Bio-orthogonal hydrogels for sustained-release drug delivery

Abstract

The present invention relates to bioorthoscopic biodegradable hydrogels for sustained-release drug delivery, methods for preparing bioorthoscopic biodegradable hydrogels, their use as implantable materials in the treatment of diseases, kits for preparing hydrogels in situ at the treatment site, and methods of treatment using hydrogels or kits, such as for treating eye diseases.

Description

[Technical Field]

[0001] The present invention relates to bioorthoscopic biodegradable hydrogels for sustained-release drug delivery, methods for preparing bioorthoscopic biodegradable hydrogels, their use as implantable materials in the treatment of diseases, kits for preparing hydrogels in situ at the treatment site, and methods of treatment using hydrogels or kits. [Background technology]

[0002] Biocompatible and / or biodegradable hydrogels are widely used in pharmaceutical and biomedical research due to their extensive applications as carriers for drug delivery and scaffolds for tissue engineering. Their highly cross-linked structure and their ability to retain large amounts of water while maintaining their 3D structure make them ideal candidates for localized sustained-release drug delivery.

[0003] Synthetic biodegradable polymers such as polyethylene glycol (PEG), poly(N-isopropylacrylamide) (PNIPAM), and poly(vinyl alcohol) (PVA), as well as naturally occurring polymers and polysaccharides such as agarose, collagen, and fibrin, are the most common types of polymer hydrogels.

[0004] Reversible physical interactions such as hydrogen bonding, ion association, and hydrophobic interactions can induce gelation through the formation of weak crosslinks, but strong chemical bonding of polymer fractions is preferred to provide desired mechanical properties. These chemical crosslinks can be achieved by redox reactions and photopolymerization, Michael addition, Schiff base formation, epoxide coupling, disulfide exchange, and several other reactions. For example, EP3613413A1 describes zero gels and hydrogels formed by chemical crosslinking of polymer precursors having hydroxysuccinimide and amino (NHS / NH2) functional groups in nucleophilic-electrophilic crosslinking reactions, and their use for drug delivery. However, biomolecules as activators delivered by these hydrogels, such as peptides and proteins, often have NHS / NH2 functional groups and terminal amines or other functional groups that interfere with crosslinking reactions, making in-situ gel formation with such drugs difficult and preventing the use of bioorthogonal gels for drug delivery of biomolecules. Therefore, a bioorthogonal drug delivery system is needed to avoid such reactivity problems.

[0005] All references disclosed herein are incorporated herein by reference in their entirety for any purpose. [Overview of the Initiative]

[0006] An object and aspect of certain embodiments of the present invention is to provide a pharmaceutically acceptable drug delivery system, such as an implant, for the sustained release of an active agent into a patient's body.

[0007] A further object and aspect of certain embodiments of the present invention is to provide a biodegradable drug delivery system, such as an implant that is degraded in vivo.

[0008] A further object and aspect of certain specific embodiments of the present invention is to provide a biodegradable drug delivery system, such as an implant or insert, for the sustained release of an active agent to a patient in need.

[0009] A further object and aspect of certain embodiments of the present invention is to provide a bioorthogonal drug delivery system for the sustained release of an active agent for in situ formation of an implant or insert at a therapeutic site in vivo.

[0010] In further objects and aspects of certain embodiments of the present invention, the active agent can be a small molecule active agent or a biomolecule active agent, such as a peptide, protein, virus, etc.

[0011] A further object and aspect of certain embodiments of the present invention is to provide a method for manufacturing such a drug delivery system.

[0012] A further object and aspect of certain embodiments of the present invention is to provide a method for treating a patient's disease / medical condition using a sustained release bioorthogonal drug delivery system.

[0013] A further object and aspect of certain embodiments of the present invention is to provide a kit comprising one or more sustained release biodegradable drug delivery systems.

[0014] In certain embodiments, the present invention is directed to a sustained release drug delivery system comprising a bioorthogonal hydrogel and at least one active agent, the bioorthogonal hydrogel comprising a biodegradable covalently crosslinked polymer network and containing at least one active agent, the polymer network comprising a plurality of polymer units and chemical bonds that can be cleaved by hydrolysis, wherein at least one of the hydrolyzable bonds is an ester bond, and crosslinks formed by click chemistry reactions.

[0015] In a particular embodiment, the polymer network forming the hydrogel includes a core unit and at least one multi-arm unit having three or more arms connected thereto, for example, 3 to 10, or 4 to 8, or 4 to 6 arms, wherein the crosslinks formed by click reactions are located on the arms, preferably at the polymer unit ends of each arm. The polymer network may include first and second multi-arm units, where the crosslinks formed by click reactions are located between the arms of the first and second multi-arm units. The first and second multi-arm units may have the same or different amounts of arms, and the first and second multi-arm units may have the same or different core units. In other embodiments, the first and second multi-arm units may have the same or different amounts of arms, or the first and second multi-arm units may have the same or different core units.

[0016] In a particular embodiment, at least one of the multi-arm units is a polymer multi-arm unit having a polymer arm containing a polymer unit. The polymer unit can be selected from at least one of the following: polyethylene glycol (PEG), polyethylene oxide, polypropylene oxide, polypropylene glycol (PPG), polyvinyl alcohol (PVA), poly(vinylpyrrolidinone) (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-coglycolic acid (PLGA), p-dioxanone, trimethylene carbonate, caprolactone, random or block copolymers, or any combination or mixture thereof, or polyamino acids, glycosaminoglycans, polysaccharides, or proteins. In a particular embodiment, the polymer unit in the hydrogel is a polyethylene glycol unit.

[0017] In certain embodiments, the hydrolyzable ester bond may be part of a linker unit located between the end of an arm of a multi-arm unit and a crosslink formed by a click reaction. The first and / or second multi-arm units may include a linker unit containing a hydrolyzable ester bond, and the linker unit L may be the same or different in the first and second multi-arm units. The linker unit L may be derived from or contain acid and amide groups such as dibasic acids, or succinic acid, glutaric acid, adipic acid, azelaic acid, dibasic acids or acid amides, or glutaramide.

[0018] In certain embodiments, in a hydrogel of a sustained-release drug delivery system, crosslinks are formed by a click chemical reaction, such as a copper-free SPAAC or IEDDA reaction, which involves reacting a first functional group of a first multi-arm precursor or unit with a complementary second functional group of a second multi-arm precursor or unit, the second complementary functional group can react with the first functional group in the click chemical reaction, and the first and second functional groups are selected from pairs including an alkyne moiety and azide such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]-nonaine (BCN), or norbornene or transcyclocten (TCO) and tetrazine (Tz) moiety, or functionalized derivatives thereof. In certain embodiments, the crosslinks in the hydrogel formed by the click chemical reaction include chemical groups selected from triazole and dihydropyridazine.

[0019] In certain embodiments of the sustained-release drug delivery system of the present invention, the activator is selected from the group consisting of therapeutic or diagnostic activators. The activator is dispersed in a hydrogel and includes steroids; nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, meclofename, mefanamic A, salsalate, sulindac, tolmetin, ketoprofen, diflunisal, piroxicam, naproxen, etodrac, fluviprofen, fenoprofen C, indomethacin, celecoxib, ketrolac, and nepafenac; intraocular pressure lowering agents; antibiotics such as ciprofloxacin; analgesics such as bupivacaine; calcium channel blockers such as nifedipine; cell cycle inhibitors such as simvastatin; proteins such as insulin; and small molecule hydrophilic drugs including carboxylates and amine salts. The activator can be selected from small molecule hydrophobic drugs, insulin, hydrophilic peptides and protein drugs such as single-chain antibody fragments, Fab fragments, IgG antibodies, and fusion antibodies; aptamers; particularly bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, antibiotics, analgesics, calcium channel blockers, cell cycle inhibitors, chemotherapeutic agents, antiviral agents, anesthetics, hormones, anticancer agents, antitumor agents, viruses, genetically modified viruses such as AAV, nanobodies, aphibodies, ankyrin, DARPins, or any combination thereof. In one embodiment, the activator is a small molecule activator or a biomolecule activator, preferably a small molecule activator, peptide, protein, or virus, with small molecule activators, peptides, or proteins being particularly preferred. In a preferred embodiment, the activator is bevacizumab. The activator must not be a cell or stem cell. In one embodiment, the activator must not be a biological sample, i.e., a tissue, cell sample, virus, or virion.

[0020] In certain embodiments, the sustained-release drug delivery system includes an activator that is a peptide selected from the group consisting of bevacizumab, compstatin, APL-1, and Fc-III-4C, Beob 15 (brolucizumab), Zimra (abacincaptado pegol), pegcetacoplan, abisipal pegol, lamparizumab, Fovista, listiganib, AXT107, elamipretide, THR149, ALM201, VGB3, and largazole. In certain preferred embodiments, the sustained-release drug delivery system includes the activator bevacizumab. In some of these embodiments, the peptide activator is included in one or more precursor compositions that form a bio-orthogonal hydrogel in situ at the site of treatment.

[0021] In certain embodiments, the present invention further relates to a method for producing a sustained-release drug delivery system comprising a bioorthogonal hydrogel and at least one activator, the method comprising the steps of: preparing a first mixture comprising a first multi-arm precursor having a first functional group suitable for forming a linkage with a second functional group by click chemistry, and at least one activator; providing a second mixture comprising a second multi-arm precursor having a second functional group suitable for forming a linkage with a first functional group by click chemistry; combining the first and second mixtures; and enabling the combined reaction mixture to gel by forming a click chemical linkage between the first and second functional groups, thereby forming a hydrogel comprising at least one activator, wherein at least one of the first or second multi-arm precursors is a polymer multi-arm precursor having polymer arms comprising polymer units, and at least one of the first or second multi-arm precursors comprises linker units L comprising hydrolyzable ester bonds.

[0022] In certain embodiments of the method, at least one third multi-arm precursor having a first or second functional group suitable for forming a click chemical connection with either the first or second multi-arm precursor can be added to one of the first or second mixtures. In some embodiments of the method, the first and / or second mixture comprises at least one solvent. The at least one solvent can be selected from water, a buffered aqueous medium such as PBS buffer or saline, or an organic solvent or any mixture thereof. In certain embodiments, the method further includes the step of removing the solvent or solvent mixture, for example, by drying the formed gel.

[0023] In embodiments of the method, the multi-arm precursor may have a core unit and at least three arms connected to the core unit, such as 3 to 10, or 4 to 8, or 4 to 6 arms, wherein functional groups suitable for forming connections by click chemistry are located at the ends of the arms. The first and second multi-arm precursors may have the same or different amounts of arms and / or the same or different core units. In the embodiment, the polymer unit is selected from at least one of polyethylene glycol (PEG), polyethylene oxide, polypropylene oxide, polypropylene glycol (PPG), polyvinyl alcohol (PVA), poly(vinylpyrrolidinone) (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-coglycolic acid (PLGA), p-dioxanone, trimethylene carbonate, caprolactone, random or block copolymers, or any combination or mixture thereof, or one or more units of polyamino acids, glycosaminoglycans, polysaccharides, or proteins. In one embodiment, the polymer units are selected from at least one of polyethylene glycol (PEG) units, polypropylene glycol (PPG) units, polyglycolic acid (PGA), polylactic acid (PLA), or polylactic acid-coglycolic acid (PLGA), random or block copolymers, and / or combinations or mixtures thereof, preferably polyethylene glycol. In a further embodiment, the covalently crosslinked polymer network comprises a plurality of hydrophobic polymer units and / or hydrophilic polymer units, wherein the hydrophobic polymer units are selected from at least one of polylactic acid (PLA) and polylactic acid-coglycolic acid (PLGA) units, and / or the hydrophilic polymer units are selected from at least one of polyethylene glycol units, polypropylene glycol units, or polyglycolic acid (PGA), preferably polyethylene glycol units.

[0024] In certain embodiments, the hydrolyzable ester bond is part of a linker unit located between the polymer unit end of an arm of the multi-arm precursor and a functional group suitable for forming a linkage by click chemistry. The first multi-arm precursor or the second multi-arm precursor, or both, may contain a linker unit L comprising a hydrolyzable ester bond, and the linker unit L may be the same or different in the first and second multi-arm precursors. The linker unit L may be derived from or contain dibasic acids or acid and amide groups such as succinic acid, glutaric acid, adipic acid, azelaic acid, dibasic acid or acid amide, or glutaramide.

[0025] In certain embodiments of the method, the first and second functional groups suitable for forming a linkage by click chemistry include moieties selected from the group consisting of alkynes such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]-nonane (BCN), cycloalkynes, strains or terminal alkenes such as norbornene or transcyclooctene (TCO), azides, or tetrazine (Tz), or functionalized derivatives thereof. The first and second functional groups suitable for forming a linkage by click chemistry may include moieties selected from dibenzocyclooctin and azides.

[0026] In further embodiments, the present invention relates to a method of treatment comprising using a sustained-release drug delivery system described herein for use as a pharmaceutical, or treating a disease or condition in a patient using a sustained-release drug delivery system described herein. The treatment may be the treatment of ocular diseases such as posterior segment diseases, such as any posterior segment ocular disease that affects the blood vessels and structures of the retina, macula, or choroid and causes visual impairment, visual loss, or blindness, in particular posterior segment disease conditions resulting from aging, trauma, or surgical intervention, such as age-related macular degeneration (AMD), cystoid macular edema (CME), diabetic macular edema (DME), posterior uveitis, and diabetic retinopathy, or other conditions described further below in the present invention.

[0027] In certain embodiments of the treatment method, the drug delivery system or hydrogel may be formulated for direct injection at the treatment site of the patient, for example, for injection into the eye, such as parenteral administration, intratumoral injection, intravitreal injection, anterior chamber injection, subconjunctival injection, retrobulbar injection, sub-Tenon's capsule injection, subretinal injection, or suprachoroidal injection. The hydrogel may be formulated for direct injection, oral application, incorporation into a gel, or incorporation into an implant, and administered by those means. In certain embodiments, the drug delivery system may contain two or more different activators, for example, for use in combination therapy involving the administration of two or more different activators.

[0028] In certain embodiments, the treatment method involves in situ formation of a hydrogel of a drug delivery system at the treatment site of the patient. The hydrogel can be formed in situ at the treatment site of the patient by combining a first formulation containing a first mixture of the above-described manufacturing method and a second formulation containing a second mixture, thereby enabling the combined formulation mixture to gel in situ at the treatment site. The first and second formulations can be combined immediately before administering the formulation mixture at the treatment site.

[0029] In certain embodiments involving in-situ formation of a hydrogel of a drug delivery system at the patient's treatment site, the hydrogel can be formed in situ by administering a suspension containing a first mixture of the above-described manufacturing method, a second mixture of the above-described manufacturing method, and a hydrophobic organic liquid at the patient's treatment site, thereby enabling the combined formulation to gel in situ at the treatment site. The hydrophobic organic liquid can be contained in at least one of the first or second mixtures and may be a biocompatible oil. In certain embodiments thereof, the activator is dissolved or dispersed in the hydrophobic organic liquid, or the activator is the hydrophobic organic liquid or forms at least a portion thereof.

[0030] In a particular embodiment, the drug delivery system may be provided as a kit for forming a hydrogel containing an activator, the kit comprising a first precursor mixture and a second precursor mixture in separate containers for mixing and gel formation, if necessary. In that embodiment, the first formulation may be supplied in a first syringe, the second formulation in a second syringe, and the two formulations may be combined in a Y-mixer before direct injection at the treatment site.

[0031] In an alternative embodiment of the treatment method, the first and second formulations are each administered at the treatment site, thereby allowing the formulations to be combined at the treatment site and the combined formulation to gel in situ at the treatment site. The first formulation may be in a first syringe, and the second formulation may be in a second syringe, and the two formulations are injected simultaneously or sequentially at the treatment site.

[0032] Further embodiments of the present invention are described below in this specification. [Brief explanation of the drawing]

[0033] [Figure 1] HPLC analysis showing the reaction of 4-arm PEG SAP with DBCO-amine in 1:1 (black) and 1:2 (blue) ratios. [Figure 2] Use of magnetic beads to remove excess DBCO-amine from the system. [Figure 3a] A graphical summary of the effects of the number of arms on gel time and swelling. [Figure 3b] A graphical summary of the effects of the number of arms on gel time and swelling. [Figure 4] Sustained-release profile of gold nanoparticles from DBCO / azide gel. [Figure 5] Summary of SDS page results for Avastin solutions treated with different multi-arm PEG. [Figure 6] Summary of SEC-HPLC data showing the presence of avastin and PEG species in non-PEGylated samples. [Figure 7] Summary of ELISA measurement results for bevacizumab. [Figure 8] Summary of SPR results showing protein binding with VEGF. [Figure 9] The effect of crosslinking density (MWc) on release dynamics. [Figure 10] Release data for DBCO / tetrazine-functionalized hydrogels. [Figure 11] Gel time of DBCO / tetrazine / azide-functionalized hydrogel. [Figure 12] Release data for DBCO / tetrazine / azide-functionalized hydrogels.

[0034] definition The term "biochorthogonal" refers to a material or object (such as a hydrogel or organogel according to the present invention) that is formed by chemical reactions that can occur substantially within a biological system without interfering with natural biochemical processes.

[0035] The term "biodegradable" refers to a material or object (such as a hydrogel according to the present invention) that is biocompatible and decomposes in vivo, i.e., when placed in the body of a human or animal, or when immersed in an aqueous solution under physiological conditions such as pH 7.2–7.4 at 37°C. In the context of the present invention, as disclosed in detail below, a hydrogel, once administered or deposited in the body of a human or animal, slowly biodegrades and is removed over time. In certain embodiments, biodegradation occurs at least partially via ester hydrolysis in the aqueous environment of the body. Biodegradation may occur by hydrolysis or enzymatic cleavage of covalent or conjugation bonds in the linker group and / or polymer arms. The hydrogel slowly disintegrates, resulting in clearance through physiological pathways. In certain embodiments, the hydrogel of the present invention is stable degradation over a long period of time (e.g., about 1 month, 3 months, or 6 months). In certain embodiments, the hydrogel simply biodegrades until, for example, the activator or at least a significant amount thereof (e.g., at least 50%, at least 75%, or at least 90%) is released therefrom.

[0036] A "hydrogel" is a three-dimensional network of one or more hydrophilic natural or synthetic polymers (as disclosed herein) that swells in water and retains a certain amount of water, while maintaining or substantially maintaining its structure, for example, by chemical or physical crosslinking of individual polymer chains. Due to their high water content, hydrogels are soft and flexible and very similar to natural tissues. In the present invention, the term "hydrogel" is used to refer to both a hydrated hydrogel when it contains water (for example, after it has been formed in an aqueous solution, or after it has been inserted into an eye or immersed in an aqueous environment by another means to be hydrated or (re)hydrated), and a dry (dried / dehydrated) hydrogel when it has been dried to a low water content, for example, 1% by weight or less.

[0037] The term "polymer network" refers to a structure formed from polymer chains that are crosslinked with each other (having the same or different molecular structures and the same or different molecular weights). Types of polymers suitable for the purposes of this invention are disclosed below in this specification.

[0038] In this specification, the term “precursor” refers to a molecule or compound that reacts with each other, thereby bonding via crosslinking, to form a polymer network and thus a hydrogel matrix. Other materials, such as activators or buffers, may be present in the hydrogel, but they are not referred to as “precursors.”

[0039] The portions of precursor molecules that still exist within the final polymer network are also referred to herein as “units.” Thus, “units” are the building blocks or components of the polymer network that forms the hydrogel. For example, a polymer network suitable for use in the present invention may contain identical or different polyethylene glycol units, as further disclosed herein.

[0040] The term "multi-arm precursor" means that the precursor is branched, i.e., nonlinear, and has a core unit and arms extending from the core, where the core refers to a continuous portion of molecules bonded to the arms.

[0041] It should be noted that multi-arm precursors may have, for example, 3 to 100 arms, each with an end, and some precursors may be dendrimers or other highly branched materials.

[0042] The arms on the precursor refer to a linear chain of chemical groups connecting the crosslinkable groups to the core. Some embodiments are precursors having 3 to 300 arms; those skilled in the art will immediately understand that all ranges and values ​​within the explicitly stated ranges, e.g., 4, 6, 8, 10, 12, 4 to 16, 8 to 100, 6, 8, 10, 12, or at least 4 arms are intended.

[0043] In the case of polymer multi-arm precursors, the precursor has arms containing a core unit and polymer units extending from the core unit, and the polymer arms often have functional groups suitable for crosslinking at the ends of the arms.

[0044] The term "small molecule" refers to organic compounds with a low molecular weight (≤1000 daltons). Therefore, small molecule multi-arm precursors are branched, have a low molecular weight (≤1000 daltons), and possess functional groups suitable for crosslinking at the arms.

[0045] The molecular weights of polymer precursors used for the purposes of the present invention and disclosed herein can be determined by analytical methods known in the art. The molecular weight of polyethylene glycol can be determined by any method known in the art, including, for example, 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 dynamic light scattering (DLS) for simultaneous particle size determination, 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 containing polyethylene glycol precursors disclosed herein is the average molecular weight (based on the molecular weight distribution of the polymer) and may therefore be expressed by various average values, including weight-average molecular weight (Mw) and number-average molecular weight (Mn). In the case of crosslinkable polymer gelators such as polyethylene glycol, the molecular weight shown herein is the number-average molecular weight (Mn) determined by gel permeation chromatography using a polystyrene standard, according to standard methods known in the art. Typically, materials, particularly multi-arm precursors, are purchased at specific molecular weights defined by the vendor. Suitable PEG precursors are available from numerous suppliers, such as Jenkem Technology.

[0046] For the purposes of this invention, the term “sustained release” means characterizing a product formulated to make the activator available over a long period of time, thereby enabling a reduction in the frequency of administration compared to immediate-release dosage forms such as a solution of the activator 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.” In the sense of this invention, the term “sustained release” includes constant activator release, tapered activator release, and any combination thereof (e.g., tapered activator release after constant activator release). In the sense of this invention, the terms “tasted” or “tasting” refer to a decrease in the release of the activator over time. Specifically, the term “sustained release” refers to the release of the activator from a drug delivery system in a predetermined manner, in contrast to immediate release such as bolus injection. Controlled release refers to the amount of activator released over the total number of days required for 100% release of the activator in an aqueous solution under in vitro physiological conditions such as pH 7.2–7.4 and 37°C.

[0047] As used herein, the term “long duration” means any period of time that a person skilled in the art would consider to be an extended treatment of a disease, in particular a period of at least about one week, or at least about one month, for example up to about 12 months, or any intermediate period, for example, about 1 to about 6 months, about 2 to about 4 months, about 2 to about 3 months, or about 3 to about 4 months.

[0048] "Zero-order" release, or "substantially zero-order" release, or "near zero-order" release, is defined as a relatively straight line in a graph of the percentage of released activator versus time. In certain embodiments of the present invention, substantially zero-order release is defined as an amount of activator released that is within 20% of the elapsed time. As used herein, 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 one day or approximately 24 hours that has already elapsed since administration of the drug delivery system.

[0049] The terms “API,” “Active(pharmaceutical) ingredient,” “Active(pharmaceutical) agent,” “Active(pharmaceutical) ingredient,” “(active) therapeutic agent,” “active drug,” and “drug” are used interchangeably herein and refer to substances used in a finished pharmaceutical product (FPP) that are intended to impart pharmacological activity or have a direct effect on the diagnosis, cure, mitigation, treatment, or prevention of a disease or on the restoration, correction, or modification of a patient’s physiological function, and substances used in the preparation of such a finished pharmaceutical product. The activator may be a small molecule activator or a biomolecule activator, such as a peptide, protein, or virus. In a preferred embodiment, the activator may be a peptide or protein. In a preferred embodiment, the activator is bevacizumab. The activator shall not be a cell or stem cell. In one embodiment, the activator shall not be a biological sample, i.e., a tissue, cell sample, virus, or virion.

[0050] The activators used in accordance with the present invention may be activators for the treatment and / or prevention of eye diseases or disorders, or diagnostic agents such as markers. In embodiments of the present invention, the activator is a low-water-solubility activator (i.e., having water solubility of less than about 1000 μg / mL or less than about 100 μg / mL). In another embodiment of the present invention, the activator is a high-water-solubility activator (i.e., having water solubility of more than about 1000 μg / mL or even more than about 10 μg / mL). This definition does not depend on drugs approved by government agencies.

[0051] For the purposes of the present invention, all possible forms of the activator can be used, including free acids, free bases, polymorphs, or any pharmaceutically acceptable salts, anhydrides, hydrates, cocrystals, prodrugs, or other solvates or derivatives such as prodrugs or conjugates. In this description or claims, whenever the activator is mentioned without further specification, it also refers to the activator in the form of any such polymorph, pharmaceutically acceptable salt, anhydride, solvate (including hydrate), or derivative, even if not explicitly stated. With respect to the activator, preferred solid forms include, without limitation, pure substance forms in any physical form known to those skilled in the art. For example, the activator may take the form of particles. For example, the activator may take the form of particles. The particles may be amorphous or crystalline, or a mixture of these two forms, and can be made of any size which can be classified without limitation as coarse particles, fine particles, or ultrafine particles, the dimensions which can be seen particularly with the naked eye or under a microscope, and can have shapes such as single grains and / or aggregates. The particles can also be micronized. As used herein, the term “micronization” refers, without limitation, to small-sized particles, particularly microscopic-scale particles, whose particle size has been reduced by, for example, jet milling, jaw crushing, hammer milling, wet milling, precipitation in a non-solvent, cryomilling (milling with liquid nitrogen or dry ice), and ball milling. The activator may also exist in a dissolved or dispersed state, for example, in a solvent or aqueous medium, for example, in the form of particles dispersed in an oil or a compatible aqueous suspension (which may optionally contain further excipients such as surfactants).

[0052] As used herein, the term “therapeutably effective” refers to the amount of activator required to produce a desired therapeutic outcome after administration. For example, in the context of the present invention, one desired therapeutic outcome is, for example, a reduction in symptoms associated with DED, as measured by in vivo tests known to those skilled in the art, such as an increase in Schirmer's tear film test score, a decrease in staining values ​​as measured by conjunctival lysamine green staining or corneal fluorescein staining, a decrease in the severity and / or frequency of eye dryness score on a visual analog scale (VAS), a decrease in the Ocular Surface Disease index and / or Standard Patient Evaluation of Eye Dryness score, and a decrease in best corrected visual acuity. In one embodiment, "therapeutably effective" refers to the amount of activator in a sustained-release intraductal insert that can achieve, in terms of therapeutic effect, a tear concentration equivalent to a cyclosporine concentration of 0.236 μg / mL (considered necessary for immunomodulation, Tang-Liu and Acheampong, Clin. Pharmacokinet. 44(3), pp. 247-261) over a long period of time, and in particular, over substantially the entire remaining period of wear of the insert once that tear concentration is achieved.

[0053] As used herein, the values ​​"d10", "d50", "d90", and "d100" refer to values ​​that characterize the proportion of particles in a particle size distribution that satisfy a particular particle size. In a given particle size distribution, 10% of the particles have a particle size of d10 or less, 50% of the particles have a particle size of d50 or less, 90% of the particles have a particle size of d90 or less, and substantially all particles have a particle size of d100 or less. The percentages can be expressed by different parameters known to those skilled in the art, for example, the percentages may be based on volume, weight, or the number of particles. Thus, d50 may exemplary be the median particle size on a volume basis, weight basis, or number basis. For example, if d90 on a volume basis is 43 μm, it means that 90% of the volume of particles have a particle size of 43 μm or less. In certain embodiments, d10, d50, and d90 are volume-based values. The particle size distribution (PSD) can generally be measured by methods known to those skilled in the art, including sieving and laser diffraction. In certain embodiments, the PSD is measured by USP <429> The particle size is measured by optical diffraction, specifically by laser diffraction. In a particular embodiment, the PSD is measured by laser diffraction using a Beckman Coulter LS 13 320, based on the optical model "Fraunhofer.rf780z" with a occlusion value in the range of 7–9%.

[0054] The term “patient” as used herein includes both human and animal patients. Therefore, the biodegradable drug delivery systems according to the present invention are suitable for human or veterinary medical use. Generally, “subject” refers to the individual (human or animal) to whom the drug delivery system according to the present invention is administered. “Patient” refers to a subject requiring treatment due to a specific physiological or pathological condition. A “patient” is not necessarily diagnosed with a specific physiological or pathological condition prior to receiving the drug delivery system.

[0055] Room temperature refers to temperatures between 20°C and 25°C, such as 20°C.

[0056] Unless otherwise explicitly defined, all percentages, "%", in this specification mean weight percentages (w / w, weight %).

[0057] As used herein, the term “approximately” in relation to a measured quantity refers to the normal variation in that measured quantity that can be expected by a person skilled in the art when performing a measurement and exercising a level of care appropriate to the purpose of the measurement and the precision of the measuring instrument.

[0058] The term "at least about" in relation to a measured quantity refers to the normal variation in the measured quantity and amounts higher than the measured quantity that a person skilled in the art would expect when performing a measurement and exercising a level of care appropriate to the purpose of the measurement and the precision of the measuring instrument.

[0059] As used herein, the singular forms "a," "an," and "the" include plural references unless otherwise explicitly indicated by the context.

[0060] In this specification, 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".

[0061] Open terms such as "include," "including," "contain," and "containing" all mean "comprising." These unrestricted transition clauses are used to introduce an unrestricted list of elements, method steps, etc., that do not exclude additional unlisted elements or method steps.

[0062] Where used herein in conjunction with a specific value or number, the term “maximum” means including that respective value or number.

[0063] The terms "A~B (from A to B)," "A~B (of A to B)," and "A~B (of A to B)" are used synonymously in this specification and all refer to a range from A to B, including an upper limit A and a lower limit B.

[0064] Throughout this disclosure, various aspects of the invention are presented in range form. It should be understood that the range form is for convenience and brevity only and should not be interpreted as an inflexible limitation on the scope of the invention. Therefore, a range description should be considered to specifically disclose not only the individual numbers within that range, but also all possible sub-ranges. For example, a range description such as 1-6 should be considered to have specifically disclosed sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as the individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range. A stated range of numbers includes the number defining the range and each integer within the defined range.

[0065] As used herein, the abbreviation "PEG" means polyethylene glycol.

[0066] As used herein, the abbreviation "PBS" means phosphate-buffered saline, followed by a number indicating its pH.

[0067] "TBS" is an abbreviation for Tris-Buffered Saline, and the number that follows is its pH. [Modes for carrying out the invention]

[0068] In order to overcome some of the shortcomings of conventional drug delivery gels in terms of biocompatibility and versatility for delivering various types of drugs, including biomolecules such as peptides, proteins, and antibodies, embodiments of the present invention have developed hydrogels formed by click chemical reactions. In certain embodiments, the present invention relates to a hydrogel containing an activator containing biomolecules for delivering the activator to a treatment site in a patient, wherein the hydrogel releases the activator over time and biodegrades before being removed from the body. The hydrogel comprises a biodegradable covalently crosslinked polymer network and contains at least one activator. The term "containing an activator" means that the hydrogel contains an activator that does not have a functional group of the activator that reacts with the hydrogel, or the activator is covalently conjugated to the hydrogel. In one embodiment, the activator is contained in the hydrogel and optionally at least partially further covalently conjugated to the hydrogel. In a preferred embodiment, the activator is contained in the hydrogel without being covalently conjugated to the hydrogel. In certain preferred embodiments, the activator is contained in the hydrogel without being covalently conjugated to the hydrogel via oligonucleotides. The polymer network forming the hydrogel comprises multiple polymer units that are crosslinked by click chemical reactions when the hydrogel is formed. Furthermore, the hydrogel contains chemical bonds that can be cleaved by hydrolysis, such as ester bonds, so that the hydrogel can be broken down into smaller units that can be removed from the body under physiological conditions.

[0069] Click chemistry, encompassing a series of reactions that combine two specific precursor molecules, provides a means for obtaining different types of bio-orthogonal hydrogels. Click reaction chemistry is fast, spontaneous, and selective with high yields, and does not produce byproducts under mild conditions in an aqueous environment. Specifically, copper-free strain-promoted azido-alkyne cycloaddition (SPAAC) and reverse electron-required Diels-Alder reactions (IEDDA) are click chemical reactions that can be used in embodiments of the present invention for hydrogel formation and gel-precursor crosslinking by forming triazole or dihydropyridazine covalent crosslinks. Since these crosslinks are stable bonds that are not readily biodegradable, in embodiments of the present invention, hydrogels for use as implants and drug delivery materials require the presence of further hydrolyzable bonds within the polymer network. These hydrolyzable bonds can be introduced in certain embodiments of the present invention by introducing linkers that form hydrolyzable bonds, such as ester bonds, amide bonds, and carboxamide bonds, with polymer units and functional groups for crosslinking at various positions in the polymer network formed during gelation or the gel precursor.

[0070] Hydrogel A drug delivery system according to a particular embodiment of the present invention provides sustained release of an activator from a biodegradable and biocompatible hydrogel. At least one activator is dispersed in the hydrogel and can be released from the hydrogel under physiological conditions.

[0071] The structural framework of a hydrogel is a three-dimensional polymer network that can be formed by crosslinking polymer precursors with each other through the formation of chemical bonds. Therefore, the polymer network includes portions of precursor molecules that still exist after hydrogel formation, hereafter referred to as "units."

[0072] In this disclosure, these crosslinks are formed by click reactions. Preferred functional groups and click reactions are described in detail herein.

[0073] Polymer units and precursors In certain embodiments of this disclosure, the polymer network comprises a multi-arm unit having a core unit X and at least (≧) three arms attached to the core unit. Functional groups suitable for click reactions present in each multi-arm precursor are converted into crosslinks formed by click reactions of the polymer network during hydrogel formation. These crosslinks are located on the polymer arms or units, for example, at the polymer unit ends of each arm of the multi-arm unit, or at least a large portion of the multi-arm unit ends.

[0074] The polymer network may include first and second multi-arm units, and the crosslinks formed by click chemical reactions are located between the arms of the first and second multi-arm units. If the polymer network of the hydrogel includes one or more multi-arm units, at least one of the multi-arm units must be a polymer multi-arm unit having polymer arms containing polymer units of the hydrogel. The polymer multi-arm units may be crosslinked with non-polymer multi-arm units such as small molecule crosslinking agents.

[0075] According to certain embodiments of the present invention, the hydrogel comprises a polymer network including at least two covalently crosslinked multi-arm precursors. In some embodiments, the hydrogel comprising the polymer network comprises at least two covalently crosslinked multi-arm precursors.

[0076] Therefore, the precursor is always a "functional polymer" or "functional material," such as a crosslinking agent (e.g., low molecular weight) that can participate in click chemical crosslinking reactions with other precursors to form a covalently crosslinked polymer network (or matrix). Thus, the term "non-functional polymer" refers to a polymer that may be present in the hydrogel of the present invention but does not participate in crosslinking reactions with precursors that form the polymer network.

[0077] The term "multi-armed" precursor means that the precursor is branched, i.e., nonlinear. In the case of multi-armed polymers, the core refers to a continuous portion of molecules linked to arms extending from the core, and the arms have functional groups for click chemistry, such as azides or DBCOs, which are often at the ends of the branches. It should be noted that a precursor may have, for example, 3 to 100 arms, each arm having an end, and some precursors may be dendrimers or other highly branched materials. The arms on a precursor refer to a linear chain of chemical groups that connect crosslinkable functional groups to the polymer core. Some embodiments are precursors having 3 to 300 arms; those skilled in the art will immediately understand that all ranges and values ​​within the explicitly stated ranges, e.g., 4, 6, 8, 10, 12, 4 to 16, 8 to 100, 6, 8, 10, 12, or at least 4 arms are intended.

[0078] In certain embodiments, the polymer multi-arm units present in the hydrogel are derived from one or more multi-arm PEG precursors (or other polymer units described herein) having 3 to 10 arms, or 4 to 8 arms, or 4, 5, 6, 7, or 8 arms. The precursors used as the first or second multi-arm units in the hydrogel of certain embodiments of the present invention may have different or the same number of arms. In certain embodiments, the precursors used in the hydrogel of the present invention have 4 and / or 8 arms. Multi-arm precursors for use in embodiments of the present invention, such as PEG precursors, Tetronic®, or PLGA precursors, are commercially available, for example, from JenKem Technology USA, SinoPEG, or Sigma-Aldrich, and optionally contain a variety of functional end groups such as NHS, NH2, azide, or DBCO.

[0079] In certain embodiments, the multi-arm precursor of the present invention has a core and 3 to 10 arms, or 4 to 8 arms, or 4, 6, or 8 arms, each arm comprising polymer units and having an end. In preferred embodiments, the multi-arm precursor has 4 or 8 arms.

[0080] In some embodiments, if each precursor is multi-armed, it comprises three or more arms, and therefore three or more functional groups for click chemical crosslinking, and as a result, each functional group can react with another complementary functional group of another precursor in a click chemical reaction to form a crosslinked polymer network of hydrogels. Thus, for example, in some embodiments, the precursor has four arms, and each arm is terminated with one type of functional group suitable for click chemistry.

[0081] According to one aspect of the present invention, the hydrogel comprises at least two multi-arm precursors, including a first multi-arm precursor containing a first functional group and a second multi-arm precursor containing a second functional group. In this embodiment, the first multi-arm precursor and the second multi-arm precursor are covalently crosslinked with each other in a click reaction. In this context, multi-arm refers to at least eight arms, such as at least three arms, four arms, five arms, six arms, seven arms, or at least ten arms.

[0082] The polymer units in the precursor and / or hydrogel may be the same or different in each arm and may be selected from at least one of the following: polyethylene glycol (PEG), polyethylene oxide, polypropylene oxide, polypropylene glycol (PPG), poly(ethylene glycol)-block-poly(propylene glycol) copolymer, poloxamers such as Tetronic®, polyvinyl alcohol (PVA), polyvinyl acetate, poly(vinylpyrrolidinone) (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-coglycolic acid (PLGA), p-dioxanone, trimethylene carbonate, caprolactone, random or block copolymers, or any combination or mixture thereof, or polyamino acids, glycosaminoglycans, polysaccharides, or proteins. In certain embodiments, the polymer units are selected from at least one of polyethylene glycol (PEG) units, polypropylene glycol (PPG) units, polyglycolic acid (PGA), polylactic acid (PLA), or polylactic acid-coglycolic acid (PLGA), random or block copolymers, and / or any of these, preferably combinations or mixtures of polyethylene glycol units. In certain embodiments of the present invention, at least one hydrophobic polymer unit, such as poly(ethylene glycol) block poly(propylene glycol) copolymer, is included in a polymer network also known as a poloxamer, such as commercially available Tetronic® poloxamer.

[0083] In certain embodiments, the polymer network includes polylactic acid-coglycolic acid (PLGA) units, or a combination of polylactic acid-coglycolic acid (PLGA) units and polyethylene glycol (PEG) units, preferably a copolymer of PEG and PLGA, particularly preferably a block copolymer of multi-arm PEG copolymerized with PLGA, or a combination of polylactic acid-coglycolic acid (PLGA) units and poly(ethylene glycol)-block poly(propylene glycol) copolymer units (Tetronic®). The ratio of polylactic acid-coglycolic acid (PLGA) units to polyethylene glycol (PEG) units or poly(ethylene glycol)-block poly(propylene glycol) copolymer units (Tetronic®) can be selected to be about 2.5:1 to about 1:2.5, or about 2:1 to about 1:2, or about 1:1.

[0084] When using polylactic acid-coglycolic acid (PLGA) units, they may have L / G ratios (in %L or G units) in the range of 0:100 to 100:0, or 1:99 to 99:1, or 10:90 to 90:10, or 25:75 to 75:25, or 50:50. Polylactic acid-coglycolic acid (PLGA) refers to a polymer having repeating groups (-(C=O)-CH(CH3)-O)x-(-(C=O)-CH2-O-)x, where x is at least 3. In other embodiments, x may be 1 to 2000, for example, 1 to 1800, 1 to 1600, 1 to 1400, 1 to 1200, 1 to 1000, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to 400, or 1 to 300. In certain embodiments, x may be 1 to 500, 1 to 400, or 1 to 300. In one embodiment, the polymer multi-arm unit includes eight arms, and x is in the range of 1 to 300. In another embodiment, the polymer multi-arm unit includes four arms, and x is in the range of 1 to 500.

[0085] In certain embodiments, polymer arms included in a polymer network are made from or contain at least one polyethylene glycol unit. Polyethylene glycol (PEG), also called polyethylene oxide, refers to a polymer having repeating groups (CH2CH2O)n or (CH2CH2O)y, where n and y are at least 3, respectively. In other embodiments, n and y may be 1 to 2000, e.g., 1 to 1800, 1 to 1600, 1 to 1400, 1 to 1200, 1 to 1000, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to 400, 1 to 300, 1 to 200, 1 to 100, or 50 to 100, respectively. In yet another embodiment, n and y may be 1 to 500, 50 to 450, 100 to 400, or 100 to 300, respectively. In one embodiment, the polymer multi-arm unit includes eight arms, where n and y are in the range of 1 to 300, respectively. In another embodiment, the polymer multi-arm unit includes four arms, where n and y are in the range of 1 to 500, respectively.

[0086] In certain embodiments, polymer arms included in a polymer network are made from or include at least one polypropylene glycol unit. Polypropylene glycol (PPG) refers to a polymer having a repeating group (-CH2-CH(CH3)-O-)x, where x is at least 3. In other embodiments, x may be 1 to 2000, e.g., 1 to 1800, 1 to 1600, 1 to 1400, 1 to 1200, 1 to 1000, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to 400, 1 to 300, 1 to 200, 1 to 100, or 50 to 100. In preferred embodiments, x may be 1 to 500, 50 to 450, 100 to 400, or 100 to 300. In one embodiment, a polymer multi-arm unit includes eight arms, where x is in the range of 1 to 300. In another embodiment, the polymer multi-arm unit includes four arms, where x is in the range of 1 to 500.

[0087] In certain embodiments, the polymer arms included in the polymer network are made from or include at least one polylactic acid unit. Polylactic acid (PLA) refers to a polymer having a repeating group (-C(C=O)-CH(CH3)-O)x, where x is at least 3. In other embodiments, x may be 1 to 2000, e.g., 1 to 1800, 1 to 1600, 1 to 1400, 1 to 1200, 1 to 1000, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to 400, 1 to 300, 1 to 200, 1 to 100, or 50 to 100. In preferred embodiments, x may be 1 to 500, 50 to 450, 100 to 400, or 100 to 300. In one embodiment, the polymer multi-arm unit includes 8 arms, where x is in the range of 1 to 300. In another embodiment, the polymer multi-arm unit includes four arms, where x is in the range of 1 to 500.

[0088] Each polymer unit or arm may have an average molecular weight (Mn) in the range of approximately 1,000 to approximately 100,000 daltons, or approximately 10,000 to approximately 60,000 daltons, or approximately 15,000 to approximately 50,000 daltons. Furthermore, the polymer arms of the polymer multi-arm precursor may contain polyethylene glycol (PEG) having an average molecular weight (Mn) in the range of approximately 1,000 to approximately 100,000 daltons, or approximately 10,000 to approximately 60,000 daltons, or approximately 15,000 to approximately 50,000 daltons.

[0089] Considering the average molecular weight (Mn) of each arm, the polymer multi-arm precursor may have an average molecular weight of 5,000 to 100,000 daltons, such as 5,000, 10,000, 15,000, 20,000, or 40,000 daltons, in certain embodiments.

[0090] In certain embodiments, the average molecular weight (Mn) of the polymer arms of a first multi-arm unit may be the same as or different from that of the polymer arms of a second multi-arm unit. For example, the average molecular weight of the polymer arms of the first multi-arm unit may be higher or lower than that of the polymer arms of the second multi-arm unit.

[0091] In a particular embodiment of the present invention, the polymer multi-arm unit includes a polyethylene glycol unit with 3 to 10 arms, such as a polyethylene glycol unit with 4 to 8 arms or a polyethylene glycol unit with 4 arms.

[0092] In a particular embodiment, the polymer network comprises at least first and second multi-arm units, one of which is a small molecule multi-arm unit having a molecular weight of less than 1000 daltons, and the other multi-arm unit is a polymer multi-arm unit having an average molecular weight (Mn) of more than 5000 daltons.

[0093] In some embodiments, the hydrogel is composed of precursors such as multi-armed PEG precursors, each having an average molecular weight (Mn) of less than 50,000 daltons, such as less than 45,000 daltons, less than 40,000 daltons, less than 35,000 daltons, or less than 30,000 daltons. When the hydrogel is degraded in vivo, the degradation fragments obtained after cleaving the hydrolyzable (ester) bonds are of a size that allows for clearance, such as renal excretion from the body.

[0094] The core unit X is located at the center of the multi-arm unit from which the arms emanate. The core unit X has at least 3 bonding c groups, each to which an arm is connected, i.e., covalently. In certain embodiments, the core unit X has 3 to 10, or 4 to 8, or 4 to 6, or 4 bonding c groups. The core unit X may originate from a molecule or chemical structure having several c functional groups to which the arms are connected. For example, in certain embodiments, the core unit X originates from a polyol having at least 3 hydroxyl groups, or 4, 5, 6, 7, 8, 9, or 10 hydroxyl groups. In such embodiments, the polyol can be selected from glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, hexaglycerol, isomalt, lactitol, maltitol, mannitol, or sorbitol.

[0095] In other embodiments, the core unit X is derived from polyamines such as methylenediamine, ethylenediamine, 1,3-diaminopropane, putrescine, cadaverine, hexamethylenediamine, or 1,2-diaminopropane; triamines such as diethylenetriamine, propane-1,2,3-triamine, propane-1,1,3-triamine, pentane-1,3,5-triamine; or other polyamines such as triethylenetramine, butane-1,2,3,4-tetramine, or butane-1,1,4,4-tetramine.

[0096] In certain embodiments, a core unit derived from a polyol or polyamine is ethoxylated at each of its hydroxyl groups to form a multi-arm unit, the arms being polymer PEG arms end-capped by crosslinks formed by click chemical reactions. An exemplary core unit structure derived from a polyol having a bonding affinity of 3 can be described by the following formula 1, where the bonding affinity c is denoted as OH: [ka]

[0097] Therefore, the core unit of a multi-arm unit in a particular embodiment of the present invention is a suitable structure for providing a desired number of arms of a precursor. For example, in the case of a four-arm unit, the core unit can be a pentaerythritol or ethylenediamine structure, while in the case of an eight-arm unit, the core unit can be a hexaglycerol or butane-tetramine structure. In a particular embodiment of the present invention, the core unit is pegylated with its bonding c in polyethylene glycol arms, such as the structure described below. The terminal bonding is indicated as an OH group, which can connect linkers or crosslinks formed by a click reaction. [ka] In the formula, n defines the number of ethoxy groups in the PEG unit, and n can be between 1 and 2300. In other embodiments, n can be between 1 and 2000, for example, 1 to 1800, 1 to 1600, 1 to 1400, 1 to 1200, 1 to 1000, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to 400, 1 to 300, 1 to 200, 1 to 100, or 50 to 100. In yet another embodiment, n can be between 1 and 500, 50 to 450, 100 to 400, or 100 to 300.

[0098] In a particular embodiment, the polymer multi-arm unit is given by formula (ii): [ka] It can be represented as follows: in the formula, X is the core unit, Y is a polymer unit such as PEG, L is a linker unit containing hydrolyzable ester bonds, A is a crosslink formed by a click chemical reaction, p is either 0 or 1, and q is an integer between 2 and 10, or between 3 and 10.

[0099] Linker unit By incorporating linker groups that are unstable to hydrolysis (hydrolyzable bonds) into the polymer network, biodegradation of the hydrogel becomes possible under physiological conditions. By cleaving the hydrolyzable bonds, the hydrogel can be broken down into smaller constituent units with lower molecular weights, which can then be removed from the body through normal physiological pathways.

[0100] A polymer network of a hydrogel according to a particular embodiment of the present invention comprises chemical bonds that can be cleaved by hydrolysis, at least one of which is an ester bond. In a particular embodiment, this hydrolyzable ester bond is provided by linker units L located between the ends of the arms of a multi-arm unit and a crosslink formed by a click chemical reaction. In a particular embodiment, a first multi-arm unit and a second multi-arm unit each contain linker units L containing a hydrolyzable ester bond, and the linker units L may be the same or different in the first and second multi-arm units. In another embodiment, only the first or second multi-arm unit contains linker units L containing a hydrolyzable ester bond.

[0101] The linker unit L is derived from or may consist of dibasic acids, or acid and amide groups such as succinic acid (S), glutaric acid (G), adipine (AP), azelaic acid (AZ), or acid amides, or glutaramide (GA).

[0102] In a particular embodiment, the linker unit L is given by equation (i): [ka] It includes a structure represented by the formula, where U 1 and U 2 These are independently NH or O, and can be the same or different, U 1 and U 2At least one of them is O, and t is an integer from 0 to 10. In other embodiments, t is an integer from 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, or 0 to 1. In other embodiments, t is 0, t is 1, t is 2, t is 3, t is 4, t is 5, t is 6, t is 7, t is 8, t is 9, or t is 10.

[0103] The biodegradability of the hydrogel can be adjusted to have the desired stability intended for a particular use by selecting an appropriate linker unit L and / or the amount and / or position of the linker unit L. For example, the amount of linker units in the hydrogel can affect the stability of the corresponding hydrogel in an aqueous environment over time.

[0104] In certain embodiments, only the first or second multi-arm precursor contains a linker unit L having a hydrolyzable ester bond. In another embodiment, the first and second multi-arm precursors include a linker unit L having a hydrolyzable ester bond.

[0105] Furthermore, the rate of biodegradation / hydrolysis of the ester bonds contained in the linker unit L increases from a longer chain length (a larger t such as 6 in the azelaic acid linker unit) to a shorter chain length (a smaller t such as 1 in the succinic acid linker unit). The shorter the length of the linker chain, the faster the ester bond is hydrolyzed.

[0106] In certain embodiments, the chain length (t1) of the linker unit of the first multi-arm precursor is shorter (t1 < t2), longer (t1 > t2), or the same (t1 = t2) than the chain length (t2) of the linker unit of the second multi-arm precursor.

[0107] Hydrogel formation Several methods for producing hydrogels are known to those skilled in the art, and these methods are mainly applicable and can be suitably adapted in embodiments of the present invention.

[0108] In embodiments of the present invention, a) A step of preparing a first mixture containing a first multi-arm precursor having a second functional group and a first functional group suitable for forming a bond by click chemistry, b) Providing a second mixture containing a second multi-arm precursor having a second functional group suitable for forming a link with a first functional group by a click chemical reaction, c) The step of combining the first and second mixtures, d) Adding at least one activator to the first, second, or a combined mixture of the first and second agents, e) A method is provided for synthesizing a drug delivery system, comprising the step of enabling the combined reaction mixture to gel by forming a click chemical linkage between a first functional group and a second functional group, thereby forming a hydrogel containing at least one activator.

[0109] In this embodiment, at least one of the first or second multi-arm precursor is a polymer multi-arm precursor having polymer arms containing polymer units such as PEG. The first and second multi-arm precursors used as starting materials in the method of the present invention are known in the art or can be obtained as described herein.

[0110] Furthermore, at least one of the first or second multi-arm precursors contains linker units L, which include hydrolyzable ester bonds. By including the linker units, the resulting hydrogel becomes biodegradable, as described above in the chapter on "Linker Units".

[0111] Since the above-mentioned multi-arm units originate from their respective multi-arm precursors, the parts of the multi-arm precursor that do not participate in the crosslinking reaction, namely the linker, polymer arm, and core unit, remain unchanged and are therefore defined as described above for the multi-arm units.

[0112] In some embodiments, the activator is added to both the first and second mixtures. In other embodiments, the activator is added to a combined mixture of the first and second mixtures. Each of the first or second mixtures may contain a solvent, such as water or an aqueous solvent such as the aqueous buffer described herein. Each of the first or second mixtures may contain additional components such as solvents, pharmaceutically active ingredients, buffers, additional therapeutic or diagnostic agents, thickeners, viscosity enhancers, stabilizers, and auxiliaries. The second mixture may further contain one or more activators.

[0113] In certain embodiments, the first and / or second mixture comprises at least one solvent. The at least one solvent can be selected from acetone, acetonitrile, benzyl alcohol, chloroform, dichloromethane (DCM), dioxane, dimethyl carbonate (DMC), dimethyl sulfoxide (DMSO), methanol, ethanol or propanol, ethyl acetate, ethyl formate, ethyl propionate, glycoflore, hexafluoroisopropanol, isorpide dimethyl ether, isopropanol, methyl chloride, methylene chloride, methyl ethyl ketone, N-methylpyrrolidone, propylene carbonate, triethylamine, or tetrahydrofuran; a buffered aqueous medium such as water, PBS buffer or saline; or any mixture thereof. In certain embodiments, such as for the in situ preparation of a drug delivery system, the at least one solvent can be selected from a buffered aqueous medium such as water, PBS buffer or saline, or any mixture thereof mixed with a biocompatible organic solvent, at an optional choice.

[0114] The method of a particular embodiment may further include the step of removing the solvent or solvent mixture. The removal of the solvent can be carried out by any suitable drying method, which may be carried out by hot air convection, drying in an inert gas atmosphere or in a flow of an inert gas such as nitrogen or argon, or by direct drying, indirect or contact drying, spray drying, dielectric drying, vacuum drying, freeze drying, supercritical or superheated vapor drying, or any combination thereof.

[0115] In one embodiment, when the first and second mixtures are combined into a combined reaction mixture, the precursors react in a click chemical reaction to form a covalently crosslinked hydrogel polymer network. The reaction may be initiated or accelerated by heating, or it may occur under ambient conditions, even in vivo.

[0116] The formed hydrogel may be molded, and molding may involve molding, extruding, or casting the reaction mixture before complete gelation of the hydrogel, then allowing the mixture to gel, and optionally drying and completely or at least partially removing the solvent to obtain a zero gel. Molding may be done by filling the reaction mixture into a mold or tube before complete gelation of the hydrogel, allowing the mixture to gel, and optionally drying. In some embodiments, the reaction mixture may be filled into a small-diameter tube or needle to prepare hydrogel chains that can be used directly or dried before use. The reaction mixture may also be applied as a coating on a substrate. As part of the process, the crosslinked hydrogel may be deformed and cured to allow injection through the needle lumen, and the rigidity and reshaping are reversible upon contact with the warmth and / or moisture of the tissue. Rigidity may be provided through crystallization, a secondary crosslinking mechanism, or through water-soluble primary structural components, such as PEG fibers.

[0117] The combined reaction mixture, including the precursors mixed therein, can be prepared with a viscosity suitable for introduction through a small gauge needle using manual force. The small gauge needle has a diameter smaller than that of a 27 gauge needle, for example, 28, 29, 30, 31, 32, or 33 gauge needle, and the gauge is specific to the inner and / or outer diameter. Furthermore, material can be delivered to the implantation site to form a drug delivery device in situ, including those with inner and / or outer diameters equivalent to or smaller than that of the small gauge needle, using hollow tubular wires, such as those used in endovascular techniques. Thus, viscosities of about 1 to about 100,000 mPa·s can be used, and those skilled in the art will immediately understand that all ranges and values ​​within the explicitly stated ranges are intended, for example, about 10 to about 10,000 mPa·s, less than about 5 to about 10,000 mPa·s, less than about 100 or about 500 mPa·s, or about 1 to about 100 mPa·s. Viscosity can be controlled, for example, by selecting a suitable precursor, adjusting the solid and / or solvent concentrations, and by the reaction dynamics. Generally, lower concentrations of precursors and / or lower molecular weights are preferred for lower viscosity.

[0118] Viscosity enhancers may be used in conjunction with precursors. In certain embodiments, the viscosity enhancer does not react with the precursor to form a covalent bond. It is understood that precursors that generally do not contain such bonds may sometimes be involved in undesirable side reactions, but these have little effect on the hydrogel, as the precursor does not "contain" such reactions. For example, while the precursor reacts by a click chemical reaction, the viscosity enhancer does not have to contain functional groups that are reactive in click chemistry, which can form a covalent bond with the functional groups of the precursor, even if some low levels of undesirable side reactions are present. The viscosity enhancer may be a hydrophilic polymer with a molecular weight, e.g., at least 20,000, or about 10,000 to about 500,000 Daltons. Those skilled in the art will immediately understand that all values ​​and ranges between these explicitly stated values, e.g., at least about 100,000 or 200,000, are also stated. For example, concentrations of about 1% to about 40%, or about 5% to about 25% w / w, may be used. PEG (e.g., MW 100,000-250,000) is useful, for example. Viscosity enhancers may include one or more biodegradable bonds as described herein for the precursor. Viscosity enhancers can be useful in preventing the precursor from flowing out of the tissue site before the precursor cross-links to form a gel.

[0119] The method provided is not limited to two multi-arm precursors and may further include the addition in one of steps a) to c) a first or second functional group suitable for forming a click chemical connection with either the first or second multi-arm precursor.

[0120] In one embodiment, the method of the present invention comprises a portion selected from the group consisting of alkynes such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonane (BCN), strains or terminal alkenes such as norbornene or transcyclooctene (TCO), azides, or tetrazines (Tz).

[0121] In a preferred embodiment of the method of the present invention, the first and second functional groups suitable for forming a linkage by click chemistry include portions selected from dibenzocyclooctin and azide.

[0122] In one embodiment of the method of the present invention, the first multi-arm precursor including an azide (N3) portion is [ka] Selected from the group consisting of, where L is a linker, n is between 1 and 2300, t is an integer between 0 and 10, and / or a corresponding structure having 6-arm or 8-arm PEG cores.

[0123] In one embodiment of the method of the present invention, the first multi-arm precursor containing an azide (N3) moiety is selected from the group consisting of 8a5kPEG-N3, 8a10kPEG-N3, 8a15kPEG-N3, 8a20kPEG-N3, 4a20kPEG-N3, 4a20kPEG-PEG2N3S, 4a20kPEG-PEG2N3G, 4a20kPEG-PEG2N3AP, and 4aPEG-PEG2N3AZ.

[0124] In one embodiment of the method of the present invention, a second multi-arm precursor containing a dibenzocyclooctin (DBCO) moiety is [ka] Selected from the group consisting of, where L is a linker, n is between 1 and 2300, t is an integer between 0 and 10, and / or a corresponding structure having 6-arm or 8-arm PEG cores.

[0125] In one embodiment of the method of the present invention, the second multi-arm precursor containing a dibenzocyclooctin (DBCO) moiety is 4a20kPEG-D, 6a20kPEG-D, 8a20kPEG-D, 4a20kPEG-DS, 8a10kPEG-DG, 8a15kPEG-DG, 4a20kPEG-DG, 4a20kPEG-DAP, 4a20kPEG-DAZ, 4a20kPEG-DPEG 23 The group is selected from AZ and 4a20kPEG-sDG.

[0126] In one embodiment of the method of the present invention, a first multi-arm precursor comprising an azide (N3) moiety and a second multi-arm precursor comprising a dibenzocyclooctin (DBCO) moiety are [ka] [ka] Selected from the group consisting of, where L is a linker, n is between 1 and 2300, t is an integer between 0 and 10, and / or a corresponding structure having 6-arm or 8-arm PEG cores.

[0127] In one embodiment of the method of the present invention, the first multi-arm precursor containing an azide (N3) moiety is selected from the group consisting of 8a5kPEG-N3, 8a10kPEG-N3, 8a15kPEG-N3, 8a20kPEG-N3, 4a20kPEG-N3, 4a20kPEG-PEG2N3S, 4a20kPEG-PEG2N3G, 4a20kPEG-PEG2N3AP, and 4aPEG-PEG2N3AZ, and the second multi-arm precursor containing a DBCO moiety is selected from the group consisting of 4a20kPEG-D, 6a20kPEG-D, 8a20kPEG-D, 4a20kPEG-DS, 8a10kPEG-DG, 8a15kPEG-DG, 4a20kPEG-DG, 4a20kPEG-DAP, 4a20kPEG-DAZ, and 4a20kPEG-DPEG 23 The group is selected from AZ, 4a20kPEG-sDG, and 4a20kPEG-PEG2BG.

[0128] In one embodiment of the method of the present invention, a first multi-arm precursor comprising an azide (N3) moiety and a second multi-arm precursor comprising a dibenzocyclooctin (DBCO) moiety are [ka] [ka] Selected from the group consisting of the following, where x and y define the number of PPG, PEG, PLA, or PGA units, and t is an integer from 0 to 10, and / or the corresponding structure having 6-arm or 8-arm PEG cores.

[0129] In one embodiment of the method of the present invention, the first multi-arm precursor comprises a tetrazine (TET) moiety selected from the group consisting of 8a5kPEG-TET, 8a10kPEG-TET, 8a15kPEG-TET, 8a20kPEG-TET, 8a20kPEG-PEG5TET, 4a20kPEG-TET, or ester-linked 4a20kPEG-PEG5TETS, 4a20kPEG-PEG5TETG, 4a20kPEG-PEG5TETAP, and 4aPEG-PEG5TETAZ, and the second multi-arm precursor comprises 4a20kPEG-D, 6a20kPEG-D, 8a20kPEG-D, 4a20kPEG-DS, 8a10kPEG-DG, 8a15kPEG-DG, 4a20kPEG-DG, 4a20kPEG-DAP, 4a20kPEG-DAZ, and 4a20kPEG-DPEG 23 It includes a DBCO portion selected from the group consisting of AZ, 4a20kPEG-sDG, and 4a20kPEG-PEG2BG.

[0130] In one embodiment of the method of the present invention, the first multi-arm precursor comprises a tetrazine (TET) moiety selected from the group consisting of 8a5kPEG-TET, 8a10kPEG-TET, 8a15kPEG-TET, 8a20kPEG-TET, 8a20kPEG-PEG5TET, 4a20kPEG-TET, or ester-linked 4a20kPEG-PEG5TETS, 4a20kPEG-PEG5TETG, 4a20kPEG-PEG5TETAP, and 4aPEG-PEG5TETAZ, and the second multi-arm precursor comprises a norbornene (Nor) moiety selected from the group consisting of 4a20kPEG-Nor, 6a20kPEG-Nor, 8a20kPEG-Nor, 4a20kPEG-NorS, 8a10kPEG-NorG, 8a15kPEG-NorG, 4a20kPEG-NorG, 4a20kPEG-NorAP, 4a20kPEG-NorAZ, 4a20kPEG-NorPEG 23 AZ, and 4a20kPEG-PEG2NorG.

[0131] In one embodiment of the method of the present invention, the first multi-arm precursor comprising an azide (N3) moiety is selected from the group consisting of 8a5kPEG-N3, 8a10kPEG-N3, 8a15kPEG-N3, 8a20kPEG-N3, 4a20kPEG-N3, 4a20kPEG-PEG2N3S, 4a20kPEG-PEG2N3G, 4a20kPEG-PEG2N3AP, and 4aPEG-PEG2N3AZ, and the second multi-arm precursor comprising a bicyclononine (BCN) moiety is selected from the group consisting of 4a20kPEG-D, 6a20kPEG-BCN, 8a20kPEG-BCN, 4a20kPEG-BCNS, 8a10kPEG-BCNG, 8a15kPEG-BCNG, 4a20kPEG-BCNG, 4a20kPEG-BCNAP, 4a20kPEG-BCNAZ, 4a20kPEG-BCNPEG 23 AZ, and 4a20kPEG-PEG2BCNG.

[0132] In one embodiment of the method of the present invention, the first multi-arm precursor containing an azide (N3) moiety is selected from the group consisting of 4a18Tetronic1307-PEG2N3, 4a20kPLA-PEG2N3, 8a20k50:50PLGA-PEG2N3, and 4a20kPLA-PEG2N3S.

[0133] In one embodiment of the method of the present invention, the second multi-arm precursor containing a dibenzocyclooctin (DBCO) moiety is selected from the group consisting of 4a20kPEG-DAZ, 8a20kPEG-D, and 4a18Tetronic1307-DAZ.

[0134] In one embodiment of the method of the present invention, a first multi-arm precursor containing an azide (N3) moiety is selected from the group consisting of 4a18Tetronic1307-PEG2N3, 4a20kPLA-PEG2N3, 8a20k50:50PLGA-PEG2N3, and 4a20kPLA-PEG2N3S, and a second multi-arm precursor containing a dibenzocyclooctin (DBCO) moiety is selected from the group consisting of 4a20kPEG-DAZ, 8a20kPEG-D, and 4a18Tetronic1307-DAZ.

[0135] Further first and second multi-arm precursors suitable as starting materials in the method of the present invention are described herein, for example, in the chapters “Polymer Units and Precursors” and “Functional Groups Suitable for Click Chemical Reactions.”

[0136] The amounts of different functionalized PEG multi-arm precursors used for hydrogel formation can be calculated, for example, by converting moles to grams and using stoichiometric equations for molar ratios, as described in principle in WO2024 / 035690A1, paragraphs 132-134, which is incorporated herein by reference.

[0137] The molecular weight between crosslinks, MWc, and parameters defining the mesh size within the hydrogel, or crosslink density, can be calculated by summing the average arm lengths of each multi-arm PEG precursor (PEG, MW = molecular weight).

number

[0138] For example, hydrogels from equimolar amounts of 8a15kDG and 8a20kN3 (see Example 7) have a MWc of 15kDa / 8 + 20kDa / 8 = 1,875Da + 2,500Da = 4,375Da. Similar calculations can be performed for other types of polymers described herein.

[0139] In a particular embodiment of the present invention, the sustained-release drug delivery system has a MWc of at least 2,500 Da, such as at least 3,000 Da, 4,000 Da, 5,000 Da, 6,000 Da, 7,000 Da, 8,000 Da, 9,000 Da, 10,000 Da, 12,000 Da, or at least 15,000 Da, or less than 25,000 Da, such as up to 20,000 Da or up to 18,000 Da.

[0140] Functional groups suitable for click chemical reactions The three-dimensional polymer network of the hydrogel is formed by reacting at least one first multi-arm precursor having functional groups suitable for click chemistry with at least one second multi-arm precursor having functional groups suitable for forming click chemical bonds with the functional groups of the first multi-arm precursor.

[0141] Suitable functional groups for click chemistry are those that enable click chemical reactions such as strain-enhanced alkyne-azide ring addition (SPAAC), also known as Cu-free click reactions or reverse electron-required Diels-Alder ligation (IEDDA) type click chemical coupling reactions. An overview of this type of reaction is provided by reference in H. Kolb; M.G. Finn; K.B. Sharpless (2001). “Click Chemistry: Diverse Chemical Function from a Few Good Reactions”, Angewandte Chemie International Edition, 40 (11): 2004-2021).

[0142] Other suitable click chemical reactions include aldehyde / ketone condensation, cyanobenzothiazole condensation, strain-accelerated oxidation-controlled cyclooctin-1,2-quinone ring addition (SPOCQ), 1,3-dipolar ring addition, [3+2] ring addition such as alkene-nitrone ring addition or alkyne-nitrone ring addition, [4+2] ring addition, and hetero-Diels-Alder reactions.

[0143] SPAACs require cyclic alkynes such as dibenzocyclooctyne (DBCO) and bicyclo[6.1.0]nonine (BCN) to react with aliphatic azides. This strain reaction proceeds efficiently without the need for the copper catalyst required for copper(I) catalyzed azide-alkyne click reactions (CuAAC). Similarly, IEDDAs do not require a catalyst, but require the reaction of norbornene or DBCO and tetrazine. Therefore, the advantage of SPAACs and IEDDAs over electrophilic-nucleophilic reactions such as CuAACs and NHS-NH2s is that no catalyst is needed after the reaction is complete and there are no byproducts.

[0144] SPAAC and IEDDA coupling reactions are bioorthogonal reactions with selective and quantitative yields under mild conditions and can occur within biological systems without interfering with natural biochemical processes. These click reactions utilize a pair of functional groups, such as cyclooctyne and azide, that react mutually and efficiently while remaining inert to naturally occurring functional groups. Scheme 1: [ka] In the formula, R1 and R2 are polymer arms that optionally include linker L.

[0145] This reaction is suitable for forming covalent crosslinks in a hydrogel polymer network from a correspondingly functionalized multi-arm precursor, as described herein. Among the many known cyclooctins, dibenzocyclooctin (DBCO) compounds belong to a class of reagents that exhibit moderately fast kinetics in the SPAAC reaction and good stability in aqueous buffers. Within the physiological temperature and pH range, the DBCO group does not react with amines or hydroxyls naturally present in many biomolecules. In addition, the reaction of the DBCO group with an azide group is remarkably fast.

[0146] The advantage of DBCO-based SPAACs is their biocompatibility, for example, because they do not require cytotoxic copper catalysts that may leave undesirable traces in the hydrogel. Another advantage is the use of mild reaction conditions: coupling the arms of the multi-arm precursor is possible under physiological conditions in buffered aqueous media or common organic solvents. Furthermore, the DBCO and azide moieties are long-term stable and possess high selectivity and specificity because the azide group reacts only with DBCO in the presence of amines, hydroxyl, thiol, and acid groups, as well as other protein functional groups. The reaction also produces a stable triazole in quantitative yield at a high reaction rate with no byproducts. Similar advantages are provided by IEDDA coupling reactions and click chemical reactions that do not involve other types of catalysts as described herein.

[0147] Since click reactions utilize a pair of functional groups that react mutually and efficiently, a single multi-arm precursor can have only a portion of the functional groups in that pair (e.g., simply an azide or simply a DBCO). Otherwise, an intramolecular click reaction would occur, and the multi-arm precursor would not be suitable for constructing the hydrogel of the present invention, which includes a polymer network.

[0148] Therefore, a first multi-arm precursor having a first functional group suitable for click chemistry must react with a second multi-arm precursor having a second functional group suitable for forming a click chemical bond with the first functional group of the first multi-arm precursor.

[0149] The polymer multi-arm precursor is given by formula (iii): [ka] It can be represented as follows, where X is the core unit, Y is a polymer unit such as PEG, L is a linker unit containing a hydrolyzable ester bond, B is a functional group suitable for forming a bond by click chemistry, p is either 0 or 1, and q is an integer from 3 to 10.

[0150] In certain embodiments, the preferred functional group (B) includes a portion selected from the group consisting of alkynes, cycloalkynes such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonine (BCN), strained alkynes or terminal alkynes such as norbornene or transcyclooctene (TCO), azides, or tetrazines (Tz).

[0151] For example, in exemplary embodiments, a first multi-arm precursor having a functional group containing an azide moiety can react with a second multi-arm precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety to form a polymer network of the hydrogel of exemplary embodiments of the present invention. In one embodiment, the polymer network of the hydrogel is formed by simply reacting a first multi-arm precursor having a functional group containing an azide (N3) moiety with a second precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety, i.e., no additional reaction with other multi-arm precursors containing functional groups other than the azide or DBCO moiety is performed to form the polymer network of the hydrogel. In one embodiment, the first multi-arm precursor having a functional group containing an azide moiety does not originate from pentaerythritol tetrakis(2-bromoisobutyrate).

[0152] In one embodiment, the polymer network of the hydrogel is formed by reacting a first multi-arm precursor having a functional group containing an azide (N3) moiety with a second multi-arm precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety, and the activator is a small molecule activator, peptide, protein, or virus.

[0153] In another embodiment, the polymer network of the hydrogel is formed by reacting a first multi-arm precursor having a functional group containing an azide (N3) moiety with a second multi-arm precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety, wherein the activator is contained in the hydrogel without being covalently conjugated to the hydrogel, preferably the activator is contained in the hydrogel without being covalently conjugated to the hydrogel via an oligonucleotide, inserted after claim 26; WO2021113115, WO2013036847, ELSchneider et al., GWAshley et al, Limitations on Polymer-Drug Conjugates

[0154] In certain preferred embodiments, the polymer network of the hydrogel is formed by reacting a first multi-arm precursor having a functional group containing an azide (N3) moiety with a second multi-arm precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety, preferably the first multi-arm precursor having a functional group containing an azide moiety is not derived from pentaerythritol tetrakis(2-bromoisobutyrate), the activator is a small molecule activator, peptide, protein, or virus, and the activator is contained in the hydrogel without being covalently conjugated to the hydrogel, preferably the activator is contained in the hydrogel without being covalently conjugated to the hydrogel via an oligonucleotide.

[0155] Exemplary polymer four-arm precursors having functional groups suitable for click chemistry and lacking or having linker units L are as follows. The abbreviation for multi-arm precursors in this disclosure is generally the following formula qM w In accordance with Y-BL, where q is the number of arms, M w is the average molecular weight of the precursor, Y is the polymer unit, B is the functional group, and L is the linker.

[0156] The following functional groups B are abbreviated as dibenzocyclooctin (D), dibenzocyclooctinsulfamine (sD), azide (N3), bicyclo[6.1.0]nonine (B), norbornene (N), transcyclooctene (TCO), tetrazine (T), amine (NH2), and N-hydroxysuccinimidyl (NHS). In general structures where the molecular weight is not defined, Y is Y 個の繰り返し単位 When described as such, and the linker unit L as defined above is shown without a specified t, the abbreviation L t This is used. The magnificent polymer four-arm precursor is Without linker: [ka] Linker L present: [ka] In the formula, n and t are as defined herein above.

[0157] In one embodiment, a first multi-arm precursor having a functional group containing an azide (N3) moiety is [ka] A selection is made from the group consisting of the following, where n and t are as defined herein above.

[0158] In one embodiment, a second multi-arm precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety is [ka] A selection is made from the group consisting of the following, where n and t are as defined herein above.

[0159] In one embodiment, the polymer network of the bioorthogonal hydrogel is formed by reacting at least one, preferably one, of the following first multi-arm precursors having a functional group containing an azide (N3) moiety with at least one, preferably one, of the following second multi-arm precursors having a functional group containing a dibenzocyclooctin (DBCO) moiety: [ka] [ka] In the formula, n is an integer between 1 and 2300, and t is an integer between 0 and 10.

[0160] In one embodiment, the first multi-arm precursor having a functional group containing an azide (N3) moiety is 8a10kPEG-N 3、The compounds are selected from the group consisting of 4a20kPEG-N3, 4a20kPEG-PEG2N3S, 4a20kPEG-PEG2N3G, 4a20kPEG-PEG2N3AP, and 4aPEG-PEG2N3AZ. All compounds are commercially available.

[0161] In one embodiment, the second multi-arm precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety is 4a20kPEG-D, 6a20kPEG-D, 8a20kPEG-D, 4a20kPEG-DS, 8a10kPEG-DG, 4a20kPEG-DG, 4a20kPEG-DAP, 4a20kPEG-DAZ, 4a20kPEG-DPEG 23 The compounds are selected from the group consisting of AZ and 4a20kPEG-sDG. All compounds are commercially available.

[0162] In one embodiment, the polymer network of the bio-orthogonal hydrogel is 8a10kPEG-N 3、 At least one, preferably one, first multi-arm precursor selected from the group consisting of 4a20kPEG-N3, 4a20kPEG-PEG2N3S, 4a20kPEG-PEG2N3G, 4a20kPEG-PEG2N3AP, and 4aPEG-PEG2N3AZ is used in the following process: 4a20kPEG-D, 6a20kPEG-D, 8a20kPEG-D, 4a20kPEG-DS, 8a10kPEG-DG, 4a20kPEG-DG, 4a20kPEG-DAP, 4a20kPEG-DAZ, 4a20kPEG-DPEG 23 It is formed by reacting it with at least one, preferably one, second multi-arm precursor selected from the group consisting of AZ, 4a20kPEG-sDG, and 4a20kPEG-PEG2BG.

[0163] In one embodiment, the polymer network of the bio-orthogonal hydrogel consists of a first multi-arm precursor 4a20kPEG-N3, 4a20kPEG-DG, 4a20kPEG-sDG, 4a20kPEG-PEG2BG, 4a20kPEG-DAZ, and 4a20kPEG-PEG 23It is formed by reacting it with at least one, preferably one, second multi-arm precursor selected from the group consisting of DAZs.

[0164] In one embodiment, the polymer network of the bioorthogonal hydrogel is formed by reacting at least one, preferably one, of the following first multi-arm precursors having a functional group containing an azide (N3) moiety with at least one, preferably one, of the following second multi-arm precursors having a functional group containing a dibenzocyclooctin (DBCO) moiety: [ka] [ka] In the formula, x and y define the number of PPG, PEG, PLA, or PGA units, and t is as defined herein.

[0165] In one embodiment, the first multi-arm precursor having a functional group containing an azide (N3) moiety is selected from the group consisting of 4a18Tetronic1307-PEG2N3, 4a20kPLA-PEG2N3, 8a20k50:50PLGA-PEG2N3, and 4a20kPLA-PEG2N3S. All compounds are commercially available.

[0166] In one embodiment, the second multi-arm precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety is selected from the group consisting of 4a20kPEG-DAZ, 8a20kPEG-D, and 4a18kTetronic1307-DAZ. All compounds are commercially available.

[0167] The polymer network of the bio-orthogonal hydrogel is formed by reacting at least one, preferably one, first multi-arm precursor having a functional group containing an azide (N3) moiety selected from the group consisting of 4a18Tetronic1307-PEG2N3, 4a20kPLA-PEG2N3, 8a20k50:50PLGA-PEG2N3, and 4a20kPLA-PEG2N3S with at least one, preferably one, second multi-arm precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety selected from the group consisting of 4a20kPEG-DAZ, 8a20kPEG-D, and 4a18Tetronic1307-DAZ.

[0168] In one embodiment, the polymer network of the present invention is composed of the following compounds: [ka] It is not formed by the reaction of , where n is 1 to 400 and t is 2.

[0169] As described above, in certain embodiments, at least one of the two multi-arm precursors is a polymer multi-arm precursor. Therefore, the other multi-arm precursor can be either another polymer multi-arm precursor as described above, or a small molecule multi-arm precursor having a molecular weight of less than 1000 Daltons. The small molecule multi-arm precursor can be defined in the same manner as the polymer multi-arm precursor, except that the arms do not contain polymer units.

[0170] Following a click reaction between the first and second functional groups, a covalent bond is formed. In certain embodiments, this covalent bond may comprise a chemical group selected from triazoles and dihydropyridazines. The triazole moiety is obtained when a functional group containing an azide moiety reacts with a functional group containing an alkyne moiety. The dihydropyridazine moiety is obtained when a functional group containing a tetrazine moiety reacts with a functional group containing a terminal or strained alkene moiety.

[0171] Multi-arm precursors having nucleophilic or electrophilic functional groups at the ends of their arms are known in the art. Click chemical linkers can be used to convert these nucleophilic or electrophilic functional groups into functional groups suitable for forming click chemical links. These click chemical linkers contain, for example, functional groups suitable for forming click chemical links, such as azides or cyclooctine, and nucleophilic or electrophilic functional groups, such as amines (nucleophilic) or succinimidyl esters (electrophilic). In the conversion, the nucleophilic or electrophilic functional groups at the ends of the arms of the multi-arm precursor react with the corresponding electrophilic or nucleophilic functional groups of the click chemical linker.

[0172] For example, this conversion can be carried out by reacting the NHS group with a DBCO-amine click chemical linker such as DBCO-NH2 having the following structure, thereby converting a polymer arm having an NHSS (succinimidyl succinate), NHSG (succinimidyl glutarate), NHSAP (succinimidyl adipate), or NHSAZ (succinimidyl azelaic acid) terminal group to a DS (dibenzocyclooctinamide succinate), DG (dibenzocyclooctinamide glutarate), DAP (dibenzocyclooctinamide adipic acid), or DAZ (dibenzocyclooctinamide azilaic acid) group. Scheme 2: [ka]

[0173] Other click chemical linkers for converting NHS-terminated polymer precursors into functionalized precursors suitable for click chemistry include, for example, more hydrophilic sulfo-DBCO-amines (sD-NH2) having the following structure: Scheme 3: [ka] or DBCO-PEG 23-PEG-extended DBCO-amines such as amines, or bicyclo[6.1.0]nonine (BCN)amines such as BCN-PEG2-NH2 having the following structure: Scheme 4: [ka] and similar commercially available linkers having ethoxylations of various lengths.

[0174] Similarly, the conversion of PEG-NHS-terminated PEG arms to azide-terminated PEG arms can be carried out by reacting the NHS group with an azidoamine click chemical linker such as azido-PEG2-NH2. Such azidoamine click chemical linkers are commercially available from several vendors and have the following structures: Scheme 5: [ka] n defines the number of repeating ethylene glycol units.

[0175] The following is a schematic reaction between an exemplary first multi-arm precursor and an exemplary second multi-arm precursor, thereby forming a hydrogel: Scheme 6: [ka] In the formula, n is as previously defined herein.

[0176] Because hydrogels contain hydrolyzable ester bonds, they are biodegradable in aqueous environments. The following is an exemplary schematic hydrolysis reaction of hydrogel degradation products. Scheme 7: [ka] In the formula, n is as previously defined herein.

[0177] Activating agent The activator according to the present invention may be a therapeutic activator, a diagnostic activator, or a combination thereof. It may be a single activator or a plurality of activators.

[0178] Therapeutically active drugs include steroids; non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, meclofename, mefanamic A, salsalate, sulindac, tolmetin, ketoprofen, diflunisal, piroxicam, naproxen, etodrug, fluviprofen, fenoprofen C, indomethacin, celecoxib, ketrolac, and nepafenac; intraocular pressure lowering agents; antibiotics such as ciprofloxacin; analgesics such as bupivacaine; calcium channel blockers such as nifedipine; cell cycle inhibitors such as simvastatin; proteins such as insulin; and small molecule hydrophilic 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, and fusion antibodies; aptamers; in particular bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, antibiotics, analgesics, calcium channel blockers, cell cycle inhibitors, chemotherapeutic agents, antiviral agents, anesthetics, hormones, anticancer agents, antitumor agents, viruses, genetically modified viruses such as AAV, protein binders such as nanobodies, aphibodies, ankyrin, and DARPins, or any combination thereof may be selected.

[0179] In some embodiments, the steroid may be selected from corticosteroids that include hydrocortisone, loteprednol, cortisol, cortisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, aldosterone, or fludrocortisone.

[0180] In some embodiments, the NSAID may include diclofenac (e.g., diclofenac sodium), fluviprofen (e.g., fluviprofen sodium), ketrolac (e.g., ketrolactromethamine), bromfenac, or nepafenac.

[0181] In some embodiments, the IOP-reducing agent and / or glaucoma agent may include prostaglandin analogs (e.g., bimatoprost, latanoprost, travoprost, or latanoprostembunod), Rho kinase inhibitors (e.g., netaludil), adrenergic agonists (epinephrine or dipibrin), beta-adrenergic agonists also known as beta-blockers (e.g., timolol, levobunolol, metipranolol, carteolol, or betaxolol), alpha-2-adrenergic agonists (e.g., apraclonidine, brimonidine, or brimonidine tartrate), carbonic anhydrase inhibitors (e.g., brinzolamide, dichlorfenamide, metazolamide acetazolamide, acetazolamide, or dorzolamide), pilocarpine, ecothiophate, demel potassium, physostigmine, and / or isofluorophate.

[0182] In some embodiments, the antiinfective agent may include antibiotics such as 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, natamycin, voriconazole, fluconazole, miconazole, clotrimazole, ketoconazole, posaconazole, echinocandin, caspofungin, and / or micafungin.

[0183] In some embodiments, the antimetabolite may include methotrexate, mycophenolic acid, or azathioprine. In some embodiments, the antifibrotic agent may include mitomycin C or 5-fluorouracil.

[0184] In some embodiments, the angiogenesis inhibitor may include anti-VEGF agents (e.g., aflibercept, ranibizumab, bevacizumab), PDGF-B inhibitors (e.g., Fovista®), complement antagonists (e.g., eculizumab), tyrosine kinase inhibitors (e.g., sunitinib, axitinib), and / or integrin antagonists (e.g., natalizumab and vedolizumab). In certain embodiments, the activator may be selected from peptides selected from the group consisting of compstatin, APL-1, Fc-III-4C, Beob (brolucizumab), Zimura (abacincaptado pegol), pegcetacoplan, abisipal pegol, lamparizumab, Fovista, listiganib, AXT107, elamipretide, THR149, ALM201, VGB3, and largazole.

[0185] In some embodiments, nanobodies can be incorporated into hydrogels. Nanobodies are described, for example, in Yang et al. (2020), Nanobodies: Next Generation of Cancer Diagnostics and Therapeutics, Front. Oncol. 10:1182 (the entire text is incorporated herein by reference). Nanobodies are, 68 GaNOTA-Anti-HER2-VHH1, 68 GaNOTA-Anti-HER2-VHH1, 99m Tc-NM-02, 131 I-SGMIB-Anti-HER2-VHH1, 68 GaNOTA-Anti-MMR-VHH2, 99m The following can be selected: Tc-Anti-PD-L1, L-DOS47 + doxorubicin, L-DOS47 + cisplatin / vinorelbine, KN035 + trastuzumab / docetaxel, KN035, KN044, TC-210 T cells, CD19 / CD20 bispecific CART cells, BCMA CART cells, or TAS266 nanobodies.

[0186] In some embodiments, non-immunoglobulin affinity proteins such as affibody proteins can be included in the hydrogel delivery system. Affibody molecules are described, for example, in Stahl et al., Affibody Molecules in Biotechnological and Medical Applications, Trends in Biotechnology 2017, 35(8) pp. 691-712 (the entire article is incorporated herein by reference).

[0187] In some embodiments, binding proteins such as ankyrin and DARPin can be included in the hydrogel. Ankyrin and DARPin are described, for example, in Caputi et al., Current Opinion in Pharmacology 2020, 51:93-101 (the entire text of which is incorporated herein by reference). Ankyrin and DARPin may be selected from MP0250, a 3-specific DARPin drug candidate capable of binding 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), avisipalpegol (MP0112 or AGN-150998), brolucizumab, ranibizumab, or aflibercept.

[0188] In some embodiments, the cytoprotective agent may include ebselen, sulforaphane, ortiplasm, or dimethyl fumarate. In some embodiments, the neuroprotective agent may include ursodiol, memantine, or acetylcysteine. In some embodiments, the anesthetic may include lidocaine, propalacaine, or bupivacaine.

[0189] In some embodiments, the activator may be dexamethasone, ketorolac, diclofenac, vancomycin, moxifloxacin, gatifloxin, becifloxacin, travoprost, 5-fluorouracil, methotrexate, mitomycin C, prednisolone, bevacizumab (Avastin®), ranibizumab (Lucentis®), sunitinib, pegaptanib (Macugen®), timolol, latanoprost, brimonidine, nepafenac, bromfenac, triamcinolone, difluprednate, fluocinolide, aflibercept, or a combination thereof. In some embodiments, the drug may be dexamethasone, ketorolac, diclofenac, moxifloxacin, travoprost, 5-fluorouracil, or methotrexate.

[0190] In alternative embodiments, active agents that can be used in the implants and methods of the present invention include, but are not limited to, immunosuppressants, complement inhibitors (e.g., C5 inhibitors such as eculizumab or abasin captado pegol), steroids, anti-inflammatory agents such as steroids and non-steroidal anti-inflammatory drugs (e.g., COX1 or COX2 inhibitors), antiviral agents, antibiotics, antiglaucoma agents, anti-VEGF agents, analgesics, tyrosine kinase inhibitors, integrin inhibitors, IL-6 blockers, reactive aldehyde species (RASP) inhibitors, nitric oxide donors (PgA), antihistamines, mast cell stabilizers, rho kinase inhibitors, plasma kallikrein inhibitors, BCL-2 blockers, semaphorin antagonists, HtRA1 blockers, IGF-1R inhibitors, VEGF combination agents (multispecific anti-angiogenic agents), and combinations thereof.

[0191] 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, mycophenolate mofetil, 15-deoxysperguarin, thalidomide, glatiramer, leflunomide, vincristine, cytarabine, pharmaceutically acceptable salts thereof, and combinations thereof.

[0192] Nonsteroidal anti-inflammatory compounds (NSAIDs) include inhibitors of cyclooxygenase (COX) enzymes, such as cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) isozymes. A common class of NSAIDs includes salicylates, propionic acid derivatives, acetic acid derivatives, enolic acid derivatives, and anthranilic acid derivatives. Examples of nonsteroidal anti-inflammatory compounds include acetylsalicylic acid, diflunisal, salsalate, ibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen, dexketoprofen, fluviprofen, oxaprozin, loxoprofen, indomethacin, tolmetine, sulindac, etodolac, ketrolac, diclofenac, aceclofenac, nabumetone, piroxicam, tenoxicam, tenoxicam, loroxicam, phenylbutazone, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, celecoxib, pharmaceutically acceptable salts thereof, and combinations thereof.

[0193] The anti-inflammatory agents that can be used in the implants 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.

[0194] Analgesics that can be used in the implants and methods of the present invention include acetaminophen, acetaminosarol, aminochlortenoxazine, acetylsalicylic acid 2-amino-4-picolinic acid, acetylsalicylsalicylic acid, anilelysine, benoxaprofen, benzylmorphine, 5-bromosalicylic acid, busetin, buprenorphine, butorphanol, capsaicin, syncophene, silamdol, clomethacin, clonixin, codeine, desomorphine, dezosine, dihydrocodeine, dihydromorphine, dimefeptanol, dipylocetyl, eptazosine, etoxazene, ethylmorphine, eugenol, and f This includes loctaphenine, phosphosal, graphenine, hydrocodone, hydromorphone, hydroxypethidine, ibufenac, p-lactophenetide, levorphanol, meptazinol, metazosin, methopone, morphine, nalbuffine, nicomorphine, norlevorphanol, normorphine, oxycodone, oxymorphone, pentazocine, phenazosin, phenocol, phenoperidine, phenylbutazone, phenylsalicylic acid, phenyllamidol, salicin, salicylamide, thiorphan, tramadol, diaselein, actarit, pharmaceutically acceptable salts thereof, and combinations thereof.

[0195] Antibiotics that can be used in the implants and methods of the present invention include aminoglycosides, penicillins, cephalosporins, fluoroquinolones, macrolides, and combinations thereof. Aminoglycosides may include tobramycin, kanamycin A, amikacin, dibekacin, gentamicin, shisomycin, netylmycin, neomycin B, neomycin C, neomycin E, streptomycin, paramomycin, pharmaceutically acceptable salts thereof, and combinations thereof. Penicillins may include amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, pivampicillin, pivmecillinum, ticarcillin, pharmaceutically acceptable salts thereof, and combinations thereof. Cephalosporins include cefatril, cefadroxil, cephalexin, cephaloglysin, cephalonium, cephaloridine, cephalothin, cefapillin, cefatoridine, cefazflur, cefazedone, cefazolin, cefradin, ceffloxazine, ceftezol, cefaclor, cefamandol, cefmetazole, cefonisid, cefotetan, cefoxitin, cefprodil, cefuroxime, cefzonam, cefcapene, cefdaroxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefozidime, cefotaxime, cefpimisole, cefpodoxime, and This may include fteram, ceftibutene, ceftiofur, cefthiolen, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclizine, cefepime, ceffluprenum, cefoselis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftarolin, cefaclomedin, cephaloram, cefparol, cefcanel, cefedrol, cefenpidone, cefetrizole, cefibitril, cefmethylene, cefmepidium, cefobesin, cefoxazole, cefrotyl, cefsmid, cefrastim, ceftioxide, pharmaceutically acceptable salts thereof, and combinations thereof.Fluoroquinolones may include ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, ofloxacin, norfloxacin, their pharmaceutically acceptable salts, and combinations thereof. Macrolides may include azithromycin, erythromycin, clarithromycin, dilithromycin, oxythromycin, telithromycin, their pharmaceutically acceptable salts, and combinations thereof.

[0196] Antiviral agents that can be used in the implants 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 drugs include abacavir, acyclovir, adefovir, amantadine, amprenavir, amprigen, arbidol, atazanavir, boceprevir, cidofovir, darunavir, delavirdin, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfvirtide, entecavir, famciclovir, homivirsen, fosamprenavir, foscarnet, phosphonet, ganciclovir, ivacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, and This includes, but is not limited to, virid, maraviroc, moloxidine, methisazone, nelfinavir, nevirapine, nexavir, oseltamivir, pegylated interferon alfa-2a, penciclovir, peramivir, preconalil, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, pyramiding saquinavir, stabudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, tolvada, valacyclovir, valganciclovir, bicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine, their pharmaceutically acceptable salts and combinations thereof.

[0197] Steroidal anti-inflammatory agents that can be used in the implants and methods of the present invention include dexamethasone, budesonide, triamcinolone, hydrocortisone, fluocinolone, loteprednol, prednisolone, mometasone, fluticasone, rimexolone, fluorometholone, beclomethasone, flunisolide, pharmaceutically acceptable salts thereof, and combinations thereof.

[0198] Antiglaucoma agents that can be used in the implants and methods of the present invention include: β-blockers such as atenolol, propranolol, metipranolol, betaxolol, carteolol, levobetaxolol, levovunolol, timolol, pharmaceutically acceptable salts thereof, and combinations thereof; adrenergic or sympathomimetic agents such as epinephrine, dipibrin, clonidine, aparclonidine, brimonidine, pharmaceutically acceptable salts thereof, and combinations thereof; parasympathomimetic or cholinergic agents such as pilocarpine, carbachol, phospholine iodine, physostigmine, pharmaceutically acceptable salts thereof, and combinations thereof; acetozolamide, brinzolamide, dorzolamide Examples include carbonic anhydrase inhibitors, including topical or systemic agents such as metazolamide, ethoxyzolamide, dichlorphenamide, pharmaceutically acceptable salts thereof, and combinations thereof; mydriatic-cycloplegic agents such as atropine, cyclopentolate, succinylcholine, homatropin, phenylephrine, scopolamine, tropicamide, pharmaceutically acceptable salts thereof, and combinations thereof; prostaglandins such as prostaglandin F2 alpha, anti-prostaglandins, prostaglandin precursors, or prostaglandin analogs such as bimatoprost, latanoprost, travoprost, unoprostone, tafluprost, pharmaceutically acceptable salts thereof, and combinations thereof.

[0199] Anti-VEGF agents that can be used in the implants and methods of the present invention include bevacizumab, pegaptanib, ranibizumab, brolucizumab, convercept, aflibercept, pharmaceutically acceptable salts thereof, and combinations thereof.

[0200] Tyrosine kinase inhibitors that can be used in the implants and methods of the present invention include duclavacitinib, axitinib, avapritinib, capmatinib, pegmatinib, ripretinib, serpercatinib, selumetinib, tucatinib, entrectinib, erdaftinib, fedratinib, pexidartinib, upadacatinib, zanubrutinib, Baricitinib, Binimetinib, Dacomitinib, Fostamatinib, Gilteritinib, Lalotrectinib, Lorlatinib, Acalabrutinib, Brigutinib, Midostaurin, Neratinib, Alectinib, Cobimetinib, Lenvatinib, Osimertinib, Ceritinib, Nintedanib, Afatinib, Ibrutinib, Trametinib, Bosutinib, Caboza Ntinib, ponatinib, regorafenib, tofacitinib, crizotinib, ruxolitinib, vandetanib, pazopanib, lapatinib, nilotinib, dasatinib, sunitinib (bororanib), sorafenib, erlotinib, gefitinib, imatinib, afatinib, bosutinib, cabozantinib, sediranib, ceritinib, crizotinib, da Examples include blafenib, dasatinib, erlotinib, everolimus, gefitinib, imatinib, restaurtinib, nilotinib, palbociclib, pazopanib, ponatinib, regorafenib, ruxolitinib, semananib, sirolimus, sorafenib, temsirolimus, tofacitinib, trametinib, vandetanib, and vemurafenib.In another embodiment, the tyrosine kinase inhibitor is, but is not limited to, A419259, AP23451, AP23464, AP23485, AP23588, AZD0424, AZM475271, BMS354825, CGP77675, CU201, ENMD2076, KB SRC4, KX2361, KX2-391, MLR1023, MNS, PCI-32765, PD166285, PD180970, PKC-412, PKI166, PP1, PP2, SRN004, SU6656, TC-S7003, TG100435, TG100948, TX-1123, VAL201, WH-4-023, XL228, Artenacin, Bosutinib, Damnacanthal, Dasatinib, Herbimycin A, Indirubin, Neratinib, La These are Src family tyrosine kinase inhibitors such as bendastine A, peritinib, piceatannol, salakatinib, SrcI1, foretinib, motesanib, tivozanib, LY2457546, MGCD-265, MGCD-510, tivantinib, AMG458, JNJ-3887, EMD1214063, BMS794833, PHI1665752, SGX-523, INCB280, their pharmaceutically acceptable salts, and combinations thereof.

[0201] Complement pathway modulators that can be used in the implants and methods of the present invention include, for example, those that target C1 / C1q, C3, C3 convertase, C5, C5 convertase, C5a, C5aR, C6, C7, C8, C9, CD59, factor B, factor D, factor H, factor P, or combinations thereof. Specific drugs include Synrise, Berinart, Luconest, Stirimulimab, Pegcetacoplan (GA), Eculizumab, Laburizumab, Abacopan, Pozelimab, Nomacopan, Zircopan, Viroberimab, Clobarimab, Abasin Capted Pegol), Semdisilan, BDB-001, Tesidromab, Abdularimab, MOR210, ALXN1720, Danicopan, Bemilcopan, A Examples include CH-5228, ACH-5548, BCX-9330, AMY-101, ANX005, ANX007, nalsoprimab, iptacopan, CLG561, GT103, ARGX-117, ALXN1820, NGM621, lampalizumab, NGM621, IONIS-FB-Lrx, GEM103, CLG561, their pharmaceutically acceptable salts, and combinations thereof.

[0202] Integrin inhibitors that can be used in the implants and methods of the present invention include rifitegrast, vedolizumab, natalizumab, efalizumab, tyrofiban, eptifivatide, absiximab, IDL-2965, PLN-74809, PLN-1474, PN-943, 7HP349, MORF-057, OS2966, OTT166, AXT-107, JSM-6427, listiganib, THR-687(D / ced), pharmaceutically acceptable salts thereof, and combinations thereof.

[0203] Antihistamines that can be used in the implants and methods of the present invention include loratadine, hydroxyzine, diphenhydramine, chlorpheniramine, brompheniramine, cyproheptadine, terfenadine, clemastine, triprolidine, carbinoxamine, diphenylpyraline, phenyndamine, azatadine, triperenamine, dexchlorpheniramine, dexbrompheniramine, methidilazine, and trimprazine doxylamine, pheniramine, pyriramine, chlorcyclidine, tondylamine, pharmaceutically acceptable salts thereof, and combinations thereof.

[0204] IL-6 inhibitors that can be used in the implants and methods of the present invention include sarilumab, tocilizumab, RG6179, pharmaceutically acceptable salts thereof, and combinations thereof.

[0205] HtrA1 inhibitors that can be used in the implants and methods of the present invention include IC-500, FHTR2163, RG6147, pharmaceutically acceptable salts thereof, and combinations thereof.

[0206] Examples of RASP inhibitors that can be used in the implants and methods of the present invention include reproxalap and its pharmaceutically acceptable salts.

[0207] Rho kinase inhibitors that can be used in the implants and methods of the present invention include netarducyl, ripasudil, HA-1077, Y-27632, H-1152P, INS-115644, Y-39983, SB772077BS, LX71D1, AR-12286, AMA-0076, AR-13533, pharmaceutically acceptable salts thereof, and combinations thereof.

[0208] Plasma kallikrein inhibitors that can be used in the implants and methods of the present invention include ecalantide, lanadermab, velotralstat, ATN-249, KVD900, KVD824, THR-149, pharmaceutically acceptable salts thereof, and combinations thereof.

[0209] Examples of nitric oxide donors PgA that can be used in the implants and methods of the present invention include latanoprostenbunod, NCX470, NCX125, pharmaceutically acceptable salts thereof, and combinations thereof.

[0210] Examples of mast cell stabilizers that can be used in the implants and methods of the present invention include rhodoxamide, nedocromil, pemirolast, cromoglycic acid (e.g., sodium cromoglycate), pharmaceutically acceptable salts thereof, and combinations thereof.

[0211] IGF-1R inhibitors that can be used in the implants and methods of the present invention include teprotutumab, VRDN-001, VRDN-002, VRDN-003, ganitumab, figtumumab, MEDI-573, cyclotumumab, darotuzumab, lobatumumab, AVE1642, BIIB022, xentuzumab, istilazumab, lincitinib, picropodophyllin, BMS-754807, BMS-536924, and BMS-5 Examples include 54417, GSK1838705A, GSK1904529A, NVP-AEW541, NVP-ADW742, GTx-134, AG1024, KW-2450, PL-2258, NVP-AEW541, NSM-18, AZD3463, AZD9362, B1I885578, B1893923, TT-100, XL-228, A-928605, their pharmaceutically acceptable salts, and combinations thereof.

[0212] TRPV1 antagonists that can be used in the implants and methods of the present invention include AshibaTrep, V116517, for example, U.S. Patent Application No. 2004 / 0157849, U.S. Patent Application No. 2004 / 0209884, U.S. Patent Application No. 2005 / 0113576, International Patent Application No. WO05 / 016890, U.S. Patent Application No. 2004 / 0254188, U.S. Patent Application No. 2005 / 0043351, International Patent Application No. WO05 / 040121, U.S. Patent Application No. 2005 / 0085512, and Gomtsyan et al. Condensed azabicyclic compounds, heterocyclic compounds, and amide compounds as described in al., 2005, J. Med. Chem. 48:744-752; for example, condensed pyridine derivatives as described in U.S. Patent Application No. 2004 / 0138454; for example, Swanson et al., 2005, J. Med. Chem. 48:1857-1872 and U.S. Patent Application No. 2005 / 0049241, as well as AMG8163 (Bannon et al., 2005, 11th World Congress on Pain) and BCTC (Sun et al. Pyridylpiperazinyl urea as described in (al., 2003, Chem. Lett. 13:3611-3616); 2-(piperazin-1-yl)-1H-benzimidazole; pyridadinylpiperazine; urea derivatives as 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); SB-366791 (Gunthorpe et al., 2004, Neuropharmacology 46:133-149) and AMG9810 (Gawa et al. Cinnamide (including al., 2005, J. Pharmacol. Exp. Ther. 313:474-484) is one example.

[0213] In some embodiments, TRPV1 antagonists useful in the methods and compositions disclosed herein include, for example, capsazepine, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide (commercially available as AMG9810 from Tocris Bioscience, Bristol, United Kingdom), and 4-tert-butylcyclohexane (Symrise, Holzminden, Germany). Examples include TRPV1 antagonists, including those marketed as SYMSITIVE1609 from GmbH, and TRPV1 antagonists disclosed in U.S. Patent Nos. 8,815,930, 6,933,311, 7,767,705, and U.S. Patent Publication Nos. 2010 / 0249203 and 2011 / 0104301, and International Patent Application No. WO / 2008 / 013861.

[0214] In some embodiments, TRPV1 antagonists useful in the methods, compositions, and devices disclosed herein include AMG-517 and AMG-628 (Amgen Inc., Thousand Oaks, Calif.). TRPV1 antagonists useful in this application are also described, for example, in International Patent Application WO2006065484, International Patent Application WO2003070247, U.S. Patent Application US2005080095, and International Patent Application WO2005007642. Additional TRPV1 antagonists useful for the methods, compositions, and devices disclosed herein include: TRPV1 antagonist: ABT-102, AMG8562, AMG9810, BCTC, SB366791, JNJ17203212, I-TTX, JYL-1421, A-425619, N-[4-[6-[4(trifluoromethyl)phenyl)pyrimidine-4-yloxy]benzothiazole-2 Examples include (R)-N-(4-(6-(4-(1-(4-(fluorophenyl)ethyl)piperazine-1-yl)pyrimidine-4-yloxy)benzo[d]thiazole-2-yl)acetamide (also known as AL-49976, AMG-628), pharmaceutically acceptable salts thereof, and combinations thereof.

[0215] Other TRPV1 antagonists useful in the methods, compositions and devices disclosed herein include, for example, 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)ureido)methyl)-5-(trifluoromethyl)phenyl)butanoate; 1-(2-(4-hydroxy-3,3-dimethylbutyl)-4-(trifluoromethyl)benzyl)-3-(1-methyl-1H-indazol-4-yl)urea; 2,2-dimethyl-4-(2-((3-(1-methyl-1H-indazol-4-yl)ureido)methyl-5-trifluoromethyl)phenyl)butanoic acid; 1-[4-chloro-3-(3,3-dimethylbutyl)benzyl]-3-(1-methyl-1H-indazol-4-yl)urea-; 1-(2-isobutyl-4-(trifluoromethyl)benzyl)-3-(1-methyl-1H-indazol-4-yl)urea, 1-(2-isopropyl-4-(trifluoromethyl)benzyl)-3-(1-methyl-1H-indazol-4-yl)urea, 1-(4-chloro-3-isopropylbenzyl)-3-(1-methyl-1H-indazol-4-yl)urea, their pharmaceutically acceptable salts, and combinations thereof, which have low inhibitory activity against CYP3A4.

[0216] TrkA antagonists that can be utilized in the implants and methods of the present invention include VM902A, larotrectinib, entrectinib, selitrectinib (LOXO-195, BAY2731954), repotrectinib (TPX-0005), their pharmaceutically acceptable salts, and combinations thereof.

[0217] For the purposes of the present invention, the active agent includes all possible forms thereof, including free acids, free bases, polymorphs, pharmaceutically acceptable salts, anhydrides, hydrates, other solvates, stereoisomers, crystalline forms, co-crystals, prodrugs, conjugates (e.g., pegylated compounds), their complexes and mixtures.

[0218] Diagnostic activators may be, for example, contrast agents, markers, or visualization agents. Generally, diagnostic agents may be substances used to examine the body to detect impairments in the normal functioning of the body. In some cases, diagnostic agents may be agents with a functional purpose, such as being used to detect deformities, diseases, and pathophysiological aspects of the eye. For example, diagnostic agents may be important and effective diagnostic aids such as dyes to aid in the visualization of ocular tissue (e.g., dark quenchers such as fluorescein dye, indocyanine green, trypan blue, cyanine dyes, azo dyes, acridine, fluoron, oxazine, phenanthridine, naphthalimide, rhodamine, benzopyrone, perylene, benzantrone, and prabenzoxanthrone). Diagnostic agents may include paramagnetic molecules, fluorescent compounds, magnetic molecules, radionuclides, X-ray imaging agents, and / or contrast agents. In some embodiments, the diagnostic agent may include radiopharmaceuticals, contrast agents used in imaging techniques, allergen extracts, activated carbon, various test strips (e.g., cholesterol, ethanol, and glucose), pregnancy tests, breath tests with urea 13C, and various stains / markers. In some embodiments, the labeling portion is a fluorescent dye or dark quencher selected from the group consisting of coumarin, cyanine dyes, azo dyes, acridine, fluorone, oxazine, phenanthridine, naphthalimide, rhodamine, benzopyrone, perylene, benzantrone, and benzoxanthrone. In certain non-limiting embodiments, the fluorescent dye is a compound selected from the group consisting of coumarin, fluorescein, cyanine 3 (Cy3), cyanine 5 (Cy5), cyanine 7 (Cy7), Alexa dye, bodipy derivatives, (E)-2-(4-(phenyldiazenyl)phenoxy)acetic acid, 3-(3',3'-dimethyl-6-nitrospiro[chromen-2,2'-indoline]-1'-yl)propanoate (spiropyran), 3,5-dihydroxybenzoate, and (E)-2-(4-(phenyldiazenyl)phenoxy)acetic acid, or a combination thereof, or a residue thereof.

[0219] In certain embodiments of the present invention, the active agent can be dispersed, embedded, or encapsulated in the hydrogel. In certain of those embodiments, the active agent can be in particulate form. In certain embodiments, the active agent can be conjugated by covalent bond to click chemical functional groups within the polymer network, such as being unreacted or having additional functional groups additionally provided to the arms of the polymer units.

[0220] In embodiments where the active agent is used in particulate form, the particles of the active agent can be micronized particles having, for example, a D50 particle size of less than about 15 μm or less than 10 μm, and / or a D99 particle size of less than about 100 μm or less than about 50 μm, or a D90 particle size of less than or equal to about 50 μm or less than or equal to about 5 μm, and / or a D98 particle size of less than or equal to about 10 μm. In another embodiment, the active agent particles can be nano-sized particles, for example, having a D50 particle size of less than about 100 nm, or less than about 50 nm, and / or a D99 particle size of less than about 50 nm, or a D90 particle size of less than or equal to about 5 nm, and / or a D98 particle size of less than or equal to about 10 nm. The particle size is determined as disclosed in the "Definitions" section of this specification.

[0221] According to the present invention, a drug delivery system comprising a hydrogel can be designed as desired for the desired use and therapeutic use. In certain embodiments, the hydrogel comprises 1 to 50% by weight of the active agent, or 5 to 50% by weight, 5 to 40% by weight, 10 to 30% by weight, or 10 to 25% by weight, and the weight percentages are each based on the total dry weight of the hydrogel or the drug delivery system.

[0222] In a particular embodiment, the hydrogel drug delivery system according to the present invention in a dry state contains about 15% to about 80% by weight of activator and about 20% to about 60% by weight of polymer units based on the total weight of the hydrogel, or about 30% to about 65% by weight of activator and about 25% to about 50% by weight of polymer units based on the total weight of the insert, or about 45% to about 55% by weight of activator and about 37% to about 47% by weight of polymer units based on the total weight of the hydrogel.

[0223] In one further specific embodiment, on a dry weight basis, the activator to PEG or polymer ratio is approximately 50% to 60% by weight of activator to approximately 40% by weight of PEG or polymer, based on the total weight of the insert, with the remainder being phosphates and other excipients.

[0224] In one embodiment, the hydrogel, after being formed and before being dried, i.e., in a wet state, contains about 3% to about 20% by weight of polyethylene glycol, and represents the weight of polyethylene glycol divided by the fluid weight × 100. In one embodiment, the hydrogel in a wet state contains about 7.5% to about 15% by weight of polyethylene glycol, and represents the weight of polyethylene glycol divided by the fluid weight × 100.

[0225] In certain embodiments, the hydrogel is dried after production, and the water content of the dried (dehydrated / dried) hydrogel may be very low, such as 1% by weight or less of water. In other words, in certain embodiments, the dried hydrogel contains about 1% by weight or less of water. In certain embodiments, the water content may also be lower than that, for example, 0.25% by weight or less or 0.1% by weight or less, based on the total weight of the hydrogel.

[0226] Characteristic evaluation Characterization and purification of click chemically functionalized precursors are performed. 1This can be performed by 1H-NMR (1H-nuclear magnetic resonance) and HPLC-UV (high-pressure liquid chromatography with ultraviolet absorption detection). In one embodiment of the method of the present invention, NHS-functionalized magnetic beads can be used to remove residual unreacted DBCO-amine from the functionalization precursor product.

[0227] Other purification methods for hydrogel precursors obtained by the synthesis methods described herein include, for example, dialysis, SEC column filtration, or (U)HPLC.

[0228] Dialysis is a common purification method based on separating molecules in a solution through differences in diffusion rates across a semipermeable membrane, such as a dialysis tube. Dialysis tubes are commercially available, for example, from Spectra / Por® Float-A-Lyzer G2 Dialysis Devices and Spectrum® Laboratories, and have several different molecular weight cutoffs as needed for specific separation tasks. By selecting appropriate molecular weight cutoff dialysis tubes and membranes, it is possible to remove most impurities, such as excess amounts of unreacted precursors, from the product through a series of separation steps using different cutoff dialysis tubes as needed.

[0229] Another purification method that can be applied to certain aspects of the present invention is the use of size exclusion chromatography (SEC), such as an SEC column. For example, Zeba® Spin Desalting columns (from Thermo Fisher Scientific), designed for protein purification to remove salts and small-sized impurities, can be used to purify hydrogel precursor reaction mixtures. Columns with different pore sizes can be used. The purification mechanism is based on size exclusion chromatography, in which small particles are trapped in the pores on the static phase material, while larger particles elute through the column and are collected in a purified form.

[0230] The resulting purified product can be characterized by ultra-high performance liquid chromatography (UHPLC), an efficient technique that provides more sensitive analysis with good chromatographic separation and resolution of the analyte. It offers advantages including rapid analysis, high-resolution separation, reduced solvent and sample usage, and improved sensitivity and accuracy. Based on calibration curves 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.

[0231] Another parameter of particular importance for the in-situ gel-forming drug delivery systems of embodiments of the present invention is the gel time, i.e., the time from mixing the reaction precursor solution (and at least one activator) together at room temperature for about 10 seconds to the final formation of the (solid) gel. For in-situ applications, the processing time to ensure uniform mixing, minimize air bubbles, and load the needle into the syringe should be about 1-2 minutes, such that the gelation time is greater than this processing time. After processing, the gel time can be controlled in certain embodiments, as further described herein, to provide an additional gel time (in addition to the mixing and processing time) as follows: - When injected into liquid tissue (such as vitreous humor), the injection is very fast (0.5 seconds to 1 minute). In this way, the gel forms a shell very quickly and is not diluted in the liquid environment. - For injections into soft tissue, moderate duration (1-2 minutes) - For injections into harder tissues, the injection time is slower (1-5 minutes).

[0232] Therefore, in embodiments including in situ applications, the total gel time of the reaction mixture should be 2 to 9 minutes, or at least about 1 to 10 minutes, such as about 1.5, 2, 3, 4, 5, 6, 7, or 8 minutes.

[0233] Interpenetration network The use of click chemistry concepts in hydrogel synthesis allows for the combination of two types of gel crosslinking chemistry in a one-pot reaction. For example, in certain embodiments of the present invention, it is possible to create an interpermeable network between a hydrogel formed from a precursor crosslinked by click chemistry, such as DBCO-azide chemistry as described herein, and a hydrogel formed from a precursor crosslinked by NHS-NH2 electrophilic-nucleophilic chemistry.

[0234] Since both crosslinking functional groups do not react with each other, according to these crosslinking reactions, the provision of four precursors, each having a different type of functional group, crosslinks into two different crosslinked gels that interpenetrate each other.

[0235] Different types of networks can have different gel times and molecular weights between crosslinking with NHS-NH2 in another PEG network having click chemical crosslinking. The NHS-NH2 chemistry does not interact with DBCO-azide, creating two different hydrogel substrates with different mechanical properties that are entangled with each other. The entanglement of the two types of crosslinked macromolecules in the gel allows for building blocks with lower molecular weights with the same number of arms of the precursor used, or allows for the use of fewer arm precursors with the same or higher overall molecular weight in the interpenetration network gel. In certain embodiments, the interpenetration embodiment has a swelling rate approximately equal to the average of two distinct gels at the same polymer concentration. In certain embodiments, the interpenetration network is used for antibody delivery and release.

[0236] Furthermore, in embodiments of the present invention, the interpenetration network can be formed from two or more click-chemically reactive group-functionalized precursors. For example, the DBCO-functionalized multi-arm PEG first precursor may react with two or more different second precursors, each of which has another DBCO-reactive functional group, such as an azide and a tetrazine functional group. In one embodiment, the DBCO-functionalized multi-arm PEG first precursor reacts with a mixture of tetrazine-functionalized multi-arm PEG second precursors in the presence of an azide-functionalized multi-arm PEG third precursor. As shown in Example 10 below, such a ternary system allows for easy control of the gel time by varying the concentration of the third precursor, such as the azide precursor concentration. While we do not wish to be constrained by theory, it is conceivable that such a system having more than two reactive species forms an interpenetration network of different clicked precursors in a competitive reaction. Therefore, the third precursor can be used to delay or accelerate the gel time of a sustained-release drug delivery system reaction mixture, so that the adjusted gel time can be provided for different in-situ gel formation applications.

[0237] The use of a third (or more) click-reactive precursor can provide control over the viscosity of the solution and the gelation time. Fast gel times (e.g., about 10 seconds to 1 minute) are suitable for in-situ gel-forming injections into liquid tissues such as vitreous humor or blood, without dilution of the gel in a liquid environment. Shorter to moderate gel times (e.g., about 1 to 2 or 1 to 3 minutes) are preferred for injections into soft tissues, while slower gel times (e.g., about 1 to 8 or 2 to 5 minutes) may be more preferable for in-situ gel-forming injections into harder tissues.

[0238] Release kinetics In certain embodiments, the hydrogels of the present invention can be used for sustained drug delivery. The hydrogel drug delivery system can be adapted by several means to modify the release of the active agent dispersed in the hydrogel. For example, adjusting or suitably selecting the precursor components such as the length and molecular weight of the polymer arms, the number of arms, the type of polymer units such as hydrophobic and hydrophilic polymer units, and their combinations, and the type and length of the linkers used can affect the release of the active agent.

[0239] In certain embodiments, the linker of formula (i) used in the hydrogel structure introduces a hydrolyzable bond into the hydrogel, which can be used to change the degradation rate of the hydrogel and / or the release rate of the active agent from the hydrogel when the hydrogel degrades in vivo. For example, the biodegradation / hydrolysis rate of the ester bonds in these linkers decreases from succinate (C4) to azelate (C9) linkers. The shorter the length of the dibasic acid linker chain, the faster the formed ester bond is hydrolyzed. Thus, the hydrolysis rate decreases in the order of SS > SG > SAP > SAZ > SGA ester bonds. In embodiments of the present invention, this can be used to control the degradation rate of the hydrogel and / or the release of the active agent dispersed therein. For example, esters formed from succinimidyl succinate (NHSS) groups can degrade in days, while esters of succinimidyl glutarate (NHSG) groups degrade in weeks. Different linkers can be used at different positions within the hydrogel to control the degradation rate between crosslinks within the hydrogel and thus control the release of the active agent from the hydrogel.

[0240] In the case of crosslinking formed by click chemical reactions, spacer structures between the DBCO / azide functional group and the functional group to which it bonds to the polymer arm of the precursor, such as the extension of alkylene chains or pegylation, can also be used in embodiments of this disclosure to slow the hydrolysis of adjacent ester bonds. The greater the distance between the DBCO / azide functional group (or other click chemical bonds described herein) and the next hydrolyzable ester group, the slower the hydrolysis of the ester occurs. Different linkers within the hydrogel precursor can be used to enable degradation control. This can be used to control the half-life of the activator and change the release rate.

[0241] In certain embodiments, hydrophobic organic liquids such as oils can be incorporated into the polymer network of the gel, thus forming an organic gel containing the hydrophobic organic liquid as a continuous phase. Examples of hydrophobic organic liquids include triethyl citrate, acetyl triethyl citrate (ATEC), acetyl tributyl citrate (ATBC), α-tocopherol (vitamin E), α-tocopherol acetate; plant or vegetable oils such as sesame oil, olive oil, soybean oil, sunflower oil, coconut oil, canola oil, and rapeseed oil; nut oils such as hazelnut, walnut, pecan, and almond oil; cottonseed oil, corn oil, linseed oil, ethyl oleate, castor oil, and its derivatives (Cremofor®). ), lipids that are liquid at temperatures below 37°C, such as saturated or unsaturated fatty acids, monoglycerides, diglycerides, triglycerides (Myglyols®), phospholipids, glycerophospholipids, sphingolipids, sterols, prenols, and polyketides; and biocompatible oils selected from the group consisting of hydrophobic biodegradable liquid polymers (such as low molecular weight PLGA, PGA, or PLA), low melting point waxes such as plant, animal, or synthetic waxes, lanolin, jojoba oil, or combinations thereof.

[0242] In certain embodiments, the hydrophobic organic liquid is a liquid at body temperature, such as about 37°C or below, or in the range of 0°C to 40°C, or 10°C to 38°C, or 15°C to 37°C, or 25°C to 37°C, or 37°C, and may have a glass transition temperature and / or a melting temperature of 45°C or below, or 37°C or below. The term “liquid” may include viscous fluids having a creamy or waxy appearance but not a solid. Also, in the case of some hydrophobic organic liquids that undergo hydration in aqueous embodiments such as body fluids, the melting point at a certain temperature may differ in the hydrated material from that of the unhydrated material. In certain embodiments of the present invention, the hydrated form of such material is a liquid under the above conditions.

[0243] In certain embodiments, the hydrophobic organic liquid is non-volatile, non-toxic, and / or biocompatible at 37°C and ambient pressure, and / or can be removed from the implantation site, metabolized, and / or removed from the body without change.

[0244] The activator can be dissolved in a hydrophobic organic liquid or dispersed in a hydrophobic organic liquid in form or particles. In certain embodiments, if the activator is an oil itself, such as travoprost, the activator is the hydrophobic organic liquid itself or forms at least a part of it. In such embodiments, the oil can be used as a carrier to control the release of the activator by the diffusion of the oil into the internal environment.

[0245] In certain embodiments, the sustained-release drug delivery hydrogel of the present invention is formulated to make the activator available over a long period of time, thereby enabling a reduction in the frequency of administration compared to immediate-release dosage forms, such as a solution of the activator applied topically to the eye (i.e., eye drops). In certain embodiments, the release of the activator includes constant activator release, gradually decreasing activator release, and any combination thereof (e.g., gradually decreasing activator release after constant activator release). "Sustained release" can be measured in vitro in an aqueous solution under physiological conditions such as pH 7.2-7.4 and 37°C, and is considered to be the same or substantially the same when the sustained-release drug delivery hydrogel is administered in vivo to a subject.

[0246] In various embodiments of the present invention, the activator release follows zero-order release dynamics or substantially zero-order release dynamics, preferably without a "burst" of activator at the start of the period.

[0247] Embodiments of the present invention may provide the release of a therapeutically effective amount of the activator for a period of up to 1 year, up to 9 months, up to 6 months, up to 3 months, up to 1 month, or up to about 25 days after administration. Another embodiment of the present invention may provide the release of a therapeutically effective amount of the activator for a period of up to about 14 days, or up to about 21 days, or for a period of about 6 hours or more, or for a period of about 12 hours, or 24 hours or more, or for a period of about 48 hours or more, or for a period of about 72 hours or more, or for a period of about 7 days or more, or for a period of about 10 days or more after administration. The present invention intends to achieve all of the above shorter and longer periods in any combination of ranges.

[0248] Some aspects of the present disclosure relate to a pharmaceutically acceptable hydrogel drug delivery system for controlled release of an activator dispersed therein, characterized in that the amount of activator released on day 1 is 0 to 50% of the total amount of activator, the amount of activator released per day from day 2 to the final day of release is 0 to 50% of the total amount of activator, and / or the number of days required for 100% release of the total amount of activator is at least 2 days.

[0249] In one embodiment, controlled release of the activator is characterized in that the amount of activator released on day 1 is 0-50% of the total amount of activator, the amount of activator released per day from day 2 to the last day of release is 0-50% of the total amount of activator, and / or the number of days required for 100% release of the total amount of activator is at least 2 days. In another embodiment, the amount of activator released on day 1 is 0-25%, 0-20%, 0-10%, 0-5%, or about 0% of the total amount of activator, the amount of activator released per day from day 2 to the last day of release is 0-50% of the total amount of activator, and / or the number of days required for 100% release of the total amount of activator is at least 3 days, but 30 days, 25 days or less, or 16 days or less.

[0250] Upon contact with aqueous bodily fluids, hydrogels of certain embodiments of the present invention swell by incorporating water, particularly in the case of dry hydrogels / zerogels. The degree of swelling is primarily determined by the hydrophilic gel-forming component. The swelling can result in an increase of up to 2000%, 1000%, 100%, 95%, 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% in the length and / or diameter dimensions of hydrogels of the form according to the present invention.

[0251] However, even after swelling, in certain embodiments, the drug delivery system of the present invention maintains its shape or substantial shape over a long period of time due to the crosslinking of the polymer precursor. In certain embodiments, the polymer network of the hydrogel substantially decomposes only after all of the activator has been released, or after at least the majority of the activator has been released, for example, at least 50%, 60%, 70%, 80%, 90%, 99%, or 100% by weight of the activator.

[0252] In certain embodiments, swelling, for example, leads to an increase in the size of the hydrogel implant, but is also thought to affect the release of the activator and / or the biodegradation of the gel matrix. As the amount of water entering the gel increases due to the swelling of the hydrogel, the outward diffusion of the activator may be accelerated and replaced by water.

[0253] For example, water that diffuses into the gel and / or replaces the organic hydrophobic liquid within the gel over time can steadily dissolve the hydrophilic activator dispersed therein, which can be used to control activator release. In this embodiment, activator release is primarily or entirely controlled diffusion of the activator into the surrounding tissue. In certain embodiments, if the activator release rate is largely independent of the diffusion rate of the hydrophobic liquid, for example, if the activator is a hydrophilic agent dispersed as solid particles in the hydrophobic liquid, another factor that affects or determines the release of the activator dispersed in the hydrophobic liquid is the diffusion rate of water into the gel and / or hydrophobic liquid, thereby subsequently dissolving the activator from the organogel and eluting it into the surrounding aqueous environment.

[0254] Furthermore, in certain embodiments, water may slowly diffuse into the hydrogel, and although the hydrogel remains cross-linked and maintains its shape, it may swell, so that after the drug delivery system has depleted the activator, the gel may become more susceptible to (bi)degradation by hydrolysis and / or enzymatic reactions.

[0255] The overall release of the activator is controlled by at least one of these release mechanisms, or by a combination thereof.

[0256] Delivery system The sustained-release drug delivery system of the present invention may be in the form of a medical implant or a pharmaceutically acceptable implant, an implant coating, or an oral dosage form. The drug delivery system may also be provided in the form of a kit, for example, for forming an implant in situ, as further defined below.

[0257] A hydrogel drug delivery system of a particular embodiment of the present invention offers many advantages as a carrier system and can therefore be used for drug delivery to patients, and for example, for ophthalmic drug delivery. Hydrogel drug delivery systems can be used for drug delivery in ophthalmology, gene delivery, antioxidant delivery, peptide delivery, biomedical imaging, and genetic testing.

[0258] In the case of sustained release, the hydrogel drug delivery system is provided in the form of an implant, which may be an intraocular implant, an intracavitary implant, an anterior chamber implant, an implant for introduction into the anterior chamber, the vitreous humor, episclera, posterior subtenon's space (inferior fornix), subconjunctival, intra-anterior chamber, periocular, posterior, subtenon's space, retina, subretinal, intracanaliculi, intravitreous, intrascleral, choroid, suprachoroidal, retina, subretinal, or the surface of the lens, cornea or conjunctiva, lacrimal punctum (lacrimal canaliculi, superior / inferior canaliculi), orbital fornix, superior / inferior orbital fornix, subtenon's space, choroid, superior choroid, Tenon's capsule, cornea, cancerous tissue, organs, prostate, breast, joint cavity, subdural, dental, subcutaneous, carpal tunnel, perivascular, surgically created cavities or injuries, spaces, and potential spaces.

[0259] In a particular embodiment of the present invention, the sustained-release drug delivery system may be formulated for administration via a variety of routes, such as oral, parenteral, surgical insertion, or injection. The oral dosage form may consist of the hydrogel of the present invention, which may be optionally enterically coated, or in the form of small particles filled into capsules or the like.

[0260] In embodiments where in-situ gel formation is desired, it is necessary to avoid pegylation reactions between the activator and the precursor material, as pegylation or covalent bonding of the activator to the hydrogel precursor interferes with or disrupts in-situ gel formation and / or results in reduced activator recovery from the hydrogel. For example, hydrogels with sulfo-DBCO functionalized precursors, sometimes used to improve water solubility, may not be suitable for delivering peptides or proteins because these agents exhibit strong charge interactions with sulfonic acid groups, reducing protein recovery. However, for small molecule APIs, these hydrogel precursors can be a suitable option. Furthermore, residual N-hydroxysuccinimide groups on multi-arm precursors that are not fully functionalized with click chemically reactive groups can also reduce protein or peptide recovery by covalently bonding these agents to the hydrogel precursor. In certain embodiments, it is preferable to use a precursor that forms a hydrogel, and the API is dispersed / contained in the hydrogel, preferably not covalently bonded to the hydrogel or its components.

[0261] For in-situ gel-forming sustained delivery systems, it is preferable to select precursors to achieve a gel time of 1 to 10 minutes, preferably 2 to 4 minutes at 37°C, as further disclosed herein. