPR) coupling analysis methods obtained from Mosaic Biosciences, Inc. were used.

[0360] A Biacore 3000 instrument was used to detect the SPR signal. C3 and C3b were typically immobilized at high density (approximately 20 kRU) on the sensor chip surface. Buffered saline at pH 7.4 was flowed through the device at a flow rate of 30 μL / min at 25°C. The hyperbranched polymer-peptide conjugates of the embodiments of the present invention were injected at concentrations ranging from 1 nM to 300 nM (APL-1 derivative) or 200 nM to 50 μM (compstatin derivative). Association was monitored for 4 minutes and dissociation for 10 minutes. Equilibrium analysis was performed for the compstatin analog, and kinetic analysis was performed for the mass transport of the APL-1 analog.

[0361] Binding affinity of free peptides Figures 11 and 12 show the C3 and C3b binding of different types of compstatin, and Figure 13 shows the C3 and C3b binding of different types of APL-1. The KD results are summarized in Table 15. These results clearly show that the free peptides compstatin and APL-1 exhibit KD values ​​very similar to those reported. This result is consistent with previous measurements using APL-1, but is lower than the affinity (200 pM) reported by Apellis for APL-2, which is thought to be due to the avidity effect on divalent APL-2.

[0362] [Table 18]

[0363] Similar experiments were conducted for Fc-III 4C. For comparison, another peptide, Fc-III, was also evaluated under the same conditions. Fc-III has a similar peptide sequence to Fc-III 4C, but lacks one Cys-Cys crosslink. Its amino acid sequence structure is as follows:

[0364] Scheme 6 [ka]

[0365] This structural difference leads to significant differences in antibody binding affinity, with Fc-III 4C showing approximately eight times greater sensitivity to the antibody (KD of 2.45 nM vs. 16 nM). SPR results (Figure 13 and Table 16) also showed that the three types of Fc-III 4C exhibited much lower binding affinity KD than Fc-III.

[0366] [Table 19]

[0367] Binding affinity of superbranched polymer conjugate peptides The same experiment was applied to test the binding affinity of hyperbranched polymer conjugate peptides, with C3 immobilized on a chip and hyperbranched polymer peptide conjugates flowing at various concentrations.

[0368] Figure 15 shows a comparison between free compstatin and polyvalent compstatin (4-arm 40k PEG-SGA-(compstatin)4). When both samples flowed over a C3-coated tip, both showed very fast binding velocities. After interaction, buffer was flowed through the tip to wash away the bound samples on the tip. During this dissociation time, the response from free compstatin decreased rapidly, at a rate equal to the association rate. On the other hand, the hyperbranched polymer-compstatin conjugate showed a very slow dissociation rate. This is due to multiple interactions between the peptide on the hyperbranched polymer and the receptor.

[0369] Figures 16a-c) show SPR results based on a comparison of three different hyperbranched polymer-compstatin conjugates and free compstatin. The hyperbranched polymer-conjugate compstatin appears to contain both fast-dissociating and slow-dissociating components, and the slower dissociation phase associated with these structures indicates that the polyvalent hyperbranched polymer conjugates are cooperatively bound to the C3 surface.

[0370] The sample in Figure 16d) is compstatin after hydrolysis of 4a 40k PEG-SS-(compstatin)4. This hydrolyzed peptide contains a succinic acid ester bond due to the decomposition of the conjugation linker group, and according to similar SPR signals, it shows no effect on the C3 bond. This strongly suggests that the ester bond did not alter the biological activity of the peptide.

[0371] The same high-purity sample set was detected again, and quantitative KD analysis was performed. The KD results are shown in Table 17 and plotted against the number of peptide substitutions for each corresponding sample. It can be seen that the KD decreases as the number of peptides substituted with hyperbranched polymers increases.

[0372] [Table 20]

[0373] Example 10 Alternative Route (AP) Hemolysis Assay for IC50 Measurement IC50 (median inhibitory concentration) was measured by an alternative pathway (AP) hemolysis assay. Sample numbers REA638(4a 40k-PEG-SGA-(compstatin)) contained 2.6 and 2.5 compstatin molecules per ultrabranched polymer, respectively. n TFA salt) and REA639 (4a-40k-PEG-SGA-(compstatin) n Sample No. REA640 (4a 40k-PEG-[4a-10kPEG-(compstatin)) contains 10.8 and 7.1 compstatin molecules per ultrabranched polymer, respectively. n ]4) and REA641 (4a-40k-PEG-[4a-20k-PEG-(compstatin) n ]4) Four compstatin conjugate hyperbranched polymers are used (see Tables 10 and 15).

[0374] In a 96-well plate assay (see Figure 18), the inhibitor (50 μL) was diluted with GVBo (GVBo: 0.1% gelatin, 5 mM barbiturates, 145 mM NaCl, 0.025% NaN3, pH 7.3) and incubated with normal human serum:GVBo in a 1:2 ratio at various concentrations at room temperature for 30 minutes. Rabbit RBCs (CompTech) were centrifuged at 500 × g for 3 minutes and resuspended in MgEGTA (MgEGTA: 0.1 M MgCl2, 0.1 M EGTA, pH 7.3) at a concentration of 5.0 × 10⁸ cells / mL. Rabbit RBCs (20 μL) were added and incubated at 37°C for 60 minutes. The reaction was stopped by adding 200 μL of GVBE (GVBE: 0.1% gelatin, 5 mM barbiturate, 145 mM NaCl, 10 mM EDTA, 0.025% NaN3, pH 7.3). The cells were centrifuged at 500 × g for 5 minutes, and the supernatant (150 μL) was transferred to a new 96-well plate. The hemolysis rate (%) was calculated as (A412 inhibitor / no A412 inhibitor) × 100, and fitted using 4PL curve fitting to obtain IC. 50 The result is determined and shown in Table 18.

[0375] [Table 21]

[0376] The results (Figure 19 and Table 18) show that the G0 4-arm PEG compstatins (REA638 and REA639, containing 2.6 and 2.5 compstatins per ultrabranched polymer molecule, respectively) have lower IC50 per molecule compared to free compstatin. This is not surprising, as these molecules contain approximately 2 compstatins per molecule, making it unlikely that binding activity can be achieved at such a low degree of substitution. On the other hand, the G1 PEG ultrabranched polymer compstatins (REA640 and REA641, containing 10.8 and 7.1 compstatins per ultrabranched polymer molecule, respectively) have improved IC50 compared to free compstatin, suggesting enhanced efficacy due to binding activity. Furthermore, the slopes of these curves are lower compared to free compstatin, REA638, and REA639, suggesting that multiple binding events contribute to inhibition. Interestingly, REA641, containing 7.1 compstatin molecules, performed better than REA640, containing 10.8 compstatin molecules, despite the latter having a higher valency. The 20k PEG dendrons in REA641 are thought to offer greater flexibility in interacting with C3 than the shorter 10k PEG dendrons in REA640.

[0377] Classical Pathway (CP) Hemolysis Assay IC50 (median inhibitory concentration) can also be measured using the same ultrabranched polymer conjugate as in the AP hemolysis assay described above, but in the classical pathway (CP) hemolysis assay. This assay is similar in principle to the AP hemolysis assay, but since the classical pathway is initiated by antibody binding to cells, sheep-sensitized red blood cells are used.

[0378] Serial dilutions of the inhibitor (50 μL) were placed in a 96-well plate, and Mg 2+ and Ca 2+Gelatin Veronal buffer containing GVB++ (GVB++: 0.1% gelatin, 5 mM barbiturate, 145 mM NaCl, 0.025% NaN3, pH 7.3, 0.15 mM calcium chloride, and 0.5 mM magnesium chloride) was prepared. C3-depleted human serum supplemented with 12 nM human C3 was diluted 1:2 with GVB++ (30 μL) and added to each well, and incubated for 30 minutes. Sheep erythrocytes sensitized with anti-sheep pAb were added in 5.0 × 10⁶ cells in GVB++ (20 μL). 8 Cells were added at a concentration of 1 / mL and incubated at 37°C for 30 minutes. The reaction was stopped by adding EDTA-added gelatin veronal buffer (GVBE, 200 μL). The cells were centrifuged at 500 × g for 5 minutes, and the supernatant (150 μL) was transferred to a new 96-well plate. Absorbance at 412 nm was measured using a Molecular Devices SpectraMax M5 plate reader, and hemolysis (%) was calculated as hemolysis (%) = (A412 阻害剤 / A412 阻害剤なし The calculation was performed using ) × 100. IC50 was determined using GraphPad Prism's 4PL curve fitting. To measure background hemolysis, this assay was also performed in the absence of C3. The results are shown in Table 19. [Table 22]

[0379] The IC50 of free compstatin was 142 μM. Increasing the valence of the hyperbranched polymers REA638 and REA639 to 2.6 and 2.5 improved the IC50 to 56.0 μM and 73.3 μM, respectively. Furthermore, increasing the valence of the hyperbranched polymers to 10.8 or 7.1 compstatin (REA640 and REA641) improved the IC50 to 30.8 μM and 34.6 μM, respectively. Taken together, these data suggest that the high-valence compstatin of the present invention is more effective in inhibiting CP hemolysis compared to free compstatin alone.

[0380] Similarly to the above, APL-1 was used instead of compstatin, and a sample of the 12-arm G1 hyperbranched polymer 4a40k-PEG(SGA)-[4a20k PEG(SG)-(APL-1)3]4, prepared by convergent synthesis similar to Example 4, was used to measure the IC50 (median inhibitory concentration) by classical pathway (CP) hemolysis assay. This product had a substitution rate of approximately 35% of the 12 terminal groups, which was also used in Examples 11 and 12 below. 32 mg x 2 lyophilized dendrimers (equivalent to 20 mg x 2 dendrimers) were reconstituted in aqueous solutions of 90 mM sodium phosphate and 360 mM NaCl, and a 50 μL aliquot (1.25 mg / eye) was injected into the eyes of animals (New Zealand white rabbits) for one week. Vitreous fluid was collected from two animals on days 1, 3, 5, and 7 post-administration, and the IC50 was measured over time using classical pathway (CP) hemolysis assay. The IC50 values ​​of the dendrimers were measured as a control. The results are summarized in Table 20 below. [Table 23]

[0381] The results indicate that APL-1 bound to dendrimers maintains its stability and activity in vivo for more than 7 days.

[0382] Example 11 Correlation between hydrodynamic radius and in vivo half-life Hydrodynamic radius R of linear pegylated proteins (IgG) of different sizes (IgG 2x40k PEG and 2x20k PEG) h And, the half-life T in New Zealand white rabbit vitreous fluid (NZWVH) 1 / 2 We measured and compared these values ​​with free protein, and used several non-conjugated active ingredients (APIs) to create a calibration curve that could estimate the T1 / 2 of APL-1 conjugated to a 12-arm 120kDa PEG dendrimer based on Rh determined by SEC. The results are shown in Table 21 below. [Table 24]

[0383] As can be seen from Table 20 and Figure 20, the hydrodynamic radius R measured by SEC h This allows for reliable estimation of the half-life of the dendrimer-drug conjugate in embodiments of the present invention by adjusting the sustained-release characteristics, which depend on the size of the dendrimer.

