Dendrimer-like hyperbranched macromolecule based on poly (ethylene glycol), and preparation method and application thereof

Abstract

In certain embodiments, the present invention relates to dendrimer-like hyperbranched macromolecules for use in several uses, such as medical or biopharmaceutical applications, or non-medical or industrial applications, such as antibody purification, cosmetic applications, catalytic applications, applications in electronics, agriculture, food, filtration, and other applications. In an embodiment, the present invention relates to a hyperbranched macromolecule for drug delivery comprising a polyethylene glycol (PEG) unit and at least one active agent conjugated to the hyperbranched macromolecule. In addition, methods for synthesizing, purifying and characterizing such hyperbranched macromolecules and methods of treating medical conditions, such as treating ocular diseases, are provided.

Description

Invention Field

[0001] In some embodiments, the present invention relates to dendritic polymer-like hyperbranched macromolecules for various uses, such as medical or biopharmaceutical applications, or non-medical or industrial uses, such as antibody purification, cosmetic applications, catalytic applications, applications in electronics, agriculture, food, filtration, and other applications. In medical applications, the hyperbranched molecules are conjugated with active agents such as pharmaceuticals, peptides, or proteins. Additionally, in some embodiments, the present invention relates to a hyperbranched macromolecule for drug delivery comprising polyethylene glycol (PEG) units and at least one active agent conjugated to the hyperbranched macromolecule. In some other embodiments, the present invention relates to methods for synthesizing, purifying, and characterizing such dendritic polymer-like hyperbranched macromolecules. In some embodiments, the invention also relates to methods for treating medical conditions, such as treating eye diseases. Background of the Invention

[0003] In recent years, controlled delivery and stabilization of therapeutic agents has become a major area of ​​research. Controlled delivery can improve therapy, facilitate administration, and lead to enhanced efficacy, better compliance, fewer side effects, and better overall treatment outcomes.

[0004] The eye is a perfectly formed and complex organ, a microcosm of the body in many ways. It offers a significant opportunity for nanomedicine due to its readily available resources, allowing for direct drug / gene delivery to maximize therapeutic efficacy and minimize side effects. Developing appropriate delivery systems that can sustainably deliver therapeutic agents to target tissues is a key challenge that nanotechnology can address. Current delivery systems used for anterior segment conditions (e.g., punctal plugs, micron and nanoparticle encapsulation, microneedle systems, iontophoresis, various types of intravitreal implants, etc.) represent the most advanced tools for sustained and controlled drug release in the eye.

[0005] Over the past two decades, dendritic and hyperbranched polymers have attracted the attention of scientists in the fields of drug and gene delivery due to their versatility, complexity, and multibranching properties. Dendritic polymers are tree-like, highly symmetrical, monodisperse, branched nanostructured polymers with well-defined dimensions, customizable structures, and potentially favorable ocular biodistribution as reproducible building blocks. Dendritic polymers have been extensively explored as a novel platform for delivering bioactive agents due to their unique biological properties (e.g., high drug loading, lipid bilayer interactions, targeting potential, plasma retention time, filtration, intracellular internalization, biodistribution, transfection, good colloid properties, and biostability). Many dendritic polymers have been explored for drug delivery, including polymer-based dendritic polymers such as polyamide amine (PAMAM), poly(propyleneimide) (PPI), polyester, polyether, poly-L-lysine, triazine, melamine, poly(glycerol-co-succinic acid), poly(glycerol), and poly[2,2-bis(hydroxymethyl)propionic acid] dendritic polymers, as well as other types of dendritic polymers made from peptides, liquid crystal-formed dendritic polymers, carbosilanes, etc. (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, XinQiao, and Hu Yang, Journal of Biological Engineering (2022) 16:18).

[0006] The application of dendritic polymers in drug delivery is of particular interest in the treatment and management of ocular diseases. Ocular drug therapy has several significant drawbacks, including frequent administration, poor penetration, and / or rapid elimination. The use of dendritic polymers as a strategy to overcome the barriers of traditional treatments for ocular diseases shows promising progress in this field, and ocular safety approaches using dendritic polymers are expected to be the most advanced science to date. Several ocular applications of dendritic polymers and dendritic polymer delivery systems are known; see “Dendrimer as nanocarrier fordrug delivery”, Prashant Kesharwani, Keerti Jain, Narendra Kumar Jain, Progress in Polymer Science 39 (2014) 268-307. However, most of these applications remain in the early stages of laboratory exploration, and to date, only a very small number of commercial products using dendritic polymers for the delivery of treatments for ocular diseases are known.

[0007] Therefore, the industry needs to provide strategies for optimizing drug delivery and site-specific targeting. It also needs to provide delivery systems for sustained drug delivery, allowing for increased half-life of active agents and enhanced efficacy and bioavailability. For the delivery of biomolecules such as peptides and proteins, it is also necessary to improve receptor-binding affinity and prolong half-life by providing multivalent delivery possibilities.

[0008] In addition, dendritic polymers have a variety of applications in non-medical or industrial fields, such as antibody purification, cosmetics, catalysis, electronics, agriculture, food, filtration, energy storage, building materials, and other applications.

[0009] All references cited in this article are incorporated herein by reference for all purposes. Summary of the Invention

[0010] Therefore, some embodiments of the present invention aim to provide a dendritic polymer platform based on polyethylene glycol building blocks, which are highly variable and flexible, suitable for a number of uses and applications, and easy to synthesize.

[0011] Another objective of certain embodiments of the present invention is to provide a system with optimized drug delivery and site-specific targeting, specifically for the treatment of eye diseases.

[0012] Another objective of certain embodiments of the present invention is to provide a delivery system for sustained-release drug delivery that allows for increased half-life of the active agent, thereby enhancing the efficacy, affinity, and bioavailability of the active agent.

[0013] Another objective of certain embodiments of the present invention is to provide a biodegradable drug delivery system that can utilize a wide range of different biodegradable molecular groups (including hydrolyzable groups and links constructed in the molecular structure of the drug delivery system) to modulate its degradation rate and active agent release rate. The biodegradable drug delivery system of certain embodiments should be fully absorbable and degradable into initial building blocks that can be readily cleared from local tissues and ultimately eliminated from the body.

[0014] Another objective of certain embodiments of the present invention is to provide a biodegradable drug delivery system that enhances the binding affinity and / or affinity of active agents (e.g., biomolecules, such as peptides or proteins) to biological targets.

[0015] Another objective and aspect of certain embodiments of the present invention is to provide a method for treating a patient’s disease / medical ailment.

[0016] Another objective of certain embodiments of the present invention is to provide dendritic polymers that are also applicable to non-medical or industrial uses, such as antibody purification, cosmetic applications, catalytic applications, applications in electronics, agriculture, food, filtration, energy storage, building materials, coatings, adhesives, water purification, oil extraction, fragrance release, papermaking, environmental sensing and release systems, membranes, textiles, printing inks, surface chemistry applications, thickeners, detergents, rheology modifiers, scaffolds, or 3D printing.

[0017] The aforementioned objective is achieved by an invention as described in the independent claim. Advantageous modifications are disclosed in the dependent claims.

[0018] Some aspects of this disclosure are directed to hyperbranched macromolecules comprising a core unit having at least three linkages (c), a plurality of polymer arms connected to the core unit at the linkages (c), at least one polymer arm being connected by a hydrolyzable bond to a dendritic repeating unit (DCRU), the DCRU comprising branch units connected to at least two polymer arms, each of the at least two polymer arms comprising an end group or connected to a next dendritic repeating unit, the next dendritic repeating unit being reconnected to other dendritic repeating units, the polymer arms of the outermost dendritic repeating unit each comprising an end group; wherein the polymer arms are composed of polyethylene glycol (PEG) units; wherein optionally at least one active agent is conjugated to at least one outermost polymer arm. In some embodiments, at least 10%, preferably about 20% to 100%, of the linked chemical bonds can be cleaved by hydrolysis. The bonds that can be cleaved by hydrolysis are preferably ester bonds. In some embodiments, the ester bonds are introduced by using a linker derived from an organic diacid.

[0019] In some embodiments, the building blocks or fragments of the hyperbranched macromolecule obtained / available after cleaving all hydrolyzable bonds have an average molecular weight (Mn) of less than 50,000 Daltons. The active agent can be covalently or non-covalently bound to the hyperbranched macromolecule. In some embodiments, the active agent is covalently conjugated to the hyperbranched macromolecule.

[0020] In some aspects, the hyperbranched macromolecule is a G0 generation dendritic polymer-like branched macromolecule, wherein the surface end groups of the branched macromolecule are end groups attached to polymer arms that are connected to the core unit but not further connected to the DCRU. In other aspects, the hyperbranched macromolecule is a higher generation Gx dendritic polymer-like hyperbranched macromolecule, where x is an integer from 1 to 10, defining the number of continuously connected dendritic repeating units in the hyperbranched macromolecule, each polymer arm of the outermost dendritic repeating unit containing an end group, wherein at least one active agent is conjugated to at least one outermost polymer arm.

[0021] In some aspects of this disclosure, the core unit and branch units of the hyperbranched macromolecule are the same or different and independently have 3 to 10, 4 to 8, 4 to 6, or 4 linkages c. In some aspects, the core unit and branch units are the same or different and each is derived from a polyol having at least 3 hydroxyl groups. In some aspects, the polyol is selected from the group consisting of glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomaltitol, lactitol, maltitol, mannitol, or sorbitol.

[0022] In some aspects of this disclosure, the polymer arms of the hyperbranched macromolecule comprise PEG units with an average molecular weight (Mw) ranging from about 1,000 Daltons to about 80,000 Daltons, or from about 10,000 Daltons to about 60,000 Daltons, or from about 15,000 Daltons to about 50,000 Daltons. In some aspects, the average molecular weight of the polymer arm PEG units attached 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 attached to the core may be higher or lower than the average molecular weight of the polymer arms in the dendritic repeating unit. For higher-generation Gx hyperbranched macromolecules, where 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.

[0023] In some aspects of this disclosure, the end group attached to the outermost polymer arm is grafted directly or via a bifunctional linker to the end of the polymer arm, the bifunctional linker comprising a hydrolyzable bond such as a carboxyl, dicarboxyl, carboxamide, dicarboxamide, functionalized aliphatic, heteroaliphatic, or aromatic or heteroaromatic group. In other embodiments, the end group attached to the outermost polymer arm is a functional group selected from: electrophiles, such as activated esters, such as succinimidyl esters, succinimidyl carbonate; nitrophenyl carbonate, aldehydes, ketones, acrylates, acrylamide, maleimide, vinyl sulfone, iodoacetamide, alkenes, alkynes, azides, norbornene, epoxides, methanesulfonates, toluenesulfonates, trifluoroethylsulfonyl (tresyl), cyanurate, ortho-di(2 ... Thiopyridine or halogen; nucleophile, such as amine (e.g., primary amine), hydroxyl, alcohol, thiol, azide anion, and carboxyl; functional group for click chemistry; functional group for cycloaddition (such as 1,3-dipolar cycloaddition, [3+2] cycloaddition, [4+2] cycloaddition such as olefin-nitroketone cycloaddition or alkyne-nitroketone cycloaddition); functional group for thiol-alkene reaction; functional group for hetero-Diels-Alder cycloaddition; functional group for nucleophilic ring opening; functional group for non-aldecorative carbonyl reaction; functional group for carbon-carbon multiple bond addition reaction; polymerizable vinyl group or combination thereof.

[0024] In some aspects of this disclosure, the end group attached to the outermost polymer arm is a linker spacer functional group selected from the following: succinimide succinate (SS), succinimide glutarate (SG), succinimide adipate (SAP), succinimide azelate (SAZ), and succinimide glutaramide (SGA).

[0025] In some aspects of this disclosure, the end group attached to the outermost polymer arm is selected from the functional groups of: alkynes such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]-nonyne (BCN); or norbornene or trans-cyclooctene (TCO); azides, 3,4-dihydroxyphenylacetic acid (DHPA) or tetrazine (Tz).

[0026] In some aspects of this disclosure, the connection between the polymer arm attached to the core unit and the first dendritic repeating unit and / or the connection between successive dendritic repeating units is formed by click chemistry.

[0027] In some aspects of this disclosure, the link is formed via click chemistry, wherein the link is formed by reacting a polymer arm partially functionalized with an alkyne, cycloalkyne, or strained or terminal olefin in a SPAAC or IEDDA-type click chemical coupling reaction with a polymer arm partially functionalized with an azide or tetrazine. In some aspects, the alkyne moiety is a dibenzocyclooctyne moiety.

[0028] In some aspects of this disclosure, the connection between the polymer arm connected to the core unit and 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 branch 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.

[0029] In some aspects of this disclosure, the active agent conjugated to at least one end group located on the surface of a hyperbranched macromolecule is selected from the group consisting of therapeutic or diagnostic active agents.

[0030] In certain aspects of this disclosure, the active agent conjugated to at least one end group located on the surface of the dendritic polymer is selected from steroids; nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, ibuprofen, meclofenamate, mefenamic A, salicylate, sulindac, tolmetin, ketoprofen, diflunisal, piroxicam, naproxen, etodolac, flurbiprofen, and fenoprofen C. C) Indomethacin, celecoxib, ketorolac, nepafenac; intraocular pressure-lowering drugs; antibiotics, such as ciprofloxacin; pain relievers, 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; small molecule hydrophobic drugs, hydrophilic peptides and protein drugs, such as insulin, single-chain antibody fragments, F ab fragments, IgG antibodies, fusion antibodies, etc.; aptamers; especially bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, antibiotics, pain relievers, calcium channel blockers, cell cycle inhibitors, chemotherapy agents, antiviral drugs, anesthetics, hormones, anticancer drugs, antitumor agents, viruses, gene-delivering viruses (such as AAV), protein binding agents (such as nanobodies, affinity molecules, ankylosing smears, DARPin, etc.) or any combination thereof.

[0031] In certain aspects of this disclosure, the active agent covalently or non-covalently conjugated to at least one end group located on the surface of a hyperbranched macromolecule is a peptide selected from the group consisting of: compstatin, APL-1 and Fc-III-4C, Beovu (brolucizumab), Zimura (avacincaptadpegol), Pegcetacoplan, Abicipar Pegol, Lampalizumab, Fovista, Risuteganib, AXT107, Elamipretide, THR149, ALM201, VGB3, and Largazole.

[0032] In some aspects of this disclosure, the active agent is covalently bonded to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% of the end groups located on the surface of the hyperbranched macromolecule.

[0033] In some embodiments of this disclosure, the dendritic repeating unit is represented by equation (i):

[0034]

[0035] Where A is the connection to the polymer arm attached to the core unit, or A is the connection to B of the previous dendritic repeating unit represented by equation (i), L A It is a connector, m is 0 or 1, n is an integer from 20 to 2000, o is an integer from 20 to 2000, n and o can be different or the same, X is a branching unit (such as a polyol derivatization unit), L B It is a linker, p is 0 or 1, B contains end groups located on the surface of hyperbranched macromolecules or links to A forming continuous dendritic repeating units or links to surfactants, L A and L B They can be different or the same, m and p can be different or the same, and y is an integer from 2 to 9, where y = c - 1, c is the connectivity of the branching unit X; and the dendritic constituent units in the hyperbranched macromolecule can be the same or different.

[0036] In some other respects, A contains functional groups (such as triazoles or dihydropyrazines) formed by click chemistry, and / or linkers L A and / or L B It contains diacid and / or acid diamide groups, carboxyl and / or carboxamide groups (such as succinate, glutarate, adipate, azelaate, or glutaramide). In some embodiments, the linker LA and / or L B Includes the structure represented by equation (ii):

[0037]

[0038] Among them U 1 and U 2 Independently, it can be NH or O and may be the same or different, where t is an integer from 0 to 10. Connector L A and / or L B A polyethylene glycol unit may be further included between the bond to B and the carboxyl, carboxamide, or structural group of formula (ii).

[0039] In certain embodiments of this disclosure, the present invention provides a method for manufacturing hyperbranched macromolecules as described herein by divergent synthesis, comprising the steps of: (a) providing a core unit having at least three connectivity c, a plurality of polymer arms connected to the core unit having functional groups at the ends of the polymer arms suitable for click chemistry; (b) providing a dendritic constituent repeating unit precursor comprising a polymer arm containing functional groups (such as azides, alkynes, alkenes, or tetrazines) suitable for forming a link with corresponding functional groups of the polymer arms connected to the core via click chemistry, and at least two polymer arms containing functional groups that are non-reactive in click chemistry; (c) forming a link between the polymer arm connected to the core and the polymer arm of the dendritic constituent repeating unit precursor via click chemistry; (d) optionally converting the functional groups of the at least two polymer arms containing functional groups that are non-reactive in click chemistry into functional groups suitable for click chemistry; and (e) conjugating an active agent containing functional groups to the outermost polymer arm by reacting with the functional groups of the outermost polymer arm, thereby forming a hyperbranched macromolecule-active agent conjugate. For higher-generation Gx hyperbranched macromolecules, where x is an integer from 2 to 10, step (d) can be mandatory, and prior to the conjugation of the active agent in step (f), other continuous dendritic constituent repeating unit precursors are linked to the functional groups suitable for click chemistry obtained in step (d) to be linked to the hyperbranched macromolecule via click chemistry.

[0040] In some embodiments of the method, the dendritic constituent repeating unit precursor in step (c) is represented by equation (iii):

[0041]

[0042] Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines), D contains functional groups not suitable for click chemistry (such as succinimide groups), and L A m, n, X, o, L Bp and y are as defined above, and the repeating units of the dendritic structures can be the same or different.

[0043] In another embodiment of the method, the present invention relates to a method for manufacturing hyperbranched macromolecules as described herein by aggregation synthesis, comprising the steps of: (I) providing a dendritic repeating unit precursor comprising a polymer arm containing functional groups (such as azides, alkynes, alkenes, or tetrazines) suitable for linking via click chemistry, and at least two polymer arms containing functional groups that are non-reactive in click chemistry; (II) conjugating an active agent containing functional groups to at least one of the at least two polymer arms containing functional groups that are non-reactive in click chemistry of the dendritic repeating unit precursor; (III) providing a core unit having at least three linkages c, to which a plurality of polymer arms are connected, having at the ends of the polymer arms functional groups (such as azides, alkynes, alkenes, or tetrazines) suitable for click chemistry; and (IV) forming a link via click chemistry between the polymer arm connected to the core provided in step III) and the polymer arm containing functional groups suitable for linking via click chemistry of the dendritic repeating unit precursor obtained in step II), thereby forming a hyperbranched macromolecule-active agent conjugate. In an implementation of this method, the dendritic constituent repeating unit precursor in step I) is represented by equation (iii) as described above.

[0044] For higher-generation Gx hyperbranched macromolecules, 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 a reverse dendritic repeating unit precursor, which comprises a polymer arm containing a functional group that is non-reactive in click chemistry, and at least two polymer arms containing functional groups suitable for click chemistry (such as azides, alkynes, alkenes, or tetrazines). The non-reactive functional group of one polymer arm is subsequently converted into a functional group suitable for click chemistry before being linked to other reverse dendritic repeating unit precursors or before being linked by click chemistry to the polymer arm linked to the core in step IV), thereby forming a higher-generation hyperbranched macromolecule. In one aspect, dendritic repeating unit precursors with different active agents conjugated to polymer arms can be obtained by performing steps I) and II) on each active agent conjugated dendritic repeating unit precursor, and then the mixture of the obtained active agent conjugated dendritic repeating unit precursors is used in step IV), thereby forming hyperbranched macromolecule-active agent conjugates with different active agents in different regions of the hyperbranched macromolecule surface.

[0045] In another embodiment, the present invention relates to hyperbranched macromolecules as described herein, which are used as pharmaceuticals. In another embodiment, the present invention relates to treatment methods, wherein the methods comprise treating a patient's disease or medical condition with the hyperbranched macromolecules of the embodiments of the present invention. In one aspect, the hyperbranched macromolecules are used for ocular treatments, such as treating ocular diseases, such as posterior ocular diseases, such as any posterior segment ocular disease affecting the retinal, macular, or choroidal vascular system and integrity, resulting in visual acuity impairment, vision loss, or blindness, particularly posterior segment disease states caused by age, trauma, or surgical intervention, such as age-related macular degeneration (AMD), cystic macular edema (CME), diabetic macular edema (DME), posterior uveitis, and diabetic retinopathy.

[0046] In other areas, hyperbranched macromolecules are used to treat ocular diseases selected from the group consisting of: retinal neovascularization, choroidal neovascularization, wet AMD, dry AMD, retinal vein occlusion, diabetic macular edema, retinal degeneration, anterior chamber hemorrhage, presbyopia, corneal graft 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 squamous pigment epithelial lesions, and Behcet's disease. Diseases including shotgun retinal choroidal lesions, posterior uveitis, posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusive disease, central retinal vein occlusion, disseminated intravascular coagulation, retinal branch vein occlusion, hypertensive fundus changes, ocular ischemia syndrome, retinal artery microaneurysms, Coat's disease, parafoveal telangiectasia, hemiretinal vein occlusion, optic papillary phlebitis, carotid artery disease (CAD), frost-like dendritic vasculitis, sickle cell retinopathy, vascular streaks, familial exudative vitreoretinopathy, and Eales' disease. Diseases including proliferative vitreoretinopathy, diabetic retinopathy, tumor-associated retinal diseases, congenital hypertrophy of retinal pigment epithelium (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, combined retinal and retinal pigment epithelial hamartoma, retinoblastoma, fundus angiogenesis tumors, retinal astrocytomas, intraocular lymphomas, myopic retinal degeneration, acute retinal pigment epitheliitis, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal carcinoma, retinitis pigmentosa, Leber's congenital amaurosis, choroidal agenesis, X-linked retinitis pigmentosa, best vitelliform macular dystrophy, X-linked retinoschisis, CNGA3 achromatopsia, CNGB3 achromatopsia, LHON, Stargardt disease, Usher syndrome, Norrie disease. Diseases, Bardet-Biedl syndrome, and red-green color blindness.

[0047] In some implementations, hyperbranched macromolecules are formulated for direct injection at the patient's treatment site, such as via parenteral administration, intratumoral injection, or injection into the eye, such as intravitreal, intraanterior chamber, subconjunctival, retrobulbar, subtenon, subretinal, or suprachoroidal injection. Hyperbranched macromolecules can be administered via direct injection, oral administration, incorporation into gels, or incorporation into implants.

[0048] definition

[0049] The terms “hyperbranched macromolecule” or “hyperbranched polymer” or simply “branched polymer” or “branched macromolecule” are used interchangeably herein to refer to dendritic polymer-like branched macromolecules or polymers having a tree-like structure (such as dendritic polymers), and the term “dendritic polymer” is used herein as a synonym for it. However, while dendritic polymers are precisely defined monodisperse, highly symmetrical molecules, the hyperbranched macromolecules of the present invention are polydisperse molecules because they include polyethylene glycol arms or units that have a certain degree of polydispersity, similar to the structures of most synthetic polymers. PEG chains and the precursor molecules comprising them may have lower polydispersity, but they also impart polydispersity to dendritic polymer-like hyperbranched macromolecules as described herein.

[0050] Polydispersion is given by the polydispersion index D, where D = M w / M n M w It is the weight-average molar mass and M n It is the number-average molar mass, determined by gel permeation chromatography. For most polyethylene glycol (PEG) materials, the polydispersity index is a parameter given by the manufacturer in the product specifications as an indicator of material uniformity and quality. The polydispersity of PEG multi-arm precursors can be less than 1.3, less than 1.2, or less than 1.1.

[0051] The term "biodegradable" refers to a material or object (such as the hyperbranched macromolecules according to the invention) that degrades in vivo (i.e., when placed in a human or animal body) or in vitro when immersed in an aqueous solution under physiological conditions (e.g., pH 7.2-7.4, 37°C). In the context of this invention, as detailed below, the hyperbranched macromolecules immediately and slowly biodegrade upon application or deposition in a human or animal body and are cleared over time. In some embodiments, biodegradation occurs at least partially via ester hydrolysis in the body's aqueous environment. Biodegradation can also occur within linking groups and / or polymer arms via hydrolysis of covalently linked or conjugated bonds or enzymatic cleavage. The hyperbranched macromolecules slowly disintegrate, thereby being cleared through physiological pathways. In some embodiments, the hyperbranched macromolecules of the invention are degradable and stable over extended time periods (e.g., about 1 month, 3 months, or 6 months). In some embodiments, the hyperbranched macromolecules only biodegrade after, for example, an active agent or at least a majority (e.g., at least 50%, at least 75%, or at least 90%) of it has been released therefrom.

[0052] The terms “precursor” or “component” or “building block” in this document refer to those molecules or compounds that react with each other and are thus linked by covalent bonds to form hyperbranched macromolecules.

[0053] The portion of the precursor molecule that remains in the final dendritic polymer-like hyperbranched macromolecule is also referred to herein as a “unit” or “polymer arm.” Therefore, a “unit” or “polymer arm” is a major building block or component of the hyperbranched polymer macromolecule. For example, in addition to the core unit and branching unit as further disclosed herein, the hyperbranched macromolecules suitable for use in this invention may contain the same or different polyethylene glycol units or arms.

[0054] As used herein, the term "core unit" refers to a constituent unit emanating from the center of a hyperbranched macromolecule, either a polymer arm or a dendritic repeating unit (DCRU) or a dendron. The core unit has at least three linkages (or valences), from which the polymer arm or dendritic repeating unit is covalently attached, to each of these linkages. For example, in a G0 generation multi-arm PEG branched macromolecule, the core unit may be derived from a polyol compound in which each hydroxyl group is poly(ethoxylated).

[0055] An exemplary core unit structure with three connectivityes is shown below, where connectivity c is shown as OH:

[0056]

[0057] As used herein, the term "branching unit" or "branch point" refers to a constituent unit within a dendritic repeating unit having at least three connectivity c' (or valences), to which a polymer arm or another dendritic repeating unit is connected. The branching unit may have the same or different chemical structure as the core unit.

[0058] As used herein, the term "dendritic constituent repeating unit" (DCRU, sometimes referred to as "dendritic motif") refers to a constituent repeating unit with connectivity c' ≥ 3, including branch points and polymer arms emanating from them. It can connect to a total of c polymer arms continuously emanating from the core unit and / or other DCRUs to form a dendritic polymer-like hyperbranched structure.

[0059] As used herein, the term "terminal group" refers to a constituent unit, such as a functional group, located at the end of a polymer arm or DCRU. In hyperbranched macromolecules, terminal groups on the outermost hyperbranched macromolecular surface can be used to conjugate or bond surfactant molecules to the hyperbranched macromolecule. Terminal groups can consist of linkers having hydrolyzable groups attached to the terminal functional group.

[0060] The term "generation" (abbreviated as "G") refers to a set of dendritic repeating units separated from free-valence dendritic units by the same number of dendritic repeating units.

[0061] As used herein, the term "dendritic motif" refers to a portion of a hyperbranched macromolecule that has only one free valence, contains only DCRUs and end groups, and in which each pathway from the free valence to any end group contains the same number of constituent repeating units.

[0062] As used herein, the term "conjugated" includes covalent or non-covalent bonding of an active agent to a hyperbranched macromolecule. Conjugation includes non-covalent bonding, such as non-covalent bonding to a hyperbranched macromolecule end group that has an affinity for the active agent molecule, which can also be a way of linking an active agent molecule to a hyperbranched macromolecule.

[0063] As used herein, the term "release" (and the corresponding terms "released," "releasing," etc.) refers to the chemical separation of the active agent from the hyperbranched macromolecule of the present invention and its delivery to the surrounding environment. The released agent may or may not have molecular fragments of the hyperbranched macromolecule still bound to it. The surrounding environment may be an in vitro or in vivo environment as described herein. In certain specific embodiments, the surrounding environment is vitreous fluid and / or ocular tissues, such as the retina and choroid. The link between the API and the hyperbranched macromolecule may be a covalent link, wherein the API can detach from the hyperbranched macromolecule via a chemical event, such as hydrolysis of the linker group. Alternatively, a variety of hydrolyzable linker chemistry can be employed to release the API from the same or blended hyperbranched macromolecules at multiple rates to achieve a desired release profile. Furthermore, the hyperbranched macromolecule can be functionalized with non-covalently bound API end groups to release the API according to the binding affinity kinetics of the hyperbranched macromolecule end-API pair. Multiple non-covalently bound end-API pairs can be employed to achieve a desired release profile.

[0064] The term "100% release of the active agent" should be understood as 95% to 100%. This controlled release is achieved through a variety of parameters specific to drug delivery systems as disclosed herein. Each of these characteristic features of a drug delivery system may be responsible for controlled release, either individually or in combination with each other.

[0065] The term "sustained release" used for the purposes of this invention is intended to characterize products such as biodegradable hyperbranched macromolecules, formulated to make the active agent available over an extended period of time, thereby allowing for a lower dosing frequency compared to immediately released dosage forms (e.g., solutions of active agents applied topically to the eye, i.e., eye drops). Other terms that may be used interchangeably with "sustained release" herein are "extended release" or "controlled release." Within the meaning of this invention, the term "sustained release" includes constant active agent release, gradually decreasing active agent release, incremental active agent release, and any combination thereof, such as constant active agent release followed by gradually decreasing active agent release. Within the meaning of this invention, the term "gradually decreasing" or "gradually decreasing" refers to a decrease in active agent release over time. Specifically, the term "sustained release" refers to the release of the active agent from a hyperbranched macromolecule or a drug delivery system comprising it in a predetermined manner, in contrast to immediately released (e.g., bolus injection). In some implementations, controlled release refers to the amount of active agent released within the total number of days required to achieve 100% release of the active agent in aqueous solution under in vitro physiological conditions (e.g., at pH 7.2–7.4 and 37°C).

[0066] As used herein, the term “extended time period” means any period of time that a person skilled in the art would consider to be an extension of the treatment of a disease, and specifically refers to a period of at least about one week, or at least about one month or longer (e.g., up to about 12 months), or any intermediate period of about one month to about six months, about two months to about four months, about two months to about three months, or about three months to about four months, or other periods as disclosed herein.

[0067] "Zero-order" release, or "substantially zero-order" release, or "near-zero-order" release, is defined as a relatively linear relationship in a graphical representation of the percentage of active agent released versus time. In some embodiments of the invention, substantially zero-order release is defined as the amount of active agent released being proportional to the amount of time elapsed within 20%.

[0068] The terms “API,” “active (drug) ingredient,” “active (drug) agent,” “active (drug) substance,” “(active) therapeutic agent,” “active substance,” and “drug” are used interchangeably in this document and refer to substances used in finished pharmaceutical products (FPPs) and substances used in the preparation of such finished pharmaceutical products to provide pharmacological activity, or otherwise to have a direct effect in the diagnosis, cure, relief, treatment, or prevention of a disease, or to restore, correct, or alter the physiological function of a patient.

[0069] The active agent used according to the present invention can be an active agent for treating and / or preventing diseases or conditions, or a diagnostic agent such as a marker. In embodiments of the present invention, the active agent is an active agent with low water solubility (i.e., having a water solubility of less than about 1000 μg / mL or less than about 100 μg / mL). In other embodiments of the present invention, the active agent is an active agent with high water solubility (i.e., having a water solubility of greater than about 1000 μg / mL or even greater than 10 mg / mL). This definition does not depend on agents approved by government agencies.

[0070] For the purposes of this invention, all possible forms of active agents may be used, including free acids, free bases, polymorphs, or any pharmaceutically acceptable salts, anhydrous forms, hydrates, cocrystals, or other solvates or derivatives, such as prodrugs or conjugates. For conjugation with hyperbranched macromolecules, the active agent may need to be functionalized unless it already contains functional groups suitable for conjugation. Whenever an active agent is mentioned in this specification or claims without further description, it refers to the active agent in the form of any such polymorph, pharmaceutically acceptable salt, anhydrous form, or solvate (including hydrates), even if not explicitly stated. Suitable solid forms of active agents include (but are not limited to) pure forms of any physical form known to those skilled in the art.

[0071] As used herein, the term "therapeuticly effective" refers to the amount of active agent required to produce the desired therapeutic outcome after application. For example, in the context of this invention, a desired therapeutic outcome would be a reduction in symptoms associated with dry eye disease (DED), such as an increase in Schirmer's tear test score as measured by in vivo tests known to those skilled in the art, a decrease in staining values ​​as measured by conjunctival lissamine green staining or corneal fluorescein staining, a decrease in scores for the severity and / or frequency of dry eye on the Visual Analogue Scale (VAS), a decrease in the Ocular Surface Disease Index and / or Standard Patient Dry Eye Assessment score, and a decrease in best-corrected visual acuity. In one implementation, “therapeutic efficacy” means that the amount of active agent in the continuously released intratubular insert is sufficient to achieve a tear concentration equivalent in therapeutic effect to 0.236 μg / mL of cyclosporine (considered essential for immunomodulation, Tang-Liu and Acheampong, Clin. Pharmacokinet. 44(3), pp. 247-261), over an extended period of time and specifically over substantially the entire remaining wear time of the insert after that tear concentration is achieved.

[0072] The term "patient" herein includes both human and animal patients. Therefore, the biodegradable drug delivery system of the present invention is suitable for human or veterinary medical applications. Generally, a "subject" is an individual (human or animal) to whom the drug delivery system according to the present invention is administered. A "patient" is a subject who requires treatment due to a specific physiological or pathological condition. A "patient" does not necessarily need to be diagnosed with a specific physiological or pathological condition before receiving the drug delivery system.

[0073] The molecular weights of hyperbranched macromolecules, polymer precursors, polymer units, arms, etc., used for the purposes of this invention and as disclosed herein, can be determined by analytical methods known in this art. The molecular weight of polyethylene glycol can be determined, for example, by any method known in this art, including gel electrophoresis (such as SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)), gel permeation chromatography (GPC, including GPC with a static light scattering detector (SLS) or a dynamic light scattering detector (DLS)), liquid chromatography (LC), and mass spectrometry (such as matrix-assisted laser desorption / ionization-time-of-flight (MALDI-TOF) spectroscopy or electrospray ionization (ESI) mass spectrometry). The molecular weight of polymers (including polyethylene glycol precursors as disclosed herein) is an average molecular weight (based on the molecular weight distribution of the polymer) and can therefore be indicated by various average values, including weight average molecular weight (Mw) and number average molecular weight (Mn). In the case of multi-arm PEG precursors, as used in some aspects of this invention, the molecular weight indicated herein is the number average molecular weight (Mn) determined by gel permeation chromatography using a suitable molecular weight standard (such as a polyethylene glycol or polystyrene standard) according to standard methods known in the art. Typically, the purchased materials, especially multi-arm precursors, have a specified molecular weight and polydispersity defined by the supplier. Suitable PEG precursors are available from, for example, several suppliers (such as Jenkem Technology, Xiamen SinoPeg Biotech Co., Ltd., and others).

[0074] As used in this article, the term "Day 1" refers to the point in time immediately following "Day 0". Therefore, whenever "Day 1" is used, it refers to a period of time that has elapsed, one day or approximately 24 hours after the administration of the drug delivery system.

[0075] As used in this article, the term “about” refers to the normal variation of the measured quantity, as would be expected by a person skilled in the art when performing measurements and applying a level of caution commensurate with the accuracy of the measurement target and measuring equipment.

[0076] The term “at least about” in conjunction with the measurement quantity refers to normal variations in the measurement quantity, such as those expected by a person skilled in the art when performing the measurement and applying a level of caution commensurate with the accuracy of the measurement target and measuring equipment, as well as any quantity above that level.

[0077] As used in this article, the term "average" refers to the central or typical value in a set of data (points), which is calculated by dividing the sum of the data (points) in the set by its quantity (i.e., the average of a set of data).

[0078] Unless the context clearly indicates otherwise, the singular forms “a” and “the” as used herein include multiple indicators.

[0079] The term “and / or” as used in phrases such as “A and / or B” is intended to include both “A and B” and “A or B”.

[0080] Open terms (such as "include", "including", "contain", "containing", etc.) mean "comprising". These open transition phrases are used to introduce an open list of elements, methods, etc., which does not exclude additional, unlisted elements or methods.

[0081] The term “high” is intended to include individual values ​​or numbers when used in conjunction with a value or number in this text.

[0082] The terms “from A to B”, “from A to B”, and “from A to B” are used interchangeably in this document and all refer to the range from A to B, including the upper limit A and the lower limit B.

[0083] Throughout this disclosure, various aspects of the invention are presented in a scope format. It should be understood that the scope format is for convenience and brevity only and should not be construed as an inflexible limitation of the scope of the invention. Therefore, the description of scope should be considered as the specific disclosure of all possible sub-scopes and individual numerical values ​​within those scopes. For example, a description of a scope such as 1 to 6 should be considered as the specific disclosure of sub-scopes (such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.) and individual numbers within that scope (e.g., 1, 2, 3, 4, 5, and 6). This applies regardless of the breadth of the scope. The enumerated numerical ranges include the numbers defining the range and include every integer within the defined range.

[0084] The abbreviation "PBS" as used in this article refers to phosphate-buffered saline.

[0085] The abbreviation "PEG" in this article refers to polyethylene glycol. Attached Figure Description

[0086] Figure 1 The schematic diagrams illustrate the different synthetic methods for dendritic polymers or hyperbranched macromolecules a)-c).

[0087] Figure 2 A schematic diagram illustrating the generations of dendritic polymers or hyperbranched macromolecules.

[0088] Figure 3This diagram shows a) a schematic of the formation of hyperbranched PEG macromolecules via DBCO-azide coupling; and b) a schematic illustration of a 3D model of an 8-arm PEG core with eight 4-arm PEG branched repeating units conjugated with peptides.

[0089] Figure 4 The diagram illustrates the structures of the peptides campstatin, APL-1 (Mod2), and APL-1 (Mod3).

[0090] Figure 5 The illustrations illustrate the purification setups of Examples 6 and 7, using a) dialysis and b) SEC column filtration.

[0091] Figure 6 This is a chromatogram of UHPLC analysis of the hyperbranched macromolecule-campatidine conjugate purified by dialysis in Example 6.

[0092] Figure 7 This is a chromatogram of UHPLC analysis of the hyperbranched macromolecule-campatidine conjugate purified by SEC column filtration in Example 7.

[0093] Figure 8 shows the following UHPLC chromatograms: a) standard curves of the campstatin samples with calibration plots (12.5 µg / mL, 25 µg / mL, 50 µg / mL, 100 µg / mL, 200 µg / mL), and b) the 4-arm G0 hyperbranched macromolecule-campstatin conjugate of Example 1.

[0094] Figure 9 A graph showing a comparison of the substitution rates of different linkers PEG used in Example 8, specifically for campstatin and campstatin-lysine.

[0095] Figure 10 This is a graph showing the optimized conditions for the campstatin conjugation from Example 8.

[0096] Figure 11 illustrates the SPR results of C3 binding in Example 9: a1-4) KD of four campstatin samples from different suppliers; b) Equilibrium analysis.

[0097] Figure 12 illustrates the SPR results of C3b binding in Example 9: a1-4) KD of four campstatin samples from different suppliers; b) Equilibrium analysis.

[0098] Figure 13 Illustrated Example 9: SPR results of C3 and C3b binding: a) APL-1 (amine acetylation), b) APL-1 (amine, acetate), c) APL-1 (lysine terminus).

[0099] Figure 14The diagram illustrates the SPR results of IgG binding: ac) Fc-III 4C, and d) Fc-III.

[0100] Figure 15 A diagram illustrating the SPR comparison between free campstatin and multivalent campstatin.

[0101] Figure 16 The diagram illustrates the SPR results of a) 8a-40k-PEG-[(4a-2kPEG-(campatamine)3]8, b) 4a-40kPEG-SGA-(campatamine)4, c) 4a-40kPEG-SS-(campatamine)4, and d) SS-campatamine (hydrolyzed) compared to free campatamine.

[0102] Figure 17 It is the dissociation constant K of the hyperbranched macromolecule-campatine conjugate. D A graph showing the number of substitutions for the corresponding peptides.

[0103] Figure 18 This is a graphical illustration of the alternative pathway (AP) hemolysis analysis.

[0104] Figure 19 IC50 of hyperbranched macromolecule-campatamine conjugate 50 The results of AP hemolysis.

[0105] Figure 20 The hydrodynamic radius R was determined in the vitreous fluid of a New Zealand White Rabbit. h For half-life T 1 / 2 The calibration curves were used to predict the sustained release of dendritic polymer drug conjugates.

[0106] Figures 21 a) to c) show the degradation effect at a constant pH of 7.4 as the temperature changes from 35°C to 39°C.

[0107] Figures 22 a) to c) show the degradation effect of pH changing from pH 7.0 to pH 8.5 at a constant temperature of 37°C. Detailed Implementation

[0108] In some aspects, the present invention is directed to hyperbranched macromolecules (dendritic polymers) comprising polyethylene glycol polymer units and an active agent covalently bonded or conjugated to at least one outermost arm of the hyperbranched macromolecule. Conjugation includes both covalent and non-covalent bonding, such as covalent and non-covalent bonding to the terminal group of a peptide hyperbranched macromolecule having an affinity for the active agent molecule; this can also be a way of linking the active agent to the hyperbranched macromolecule.

[0109] Dendritic polymers are monodisperse macromolecules with several reactive end groups on their surface. They are often compared to tree-like structures (i.e., branched molecular architectures that offer numerous possible end groups and anomalous structural control). Elements are added to the dendritic polymer structure via a series of chemical reactions, constructing branched, spherical structures from starting atoms or core units. The central core unit has at least two or three reactive functional groups, and repeating branches are organized into a series of "radially concentric layers," called "generations." Hyperbranched macromolecules can have the same molecular architecture as dendritic polymers without being monodisperse, as they can be constructed using polydisperse precursors or units.

[0110] The inventors have discovered that strategies for optimizing drug delivery and site-specific targeting using dendritic polymer-like hyperbranched macromolecules, as described herein, offer several advantages in drug delivery, and that the advantages of dendritic polymers can also be applied to hyperbranched macromolecules having a similar structure to dendritic polymers but not being monodisperse molecules. For example, dendritic polymer-like hyperbranched macromolecules provide a variety of terminal functional groups that can be used to modulate the hydrophobicity / hydrophilicity of hyperbranched macromolecules used as active agent carriers, or can be used as conjugation precursors of target molecules to enhance the interaction between APIs and hyperbranched macromolecules, for example, by multivalent binding to receptors and / or modifying the affinity of the conjugated biomolecule (such as a peptide or protein).

[0111] Multiple surface groups on hyperbranched macromolecules can be used to anchor more APIs using desired bonding methods and achieve controlled release through different degradation conditions or degradation kinetics. Compared to free drugs, hyperbranched macromolecule-drug conjugates can enhance the stability and solubility of the intended therapeutic agent, reduce systemic effects, and increase efficacy at the target site. Furthermore, hyperbranched macromolecules can possess symmetrical structures, providing multiple intramolecular cavities to trap unbound API molecules. Additionally, the large external hydration radius of specific PEG-based dendritic polymer structures prolongs the in vivo (e.g., in the vitreous) half-life of dendritic polymer drug conjugates, which can be used to control and modulate the sustained release of the active agent.

[0112] Furthermore, biodegradable synthetic dendritic polymers offer the advantage of built-in controllable degradable functional groups (such as hydrolyzable or enzymatically cleavable bonds), which, upon degradation, produce smaller molecular weight fragments with lower hydration radii and different half-lives, thus determining their clearance from the body. The built-in degradable groups can be used to tune and control the release rate of active agents associated with the dendritic polymer.

[0113] hyperbranched macromolecules

[0114] In some aspects of the invention, a hyperbranched macromolecule is provided, which is formed of several building blocks (e.g., core unit, polymer arms, branch units, linkers and / or extensions, and dendritic repeating units) of a dendritic structure (excluding an active agent). In embodiments, a hyperbranched macromolecule is provided comprising a core unit having at least three linkages c, a plurality of polymer arms connected to the core unit at linkages c, each polymer arm comprising an end group or connected to a dendritic repeating unit, the dendritic repeating unit comprising branch units connected to at least two polymer arms, each of the at least two polymer arms comprising an end group or connected to a next dendritic repeating unit, the next dendritic repeating unit being reconnectable to other dendritic repeating units, the polymer arms of the outermost dendritic repeating unit each comprising an end group; wherein the polymer arms comprise or are composed of linear polyethylene glycol (PEG) units; and wherein at least one active agent is conjugated to at least one end group of the outermost polymer arm of the hyperbranched macromolecule. The hyperbranched macromolecule includes chemical bonds that can be cleaved by hydrolysis, thereby making the hyperbranched macromolecule biodegradable in an aqueous environment.

[0115] In the implementation, the hyperbranched molecule is formed from building blocks at least partially linked by hydrolyzable bonds or linkages located at positions such that complete hydrolysis of all hydrolyzable bonds in the macromolecule produces hydrolytic fragments, each with a molecular weight less than 40 kDa. This can be achieved by selecting suitable building blocks or precursors with a molecular weight less than 40 kDa and linking them by hydrolyzable, typically acid-labile chemical bonds (such as ester or amide bonds as further described herein). For example, including diacid linkers to link constituent repeating units, each with a molecular weight less than 40 kDa, allows hydrolytic degradation to produce hydrolytic fragments that each meet the desired molecular weight limit. If non-hydrolyzable linkages are used, such as those formed through some click chemistry (such as alkyne-azide coupling), they should be located between building blocks that together meet the molecular weight limit of hydrolytic fragments less than 40 kDa.

[0116] In some aspects of this invention, the overall molecular size and number of surface groups of the hyperbranched macromolecule gradually increase with the addition of successive monomer layers, referred to as a first generation. Biodegradable hyperbranched macromolecules can be synthesized by divergent or convergent synthesis or a combination of both; see [link to relevant documentation]. Figure 1 The divergent approach involves adding monomers, or so-called dendritic repeating units (DCRUs), in a repeating sequence, increasing the number of branches from the multivalent core to the surface molecules. Molecular size and the number of surface groups increase progressively with the addition of successive monomer layers, termed generations. While the convergent approach involves synthesizing hyperbranched macromolecules from the surface to the core and results in the formation of cone-wedge units or dendritic motifs, these units or dendritic motifs are linked to the multivalent core in a final step.

[0117] The hyperbranched macromolecules of certain embodiments of the present invention include a core unit as the main building unit and multiple branch units optionally derived from polyols, multiple polymer arms comprising polyethylene (PEG) units, optionally present hydrolyzable linking groups, linking groups between building units, terminal groups, and conjugating agents (such as peptides). All these components or building units are further described below.

[0118] By introducing suitable linking groups between the PEG arms and functional groups that connect different units to form hyperbranched macromolecules, the links formed between different polymer arms in a hyperbranched macromolecule can include hydrolyzable bonds. Such linkers forming hydrolyzable bonds promote in vivo biodegradation in aqueous environments, such as the human or animal body. For example, the hydrolyzable chemical bonds can be acid-labile to facilitate cleavage in more acidic environments, such as those found at the cellular level within tumors.

[0119] Hydrolyzable chemical bonds can include bonds or links selected from the group consisting of amines, amides, carbamates, esters, acid anhydrides, ethers, acetals, ketals, nitriles, isonitriles, isothiocyanates, or imine bonds and combinations thereof. These bonds are typically formed during the synthesis of hyperbranched macromolecules via condensation reactions or click chemistry of suitable functionalized precursors. In some 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 homologs).

[0120] Exemplary structures of hyperbranched macromolecules according to certain embodiments of the present invention are shown below:

[0121]

[0122] In this formula for a G1 generation hyperbranched macromolecule, X represents a core unit or a branch unit derived from a polyol (such as glycerol). Each core or branch unit is connected to three polyethylene glycol (PEG) arms, and the branch unit is connected to one PEG arm of the central core unit via a linker Y. Linker Y contains hydrolyzable bonds, such as ester or amide bonds as further defined herein. n represents the number of repeating PEG units in the polymer arm.

[0123] The building blocks forming hyperbranched macromolecules comprise a core unit, polymer arms (such as arms composed of polyethylene glycol (PEG), bifunctional linkers or linkers, bifunctional extensions, dendritic repeating units comprising branching units, and functional end groups. In embodiments of the invention, the building blocks have an average molecular weight (Mn) of less than 50,000 Daltons (e.g., less than 45,000 Daltons, or less than 40,000 Daltons, or less than 35,000 Daltons, or less than 30,000 Daltons).

[0124] Core Unit

[0125] The core unit is the center of the hyperbranched macromolecule from which polymer arms or dendritic repeating units (DCRUs) or dendritic motifs emanate. The core unit has at least three connectivity 'c's (or valences), and the polymer arms or dendritic repeating units are covalently bonded to each of these connectivityes. The polymer arms can be linked to the core unit via hydrolyzable bonds, preferably via non-hydrolyzable bonds (such as ether bonds).

[0126] In some embodiments, the core unit has 3 to 10, or 4 to 8, or 4 to 6, or 4 linkages (c). The core unit may be derived from a molecular or chemical structure having multiple c functional groups that bind to the polymer arm. For example, in some 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.

[0127] In such embodiments, the polyol may be selected from glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomaltitol, lactitol, maltitol, mannitol, or sorbitol. In some embodiments, the core unit derived from the polyol is ethoxylated at each of its hydroxyl groups to form a multi-arm precursor, wherein the arms are polymeric PEG arms capped with end groups or functional groups.

[0128] An exemplary core unit structure with three connectivityes can be represented by the following formula, where connectivity c is represented as OH:

[0129]

[0130] Therefore, the core unit that can be used to form multi-arm precursors of hyperbranched macromolecules in certain embodiments of the present invention is a structure suitable for providing the desired number of arms of the precursor. For example, for a 4-arm precursor, the core unit may be a pentaerythritol or ethylenediamine structure, while for an 8-arm precursor, the core unit may be a hexaglycerol structure. In some embodiments of the present invention, the core unit is PEGylated at its connection point with the polyethylene glycol arm, as in the structures illustrated below. The terminal connection is again shown as an OH group, which may connect a linker, a functional group, or another DCRU.

[0131]

[0132] The terminal connectivity or end group is again shown as an OH group, which can be connected to a linker, functional group, extension or another DCRU for other hyperbranched macromolecular generations.

[0133] In embodiments of the invention, building blocks composed of multi-arm PEG precursors, further defined below, derived from ethoxylated polyol core units, are used, and these multi-arm PEG precursor building blocks have an average molecular weight (Mn) of less than 50,000 Daltons (e.g., less than 45,000 Daltons, or less than 40,000 Daltons, or less than 35,000 Daltons, or less than 30,000 Daltons).

[0134] generations

[0135] Similar to that in dendritic polymers, the sequence of repeating units in the hyperbranched macromolecules of this invention can be represented by generations. In some embodiments, the hyperbranched macromolecule can be a G0 hyperbranched macromolecule, or G1 to G10 hyperbranched macromolecules, such as G1, G2, G3, G4, or G5 hyperbranched macromolecules, generally referred to as Gx hyperbranched macromolecules, where x is an integer from 1 to 10. The abbreviation G refers to a generation, and the number represents the total number of dendritic repeating units that are continuously bonded to each other in rows.

[0136] like Figure 2 As shown in the data, the number of branches and the outermost terminal bases increases with each generation.

[0137] In an exemplary embodiment, the hyperbranched macromolecule is a G0-branched macromolecule, wherein the end groups located on the surface of the hyperbranched macromolecule are end groups of polymer arms attached to the core unit. The G0-branched macromolecule can also be described as a multi-armed PEG molecule, wherein the activator is covalently bonded to at least one of its arms.

[0138] In another exemplary embodiment, the hyperbranched macromolecule is a higher-generation Gx hyperbranched macromolecule, where x is an integer from 1 to 10, defining the number of continuously connected dendritic repeating units in the hyperbranched macromolecule, each polymer arm of the outermost dendritic repeating unit containing an end group, wherein at least one active agent is conjugated to at least one outermost polymer arm.

[0139] Exemplary embodiments of the present invention include hyperbranched macromolecules of G1 to G10, such as G1 to G8, G1 to G6 or G1 to G4, such as G1, G2, G3 or G4 hyperbranched macromolecules.

[0140] Different generations of DCRUs can be formed in hyperbranched macromolecules using the same or different DCRUs, such as DCRUs with different molecular weights (attributed to different PEG arm lengths) or different numbers of arms. Furthermore, different generations of DCRUs within a hyperbranched macromolecule can be connected to each other using the same linker and functional groups or using different linkers and functional groups, for example, to control the degradation rate at different junctions within the hyperbranched macromolecule.

[0141] Branch unit

[0142] The branching unit can be selected from the same chemical entity as the core unit described above. Like the core unit, the branching unit is a branched chemical structure that includes branch points and multiple connections.

[0143] Although the core unit is located at the center of the hyperbranched macromolecule and appears only once, branching units appear in the dendritic repeating units (DCRUs) of the hyperbranched macromolecule. G0 hyperbranched macromolecules include a core unit but not branching units. Higher-generation hyperbranched macromolecules, such as Gx, include multiple branching units. Branching units in a hyperbranched macromolecule can have the same chemical structure as the core unit or they can be different. For example, the core unit of a G1 hyperbranched macromolecule can be derived from pentaerythritol with a linkage c = 4. The hyperbranched macromolecule can include 4 DCRUs, and each branching unit can be derived from glycerol with a linkage c' = 3, so that the G1 hyperbranched macromolecule has a total of 8 terminal (outermost) end groups. If the 4 DCRUs have branching units also derived from pentaerythritol, then, like the core unit, the hyperbranched macromolecule will have a total of 12 outermost end groups.

[0144] polymer arm

[0145] In some embodiments, the polymer arms of the hyperbranched macromolecule are composed of polyethylene glycol (PEG) polymer units. In G0 generation branched macromolecules, the polymer arms are connected to the core unit, for example, via ether bonds, and have terminal groups located on the surface of the branched macromolecule. The surfactant is covalently bonded to at least some of these terminal groups. In higher generation Gx hyperbranched macromolecules, the polymer arms additionally appear in continuously connected dendritic repeating units.

[0146] Therefore, in some embodiments, the polymer arms contained in the hyperbranched macromolecule are composed of or include at least one polyethylene glycol unit. Polyethylene glycol (PEG, also known as polyethylene oxide) refers to a polymer having a repeating group (CH2CH2O)n, wherein n is at least 3.

[0147] Therefore, the polymer arm having polyethylene glycol has at least three of the repeating groups linked to each other in a linear series. The PEG polymer arm terminates at an end group, such as a nucleophile or electrophile, dibenzocyclooctylene (or other strained alkynes), strained olefin, tetrazine, or azide, and can be used for conjugation with surfactants or for linking with DCRU precursors to construct next-generation hyperbranched macromolecules.

[0148] The polymer arm may contain PEG units with an average molecular weight (Mn) ranging from about 1,000 Daltons to about 100,000 Daltons, or from about 10,000 Daltons to about 60,000 Daltons, or from about 15,000 Daltons to about 50,000 Daltons.

[0149] In some embodiments, the average molecular weight (Mn) of the polymer arm PEG units attached to the core can be the same as or different from the average molecular weight of the polymer arms in the dendritic constituent repeating unit. For example, the average molecular weight of the polymer arm PEG units attached to the core can be higher or lower than the average molecular weight of the polymer arms in the dendritic constituent unit. In one aspect, for higher generation Gx biodegradable hyperbranched macromolecules, where x is an integer from 2 to 10, the average molecular weight of the polymer arm PEG units can decrease or increase from the innermost polymer arm to the outermost polymer arm. For example, the polymer arms attached to the core unit can have a large molecular weight, and the polymer arms of the DCRU can have a shorter molecular weight, or vice versa. The molecular weight of the polymer arms can also vary depending on the generation of the DCRU. As an example, a G2 hyperbranched macromolecule with 24 outermost conjugation sites can be constructed from a 4-arm 40k PEG core attached to four 4-arm 20k PEG DCRUs, which can then be reattached to twelve 3-arm 30k PEGs. In this context, K refers to kilodaltons (kDa), so 4-arm 40k PEG has 4 polymer PEG arms and a total molecular weight of 40 kDa.

[0150] Dendritic repeating units

[0151] Dendritic constituent repeating units (DCRUs) are partial structures within higher-generation Gx hyperbranched macromolecules as defined herein, possessing connectivity c' ≥ 3 and comprising branch points and polymer arms emanating from them. They can connect to a total of c' polymer arms continuously emanating from the core unit and / or other DCRUs to form a tree-like dendritic polymer structure.

[0152] In some embodiments, the dendritic constituent repeating units in the dendritic polymer can be represented by general formula (i):

[0153]

[0154] In equation (i), A is the connection to the polymer arm attached to the core unit, or A is the connection to B of the previous dendritic repeating unit represented by equation (i), L A X is the linker group, m is 0 or 1, indicating that the linker may or may not exist, n is an integer from 3 to 2000 or from 20 to 2000, o is an integer from 3 to 2000 or from 20 to 2000, and n and o may be different or the same, X is the branching unit, L B It is a linker group, p is 0 or 1, meaning that the linker may or may not exist. B contains end groups located on the surface of hyperbranched macromolecules or links to A forming continuous dendritic repeating units or to surfactants. L A and LB The connections between A and B can be different or the same, m and p can be different or the same, and y is an integer from 2 to 9, where y = c'-1, and c' is the connectivity c' of the branching unit X. In some embodiments of the hyperbranched macromolecule including the DCRU of formula (i), the connection between A and B can contain functional groups formed by click chemistry, such as triazoles or dihydropyrazines.

[0155] The branching unit can be derived from polyols such as glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomaltitol, lactitol, maltitol, mannitol, or sorbitol. In formula (i) above, the branching unit is ethoxylated at all its linkage c, thus it is linked to a PEG polymer arm via an ether linker, which optionally is linked by a linking group L. A Connected to A; and y PEG polymer arms, each of which optionally connects to a linking group L. B Connected to B. The same or different DCRUs can be used in hyperbranched macromolecules, such as DCRUs with different molecular weights (attributed to different PEG arm lengths) or different numbers of arms.

[0156] Linking group

[0157] The inclusion of hydrolyzably unstable linker groups within the dendritic constituent units allows for the biodegradation of hyperbranched macromolecules under physiological conditions. High-molecular-weight hyperbranched macromolecular conjugates can degrade into smaller constituent units with lower molecular weights and be eliminated from the body via normal physiological pathways.

[0158] In some embodiments of hyperbranched macromolecules including DCRU of formula (i), the linking group L A and / or L B The polymer comprises dicarboxyl groups and / or carboxamide moieties of varying chain lengths, or combinations thereof, and these groups may be derived from diacid groups, such as succinate, glutarate, adipate, azelaate, or acid diamide groups (e.g., glutaramide). These groups can be linked to PEG polymer arms A and / or B via ester or amide bonds, which can be hydrolyzed in vivo at different rates under physiological conditions, depending on the acid chain length. In some embodiments, the linker forms an ester bond and is derived from a diacid.

[0159] In some implementations, connector L A and / or L B Includes the structure represented by equation (ii):

[0160]

[0161] Among them U 1 and U 2Independently, it can be NH or O and may be the same or different, and where t is an integer from 0 to 10. For example, in succinate linkers, U 1 and U 2 All are oxygen, and t is 2. For the linker-terminal group, the linker of formula (ii) contains a terminal functional group at one end. For the above examples of succinate linkers, reaction with an N-hydroxysuccinimide group produces a succinate succinimide ester linker-terminal group, which can be used to conjugate amine-functionalized activators with hyperbranched macromolecules having this linker-terminal group on the outermost polymer arm. In some embodiments, the linker L... A and / or L B The bond with B further includes a polyethylene glycol unit between the carboxyl group, carboxamide group, or structure of formula (ii).

[0162] The linkers of formula (ii) introduce hydrolyzable bonds into hyperbranched macromolecules, which can be used to modulate the degradation rate of the hyperbranched macromolecules and / or the release rate of the conjugated active agent from the hyperbranched macromolecules. For example, the biodegradation / hydrolysis rate of the ester bonds at these linkers decreases from succinate (C4) to azelaate (C9). In embodiments of the invention, this can be used to control the degradation rate of hyperbranched macromolecules and / or the release of active agents conjugated to the hyperbranched macromolecules via these linkers. For example, succinate succinimide ester groups (SS) can degrade within days, while glutarate succinimide ester groups (SG) degrade within weeks.

[0163] In the linking group L A and L B At the other end, an end group (such as an ester) can be attached, for example by esterifying the linker acid group with an N-hydroxysuccinimide or a click chemical functional group (such as DBCO or an azide) to form a succinimide group (NHS), as further described below.

[0164] extension

[0165] In addition to linkers or alternative linkers, bifunctional extension units can be incorporated as additional building blocks to extend the length of polymer arms, for example, between arms connected to the core and branching units, to provide further flexibility and / or to provide additional hydrolytic cleavage points in dendritic polymers. Such extensions are typically linear bifunctional polymer chains, such as linear PEG extensions.

[0166] In some embodiments, the hyperbranched macromolecule comprises at least one extension unit containing polyethylene glycol (PEG) units or composed thereof, wherein the extension unit is a linear, bifunctional polymer arm connected to a dendritic repeating unit, or a polymer arm connected to a core unit and connected to an end group or polymer arm of the next dendritic repeating unit.

[0167] In its embodiments, the extension unit includes at least one linker, wherein the linker may be located at either end or both ends of the extension unit and is a bifunctional linker containing a hydrolyzable bond, the 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.

[0168] Extenders can be used to increase the hydration radius of dendritic polymers and increase the in vivo half-life of hyperbranched molecules.

[0169] PEG precursors and DCRU precursors of G0 branched macromolecules

[0170] The core element of the hyperbranched macromolecule in certain embodiments of the present invention may comprise one or more multi-arm PEG precursors having 2 to 10 arms, or 4 to 8 arms, or 4, 5, 6, 7, or 8 arms. It should be noted that, because multi-arm precursors have a core, for example, a 2-arm PEG precursor differs from a simple linear PEG in the presence of a core structure. The PEG precursors used in the hyperbranched macromolecule can have different or the same number of arms. In some embodiments, the PEG precursors used in the hyperbranched macromolecule of the present invention have 3, 4, and / or 8 arms. In some embodiments, combinations of 4-arm and 3-arm PEG precursors, or combinations of 4-arm and 8-arm PEG precursors, and any combination thereof are used. For example, an 8-arm core unit may be combined with eight 4-arm DCRU precursors, which may again be linked with 24 precursors having 3 arms, to produce a hyperbranched macromolecule with 48 conjugate sites on the outermost arm. In another exemplary embodiment, the 4-arm core unit can be combined with four 3-arm precursors, which can then be reconnected to eight 4-arm precursors to produce a hyperbranched macromolecule with 24 conjugation sites on the outermost arm. In embodiments of the invention, the multi-arm PEG precursors for G0 branched macromolecules and DCRUs can be available from, for example, JenKem Technology USA, SinoPEG, or Sigma-Aldrich, optionally including various functional end groups for further derivatization.

[0171] In some embodiments of the invention, the polyethylene glycol (PEG) unit used as a core building block or DCRU precursor has an average molecular weight in the range of about 1,000 Daltons to about 80,000 Daltons, or between about 10,000 Daltons to about 60,000 Daltons, or between about 15,000 Daltons to about 50,000 Daltons. In some embodiments, the PEG unit has an average molecular weight in the range of about 10,000 Daltons to about 40,000 Daltons, or about 20,000 Daltons. PEG precursors with the same average molecular weight can be used, or PEG precursors with different average molecular weights can be combined with each other. The average molecular weight of the PEG precursors used in this invention is given as a number average molecular weight (Mn), which in some embodiments can be determined according to a standardized method by gel permeation chromatography against a polystyrene standard.

[0172] In a 4-arm PEG, each arm can have an average arm length (or molecular weight) of the total molecular weight of PEG divided by 4. Therefore, the 4a20kPEG precursor (which is one such precursor used in this invention) has four arms, each with an average molecular weight of approximately 5,000 Daltons (+ / - 500) linked to a pentaerythritol core unit. Therefore, in addition to the 4a20kPEG precursor, the 8a20kPEG precursor used in this invention has eight arms, each with an average molecular weight of 2,500 (+ / - 250) Daltons linked to a tripentaerythritol or hexaglycerol core unit.

[0173] Generally, when referring to a polymer precursor having a certain average molecular weight (such as a 15k PEG precursor), the indicated average molecular weight (i.e., Mn of 15,000 or 20,000) refers to the polymeric unit portion of the precursor before the addition of end groups (“20k” here means 20,000 Daltons (+ / - 2,000 Da), and “15k” means 15,000 Daltons (+ / - 1,500 Da) – the same abbreviation is used herein for other average molecular weights of PEG or other polymer precursors). In some embodiments, the Mn of the polymeric unit portion of the precursor is determined against a polystyrene standard by gel permeation chromatography according to a standardized method. The degree of substitution with end groups as disclosed herein can be determined after end group functionalization by means of… 1 Measured by H-NMR.

[0174] In some implementations, the precursor suitable for forming the DCRU is typically represented by formula (iii):

[0175]

[0176] Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines), D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L A X is the linker group, m is 0 or 1, indicating that the linker may or may not exist, n is an integer from 3 to 2000 or from 20 to 2000, o is an integer from 3 to 2000 or from 20 to 2000, and n and o may be different or the same, X is the branching unit, L B It is a linker group, p is 0 or 1, meaning that the linker may or may not exist, L A and L B They can be different or the same, m and p can be different or the same, and y is an integer from 2 to 9, where y = c' - 1, and c' is the connectivity of the branch unit X.

[0177] In some embodiments, the PEG precursor that can be used to form the hyperbranched macromolecule DCRU is an NHS dicarboxylate-terminated multi-arm PEG precursor derived from commercially available multi-arm PEG compounds, such as formula (iv), which is an example of a 4-arm structure derived from pentaerythritol.

[0178]

[0179] The PEG precursor that can be used to form a DCRU in certain implementations can be represented by the following formula (v):

[0180] (v)

[0181] Where n is determined based on the molecular weight of each individual 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 (and therefore can be, for example, 2, 4, 8, etc., see above). When m is 1, each arm is capped with a succinimide succinate (SS) end group; when m is 2, each arm is capped with a glutaryl succinate succinate (SG) group; when m is 3, each arm is capped with an adipate succinate succinate (SAP) group; and when m is 6, each arm is capped with an azelaic acid succinate succinate (SAZ) group. Using these specific electrophilic end groups, the multi-arm PEG unit can be abbreviated as, for example, 4a20kPEG-SAP, which refers to a 4-arm PEG with an adipate succinate succinate end group and a molecular weight of 20,000 Da. In the above formula, R is the core unit structure suitable for providing the desired number of arms. For the 4-arm PEG unit and precursor shown in the above formula, R can be a pentaerythritol structure, while for the 8-arm PEG unit and precursor, R can be a hexaglycerol structure.

[0182] In some implementations, the PEG precursor used is 4a20kPEG-SG or 4a20kPEG-SAP.

[0183] Precursors with nucleophilic terminals can also be used instead of electrophilic terminals. In some embodiments, the nucleophilic terminal used as a precursor for hyperbranched macromolecule PEG is an amine (denoted as "NH2") terminal. Thiol (-SH) terminals or other nucleophilic terminals are also possible.

[0184] In some embodiments, the hyperbranched macromolecules of the present invention can be formed using four-armed PEG with an average molecular weight of about 20,000 Daltons and four-armed PEG with an average molecular weight of about 40,000 Daltons.

[0185] Functional groups used to connect hyperbranched macromolecular building blocks

[0186] To synthesize hyperbranched macromolecules, polymer arms or precursors have paired functional groups that react with each other. That is, the first functional group on the first polymer arm or precursor can react with the second functional group on the second polymer arm or precursor of different DCRU precursors.

[0187] In the implementation, a first multi-arm precursor, including a core unit and a PEG arm connected thereto, comprises a first functional group, and a second multi-arm precursor DCRU comprises a second functional group capable of reacting with the first functional group, while all other end groups of the second DCRU precursor do not react with the first functional group, which is located at the end of the arm of the precursor or DCRU. The first and second functional groups can be grafted to the arm ends directly or via a linker, preferably a hydrolyzable linker as defined elsewhere herein. The functional groups are capable of reacting with each other and forming covalent bonds, for example in click chemistry or electrophilic-nucleophilic reactions, or configured to participate in other chemical crosslinking reactions as described below.

[0188] In some embodiments of the invention, the first and second functional groups are selected from electrophiles and nucleophiles, functional groups for click chemistry, functional groups for cycloaddition (especially 1,3-dipolar cycloaddition, heterodiels-Alder cycloaddition), functional groups for nucleophilic ring-opening, functional groups for non-aldecosyl reactions, functional groups for addition reactions of carbon-carbon multiple bonds, polymerizable vinyl groups, or combinations thereof. Those skilled in the art will appreciate that certain paired functional groups can be classified into more than one of these groups. For example, in click chemistry, azides reacting with dibenzocyclooctyne can also be considered an electrophile-nucleophile reaction pair.

[0189] In certain embodiments of the hyperbranched macromolecules of the present invention, the connections between different parts of the hyperbranched macromolecule (such as polymer arms attached to the core unit and DCRUs) are formed via click chemistry reactions (such as strain-promoted alkyne-azide cycloaddition (SPAAC, also known as Cu-free click reactions), or inverse electron-demanding Diels-Alder linkage (IEDDA) type click chemical coupling reactions). An overview of such reaction types is given in HC Kolb; MG Finn; KBSharpless (2001). “Click Chemistry: Diverse Chemical Function from a Few GoodReactions”, Angewandte Chemie International Edition, 40 (11): 2004-2021, which is incorporated herein by reference.

[0190] Other click chemistry reactions suitable for linking the constituent units of hyperbranched macromolecules in certain embodiments include aldehyde / ketone condensation, cyanobenzothiazole condensation; strain-promoted oxidation-controlled cyclooctyne-1,2-quinone cycloaddition (SPOCQ); 1,3-dipolar cycloaddition, [3+2] cycloaddition (such as olefin-nitroketone cycloaddition or alkyne-nitroketone cycloaddition), [4+2] cycloaddition; and heterodiels-Alder reactions.

[0191] SPAAC requires the reaction of cyclic alkynes (such as dibenzylcyclooctyne (DBCO) and bicyclic [6.1.0]nonyne (BCN)) with aliphatic azides. This strain chemistry allows the reaction to occur efficiently without the copper catalyst required in copper(I)-catalyzed azide-alkyne click chemistry (CuAAC). Similarly, IEDDA requires norbornene and tetrazine to react without a catalyst. Therefore, the advantages of SPAAC and IEDDA over CuAAC and electrophilic-nucleophilic reactions (such as NHS-NH2) are that no catalyst is required and no byproducts are produced after the reaction is complete.

[0192] The SPAAC and IEDDA coupling reaction is a bioorthogonal reaction with selective and quantitative yields under mild conditions, which can occur even within living systems without interfering with native biochemical processes. These click chemistry reactions utilize a pair of reagents, such as cyclooctyne and an azide, that react specifically and efficiently with each other while remaining inert to native functional groups.

[0193] Option A:

[0194]

[0195] R1 and R2 are any residues that are the same or different.

[0196] This reaction is applicable to the formation of hyperbranched macromolecules according to embodiments of the present invention from the corresponding functionalized precursors and DCRUs as described herein. Among a large number of known cyclooctynes, dibenzocyclooctyne (DBCO) compounds are a class of reagents containing relatively fast kinetics and good stability in aqueous buffers. Within physiological temperature and pH ranges, the DBCO group will not react with amines or hydroxyl groups naturally present in many biomolecules, or exist as different functional groups on the hyperbranched macromolecular moiety. Furthermore, the reaction of the DBCO group with azide groups is significantly faster and in higher yields.

[0197] The advantages of DBCO-based SPAACs include, for example, their biocompatibility, as they do not require cytotoxic copper catalysts, which can leave undesirable traces in hyperbranched macromolecules. Another advantage is the use of mild reaction conditions: it is possible to link DCRUs or conjugated activators under physiological conditions in aqueous buffer media or common organic solvents. Furthermore, both the DBCO and azide moiety are long-term stable and exhibit high selectivity and specificity, as the azide group reacts with DBCO only in the presence of amines, hydroxyl groups, thiols, acid groups, and other protein functional groups. Similarly, the reaction results in the formation of stable triazoles in quantitative yields at high reaction rates without leaving byproducts. The aforementioned IEDDA coupling reaction and other types of catalyst-free click chemistry reactions offer similar advantages.

[0198] In an exemplary implementation, hyperbranched macromolecules including diacid-derived hydrolyzable linkers can be formed using the following click chemistry precursors:

[0199] and

[0200] or

[0201] ;or

[0202] and or

[0203] ;or

[0204] and

[0205] or

[0206] ;or

[0207] and

[0208] or

[0209] and ,

[0210] Where t is m, and n and m are as defined above for equation (v).

[0211] Other core / branch unit connectivity can also be used instead of the 4-arm PEG, as described herein. Similarly, in other embodiments, the precursor described above may include hydrolyzable links, including carboxamide bonds instead of ester bonds, or ester and amide bonds, such as in the SGA linker unit further described above.

[0212] In some embodiments, using click chemistry functional groups, the links in the hyperbranched macromolecule can be selectively formed using DCRU precursors (such as the aforementioned DCRU precursors comprising a functional group for click chemistry bond formation), while other terminal functional groups of the DCRU remain unreactive and can subsequently be used for other follow-up reactions, such as hyperbranched macromolecule growth or conjugation. In other embodiments, the links in the hyperbranched macromolecule can be selectively formed using electrophilic-nucleophilic precursors or other functional groups that are unreactive in click chemistry, while other terminal functional groups of the DCRU, including those for click chemistry bond formation, remain unreactive and can subsequently be used for other follow-up reactions, such as hyperbranched macromolecule growth or conjugation under click chemistry.

[0213] Functional group pairs used in click chemistry can be selected for cycloaddition, especially 1,3-dipolar cycloaddition, [3+2] cycloaddition (such as olefin-nitroketone cycloaddition or alkyne-nitroketone cycloaddition), [4+2] cycloaddition, heterodiels-Alder cycloaddition; functional groups for thiol-alkene reactions; functional groups for nucleophilic ring opening; functional groups for non-aldehyde-alcohol carbonyl reactions; functional groups for carbon-carbon multiple bond addition reactions; and functional groups for Michael-type addition.

[0214] For example, the first functional group is an alkyne compound, such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]-nonyne (BCN); or norbornene or trans-cyclooctyne (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 can be grafted to the end of the multi-arm precursor via hydrolyzable linkers (such as acid groups, diacid groups, amide groups, functionalized aliphatic, heteroaliphatic, or aromatic or heteroaromatic groups), or can be directly linked to PEG.

[0215] In another embodiment, the first and second functional groups are selected for [3+2] cycloaddition reactions, such as olefin-nitroketone cycloaddition or alkyne-nitroketone cycloaddition. In another embodiment, the first and second functional groups are selected for [4+2] cycloaddition reactions, particularly heterodiels-Alder reactions, wherein 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 nitroso-olefin compound. In another embodiment, the first and second functional groups are selected for nucleophilic ring-opening reactions, wherein the first functional group is selected from epoxides, thiocyclopropanes, aziridines, or lactams, and the second functional group is a nucleophile as mentioned above. In another embodiment, the first and second functional groups are selected for non-aldecanol carbonyl reactions, wherein the first functional group is an aldehyde or ketone compound, and the second functional group is a primary amine, acylhydrazine, acylhydrazine, or aminooxy compound to form an imine, amide, isourea, hydrazone, acylhydrazone, or oxime linker.

[0216] Conjugation of surfactants

[0217] The active agent can also be bonded or conjugated to the outermost polymer arm of the hyperbranched macromolecule via click chemistry as described above for connecting hyperbranched macromolecular building blocks, or via electrophilic-nucleophilic reactions and other types of coupling reactions as mentioned herein.

[0218] Therefore, in one embodiment, the first functional group on the outermost polymer arm of the hyperbranched macromolecule can be a nucleophile and the second functional group on the activator can be an electrophile, or vice versa, and the reaction between the first and second functional groups is an electrophile-nucleophile reaction that forms a covalent bond.

[0219] The nucleophile can be selected from amines (e.g., primary amines), hydroxyl groups, thiols, carboxyl groups, or hydrazide groups. In some embodiments, a functional group comprises a nucleophile, such as a primary amine.

[0220] The electrophile that can be used in embodiments of the present invention may be selected from activated ester groups, such as succinimidyl esters, succinimidyl carbonate; nitrophenyl carbonate, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, norbornene, epoxides, methanesulfonates, toluenesulfonates, trifluoroethylsulfonyl, cyanurates, o-dithiopyridine, or halogens. These electrophiles contain functional groups that participate in electrophile-nucleophile reactions, and preferably additionally include reactive groups that form PEG linkers, said PEG linkers comprising hydrolyzable groups or bonds, such as glutarate esters. For example, in embodiments of the present invention, succinimidyl esters may contain reactive groups such as succinimidyl succinate (SS), succinimidyl glutarate (SG), succinimidyl adipate (SAP), succinimidyl azelate (SAZ), or succinimidyl glutaramide. Such electrophilic-nucleophilic reactions for connecting multi-arm PEG precursors are described, for example, in US 2002 / 0042473A1, which is incorporated herein by reference.

[0221] Surfactants can be suitably derived using the functional groups mentioned above, unless they already possess functional groups suitable for linking with hyperbranched macromolecules. For example, peptides with primary amino groups can be conjugated to hyperbranched macromolecules with activated ester groups on their surface via electrophilic-nucleophilic reactions.

[0222] In some embodiments, the active agent, particularly the peptide, can be conjugated to a hyperbranched macromolecule via a click chemistry reaction. In one embodiment, the active agent or peptide having a terminal primary amino group first reacts with a DBCO-NHS or azide-NHS compound to produce an active agent or peptide functionalized with a DBCO or azide group, which is suitable for reacting with the corresponding functional group at the end of its hyperbranched macromolecule, thereby generating a conjugate with high reproducibility.

[0223] Reactants suitable for click chemistry functionalization of surfactants or peptides with terminal primary amino groups are, for example, N-hydroxysuccinimide azidoacetate (NHS-azide), N-hydroxysuccinimide azidobutyrate, or other azidoacid-NHS esters, and dibenzocyclooctyne-N-hydroxysuccinimide esters with varying acid chain lengths (DBCO-NHS). Both azidoacid-NHS esters and DBCO-NHS esters can be used with acids of varying chain lengths (e.g., as discussed above as linkers) to alter the biodegradation rate and surfactant release of hyperbranched macromolecules. Such reagents for click chemistry are available from suppliers such as Sigma-Aldrich, Thermo Fisher Scientific, and others.

[0224] In some embodiments, an active agent or peptide having a thiol functional group (such as a cysteine ​​thiol group) for conjugation can be conjugated to a hyperbranched macromolecule via a maleimide-thiol click chemistry reaction according to the following reaction scheme:

[0225] Option B:

[0226]

[0227] R1 is the terminal of a hyperbranched macromolecule, and R2 is a peptide or activator. The thiol-maleimide reaction is a Michael addition reaction of thiols that produces thio-succinimide linkages. This reaction is relatively fast and exhibits chemoselectivity for thiols at pH 6.5 to pH 7.5.

[0228] For example, maleimide-functionalized ends of hyperbranched macromolecules can be used to conjugate peptides or surfactants via a maleimide-thiol reaction. In another embodiment, the DBCO or azide-functionalized ends of hyperbranched macromolecules can be provided with maleimide-terminal functionalization by reacting with a click chemistry linker having an azide or DBCO functional group and a maleimide group at its other end, which can then be used for conjugation with a thiol group at the peptide or surfactant. Suitable DBCO-maleimide or azide-maleimide linkers can optionally be partially extended with PEG and are available from Sigma-Aldrich, TCI, Thermo Fisher, etc.

[0229] Examples are compounds such as DBCO-maleimide, DBCO-PEG3-maleimide, DBCO-PEG4-maleimide, and azido-PEG3-maleimide, which have the following exemplary structures:

[0230] , , .

[0231] In some embodiments, the surfactant is bound to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% of the outermost polymer arm. The average substitution rate of the surfactant conjugated to the surface end groups of the hyperbranched macromolecule can be determined by UHPLC as further described herein.

[0232] Surfactant:

[0233] The active agent in the biodegradable microparticles of the embodiments of the present invention can be a therapeutic active agent, a diagnostic active agent, or a combination thereof. It can be a single active agent or multiple active agents.

[0234] In some embodiments, the hyperbranched macromolecule contains two or more different active agents in different dendritic units or regions on its surface. Each of the two or more active agents can be linked to the same or different hydrolyzable groups to control the release of the active agents at different rates. Alternatively, the active agents can be linked to the dendritic polymer with or without hydrolyzable links or arms / extensions or combinations thereof to control the release of the active agents at different rates.

[0235] In some embodiments, the active agent conjugated to at least one outermost polymer arm of the hyperbranched macromolecule is a peptide selected from the group consisting of: campstatin, APL-1, Fc-III-4C, Beovu (busizumab), Zimura (avocado polyethylene glycol), pecicoplanin, arbexipa polyethylene glycol, lampalizumab, Fovista, risunib, AXT107, iramitide, THR149, ALM201, VGB3, and lagozopran.

[0236] Therapeutic agents can include steroids; nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, meclofenamic acid, meclofenamic acid A, disalicylate, sulindac, tometidine, ketoprofen, diflunisal, piroxicam, naproxen, etodoxacin, flurbiprofen, fenofibrate C, indomethacin, celecoxib, ketorolac, and nepafenamide; intraocular pressure-lowering drugs; antibiotics such as ciprofloxacin; pain relievers 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 molecule hydrophobic drugs, hydrophilic... Aqueous peptides and protein drugs, such as insulin, single-chain antibody fragments, Fab fragments, IgG antibodies, fusion antibodies, etc.; aptamers; especially bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, antibiotics, pain relievers, calcium channel blockers, cell cycle inhibitors, chemotherapeutic agents, antiviral drugs, anesthetics, hormones, anticancer drugs, antitumor agents, viruses, gene-delivering viruses (such as AAV), protein binding agents (such as nanobodies, affinity molecules, ankylosing smears, DARPin, etc.) or any combination thereof.

[0237] In some embodiments, the steroid may be a corticosteroid, which may include hydrocortisone, loteprednol, cortisol, cortisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, aldosterone, or fludrocortisone.

[0238] In some implementations, NSAIDs may include diclofenac (e.g., diclofenac sodium), flurbiprofen (e.g., flurbiprofen sodium), ketorolac (e.g., ketorolac tromethamine), bromfenac, or naprafenamide.

[0239] In some embodiments, IOP-lowering agents and / or glaucoma medications may include prostaglandin analogs (e.g., bimatoprost, latanoprost, travoprost, or latanoprostene bunod), rho kinase inhibitors (e.g., netarsudil), adrenergic agonists (e.g., adrenaline or dipivefrin), β-adrenergic antagonists (also known as β-blockers, such as timolol, levobunolol, metipranolol, carteolol, or betaxolol), and α2-adrenergic agonists (e.g., apraclonidine, brimonidin). e) or brimonidine tartrate), carbonic anhydrase inhibitors (e.g., brinzolamide, dichlorphenamide, methazolamide, acetazolamide, acetazolamide or dorzolamide), pilocarpine, echothiophate, demercarium, physostigmine and / or isofluorophate).

[0240] In some implementations, the anti-infective agent may include antibiotics, including ciprofloxacin, tobramycin, erythromycin, ofloxacin, gentamicin, fluoroquinolone antibiotics, moxifloxacin, and / or gatifloxacin; antiviral agents, including ganciclovir, idoxuridine, vidarabine, and / or trifluridine; and / or antifungal agents, including amphotericin B. B) Natamycin, voriconazole, fluconazole, miconazole, clotrimazole, ketoconazole, posaconazole, echinocandin, caspofungin, and / or micafungin.

[0241] In some embodiments, the antimetabolite may include methotrexate, mycophenolate, or azathioprine. In some embodiments, the antifibrotic agent may include mitomycin C or 5-fluorouracil.

[0242] In some implementations, angiogenesis inhibitors may include anti-VEGF agents (e.g., aflibercept, ranibizumab, bevacizumab) and 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).

[0243] In some implementations, nanobodies can be conjugated to hyperbranched macromolecules. Nanobodies are described, for example, in Yang et al. (2020), Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics, Front. Oncol. 10:1182, the full text of which is incorporated herein by reference. Nanobodies can be selected from... 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 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, BCMACAR T cells, or TAS266 nanobody.

[0244] In some implementations, non-immunoglobulin affinity proteins (such as affinity molecules) can be conjugated to hyperbranched macromolecules. Affinity molecules are described, for example, in Ståhl 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.

[0245] In some implementations, binding proteins (such as ankyrin and DARPin) can be conjugated to hyperbranched macromolecules. Ankyrin and DARPin are described, for example, in a review by Caputi et al., Current Opinion in Pharmacology 2020, 51:93-101, the full text of which is incorporated herein by reference. Ankyrin and DARPin can be selected from MP0250, a trispecific DARPin candidate that binds to VEGF-A and hepatocyte growth factor (HGF) as well as one molecule of MP0250 that binds to two molecules of human serum albumin (HSA); azithromycin PEGylated (MP0112 or AGN-150998); busizumab, ranibizumab, or aflibercept.

[0246] In some embodiments, the cytoprotective agent may include ebselen, sulforaphane, oltipraz, or dimethyl fumarate. In some embodiments, the neuroprotective agent may include ursodeoxycholic acid, memantine, or acetylcysteine. In some embodiments, the anesthetic agent may include lidocaine, proparacaine, or bupivacaine.

[0247] In some embodiments, the active agent may be dexamethasone, ketoroxyprostol, diclofenac, vancomycin, moxifloxacin, gatifloxacin, besifloxacin, travoprost, 5-fluorouracil, methotrexate, mitomycin C, prednisolone, bevacizumab (Avastin®), ranibizumab (Lucentis®), sunitinib, pegaptanib (Macugen®), timolol, latanoprost, brimonidine, napafenamide, bromofenac, triamcinolone, difluprednate, fluocinolone, aflibercept, or combinations thereof. In some embodiments, the agent may be dexamethasone, ketoroxyprostol, diclofenac, moxifloxacin, travoprost, 5-fluorouracil, or methotrexate.

[0248] In alternative embodiments, active agents that can be used with the dendritic polymers and methods of the present invention include (but are not limited to) immunosuppressants, complement inhibitors (e.g., C5 inhibitors, such as eculizumab or avacalide polyethylene glycol), steroids, anti-inflammatory drugs (e.g., steroid and non-steroidal anti-inflammatory drugs, such as COX1 or COX2 inhibitors), antiviral drugs, antibiotics, antiglaucoma agents, anti-VEGF agents, analgesics, tyrosine kinase inhibitors, integrin inhibitors, IL-6 blockers, reactive aldehyde (RASP) inhibitors, nitric oxide donor PgA, antihistamines, mast cell stabilizers, rho kinase inhibitors, plasma kallikrein inhibitors, BCL-2 blockers, semaphorin antagonists, HtRA1 blockers, IGF-1R inhibitors, VEGF combinations (multispecific anti-angiogenic agents), and combinations thereof.

[0249] 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, etoposide, thiotepa, methotrexate, azathioprine, mercaptopurine, interferon, infliximab, etanercept, and mycophenolate mofetil. mofetil, 15-deoxyguanidin, thalidomide, glatiramer, leflunomide, vincristine, cytarabine, their pharmaceutically acceptable salts, and combinations thereof.

[0250] Nonsteroidal anti-inflammatory compounds include inhibitors of cyclooxygenases (COX, such as cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) isoenzymes). General categories of nonsteroidal anti-inflammatory compounds include salicylates, propionic acid derivatives, acetic acid derivatives, enolic acid derivatives, and anthranilic acid derivatives. Examples of nonsteroidal anti-inflammatory compounds include acetylsalicylic acid, diflunisal, disalicylate, ibuprofen, dex-ibuprofen, naproxen, fenofoprofen, ketoprofen, dex-ketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, tometidine, sulindac, etodoxacin, ketoroxylic acid, diclofenac, aceclofenac, nabumetone, piroxicam, tenoxicam, loroxicam, phenylbutazone, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, their pharmaceutically acceptable salts, and combinations thereof.

[0251] Anti-inflammatory agents that can be used with the dendritic polymers 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.

[0252] Analgesics that can be used with the dendritic polymers and methods of the present invention include acetaminophen, acetaminosalol, aminochlorthenoxazin, acetylsalicylic acid 2-amino-4-methylpyridine, acetylsalicylic acid, anileridine, benzoxaprofen, benzylmorphine, 5-bromosalicylic acid acetate, bucetin, buprenorphine, butorphanol, and other similar products. Capsaicin, cinchophen, ciramadol, clometacin, clonixin, codeine, desomorphine, dezocine, dihydrocodeine, dihydromorphine, dimepheptanol, dipyrocetyl, eptazocine, ethoxazene, ethylmorphine, eugenol, floctafenine, phosphorus Salicylic acid (fosfosal), glafenine, hydrocodone, hydromorphone, hydroxypethidine, ibufenac, p-lactophenetide, levorphanol, meptazinol, metazocine, metopon, morphine, nalbuphine, nicomorphine, norlevophenol rphanol), normorphine, oxycodone, oxymorphone, pentazocine, phenazocine, phenocoll, phenoperidine, phenylbutazone, phenyl salicylate, phenylramidol, salicin, salicylamide, tiorphan, tramadol, diacerein, actarit, their pharmaceutically acceptable salts and combinations thereof.

[0253] Antibiotics that can be used with the dendritic polymers and methods of the present invention include aminoglycosides, penicillin, cephalosporins, fluoroquinolones, macrolides, and combinations thereof. Aminoglycosides may include tobramycin, kanamycin A, amikacin, dibekacin, gentamicin, sisomicin, netilmicin, 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, pivmecillinam, ticarcillin, their pharmaceutically acceptable salts, and combinations thereof. Cephalosporins can include cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, and cefazolin. Cefradine, cefroxadine, ceftezole, cefaclor, cefamandole, cefmetazole, cefonicid, cefotetan, cefoxitin, cefprozil, cefuroxime, cefzonam, cefcapeneCefodaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefpimizole, cefpodoxime, cefteram, cefbufenozide Ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime, ceffluprenam, cefoselis, and more. Cefozopran, cefpirome, cefquinome, ceftobiprole, cefftaroline, cefclomezine, cefloram, cefparole, cefcanel, cefdrolor, cefempidone, ceftrizole, cefvitril, cefmatilen, cefmepidium, cefvecin, cefoxazole, cefrotil, cefsumide, ceffuracetime, ceftioxide, their pharmaceutically acceptable salts, and combinations thereof. Fluoroquinolones can include ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, ofloxacin, norfloxacin, their pharmaceutically acceptable salts, and combinations thereof. Macrolides can include azithromycin, erythromycin, clarithromycin, dirithromycin, oxithromycin, telithromycin, their pharmaceutically acceptable salts, and combinations thereof.

[0254] Antiviral agents that can be used with the dendritic polymers and methods of 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 agents include (but are not limited to) abacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, boceprevir, cidofovir, darunavir, and delaviridine. virdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir Ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, type III interferon, type II interferon, type I interferon, interferon, lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine Nexavir, oseltamivir, pegylated interferon alpha-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, pyramidal saquinavirsaquinavir, stavudine, tenofovir, tenofovir disoproxil, tipranavir, trizivir, tromantadine, truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, veramidine, zalcitabine, zanamivir, zidovudine, their pharmaceutically acceptable salts and combinations thereof.

[0255] Steroid anti-inflammatory agents that can be used with the dendritic polymers and methods of the present invention include dexamethasone, budesonide, triamcinolone, hydrocortisone, fluocinolone, lorteprednisolone, prednisolone, mometasone, fluticasone, rimexolone, fluorometholone, beclomethasone, flunisolide, pharmaceutically acceptable salts thereof, and combinations thereof.

[0256] Antiglaucoma agents that can be used with the dendritic polymers and methods of the present invention include β-blockers such as atenolol, propranolol, metenolol, betalol, carteolol, levobetaxolol, levobenolol, timolol, pharmaceutically acceptable salts thereof, and combinations thereof; adrenergic agonists or sympathomimetic agents such as adrenaline, dipiformin, clonidine, aparclonidine, brimonidine, pharmaceutically acceptable salts thereof, and combinations thereof; and parasympathomimetic agents or cholinergic agonists such as pilucarpine, carbachol, phosphorylcholine iodide, and physostigmine. Pharmaceutically acceptable salts and combinations thereof; carbonic anhydrase inhibitors, including local or systemic agents such as acetozolamide, bullinzoamide, dazolamide; acetozolamide, ethoxzolamide, diclofenac, pharmaceutically acceptable salts and combinations thereof; mydriatic-cycloplegic agents such as atropine, cyclopentolate, succinylcholine, homatropine, phenylephrine, scopolamine, tropicamide, pharmaceutically acceptable salts and combinations thereof; prostaglandins such as prostaglandin F2α, antiprostaglandins, prostaglandin precursors or prostaglandin analogs such as bimatoprost, latanoprost, travoprost, unoprostone, tafluprost, pharmaceutically acceptable salts and combinations thereof.

[0257] Anti-VEGF agents that can be used with the dendritic polymers and methods of the present invention include bevacizumab, pilgatanib, ranibizumab, buxizumab, conbercept, aflibercept, pharmaceutically acceptable salts thereof, and combinations thereof.

[0258] Tyrosine kinase inhibitors that can be used with the dendritic polymers and methods of the present invention include deucravacitinib, axitinib, avapritinib, capmatinib, pegmatinib, ripretinib, selpercatinib, selumetinib, tucatinib, entrectinib, erdaftinib, fedratinib, and pexidatinib. rtinib, upadacatinib, zanubrutinib, baricitinib, binimetinib, dacomitinib, fostamatinib, gilteritinib, larotrectinib, lorlatinib, acalabrutinib, brigatinib, midostaurin, neratinib, acalabrutinib alectinib, cobimetinib, lenvatinib, osimertinib, ceritinib, nintedanib, afatinib, ibrutinib, trametinib, bosutinib, cabozantinib, ponatinib, regorafenib, tofacitinib, crizotinib b) Ruxolitinib, vandetanib, pazopanib, lapatinib, nilotinib, dasatinib, sunitinib (voronib), sorafenib, erlotinib, gefitinib, imatinib, afatinib, bosutinib, cabozantinib, cediranib, ceritinib, crizotinib, dabrafenib, dasatinibErlotinib, everolimus, gefitinib, imatinib, lestaurtinib, nilotinib, palbociclib, pazopanib, ponatinib, regoratinib, ruxotinib, semananib, sirolimus, sorafenib, tesimolimus, tofacitinib, trametinib, vandetanib, and vemurafenib. In another embodiment, the tyrosine kinase inhibitor is a Src family tyrosine kinase inhibitor, such as (but not limited to) A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD 2076, KB SRC 4, KX2361, KX2-391, MLR1023, 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. Altenusin, Bosutinib, Damnacanthal, Dasatinib, Herbimycin A, Indirubin, Lenatinib, Lavandustin A, Pelitinib, Piceatannol, Saracatinib, 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.

[0259] Complement pathway modulators that can be used with the dendritic polymers and methods of the present invention include complement pathway modulators that target, for example, 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 modulators may include cinryze, berinert, ruconest, sutimlimab, percicobulin (GA), eculiziumab, ravuilizumab, avacopan, pozelimab, nomacopan, zilucopan, vilobelimab, crovalimab, and avacincapeptide. pegol), cemdisiran, BDB-001, tesidolumab, avdoralimab, MOR210, ALXN1720, danicopan, vemircopan, ACH-5228, ACH-5548, BCX-9330, AMY-101, ANX005, ANX007, narsoplimab, iptacopan, CLG561, GT103, ARGX-117, ALXN1820, NGM621, lamparibumab, NGM621, IONIS-FB-Lrx, GEM103, CLG561, their pharmaceutically acceptable salts and combinations thereof.

[0260] Integrin inhibitors that can be used with the dendritic polymers and methods of the present invention include lifitegrast, vedolizumab, natezumab, efalizumab, tirofiban, eptifibatide, abciximab, IDL-2965, PLN-74809, PLN-1474, PN-943, 7HP349, MORF-057, OS2966, OTT166, AXT-107, JSM-6427, risunib, THR-687 (D / ced), their pharmaceutically acceptable salts, and combinations thereof.

[0261] Antihistamines that can be used with the dendritic polymers and methods of the present invention include loradatine, hydroxyzine, diphenhydramine, chlorpheniramine, brompheniramine, cyproheptadine, terfenadine, clemastine, triprolidine, carbinoxamine, diphenylpyraline, and benzoindene. Phenindamine, azatadine, tripelennamine, dexchlorpheniramine, dexbrompheniramine, methdilazine and trimrazine, doxylamine, pheniramine, pyrilamine, chiorcyclizine, thonzylamine, their pharmaceutically acceptable salts and combinations thereof.

[0262] IL-6 inhibitors that can be used with the dendritic polymers and methods of the present invention include sarilumab, tocilizumab, RG6179, pharmaceutically acceptable salts thereof, and combinations thereof.

[0263] HtrA1 inhibitors that can be used with the dendritic polymers and methods of the present invention include IC-500, FHTR2163, RG6147, their pharmaceutically acceptable salts, and combinations thereof.

[0264] RASP inhibitors that can be used with the dendritic polymers and methods of the present invention include reproxalap and its pharmaceutically acceptable salts.

[0265] Rho kinase inhibitors that can be used with the dendritic polymers and methods of the present invention include netardil, ripasudil, HA-1077, Y-27632, H-1152P, INS-115644, Y-39983, SB772077BS, LX71D1, AR-12286, AMA-0076, AR-13533, their pharmaceutically acceptable salts, and combinations thereof.

[0266] Plasma kallikrein inhibitors that can be used with the dendritic polymers and methods of the present invention include ecallantide, lanadelumab, berotopralstat, ATN-249, KVD900, KVD824, THR-149, their pharmaceutically acceptable salts, and combinations thereof.

[0267] Nitric oxide donors PgA that can be used with the dendritic polymers and methods of the present invention include latanoprostone bronode, NCX470, NCX125, their pharmaceutically acceptable salts, and combinations thereof.

[0268] Mast cell stabilizers that can be used with the dendritic polymers and methods of the present invention include lodoxamide, nedocromil, pemirolast, cromoglycine (e.g., sodium cromoglycine), pharmaceutically acceptable salts thereof, and combinations thereof.

[0269] IGF-1R inhibitors that can be used with the dendritic polymers and methods of the present invention include teprotumumab, VRDN-001, VRDN-002, VRDN-003, ganitumab, figitumumab, MEDI-573, cixutumab, dalotuzumab, robatumumab, AVE1642, BIIB022, xentuzumab, istiramumab, and lincitidine. Linsitinib, picopodophyllin, BMS-754807, BMS-536924, BMS-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 pharmacologically acceptable salts and combinations thereof.

[0270] TRPV1 antagonists that can be used with the dendritic polymers and methods of the present invention include asivatrep; V116517; such as, 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. Chem. Fused azabicyclic, heterocyclic, and amide compounds as described in 48:744-752; fused pyridine derivatives as described, for example, in U.S. Patent Application No. 2004 / 0138454; pyridylhexahydropyrazinylurea as described, for example, in Swanson et al., 2005, J. Med. Chem. 48:1857-1872 and U.S. Patent Application No. 2005 / 0049241; and AMG8163 (Bannon et al., 2005, 11. sup. World Congress on Pain) and BCTC (Sun et al., 2003, Chem. Lett.). 13:3611-3616); 2-(hexahydropyrazin-1-yl)-1H-benzimidazole; pyridazinylhexahydropyrazine; urea derivatives such as those described in, for example, U.S. Patent Application No. 2005 / 0107388, U.S. Patent Application No. 2005 / 0187291 and U.S. Patent Application No. 2005 / 0154230, and A-425619 (El Kouhen et al., 2005, J. Pharmacol. Exp. Ther. 314:400-409); cinnamamides, including SB-366791 (Gunthorpe et al., 2004, Neuropharmacology 46:133-149) and AMG 9810 (Gawa et al., 2005, J. Pharmacol. Exp. Ther. 313:474-484).

[0271] In some embodiments, TRPV1 antagonists that can be used in the methods and compositions disclosed herein include, for example, TRPV-1 antagonists including capsazepine, (E)-3-(4-tert-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)acrylamide (available, for example, from Tocris Bioscience, Bristol, United Kingdom, AMG9810) and 4-tert-butylcyclohexane (available from Symrise GmbH of Holzminden, Germany, SYMSITIVE 1609), and TRPV1 antagonists as disclosed in U.S. Patent Nos. 8,815,930, 6,933,311, 7,767,705 and U.S. Patent Application Publications Nos. 2010 / 0249203 and 2011 / 0104301, and International Application WO / 2008 / 013861.

[0272] In some embodiments, TRPV1 antagonists that can be used in the methods, compositions, and apparatuses disclosed herein include AMG-517 and AMG-628 (Amgen Inc., Thousand Oaks, Calif.). TRPV1 antagonists that can be used in this application are also described, for example, in International Patent Application No. WO 2006065484; International Patent Application No. WO2003070247; U.S. Patent Application No. US 2005080095; and International Patent Application No. WO 2005007642. Other TRPV1 antagonists that can be used in the methods and compositions and devices disclosed herein include TRPV1 antagonists: ABT-102, AMG8562, AMG9810, BCTC, SB366791, JNJ17203212, I-TTX, JYL-1421, A-425619, N-[4-[6-[4(trifluoromethyl)phenyl)pyrimidin-4-yloxy]benzo[d]thiazolyl]acetamide (also known as AL-49975 or AMG-517), (R)-N-(4-(6-(4-(1-(4-fluorophenyl)ethyl)hexahydropyrazin-1-yl)pyrimidin-4-yloxy)benzo[d]thiazolyl)acetamide (AL-49976, also known as AMG-628), their pharmaceutically acceptable salts, and combinations thereof.

[0273] Other TRPV1 antagonists that can be used in the methods and compositions and devices disclosed herein are TRPV1 antagonists with low inhibitory activity against CYP3A4, such as 1-(2-(3,3-dimethylbutyl)-4-(trifluoromethyl)benzyl)-3-(1-methyl-1H-indazol-4-yl)urea; methyl 2,2-dimethyl-4-(2-((3-(1-methyl-1H-indazol-4-yl)ureo)methyl)-5-(trifluoromethyl)phenyl)butyrate; methyl 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)ureoyl)methyl)-5-trifluoromethyl)phenyl)butyric 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, their pharmaceutically acceptable salts and combinations thereof.

[0274] TrkA antagonists that can be used with the dendritic polymers and methods of the present invention include VM902A, larotrectinib, entrectinib, selitrectinib (LOXO-195, BAY 2731954), repotrectinib (TPX-0005), their pharmaceutically acceptable salts, and combinations thereof.

[0275] For the purposes of this invention, the active agent includes all possible forms thereof, including free acids, free bases, polymorphs, pharmaceutically acceptable salts, anhydrite, hydrates, other solvates, stereoisomers, crystalline forms, cocrystals, prodrugs, conjugates (e.g., polyethylene glycolated compounds), complexes, and mixtures thereof.

[0276] Diagnostic agents can be, for example, imaging agents, markers, or visualization agents. Typically, diagnostic agents are used to examine the body to detect substances that impair its normal function. In some cases, diagnostic agents can be functional agents, such as those used to detect ocular malformations, pain, and pathophysiological aspects. For example, diagnostic agents can be important and effective diagnostic adjuvants, such as dyes (e.g., fluorescein dyes, indigo green, trypan blue), dark quenchers such as anthocyanin dyes, azo dyes, acridine, fluorene, oxazine, phenanthrene, naphthalenediimide, rhodamine, benzopyrone, perylene, benzanthrone, p-benzanthrone) to aid in the visualization of ocular tissues. Diagnostic agents can include paramagnetic molecules, fluorescent compounds, magnetic molecules, radionuclides, X-ray imaging agents, and / or contrast media. In some embodiments, diagnostic agents may include radiopharmaceuticals, contrast agents for imaging techniques, allergen extracts, activated charcoal, various test strips (e.g., cholesterol, ethanol, and glucose), pregnancy tests, 13C urea breath tests, and various staining agents / markers. In some embodiments, the marking portion is a fluorescent dye or dark quencher selected from the group consisting of: coumarin, anthocyanin dyes, azo dyes, acridine, fluorene, oxazine, phenanthridine, naphthalenediimide, rhodamine, benzopyranone, perylene, benzanthrone, and benzanthrphenolone. In a specific non-limiting embodiment, the fluorescent dye is a compound selected from the group consisting of or residues of the following: coumarin, fluorescein, cyanine 3 (Cy3), cyanine 5 (Cy5), cyanine 7 (Cy7), Alexa dye, bodipy derivative, (E)-2-(4-(phenyldiazeninyl)phenoxy)acetic acid, 3-(3′,3′-dimethyl-6-nitrospiro[benzopyran-2,2′-indoline]-1′-yl)propionate (spiropyran), 3,5-dihydroxybenzoate and (E)-2-(4-(phenyldiazeninyl)phenoxy)acetic acid or combinations thereof.

[0277] In some embodiments of the invention, the surfactant may be further dispersed, embedded, or encapsulated within the voids of hyperbranched macromolecules. In some embodiments, the surfactant may be in particulate form.

[0278] synthesis

[0279] Those skilled in the art know several methods for manufacturing hyperbranched macromolecules, and these methods can be applied primarily to embodiments of the present invention, with appropriate modifications.

[0280] In some embodiments of the invention, synthetic methods have been developed to produce dendritic polymers, among which divergent synthesis and convergent synthesis are two of the most common and general methods used by chemists. In principle, these methods can also be used to synthesize hyperbranched macromolecules of some embodiments of the invention. Divergent methods involve adding monomers in a repeating sequence, increasing the number of branches from a multivalent core to surface molecules. The molecular size and number of surface groups increase progressively with the addition of successive monomer layers, referred to as generations (see [link to documentation]). Figure 2 Although the convergence method involves synthesizing hyperbranched macromolecules from the surface to the core and resulting in the formation of cone-shaped wedge-shaped units or dendritic motifs, these units or dendritic motifs are linked to a multivalent core in the final step. Alternatively, a combined divergence / convergence method can also be employed in embodiments of the invention. For example, in embodiments of combined divergence / convergence synthesis, such as... Figure 1 As shown, the first-generation DCRU is connected to the core unit, and the second-generation to higher-generation DCRUs are first connected to each other, and then connected to the first-generation DRCU. Depending on the specific hyperbranched macromolecular structure targeted, any variation of the combined divergent and convergent synthesis steps can be used in embodiments of the invention.

[0281] In an embodiment of the present invention, a method for divergent synthesis of hyperbranched macromolecules is provided, comprising the following steps:

[0282] (a) A core unit having at least three connectivity c, a plurality of polymer arms connected to the core unit having functional groups suitable for click chemistry at the ends of the polymer arms;

[0283] (b) Providing a dendritic constituent repeating unit precursor, which comprises

[0284] - A polymer arm comprising functional groups (such as azides, alkynes, alkenes, or tetrazides) suitable for linking with corresponding functional groups of the polymer arm connected to the core via click chemistry, and

[0285] - At least two polymer arms containing functional groups that are non-reactive in click chemistry.

[0286] (c) A connection is formed between the polymer arms attached to the core and the polymer arms that form the repeating unit precursors of the dendritic structure through click chemistry.

[0287] (d) Optionally, converting the functional groups of at least two polymer arms containing functional groups that are non-reactive in click chemistry into functional groups suitable for click chemistry, and

[0288] (e) An active agent containing a functional group is conjugated to the outermost polymer arm by reacting with the functional group of the outermost polymer arm, thereby forming a hyperbranched macromolecule-active agent conjugate.

[0289] In some embodiments for higher-generation Gx hyperbranched macromolecules, where x is an integer from 2 to 10, step (d) is mandatory, and prior to the conjugation of the active agent in step (f), other successive dendritic constituent repeating unit precursors are linked to the functional groups suitable for click chemistry obtained in step (d) to be linked to the hyperbranched macromolecule via click chemistry.

[0290] For example, step d) can be carried out by reacting the NHS group with a DBCO-amine click linker, such as the following, to convert a PEG arm having an NHS end group of SS (succinimide succinate), SG (succinimide glutarate), SAP (succinimide adipate), or SAZ (succinimide azelaate) to a DS (dibenzocyclooctylamide succinate), DG (dibenzocyclooctylamide glutarate), DAP (dibenzocyclooctylamide adipate), or DAZ (dibenzocyclooctylamide azelaate) group:

[0291] .

[0292] Similarly, the conversion of PEG-NHS ends into PEG arms capped with azide groups can be achieved by reacting the NHS groups with an azide-amine click linker (such as azide-PEG2-NH2). Such azide-amine click linkers are available from several suppliers and have the structures shown below:

[0293] , where n defines the number of repeating units in PEG.

[0294] In the synthesis method, the dendritic constituent repeating unit precursor in step (c) can be represented by equation (iii):

[0295]

[0296] Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazides), D contains functional groups that are non-reactive in click chemistry (such as succinimide groups or primary amines), and L... A X is the linker group, m is 0 or 1, indicating that the linker may or may not exist, n is an integer from 3 to 2000 or from 20 to 2000, o is an integer from 3 to 2000 or from 20 to 2000, and n and o may be different or the same, X is the branching unit, L B It is a linker group, p is 0 or 1, meaning that the linker may or may not exist. B contains an end group located on the surface of the hyperbranched macromolecule or contains a bond connected to A or an active agent that is linked to a continuous dendritic repeating unit. L Aand L B They can be different or the same, m and p can be different or the same, and y is an integer from 2 to 9, where y = c' - 1, c' is the connectivity c' of the branching unit X, and the DCRU precursors used to synthesize hyperbranched macromolecules can be the same or different.

[0297] Exemplary precursors with four arms are 4-aPEG-NHS(3) azide(1) or 4-arm PEG-NHS(3)DBCO(1) compounds and similar structures, with or without hydrolyzable linking groups, such as those linked by ester bonds, amide bonds or a combination of both, see structures such as those below.

[0298] and ,or

[0299] and

[0300] and ,or

[0301] and ,or

[0302] and ,or

[0303] and ,or

[0304] and ,or

[0305] and ,

[0306] Or the following exemplary precursor pairs:

[0307] and

[0308] or

[0309] ;or

[0310] and or

[0311] ;or

[0312] and

[0313] or

[0314] ;or

[0315] and

[0316] or

[0317] and

[0318] , where t is m, and n and m are as defined above for equation (v).

[0319] refer to Figure 3 This shows a G1 hyperbranched macromolecule with peptide conjugation of 4-4 arm PEG units according to certain embodiments. Figure 3 a)) and an exemplary synthetic scheme for 8-4 arm PEG hyperbranched macromolecules. In a general embodiment, a multi-arm PEG with terminal functional groups (such as DBCO or azides) for click chemistry can be used as the core of the hyperbranched macromolecule. Another branched PEG with functional groups is then reacted with the core PEG via click chemistry. The branched PEG will contain two types of functional groups: one functional group (such as azides or DBCO) can be coupled to the core PEG in the click chemistry reaction for hyperbranched macromolecule growth, while the remainder of the branched PEG (i.e., DCRU) is inert to the core PEG and can be used for next-generation hyperbranched macromolecule growth or as a precursor for terminal bioconjugation. Figure 3 a) Based on this general method, multi-generational hyperbranched macromolecules can be synthesized to achieve different numbers of terminal functional groups. Figure 3 b) Shows the 3D structure of a hyperbranched macromolecule that begins with an 8-arm PEG core and is coupled with eight 4-arm PEG branches to achieve 24 end groups on the surface, and is ultimately conjugated with up to 24 peptides.

[0320] As shown in the examples, the two cyclic peptides, campstatin and APL-1, which are C3 binding inhibitors, and the peptide ligand Fc-III-4C, which binds immunoglobulin G (IgG), can be exemplary used as APIs conjugated to PEG hyperbranched macromolecules. The primary amine group on the peptide can act as a nucleophile to react with the electrophilic NHS group on the outermost polymer arm of the hyperbranched macromolecule.

[0321] Alternatively, the activator can be conjugated in the click chemistry reaction of the outermost polymer arm of a hyperbranched macromolecule by converting the functional groups of the outermost polymer arm containing non-reactive functional groups in click chemistry to functional groups suitable for click chemistry, and by functionalizing the activator (such as peptides) with functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines).

[0322] In certain embodiments, suitable ester groups (such as succinate (S-), glutarate (G-), adipate (AP-), and azelaate (AZ-)) on the outermost polymer PEG arm of the hyperbranched macromolecule can be hydrolyzed 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.

[0323] In an alternative embodiment, the aggregation synthesis of the hyperbranched macromolecules of the present invention is provided, comprising the following steps:

[0324] I) Provides a dendritic constituent repeating unit precursor, which contains

[0325] - A polymer arm containing functional groups (such as azides, alkynes, alkenes, or tetrazides) suitable for linkage via click chemistry, and

[0326] - At least two polymer arms containing functional groups that are non-reactive in click chemistry.

[0327] II) Conjugating an active agent containing a functional group to at least one of at least two polymer arms containing a functional group that is non-reactive in click chemistry of a dendritic constituent repeating unit precursor.

[0328] III) Provides a core unit having at least three connectivity c, and a plurality of polymer arms connected to the core unit, wherein the ends of the polymer arms have functional groups suitable for click chemistry (such as azides, alkynes, alkenes, or tetrazines), and

[0329] IV) A link is formed between the polymer arm connected to the core provided in step III) and the polymer arm containing functional groups suitable for linking by click chemistry of the dendritic constituent repeating unit precursor obtained in step II), thereby forming a biodegradable hyperbranched macromolecule-activator conjugate.

[0330] In this method, the dendritic constituent repeating unit precursor in step I) is represented by equation (iii) as described above.

[0331] In certain embodiments of higher-generation Gx biodegradable hyperbranched macromolecules, where x is an integer from 2 to 10, the active agent-conjugated dendritic repeating unit precursor obtained in step II) is linked by click chemistry to a reverse dendritic repeating unit precursor, the reverse dendritic repeating unit precursor comprising a polymer arm containing a functional group that is non-reactive in click chemistry, and at least two polymer arms containing functional groups suitable for click chemistry (such as azides, alkynes, alkenes, or tetrazines). The non-reactive functional group of one polymer arm is subsequently converted into a functional group suitable for click chemistry before being linked to other reverse dendritic repeating unit precursors or before being linked by click chemistry to the polymer arm linked to the core in step IV), thereby forming a higher-generation biodegradable hyperbranched macromolecule.

[0332] Compared to divergent synthesis methods, convergence methods also allow the synthesis of hyperbranched macromolecules with two or more different active agents in different dendritic units or regions on the surface of hyperbranched macromolecules. This allows more than one active agent to aggregate on the surface of hyperbranched macromolecules. In some embodiments of the convergence method of the present invention, dendritic constituent repeating unit precursors having different active agents conjugated to polymer arms can be obtained by performing steps I) and II) on each active agent-conjugated DCRU precursor, and the resulting mixture of active agent-conjugated DCRU precursors is used in step IV), thereby forming biodegradable hyperbranched macromolecule-active agent conjugates with different active agents in different regions on the surface of the hyperbranched macromolecule. Such aggregated hyperbranched macromolecules can be used, for example, in combination therapies involving the administration of more than one active agent.

[0333] In alternative embodiments of the divergent and convergent synthesis methods discussed above, the methods can also be implemented using reverse-exchange functional groups, i.e., using other reactions and functional groups to form links within the hyperbranched macromolecule and using click chemistry functional groups for terminal conjugation. In such "reverse" embodiments of the synthesis methods, links in the hyperbranched macromolecule can be selectively formed using electrophilic-nucleophilic precursors or other functional groups that do not react with the click chemistry functional groups, while all other terminal functional groups of the DCRU that do not participate in the connection to the core or previous DCRU include functional groups used for click bond formation and remain unreacted in the link-forming reaction. These terminal click chemistry functional groups can then be used for other subsequent reactions, such as the growth or conjugation of hyperbranched macromolecules under click chemistry.

[0334] Therefore, in another embodiment of the present invention, a method for divergent synthesis of hyperbranched macromolecules is provided, comprising the following steps:

[0335] (a) A core unit having at least three connectivity c is provided, wherein a plurality of polymer arms connected to the core unit have functional groups at the ends of the polymer arms that are non-reactive in click chemistry;

[0336] (b) Providing a dendritic constituent repeating unit precursor, which comprises

[0337] - A polymer arm comprising functional groups (such as electrophiles or nucleophiles, e.g., amines, NHS) suitable for forming links with corresponding functional groups of the polymer arm attached to the core in reactions other than click chemistry, and

[0338] - At least two polymer arms containing functional groups suitable for click chemistry.

[0339] (c) By enabling functional groups that are non-reactive in click chemistry to react, a connection is formed between the polymer arm attached to the core and the polymer arm that forms the repeating unit precursor of the dendritic structure.

[0340] (d) Optionally, the functional groups of at least two polymer arms containing functional groups suitable for click chemistry are converted into functional groups that are non-reactive in click chemistry, and

[0341] (e) An active agent containing a functional group is conjugated to the outermost polymer arm by reacting with the functional group of the outermost polymer arm, thereby forming a hyperbranched macromolecule-active agent conjugate.

[0342] In some embodiments for higher-generation Gx hyperbranched macromolecules, where x is an integer from 2 to 10, step (d) is mandatory, and prior to the conjugation of the active agent in step (f), other successive dendritic constituent repeating unit precursors are attached to the click chemistry-unreactive functional groups obtained in step (d) to attach to the hyperbranched macromolecule.

[0343] In the synthesis method, the dendritic constituent repeating unit precursor in step (c) can be represented by equation (iii) as described above.

[0344] In another alternative embodiment of the aggregation method, the aggregation synthesis of the hyperbranched macromolecules of the present invention is provided, comprising the following steps:

[0345] I. Provides a precursor for a dendritic repeating unit, which contains

[0346] - A polymer arm containing functional groups that are non-reactive in click chemistry, and

[0347] - At least two polymer arms containing functional groups (such as azides, alkynes, alkenes, or tetrazines) suitable for linking via click chemistry.

[0348] II. Conjugating an active agent 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.

[0349] III. A core unit having at least three connectivity c is provided, wherein a plurality of polymer arms connected to the core unit have functional groups at their ends that are non-reactive in click chemistry, and

[0350] IV. A connection is formed between the polymer arm connected to the core provided in step III) and the polymer arm containing non-reactive functional groups in the click chemistry of the dendritic constituent repeating unit precursor obtained in step II), thereby forming a biodegradable hyperbranched macromolecule-activator conjugate.

[0351] Exemplary reaction conditions for forming dendritic polymers involve reacting the core precursor and DCRU in a suitable solvent (such as DMF) at relatively mild temperatures (such as 10°C to 50°C, or 30°C to 45°C) for several hours (such as overnight or up to 24 hours or even 48 hours).

[0352] Purification and characterization

[0353] Purification of the hyperbranched macromolecular reaction mixture obtained by the synthetic method described herein can be performed, for example, by filtration, dialysis, SEC column filtration, centrifugation, or UHPLC.

[0354] In one exemplary embodiment, the synthetic reaction mixture of a hyperbranched molecule optionally conjugated with an active agent (such as a peptide) is diluted, filtered (e.g., to a 0.45 µm mesh size), and then purified by ultracentrifugation filtration (e.g., using a 100 kDa membrane). The final product can then be obtained by lyophilization after the addition of a sugar buffer. For administration in a therapeutic manner, the lyophilized product can be reconstituted by adding a solvent (optionally including another sugar buffer).

[0355] Before or after lyophilization, a sugar buffer may be added as needed to improve the solubility and stability of dendritic peptide or protein conjugates, for example, by preventing peptide precipitation. Even when using non-peptide dendritic polymer conjugates, the addition of a sugar buffer improves stability and solubility because the PEG-based dendritic polymers of the present invention exhibit behavior similar to synthetic proteins. Exemplary sugar buffer formulations for use with embodiments of the present invention may comprise aqueous solutions of sugars (such as trehalose), monophosphates, and diphosphates at suitable concentrations (such as 3% or 30 mg / mL) and a pH of about 6.4.

[0356] Dialysis is a common purification method based on the difference in the rate at which molecules diffuse through a semipermeable membrane (such as a dialysis tube) in solution to separate the molecules. To purify a hyperbranched macromolecular reaction mixture, which can be a solution containing molecules of different sizes (such as free peptides (MW of, for example, about 1.5 kDa), free PEG / DCRU precursors (MW of, for example, about 10-40 kDa), small hyperbranched macromolecular conjugates (such as G0, MW of, for example, about 20-50 kDa), and large conjugates (such as higher-generation Gx hyperbranched macromolecular conjugates, for example, about 50 kDa and above)), this solution can be loaded into a dialysis tube with a membrane of a specific pore size having a defined cutoff value and immersed in a large volume of solvent. Molecules smaller than the pore size will elute from the tube into the solvent, while molecules larger than the pore size will remain inside the tube. Dialysis tubes are available from companies such as Spectra / Por® Float-A-Lyzer G2Dialysis Devices and Spectrum® Laboratories, and come with several different molecular weight cutoff values ​​depending on the specific separation task required. Figure 5 a) Show the corresponding experimental setup for purification by dialysis.

[0357] By selecting dialysis tubes and membranes with appropriate molecular weight cutoff values, it may be possible to remove most impurities from the product, such as excess peptides and unreacted precursors and small-sized intermediates, in a series of separation steps using dialysis tubes with different cutoff values, if necessary.

[0358] Another purification method applicable to certain aspects of this invention is the use of size exclusion chromatography (SEC), such as SEC columns. For example, hyperbranched macromolecular conjugates can be purified using a Zeba™ rotary desalting column (from ThermoFisher Scientific) designed for protein purification to remove salts and small-size impurities. Columns with different pore sizes (e.g., 7 kDa and 40 kDa) can be used. The purification mechanism is based on size exclusion chromatography, where small particles are trapped in pores on a stationary phase material, and particles with larger sizes (such as hyperbranched macromolecular conjugates of certain embodiments of this invention) are eluted through the column and collected in purified form. Figure 5 b) Shows the corresponding experimental setup for purification via SEC column filtration.

[0359] The purified product can be characterized by ultra-high performance liquid chromatography (UHPLC), a highly efficient technique that provides more sensitive analysis with good chromatographic separation and resolution of the analyte. It offers benefits including rapid analysis, high-resolution separation, reduced solvent and sample usage, and enhanced sensitivity and accuracy. Based on calibration curves using the free surfactant and free precursor units, and a comparison of solutions before and after purification, the amount of the desired product in the purified solution can be determined by peak area integration.

[0360] Purification by dialysis yields a product solution containing greater than 99% of the hyperbranched macromolecular-peptide-conjugate (see Example 6), as determined by UHPLC based on peak area integral. Purification by SEC column yields a product solution containing greater than 98% of the hyperbranched macromolecular-peptide-conjugate (see Example 7), as determined by UHPLC based on peak area integral. Both purification methods demonstrate extremely high purification efficiency.

[0361] To determine the molecular weight of hyperbranched macromolecular conjugates in certain embodiments of the present invention, SDS-PAGE can be used. SDS-PAGE is an analytical technique that separates materials based on their molecular weight. When a sample is separated by electrophoresis at a potential through a gel matrix, smaller compounds migrate faster due to less resistance from the gel matrix, while larger molecules migrate more slowly. Sodium dodecyl sulfate (SDS) is a surfactant that can strip macromolecules (such as proteins) and eliminate the influence of their structure and charge to separate compounds based solely on molecular size.

[0362] In embodiments of the invention, lyophilized hyperbranched molecules are used to provide a storage-stable formulation that can be reconstituted with a suitable solvent prior to therapeutic use.

[0363] Multivalent receptor binding

[0364] For bioefficacy, it is desirable that biomolecules conjugated to the hyperbranched macromolecules of the present invention exhibit the same or similar affinity for the receptor. Furthermore, efficacy can be improved by extending the half-life of receptor-binding biomolecules through multivalent binding. For example, in antigen binding, affinity is defined as the strength required for interaction between the antigen-binding site at the antibody and the antigenic epitope. Affinity is the total strength required for interaction between a multivalent antibody and multiple antigenic epitopes. This definition can also be applied to other biomolecules binding to specific target or receptor sites. Therefore, multivalent binding can improve affinity. The concept of multivalent binding, the resulting concept of affinity, and the model for quantifying affinity have been described by Kitov et al., “On the Nature of the Multivalency Effect: A Thermodynamic Model”, JACS 2003, 125, 16271-16284, the entire text of which is incorporated herein by reference.

[0365] Kitov describes the interaction between Shiga-like toxins and a series of multivalent oligosaccharide ligands conjugated to dendritic polymers with different multivalent PANAM structures. Kitov specifically found that even though the additional branches of the multivalent ligand dendritic polymers do not logically interact with the receptor, they still increase the probability of receptor interaction. Furthermore, Kitov concludes that "in cases where inhibition of all binding sites is required to achieve the desired effect, the fraction of uninhibited binding sites can be precisely controlled by assembling multivalent inhibitors with an appropriate number of branches." Therefore, using multivalent inhibitor systems as an example, additional branches with other conjugated inhibitor molecules can guarantee a higher degree of inhibition, but individual inhibitors may not interact specifically with the receptor, resulting in prolonged half-life, improved efficacy, and enhanced affinity even through multivalent binding probability.

[0366] Khalili et al., “Fab-PEG-Fab as a Potential Antibody Mimetic”, Bioconjugate Chem. 2013, 24, 1870-1882 (the full text of which is incorporated herein by reference), illustrate the example of divalent PEG conjugated to protein-binding ligands, demonstrating the improved affinity of divalent PEG.

[0367] Some embodiments of the present invention utilize the following examples 8, 9, and... Figure 18 and Figure 19 The concepts shown.

[0368] Embodiments of the present invention relate to methods of treating diseases with antibodies bound to dendritic polymers as described herein. For example, the dendritic polymers of embodiments of the present invention can be used to improve the pharmacokinetics of antibodies delivered as delivery targets bound to or conjugated to hyperbranched molecules as described herein. Suitable delivery targets are, for example, selected from anti-VEGF, aflibercept, faricimab, bevacizumab, anti-TNF-α, infliximab, etanercept, adalimumab, anti-IL-6R, cerilurumab, anti-IL-6, siltuximab, anti-C5, rivulizumab, eculizumab, anti-CD20, ocrelizumab, rituximab, anti-IGF-1R, or tetuximab. These antibodies, bound to and delivered by the dendritic polymer, are non-covalently bound to the antibody drug and, when administered intravenously or injected into the vitreous fluid (IVT), prolong their half-life in the blood. The high molecular weight of dendritic polymer-antibody conjugates prevents the bound antibodies from being cleared from the blood via the kidneys and slows their diffusion from therapeutic targets such as vitreous humor.

[0369] The antibody retains its function while binding to the dendritic polymer of an embodiment of the invention. Gradual release from the dendritic polymer allows for unimpeded delivery of the antibody to the target tissue. The dendritic polymer can be programmed to degrade into smaller molecular weights, such as fragments having less than 50,000 kDa as described herein, for eventual clearance by the kidneys.

[0370] The nanoscale size of the dendritic polymer-antibody conjugates in this invention further allows for passive targeting of leaking blood vessels, such as tumors or choroidal neovascularization (CNV), through an enhanced permeability and retention (EPR) effect. EPR enables subcutaneous (SC) or intravenous (IV) administration routes by reducing off-target effects. This allows for SC or IV delivery to CNV areas in the eye.

[0371] Exemplary diseases that can be treated with dendritic antibody conjugates in certain embodiments include wet AMD, cancer (e.g., with an anti-VEGF dendritic conjugate, IVT, or SC); RA, PsA, COPD (e.g., with an anti-TNF-α dendritic conjugate, IV or SC); PNH, aHUS, MG, glomerulonephritis, GA (e.g., with a dendritic conjugate containing anti-C5, rivulizumab, or eculizumab, IV, IVT, or SC); RA (e.g., with a dendritic conjugate containing rituximab, IV); or TED (e.g., with a dendritic conjugate containing anti-IGF-1R, IV or SC).

[0372] Other embodiments of the invention relate to methods of treating diseases with peptides bound to dendritic polymers as described herein. For example, the dendritic polymers of the embodiments of the invention can be used to improve the pharmacokinetics of peptides delivered as delivery targets bound to or conjugated to hyperbranched molecules as described herein. Suitable delivery targets are, for example, selected from anti-C3, C3B, syfovre, GLP-1RA, liraglutide, victosa, saxenda, semaglutide, Ozempic, rybelsus, wegovy, exenatide, hormone therapy, HGH (somatotriplin), insulin, estrogen, etc.

[0373] Compared to larger proteins, peptides have the advantages of lower immunogenicity and better stability. However, peptides have the disadvantage of rapid clearance and can have low solubility, which may limit their usefulness in therapy. In embodiments of the present invention, conjugation with dendritic polymers may be a successful strategy for peptide delivery in therapeutic treatments (e.g., dendritic polymer Syfovre conjugates) to increase solubility and prolong half-life. Syfovre also benefits from divalent conjugation for improved binding affinity. As in embodiments of the present invention, conjugation with dendritic polymers can go beyond simple PEG conjugation to provide higher molecular weight, longer half-life, and higher valence—higher affinity. The stepwise biodegradation of dendritic polymers into smaller fragments allows for the clearance of these high molecular weight molecules and prevents their accumulation in the body.

[0374] Exemplary diseases that can be treated with dendritic peptide conjugates in certain embodiments include GA, PNH (e.g., with anti-C3, C3B dendritic conjugates, IVT, IV, or SC); T2D, obesity (e.g., with GLP-1RA dendritic conjugates); and hormone deficiency syndromes (e.g., with hormone dendritic conjugates, inhaled, IV, or SC).

[0375] Other embodiments of the invention relate to methods of treating diseases using aptamers bound to dendritic polymers as described herein. For example, the dendritic polymers of the present invention can be used to improve the pharmacokinetics of aptamers delivered as delivery targets bound to or conjugated to hyperbranched molecules as described herein. Suitable delivery targets are, for example, selected from anti-C5, Izervay, anti-VEGF165, Macugen, anti-CXCL12 / SDF-1, or NOX-A12.

[0376] In terms of low immunogenicity, aptamers are similar to peptides. However, in vivo stability has become an issue, which can be addressed by conjugation with dendritic polymers as described herein. Aptamers also exhibit good water solubility. Conjugation with PEG has been a successful strategy for extending the half-life of aptamers (e.g., Macugen and Izervay). Conjugation with dendritic polymers as described herein goes beyond simple PEG conjugation to provide higher molecular weight, longer half-life, and higher valence—higher affinity. The stepwise biodegradation of dendritic polymers into smaller fragments allows for the clearance of these high molecular weight molecules and prevents their accumulation in the body.

[0377] Exemplary diseases that can be treated with dendritic aptamer conjugates in certain embodiments include wet AMD (e.g., with an anti-VEGF165 dendritic conjugate); PNH, aHUS, MG, glomerulonephropathy, GA (e.g., with an anti-C5 or izervay dendritic conjugate); CLL, pancreatic cancer (e.g., with an anti-CXCL12 / SDF-1 dendritic conjugate).

[0378] To determine the bioefficacy of the hyperbranched macromolecular conjugates in certain embodiments of the present invention, binding assays can be performed to analyze the binding affinity of the peptides conjugated to the hyperbranched macromolecular conjugates.

[0379] Complement activation is essential for the development of a normal inflammatory response against exogenous pathogens; however, inappropriate activation has become a cause of tissue damage in many disease states. Complement component C3 is a common pathway for complement activation, encompassing the classical, alternative, and lectin pathways. Uncontrolled complement activation can lead to a range of life-threatening or debilitating conditions.

[0380] Campstatin is a disulfide-bridged 13-mer peptide (le-Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys-Thr-NH2), a novel and promising inhibitor of complement system activation, which was initially isolated from a library of random peptides displayed by phages selected for C3b.

[0381] APL-1 (le-Cys-Val-MeTrp-Gln-Asp-Trp-Gly-Ala-His-Arg-Cys-Thr-NH2) has a structure similar to that of campstatin, but with two distinct amino acids in its sequence. As reported in the literature, the dissociation constant K of APL-1 at C3 is... D It is 10 nM, while the K of campstatin D The concentration is 13 µM, and its C3 binding affinity differs by approximately 100-fold. The structures of the campstatin and APL-1 peptide sequences are shown below:

[0382] Option C:

[0383]

[0384] Fc-III-4C is an immunoglobulin G (IgG) binding peptide ligand composed of 15 residues, of 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:

[0385] Option D:

[0386]

[0387] The binding affinity of the Fc-III-4C peptide to human IgG was determined to be 2.45 nM (K). D The Fc-III-4C peptide exhibits high affinity for various IgGs from different species, and has therefore been reported as a peptide-based antibody affinity tag.

[0388] Using a surface resonance plasmon setup at Mosaic Biosciences, Inc., USA, the three peptides mentioned above can be used to analyze the binding affinity of their hyperbranched macromolecular conjugates. Surface plasmon resonance (SPR) binding assays can be used to study molecular interactions. SPR is an optical technique used to detect the interaction of two different molecules, one of which is mobile and the other is immobilized on a thin film. In this type of analysis, a C3 target is immobilized on the surface of a thin film or chip, and a solution of the hyperbranched macromolecular-peptide conjugate is passed through it. Differences in signal are monitored as the hyperbranched macromolecular-peptide conjugate associates / dissociates on the C3 target.

[0389] These analyses confirm that the C3 and C3b bindings of free campstatin, APL-1, and Fc-III 4C are related to the dissociation constant K reported in the literature. D The values ​​match well, therefore the chosen analysis is a reliable tool.

[0390] The same analysis can be applied to test the binding affinity of hyperbranched macromolecules to peptides, where C3 is immobilized on an analytical chip and a solution of the hyperbranched macromolecule-peptide conjugate is passed through it at a series of concentrations. If multiple peptides conjugated to the hyperbranched macromolecule undergo multivalent binding, the hyperbranched macromolecule should dissociate very slowly, while the free peptide is expected to dissociate rapidly. This measurement allows for comparison of dissociation rates between different generations of hyperbranched macromolecules.

[0391] This theoretical prediction has been confirmed by SPR measurements. A comparison of the binding affinity of hyperbranched macromolecular conjugates of campstatin, APL-1, and Fc-III 4C from certain embodiments of the present invention in the same analysis showed rapid association rates. During dissociation, the reaction from free campstatin decreased rapidly, at the same rate as association, as expected. On the other hand, the hyperbranched macromolecular-peptide conjugates showed much slower dissociation rates. Not wishing to be limited by theory, it is thought that this is due to multiple interactions (e.g., multivalent binding) of several peptides conjugated on the hyperbranched macromolecule. The slower dissociation-associated phase indicates the co-binding of the multivalently conjugated hyperbranched macromolecular-peptide to the C3 surface.

[0392] Similar experiments have shown that peptides hydrolyzed from hyperbranched macromolecules during biodegradation possess approximately the same binding affinity as free peptides. Hydrolyzed peptides contain ester links from the degradation process (part of the linkers on the hyperbranched macromolecules), and based on similar SPR signals, this indicates no effect on their C3 binding. Therefore, it is assumed that ester links do not alter the peptide's biological activity.

[0393] In addition, in quantitative K D The analysis shows that when a polypeptide is substituted onto a hyperbranched macromolecule, the K of the hyperbranched macromolecular conjugate increases. D The display shows a decrease.

[0394] refer to Figure 18 and Figure 19 The IC50 (half-maximal inhibitory concentration) of four different hyperbranched macromolecules of the present invention, as described in Example 10, was measured using an alternative pathway (AP) hemolysis assay. It was observed that G1 PEG hyperbranched macromolecules of the campstatin exhibited a modified IC50 relative to free campstatin, indicating improved potency through affinity modification. Not wishing to be limited by theory, it is thought that the use of higher-generation hyperbranched macromolecule peptide conjugates, with multiple binding events or multivalent binding, is possible, which contributes to receptor inhibition and increases efficiency. Furthermore, hyperbranched macromolecule conjugates with longer polymer arms conjugated to the peptide appear to have a modified IC50, possibly due to the greater flexibility in receptor interaction compared to shorter polymer arms which may have steric hindrance issues. These results were confirmed using a classical pathway (CP) hemolysis assay, demonstrating that higher-valent campstatin conjugates of certain embodiments of the present invention are more effective in inhibiting CP hemolysis compared to free campstatin alone. In addition, as shown in Example 10, dendritic polymer binders (such as APL-1 used in this example) can maintain their in vivo stability and activity over extended periods of time.

[0395] Release kinetics

[0396] In some embodiments, the hyperbranched macromolecules of the present invention can be used for sustained-release drug delivery. Generally, the conjugation of a therapeutic active agent to a hyperbranched macromolecule can prolong the in vivo half-life of the agent. The hyperbranched macromolecular structure can be adapted to modify the release of the active agent conjugated at the hyperbranched macromolecule through several measures to provide a hyperbranched macromolecule-based drug delivery system. For example, adjusting or appropriately selecting the precursor components forming the hyperbranched macromolecule and the DCRU (such as the length and molecular weight of the polymer arms, the type of linker used, and the links formed between the hyperbranched macromolecular portions for conjugation, etc.) has an impact on the release of the active agent.

[0397] Furthermore, the release of surfactants with multiple dendritic polymer binding sites can be mitigated by intramolecular and / or intermolecular multiple binding of the surfactant to the functional end groups of the dendritic polymer (i.e., the surfactant connecting two or more dendritic polymers through a single multiple bond). For example, multiple binding with the dendritic polymer can be used to increase the half-life of the surfactant, since the release of the surfactant from the dendritic polymer requires the cleavage of more than one bond to fully release the surfactant.

[0398] Dendritic polymers used for drug delivery can be considered as large supports or carrier media. Due to their highly symmetrical and regular spherical structure, they can be used to extend the hydration or hydrodynamic radius R of the active agent bound to them. h The large hydrodynamic radius of specific PEG-based dendritic polymer structures can be used to further extend the in vivo (e.g., in the vitreous) half-life of dendritic drug conjugates, which can be used to control and regulate the sustained release of the active agent. This can be estimated using the Stokes-Einstein equation.

[0399]

[0400] Given that temperature T and viscosity η are relatively constant in physiological fluids within the body, the diffusion rate D of spherical particles is approximately equal to the particle's hydrodynamic radius R. h Inversely proportional. Therefore, the larger the radius of the dendritic polymer drug conjugate, the slower the diffusion rate, and the longer the in vivo half-life T of the active agent. 1 / 2 The longer.

[0401] Therefore, embodiments of the present invention utilize large-sized dendritic polymers to delay the release of the active agent in vivo by appropriately adjusting the overall size of the dendritic polymer drug conjugate. Due to the hydrodynamic radius R... hIt can be easily determined, for example, by size exclusion chromatography (SEC), so the release rate or half-life of the active agent bound to the dendritic polymer can be predictively adjusted based on calibration information from the SEC measurement. Example 11 below exemplarily shows a way to correlate dendritic polymer size with release rate.

[0402] Therefore, the biodegradable synthetic dendritic polymers of the embodiments of the present invention offer the advantage of built-in controlled degradable functional groups that generate a gradually decreasing hydrodynamic radius R during degradation. h Smaller fragments with different half-lives determine their mobility and / or clearance from the body. As an example, a first-generation (G1) dendritic polymer (e.g., 4a40k-PEG(SGA)-[4a20k-PEG(SG)-(Fc-III-4C)3]4) constructed from a 4-arm 40 kDa PEG core and four 4-arm 20 kDa dendritic motifs conjugated with 12 peptides or proteins, each 1.7 kDa, has a molecular weight of approximately 145 kDa. Cleavage of one, two, three, or all four dendritic motifs progressively reduces the molecular weight to produce fragments of approximately 115 kDa, 90 kDa, and 65 kDa, ultimately leaving a 40 kDa core and four dendritic motifs, each approximately 25 kDa, with each fragment having a different hydrodynamic radius and diffusion rate. If a 20 kDa linear PEG extension is constructed in the same macromolecule between the core and branching unit arms, the molecular weight cascade includes a dendritic polymer of about 22 kDa and degradation fragments of about 175 kDa, 130 kDa, 85 kDa, 45 kDa, 40 kDa, 25 and 20 kDa. This dendritic polymer will have different extended release rates and a larger fragment half-life distribution.

[0403] The multiple half-life aspects of the biodegradable dendritic polymers in the embodiments of the present invention are based on the initial R of the dendritic polymer itself. h (G1, G2, etc.), followed by another half-life based on degradable fragments (dendritic units, dendritic unit-like fragments, or dendritic unit-like fragments with linear PEG extensions) formed by breaking hydrolyzable bonds within the hyperbranched macromolecular structure. These various sequentially degradable substances have different R values ​​as they degrade and detach from the initial dendritic polymer structure. h This results in a series of substructures with different hydrodynamic radii, leading to varying clearance rates and half-lives. The R values ​​of these different substances... hAssociated with different building blocks of varying molecular weights, arm numbers, linear PEG extensions, and / or linkers. By constructing hyperbranched dendritic polymer macromolecules from building blocks of varying molecular weights and arm numbers using bifunctional linkers of varying lengths between linear PEG extensions and hydrolyzable bonds, the dendritic polymers of embodiments of the present invention can be designed for each individual active agent and / or therapeutic purpose / or form of administration, degrading and clearing fragments at multiple rates and half-lives.

[0404] Specifically, built-in degradable linker groups (such as diacid-derived esters based on linkers of succinimide succinate (SS), succinimide glutarate (SG), succinimide adipate (SAP), and succinimide azelaate (SAZ), such as diester succinate (S), diester glutarate (G), diester adipate (AP), or diester azelaate (AZ)) can thus be used to tune and control the release rate of the active agent conjugated or associated with the dendritic polymer. For example, in one embodiment, the dendritic building blocks can be made using different degradable linkers (such as SS, SG, SAP, SAZ, etc.), resulting in a homogeneous dendritic polymer where all dendritic units degrade at the same rate if the ester linkages are identical.

[0405] In another embodiment, the dendritic building blocks can be made using different degradable links (such as S, G, AP, AZ, etc.), resulting in heterogeneous dendritic polymers where the dendritic units degrade at different rates if the ester links are different. For example, in one embodiment, the dendritic polymer can be made using S, G, AP, or AZ dendritic units or mixtures of such dendritic units by clicking chemical links onto the core structure.

[0406] In other embodiments, a mixture of several homogeneous dendritic polymers can be blended by dry or wet blending to adjust a specific release profile and regulate half-life clearance.

[0407] The PEG-based embodiments of the present invention can also be described as nanodroplets due to their large hydrodynamic radius at low solids content. Dendritic polymers exhibit much higher hydrodynamic radii at similar weights or solids contents to the agent or peptide itself (see [link to original text]). Figure 20 And in Example 11), and therefore the diffusion rate is slower, and the in vivo half-life T of the conjugated active agent is longer. 1 / 2 Longer.

[0408] In some embodiments, the linkers of formula (ii) used in the hyperbranched macromolecular structure introduce hydrolyzable bonds into the hyperbranched macromolecular, which can be used to alter the degradation rate of the hyperbranched macromolecular and / or the release rate of the conjugated active agent from the hyperbranched macromolecular. For example, the biodegradation / hydrolysis rate of the ester bonds at these linkers increases from succinate (C4) to azelate (C9). The shorter the chain length of the diacid linker, the faster the hydrolysis of the ester bonds formed. Therefore, the hydrolysis rate decreases from SS>SG>SAP>SAZ>SGA ester bonds. In embodiments of the invention, this can be used to control the degradation rate of the hyperbranched macromolecular and / or the release of active agents conjugated to the hyperbranched macromolecular via these linkers. For example, esters formed from succinate succinimide (SS) groups can degrade within days, while esters from glutarate succinimide (SG) groups degrade within weeks. Different linkers can be used within hyperbranched macromolecules to control the degradation rate at the junctions of different generations of DCRUs in the hyperbranched macromolecules, and can be used to conjugate surfactants to control the release of surfactants from the hyperbranched macromolecules.

[0409] For links formed via click chemistry, embodiments of this disclosure may also utilize the extension of the spacer structure (e.g., alkylene chains or polyethylene glycolation) between the DBCO / azide functional group and the functional group bound to the polymer arm to delay the hydrolysis of adjacent ester links. The greater the distance between the DBCO / azide functional group and the next hydrolyzable ester group, the slower the ester hydrolysis occurs. Degradation control can be achieved by using different linkers within the hyperbranched macromolecule and at the conjugation site of the surfactant. By using short-chain linking groups (such as succinates) for DCRU links and long-chain linkers (such as SAZs) at the conjugation site, it is possible to first cleave the ester group within the hyperbranched macromolecule during degradation, followed by the DCRU-surfactant conjugation. Conversely, the conjugation site for surfactant release can be intentionally designed to first cleave the surfactant release with a short-chain linker. This can be used to control the surfactant's half-life and alter release kinetics.

[0410] Furthermore, depending on the chain length of the diacid linker, the hydrolysis of the ester bond will depend on the pH and / or temperature of the environment. This can be used in some embodiments to control the release of the active agent, such as site-specific release in certain tumor cells with a pH higher than that of the surrounding cells.

[0411] In some embodiments, the sustained-release drug delivery hyperbranched macromolecules of the present invention are formulated to make the active agent available over an extended period of time, thereby allowing for a lower dosing frequency compared to immediately released dosage forms (such as solutions of the active agent applied topically to the eye, i.e., eye drops). In some embodiments, the release of the active agent includes constant release, gradually decreasing release, and any combination thereof, such as constant release followed by gradually decreasing release. "Sustained release" can be measured in vitro in aqueous solution under physiological conditions (such as at pH 7.2-7.4 and 37°C) and considered the same or substantially the same when the hyperbranched macromolecule is administered in vivo to a subject.

[0412] In various embodiments of the invention, the release of the active agent follows zero-order release kinetics or substantially zero-order release kinetics, and preferably does not have a “burst” of active agent in the initial period.

[0413] Embodiments of the present invention may provide the release of a therapeutically effective amount of the active agent over a period of time following application (e.g., 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). Other embodiments of the present invention may provide the release of a therapeutically effective amount of the active agent over a period of up to about 14 days or up to about 21 days following application, or the release of a therapeutically effective amount of the active agent over a period of about 6 hours or longer following application, or about 12 hours, or 24 hours or longer, or about 48 hours or longer, or about 72 hours or longer, or about 7 days or longer, or about 10 days or longer following application. All of the lower and higher time periods described above are included in any combination of the scope of the present invention.

[0414] Some aspects of this disclosure relate to the controlled release of an active agent conjugated to a pharmaceutically acceptable hyperbranched macromolecule, wherein the controlled release is characterized by: the amount of active agent released on day 1 being 0 to 50% of the total amount of active agent; the amount of active agent released each day from day 2 until the last day of release being 0 to 50% of the total amount of active agent; and / or the number of days required for 100% release of the total amount of active agent being at least 2 days.

[0415] In one embodiment, controlled release of the surfactant is characterized by the release of 0 to 50% of the total surfactant on day 1, the release of 0 to 50% of the total surfactant daily from day 2 until the last day of release, and / or the number of days required for 100% release of the total surfactant is at least 2 days. In another embodiment, the release of the surfactant on day 1 is 0 to 25%, 0 to 20%, 0 to 10%, 0 to 5%, or about 0% of the total surfactant, the release of 0 to 50% of the total surfactant daily from day 2 until the last day of release, and / or the number of days required for 100% release of the total surfactant is at least 3 days, but not more than 30 days, 25 days, or 16 days.

[0416] Sustained-release drug delivery systems and administration

[0417] The hyperbranched macromolecules of certain embodiments of the present invention can be used for drug delivery to patients, and for example for ophthalmic drug delivery, because they offer several advantages as a carrier system. Hyperbranched macromolecules can be used for drug delivery, gene delivery, antioxidant delivery, peptide delivery, biomedical imaging, and genetic testing in ophthalmology.

[0418] Hyperbranched macromolecules are capable of transporting in and out of cells. Drug delivery of hyperbranched macromolecules can be achieved using various ocular administration routes, and their tunable properties (such as water solubility, permeability, bioavailability, and biocompatibility) can vary widely depending on the specific needs of different medical applications.

[0419] In some embodiments, a sustained-release biodegradable drug delivery system is provided, comprising hyperbranched macromolecules as described herein. In some embodiments of the invention, the hyperbranched macromolecules or drug delivery systems comprising them can be formulated for direct or indirect administration via a variety of routes, such as oral, parenteral, or surgical insertion or injection.

[0420] To formulate a drug delivery system, hyperbranched macromolecules can be incorporated into a suitable carrier (such as a solvent or solvent mixture), or they can be incorporated into a hydrogel or organic gel.

[0421] In some implementations, hyperbranched macromolecules are formulated for direct injection at the patient's treatment site, such as via parenteral administration or intratumoral injection, or injection into the eye, such as intravitreal, intraanterior chamber, subconjunctival, retrobulbar, subtenon, subretinal, and suprachoroidal injection. It can be formulated for injection into the anterior chamber, vitreous body, extrasclera, posterior subglobose space (inferior fornix), subconjunctiva, anterior chamber, periorbital, posterior to the eye, subglobose, retina, subretinal, tubules, vitreous body, intrasceleral, choroid, suprachoroidal, retina, subretinal or lens, corneal or conjunctival surface, lacrimal puncta (tubules, superior / inferior tubules), fornix, superior / inferior fornix, subglobose space, choroid, suprachoroidal, bulbar fascia, cornea, cancerous tissue, organs, prostate, breast, joint spaces, subdural, teeth, subcutaneous, carpal tunnel, perivascular space, surgically created spaces or injuries, cavities, and potential spaces.

[0422] In embodiments of the present invention, a drug delivery system is used to generate or form a medical implant in which hyperbranched macromolecules are embedded or dispersed in a hydrogel or organic gel matrix.

[0423] Treatment

[0424] According to certain embodiments of the invention, hyperbranched macromolecules or biodegradable drug delivery systems containing hyperbranched macromolecules are configured for use as drugs, such as for treating a patient’s disease or medical ailment.

[0425] In one embodiment, a method of treating a patient’s disease or medical ailment includes administering a hyperbranched macromolecule to the patient to release the active agent over an extended period of time.

[0426] The treatment methods of this invention include ocular treatments. In such treatments, hyperbranched macromolecules are used to release an active agent in the eye over an extended period of time. In its embodiments, the disease or medical condition to be treated is an eye disease, an ocular disease, such as a posterior ocular disease, such as any posterior segment ocular disease affecting the retinal, macular, or choroidal vascular system and integrity, resulting in visual acuity impairment, vision loss, or blindness, particularly posterior segment disease states caused by age, trauma, or surgical intervention, such as age-related macular degeneration (AMD), cystic macular edema (CME), diabetic macular edema (DME), posterior uveitis, and diabetic retinopathy.

[0427] Treatment methods can also include glaucoma, high intraocular pressure, anterior chamber hemorrhage, presbyopia, cataracts, retinal vein occlusion, inflammation, miosis, mydriasis, conjunctivitis, intraocular infections, choroidal neovascularization (CNV), intraocular tumors, and retinal nerve inflammation.

[0428] Eye diseases can further include one of the following: retinal neovascularization, choroidal neovascularization, wet AMD, dry AMD, retinal vein occlusion, diabetic macular edema, retinal degeneration, corneal graft rejection, retinoblastoma, melanoma, glaucoma, autoimmune uveitis, uveitis, proliferative vitreoretinopathy and corneal degeneration, acute and chronic macular neuroretinopathy, central serous chorioretinopathy, macular edema, acute multifocal squamous epiretinal disease. Skin lesions, Behcet's disease, shotgun retinal choroidal lesions, posterior uveitis, posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusive disease, central retinal vein occlusion, disseminated intravascular coagulation, retinal branch vein occlusion, hypertensive fundus changes, ocular ischemia syndrome, retinal artery microaneurysms, Koch's disease, parafoveal telangiectasia, hemiretinal vein occlusion, papillary phlebitis, carotid artery disease (CAD), frost-like dendritic vasculitis, sickle cell retinopathy, vascular streaks, familial exudative vitreoretinopathy, Ilse's disease, proliferative vitreoretinopathy, diabetic retinopathy, tumor-associated retinal diseases, congenital hypertrophy of retinal pigment epithelium (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, combined retinal and retinal pigment epithelium hamartoma, retinoblastoma, fundus vascular proliferative tumors Tumors, retinal astrocytomas, intraocular lymphomas, myopic retinal degeneration, acute retinal pigment epithelial inflammation, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal carcinoma, retinitis pigmentosa, Leber congenital amaurosis, choroidal agenesis, X-linked retinitis pigmentosa, best yolk sac macular dystrophy, X-linked retinoschisis, achromatopsia CNGA3, achromatopsia CNGB3, LHON, Sturges disease, Arthur syndrome, Norrie disease, Barby-Bisson syndrome, and red-green color blindness.

[0429] The methods described in this section may also include the administration of a combination of hyperbranched macromolecules and another agent, also known as "combination therapy".

[0430] In one embodiment, the combination therapy comprises administering a hyperbranched macromolecule in combination with one or more other agents on the same or different days. In one embodiment, the other agent to be administered in the combination therapy may be a liquid formulation of the agent, or may be contained in an oral dosage form. Thus, the other agent may be any small molecule, macromolecule, protein, nanoparticle, or any other active agent described herein. In another embodiment, a hyperbranched macromolecule having more than one active agent conjugated thereto (such as those hyperbranched macromolecules that can be obtained by the aggregation synthesis described above) may be used in combination therapies involving the administration of more than one active agent. Using the hyperbranched macromolecule of certain embodiments may allow different regions on the surface of the hyperbranched macromolecule to be conjugated with different agents.

[0431] Treatment methods including the application of hyperbranched macromolecules can include any of the following: intravitreal, intraanterior chamber, subconjunctival, retrobulbar, subfollicular, subretinal, and suprachoroidal injections. Administration can also be local or oral.

[0432] The active agent or other agent to be used in combination therapy can also be a diagnostic agent. Diagnostic agents, as described above, can be substances used to examine the body to detect impairment of its normal function. In some cases, diagnostic agents can be agents with functional purposes, such as those used to detect ocular malformations, pain, and pathophysiological aspects.

[0433] Exemplary embodiments of the medical treatment of the drug-conjugated dendritic polymers relating to the present invention are summarized in Table A below.

[0434] Table A

[0435]

[0436]

[0437] Industrial and non-medical applications

[0438] In embodiments of the invention, the hyperbranched molecules / dendritic polymers can also be used in non-medical or industrial applications. In some embodiments, the dendritic polymers do not include hydrolyzable bonds. In other embodiments, the dendritic polymers may include hydrolyzable bonds as described herein.

[0439] Table B below provides an overview of the non-medical and industrial applications and exemplary uses of the dendritic polymers of the present invention.

[0440] Table B:

[0441]

[0442]

[0443]

[0444] Other exemplary applications and use cases include those listed in Table C:

[0445] Table C:

[0446]

[0447] Example

[0448] The following embodiments are included to illustrate certain aspects and embodiments of the invention as described in the claims. However, those skilled in the art will understand that the following description is merely illustrative and should not be construed as limiting the invention in any way.

[0449] Materials and abbreviations used in the examples:

[0450] The biodegradable bifunctional 4-arm PEG-(NHS)3-(azide)1 (10k, 20k, and 40 kDa) and 4-arm PEG-DBCO (10k, 20k, and 40 kDa) were purchased from XIAMEN SINOPEG BIOTECH Co. Ltd. All NHS-terminated PEGs were purchased from JenKem Technology USA.

[0451] Campstatin (ICVVQDWGHHRCT, disulfide bridge: Cys2-Cys12, TFA and acetate forms) was purchased from MedChemExpress. APL-1 (ICV{L-1-Me-Trp}QDWGAHRCT, disulfide bridge: Cys2-Cys12, TFA and acetate forms) and Fc-III 4C (CDCAWHLGELVWCTC, disulfide bridges: Cys1-Cys15, Cys3-Cys13, TFA, and acetate forms) was purchased from Alan Scientific. The structure is shown in... Figure 4 middle.

[0452] Solvents and other reagents (including methanol, acetonitrile, PBS buffer, and triethylamine) were purchased from VWR.

[0453] Example 1

[0454] Divergent synthesis of branched macromolecular G0-peptide conjugates:

[0455] Option 1:

[0456]

[0457] A measured amount of peptide was weighed and dissolved in anhydrous methanol. A 4-arm PEG-SS-NHS (MW=40 kDa) in a molar ratio of 1:6 to the peptide was slowly added to the peptide (campatine) solution under 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 collected and purified by dialyzing in methanol for 24 hours using a 10 kDa molecular weight cutoff tube. The purified hyperbranched macromolecular G0-peptide conjugate was collected from the dialysate tube and the solvent was removed using a rotary evaporator. The dry powder was stored at -20°C for characterization. The formulation is shown in Table 1.

[0458] Table 1:

[0459]

[0460] Example 2

[0461] Divergent synthesis of hyperbranched macromolecular G1-peptide conjugates:

[0462] Option 2:

[0463]

[0464] Terminal functional group transformation of 4-arm PEG-DBCO:

[0465] A certain amount of 4-arm PEG-NHS (MW=40 kDa) was dissolved in anhydrous methanol. DBCO-amine, weighed at a molar ratio of 1:1 to 4-arm PEG-NHS, was dissolved in anhydrous acetonitrile. The DBCO-amine solution was added dropwise to the 4-arm PEG-NHS solution under 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 for the next synthesis step. The formulation is shown in step 1 of Table 2.

[0466] Formation of hyperbranched macromolecules of 4-arm PEG-[4-arm PEG-(NHS)3]4 (G1):

[0467] A certain amount of 4-arm PEG-DBCO was dissolved in anhydrous methanol:acetonitrile = 1:1 mixed solvent. A 4-arm PEG-(N3)1(NHS)3 (MW = 20 kDa) with a molar ratio of 1:1 to 4-arm PEG-DBCO was weighed and dissolved in anhydrous methanol. The 4-arm PEG-(N3)1(NHS)3 solution was slowly added to the 4-arm PEG-DBCO solution under vigorous stirring. The reaction was carried out at room temperature for 1–4 hours, and the solvent was removed on a rotary evaporator. If necessary, the dried crude product could be further purified by dialysis. The formulation is shown in step 2 of Table 2.

[0468] Peptide conjugation of hyperbranched macromolecular G1-peptide conjugates:

[0469] Weigh a certain amount of peptide and dissolve it in anhydrous methanol. Add 4-arm PEG-[4-arm PEG-(NHS)3]4 (G1) at a molar ratio of 1:18 to the peptide solution slowly under vigorous stirring. Add a small amount of triethylamine (5-10 µL) dropwise to the reaction mixture. Incubate the reaction at room temperature for 1-4 hours. Collect the final product and purify it by dialyzing in methanol for 24 hours against a 40 kDa molecular weight cutoff tube. Collect the purified hyperbranched macromolecular G1-peptide conjugate from the dialysate tube and remove the solvent using a rotary evaporator. Store the dry powder at -20°C for characterization. The formulation is shown in step 3 of Table 2.

[0470] Table 2

[0471]

[0472]

[0473]

[0474] Example 3

[0475] Divergent synthesis of hyperbranched macromolecular G2-peptide conjugates:

[0476] Option 3:

[0477]

[0478] Terminal functional group transformation of 4-arm PEG-[4-arm PEG (DBCO)3]4:

[0479] A certain amount of the 4-arm PEG-[4-arm PEG-(NHS)3]4 (G1) obtained in step 2 of Example 2 was dissolved in anhydrous methanol. DBCO-amine, weighed at a molar ratio of 1:1 to 4-arm PEG-[4-arm PEG-(NHS)3]4 (G1), was dissolved in anhydrous acetonitrile. The DBCO-amine solution was added dropwise to the 4-arm PEG-[4-arm PEG-(NHS)3]4 (G1) solution under vigorous stirring. The reaction was carried out at room temperature for 1–4 hours, and the solvent was removed on a rotary evaporator. The dried crude product was used for the next synthesis. The formulation is shown in step 1 of Table 3.

[0480] Formation of hyperbranched macromolecules of 4-arm PEG-{4-arm PEG-[4-arm PEG (NHS)3]3}4

[0481] A certain amount of 4-arm PEG-[4-arm PEG (DBCO)3]4 was dissolved in anhydrous methanol:acetonitrile = 1:1 mixed solvent. 4-arm PEG-(N3)1(NHS)3 (MW = 10 kDa) with a molar ratio of 1:1 to 4-arm PEG-[4-arm PEG (DBCO)3]4 was weighed 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 under vigorous stirring. The reaction was carried out at room temperature for 1–4 hours, and the solvent was removed on a rotary evaporator. If necessary, the dried crude product could be further purified by dialysis. The formulation is shown in step 2 of Table 3.

[0482] Peptide conjugation of hyperbranched macromolecular G2-peptide conjugates:

[0483] Weigh a certain amount of peptide and dissolve it in anhydrous methanol. Add 4-arm PEG-{4-arm PEG-[4-arm PEG (NHS)3]3}4 (G2) at a molar ratio of 1:18 to the peptide solution slowly under vigorous stirring. Add a small amount of triethylamine (5-10 µL) dropwise to the reaction mixture. Incubate the reaction at room temperature for 1-4 hours. Collect the final product and purify it by dialyzing in methanol for 24 hours against a 40 kDa molecular weight cutoff tube. Collect the purified hyperbranched macromolecular G2-peptide conjugate from the dialysate tube and remove the solvent on a rotary evaporator. Store the dry powder at -20°C for characterization. The formulation is shown in step 3 of Table 3.

[0484] Table 3:

[0485]

[0486]

[0487]

[0488] Example 4

[0489] Convergence synthesis of hyperbranched macromolecular G1-peptide conjugates:

[0490] Option 4:

[0491]

[0492] Peptide conjugation of PEG-(N3)1(peptide)3 in 4 arms:

[0493] A measured amount of peptide was weighed and dissolved in anhydrous methanol. Four-arm PEG-(N3)1(NHS)3 (MW=20 kDa) at 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 for the next synthesis step. The formulation is shown in step 1 of Table 4.

[0494] Formation of hyperbranched macromolecules in G1-peptide conjugates:

[0495] A certain amount of 4-arm PEG-(N3)1(peptide)3 was dissolved in anhydrous methanol. A 4-arm PEG-DBCO (MW=40 kDa) with a molar ratio of 1:1 to PEG-(N3)1(peptide) was weighed and dissolved in anhydrous methanol. The 4-arm PEG-(N3)1(peptide) solution was slowly added to the 4-arm PEG-DBCO solution under vigorous stirring. The reaction was carried out at room temperature for 1–4 hours. The conjugate was collected and purified in methanol for 24 hours in a dialysis tube with a molecular weight cutoff of 40 kDa. The solvent was removed on a rotary evaporator and the dry powder was stored at -20°C for characterization. The formulation is shown in step 2 of Table 4.

[0496] Table 4:

[0497]

[0498]

[0499] Example 5

[0500] Peptide transformation and G0 conjugation of hyperbranched macromolecules:

[0501] Option 5:

[0502]

[0503] Method 1:

[0504] Peptide-DBCO Conversion: Step 1. Dissolve a certain amount of peptide (campatine, MW = 1.5 kDa) in 1 mL of a mixed solvent (anhydrous methanol: acetonitrile = 1:1). Weigh DBCO-NHS and dissolve it in 1 mL of a mixed solvent (anhydrous methanol: acetonitrile = 1:1). Add the peptide solution dropwise to the DBCO-NHS solution under vigorous stirring. The reaction is carried out at room temperature for 1–4 hours. Use the dried crude product for the next synthesis. The formulation is shown in Step 1 of Table 5.

[0505] Dendritic polymer 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 then slowly added to the peptide-DBCO solution with vigorous stirring. The reaction was carried out at room temperature for 1–4 hours, and the solvent was removed on a rotary evaporator. If necessary, the dried crude product could be further purified by dialysis. The formulation is shown in Step 2 of Table 5.

[0506] Table 5:

[0507]

[0508]

[0509] Approach 2:

[0510] Peptide-Azide Conversion: Step 1. Dissolve a certain amount of peptide (campatine, MW = 1.5 kDa) in 1 mL of a mixed solvent (anhydrous methanol: acetonitrile = 1:1). Weigh the azido-NHS and dissolve it in 1 mL of a mixed solvent (anhydrous methanol: acetonitrile = 1:1). Add the peptide solution dropwise to the azido-NHS solution under vigorous stirring. The reaction is carried out at room temperature for 1–4 hours. Use the dried crude product for the next synthesis. The formulation is shown in Step 1 of Table 6.

[0511] Dendritic polymer 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 then slowly added to the peptide-azide solution under vigorous stirring. The reaction was carried out at room temperature for 1–4 hours, and the solvent was removed on a rotary evaporator. If necessary, the dried crude product could be further purified by dialysis. The formulation is shown in Step 2 of Table 6.

[0512] Table 6:

[0513]

[0514]

[0515] Example 6

[0516] Purification of hyperbranched macromolecules and peptide conjugates by dialysis.

[0517] The products obtained as described in Examples 1 to 5 were dissolved in methanol to achieve a concentration of 10 mg / mL or higher, and the solution (1-5 mL) was loaded into dialysis tubes (Spectra / Por) with a specific molecular weight cutoff value. ®Float-A-Lyzer G2 dialysis device, Spectrum ® (Laboratories). The tube was then placed in 500 mL of methanol in a beaker at room temperature for 24 hours to separate the lower molecular weight components diffusing from the tube. See [link to relevant documentation]. Figure 5 a) After processing, the product is removed from the tube and collected in a glass bottle for characterization.

[0518] Table 7: Dialysis tubing selection

[0519]

[0520] The product was then characterized by UHPLC. Figure 6 UHPLC analysis of the G1 4-arm 40k PEG-[4-arm 20k PEG-SG-(campatamine)3]4 conjugate from Example 2 is shown. This conjugate was purified by dialysis in methanol with a molecular weight cutoff of 8-10 kDa, primarily removing low molecular weight peptides (MW approximately 1.5 kDa). The blue line represents before purification, and the green line represents after purification. It can be seen that, based on peak area integral, the peak at a retention time of approximately 4 minutes in the sample (attributed to free campatamine) decreased from 45% to less than 1%. On the other hand, the starting material in the mixture, namely the 4-arm 40k PEG-SG-(DBCO)4 with a molecular weight greater than 8-10 kDa (retention time approximately 9 minutes, MW approximately 40 kDa), was retained in the tube. Additionally, the hyperbranched macromolecular conjugate (MW approximately 120 kDa) was retained in the tube, with the peak shifting slightly to the left from approximately 8.5 minutes to 9 minutes. The separation of hyperbranched macromolecular conjugates from PEG-DBCO precursors can be achieved by using another dialysis tube with a higher molecular weight cutoff value.

[0521] Table 8: Products of Example 2 before and after dialysis purification (data based on UHPLC peak area integral).

[0522]

[0523] These data show that dialysis can achieve highly effective purification of hyperbranched macromolecule-peptide conjugates with a purity greater than 99%.

[0524] Example 7

[0525] Purification of hyperbranched macromolecules and peptide conjugates using SEC column filtration.

[0526] Open the SEC column (Zeba™ rotary desalting column) packaging and rinse the column twice with 1 mL of methanol. Dissolve the product obtained as described in Examples 1 to 5 in methanol to achieve a concentration of 10 mg / mL or higher (1-2 mL), and load the solution onto the column, allowing it to flow through the column by gravity. See also Figure 5 b). The eluent was collected in glass vials for characterization.

[0527] Table 9: SEC Column Selection

[0528]

[0529] Figure 7 UHPLC analysis of the G0 4-arm 40k PEG-SS-campatamine conjugate of Example 1 before purification (black line) and after purification (blue line) is shown. Based on peak area integration, the content of free campatamine decreased from 38.8% to 1.6%, demonstrating very efficient purification capability.

[0530] Table 10: Content changes before and after SEC column filtration (data based on UHPLC peak area integral).

[0531]

[0532] These data show that SEC column filtration can achieve highly effective purification of hyperbranched macromolecular peptide conjugates with a purity greater than 98%.

[0533] Example 8

[0534] Purity and substitution rate by UHPLC

[0535] Ultra-high performance liquid chromatography (UHPLC) is a highly efficient technique that provides more sensitive analysis with good chromatographic separation and resolution of analytes. It offers numerous benefits, including rapid analysis, high-resolution separation, reduced solvent and sample usage, and enhanced sensitivity and accuracy. WatersXBridge BEH300 C18 columns (3.5 µm, 2.1 × 100 mm, PN1860036080) were used to characterize the hyperbranched macromolecules and hyperbranched macromolecule-peptide conjugates of Examples 1 through 5, with mobile phases A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile).

[0536] For UHPLC analysis, peptide powder was dissolved in PBS:methanol = 9:1 at concentrations of 12.5 µg / mL, 25 µg / mL, 50 µg / mL, 100 µg / mL, and 200 µg / mL and injected into the UHPLC. The peak area for each sample was integrated and used as the standard for calculating peptide concentration (see [link to UHPLC analysis]). Figure 8a The inset is a standard curve of peptide concentration versus peak integral area.

[0537] The dried G0 PEG hyperbranched macropeptide was dissolved in PBS:methanol = 9:1 at a concentration of 1 mg / mL and analyzed by UHPLC. Figure 8b The typical UHPLC chromatogram of the G0 4-arm PEG-hyperbranched macromolecule-campastatin conjugate of Example 1 is shown, where the peak with a retention time of 22 min is from free campastatin, and the peak concentrated at 42 min is from the 4-arm PEG-hyperbranched macromolecule-campastatin conjugate. Using the campastatin standard curve, the concentration of each component in the product can be calculated, and peptide substitution can be estimated by dividing the molar number of hyperbranched macromolecule conjugated peptides by the molar number of PEG, as shown in the following equation:

[0538]

[0539] In this equation, C(conjugated peptide) is the concentration of the conjugated peptide, C(free peptide) is the concentration of the free peptide, and C(total sample) is the concentration of total solids in the prepared PEG-hyperbranched macromolecule-peptide conjugate sample, which includes conjugated peptides, free peptides, and free macromolecules (non-conjugated). The equation yields the number of moles of conjugated peptide per mole of macromolecule.

[0540] This method provides a practical way to compare and optimize reaction methods. Campstatin peptide samples from different suppliers (Ambeed, Genscript, and MCE) in different salt forms (trifluoroacetate (TFA), acetate, and lysine) were reacted with the same 4-arm 40k PEG-SGA-NHS under the same reaction conditions. Table 11 lists the conjugation results. In summary, the substitution of each peptide on the G0 4-arm PEG was approximately 80%, with variations of ±10% from one another.

[0541] Table 11:

[0542]

[0543] Campstatin and campstatin-lysine substitution on different PEGs (such as 4-arm 40k PEG-SGA and 4-arm 40k PEG-SS) have been investigated using the same analytical methods. These reactions were run under identical conditions, with the molar ratio of each compound being the same, only the PEG being varied. Each reaction was repeated three times, and according to… Figure 9 These reactions showed good reproducibility, with an average of about 3 peptides substituted on the 4-arm PEG.

[0544] Other reaction conditions (such as reaction time, solvent, and catalyst used) have been investigated, and the results show that... Figure 10Based on the results, it is evident that the reaction in methanol using triethylamine as a catalyst is the optimal condition for achieving the highest peptide substitution, while reaction time plays a secondary role in the change of substitution number.

[0545] Table 12 below lists various hyperbranched macromolecules, from G0 to G2, synthesized via convergence or divergence methods. For the G2 hyperbranched macromolecule, the highest molecular weight hyperbranched macromolecule is approximately 240 kDa, with about 36 terminal functional groups. In most conjugation results, peptide substitution was greater than 50%, indicating good reproducibility of these methods.

[0546] Table 12:

[0547]

[0548] The same synthetic approach was applied to the conjugation of hyperbranched macromolecules using APL-1 and Fc-III 4C, and the substitution results are shown in Tables 13 and 14. The substitution of APL-1 and Fc-III 4C was less than that of campstatin.

[0549] Table 13:

[0550]

[0551] Table 14:

[0552]

[0553] Example 9

[0554] Combined analysis

[0555] We used surface plasmon resonance (SPR) combined with analytical methods obtained from Mosaic Biosciences, Inc. to study molecular interactions.

[0556] The SPR signal was detected using a Biacore 3000 instrument. Typically, C3 and C3b were immobilized at a high density (approximately 20 kRU) on the sensor chip surface. A buffered saline solution at pH 7.4 was flowed through the device at 30 µL / min at 25°C. The hyperbranched macromolecular-peptide conjugate of the present invention was injected at a concentration ranging from 1 nM to 300 nM (APL-1 derivative) or 200 nM to 50 µM (campatine derivative). Association was monitored for 4 minutes and dissociation for 10 minutes. Equilibrium analysis was performed on the campatine analogue, and mass transfer kinetics analysis was performed on the APL-1 analogue.

[0557] Binding affinity of free peptides

[0558] Figures 11 and 12 show the C3 and C3b binding of different types of campstatin, and Figure 13 The results show the C3 and C3b binding of different types of APL-1. KD results are summarized in Table 15. It is evident from these results that the free peptide, campstatin, and APL-1 show KD values ​​remarkably similar to reported results. This result is consistent with previous measurements of APL-1, but the affinity is lower than the APL-2 affinity (200 pM) reported by Apellis, which may be attributed to the affinity effect on divalent APL-2.

[0559] Table 15: KD of C3 and C3b affinity for campstatin and APL-1 and comparison with reference results.

[0560]

[0561] *Samples purchased from Ambeed, **Samples purchased from Genscript, ***Samples purchased from MCE.

[0562] The same experiment was performed on Fc-III 4C. For comparison, another peptide, Fc-III, was evaluated under the same conditions. Fc-III has a similar peptide sequence to Fc-III 4C, but lacks a Cys-Cys bridge. The amino acid sequence structure is as follows:

[0563] Option 6:

[0564]

[0565] This structural difference leads to a significant difference in antibody binding affinity; Fc-III 4C is approximately 8-fold more sensitive to the antibody (KD = 2.45 nM vs. 16 nM). SPR results ( Figure 13 (Table 16) also confirms that the three types of Fc-III 4C exhibit binding affinity KD much smaller than that of Fc-III.

[0566] Table 16: KD of IgG affinity for Fc-III 4C and comparison with reference results.

[0567]

[0568] *Samples purchased from Genscript, **Samples purchased from Alan Scientific.

[0569] Binding affinity of hyperbranched macromolecular conjugated peptides

[0570] The same experiment has been applied to test the binding affinity of hyperbranched macromolecular conjugated peptides, in which C3 is immobilized on a chip and the hyperbranched macromolecular-peptide conjugate is flowed through it at a series of concentrations.

[0571] Figure 15 This paper compares free campstatin and multivalent campstatin 4-arm 40k PEG-SGA-(campstatin)4. Both samples exhibited extremely rapid association rates when passed through a C3-coated chip. After the interaction, a buffer solution was passed through the chip to wash away the associated samples. During this dissociation time, the reaction from free campstatin decreased very rapidly, at the same rate as association. On the other hand, the hyperbranched macromolecule-campstatin conjugate showed a much slower dissociation rate, which is due to the multiple interactions between the peptide and the receptor on the hyperbranched macromolecule.

[0572] Figure 16 (ac) shows the SPR comparison results of three different hyperbranched macromolecular-campitarine conjugates with free campitarine. The hyperbranched macromolecular conjugates of campitarine appear to contain both fast and slow dissociation components, with the slower dissociation-related phase of these constructs indicating the synergistic binding of the multivalent hyperbranched macromolecular conjugates to the C3 surface.

[0573] Figure 16 The sample in d) was 4a 40k PEG-SS-(campatamine)4 hydrolyzed from campatamine. Based on similar SPR signals, this hydrolyzed peptide contains a succinate-ester linker from the degradation of the conjugated linker group and showed no effect on its C3 binding. This strongly suggests that the ester linker does not alter the peptide's biological activity.

[0574] The same set of samples with high purity were re-analyzed to obtain quantitative KD analysis. The KD results are listed in Table 17 and plotted against the corresponding peptide substitution number on each sample. It can be seen that the KD decreases when more peptides are substituted on hyperbranched macromolecules.

[0575] Table 17: K values ​​of hyperbranched macromolecules-campatstatin conjugates D .

[0576]

[0577] * K D The reported association rate constant of campstatin (5e5 M⁻¹ s⁻¹) was used to fit the dissociation rate (k) of multivalent dissociation. d ) to calculate. Then K D The calculation is kd / ka. In this experiment, the K of campstatin was directly measured. D .

[0578] Example 10

[0579] Alternative Approach (AP) Hemolysis Analysis for Measuring IC50

[0580] IC50 (half-maximal inhibitory concentration) was measured using an alternative pathway (AP) hemolysis assay. Four hyperbranched macromolecules conjugated with campstatin were used in this analysis: Sample No. REA638 (4a 40k-PEG-SGA-(campstatin)) n TFA salt) and REA639 (4a-40k-PEG-SGA-(campatamine)) n Acetate), which have 2.6 and 2.5 campstatin / hyperbranched macromolecules respectively; and sample number REA640 (4a-40k-PEG-[4a-10kPEG-(campstatin)). n ]4) and REA641 (4a-40k-PEG-[4a-20k-PEG-(campatidine)) n ]4), which have 10.8 and 7.1 campstatin / hyperbranched macromolecules, respectively (see Tables 10 and 15).

[0581] In the 96-well plate analysis experiment (see...) Figure 18 The inhibitor (50 μL) was diluted in GVBo (GVBo: 0.1% gelatin, 5 mM barbital, 145 mM NaCl, 0.025% NaN3, pH 7.3) and incubated with a series of 1:2 normal human serum:GVBo concentrations for 30 min at room temperature. Rabbit RBCs (CompTech) were centrifuged at 500×g for 3 min and then centrifuged at 5.0×10⁻⁶. 8 Cells / mL were resuspended in MgEGTA (MgEGTA: 0.1 M MgCl2, 0.1 M EGTA, pH 7.3). Rabbit RBCs (20 μL) were added and incubated at 37°C for 60 min. The reaction was terminated by adding 200 μL of GVBE (GVBE: 0.1% gelatin, 5 mM barbital, 145 mM NaCl, 10 mM EDTA, 0.025% NaN3, pH 7.3). Cells were centrifuged at 500×g for 5 min and the supernatant (150 μL) was transferred to a new 96-well plate. The hemolysis % was calculated as (A412 inhibitor / A412 without inhibitor)*100 and fitted using a 4PL curve to determine the IC50. 50 The results are shown in Table 18.

[0582] Table 18: IC50 results of AP hemolysis analysis

[0583]

[0584] From the results ( Figure 19As shown in Table 18), G0 4-arm PEG-campastatins (REA638 and REA639, with 2.6 and 2.5 campastatins / hyperbranched macromolecules, respectively) exhibited poorer IC50 per molecule compared to free campastatin. This is not surprising, as these molecules have approximately 2 campastatins / molecule, thus potentially hindering affinity from this low substitution. On the other hand, G1 PEG-hyperbranched macromolecule campastatins (REA640 and REA641, with 10.8 and 7.1 campastatins / hyperbranched macromolecules, respectively) showed improved IC50s compared to free campastatin, indicating improved potency through affinity. Furthermore, the hill slope of these curves was lower than that of free campastatin and REA 638 and REA639, suggesting that multiple binding events contribute to inhibition. Interestingly, REA641, with 7.1 kcal of campstatin, outperformed REA640, which had 10.8 kcal of campstatin, despite the latter having a higher valence. It is believed that REA641's 20 kcal PEG dendrite provides greater flexibility in its interaction with C3 compared to REA640's shorter 10 kcal PEG dendrite.

[0585] Classical route (CP) hemolysis analysis

[0586] IC50 (half-maximal inhibitory concentration) was also measured using the classical pathway (CP) hemolysis analysis, employing the same hyperbranched macromolecular conjugates as the AP hemolysis analysis described above. The principle of this analysis is similar to the AP hemolysis analysis, but it uses sheep-sensitized erythrocytes because the classical pathway begins with antibody binding to cells.

[0587] Serial dilutions (50 µL) of the inhibitor were prepared in 96-well plates in gelatin veronal buffer supplemented with Mg. 2+ and Ca 2+ GVB++ (GVB++: 0.1% gelatin, 5 mM barbital, 145 mM NaCl, 0.025% NaN3, pH 7.3, containing 0.15 mM calcium chloride and 0.5 mM magnesium chloride). C3-depleted human serum supplemented with 12 nM human C3 was diluted 1:2 in GVB++ (30 µL) and added to each well and incubated for 30 minutes. The concentration was 5.0 × 10⁻⁶ in GVB++ (20 µL). 8Add sheep erythrocytes sensitized with anti-sheep pAb at 1 / mL and incubate at 37°C for 30 min. Quench the reaction by adding gelatin-florena buffer (GVBE, 200 µL) containing EDTA. Centrifuge the cells at 500×g for 5 min and transfer the supernatant (150 µL) to a new 96-well plate. Measure the absorbance at 412 nm on a Molecular Devices SpectraMax M5 plate reader and determine the hemolysis percentage (%) by (A412). 抑制剂 / A412 无抑制剂 The percentage of hemolysis was calculated by multiplying the result by 100. The IC50 was determined using 4PL curve fitting in GraphPad Prism. Analysis was also run in the absence of C3 to determine background hemolysis. The results are shown in Table 19.

[0588] Table 19: IC50 results of AP hemolysis analysis

[0589]

[0590] The IC50 of free campstatin is 142 µM. The hyperbranched macromolecular valences of REA638 and REA639 increased to 2.6 and 2.5, respectively, improving the IC50 to 56.0 µM and 73.3 µM. Further increases in the hyperbranched macromolecular valence of campstatins to 10.8 and 7.1 (REA640 and REA641) improved the IC50 to 30.8 µM and 34.6 µM, respectively. In summary, these data demonstrate that the higher valence campstatins of this invention are more effective in inhibiting CP hemolysis compared to free campstatin alone.

[0591] In the same manner as described above, IC50 (half-maximum inhibitory concentration) was also measured using a sample prepared by classical pathway (CP) hemolysis analysis, using a 12-arm G1 hyperbranched molecule 4a40k-PEG(SGA)-[4a20kPEG(SG)-(APL-1)3]4 prepared by convergent synthesis as described in Example 4, with APL-1 replacing campstatin. The substitution rate of the 12 end groups of this product was approximately 35%, which was also used in Examples 11 and 12 below. 32 mg × 2 (equivalent to 20 mg of dendritic polymer) 2) The lyophilized dendritic polymer was reconstituted in 90 mM sodium phosphate and 360 mM NaCl aqueous solution, and 50 µL aliquots (1.25 mg / eye) were injected into animal eyes (New Zealand white rabbits) and maintained for one week. Vitreous fluid was harvested from two animals / time points at 1, 3, 5, and 7 days after the dose, and analyzed by classical pathway (CP) hemolysis assay to determine the IC50 over time. The IC50 value of the dendritic polymer was measured as a control. The results are summarized in Table 20 below.

[0592] Table 20:

[0593]

[0594] The results showed that APL-1 bound to dendritic polymers maintained its in vivo stability and activity for more than 7 days.

[0595] Example 11

[0596] Relationship between hydrodynamic radius and in vivo half-life

[0597] The hydrodynamic radius R of linear PEGylated protein (IgG) of different sizes (IgG 2×40k PEG and 2×20k PEG) was determined. h and its half-life T in New Zealand white rabbit vitreous fluid (NZWVH) 1 / 2 The results were compared with those of free proteins and several non-conjugated active substances (APIs) to obtain calibration curves, allowing the estimation of T1 / 2 of APL-1 conjugated with the 12-arm 120 kDa PEG dendritic polymer based on Rh determined by SEC. The results are shown in Table 21 below:

[0598] Table 21:

[0599]

[0600] **12a120k APL-1 is a first-generation dendritic polymer conjugate with the nominal structure 4a40k-PEG(SGA)-[4a20k-PEG(SG)-(APL-1)3]4, prepared by polymerization synthesis as described in Example 4, using APL-1 instead of campstatin. The substitution rate of the 12 end groups of this product is approximately 35%.

[0601] As shown in Table 20 and Figure 20 It can be seen that the hydrodynamic radius R measured by SEC h This allows for reliable estimation of the half-life of dendritic drug conjugates according to embodiments of the invention, depending on the size of the dendritic polymer, and adjustment of their sustained release characteristics.

[0602] Example 12

[0603] Effect of dendritic polymer degradation on in vitro release kinetics

[0604] A generation 1 dendritic polymer with the nominal structure 4a-40kPEG-[4a-40kPEG-APL-1]3 (with a substitution rate of approximately 35% for the 12 end groups), prepared by aggregation synthesis as described in Example 4 using APL-1 instead of campstatin, was subjected to in vitro degradation tests under various temperature and pH conditions to determine degradation / hydrolysis behavior. HPLC chromatograms were performed in PBS to show the disappearance of the dendritic polymer and the appearance of dendritic building blocks (hydrolysis products) over time.

[0605] Figures 21 a) to c) show the effect of temperature changes from 35°C to 39°C at a constant pH of 7.4. Figure 21 a) shows the decrease in dendritic polymer concentration over time, and Figure 21 b) shows the increase in dendritic unit concentration over time. Figure 21c This paper presents the effect of temperature on the dendritic polymer loss rate (%) at pH 7.4 on a logarithmic scale based on first-order release kinetics, to determine the rate constant K and half-life T of surfactant release estimated from these degradation rates. 1 / 2 The results are shown in Table 22 below:

[0606] Table 22:

[0607]

[0608] It is evident that the degradation and consequent release rate of APL-1 from this first-generation 120k PEG dendritic polymer increases with temperature, and the trends are similar for all three temperatures within the first 26 days. In summary, temperature has a relatively small effect on the release half-life.

[0609] Figures 22 a) through c) show the effect of pH at a constant temperature of 37°C. Figure 22 a) shows the decrease in dendritic polymer concentration over time, Figure 22 b) shows the increase in dendritic unit concentration over time, and Figure 22 c) shows the effect of pH on the dendritic polymer loss rate % at pH 7.4 on a logarithmic scale based on first-order release kinetics, to determine the rate constant K and half-life T1 / 2 of surfactant release estimated from these degradation rates. The results are in Table 23 below:

[0610] Table 23:

[0611]

[0612] As can be seen, the degradation and release rate of APL-1 from this first-generation 120kPEG dendritic polymer significantly increased over time with increasing pH. In summary, pH has a significant effect on the release half-life, and follows an exponential model. The higher the pH, the shorter the release half-life.

[0613] These release experiments demonstrate that the release kinetics of active substances conjugated with dendritic polymers can be altered or delayed by the hydrolytic degradation of ester bonds between dendritic building blocks or dendritic units.

[0614] Implementation plan of specific groups

[0615] First Implementation Plan

[0616] 1. A hyperbranched macromolecule comprising:

[0617] A core unit with at least three connectivity c;

[0618] Multiple polymer arms are connected to the core unit at connection point c.

[0619] Each polymer arm contains end groups or is connected to dendritic repeating units.

[0620] The dendritic repeating unit includes branch units connected to at least two polymer arms, each of which includes an end group or is connected to a next dendritic repeating unit, which can then be connected to other dendritic repeating units.

[0621] The outermost dendritic polymer arms of hyperbranched macromolecules, which form repeating units, each contain end groups.

[0622] The polymer arm contains polyethylene glycol (PEG) units;

[0623] At least one of the active agents is conjugated to at least one outermost polymer arm; and

[0624] Hyperbranched macromolecules include chemical bonds that can be broken by hydrolysis.

[0625] 2. As in aspect 1, the hyperbranched macromolecule is a G0 generation branched macromolecule, wherein the end groups of the branched macromolecule are end groups connected to the polymer arms of the core unit.

[0626] 3. As in aspect 1, the hyperbranched macromolecule is a higher-generation Gx hyperbranched macromolecule, where x is an integer from 1 to 10, defining the number of continuously connected dendritic repeating units in the hyperbranched macromolecule.

[0627] 4. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the core unit and the branching units are the same or different and independently have connectivity of 3 to 10, or 4 to 8, or 4 to 6, or 4 c or c'.

[0628] 5. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the core unit and the branch units are the same or different and each is derived from a polyol having at least 3 hydroxyl groups.

[0629] 6. Hyperbranched macromolecules as described in aspect 5, wherein the polyols are selected from the group consisting of: glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomaltitol, lactitol, maltitol, mannitol, or sorbitol.

[0630] 7. The hyperbranched macromolecule of any of the foregoing aspects, wherein the polymer arm comprises polyethylene glycol (PEG) units with an average molecular weight (Mn) ranging from about 1,000 Daltons to about 100,000 Daltons, or from about 10,000 Daltons to about 60,000 Daltons, or from about 15,000 Daltons to about 50,000 Daltons.

[0631] 8. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the average molecular weight of the polymer arm PEG units connected to the core is the same as or different from the average molecular weight of the polymer arms in the dendritic repeating unit.

[0632] 9. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the average molecular weight of the polymer arm PEG units connected to the core is higher or lower than the average molecular weight of the polymer arms in the dendritic constituent units.

[0633] 10. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein for higher generation Gx hyperbranched macromolecules, 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.

[0634] 11. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein an end group attached to the outermost polymer arm is grafted directly or via a suitable bifunctional linker comprising a hydrolyzable bond comprising a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, heteroaliphatic, or aromatic or heteroaromatic group.

[0635] 12. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the functional groups of the end groups and / or linker-end groups attached to the outermost polymer arms are selected from the following: electrophilic groups, such as activated ester groups, such as succinimidyl esters, succinimidyl carbonate; nitrophenyl carbonate, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, alkynes, azides, norbornene, epoxides, methanesulfonates, toluenesulfonates, trifluoroethylsulfonyl groups, cyanurates, o-dithiopyridine, or halogens; nucleophilic groups, Functional groups such as amines (e.g., primary amines), hydroxyl groups, alcohols, thiols, azides, and carboxyl groups; functional groups for click chemistry; functional groups for cycloaddition (e.g., 1,3-dipolar cycloaddition, [3+2] cycloaddition, [4+2] cycloaddition of olefin-nitroketone or alkyne-nitroketone cycloaddition); functional groups for thiol-alkene reactions; functional groups for heterodiels-Alder cycloaddition; functional groups for nucleophilic ring-opening; functional groups for non-aldecanol carbonyl reactions; functional groups for carbon-carbon multiple bond addition reactions; polymerizable vinyl groups or combinations thereof.

[0636] 13. Hyperbranched macromolecules as described above, wherein the linker-terminal group attached to the outermost polymer arm is a functional group selected from the following: succinimide succinate (SS), succinimide glutarate (SG), succinimide adipic acid (SAP), succinimide azelate (SAZ), and succinimide glutaramide (SGA).

[0637] 14. A hyperbranched macromolecule of any of aspects 1 to 10, wherein the end group attached to the outermost polymer arm is a functional group selected from: alkynes, such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]-nonyne (BCN); or norbornene or trans-cyclooctylene (TCO); azides, 3,4-dihydroxyphenylacetic acid (DHPA) or tetrazine (Tz).

[0638] 15. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein the connection between the polymer arm attached to the core unit and the first dendritic repeating unit and / or the connection between successive dendritic repeating units is formed by click chemistry.

[0639] 16. Hyperbranched macromolecules as described in aspect 15, wherein the linkage is formed by reacting a polymer arm partially functionalized with an alkyne, cycloalkyne, or strained or terminal olefin with a polymer arm partially functionalized with an azide or tetrazine in a SPAAC or IEDDA-type click chemical coupling reaction.

[0640] 17. As in aspect 16, the hyperbranched macromolecule, wherein the alkyne moiety is a dibenzocyclooctyne moiety.

[0641] 18. A hyperbranched macromolecule of any of aspects 15 to 17, wherein the connection between the polymer arm connected to the core unit and the first dendritic repeating unit and / or the connection between successive dendritic repeating units is formed between the polymer arm connected to the core unit and the polymer arm connected to the branch unit of the dendritic repeating unit, and / or between the polymer arm of the dendritic repeating unit and the polymer arm of the successive dendritic repeating unit.

[0642] 19. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein the active agent conjugated to at least one outermost polymer arm is selected from the group consisting of therapeutic or diagnostic active agents.

[0643] 20. Hyperbranched macromolecules as described above, wherein the active agent conjugated to at least one outermost polymer arm is selected from steroids; nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, meclofenamic acid, meclofenamic acid A, disalicylate, sulindac, tometidine, ketoprofen, diflunisal, piroxicam, naproxen, etodoxacin, flurbiprofen, fenofibrate C, indomethacin, celecoxib, ketorolac, nepafenamide; intraocular pressure-lowering drugs; antibiotics such as ciprofloxacin; pain relievers such as bupivacaine; calcium channel blockers such as nifedipine; cell cycle inhibitors such as simvastatin; proteins such as insulin; small molecule hydrophilic drugs, including carboxylic acids. Salts and amine salts; small molecule hydrophobic drugs, hydrophilic peptides and protein drugs, such as insulin, single-chain antibody fragments, Fab fragments, IgG antibodies, fusion antibodies, etc.; aptamers; especially bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, antibiotics, pain relievers, calcium channel blockers, cell cycle inhibitors, chemotherapeutic agents, antiviral drugs, anesthetics, hormones, anticancer drugs, antitumor agents, viruses, gene-delivering viruses (such as AAV), protein binding agents (such as nanobodies, affinity molecules, ankylosing smears, DARPin, etc.) or any combination thereof.

[0644] 21. A hyperbranched macromolecule as described above, wherein the active agent conjugated to at least one outermost polymer arm is a peptide selected from the group consisting of: campstatin, APL-1 and Fc-III-4C, Beovu (buxizumab), Zimura (avacartide polyethylene glycol), percicobulin, arbexipa polyethylene glycol, lampalizumab, Fovista, risunib, AXT107, iramitide, THR149, ALM201, VGB3 and lagozopran.

[0645] 22. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein the active agent is bound to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% of the outermost polymer arm.

[0646] 23. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the dendritic repeating units are represented by equation (i):

[0647]

[0648] Where A is the connection to the polymer arm attached to the core unit, or A is the connection to B of the previous dendritic repeating unit represented by equation (i).

[0649] L A It is a connector.

[0650] m is 0 or 1.

[0651] n is an integer between 20 and 2000, o is an integer between 20 and 2000.

[0652] n and o can be different or the same.

[0653] X is a branch unit with connectivity c'.

[0654] L B It is a connector, where p is 0 or 1.

[0655] B contains end groups located on the surface of hyperbranched macromolecules or is linked to A or an active agent that forms a continuous dendritic repeating unit.

[0656] L A and L B They can be different or the same.

[0657] m and p can be different or the same, and

[0658] y is an integer from 2 to 9, where y = c'-1, c' is the connectivity of the branching unit X; and the dendritic constituent units in the hyperbranched macromolecule can be the same or different.

[0659] 24. Hyperbranched macromolecules as described in aspect 23, wherein the link between A and B comprises functional groups formed by click chemistry, such as triazoles or dihydropyrazines.

[0660] 25. Hyperbranched macromolecules such as those in aspects 23 or 24, wherein the linker L A and / or L B Contains diacid and / or acid diamide groups, such as succinate, glutarate, adipate, azelaate or glutaramide.

[0661] 26. Hyperbranched macromolecules such as those in any of aspects 23 to 25, wherein the linker L A and / or L B Includes the structure represented by equation (ii):

[0662]

[0663] Among them U 1 and U 2 Independently, it can be NH or O and can be the same or different, and where t is an integer from 0 to 10.

[0664] 27. Hyperbranched macromolecules such as those in aspects 25 or 26, wherein the linker L A and / or L B The bond with B further includes a polyethylene glycol unit between the carboxyl group, carboxamide group, or structure of formula (ii).

[0665] 28. A method for producing hyperbranched macromolecules such as those in aspects 1 to 27 by divergent synthesis, comprising the following steps:

[0666] (a) A core unit having at least three connectivity c is provided, wherein a plurality of polymer arms connected to the core unit have functional groups at the ends of the polymer arms suitable for click chemistry;

[0667] (b) Providing a dendritic constituent repeating unit precursor, which comprises

[0668] - A polymer arm comprising functional groups (such as azides, alkynes, alkenes, or tetrazides) suitable for linking with corresponding functional groups of the polymer arm connected to the core via click chemistry, and

[0669] - At least two polymer arms containing functional groups that are non-reactive in click chemistry.

[0670] (c) A connection is formed between the polymer arms attached to the core and the polymer arms that form the repeating unit precursors of the dendritic structure through click chemistry.

[0671] (d) Optionally, converting the functional groups of at least two polymer arms containing functional groups that are non-reactive in click chemistry into functional groups suitable for click chemistry, and

[0672] (e) An active agent containing a functional group is conjugated to the outermost polymer arm by reacting with the functional group of the outermost polymer arm, thereby forming a hyperbranched macromolecule-active agent conjugate.

[0673] 29. The method of aspect 28, wherein for a higher generation Gx hyperbranched macromolecule, where x is an integer from 2 to 10, step (d) is mandatory, and prior to the conjugation of the active agent in step (e), other successive dendritic constituent repeating unit precursors are linked to the functional groups suitable for click chemistry obtained in step (d) to be linked to the hyperbranched macromolecule via click chemistry.

[0674] 30. The method of aspect 28 or 29, wherein the dendritic constituent repeating unit precursor in step (c) is represented by equation (iii):

[0675]

[0676] Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines).

[0677] D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L A m, n, X, o, L B p and y are as defined in aspects 23 to 27; and the dendritic constituent units may be the same or different.

[0678] 31. The method of aspects 28 to 30, wherein after the penultimate step (d) of converting the functional groups of at least two polymer arms containing non-reactive functional groups in click chemistry into functional groups suitable for click chemistry, the active agent in step (e) is first functionalized with functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines), and then conjugated to the outermost polymer arm of the hyperbranched macromolecule in the click chemistry reaction.

[0679] 32. The method of aspect 31, wherein the active agent functionalized with functional groups suitable for click chemistry is a peptide.

[0680] 33. A method for synthesizing hyperbranched macromolecules such as those in aspects 1 to 27 by aggregation, comprising the following steps:

[0681] I) Provides a dendritic constituent repeating unit precursor, which contains

[0682] - A polymer arm containing functional groups (such as azides, alkynes, alkenes, or tetrazides) suitable for linkage via click chemistry, and

[0683] - At least two polymer arms containing functional groups that are non-reactive in click chemistry.

[0684] II) Conjugating an active agent containing a functional group to at least one of at least two polymer arms containing a functional group that is non-reactive in click chemistry of a dendritic constituent repeating unit precursor.

[0685] III) Provides a core unit having at least three connectivity c, wherein multiple polymer arms connected to the core unit have functional groups (such as azides, alkynes, alkenes, or tetrazines) at their ends, suitable for click chemistry.

[0686] IV) A link is formed between the polymer arm connected to the core provided in step III) and the polymer arm containing functional groups suitable for linking by click chemistry of the repeating unit precursor of the dendritic composition obtained in step II), thereby forming a hyperbranched macromolecular-active agent conjugate.

[0687] 34. As in aspect 33, the dendritic constituent repeating unit precursor in step I) is represented by equation (iii):

[0688]

[0689] Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines).

[0690] D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L A L B m, n, X, o, p, and y are as defined in aspects 23-27, and the dendritic constituent units may be the same or different.

[0691] 35. The method of aspect 33 or 34, wherein for a higher generation Gx hyperbranched macromolecule, where x is an integer from 2 to 10, the active agent-conjugated dendritic repeating unit precursor obtained in step II) is click-chemically linked to an anti-dendritic repeating unit precursor, the anti-dendritic repeating unit precursor comprising:

[0692] - A polymer arm containing functional groups that are non-reactive in click chemistry, and

[0693] - At least two polymer arms containing functional groups suitable for click chemistry (such as azides, alkynes, alkenes, or tetrazines).

[0694] Before being attached to other reverse dendritic repeating unit precursors or before being attached to the polymer arm attached to the core via click chemistry in step IV), a functional group of a polymer arm that is non-reactive in click chemistry is subsequently converted into a functional group suitable for click chemistry, thereby forming a higher-generation hyperbranched macromolecule.

[0695] 36. The method of aspect 33 or 34, wherein by performing steps I) and II) on each active agent-conjugated dendritic repeating unit precursor, a dendritic repeating unit precursor having different active agents conjugated with the polymer arm is obtained, and the mixture of the obtained active agent-conjugated dendritic repeating unit precursors is used in step IV), thereby forming a hyperbranched macromolecule-active agent conjugate with different active agents in different regions of the hyperbranched macromolecule surface.

[0696] 37. The method of any of aspects 28 to 36, wherein the outermost polymer arm of the hyperbranched macromolecule has a terminal maleimide functional group, and the peptide or active agent is conjugated thereto by a maleimide-thiol reaction.

[0697] 38. The method of aspect 37, wherein the terminal maleimide functional group is provided by reacting the terminal functional group of a hyperbranched macromolecule with a click chemical linker having an azide or DBCO functional group attached to a maleimide group, said click chemical linker being such as DBCO-maleimide, DBCO-PEG3-maleimide, DBCO-PEG4-maleimide, or azide-PEG3-maleimide.

[0698] 39. Hyperbranched macromolecules of any of aspects 1 to 27, used as pharmaceuticals.

[0699] 40. A treatment method, wherein the method comprises treating a patient’s disease or medical ailment with a hyperbranched macromolecule as described in any of aspects 1 to 27.

[0700] 41. Hyperbranched macromolecules used in aspects 39 or 40, or treatment methods such as those in aspects 39 or 40, wherein the hyperbranched macromolecules are used for ocular treatment.

[0701] 42. Hyperbranched macromolecules as used in aspects 39 to 41, or treatment methods as described in aspects 39 to 41, wherein the hyperbranched macromolecules are used to treat ocular diseases, such as posterior ocular diseases, such as any posterior segment ocular disease affecting the retinal, macular, or choroidal vascular system and integrity, resulting in visual acuity impairment, vision loss, or blindness, particularly posterior segment disease states caused by 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.

[0702] 43. Hyperbranched macromolecules as used in aspects 39 to 42, or treatment methods as described in aspects 39 to 42, wherein the hyperbranched macromolecules are used to treat ocular diseases selected from the group consisting of: retinal neovascularization, choroidal neovascularization, wet AMD, dry AMD, retinal vein occlusion, diabetic macular edema, retinal degeneration, anterior chamber hemorrhage, presbyopia, corneal graft rejection, retinoblastoma, melanoma, miosis, mydriasis, glaucoma, conjunctivitis, intraocular infection, choroidal neovascularization (CNV), intraocular tumors, retinal nerve inflammation, inflammation, autoimmune diseases. Immune uveitis, uveitis, proliferative vitreoretinopathy and corneal degeneration, acute and chronic macular neuroretinopathy, central serous chorioretinopathy, macular edema, acute multifocal squamous pigment epithelial lesions, Behcet's disease, shotgun retinal choroidal lesions, posterior uveitis, posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusive disease, central retinal vein occlusion, disseminated intravascular coagulation, retinal branch vein occlusion, hypertensive fundus changes. Ocular ischemia syndrome, retinal artery microaneurysms, Kohl's disease, parafoveal telangiectasia, hemiretinal vein occlusion, papillary phlebitis, carotid artery disease (CAD), frost-like dendritic vasculitis, sickle cell retinopathy, vascular streaks, familial exudative vitreoretinopathy, Ilse's disease, proliferative vitreoretinopathy, diabetic retinopathy, tumor-associated retinal diseases, congenital hypertrophy of retinal pigment epithelium (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, retina and retinal pigment epithelium. Retinoid epithelial combined hamartoma, retinoblastoma, fundus angiogenesis tumor, retinal astrocytoma, intraocular lymphoma, myopic retinal degeneration, acute retinal pigment epitheliitis, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal carcinoma, retinitis pigmentosa, Leber congenital amaurosis, choroidal agenesis, X-linked retinitis pigmentosa, best yolk macular dystrophy, X-linked retinoschisis, achromatopsia CNGA3, achromatopsia CNGB3, LHON, Sturges disease, Arthur syndrome, Norrie disease, Barby-Bisson syndrome, and red-green color blindness.

[0703] 44. The hyperbranched macromolecule used in any of aspects 39 to 43, or the treatment method used in any of aspects 39 to 43, wherein the hyperbranched macromolecule is formulated for direct injection at the treatment site of the patient, for example by parenteral administration, intratumoral injection, or injection into the eye, such as intravitreal, intraanterior chamber, subconjunctival, retrobulbar, subfollicular, subretinal, or suprachoroidal injection.

[0704] 45. The hyperbranched macromolecules used in any of aspects 39 to 44, or the treatment methods used in any of aspects 39 to 44, wherein the hyperbranched macromolecules are administered by direct injection, by oral administration, by incorporation into a gel, or by incorporation into an implant.

[0705] 46. ​​The hyperbranched macromolecule used in any of aspects 39 to 46, or the treatment method used in any of aspects 39 to 46, wherein different dendritic units or regions on the surface of the hyperbranched macromolecule contain two or more different active agents.

[0706] 47. Hyperbranched macromolecules as used in aspect 46 or treatment methods as used in aspect 46, which are used in combination therapies involving the administration of more than one active agent.

[0707] Second Implementation Plan

[0708] 1. A hyperbranched macromolecule comprising building blocks, said building blocks comprising:

[0709] A core unit with at least three connectivity c;

[0710] Multiple polymer arms are connected to the core unit at connection point c.

[0711] At least one polymer arm is connected to the dendritic repeating unit via a hydrolyzable bond.

[0712] The dendritic repeating unit comprises branch units connected to at least two polymer arms, each of which includes an end group or is connected to a next dendritic repeating unit via a hydrolyzable bond. The next dendritic repeating unit can then be chemically linked to other dendritic repeating units.

[0713] The outermost dendritic polymer arms of hyperbranched macromolecules, which form repeating units, each contain end groups.

[0714] The polymer arm is composed of polyethylene glycol (PEG) units.

[0715] 2. The hyperbranched macromolecule of aspect 1, wherein at least 10%, preferably about 20% to 100% of the linkages in the macromolecule can be cleaved by hydrolysis.

[0716] 3. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the linkages are non-hydrolyzable.

[0717] 4. Hyperbranched macromolecules of any of the foregoing aspects, which are higher generation Gx hyperbranched macromolecules, where x is an integer from 1 to 10, defining the number of continuously connected dendritic repeating units in the hyperbranched macromolecule.

[0718] 5. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the core unit and the branching units are the same or different and independently have connectivity of 3 to 10, or 4 to 8, or 4 to 6, or 4 c or c'.

[0719] 6. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the core unit and the branching units are the same or different, and are derived from polyols having at least 3 hydroxyl groups.

[0720] 7. Hyperbranched macromolecules such as in aspect 6, wherein the polyols are selected from the group consisting of: glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomaltitol, lactitol, maltitol, mannitol, or sorbitol.

[0721] 8. The hyperbranched macromolecule of any of the foregoing aspects, wherein the polyethylene glycol (PEG) unit of the polymer arm has an average molecular weight (Mn) in the range of about 1,000 Daltons to about 100,000 Daltons, or about 10,000 Daltons to about 60,000 Daltons, or about 15,000 Daltons to about 50,000 Daltons, or about 10,000 Daltons to about 40,000 Daltons.

[0722] 9. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the average molecular weight of the polymer arm PEG units connected to the core is the same as or different from the average molecular weight of the polymer arms in the dendritic repeating unit.

[0723] 10. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the average molecular weight of the polymer arm PEG units connected to the core is higher than the average molecular weight of the polymer arms in the dendritic constituent units.

[0724] 11. Hyperbranched macromolecules as described above, wherein the average molecular weight of the polymer arm PEG units connected to the core is lower than the average molecular weight of the polymer arms in the dendritic constituent units.

[0725] 12. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein for higher generation Gx hyperbranched macromolecules, 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 wherein the average molecular weight of the polymer arm PEG units increases from the innermost polymer arm to the outermost polymer arm.

[0726] 13. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein at least one arm connected to the core unit or branch unit is connected to the dendritic constituent unit by forming a hydrolyzable bifunctional linker, said hydrolyzable linker 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.

[0727] 14. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein the end group attached to the outermost polymer arm is directly or via a bifunctional linker comprising or forming a hydrolyzable bond to the end of the polymer arm, the hydrolyzable bond comprising a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, heteroaliphatic, or aromatic or heteroaromatic group.

[0728] 15. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the functional groups of the end groups and / or linker-end groups attached to the outermost polymer arms are selected from the following: electrophilic groups, such as activated ester groups, such as succinimidyl esters, succinimidyl carbonate; nitrophenyl carbonate, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, alkynes, azides, norbornene, epoxides, methanesulfonates, toluenesulfonates, trifluoroethylsulfonyl groups, cyanurates, o-dithiopyridine, or halogens; nucleophilic groups, Functional groups such as amines (e.g., primary amines), hydroxyl groups, alcohols, thiols, azides, and carboxyl groups; functional groups for click chemistry; functional groups for cycloaddition (e.g., 1,3-dipolar cycloaddition, [3+2] cycloaddition, [4+2] cycloaddition of olefin-nitroketone or alkyne-nitroketone cycloaddition); functional groups for thiol-alkene reactions; functional groups for heterodiels-Alder cycloaddition; functional groups for nucleophilic ring-opening; functional groups for non-aldecanol carbonyl reactions; functional groups for carbon-carbon multiple bond addition reactions; polymerizable vinyl groups or combinations thereof.

[0729] 16. Hyperbranched macromolecules as described above, wherein the (linker) end group attached to the outermost polymer arm is a functional group selected from the following: succinimide succinate (SS), succinimide glutarate (SG), succinimide adipic acid (SAP), succinimide azelate (SAZ), and succinimide glutaramide (SGA).

[0730] 17. A hyperbranched macromolecule of any of aspects 1 to 14, wherein the end group attached to the outermost polymer arm is a functional group selected from: alkynes, such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]-nonyne (BCN); or norbornene or trans-cyclooctylene (TCO); azides, 3,4-dihydroxyphenylacetic acid (DHPA) or tetrazine (Tz).

[0731] 18. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein the connection between the polymer arm attached to the core unit and the first dendritic repeating unit and / or the connection between successive dendritic repeating units is formed by click chemistry, optionally using click chemistry functionalized linkers including hydrolyzable bonds.

[0732] 19. Hyperbranched macromolecules as described in aspect 18, wherein the linking is formed by reacting a polymer arm, optionally partially functionalized with a linker, alkyne, cycloalkyne, or strained or terminal olefin, with a polymer arm partially functionalized with an azide or tetrazine in a SPAAC or IEDDA-type click chemical coupling reaction.

[0733] 20. As in aspect 19, the hyperbranched macromolecule, in which the alkyne moiety is the dibenzocyclooctyne moiety.

[0734] 21. A hyperbranched macromolecule of any of aspect 18 or 20, wherein the connection between the polymer arm attached to the core unit and the first dendritic repeating unit and / or the connection between successive dendritic repeating units is formed between the polymer arm attached to the core unit and the polymer arm attached to the branch unit of the dendritic repeating unit, and / or between the polymer arm of the dendritic repeating unit and the polymer arm of the successive dendritic repeating unit, optionally by forming at least one hydrolyzable bifunctional linker.

[0735] 22. A dendritic repeating unit represented by formula (i), or a hyperbranched macromolecule as described in any of the foregoing aspects, comprising a dendritic repeating unit represented by formula (i):

[0736]

[0737] Where A is the connection to the polymer arm attached to the core unit, or A is the connection to B of the previous dendritic repeating unit represented by equation (i).

[0738] L A It is a connector.

[0739] m is 0 or 1.

[0740] n is an integer between 20 and 2000, o is an integer between 20 and 2000.

[0741] n and o can be different or the same.

[0742] X is a branch unit with connectivity c'.

[0743] L B It is a connector, where p is 0 or 1.

[0744] B contains end groups located on the surface of hyperbranched macromolecules or is linked to A or an active agent that forms a continuous dendritic repeating unit.

[0745] L A and L B They can be different or the same.

[0746] m and p can be different or the same, and

[0747] y is an integer from 2 to 9, where y = c'-1, c' is the connectivity of the branching unit X; and the dendritic constituent units in the hyperbranched macromolecule can be the same or different.

[0748] 23. Hyperbranched macromolecules such as those in aspect 22, wherein the link between A and B comprises functional groups formed by click chemistry, such as triazoles or dihydropyrazines.

[0749] 24. Hyperbranched macromolecules such as those in aspects 22 or 23, wherein the linker L A and / or L B Contains diacid and / or acid diamide groups, such as succinate, glutarate, adipate, azelaate or glutaramide.

[0750] 25. Hyperbranched macromolecules such as those in any of aspects 22 to 24, wherein the linker L A and / or L B Includes the structure represented by equation (ii):

[0751]

[0752] Among them U 1 and U 2 Independently, it can be NH or O and can be the same or different, and where t is an integer from 0 to 10.

[0753] 26. Hyperbranched macromolecules, such as those in aspects 24 or 25, wherein the linker L A and / or L B The bond with B further includes a polyethylene glycol unit between the carboxyl group, carboxamide group, or structure of formula (ii).

[0754] 27. The hyperbranched macromolecule of any of the foregoing aspects, wherein the hyperbranched macromolecule further comprises at least one extension unit containing a polyethylene glycol (PEG) unit, wherein the extension unit is linear, bifunctional and connected to a polymer arm of a dendritic repeating unit, or a polymer arm connected to a core unit and an end group or polymer arm of the next dendritic repeating unit.

[0755] 28. The hyperbranched macromolecule of aspect 27, wherein the extended body unit comprises at least one linker, wherein the linker may be located at either end or both ends of the extended body unit and is a bifunctional linker comprising a hydrolyzable bond comprising a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, heteroaliphatic, or aromatic or heteroaromatic group.

[0756] 29. A hyperbranched molecule as described in any of the foregoing aspects, wherein the hydrolyzable bonds are selected from at least one or all of a core unit, a core unit comprising a polymer arm at the connectivity c, a dendritic repeating unit, a linker, and an extension, preferably all building blocks having a molecular weight of less than 50,000 Daltons, such as less than 45,000 Daltons, or less than 40,000 Daltons, or less than 35,000 Daltons, or less than 30,000 Daltons.

[0757] 30. A hyperbranched molecule as described in any of the foregoing aspects, wherein, after complete hydrolysis of the hydrolyzable bonds, all segments formed by said molecule have a molecular weight of less than 50,000 Daltons, such as less than 45,000 Daltons, or less than 40,000 Daltons, or less than 35,000 Daltons, or less than 30,000 Daltons.

[0758] 31. A precursor for a dendritic repeating unit, comprising:

[0759] - A polymer arm containing functional groups (such as azides, alkynes, alkenes, or tetrazides) suitable for linkage via click chemistry, and

[0760] - At least two polymer arms containing functional groups that are non-reactive in click chemistry.

[0761] The polymer arm is connected to the branch unit with connectivity c'.

[0762] The polymer portion of the polymer arm is composed of polyethylene glycol (PEG) units.

[0763] 32. The precursor of aspect 31, wherein the compound is represented by formula (iii):

[0764]

[0765] Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines).

[0766] D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L A m, n, X, o, L B p and y are as defined in the foregoing.

[0767] 33. A precursor for a reverse dendritic repeating unit, comprising:

[0768] - A polymer arm containing functional groups that are non-reactive in click chemistry, and

[0769] - At least two polymer arms containing functional groups suitable for click chemistry (such as azides, alkynes, alkenes, or tetrazines), and

[0770] - A branch unit with connectivity c';

[0771] The polymer portion of the polymer arm is composed of polyethylene glycol (PEG) units.

[0772] 34. The precursor of aspect 33, wherein the precursor is represented by equation (iv):

[0773]

[0774] Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines).

[0775] D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L A m, n, X, o, L B p and y are as defined in the foregoing.

[0776] 35. A method for producing hyperbranched macromolecules such as those in aspects 1 to 34 by divergent synthesis, comprising the following steps:

[0777] (a) A core unit having at least three connectivity c is provided, wherein a plurality of polymer arms connected to the core unit have functional groups at the ends of the polymer arms suitable for click chemistry;

[0778] (b) Providing a dendritic constituent repeating unit precursor, which comprises

[0779] - A polymer arm comprising functional groups (such as azides, alkynes, alkenes, or tetrazides) suitable for linking with corresponding functional groups of the polymer arm connected to the core via click chemistry, and

[0780] - At least two polymer arms containing functional groups that are non-reactive in click chemistry.

[0781] (c) A connection is formed between the polymer arms attached to the core and the polymer arms that form the repeating unit precursors of the dendritic structure through click chemistry.

[0782] (d) Optionally, converting the functional groups of at least two polymer arms containing functional groups that are non-reactive in click chemistry into functional groups suitable for click chemistry, and

[0783] (e) An active agent containing a functional group is conjugated to the outermost polymer arm by reacting with the functional group of the outermost polymer arm, thereby forming a hyperbranched macromolecule-active agent conjugate.

[0784] 36. The method of aspect 35, wherein for a higher generation Gx hyperbranched macromolecule, where x is an integer from 2 to 10, step (d) is mandatory, and prior to the conjugation of the active agent in step (e), other successive dendritic constituent repeating unit precursors are linked to the functional groups suitable for click chemistry obtained in step (d) to be linked to the hyperbranched macromolecule via click chemistry.

[0785] As in the method of aspect 35 or 36, the dendritic constituent repeating unit precursor in step (c) is represented by equation (iii):

[0786]

[0787] Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines).

[0788] D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L A m, n, X, o, L B p and y are as defined in the foregoing; and the dendritic constituent units may be the same or different.

[0789] 37. The method of aspects 35 to 37, wherein after the penultimate step (d) of converting the functional groups of at least two polymer arms containing functional groups that are non-reactive in click chemistry into functional groups suitable for click chemistry, the active agent in step (e) is first functionalized with functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines), and then conjugated to the outermost polymer arm of the hyperbranched macromolecule in the click chemistry reaction.

[0790] 38. The method of aspect 38, wherein the active agent functionalized with a functional group suitable for click chemistry is a peptide, such as one of campstatin, APL-1, Fc-III-4C, Beovu (bucizumab), Zimura (avacapeptide polyethylene glycol), percicobulin, arbexipa polyethylene glycol, lampalizumab, Fovista, risunip, AXT107, iramitide, THR149, ALM201, VGB3, and lagozolab, or angiogenesis inhibitors, such as 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., natezumab and vedolzumab).

[0791] 39. A method for synthesizing hyperbranched macromolecules such as those in aspects 1 to 34 by aggregation, comprising the following steps:

[0792] I) Provides a dendritic constituent repeating unit precursor, which contains

[0793] - A polymer arm containing functional groups (such as azides, alkynes, alkenes, or tetrazides) suitable for linkage via click chemistry, and

[0794] - At least two polymer arms containing functional groups that are non-reactive in click chemistry.

[0795] II) Conjugating an active agent containing a functional group to at least one of at least two polymer arms containing a functional group that is non-reactive in click chemistry of a dendritic constituent repeating unit precursor.

[0796] III) Provides a core unit having at least three connectivity c, wherein multiple polymer arms connected to the core unit have functional groups (such as azides, alkynes, alkenes, or tetrazines) at their ends, suitable for click chemistry.

[0797] IV) A link is formed between the polymer arm connected to the core provided in step III) and the polymer arm containing functional groups suitable for linking by click chemistry of the repeating unit precursor of the dendritic composition obtained in step II), thereby forming a hyperbranched macromolecular-active agent conjugate.

[0798] 40. The method of aspect 40, wherein the dendritic constituent repeating unit precursor in step I) is represented by equation (iii):

[0799]

[0800] Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines).

[0801] D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L A L B m, n, X, o, p, and y are as defined in the foregoing aspects, and the dendritic constituent units may be the same or different.

[0802] 41. The method of aspect 40 or 41, wherein for a higher generation Gx hyperbranched macromolecule, where x is an integer from 2 to 10, the active agent-conjugated dendritic repeating unit precursor obtained in step II) is click-chemically linked to a reverse dendritic repeating unit precursor, said reverse dendritic repeating unit precursor comprising:

[0803] - A polymer arm containing functional groups that are non-reactive in click chemistry, and

[0804] - At least two polymer arms containing functional groups suitable for click chemistry (such as azides, alkynes, alkenes, or tetrazines).

[0805] Before being attached to other reverse dendritic repeating unit precursors or before being attached to the polymer arm attached to the core via click chemistry in step IV), a functional group of a polymer arm that is non-reactive in click chemistry is subsequently converted into a functional group suitable for click chemistry, thereby forming a higher-generation hyperbranched macromolecule.

[0806] 42. The method of aspects 40 to 42, wherein by performing steps I) and II) on each active agent-conjugated dendritic repeating unit precursor, a dendritic repeating unit precursor having different active agents conjugated with the polymer arm is obtained, and the mixture of the obtained active agent-conjugated dendritic repeating unit precursors is used in step IV), thereby forming a hyperbranched macromolecule-active agent conjugate with different active agents in different regions of the hyperbranched macromolecule surface.

[0807] 43. The method of any of aspects 40 to 43, wherein the outermost polymer arm of the hyperbranched macromolecule has a terminal maleimide functional group, and the peptide or active agent is conjugated thereto by a maleimide-thiol reaction.

[0808] 44. The method of aspect 44, wherein the terminal maleimide functional group is provided by reacting the terminal functional group of a hyperbranched macromolecule with a click chemical linker having an azide or DBCO functional group attached to a maleimide group, said click chemical linker being such as DBCO-maleimide, DBCO-PEG3-maleimide, DBCO-PEG4-maleimide, or azide-PEG3-maleimide.

[0809] 45. Use of a hyperbranched macromolecule as described in any of aspects 1 to 34, wherein it is used in at least one application selected from: non-medical or industrial applications, such as antibody purification, cosmetic applications, catalytic applications, applications in electronics, agriculture, food, filtration, energy storage, building materials, coatings, adhesives, water purification, oil extraction, fragrance release, papermaking, environmental sensing and release systems, membranes, textiles, printing inks, surface chemistry applications, thickeners, detergents, rheology modifiers, scaffolds, or 3D printing.

[0810] Third Implementation Plan

[0811] 1. A hyperbranched macromolecule comprising building blocks, said building blocks comprising:

[0812] A core unit with at least three connectivity c;

[0813] Multiple polymer arms are connected to the core unit at connection point c.

[0814] At least one polymer arm is connected to the dendritic repeating unit via a hydrolyzable bond.

[0815] The dendritic repeating unit comprises branch units connected to at least two polymer arms, each of which includes an end group or is connected to a next dendritic repeating unit via a hydrolyzable bond. The next dendritic repeating unit can then be chemically linked to other dendritic repeating units.

[0816] The repeating unit composed of dendritic structures is represented by equation (i):

[0817]

[0818] Where A is the connection to the polymer arm attached to the core unit, the connection to the extension unit, or the connection to B of the previous dendritic repeating unit represented by equation (i).

[0819] L A It is a connector.

[0820] m is 0 or 1.

[0821] n is an integer between 20 and 2000, o is an integer between 20 and 2000.

[0822] n and o can be different or the same.

[0823] X is a branch unit with connectivity c'.

[0824] L B It is a connector, where p is 0 or 1.

[0825] B contains end groups located on the surface of hyperbranched macromolecules or is linked to A or an active agent that forms a continuous dendritic repeating unit.

[0826] L A and L B They can be different or the same.

[0827] m and p can be different or the same, and

[0828] y is an integer from 2 to 9, where y = c'-1, c' is the connectivity of the branching unit X; and the dendritic constituent units in the hyperbranched macromolecule can be the same or different;

[0829] The outermost dendritic polymer arms of the hyperbranched macromolecule, which form repeating units, each contain end groups; and

[0830] At least one of the active agents is conjugated to at least one outermost polymer arm.

[0831] 2. The hyperbranched macromolecule of aspect 1, wherein at least 10%, preferably about 20% to 100% of the linkages in the macromolecule can be cleaved by hydrolysis.

[0832] 3. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein each building block (fragment) of the hyperbranched macromolecule obtained after cleaving all hydrolyzable bonds connected in the macromolecule has an average molecular weight (Mn) of less than 50,000 Daltons, such as less than 45,000 Daltons, or less than 40,000 Daltons, or less than 35,000 Daltons, or less than 30,000 Daltons.

[0833] 4. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein at least one building block comprises a core unit or a branch unit having a plurality of polymer arms connected to the core unit or the branch unit by non-hydrolyzable bonds.

[0834] 5. A hyperbranched macromolecule of any of the foregoing aspects, which is a higher generation Gx hyperbranched macromolecule, where x is an integer from 1 to 10, defining the number of continuously connected dendritic repeating units in the hyperbranched macromolecule.

[0835] 6. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the core unit and the branching units are the same or different and independently have connectivity of 3 to 10, or 4 to 8, or 4 to 6, or 4 c or c'.

[0836] 7. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the core unit and the branching units are the same or different, and are derived from polyols having at least 3 hydroxyl groups.

[0837] 8. Hyperbranched macromolecules as described in aspect 7, wherein the polyols are selected from the group consisting of: glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomaltitol, lactitol, maltitol, mannitol, or sorbitol.

[0838] 9. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein the polyethylene glycol (PEG) unit of the polymer arm has an average molecular weight (Mn) in the range of about 1,000 Daltons to about 100,000 Daltons, or about 10,000 Daltons to about 60,000 Daltons, or about 15,000 Daltons to about 50,000 Daltons, or about 10,000 Daltons to about 40,000 Daltons.

[0839] 10. Hyperbranched macromolecules as described above, wherein the average molecular weight of the polymer arm PEG units connected to the core is the same as or different from the average molecular weight of the polymer arms in the dendritic repeating unit.

[0840] 11. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the average molecular weight of the polymer arm PEG units connected to the core is higher than the average molecular weight of the polymer arms in the dendritic constituent units.

[0841] 12. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the average molecular weight of the polymer arm PEG units connected to the core is lower than the average molecular weight of the polymer arms in the dendritic constituent units.

[0842] 13. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein for higher-generation Gx hyperbranched macromolecules, 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 wherein the average molecular weight of the polymer arm PEG units increases from the innermost polymer arm to the outermost polymer arm.

[0843] 14. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein at least one arm connected to the core unit or branch unit is connected to the dendritic constituent unit by forming a hydrolyzable bifunctional linker, said hydrolyzable linker 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.

[0844] 15. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein the end group attached to the outermost polymer arm is directly or via a bifunctional linker comprising or forming a hydrolyzable bond to the end of the polymer arm, the hydrolyzable bond comprising a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, heteroaliphatic, or aromatic or heteroaromatic group.

[0845] 16. Hyperbranched macromolecules as described in any of the foregoing aspects, wherein the functional groups of the end groups and / or linker-end groups attached to the outermost polymer arms are selected from the following: electrophilic groups, such as activated ester groups, such as succinimidyl esters, succinimidyl carbonate; nitrophenyl carbonate, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, alkynes, azides, norbornene, epoxides, methanesulfonates, toluenesulfonates, trifluoroethylsulfonyl groups, cyanurates, o-dithiopyridine, or halogens; nucleophilic groups, Functional groups such as amines (e.g., primary amines), hydroxyl groups, alcohols, thiols, azides, and carboxyl groups; functional groups for click chemistry; functional groups for cycloaddition (e.g., 1,3-dipolar cycloaddition, [3+2] cycloaddition, [4+2] cycloaddition of olefin-nitroketone or alkyne-nitroketone cycloaddition); functional groups for thiol-alkene reactions; functional groups for heterodiels-Alder cycloaddition; functional groups for nucleophilic ring-opening; functional groups for non-aldecanol carbonyl reactions; functional groups for carbon-carbon multiple bond addition reactions; polymerizable vinyl groups or combinations thereof.

[0846] 17. A hyperbranched macromolecule as described above, wherein the (linker) end group attached to the outermost polymer arm is a functional group selected from the following: succinimide succinate (SS), succinimide glutarate (SG), succinimide adipate (SAP), succinimide azelate (SAZ), and succinimide glutaramide (SGA).

[0847] 18. A hyperbranched macromolecule of any of aspects 1 to 13, wherein the end group attached to the outermost polymer arm is a functional group selected from: alkynes, such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]-nonyne (BCN); or norbornene or trans-cyclooctylene (TCO); azides, 3,4-dihydroxyphenylacetic acid (DHPA) or tetrazine (Tz).

[0848] 19. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein the connection between the polymer arm attached to the core unit and the first dendritic repeating unit and / or the connection between successive dendritic repeating units is formed by click chemistry, optionally using click chemistry functionalized linkers including hydrolyzable bonds.

[0849] 20. Hyperbranched macromolecules as described in aspect 19, wherein the linking is formed by reacting a polymer arm, optionally partially functionalized with a linker, alkyne, cycloalkyne, or strained or terminal olefin, with a polymer arm partially functionalized with an azide or tetrazine in a SPAAC or IEDDA type click chemical coupling reaction.

[0850] 21. For example, the hyperbranched macromolecule of aspect 20, wherein the alkyne moiety is the dibenzocyclooctyne moiety.

[0851] 22. A hyperbranched macromolecule of any of aspects 19 to 21, wherein the connection between the polymer arm connected to the core unit and the first dendritic repeating unit and / or the connection between successive dendritic repeating units is formed between the polymer arm connected to the core unit and the polymer arm connected to the branch unit of the dendritic repeating unit, and / or between the polymer arm of the dendritic repeating unit and the polymer arm of the successive dendritic repeating unit, optionally by forming at least one hydrolyzable bifunctional linker.

[0852] 23. The hyperbranched macromolecule of any of the foregoing aspects, wherein the active agent conjugated to at least one outermost polymer arm is selected from the group consisting of therapeutic or diagnostic active agents.

[0853] 24. Hyperbranched macromolecules as described above, wherein the active agent conjugated to at least one outermost polymer arm is selected from steroids; nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, meclofenamic acid, meclofenamic acid A, disalicylate, sulindac, tometidine, ketoprofen, diflunisal, piroxicam, naproxen, etodoxacin, flurbiprofen, fenofibrate C, indomethacin, celecoxib, ketorolac, nepafenamide; intraocular pressure-lowering drugs; antibiotics such as ciprofloxacin; pain relievers such as bupivacaine; calcium channel blockers such as nifedipine; cell cycle inhibitors such as simvastatin; proteins such as insulin; small molecule hydrophilic drugs, including carboxylic acids. Salts and amine salts; small molecule hydrophobic drugs, hydrophilic peptides and protein drugs, such as insulin, single-chain antibody fragments, Fab fragments, IgG antibodies, fusion antibodies, etc.; aptamers; especially bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, antibiotics, pain relievers, calcium channel blockers, cell cycle inhibitors, chemotherapeutic agents, antiviral drugs, anesthetics, hormones, anticancer drugs, antitumor agents, viruses, gene-delivering viruses (such as AAV), protein binding agents (such as nanobodies, affinity molecules, ankylosing smears, DARPin, etc.) or any combination thereof.

[0854] 25. A hyperbranched macromolecule as described above, wherein the active agent conjugated to at least one outermost polymer arm is a peptide selected from the group consisting of: campstatin, APL-1 and Fc-III-4C, Beovu (buxizumab), Zimura (avacartide polyethylene glycol), percicobulin, arbexipa polyethylene glycol, lampalizumab, Fovista, risunib, AXT107, iramitide, THR149, ALM201, VGB3 and lagozolaride.

[0855] 26. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein the active agent is bound to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% of the outermost polymer arm.

[0856] 27. A hyperbranched macromolecule as described in any of the foregoing aspects, wherein the link between A and B in formula (i) comprises a functional group formed by click chemistry, such as a triazole or a dihydropyrazine.

[0857] 28. Hyperbranched macromolecules, such as those in aspect 27, wherein the linker L A and / or L B Contains diacid and / or acid diamide groups, such as succinate, glutarate, adipate, azelaate or glutaramide.

[0858] 29. Hyperbranched macromolecules such as those in aspects 27 or 28, wherein the linker L A and / or L B Includes the structure represented by equation (ii):

[0859]

[0860] Among them U 1 and U 2 Independently, it can be NH or O and can be the same or different, and where t is an integer from 0 to 10.

[0861] 30. Hyperbranched macromolecules, such as those in aspects 28 or 29, wherein the linker L A and / or L B The bond with B further includes a polyethylene glycol unit between the carboxyl group, carboxamide group, or structure of formula (ii).

[0862] 31. The hyperbranched macromolecule of any of the foregoing aspects, wherein the hyperbranched macromolecule further comprises at least one extension unit containing a polyethylene glycol (PEG) unit, wherein the extension unit is linear, bifunctional and connected to a polymer arm of a dendritic repeating unit, or a polymer arm connected to a core unit and an end group or polymer arm of the next dendritic repeating unit.

[0863] 32. The hyperbranched macromolecule of aspect 31, wherein the extended body unit further comprises at least one linker, wherein the linker may be located at either end or both ends of the extended body unit and is a bifunctional linker comprising a hydrolyzable bond comprising a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, heteroaliphatic, or aromatic or heteroaromatic group.

[0864] 33. A hyperbranched molecule as described in any of the foregoing aspects, wherein the hydrolyzable bonds are selected from at least one or all of a core unit, a core unit comprising a polymer arm at the connectivity c, a dendritic repeating unit, a linker, and an extension, preferably all building blocks having a molecular weight of less than 50,000 Daltons, such as less than 45,000 Daltons, or less than 40,000 Daltons, or less than 35,000 Daltons, or less than 30,000 Daltons.

[0865] 34. A hyperbranched molecule as described in any of the foregoing aspects, wherein, after complete hydrolysis of the hydrolyzable bonds, all segments formed by said molecule have a molecular weight of less than 50,000 Daltons, such as less than 45,000 Daltons, or less than 40,000 Daltons, or less than 35,000 Daltons, or less than 30,000 Daltons.

[0866] 35. A precursor for a dendritic repeating unit, comprising:

[0867] - A polymer arm containing functional groups (such as azides, alkynes, alkenes, or tetrazides) suitable for linkage via click chemistry, and

[0868] - At least two polymer arms containing functional groups that are non-reactive in click chemistry.

[0869] The polymer arm is connected to the branch unit with connectivity c'.

[0870] The polymer portion of the polymer arm is composed of polyethylene glycol (PEG) units.

[0871] 36. The precursor of aspect 35, wherein the compound is represented by formula (iii):

[0872]

[0873] Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines).

[0874] D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L A m, n, X, o, L B p and y are as defined in the foregoing.

[0875] 37. A precursor for a reverse dendritic repeating unit, comprising:

[0876] - A polymer arm containing functional groups that are non-reactive in click chemistry, and

[0877] - At least two polymer arms containing functional groups suitable for click chemistry (such as azides, alkynes, alkenes, or tetrazines), and

[0878] - A branch unit with connectivity c';

[0879] The polymer portion of the polymer arm is composed of polyethylene glycol (PEG) units.

[0880] 38. The precursor of aspect 37, wherein the precursor is represented by equation (iv):

[0881]

[0882] Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines).

[0883] D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L Am, n, X, o, L B p and y are as defined in the foregoing.

[0884] 39. Hyperbranched macromolecules of any of aspects 1 to 34, wherein the core unit is a polyethylene glycolated pentaerythritol compound of formula (iv):

[0885]

[0886] Where n is an integer from 3 to 2,000.

[0887] 40. Hyperbranched macromolecules of any of aspects 1 to 34, wherein the core unit is a compound of formula (v):

[0888]

[0889] Where R is a core unit with x connectivity c, n is determined according to 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.

[0890] 41. Hyperbranched macromolecules, such as those in aspects 1 to 34 or 39 to 40, are formed from one of the following precursor / dendritic units:

[0891] and ,or

[0892] and

[0893] and ,or

[0894] and ,or

[0895] and ,or

[0896] and ,or

[0897] and ,or

[0898] and ,

[0899] Or formed from the following exemplary precursor pairs:

[0900]

[0901] and

[0902]

[0903] or

[0904] ;

[0905] or

[0906] and or

[0907] ;

[0908] or

[0909] and

[0910] or

[0911] ;

[0912] or

[0913] and

[0914] or

[0915] , where t is m, and n and m are as defined in aspect 40 for equation (v).

Claims

1. A hyperbranched macromolecule comprising building blocks, said building blocks comprising: A core unit with at least three connectivity c; Multiple polymer arms are connected to the core unit at the connectivity point c. At least one of the polymer arms is connected to the dendritic repeating unit via a hydrolyzable bond. The dendritic repeating unit comprises branch units connected to at least two polymer arms, each of which includes an end group or is connected to a next dendritic repeating unit via a hydrolyzable bond. The next dendritic repeating unit can then be chemically linked to other dendritic repeating units. Each polymer arm of the outermost dendritic repeating unit of the hyperbranched macromolecule contains an end group. The polymer arm is composed of polyethylene glycol (PEG) units; At least one of the active agents is conjugated to at least one of the outermost polymer arms.

2. The hyperbranched macromolecule of claim 1, wherein at least 10%, preferably about 20% to 100%, of the linkages in the macromolecule can be broken down by hydrolysis.

3. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein each of the building blocks (fragments) of the hyperbranched macromolecule obtained after cleaving all the hydrolyzable bonds connected in the macromolecule has an average molecular weight (Mn) of less than 50,000 Daltons, such as 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 hyperbranched macromolecule as claimed in any of the preceding claims, wherein at least one of the building blocks comprises a core unit or a branch unit having a plurality of polymer arms connected to the core unit or branch unit by non-hydrolyzable bonds.

5. The hyperbranched macromolecule as described in any of the preceding claims, which is a higher-generation Gx hyperbranched macromolecule, wherein x is an integer from 1 to 10, defining the number of continuously connected dendritic repeating units in the hyperbranched macromolecule.

6. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the core unit and the branch units are the same or different and independently have connectivity c or c' of 3 to 10, or 4 to 8, or 4 to 6, or 4.

7. The hyperbranched macromolecule as described in any of the preceding 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 hyperbranched macromolecule of claim 7, wherein the polyol is selected from the group consisting of: glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomaltitol, lactitol, maltitol, mannitol, or sorbitol.

9. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the polyethylene glycol (PEG) unit of the polymer arm has an average molecular weight (Mn) in the range of about 1,000 Daltons to about 100,000 Daltons, or about 10,000 Daltons to about 60,000 Daltons, or about 15,000 Daltons to about 50,000 Daltons, or about 10,000 Daltons to about 40,000 Daltons.

10. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the average molecular weight of the polymer arm PEG units connected to the core is the same as or different from the average molecular weight of the polymer arms in the dendritic repeating unit.

11. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the average molecular weight of the polymer arm PEG units connected to the core is higher than the average molecular weight of the polymer arms in the dendritic constituent unit.

12. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the average molecular weight of the polymer arm PEG units connected to the core is lower than the average molecular weight of the polymer arms in the dendritic constituent unit.

13. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein for a higher generation Gx hyperbranched macromolecule, 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 wherein the average molecular weight of the polymer arm PEG units increases from the innermost polymer arm to the outermost polymer arm.

14. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the at least one arm connected to the core unit or branch unit is connected to the dendritic constituent unit by forming a hydrolyzable bifunctional linker, the hydrolyzable linker 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 macromolecule as claimed in any of the preceding claims, wherein the end group attached to the outermost polymer arm is grafted to the end of the polymer arm directly or via a bifunctional linker comprising or forming a hydrolyzable bond, the hydrolyzable bond comprising a carboxyl group, a dicarboxyl group, a carboxamide group, a dicarboxamide group, a functionalized aliphatic, heteroaliphatic, or aromatic or heteroaromatic group.

16. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the functional groups of the end groups and / or linker-end groups attached to the outermost polymer arm are selected from the following: electrophiles, such as activated esters, such as succinimidyl esters, succinimidyl carbonate; nitrophenyl carbonate, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, alkynes, azides, norbornene, epoxides, methanesulfonates, toluenesulfonates, trifluoroethylsulfonyl (tresyl), cyanurates, o-dithiopyridine, or halogens; nucleophiles, such as amines, such as Primary amines, hydroxyl groups, alcohols, thiols, azides, and carboxyl groups; functional groups for click chemistry; functional groups for cycloaddition, such as 1,3-dipolar cycloaddition, [3+2] cycloaddition, [4+2] cycloaddition, such as olefin-nitroketone cycloaddition or alkyne-nitroketone cycloaddition; functional groups for thiol-alkene reactions; functional groups for hetero-Diels-Alder cycloaddition; functional groups for nucleophilic ring-opening; functional groups for non-aldecanol carbonyl reactions; functional groups for carbon-carbon multiple bond addition reactions; polymerizable vinyl groups or combinations thereof.

17. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the (linker) end group attached to the outermost polymer arm is a functional group selected from the group consisting of succinimide succinate (SS), succinimide glutarate (SG), succinimide adipate (SAP), succinimide azelate (SAZ), and succinimide glutaramide (SGA).

18. The hyperbranched macromolecule according to any one of claims 1 to 13, wherein the end group attached to the outermost polymer arm is a functional group selected from: alkynes, such as dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]-nonyne (BCN); or norbornene or trans-cyclooctylene (TCO); azides, 3,4-dihydroxyphenylacetic acid (DHPA) or tetrazine (Tz).

19. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the connection between the polymer arm attached to the core unit and the first dendritic repeating unit and / or the connection between successive dendritic repeating units is formed by click chemistry, optionally using click chemistry functionalized linkers including hydrolyzable bonds.

20. The hyperbranched macromolecule of claim 19, wherein the linkage is formed by reacting a polymer arm, optionally partially functionalized with a linker, alkyne, cycloalkyne, or strained or terminal olefin, with a polymer arm partially functionalized with an azide or tetrazine in a SPAAC or IEDDA type click chemical coupling reaction.

21. The hyperbranched macromolecule of claim 20, wherein the alkyne moiety is a dibenzocyclooctyne moiety.

22. The hyperbranched macromolecule of any one of claims 19 to 21, wherein the connection between the polymer arm connected to the core unit and 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 branch 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 by forming at least one hydrolyzable bifunctional linker.

23. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the active agent conjugated to at least one of the outermost polymer arms is selected from the group consisting of therapeutic or diagnostic active agents.

24. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the active agent conjugated to at least one of the outermost polymer arms is selected from steroids; non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, ibuprofen, meclofenamate, mefenamic A, salicylate, sulindac, tolmetin, ketoprofen, diflunisal, piroxicam, naproxen, etodolac, flurbiprofen, and fenofofen C. C) Indomethacin, celecoxib, ketorolac, nepafenac; intraocular pressure-lowering drugs; antibiotics, such as ciprofloxacin; pain relievers, 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; small molecule hydrophobic drugs, hydrophilic peptides and protein drugs, such as insulin, single-chain antibody fragments, Fab fragments, IgG antibodies, fusion antibodies, etc.; aptamers; especially bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, antibiotics, pain relievers, calcium channel blockers, cell cycle inhibitors, chemotherapy agents, antiviral drugs, anesthetics, hormones, anticancer drugs, antitumor agents, viruses; gene delivery viruses, such as AAV; protein binding agents, such as nanobodies, affinity molecules, ankylosing smears, DARPin, etc.; or any combination thereof.

25. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the active agent conjugated to at least one of the outermost polymer arms is a peptide selected from the group consisting of: Compstatin, APL-1 and Fc-III-4C, Beovu (Brolucizumab), Zimura (Avacincaptad Pegol), Pegcetacoplan, Abicipar Pegol, Lampalizumab, Fovista, Risuteganib, AXT107, Elamipretide, THR149, ALM201, VGB3, and Largazole.

26. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the active agent is bound to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to 100% of the outermost polymer arm.

27. The hyperbranched macromolecule as described in any of the preceding claims, wherein the dendritic repeating unit is represented by formula (i): Where A is the connection to the polymer arm connected to the core unit, or A is the connection to B of the previous dendritic repeating unit represented by equation (i). L A It is a connector. m is 0 or 1. n is an integer between 20 and 2000, o is an integer between 20 and 2000. n and o can be different or the same. X is a branch unit with connectivity c'. L B It is a connector, where p is 0 or 1. B contains end groups located on the surface of the hyperbranched macromolecule or is linked to A or an active agent that forms a continuous dendritic repeating unit. L A and L B They can be different or the same. m and p can be different or the same, and y is an integer from 2 to 9, where y = c'-1, c' is the connectivity c' of the branching unit X; and the dendritic constituent units in the hyperbranched macromolecule may be the same or different.

28. The hyperbranched macromolecule of claim 27, wherein the connection between A and B comprises a functional group formed by click chemistry, such as a triazole or a dihydropyrazine.

29. The hyperbranched macromolecule of claim 27 or 28, wherein the linker L A and / or L B Contains diacid and / or acid diamide groups, such as succinate, glutarate, adipate, azelaate or glutaramide.

30. The hyperbranched macromolecule according to any one of claims 27 to 29, wherein the linker L A and / or L B Includes the structure represented by equation (ii): Among them U 1 and U 2 Independently, it can be NH or O and can be the same or different, and where t is an integer from 0 to 10.

31. The hyperbranched macromolecule of claim 29 or 30, wherein the linker L A and / or L B The bond with B further includes a polyethylene glycol unit between the carboxyl group, carboxamide group, or structure of formula (ii).

32. The hyperbranched macromolecule as claimed in any of the preceding claims, wherein the hyperbranched macromolecule further comprises at least one extension unit containing a polyethylene glycol (PEG) unit, wherein the extension unit is linear, bifunctional and connected to the polymer arm of the dendritic repeating unit, or the polymer arm connected to the end group or polymer arm of the core unit and the next dendritic repeating unit.

33. The hyperbranched macromolecule of claim 32, wherein the extended body unit comprises at least one linker, wherein the linker may be located at any or both ends of the extended body unit and is a bifunctional linker comprising a hydrolyzable bond, wherein the hydrolyzable bond comprises 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.

34. The hyperbranched molecule as claimed in any of the preceding claims, wherein the hydrolyzable bonds are selected from at least one or all of a core unit, a core unit comprising a polymer arm at the connectivity c, a dendritic repeating unit, a linker, and an extension, preferably all building blocks having a molecular weight of less than 50,000 Daltons, such as 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 molecule as claimed in any of the preceding claims, wherein, after complete hydrolysis of the hydrolyzable bond, all fragments formed by the molecule have a molecular weight of less than 50,000 Daltons, such as 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 precursor for a dendritic repeating unit, comprising: - A polymer arm containing functional groups (such as azides, alkynes, alkenes, or tetrazides) suitable for linkage via click chemistry, and - At least two polymer arms containing functional groups that are non-reactive in click chemistry. The polymer arm is connected to a branch unit having connectivity c'. The polymer portion of the polymer arm is composed of polyethylene glycol (PEG) units.

37. The precursor of claim 36, wherein the compound is represented by formula (iii): Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines). D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L A m, n, X, o, L B p and y are as defined in claims 27 to 31.

38. A reverse dendritic constituent repeating unit precursor, comprising: - A polymer arm containing functional groups that are non-reactive in click chemistry, and - At least two polymer arms containing functional groups suitable for click chemistry (such as azides, alkynes, alkenes, or tetrazines), and - A branch unit with connectivity c'; The polymer portion of the polymer arm is composed of polyethylene glycol (PEG) units.

39. The precursor of claim 34, wherein the precursor is represented by formula (iv): Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines). D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L A m, n, X, o, L B p and y are as defined in the preceding claims.

40. A method for manufacturing hyperbranched macromolecules as described in any one of claims 1 to 39 by divergent synthesis, comprising the following steps: (a) A core unit having at least three connectivity c is provided, wherein a plurality of polymer arms connected to the core unit have functional groups at the ends of the polymer arms suitable for click chemistry; (b) Providing a dendritic constituent repeating unit precursor, which comprises - A polymer arm comprising functional groups (such as azides, alkynes, alkenes, or tetrazides) suitable for linking with corresponding functional groups of the polymer arm connected to the core via click chemistry, and - At least two polymer arms containing functional groups that are non-reactive in click chemistry. (c) A connection is formed between the polymer arm attached to the core and the polymer arm of the dendritic constituent repeating unit precursor by click chemistry. (d) Optionally, converting the functional groups of the at least two polymer arms containing functional groups that are non-reactive in click chemistry into functional groups suitable for click chemistry, and (e) An active agent containing a functional group is conjugated to the outermost polymer arm by reacting with the functional group of the outermost polymer arm, thereby forming a hyperbranched macromolecule-active agent conjugate.

41. The method of claim 40, wherein for a higher generation Gx hyperbranched macromolecule, where x is an integer from 2 to 10, step (d) is mandatory, and prior to conjugation of the active agent in step (e), other successive dendritic constituent repeating unit precursors are linked by click chemistry to the functional groups suitable for click chemistry obtained in step (d) to be linked to the hyperbranched macromolecule.

42. The method of claim 40 or 41, wherein the dendritic constituent repeating unit precursor in step (c) is represented by formula (iii): Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines). D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L A m, n, X, o, L B p and y are as defined in the preceding claims; and the dendritic constituent units therein may be the same or different.

43. The method of claims 40 to 42, wherein after the penultimate step (d) of converting the functional groups of the at least two polymer arms containing functional groups that are non-reactive in click chemistry into functional groups suitable for click chemistry, the active agent in step (e) is first functionalized with functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines), and then conjugated to the outermost polymer arm of the hyperbranched macromolecule in the click chemistry reaction.

44. The method of claim 43, wherein the active agent functionalized with functional groups suitable for click chemistry is a peptide.

45. A method for manufacturing hyperbranched macromolecules as described in any one of claims 1 to 39 by aggregation synthesis, comprising the following steps: I) Provides a dendritic constituent repeating unit precursor, which contains - A polymer arm containing functional groups (such as azides, alkynes, alkenes, or tetrazides) suitable for linkage via click chemistry, and - At least two polymer arms containing functional groups that are non-reactive in click chemistry. II) Conjugating an active agent containing a functional group to at least one of the at least two polymer arms containing a functional group that is non-reactive in click chemistry of the dendritic constituent repeating unit precursor. III) Provides a core unit having at least three connectivity c, wherein multiple polymer arms connected to the core unit have functional groups (such as azides, alkynes, alkenes, or tetrazines) at the ends of the polymer arms, and IV) A link is formed between the polymer arm connected to the core provided in step III) and the polymer arm containing functional groups suitable for linking by click chemistry of the dendritic constituent repeating unit precursor obtained in step II), thereby forming a hyperbranched macromolecular-active agent conjugate.

46. ​​The method of claim 45, wherein the dendritic constituent repeating unit precursor in step I) is represented by formula (iii): Where C contains functional groups suitable for click chemistry (such as alkynes, alkenes, azides, or tetrazines). D contains functional groups that are non-reactive in click chemistry (such as succinimide groups), and L A L B m, n, X, o, p, and y are as defined in the preceding claims, and the dendritic constituent units described therein may be the same or different.

47. The method of claim 45 or 46, wherein for a higher generation Gx hyperbranched macromolecule, where x is an integer from 2 to 10, the active agent-conjugated dendritic repeating unit precursor obtained in step II) is click-chemically linked to a reverse dendritic repeating unit precursor, the reverse dendritic repeating unit precursor comprising: - A polymer arm containing functional groups that are non-reactive in click chemistry, and - At least two polymer arms containing functional groups suitable for click chemistry (such as azides, alkynes, alkenes, or tetrazines). Before being attached to other reverse dendritic repeating unit precursors or before being attached to the polymer arm attached to the core via click chemistry in step IV), the functional group of one polymer arm that is non-reactive in click chemistry is subsequently converted into a functional group suitable for click chemistry, thereby forming a higher-generation hyperbranched macromolecule.

48. The method of claims 45 to 47, wherein by performing steps I) and II) on each active agent-conjugated dendritic repeating unit precursor, dendritic repeating unit precursors having different active agents conjugated with the polymer arm are obtained, and the mixture of the obtained active agent-conjugated dendritic repeating unit precursors is used in step IV), thereby forming hyperbranched macromolecule-active agent conjugates with different active agents in different regions of the hyperbranched macromolecule surface.

49. The method of any one of claims 40 to 48, wherein the outermost polymer arm of the hyperbranched macromolecule has a terminal maleimide functional group, and the peptide or active agent is conjugated thereto by a maleimide-thiol reaction.

50. The method of claim 49, wherein the terminal maleimide functional group is provided by reacting the terminal functional group of the hyperbranched macromolecule with a DBCO or azide functional group having an azide or DBCO functional group attached to a maleimide group, the click chemical linker being such as DBCO-maleimide, DBCO-PEG3-maleimide, DBCO-PEG4-maleimide, or azide-PEG3-maleimide.

51. The hyperbranched macromolecule as described in any one of claims 1 to 39, used as a drug.

52. A treatment method for treating a patient’s disease or medical condition using a hyperbranched macromolecule as described in any one of claims 1 to 39.

53. The hyperbranched macromolecule used in claim 51 or 52, or the treatment method as described in claim 51 or 52, wherein the hyperbranched macromolecule is used for ocular treatment.

54. The hyperbranched macromolecule used in claims 51 to 53 or the treatment method described in claims 51 to 53, wherein the hyperbranched macromolecule is used to treat ocular diseases, such as posterior ocular diseases, such as any posterior segment ocular disease affecting the retinal, macular, or choroidal vascular system and integrity, resulting in visual acuity impairment, vision loss, or blindness, particularly said posterior segment disease states caused by 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 hyperbranched macromolecules used as described in claims 51 to 54, or the treatment methods described in claims 51 to 54.The hyperbranched macromolecules described herein are used to treat ocular diseases selected from the group consisting of: retinal neovascularization, choroidal neovascularization, wet AMD, dry AMD, retinal vein occlusion, diabetic macular edema, retinal degeneration, anterior chamber hemorrhage, presbyopia, corneal graft rejection, retinoblastoma, melanoma, miosis, mydriasis, glaucoma, conjunctivitis, intraocular infection, choroidal neovascularization (CNV), intraocular tumors, retinal nerve inflammation, inflammation, autoimmune uveitis, uveitis, proliferative vitreoretinopathy and corneal degeneration, acute and chronic macular neuroretinopathy, central serous chorioretinopathy, macular edema, acute multifocal squamous pigment epithelial lesions, Behcet's disease. Diseases including shotgun retinal choroidal lesions, posterior uveitis, posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusive disease, central retinal vein occlusion, disseminated intravascular coagulation, retinal branch vein occlusion, hypertensive fundus changes, ocular ischemia syndrome, retinal artery microaneurysms, Coat's disease, parafoveal telangiectasia, hemiretinal vein occlusion, optic papillary phlebitis, carotid artery disease (CAD), frost-like dendritic vasculitis, sickle cell retinopathy, vascular streaks, familial exudative vitreoretinopathy, and Eales' disease. Diseases including proliferative vitreoretinopathy, diabetic retinopathy, tumor-associated retinal diseases, congenital hypertrophy of retinal pigment epithelium (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, combined retinal and retinal pigment epithelial hamartoma, retinoblastoma, fundus angiogenesis tumors, retinal astrocytomas, intraocular lymphomas, myopic retinal degeneration, acute retinal pigment epitheliitis, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal carcinoma, retinitis pigmentosa, Leber's congenital amaurosis, choroidal agenesis, X-linked retinitis pigmentosa, best vitelliform macular dystrophy, X-linked retinoschisis, CNGA3 achromatopsia, CNGB3 achromatopsia, LHON, Stargardt disease, Usher syndrome, Norrie disease. Diseases such as Bardet-Biedl syndrome and red-green color blindness.

56. The hyperbranched macromolecule used in any one of claims 51 to 55 or the treatment method as described in any one of claims 51 to 55, wherein the hyperbranched macromolecule is formulated for direct injection at the treatment site of a patient, for example by parenteral administration, intratumoral injection, or injection into the eye, such as intravitreal, intraanterior chamber, subconjunctival, retrobulbar, subtenon, subretinal, or suprachoroidal injection.

57. The hyperbranched macromolecule used in any one of claims 51 to 56 or the treatment method as described in any one of claims 51 to 56, wherein the hyperbranched macromolecule is administered by direct injection, by oral administration, by incorporation into a gel, or by incorporation into an implant.

58. The hyperbranched macromolecule used in any one of claims 51 to 57 or the treatment method as described in any one of claims 51 to 57, wherein the hyperbranched macromolecule comprises two or more different active agents in different dendrons or regions on the surface of the hyperbranched macromolecule.

59. The hyperbranched macromolecule used as in claim 54 or the treatment method as described in claim 54, used in combination therapy involving the administration of more than one active agent.

60. The hyperbranched macromolecule of any one of claims 1 to 39, the hyperbranched molecule used in any one of claims 51 to 59, or the treatment method of any one of claims 51 to 59, wherein the hyperbranched macromolecule contains an active agent at different positions or regions on the surface of the hyperbranched macromolecule, the active agent having different hydrolyzable groups for varying the release of the active agent at different rates.

61. The hyperbranched macromolecule of any one of claims 1 to 39, the hyperbranched molecule used in any one of claims 51 to 59, or the treatment method of any one of claims 51 to 59, wherein the hyperbranched macromolecule comprises two or more different active agents at different positions or regions on the surface of the hyperbranched macromolecule, the active agents having different hydrolyzable groups for altering the release of the same or different active agents at different rates.

62. The hyperbranched macromolecule of any one of claims 1 to 39, the hyperbranched molecule used in any one of claims 51 to 61, or the treatment method of any one of claims 51 to 61, wherein the active agent is attached to the macromolecule by means of a hydrolyzable or non-hydrolyzable link or connection, optionally by means of an extension or combination thereof.

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