[0384] Example 12 Effect of dendrimer degradation on in vitro release rate First-generation APL-1 conjugate dendrimers with the nominal structure 4a-40kPEG-[4a-40kPEG-APL-1]3 were prepared by convergent synthesis in the same manner as in Example 4, with approximately 35% of the 12 terminal groups substituted using APL-1 instead of compstatin. In vitro degradation tests were performed under various temperature and pH conditions to investigate the degradation / hydrolysis behavior. The tests were conducted in PBS, and the disappearance of the dendrimers and the appearance of dendron groups (hydrolysis products) over time were observed using HPLC chromatograms.

[0385] Figures 21a) to 21c) show the effects of varying the temperature from 35°C to 39°C while keeping the pH constant at 7.4. Figure 21a) shows the decrease in dendrimer concentration over time, Figure 21b) shows the increase in dendron concentration over time, and Figure 21c) shows the effect of temperature on the dendrimer loss rate % at pH 7.4 on a logarithmic scale. Based on first-order release kinetics, the release rate constant K and half-life T of the active substance estimated from these decomposition rates are shown. 1 / 2 The decision is made. The results are shown in Table 22 below.

[0386] [Table 25]

[0387] The degradation rate, and consequently the release rate, of APL-1 from this first-generation 120kPEG dendrimer increased with rising temperature, showing a nearly identical trend at all three temperatures during the first 26 days. Overall, the effect of temperature on the release half-life was low.

[0388] Figures 22a) to 22c) show the effect of pH value at a constant temperature of 37°C. Figure 22a) shows the decrease in dendrimer concentration over time, Figure 22b) shows the increase in dendron concentration over time, and Figure 22c) shows the effect of pH on the dendrimer loss rate % at pH 7.4 on a logarithmic scale. Based on primary release kinetics, the rate constant K and half-life T1 / 2 of active substance release estimated from these decomposition rates are determined. The results are shown in Table 23 below.

[0389] [Table 26]

[0390] The degradation and subsequent release rate of APL-1 from this first-generation 120kPEG dendrimer is found to increase significantly with increasing pH over time. Overall, the effect of pH on the release half-life is large and follows an exponential model. The higher the pH, the shorter the release half-life.

[0391] These release experiments demonstrate that the release rate of active ingredients conjugated to dendrimers can be altered or slowed using hydrolysis of ester bonds between dendritic building blocks or dendrons.

[0392] Specific set of embodiments First set of embodiments Appearance 1 It is a superbranched polymer, A core unit having at least three connecting parts c, A plurality of polymer arms connected to the core unit at the aforementioned connecting portion c, Each polymer arm contains terminal groups or is linked to a repeating dendritic structure unit. Each dendritic repeating unit includes a branched unit connected to at least two polymer arms containing terminal groups, or is connected to the next dendritic repeating unit, and the next dendritic repeating unit can be connected again to further dendritic repeating units. The polymer arms of the outermost dendritic repeating unit of the superbranched polymer each include the plurality of polymer arms, each containing an end group. The polymer arm contains polyethylene glycol (PEG) units, At least one activator is conjugated to at least one of the outermost polymer arms, The superbranched polymer comprises chemical bonds that can be cleaved by hydrolysis.

[0393] Appearance 2 The superbranched polymer according to embodiment 1, wherein the branched polymer is a generation G0 branched polymer, and the terminal groups of the branched polymer are terminal groups of the polymer arms connected to the core unit.

[0394] Appearance 3 The hyperbranched polymer according to Embodiment 1, wherein x is an integer from 1 to 10, and defines the number of continuously linked dendritic repeating units in the hyperbranched polymer.

[0395] Pattern 4 The superbranched polymer according to any one of the prior embodiments, wherein the core unit and the branching unit are the same or different and independently have 3 to 10, or 4 to 8, or 4 to 6, or 4 linking portions c or c'.

[0396] Appearance 5 The hyperbranched polymer according to any one of the prior embodiments, wherein the core unit and the branching unit are the same or different and each is derived from a polyol having at least three hydroxyl groups.

[0397] Appearance 6 The superbranched polymer according to embodiment 5, wherein the polyol is selected from the group consisting of glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomalt, lactitol, maltitol, mannitol, or sorbitol.

[0398] Appearance 7 The polymer arm comprises polyethylene glycol (PEG) units having an average molecular weight (Mw) in the range of about 1,000 to about 100,000 daltons, or about 10,000 to about 60,000 daltons, or about 15,000 to about 50,000 daltons, according to any one of the prior embodiments.

[0399] Appearance 8 The hyperbranched polymer according to any one of the prior embodiments, wherein the average molecular weight of the polymer arm PEG units bonded to the core is the same as or different from the polymer arms in the dendritic repeating units.

[0400] Appearance 9 The hyperbranched polymer according to any one of the prior embodiments, wherein the average molecular weight of the polymer arm PEG units bonded to the core is higher or lower than that of the polymer arms in the dendritic repeating units.

[0401] Appearance 10 The hyperbranched polymer according to any one of the preceding embodiments, wherein, in the case of a high-generation Gx hyperbranched polymer where x is an integer from 2 to 10, the average molecular weight of the polymer arm PEG units decreases or increases from the innermost polymer arm to the outermost polymer arm.

[0402] Appearance 11 The hyperbranched polymer according to any one of the prior embodiments, wherein the terminal groups bonded to the outermost polymer arm are grafted directly to the end of the polymer arm, or via a suitable bifunctional linker containing hydrolyzable bonds including a carboxyl group, dicarboxyl group, carboxamide group, dicarboxamide group, functionalized aliphatic, heteroaliphatic, or aromatic or heteroaromatic group.

[0403] Appearance 12 The functional groups of the terminal groups and / or linker terminal groups bonded to the outermost polymer arm are active ester groups such as succinimidyl esters and succinimidyl carbonates, electrophiles such as nitrophenyl carbonates, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, alkynes, azides, norbornene, epoxides, mesylates, tosylates, tresyl, cyanurates, orthopyridyl disulfide, or halogens, amines such as primary amines, hydroxyls, alcohols, thiols, azides, and A hyperbranched polymer according to any one of the preceding embodiments, wherein the functional group is selected from a nucleophile such as a carboxyl group, a functional group for click chemistry, a functional group for cyclization such as 1,3-dipolar cycloaddition, [3+2] cycloaddition such as alkene-nitrone cycloaddition or alkyne-nitrone cycloaddition, a functional group for 4+2] cycloaddition, a functional group for thiol-ene reactions; a functional group for hetero-Diels-Alder cycloaddition, a functional group for nucleophilic ring-opening, a functional group for non-aldol-type carbonyl reactions, a functional group for addition reactions to carbon-carbon multiple bonds, a polymerizable vinyl group, or a combination thereof.

[0404] Appearance 13 The hyperbranched polymer according to any one of the prior embodiments, wherein the linker-terminated group bonded to the outermost polymer arm is a functional group selected from succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), succinimidyl azelate (SAZ), and succinimidyl glutaramide (SGA).

[0405] Appearance 14 The hyperbranched polymer according to any one of embodiments 1 to 10, wherein the terminal group bonded to the outermost polymer arm is an alkyne compound such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]-nonine (BCN); or norbornene or trans-cyclooctene (TCO); or a functional group selected from azide, 3,4-dihydroxyphenylacetic acid (DHPA), or tetrazine (Tz).

[0406] Appearance 15 The superbranched polymer according to any one of the prior embodiments, wherein the connections between the core unit and the polymer arms linked to the first dendritic repeating unit, and / or the connections between consecutive dendritic repeating units, are formed by click chemistry.

[0407] Appearance 16 The superbranched polymer according to embodiment 15, wherein the coupling is formed by reacting a polymer arm functionalized with an alkyne, cycloalkyne, or a strained or terminal alkene moiety with a polymer arm functionalized with an azide or tetrazine moiety in a SPAAC or IEDDA type click chemistry coupling reaction.

[0408] Appearance 17 The hyperbranched polymer according to embodiment 16, wherein the alkene portion is a dibenzocyclooctin portion.

[0409] Appearance 18 The superbranched polymer according to any one of embodiments 15 to 17, wherein the connection between the core unit and the polymer arm connected to the first dendritic repeating unit, and / or the connection between consecutive dendritic repeating units, is formed between the polymer arm connected to the core unit and the polymer arm connected to the branching unit of the dendritic repeating unit, and / or between the polymer arm of the dendritic repeating unit and the polymer arm of the consecutive dendritic repeating unit.

[0410] Appearance 19 The hyperbranched polymer according to any of the prior embodiments, wherein the activator conjugated to at least one of the outermost polymer arms is selected from the group consisting of therapeutic activators or diagnostic activators.

[0411] Appearance 20 The activator conjugated to at least one of the outermost polymer arms includes steroids, nonsteroidal anti-inflammatory drugs (NSAIDs) (such as diclofenac, ibuprofen, meclofename, mefanamic A, sarsalate, sulindac, tolmetin, ketoprofen, diflunisal, piroxicam, naproxen, etodolac, flurbiprofen, fenoprofen C, indomethacin, celecoxib, ketorolac, nepafenac, etc.), intraocular pressure lowering agents, antibiotics such as ciprofloxacin, analgesics such as bupivacaine, calcium channel blockers such as nifedipine, cell cycle inhibitors such as simvastatin, proteins such as insulin, and small molecule hydrophilic molecules including carboxylates and amine salts. A hyperbranched polymer according to any of the preceding embodiments, selected from sexual drugs, small molecule hydrophobic drugs, hydrophilic peptides and protein drugs (insulin, single-chain antibody fragments, Fab fragments, IgG antibodies, fusion antibodies, etc.), aptamers, in particular bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDS), steroids, antibiotics, analgesics, calcium channel blockers, cell cycle inhibitors, chemotherapeutic agents, antiviral agents, anesthetics, hormones, anticancer drugs, antitumor drugs, viruses, gene delivery viruses such as AAV, protein binders (nanobodies, afibodies, ankyrin, DARPin, etc.), or any combination thereof.

[0412] Appearance 21 The hyperbranched polymer according to any of the prior embodiments, wherein the activator conjugated to at least one of the outermost polymer arms is a peptide selected from the group consisting of Compstatin, APL-1, Fc-III-4C, Beov (brolucizumab), Zimura (abacincaptado pegol), pegcetacoplan, abisipal pegol, lamparizumab, Fovista, listiganib, AXT107, elamipretide, THR149, ALM201, VGB3, and Largazole.

[0413] Appearance 22 The hyperbranched polymer according to any of the prior embodiments, wherein the activator is bonded to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% of the outermost polymer arm.

[0414] Appearance 23 The aforementioned dendritic repeating unit is represented by equation (i), [ka] In the formula, A is a connection to a polymer arm connected to the core unit, or A is a connection to B of a preceding dendritic repeating unit represented by formula (i), L A It is a linker, m is either 0 or 1. n is an integer between 20 and 2000, and o is an integer between 20 and 2000. n and o may be different or the same. X is a branching unit having a connecting portion c', L B is the linker, and p is either 0 or 1. B is a terminal group located on the surface of the superbranched polymer, or a continuous dendritic repeating unit or an activator linked to A. L A and L B They may be different or the same. m and p may be different or the same. y is an integer from 2 to 9, y = c’ - 1, where c’ is the connecting site c’ of the branching unit X, and the dendritic structural repeating units in the hyperbranched polymer may be the same or different, the hyperbranched polymer according to any of the preceding aspects.

[0415] Aspect 24 The connection between A and B includes a functional group formed by click chemistry such as triazole or dihydropyrazine, the hyperbranched polymer according to aspect 23.

[0416] Aspect 25 The linker L A and / or L <​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​A method for producing a hyperbranched polymer according to any one of embodiments 1 to 27 by branched synthesis, (a) Providing a core unit having at least three connecting portions c, and polymer arms connected to the core unit, each polymer arm having functional groups suitable for click chemistry at its ends; (b) A step of providing a dendritic repeating unit precursor, wherein the precursor is A polymer arm having a functional group suitable for forming a link between the polymer arm connected to the core and the corresponding functional group (e.g., azide, alkyne, alkene, or tetrazine) by click chemistry, A step of providing a dendritic repeating unit precursor comprising at least two polymer arms containing non-reactive functional groups in click chemistry, (c) The step of forming a link between the polymer arm connected to the core and the polymer arm of the dendritic structure repeating unit precursor by click chemistry, (d) optionally, converting the functional groups of at least two polymer arms containing non-reactive functional groups in click chemistry to functional groups suitable for click chemistry, (e) The method comprising the step of conjugating an activator containing functional groups to the outermost polymer arm by reacting it with the functional groups of the outermost polymer arm, thereby forming a hyperbranched polymer-activator conjugate.

[0420] Appearance 29 The method according to embodiment 28, wherein, in the case of a high-generation Gx hyperbranched polymer (where x is an integer from 2 to 10), step (d) is essential, and before conjugating the activator in step (e), further successive dendritic repeating unit precursors are linked by click chemistry to the functional groups suitable for click chemistry obtained in step (d) to form a hyperbranched polymer.

[0421] Appearance 30 The aforementioned dendritic repeating unit precursor is represented by formula (iii), [ka] In the formula, C comprises a functional group suitable for click chemistry (e.g., alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (e.g., succinimidyl), L A , m, n, X, o, L B The method according to embodiment 28 or 29, wherein p and y are as defined in embodiments 23 to 27, and the dendritic repeating units may be the same or different.

[0422] Appearance 31 The method according to embodiments 28-30, wherein, after the second-to-last step (d) which converts the functional groups of at least two polymer arms containing non-reactive functional groups in the click chemistry into click chemistry-compatible functional groups, the activator of step (e) is first functionalized with a click chemistry-compatible functional group (such as an alkyne, alkene, azide, or tetrazine) and then conjugated to the outermost polymer arm of the hyperbranched polymer in a click chemistry reaction.

[0423] Appearance 32 The method according to embodiment 31, wherein the activator functionalized with a functional group suitable for click chemistry is a peptide.

[0424] Appearance 33 A method for producing a hyperbranched polymer according to any one of embodiments 1 to 27 by convergent synthesis, I) A step of providing a dendritic repeating unit precursor, wherein the precursor is A polymer arm containing a functional group suitable for forming a linkage by click chemistry (e.g., azide, alkyne, alkene, or tetrazine), A step of providing a dendritic repeating unit precursor comprising at least two polymer arms containing non-reactive functional groups in click chemistry, II) The step of conjugating an activator containing a functional group to at least one of the at least two polymer arms of the dendritic repeating unit precursor that contain a functional group that does not react in click chemistry, III) Providing a core unit having at least three connecting portions c, and polymer arms connected to the core unit, each having a functional group suitable for click chemistry (e.g., azide, alkyne, alkene, or tetrazine) at the end of each polymer arm, IV) The method comprising forming a hyperbranched polymer-activator conjugate by click chemistry between the polymer arm linked to the core provided in step III) and the polymer arm containing a functional group suitable for forming click chemistry linkage of the activator conjugate dendritic repeating unit precursor obtained in step II).

[0425] Appearance 34 The aforementioned dendritic repeating unit precursor is represented by formula (iii), [ka] In the formula, C comprises a functional group suitable for click chemistry (e.g., alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (e.g., succinimidyl), L A , L B The method according to embodiment 33, wherein m, n, X, o, p, and y are as defined in embodiments 23 to 27, and the dendritic repeating units may be the same or different.

[0426] Appearance 35 In the case of a high-generation Gx hyperbranched polymer (where x is an integer from 2 to 10), The activator conjugate dendritic structure repeating unit precursor obtained in step II) A polymer arm containing a non-reactive functional group in click chemistry, A reverse dendritic repeating unit precursor comprising at least two polymer arms containing functional groups suitable for click chemistry (e.g., azides, alkynes, alkenes, or tetrazines) is linked by click chemistry, The method according to embodiment 33 or 34, wherein the functional group that does not react in the click chemistry of one polymer arm is subsequently converted to a click chemistry-suitable functional group before being linked to a further inverse dendritic repeating unit precursor, or before forming a click chemistry link with the polymer arm linked to the core in step IV), thereby forming a high-generation Gx hyperbranched polymer.

[0427] Appearance 36 The method according to embodiment 33 or 34, wherein dendritic repeating unit precursors having different activators conjugated to the polymer arm are obtained by performing steps I) and II) on each activator-conjugated dendritic repeating unit precursor, and a mixture of the obtained activator-conjugated dendritic repeating unit precursors is used in step IV), thereby forming a superbranched polymer-activator conjugate having different activators in different regions of the surface of the superbranched polymer.

[0428] Appearance 37 The method according to any one of embodiments 28 to 36, wherein the outermost polymer arm of the superbranched polymer has a terminal maleimide functional group, and a peptide or activator is conjugated to the terminal maleimide functional group via a maleimide-thiol reaction.

[0429] Appearance 38 The method according to embodiment 37, wherein the terminal maleimide functional group is provided by reacting a DBCO or azide-functionalized terminal functional group of the hyperbranched polymer with a click chemistry linker having an azide or DBCO functional group linked to a maleimide group (such as DBCO-maleimide, DBCO-PEG3-maleimide, DBCO-PEG4-maleimide, or azide-PEG3-maleimide).

[0430] Appearance 39 A superbranched polymer according to any one of embodiments 1 to 27, for use as a pharmaceutical agent.

[0431] Pattern 40 A treatment method, the method comprising treating a patient's disease or condition using a superbranched polymer described in any of embodiments 1 to 27.

[0432] Appearance 41 The superbranched polymer is used in the treatment of the eye. Branched polymer for use or treatment method according to embodiment 39 or 40.

[0433] Pattern 42 The superbranched polymer is used for the treatment of ocular diseases such as fundus diseases, including any posterior segment ocular disease that affects the vascular system and integrity of the retina, macula, or choroid and leads to visual impairment, visual loss, or blindness, particularly posterior segment conditions resulting from age, trauma, or surgical intervention, such as age-related macular degeneration (AMD), cystoid macular edema (CME), diabetic macular edema (DME), posterior uveitis, and diabetic retinopathy, as described in embodiments 39 to 41, or a method for treatment.

[0434] Appearance 43 The aforementioned superbranched polymer is used for retinal neovascularization, choroidal neovascularization, exudative AMD, atrophic AMD, retinal vein occlusion, diabetic macular edema, retinal degeneration, anterior chamber hemorrhage, presbyopia, corneal transplant rejection, retinoblastoma, melanoma, miosis, mydriasis, glaucoma, conjunctivitis, intraocular infection, choroidal neovascularization (CNV), intraocular tumors, retinal neuritis, inflammation, autoimmune uveitis, uveitis-related conditions, proliferative vitreoretinopathy, corneal degeneration, acute and chronic macular neuroretinopathy, central serous chorioretinopathy, and macula. Edema, acute multifocal placoid pigment epitheliopathy, Behçet's disease, birdshot chorioretinopathy, posterior uveitis, posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusion, central retinal vein occlusion, disseminated intravascular coagulation, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal artery microaneurysms, Cort's disease, parafoveal telangiectasia, unilateral retinal vein occlusion, papillary phlebitis, carotid artery disease ( CAD), dendritic vasculitis, sickle cell retinopathy, vascular streaks, familial exudative vitreoretinopathy, Eels' disease, proliferative vitreoretinopathy, diabetic retinopathy, tumor retina, congenital retinal pigment epithelial hypertrophy (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, complex hamartoma of the retina and retinal pigment epithelium, retinoblastoma, fundus vascular proliferative neoplasm, retinal astrocytoma, intraocular lymphoma, myopic retinal degeneration, acute retinitis pigmentosa, glaucoma, endophthalmitis, cytomegalovirus A superbranched polymer for use according to embodiments 39-42, or a method of treatment, used for the treatment of an eye disease selected from the group consisting of viral retinitis, retinal cancer, retinitis pigmentosa, Leber congenital amaurosis, choroidal agenesis, X-linked retinitis pigmentosa, Best vitiligo macular degeneration, X-linked retinoschisis, CNGA3 color blindness, CNGB3 color blindness, LHON, Stargardt disease, Usher syndrome, Norrie disease, Baldett-Beedl syndrome, and red-green color blindness.

[0435] 44 The superbranched polymer is formulated to be injected directly into the treatment site of a patient by injection into the eye, for example, by parenteral administration, intratumoral injection, intravitreous, anterior chamber, subconjunctival, retrobulbar, subtenon's capsule, subretinal, or choroidal injection, as described in any of embodiments 39 to 43, for use or a treatment method.

[0436] Appearance 45 The superbranched polymer is administered by direct injection, by oral administration, incorporated into a gel, or incorporated into an implant, according to any of embodiments 39 to 44, for use or a therapeutic method.

[0437] Appearance 46 The superbranched polymer comprises two or more different activators in different dendrons or regions on the surface of the superbranched polymer, according to any one of embodiments 39 to 46, for use or a therapeutic method.

[0438] Appearance 47 A hyperbranched polymer for use according to embodiment 46, or a therapeutic method, for use in combination therapy involving the administration of multiple activators.

[0439] Second set of embodiments Appearance 1 A superbranched polymer containing building blocks, wherein the building blocks are A core unit having at least three connecting parts c, A plurality of polymer arms connected to the core unit at the aforementioned connecting portion c, At least one of the polymer arms is connected to a repeating dendritic structure unit, Each dendritic repeating unit comprises a branched unit connected to at least two polymer arms containing terminal groups, or is linked to the next dendritic repeating unit by hydrolyzable bonds, and the next dendritic repeating unit can be further linked to further dendritic repeating units by chemical bonds. The polymer arms of the outermost dendritic repeating unit of the superbranched polymer each include the plurality of polymer arms, each containing an end group. The polymer arm is the hyperbranched polymer, which consists of polyethylene glycol (PEG) units.

[0440] Appearance 2 The hyperbranched polymer according to embodiment 1, wherein at least 10%, preferably about 20% to 100%, of the links in the polymer can be cleaved by hydrolysis.

[0441] Appearance 3 A hyperbranched polymer according to any one of the prior embodiments, wherein the bond is not hydrolyzed.

[0442] Pattern 4 The superbranched polymer is a high-generation Gx superbranched polymer, where x is an integer from 1 to 10, and defines the number of continuously linked dendritic repeating units in the superbranched polymer, as described in any one of the preceding embodiments.

[0443] Appearance 5 The superbranched polymer according to any one of the prior embodiments, wherein the core unit and the branching unit are the same or different and independently have 3 to 10, or 4 to 8, or 4 to 6, or 4 linking portions c or c'.

[0444] Appearance 6 The hyperbranched polymer according to any one of the prior embodiments, wherein the core unit and the branching unit are the same or different and are derived from a polyol having at least three hydroxyl groups.

[0445] Appearance 7 The superbranched polymer according to embodiment 6, wherein the polyol is selected from the group consisting of glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomalt, lactitol, maltitol, mannitol, or sorbitol.

[0446] Appearance 8 The polyethylene glycol (PEG) units of the polymer arm have an average molecular weight (Mn) in the range of about 1,000 to about 100,000 daltons, or about 10,000 to about 60,000 daltons, or about 15,000 to about 50,000 daltons, or about 10,000 to about 40,000 daltons, as described in any one of the prior embodiments.

[0447] Appearance 9 The hyperbranched polymer according to any one of the prior embodiments, wherein the average molecular weight of the polymer arm PEG units bonded to the core is the same as or different from the polymer arms in the dendritic repeating units.

[0448] Appearance 10 The hyperbranched polymer according to any one of the prior embodiments, wherein the average molecular weight of the polymer arm PEG units bonded to the core is higher than that of the polymer arms in the dendritic repeating units.

[0449] Appearance 11 The hyperbranched polymer according to any one of the prior embodiments, wherein the average molecular weight of the polymer arm PEG units bonded to the core is lower than that of the polymer arms in the dendritic repeating units.

[0450] Appearance 12 In the case of a high-generation Gx hyperbranched polymer where x is an integer from 2 to 10, the average molecular weight of the polymer arm PEG units decreases from the innermost polymer arm to the outermost polymer arm, or the average molecular weight of the polymer arm PEG units increases from the innermost polymer arm to the outermost polymer arm, according to any one of the preceding embodiments.

[0451] Appearance 13 The hyperbranched polymer according to any one of the prior embodiments, wherein the at least one arm connected to the core unit or the branching unit is linked to the dendritic repeating unit via a bifunctional linker that forms a hydrolyzable bond containing a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, a heteroaliphatic, or aromatic or heteroaromatic group.

[0452] Appearance 14 The hyperbranched polymer according to any one of the prior embodiments, wherein the terminal groups bonded to the outermost polymer arm are grafted directly to the end of the polymer arm, or via a bifunctional linker that includes or forms hydrolyzable bonds containing a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, a heteroaliphatic, or aromatic or heteroaromatic group.

[0453] Appearance 15 The functional groups of the terminal groups and / or linker terminal groups bonded to the outermost polymer arm are active ester groups such as succinimidyl esters and succinimidyl carbonates, electrophiles such as nitrophenyl carbonates, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, alkynes, azides, norbornene, epoxides, mesylates, tosylates, tresyl, cyanurates, orthopyridyl disulfide, or halogens, amines such as primary amines, hydroxyls, alcohols, thiols, azides, and A hyperbranched polymer according to any one of the preceding embodiments, wherein the functional group is selected from a nucleophile such as a carboxyl group, a functional group for click chemistry, a functional group for cyclization such as 1,3-dipolar cycloaddition, [3+2] cycloaddition such as alkene-nitrone cycloaddition or alkyne-nitrone cycloaddition, a functional group for 4+2] cycloaddition, a functional group for thiol-ene reactions; a functional group for hetero-Diels-Alder cycloaddition, a functional group for nucleophilic ring-opening, a functional group for non-aldol-type carbonyl reactions, a functional group for addition reactions to carbon-carbon multiple bonds, a polymerizable vinyl group, or a combination thereof.

[0454] Appearance 16 The hyperbranched polymer according to any one of the prior embodiments, wherein the (linker) terminal group bonded to the outermost polymer arm is a functional group selected from succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), succinimidyl azelate (SAZ), and succinimidyl glutaramide (SGA).

[0455] Appearance 17 The hyperbranched polymer according to any one of embodiments 1 to 14, wherein the terminal group bonded to the outermost polymer arm is an alkyne compound such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]-nonine (BCN); or norbornene or trans-cyclooctene (TCO); or a functional group selected from azide, 3,4-dihydroxyphenylacetic acid (DHPA), or tetrazine (Tz).

[0456] Appearance 18 The superbranched polymer according to any one of the prior embodiments, wherein the links between the core unit and the polymer arms linked to the first dendritic repeating unit, and / or the links between consecutive dendritic repeating units, are formed by click chemistry using click chemistry functionalized linkers, which optionally include hydrolyzable bonds.

[0457] Appearance 19 The hyperbranched polymer according to embodiment 18, wherein the coupling is formed by reacting a polymer arm functionalized with an alkyne, cycloalkyne, or a strained or terminal alkene moiety with a polymer arm functionalized with an azide or tetrazine moiety via an optional linker in a SPAAC or IEDDA type click chemistry coupling reaction.

[0458] Appearance 20 The hyperbranched polymer according to embodiment 19, wherein the alkene portion is a dibenzocyclooctin portion.

[0459] Aspect 21 The linkage between the core unit and the polymer arm linked to the first dendritic structure repeating unit, and / or the linkage between consecutive dendritic structure repeating units is optionally formed through a bifunctional linker that forms at least one hydrolyzable bond, between the polymer arm linked to the core unit and the polymer arm linked to the branching unit of the dendritic structure repeating unit, and / or between the polymer arm of the dendritic structure repeating unit and the polymer arm of the consecutive dendritic structure repeating unit. The hyperbranched polymer according to any one of Aspects 18 or 20.

[0460] Aspect 22 A hyperbranched polymer according to any of the preceding aspects, comprising a dendritic structure repeating unit represented by formula (i), or a dendritic structure repeating unit represented by formula (i), [Chemical formula] In the formula, A is a linkage to a polymer arm linked to the core unit, or A is a linkage to B of a preceding dendritic structure repeating unit represented by formula (i), L A is a linker, m is either 0 or 1, n is an integer from 20 to 2000, and o is an integer from 20 to 2000, n and o may be different or the same, X is a branching unit having a linking site c’, L B is a linker, and p is either 0 or 1, B includes a terminal group located on the surface of the hyperbranched polymer, or is a linkage to A of a consecutive dendritic structure repeating unit or an activator, L A and L B may be different or the same, m and p may be different or the same, The dendritic repeating unit in the superbranched polymer is an integer between 2 and 9, where y = c'-1, c' is the connecting portion c' of the branched unit X, and the dendritic repeating unit in the superbranched polymer may be the same or different.

[0461] Appearance 23 The superbranched polymer according to embodiment 22, wherein the link between A and B includes a functional group formed by click chemistry, such as triazole or dihydropyrazine.

[0462] Pattern 24 The aforementioned linker L A and / or L B The superbranched polymer according to embodiment 22 or 23, comprising a diacid and / or acid diamide group such as succinic acid, glutaric acid, adipic acid, azelaic acid, or glutaramide.

[0463] Appearance 25 The aforementioned linker L A and / or L B This includes the structure represented by formula (ii), [ka] In the formula, U 1 and U 2 A hyperbranched polymer according to any one of embodiments 22 to 24, wherein is independently NH or O, which may be the same or different, and t is an integer from 0 to 10.

[0464] Appearance 26 The aforementioned linker L A and / or L B The hyperbranched polymer according to embodiment 24 or 25, further comprising polyethylene glycol units between the bond to B and a carboxyl group, a carboxamide group, or the structure of formula (ii).

[0465] Appearance 27 The superbranched polymer further comprises at least one extender unit containing polyethylene glycol (PEG) units, The hyperbranched polymer according to any of the preceding embodiments, wherein the extender unit is linear and bifunctional, and is linked to the polymer arm of a dendritic repeating unit, or to the polymer arm linked to the core unit, and is linked to either the terminal group or polymer arm of the next dendritic repeating unit.

[0466] Appearance 28 The extension unit includes at least one linker, The hyperbranched polymer according to embodiment 27, wherein the linker can be located at either or both ends of the extender unit and is a bifunctional linker containing a hydrolyzable bond comprising a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, a heteroaliphatic, or aromatic or heteroaromatic group.

[0467] Appearance 29 A hyperbranched polymer according to any one of the preceding embodiments, wherein the building blocks, at least one or all, preferably all, selected from the core unit, the core unit including polymer arms at the connecting portion c, the dendritic repeating unit, the linker between hydrolyzable bonds, and the extender, have a molecular weight of less than 50,000 daltons, for example less than 45,000 daltons, or less than 40,000 daltons, or less than 35,000 daltons, or less than 30,000 daltons.

[0468] Appearance 30 The hyperbranched polymer according to any one of the prior embodiments, wherein when the hydrolyzable bond is completely hydrolyzed, all fragments formed from the molecule have a molecular weight of less than 50,000 daltons, for example less than 45,000 daltons, or less than 40,000 daltons, or less than 35,000 daltons, or less than 30,000 daltons.

[0469] Appearance 31 A dendritic repeating unit precursor, A polymer arm containing a functional group suitable for forming a linkage by click chemistry (e.g., azide, alkyne, alkene, or tetrazine), A polymer arm comprising at least two polymer arms containing non-reactive functional groups in click chemistry, The polymer arm is connected to a branching unit having a connecting portion c', The polymer portion of the polymer arm is a precursor of the dendritic structure repeating unit, consisting of polyethylene glycol (PEG) units.

[0470] Appearance 32 The aforementioned compound is represented by formula (iii), [ka] In the formula, C comprises a functional group suitable for click chemistry (e.g., alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (e.g., succinimidyl), L A , m, n, X, o, L B The precursor according to embodiment 31, wherein p and y are as defined in the prior embodiment.

[0471] Appearance 33 Inverse dendritic repeating unit precursor, A polymer arm containing a non-reactive functional group in click chemistry, A polymer arm comprising at least two functional groups suitable for click chemistry (e.g., azide, alkyne, alkene, or tetrazine), A branching unit having a connecting portion c', The polymer portion of the polymer arm is a precursor of the dendritic structure repeating unit, consisting of polyethylene glycol (PEG) units.

[0472] Appearance 34 The precursor is represented by formula (iv), [ka] In the formula, C comprises a functional group suitable for click chemistry (e.g., alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (e.g., succinimidyl), L A , m, n, X, o, L B The precursor according to embodiment 33, wherein p and y are as defined in the prior embodiment.

[0473] Appearance 35 A method for producing a hyperbranched polymer according to any one of embodiments 1 to 34 by branched synthesis, (a) Providing a core unit having at least three connecting portions c, and polymer arms connected to the core unit, each polymer arm having functional groups suitable for click chemistry at its ends; (b) A step of providing a dendritic repeating unit precursor, wherein the precursor is A polymer arm having a functional group suitable for forming a link between the polymer arm connected to the core and the corresponding functional group (e.g., azide, alkyne, alkene, or tetrazine) by click chemistry, A step of providing a dendritic repeating unit precursor comprising at least two polymer arms containing non-reactive functional groups in click chemistry, (c) The step of forming a link between the polymer arm connected to the core and the polymer arm of the dendritic structure repeating unit precursor by click chemistry, (d) optionally, converting the functional groups of at least two polymer arms containing non-reactive functional groups in click chemistry to functional groups suitable for click chemistry, (e) The method comprising the step of conjugating an activator containing functional groups to the outermost polymer arm by reacting it with the functional groups of the outermost polymer arm, thereby forming a hyperbranched polymer-activator conjugate.

[0474] Appearance 36 The method according to embodiment 35, wherein, in the case of a high-generation Gx hyperbranched polymer (where x is an integer from 2 to 10), step (d) is essential, and before conjugating the activator in step (e), further sequential dendritic repeating unit precursors are click-chemistry-linked to the functional groups suitable for click chemistry obtained in step (d) to form a hyperbranched polymer. The dendritic repeating unit precursor in step (c) is represented by formula (iii), [ka] In the formula, C comprises a functional group suitable for click chemistry (e.g., alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (e.g., succinimidyl), L A , m, n, X, o, L B The method according to embodiment 35 or 36, wherein p and y are as defined in the prior embodiment, and the dendritic repeating units may be the same or different.

[0475] Appearance 37 The method according to embodiments 35-37, wherein, after the second-to-last step (d) which converts the functional groups of at least two polymer arms containing non-reactive functional groups in the click chemistry into click chemistry-compatible functional groups, the activator of step (e) is first functionalized with a click chemistry-compatible functional group (such as an alkyne, alkene, azide, or tetrazine) and then conjugated to the outermost polymer arms of the hyperbranched polymer in a click chemistry reaction.

[0476] Appearance 38 The activators functionalized with functional groups suitable for click chemistry are peptides, such as Compstatin, APL-1, Fc-III-4C, Beov (brolucizumab), Zimura (abacincaptado pegol), Pegcetacoplan, Abisipal pegol, Lamparizumab, Fovista, Listeganib, AXT107, Elamipretide, THR149, ALM201, VGB3, and Largazole. The method according to embodiment 38, wherein the anti-VEGF agent is one of the angiogenesis inhibitors such as an anti-VEGF agent (e.g., aflibercept, ranibizumab, bevacizumab), a PDGF-B inhibitor (e.g., Fovista®), a complement antagonist (e.g., eculizumab), a tyrosine kinase inhibitor (e.g., sunitinib, axitinib), and / or an integrin antagonist (e.g., natalizumab and vedolizumab).

[0477] Appearance 39 A method for producing a hyperbranched polymer according to any one of embodiments 1 to 34 by convergent synthesis, I) A step of providing a dendritic repeating unit precursor, wherein the precursor is A polymer arm containing a functional group suitable for forming a linkage by click chemistry (e.g., azide, alkyne, alkene, or tetrazine), A step of providing a dendritic repeating unit precursor comprising at least two polymer arms containing non-reactive functional groups in click chemistry, II) The step of conjugating an activator containing a functional group to at least one of the at least two polymer arms of the dendritic repeating unit precursor that contain a functional group that does not react in click chemistry, III) Providing a core unit having at least three connecting portions c, and polymer arms connected to the core unit, each having a functional group suitable for click chemistry (e.g., azide, alkyne, alkene, or tetrazine) at the end of each polymer arm, IV) The method comprising forming a hyperbranched polymer-activator conjugate by click chemistry between the polymer arm linked to the core provided in step III) and the polymer arm containing a functional group suitable for forming click chemistry linkage of the activator conjugate dendritic repeating unit precursor obtained in step II).

[0478] Pattern 40 The aforementioned dendritic repeating unit precursor is represented by formula (iii), [ka] In the formula, C comprises a functional group suitable for click chemistry (e.g., alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (e.g., succinimidyl), L A , L B The method according to embodiment 40, wherein m, n, X, o, p, and y are as defined in the prior embodiment, and the dendritic repeating units may be the same or different.

[0479] Appearance 41 In the case of a high-generation Gx hyperbranched polymer (where x is an integer from 2 to 10), The activator conjugate dendritic structure repeating unit precursor obtained in step II) A polymer arm containing a non-reactive functional group in click chemistry, A reverse dendritic repeating unit precursor comprising at least two polymer arms containing functional groups suitable for click chemistry (e.g., azides, alkynes, alkenes, or tetrazines) is linked by click chemistry, The method according to embodiment 40 or 41, wherein the functional group that does not react in the click chemistry of one polymer arm is subsequently converted to a click chemistry-suitable functional group before being linked to a further inverse dendritic repeating unit precursor, or before forming a click chemistry link with the polymer arm linked to the core in step IV), thereby forming a high-generation Gx hyperbranched polymer.

[0480] Pattern 42 The method according to embodiments 40 to 42, wherein dendritic repeating unit precursors having different activators conjugated to the polymer arm are obtained by performing steps I) and II) on each activator-conjugated dendritic repeating unit precursor, and a mixture of the obtained activator-conjugated dendritic repeating unit precursors is used in step IV), thereby forming a superbranched polymer-activator conjugate having different activators in different regions of the surface of the superbranched polymer.

[0481] Appearance 43 The method according to any one of embodiments 40 to 43, wherein the outermost polymer arm of the superbranched polymer has a terminal maleimide functional group, and a peptide or activator is conjugated to the terminal maleimide functional group via a maleimide-thiol reaction.

[0482] 44 The method according to embodiment 44, wherein the terminal maleimide functional group is provided by reacting the DBCO or azide-functionalized terminal functional group of the hyperbranched polymer with a click chemistry linker having an azide or DBCO functional group linked to a maleimide group (such as DBCO-maleimide, DBCO-PEG3-maleimide, DBCO-PEG4-maleimide, or azide-PEG3-maleimide).

[0483] Appearance 45 Use of a hyperbranched polymer according to any one of embodiments 1 to 34 in at least one application selected from non-medical or industrial uses such as antibody purification, cosmetic applications, catalytic applications, electronic equipment, agriculture, food, filtration, energy storage, building materials, coatings, adhesives, water purification, oil recovery, fragrance release, papermaking, environmental sensing and release systems, membranes, fibers, printing inks, surface chemical applications, thickeners, detergents, rheological modifiers, scaffolding, or 3D printing.

[0484] Third set of embodiments Appearance 1 A superbranched polymer containing building blocks, wherein the building blocks are A core unit having at least three connecting parts c, A plurality of polymer arms connected to the core unit at the aforementioned connecting portion c, At least one of the polymer arms is linked to a dendritic repeating unit by a hydrolyzable bond, Each dendritic repeating unit comprises a branched unit connected to at least two polymer arms containing terminal groups, or is linked to the next dendritic repeating unit by hydrolyzable bonds, and the next dendritic repeating unit can be further linked to further dendritic repeating units by chemical bonds. The aforementioned dendritic repeating unit is represented by equation (i), [ka] In the formula, A is a connection to a polymer arm connected to the core unit, a connection to an extender unit, or A is a connection to B of a preceding dendritic repeating unit represented by formula (i). L A It is a linker, m is either 0 or 1. n is an integer between 20 and 2000, and o is an integer between 20 and 2000. n and o may be different or the same. X is a branching unit having a connecting portion c', L B is the linker, and p is either 0 or 1. B is a terminal group located on the surface of the superbranched polymer, or a continuous dendritic repeating unit or an activator linked to A. L A and L B They may be different or the same. m and p may be different or the same. y is an integer between 2 and 9, y = c'-1, c' is the connecting portion c' of the branched unit X, and the repeating dendritic structures in the hyperbranched polymer may be the same or different. The polymer arms of the outermost dendritic repeating unit of the superbranched polymer each include the plurality of polymer arms, each containing an end group. The hyperbranched polymer, wherein at least one activator is conjugated to at least one of the outermost polymer arms.

[0485] Appearance 2 The hyperbranched polymer according to embodiment 1, wherein at least 10%, preferably about 20% to 100%, of the links in the polymer can be cleaved by hydrolysis.

[0486] Appearance 3 The superbranched polymer according to any one of the prior embodiments, wherein each of the building blocks (fragments) of the superbranched polymer obtained after cleaving all of the hydrolyzable bonds of the linkage in the polymer has an average molecular weight (Mn) of less than 50,000 daltons, for example less than 45,000 daltons, or less than 40,000 daltons, or less than 35,000 daltons, or less than 30,000 daltons.

[0487] Pattern 4 The hyperbranched polymer according to any one of the prior embodiments, wherein at least one of the building blocks includes a core unit or a branching unit and has a plurality of polymer arms connected to the core unit or the branching unit by non-hydrolyzable bonds.

[0488] Appearance 5 A high-generation Gx hyperbranched polymer, wherein x is an integer from 1 to 10, and defines the number of continuously linked dendritic repeating units in the hyperbranched polymer, as described in any one of the preceding embodiments.

[0489] Appearance 6 The superbranched polymer according to any one of the prior embodiments, wherein the core unit and the branching unit are the same or different and independently have 3 to 10, or 4 to 8, or 4 to 6, or 4 linking portions c or c'.

[0490] Appearance 7 The hyperbranched polymer according to any one of the prior embodiments, wherein the core unit and the branching unit are the same or different and are derived from a polyol having at least three hydroxyl groups.

[0491] Appearance 8 The superbranched polymer according to embodiment 7, wherein the polyol is selected from the group consisting of glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomalt, lactitol, maltitol, mannitol, or sorbitol.

[0492] Appearance 9 The polyethylene glycol (PEG) units of the polymer arm have an average molecular weight (Mn) in the range of about 1,000 to about 100,000 daltons, or about 10,000 to about 60,000 daltons, or about 15,000 to about 50,000 daltons, or about 10,000 to about 40,000 daltons, as described in any one of the prior embodiments.

[0493] Appearance 10 The hyperbranched polymer according to any one of the prior embodiments, wherein the average molecular weight of the polymer arm PEG units bonded to the core is the same as or different from the polymer arms in the dendritic repeating units.

[0494] Appearance 11 The hyperbranched polymer according to any one of the prior embodiments, wherein the average molecular weight of the polymer arm PEG units bonded to the core is higher than that of the polymer arms in the dendritic repeating units.

[0495] Appearance 12 The hyperbranched polymer according to any one of the prior embodiments, wherein the average molecular weight of the polymer arm PEG units bonded to the core is lower than that of the polymer arms in the dendritic repeating units.

[0496] Appearance 13 In the case of a high-generation Gx hyperbranched polymer where x is an integer from 2 to 10, the average molecular weight of the polymer arm PEG units decreases from the innermost polymer arm to the outermost polymer arm, or the average molecular weight of the polymer arm PEG units increases from the innermost polymer arm to the outermost polymer arm, according to any one of the preceding embodiments.

[0497] Appearance 14 The hyperbranched polymer according to any one of the prior embodiments, wherein the at least one arm connected to the core unit or the branching unit is linked to the dendritic repeating unit via a bifunctional linker that forms a hydrolyzable bond containing a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, a heteroaliphatic, or aromatic or heteroaromatic group.

[0498] Appearance 15 The hyperbranched polymer according to any one of the prior embodiments, wherein the terminal groups bonded to the outermost polymer arm are grafted directly to the end of the polymer arm, or via a bifunctional linker that includes or forms hydrolyzable bonds containing a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, a heteroaliphatic, or aromatic or heteroaromatic group.

[0499] Appearance 16 The functional groups of the terminal groups and / or linker terminal groups bonded to the outermost polymer arm are active ester groups such as succinimidyl esters and succinimidyl carbonates, electrophiles such as nitrophenyl carbonates, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, alkynes, azides, norbornene, epoxides, mesylates, tosylates, tresyl, cyanurates, orthopyridyl disulfide, or halogens, amines such as primary amines, hydroxyls, alcohols, thiols, azides, and A hyperbranched polymer according to any one of the preceding embodiments, wherein the functional group is selected from a nucleophile such as a carboxyl group, a functional group for click chemistry, a functional group for cyclization such as 1,3-dipolar cycloaddition, [3+2] cycloaddition such as alkene-nitrone cycloaddition or alkyne-nitrone cycloaddition, a functional group for 4+2] cycloaddition, a functional group for thiol-ene reactions; a functional group for hetero-Diels-Alder cycloaddition, a functional group for nucleophilic ring-opening, a functional group for non-aldol-type carbonyl reactions, a functional group for addition reactions to carbon-carbon multiple bonds, a polymerizable vinyl group, or a combination thereof.

[0500] Appearance 17 The hyperbranched polymer according to any one of the prior embodiments, wherein the (linker) terminal group bonded to the outermost polymer arm is a functional group selected from succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), succinimidyl azelate (SAZ), and succinimidyl glutaramide (SGA).

[0501] Appearance 18 The hyperbranched polymer according to any one of embodiments 1 to 13, wherein the terminal group bonded to the outermost polymer arm is an alkyne compound such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]-nonine (BCN); or norbornene or trans-cyclooctene (TCO); or a functional group selected from azide, 3,4-dihydroxyphenylacetic acid (DHPA), or tetrazine (Tz).

[0502] Appearance 19 The superbranched polymer according to any one of the prior embodiments, wherein the links between the core unit and the polymer arms linked to the first dendritic repeating unit, and / or the links between consecutive dendritic repeating units, are formed by click chemistry using click chemistry functionalized linkers, which optionally include hydrolyzable bonds.

[0503] Appearance 20 The hyperbranched polymer according to embodiment 19, wherein the coupling is formed by reacting a polymer arm functionalized with an alkyne, cycloalkyne, or strained or terminal alkene moiety with a polymer arm functionalized with an azide or tetrazine moiety via an optional linker in a SPAAC or IEDDA type click chemistry coupling reaction.

[0504] Appearance 21 The hyperbranched polymer according to embodiment 20, wherein the alkene portion is a dibenzocyclooctin portion.

[0505] Appearance 22 The superbranched polymer according to any one of embodiments 19 to 21, wherein the connections between the core unit and the polymer arm connected to the first dendritic repeating unit, and / or the connections between consecutive dendritic repeating units, are formed between the polymer arm connected to the core unit and the polymer arm connected to the branched unit of the dendritic repeating unit, and / or between the polymer arm of the dendritic repeating unit and the polymer arm of the consecutive dendritic repeating unit, optionally via a bifunctional linker that forms at least one hydrolyzable bond.

[0506] Appearance 23 The hyperbranched polymer according to any of the prior embodiments, wherein the activator conjugated to at least one of the outermost polymer arms is selected from the group consisting of therapeutic activators or diagnostic activators.

[0507] Pattern 24 The activator conjugated to at least one of the outermost polymer arms includes steroids, nonsteroidal anti-inflammatory drugs (NSAIDs) (such as diclofenac, ibuprofen, meclofename, mefanamic A, sarsalate, sulindac, tolmetin, ketoprofen, diflunisal, piroxicam, naproxen, etodolac, flurbiprofen, fenoprofen C, indomethacin, celecoxib, ketorolac, nepafenac, etc.), intraocular pressure lowering agents, antibiotics such as ciprofloxacin, analgesics such as bupivacaine, calcium channel blockers such as nifedipine, cell cycle inhibitors such as simvastatin, proteins such as insulin, and small molecule hydrophilic molecules including carboxylates and amine salts. A hyperbranched polymer according to any of the preceding embodiments, selected from sexual drugs, small molecule hydrophobic drugs, hydrophilic peptides and protein drugs (insulin, single-chain antibody fragments, Fab fragments, IgG antibodies, fusion antibodies, etc.), aptamers, in particular bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDS), steroids, antibiotics, analgesics, calcium channel blockers, cell cycle inhibitors, chemotherapeutic agents, antiviral agents, anesthetics, hormones, anticancer drugs, antitumor drugs, viruses, gene delivery viruses such as AAV, protein binders (nanobodies, afibodies, ankyrin, DARPin, etc.), or any combination thereof.

[0508] Appearance 25 The hyperbranched polymer according to any of the prior embodiments, wherein the activator conjugated to at least one of the outermost polymer arms is a peptide selected from the group consisting of Compstatin, APL-1, Fc-III-4C, Beov (brolucizumab), Zimura (abacincaptado pegol), pegcetacoplan, abisipal pegol, lamparizumab, Fovista, listiganib, AXT107, elamipretide, THR149, ALM201, VGB3, and Largazole.

[0509] Appearance 26 The hyperbranched polymer according to any of the prior embodiments, wherein the activator is bonded to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% of the outermost polymer arm.

[0510] Appearance 27 The hyperbranched polymer according to any one of the prior embodiments, wherein the link between A and B in formula (i) comprises a functional group formed by click chemistry, such as triazole or dihydropyrazine.

[0511] Appearance 28 The aforementioned linker L A and / or L B The superbranched polymer according to embodiment 27, comprising a diacid and / or acid diamide group such as succinic acid, glutaric acid, adipic acid, azelaic acid, or glutaramide.

[0512] Appearance 29 The aforementioned linker L A and / or L B This includes the structure represented by formula (ii), [ka] In the formula, U 1 and U 2 A hyperbranched polymer according to either embodiment 27 or 28, wherein is independently NH or O, which may be the same or different, and t is an integer from 0 to 10.

[0513] Appearance 30 The aforementioned linker L A and / or L B The hyperbranched polymer according to embodiment 28 or 29, further comprising polyethylene glycol units between the bond to B and a carboxyl group, a carboxamide group, or the structure of formula (ii).

[0514] Appearance 31 The superbranched polymer further comprises at least one extender unit containing polyethylene glycol (PEG) units, The hyperbranched polymer according to any of the preceding embodiments, wherein the extender unit is linear and bifunctional, and is linked to the polymer arm of a dendritic repeating unit, or to the polymer arm linked to the core unit, and is linked to either the terminal group or polymer arm of the next dendritic repeating unit.

[0515] Appearance 32 The extension unit further comprises at least one linker, The hyperbranched polymer according to embodiment 31, wherein the linker can be located at either or both ends of the extender unit and is a bifunctional linker containing a hydrolyzable bond comprising a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarbboxamide group, a functionalized aliphatic, a heteroaliphatic, or aromatic or heteroaromatic group.

[0516] Appearance 33 A hyperbranched polymer according to any one of the preceding embodiments, wherein the building blocks, at least one or all, preferably all, selected from the core unit, the core unit including polymer arms at the connecting portion c, the dendritic repeating unit, the linker between hydrolyzable bonds, and the extender, have a molecular weight of less than 50,000 daltons, for example less than 45,000 daltons, or less than 40,000 daltons, or less than 35,000 daltons, or less than 30,000 daltons.

[0517] Appearance 34 The hyperbranched polymer according to any one of the prior embodiments, wherein when the hydrolyzable bond is completely hydrolyzed, all fragments formed from the molecule have a molecular weight of less than 50,000 daltons, for example less than 45,000 daltons, or less than 40,000 daltons, or less than 35,000 daltons, or less than 30,000 daltons.

[0518] Appearance 35 A dendritic repeating unit precursor, A polymer arm containing a functional group suitable for forming a linkage by click chemistry (e.g., azide, alkyne, alkene, or tetrazine), A polymer arm comprising at least two polymer arms containing non-reactive functional groups in click chemistry, The polymer arm is connected to a branching unit having a connecting portion c', The polymer portion of the polymer arm is a precursor of the dendritic structure repeating unit, consisting of polyethylene glycol (PEG) units.

[0519] Appearance 36 The aforementioned compound is represented by formula (iii), [ka] In the formula, C comprises a functional group suitable for click chemistry (e.g., alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (e.g., succinimidyl), L A , m, n, X, o, L B The precursor according to embodiment 35, wherein p and y are as defined in the prior embodiment.

[0520] Appearance 37 Inverse dendritic repeating unit precursor, A polymer arm containing a non-reactive functional group in click chemistry, A polymer arm comprising at least two functional groups suitable for click chemistry (e.g., azide, alkyne, alkene, or tetrazine), A branching unit having a connecting portion c', The polymer portion of the polymer arm is a precursor of the dendritic structure repeating unit, consisting of polyethylene glycol (PEG) units.

[0521] Appearance 38 The precursor is represented by formula (iv), [ka] In the formula, C comprises a functional group suitable for click chemistry (e.g., alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (e.g., succinimidyl), L A , m, n, X, o, L B The precursor according to embodiment 37, wherein p and y are as defined in the prior embodiment.

[0522] Appearance 39 The core unit is a pegylated pentaerythritol compound of formula (iv), [ka] A hyperbranched polymer according to any one of embodiments 1 to 34, wherein n is an integer between 3 and 2,000.

[0523] Pattern 40 The aforementioned core unit is a compound of formula (v), [ka] A hyperbranched polymer according to any one of embodiments 1 to 34, wherein R is a core unit having x binding sites c, n is determined by the molecular weight of each PEG arm and is 3 to 2,000 or 20 to 2,000, m is an integer from 0 to 10, and x is the number of arms and is an integer from 1 to 10.

[0524] Appearance 41 The following precursors / dendrons: [ka] [ka] Or, the following exemplary precursor pairs, [ka] And, [ka] Either one of the following, [ka] And, [ka] Either one of the following, [ka] And, [ka] Either one of the following, [ka] And, [ka] either A hyperbranched polymer according to any one of embodiments 1 to 34 or 39 to 40, formed from one of the formulas (wherein t is m, and n and m are similarly defined with respect to formula (v) in embodiment 40).

Claims

1. A superbranched polymer containing building blocks, The building block is, A core unit having at least three connecting parts c, A plurality of polymer arms connected to the core unit at the aforementioned connecting portion c, At least one of the polymer arms is linked to a dendritic repeating unit by a hydrolyzable bond, Each dendritic repeating unit comprises a branched unit connected to at least two polymer arms containing terminal groups, or is linked to the next dendritic repeating unit by hydrolyzable bonds, and the next dendritic repeating unit can be further linked to further dendritic repeating units by chemical bonds. The polymer arms of the outermost dendritic repeating unit of the superbranched polymer each include the plurality of polymer arms, each containing an end group. The polymer arm is made up of polyethylene glycol (PEG) units. The hyperbranched polymer, wherein at least one activator is conjugated to at least one of the outermost polymer arms.

2. The hyperbranched polymer according to claim 1, wherein at least 10%, preferably about 20% to 100%, of the links in the polymer can be cleaved by hydrolysis.

3. The superbranched polymer according to any one of the prior claims, wherein each of the building blocks (fragments) of the superbranched polymer obtained after cleaving all of the hydrolyzable bonds of the linkage in the polymer has an average molecular weight (Mn) of less than 50,000 daltons, for example less than 45,000 daltons, or less than 40,000 daltons, or less than 35,000 daltons, or less than 30,000 daltons.

4. The superbranched polymer according to any one of the prior claims, wherein at least one of the building blocks includes a core unit or a branching unit, and has a plurality of polymer arms connected to the core unit or the branching unit by non-hydrolyzable bonds.

5. A high-generation Gx superbranched polymer, wherein x is an integer from 1 to 10, defining the number of continuously linked dendritic repeating units in the superbranched polymer, according to any one of the prior claims.

6. The superbranched polymer according to any one of the prior claims, wherein the core unit and the branching unit are the same or different and independently have 3 to 10, 4 to 8, 4 to 6, or 4 connecting portions c or c'.

7. The hyperbranched polymer according to any one of the prior claims, wherein the core unit and the branching unit are the same or different and are derived from a polyol having at least three hydroxyl groups.

8. The superbranched polymer according to claim 7, wherein the polyol is selected from the group consisting of glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomalt, lactitol, maltitol, mannitol, or sorbitol.

9. The polyethylene glycol (PEG) units of the polymer arm have an average molecular weight (Mn) in the range of about 1,000 to about 100,000 daltons, or about 10,000 to about 60,000 daltons, or about 15,000 to about 50,000 daltons, or about 10,000 to about 40,000 daltons, according to any one of the prior claims.

10. The hyperbranched polymer according to any one of the prior claims, wherein the average molecular weight of the polymer arm PEG units bonded to the core is the same as or different from the polymer arms in the dendritic repeating units.

11. The hyperbranched polymer according to any one of the prior claims, wherein the average molecular weight of the polymer arm PEG units bonded to the core is higher than that of the polymer arms in the dendritic repeating units.

12. The hyperbranched polymer according to any one of the prior claims, wherein the average molecular weight of the polymer arm PEG units bonded to the core is lower than that of the polymer arms in the dendritic repeating units.

13. In the case of a high-generation Gx hyperbranched polymer where x is an integer from 2 to 10, the average molecular weight of the polymer arm PEG units decreases from the innermost polymer arm toward the outermost polymer arm, or the average molecular weight of the polymer arm PEG units increases from the innermost polymer arm toward the outermost polymer arm, according to any one of the prior claims.

14. The hyperbranched polymer according to any one of the prior claims, wherein the at least one arm connected to the core unit or the branching unit is linked to the dendritic repeating unit via a bifunctional linker that forms a hydrolyzable bond comprising a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic group, a heteroaliphatic group, or an aromatic or heteroaromatic group.

15. The hyperbranched polymer according to any one of the prior claims, wherein the terminal groups bonded to the outermost polymer arm are grafted directly to the end of the polymer arm, or via a bifunctional linker that includes or forms hydrolyzable bonds containing a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, a heteroaliphatic, or aromatic or heteroaromatic group.

16. The functional groups of the terminal groups and / or linker terminal groups bonded to the outermost polymer arm are active ester groups such as succinimidyl esters and succinimidyl carbonates, electrophiles such as nitrophenyl carbonates, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, alkynes, azides, norbornene, epoxides, mesylates, tosylates, tresyl, cyanurates, orthopyridyl disulfide, or halogens, amines such as primary amines, hydroxyls, alcohols, thiols, azides, and A hyperbranched polymer according to any one of the prior claims, wherein the functional group is selected from a nucleophile such as a carboxyl group, a functional group for click chemistry, a functional group for cyclization such as 1,3-dipolar cycloaddition, alkene-nitrone cycloaddition or alkyne-nitrone cycloaddition, [3+2] cycloaddition, [4+2] cycloaddition, a functional group for thiol-ene reactions; a functional group for hetero-Diels-Alder cycloaddition, a functional group for nucleophilic ring-opening, a functional group for non-aldol-type carbonyl reactions, a functional group for addition reactions to carbon-carbon multiple bonds, a polymerizable vinyl group, or a combination thereof.

17. The hyperbranched polymer according to any one of the prior claims, wherein the (linker) terminal group bonded to the outermost polymer arm is a functional group selected from succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), succinimidyl azelate (SAZ), and succinimidyl glutaramide (SGA).

18. The hyperbranched polymer according to any one of claims 1 to 13, wherein the terminal group bonded to the outermost polymer arm is an alkyne compound such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]-nonine (BCN); or norbornene or trans-cyclooctene (TCO); or a functional group selected from azide, 3,4-dihydroxyphenylacetic acid (DHPA), or tetrazine (Tz).

19. The superbranched polymer according to any one of the prior claims, wherein the connections between the core unit and the polymer arms linked to the first dendritic repeating unit, and / or the connections between consecutive dendritic repeating units, are formed by click chemistry using click chemistry functionalized linkers which optionally include hydrolyzable bonds.

20. The superbranched polymer according to claim 19, wherein the linkage is formed by reacting a polymer arm functionalized with an alkyne, cycloalkyne, or a strained or terminal alkene moiety with a polymer arm functionalized with an azide or tetrazine moiety via an optional linker in a SPAAC or IEDDA type click chemistry coupling reaction.

21. The superbranched polymer according to claim 20, wherein the alkene portion is a dibenzocyclooctin portion.

22. The superbranched polymer according to any one of claims 19 to 21, wherein the connections between the core unit and the polymer arm connected to the first dendritic repeating unit, and / or the connections between consecutive dendritic repeating units, are formed between the polymer arm connected to the core unit and the polymer arm connected to the branched unit of the dendritic repeating unit, and / or between the polymer arm of the dendritic repeating unit and the polymer arm of the consecutive dendritic repeating unit, optionally via a bifunctional linker that forms at least one hydrolyzable bond.

23. The hyperbranched polymer according to any of the prior claims, wherein the activator conjugated to at least one of the outermost polymer arms is selected from the group consisting of therapeutic activators and diagnostic activators.

24. The activator conjugated to at least one of the outermost polymer arms includes steroids, non-steroidal anti-inflammatory drugs (NSAIDs) (such as diclofenac, ibuprofen, meclofename, mefanamic A, sarsalate, sulindac, tolmetin, ketoprofen, diflunisal, piroxicam, naproxen, etodolac, flurbiprofen, fenoprofen C, indomethacin, celecoxib, ketorolac, and nepafenac), intraocular pressure lowering agents, antibiotics such as ciprofloxacin, analgesics such as bupivacaine, calcium channel blockers such as nifedipine, cell cycle inhibitors such as simvastatin, proteins such as insulin, and small molecule hydrophilic molecules including carboxylates and amine salts. A hyperbranched polymer according to any of the prior claims, selected from sexual drugs, small molecule hydrophobic drugs, hydrophilic peptides and protein drugs (insulin, single-chain antibody fragments, Fab fragments, IgG antibodies, fusion antibodies, etc.), aptamers, in particular bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, antibiotics, analgesics, calcium channel blockers, cell cycle inhibitors, chemotherapeutic agents, antiviral agents, anesthetics, hormones, anticancer drugs, antitumor drugs, viruses, gene delivery viruses such as AAV, protein binders (nanobodies, aphibodies, ankyrin, DARPin, etc.), or any combination thereof.

25. The hyperbranched polymer according to any of the prior claims, wherein the activator conjugated to at least one of the outermost polymer arms is a peptide selected from the group consisting of Compstatin, APL-1, Fc-III-4C, Beov (brolucizumab), Zimura (abacincaptadopegol), pegcetacoplan, abisipalpegol, lamparizumab, Fovista, listiganib, AXT107, elamipretide, THR149, ALM201, VGB3, and Largazole.

26. The hyperbranched polymer according to any of the prior claims, wherein the activator is bonded to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% of the outermost polymer arm.

27. The aforementioned dendritic repeating unit is represented by equation (i), 【Chemistry 1】 In the formula, A is a connection to a polymer arm connected to the core unit, or A is a connection to B of a preceding dendritic repeating unit represented by formula (i), L Ａ It is a linker, m is either 0 or 1. n is an integer between 20 and 2000, and o is an integer between 20 and 2000. n and o may be different or the same. X is a branching unit having a connecting portion c', L Ｂ is a linker, and p is either 0 or 1. B is a linkage to A that includes terminal groups located on the surface of the superbranched polymer, or a continuous dendritic repeating unit or an activator. L Ａ and L Ｂ They may be different or the same. m and p may be different or the same. The hyperbranched polymer according to any of the prior claims, wherein y is an integer from 2 to 9, y = c' - 1, c' is the connecting portion c' of the branching unit X, and the repeating dendritic structure units in the hyperbranched polymer may be the same or different.

28. The superbranched polymer according to claim 27, wherein the link between A and B includes a functional group formed by click chemistry, such as triazole or dihydropyrazine.

29. The aforementioned linker L Ａ and / or L Ｂ The superbranched polymer according to claim 27 or 28, comprising a diacid and / or acid diamide group such as succinic acid, glutaric acid, adipic acid, azelaic acid, or glutaramide.

30. The aforementioned linker L Ａ and / or L Ｂ This includes the structure represented by formula (ii), 【Chemistry 2】 where U １ and U ２ are independently NH or O, may be the same or different, t is an integer from 0 to 10, the hyperbranched polymer according to any one of claims 27 to 29.

31. The aforementioned linker L Ａ and / or L Ｂ The hyperbranched polymer according to claim 29 or 30, further comprising polyethylene glycol units between the bond to B and a carboxyl group, a carboxamide group, or the structure of formula (ii).

32. The superbranched polymer further comprises at least one extender unit containing polyethylene glycol (PEG) units, The hyperbranched polymer according to any of the prior claims, wherein the extender unit is linear and bifunctional, and is linked to the polymer arm of a dendritic repeating unit, or to the polymer arm linked to the core unit, and is linked to either the terminal group or polymer arm of the next dendritic repeating unit.

33. The extension unit includes at least one linker, The superbranched polymer according to claim 32, wherein the linker can be located at either or both ends of the extender unit and is a bifunctional linker comprising a hydrolyzable bond containing a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarbboxamide group, a functionalized aliphatic group, a heteroaliphatic group, or an aromatic or heteroaromatic group.

34. A hyperbranched polymer according to any one of the prior claims, wherein the building blocks, at least one or all, preferably all, selected from the core unit, the core unit including polymer arms at the connecting portion c, the dendritic repeating unit, the linker between hydrolyzable bonds, and the extender, have a molecular weight of less than 50,000 daltons, for example less than 45,000 daltons, or less than 40,000 daltons, or less than 35,000 daltons, or less than 30,000 daltons.

35. The hyperbranched polymer according to any one of the prior claims, wherein when the hydrolyzable bond is completely hydrolyzed, all fragments formed from the molecule have a molecular weight of less than 50,000 daltons, for example less than 45,000 daltons, or less than 40,000 daltons, or less than 35,000 daltons, or less than 30,000 daltons.

36. A dendritic repeating unit precursor, A polymer arm containing a functional group suitable for forming a linkage by click chemistry (e.g., azide, alkyne, alkene, or tetrazine), It comprises at least two polymer arms containing functional groups that do not react in click chemistry, The polymer arm is connected to a branching unit having a connecting portion c', The polymer portion of the polymer arm is a precursor of the dendritic structure repeating unit, consisting of polyethylene glycol (PEG) units.

37. The aforementioned compound is represented by formula (iii), 【Transformation 3】 In the formula, C comprises a functional group suitable for click chemistry (e.g., alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (e.g., succinimidyl), L Ａ , m, n, X, o, L Ｂ The precursor according to claim 36, wherein p and y are as defined in claims 27 to 31.

38. Inverse dendritic repeating unit precursor, A polymer arm containing a non-reactive functional group in click chemistry, A polymer arm comprising at least two functional groups suitable for click chemistry (e.g., azide, alkyne, alkene, or tetrazine), A branching unit having a connecting portion c', The polymer portion of the polymer arm is a precursor of the dendritic structure repeating unit, consisting of polyethylene glycol (PEG) units.

39. The aforementioned precursor is represented by formula (iv), 【Chemistry 4】 In the formula, C comprises a functional group suitable for click chemistry (e.g., alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (e.g., succinimidyl), L Ａ , m, n, X, o, L Ｂ The precursor according to claim 34, wherein p and y are as defined in the prior claims.

40. A method for producing a hyperbranched polymer according to any one of claims 1 to 39 by branched synthesis, (a) Providing a core unit having at least three connecting portions c, and polymer arms connected to the core unit, each polymer arm having functional groups suitable for click chemistry at its ends; (b) A step of providing a dendritic repeating unit precursor, wherein the precursor is A polymer arm having a functional group suitable for forming a link between the polymer arm connected to the core and the corresponding functional group (e.g., azide, alkyne, alkene, or tetrazine) by click chemistry, A step of providing a dendritic repeating unit precursor comprising at least two polymer arms containing non-reactive functional groups in click chemistry, (c) The step of forming a link between the polymer arm connected to the core and the polymer arm of the dendritic structure repeating unit precursor by click chemistry, (d) Optionally, a step of converting the functional groups of at least two polymer arms, which include functional groups that do not react in click chemistry, to functional groups suitable for click chemistry, (e) The method comprising the step of conjugating an activator containing functional groups to the outermost polymer arm by reacting it with the functional groups of the outermost polymer arm, thereby forming a hyperbranched polymer-activator conjugate.

41. The method according to claim 40, wherein, in the case of a high-generation Gx hyperbranched polymer (where x is an integer from 2 to 10), step (d) is essential, and before conjugating the activator in step (e), further continuous dendritic repeating unit precursors are click-chemistry-linked to the functional groups suitable for click chemistry obtained in step (d) to form a hyperbranched polymer.

42. The dendritic repeating unit precursor in step (c) is represented by formula (iii), 【Transformation 5】 In the formula, C comprises a functional group suitable for click chemistry (e.g., alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (e.g., succinimidyl), L Ａ , m, n, X, o, L Ｂ The method according to claim 40 or 41, wherein p and y are as defined in the prior claims, and the dendritic repeating units may be the same or different.

43. The method according to claims 40 to 42, wherein, after the second-to-last step (d) which converts the functional groups of at least two polymer arms containing non-reactive functional groups in the click chemistry into click chemistry-compatible functional groups, the activator of step (e) is first functionalized with a click chemistry-compatible functional group (such as an alkyne, alkene, azide, or tetrazine) and then conjugated to the outermost polymer arms of the hyperbranched polymer in a click chemistry reaction.

44. The method according to claim 43, wherein the activator functionalized with a functional group suitable for click chemistry is a peptide.

45. A method for producing a hyperbranched polymer according to any one of claims 1 to 39 by convergent synthesis, I) A step of providing a dendritic repeating unit precursor, wherein the precursor is A polymer arm containing a functional group suitable for forming a linkage by click chemistry (e.g., azide, alkyne, alkene, or tetrazine), A step of providing a dendritic repeating unit precursor comprising at least two polymer arms containing non-reactive functional groups in click chemistry, II) The step of conjugating an activator containing a functional group to at least one of the at least two polymer arms of the dendritic repeating unit precursor that contain a functional group that does not react in click chemistry, III) Providing a core unit having at least three connecting portions c, and polymer arms connected to the core unit, each having a functional group suitable for click chemistry (e.g., azide, alkyne, alkene, or tetrazine) at the end of each polymer arm, The method comprising the step of forming a hyperbranched polymer-activator conjugate by forming a click chemistry linkage between the polymer arm connected to the core provided in step III) and the polymer arm containing a functional group suitable for forming a click chemistry linkage of the activator conjugate dendritic repeating unit precursor obtained in step II).

46. The dendritic repeating unit precursor in step I) is represented by formula (iii), 【Transformation 6】 In the formula, C comprises a functional group suitable for click chemistry (e.g., alkyne, alkene, azide, or tetrazine), D contains a functional group that does not react in click chemistry (e.g., succinimidyl), L Ａ , L Ｂ The method according to claim 45, wherein m, n, X, o, p, and y are as defined in the prior claims, and the dendritic repeating units may be the same or different.

47. In the case of a high-generation Gx hyperbranched polymer (where x is an integer from 2 to 10), The activator conjugate dendritic structure repeating unit precursor obtained in step II) A polymer arm containing a non-reactive functional group in click chemistry, A repeating inverse dendritic structure precursor comprising at least two polymer arms containing functional groups suitable for click chemistry (e.g., azide, alkyne, alkene, or tetrazine) is linked by click chemistry, The method according to claim 45 or 46, wherein the functional group that does not react in the click chemistry of one polymer arm is subsequently converted to a click chemistry-suitable functional group before being linked to a further inverse dendritic repeating unit precursor, or before forming a click chemistry link with the polymer arm linked to the core in step IV), thereby forming a high-generation Gx hyperbranched polymer.

48. The method according to claims 45 to 47, wherein dendritic repeating unit precursors having different activators conjugated to the polymer arm are obtained by performing steps I) and II) on each activator-conjugated dendritic repeating unit precursor, and a mixture of the obtained activator-conjugated dendritic repeating unit precursors is used in step IV), thereby forming a superbranched polymer-activator conjugate having different activators in different regions of the surface of the superbranched polymer.

49. The method according to any one of claims 40 to 48, wherein the outermost polymer arm of the superbranched polymer has a terminal maleimide functional group, and a peptide or activator is conjugated to the terminal maleimide functional group via a maleimide-thiol reaction.

50. The method according to claim 49, wherein the terminal maleimide functional group is provided by reacting a DBCO or azide-functionalized terminal functional group of the ultrabranched polymer with a click chemistry linker having an azide or DBCO functional group linked to a maleimide group (such as DBCO-maleimide, DBCO-PEG3-maleimide, DBCO-PEG4-maleimide, or azide-PEG3-maleimide).

51. A superbranched polymer according to any one of claims 1 to 39, for use as a pharmaceutical agent.

52. A treatment method for treating a patient's disease or condition using a superbranched polymer according to any one of claims 1 to 39.

53. The branched polymer for use or therapeutic method according to claim 51 or 52, wherein the superbranched polymer is used for the treatment of the eye.

54. The superbranched polymer for use, or method of treatment, according to claims 51 to 53, is used to treat ocular diseases such as fundus diseases, including any posterior segment eye disease that affects the vascular system and integrity of the retina, macula, or choroid and leads to visual impairment, visual loss, or blindness, particularly posterior segment conditions resulting from age, trauma, or surgical intervention, such as age-related macular degeneration (AMD), cystoid macular edema (CME), diabetic macular edema (DME), posterior uveitis, and diabetic retinopathy.

55. The aforementioned superbranched polymer is used in the following conditions: retinal neovascularization, choroidal neovascularization, exudative AMD, atrophic AMD, retinal vein occlusion, diabetic macular edema, retinal degeneration, anterior chamber hemorrhage, presbyopia, corneal transplant rejection, retinoblastoma, melanoma, miosis, mydriasis, glaucoma, conjunctivitis, intraocular infection, choroidal neovascularization (CNV), intraocular tumors, retinal neuritis, inflammation, autoimmune uveitis, uveitis-related conditions, proliferative vitreoretinopathy, corneal degeneration, acute and chronic macular neuroretinopathy, central serous chorioretinopathy, and macula. Edema, acute multifocal placoid pigment epitheliopathy, Behçet's disease, birdshot chorioretinopathy, posterior uveitis, posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusion, central retinal vein occlusion, disseminated intravascular coagulation, branch retinal vein occlusion, hypertensive fundus changes, ocular ischemic syndrome, retinal artery microaneurysms, Cort's disease, parafoveal telangiectasia, unilateral retinal vein occlusion, papillary phlebitis, carotid artery disease (C AD), dendritic vasculitis, sickle cell retinopathy, vascular streaks, familial exudative vitreoretinopathy, Eels' disease, proliferative vitreoretinopathy, diabetic retinopathy, tumor retina, congenital retinal pigment epithelial hypertrophy (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, complex hamartoma of the retina and retinal pigment epithelium, retinoblastoma, fundus vascular proliferative neoplasm, retinal astrocytoma, intraocular lymphoma, myopic retinal degeneration, acute retinitis pigmentosa, glaucoma, endophthalmitis, cytomegalovirus A superbranched polymer for use according to claims 51 to 54, or a method of treatment, used for the treatment of an eye disease selected from the group consisting of illus retinitis, retinal cancer, retinitis pigmentosa, Leber congenital amaurosis, choroidal agenesis, X-linked retinitis pigmentosa, Best vitiligo macular degeneration, X-linked retinoschisis, CNGA-3 color blindness, CNGB-3 color blindness, LHON, Stargardt disease, Usher syndrome, Norrie disease, Baldett-Beedl syndrome, and red-green color blindness.

56. The superbranched polymer is formulated to be injected directly into the treatment site of a patient by, for example, parenteral administration, intratumoral injection, intravitreous, anterior chamber, subconjunctival, retrobulbar, subtenon's capsule, subretinal, or suprachoroidal injection, according to any one of claims 51 to 55, a superbranched polymer for use or a treatment method.

57. The superbranched polymer for use or therapeutic method according to any one of claims 51 to 56, wherein the superbranched polymer is administered by direct injection, by oral administration, incorporated into a gel, or incorporated into an implant.

58. The superbranched polymer for use or therapeutic method according to any one of claims 51 to 57, wherein the superbranched polymer comprises two or more different activators in different dendrons or regions on the surface of the superbranched polymer.

59. A superbranched polymer for use according to claim 54, or a therapeutic method, for use in combination therapy involving the administration of multiple activators.

60. The superbranched polymer comprises one activator at different positions or regions on the surface of the superbranched polymer, wherein the activator has different hydrolyzable groups to vary the rate at which the activator is released, according to any one of claims 51 to 59, a superbranched polymer for use, or a therapeutic method.

61. The superbranched polymer comprises two or more activators at different positions or regions on the surface of the superbranched polymer, wherein the activators have different hydrolyzable groups to change the release of the same or different activators at different rates, the superbranched polymer according to any one of claims 1 to 39, the superbranched molecule for use according to any one of claims 51 to 59, or a therapeutic method.

62. The activators (or multiple activators) are bonded to the polymer by hydrolyzable or non-hydrolyzable links or ligatures, optionally via extenders, or in combination thereof, according to any one of claims 1 to 39, the superbranched polymer according to any one of claims 51 to 61, or the therapeutic method.

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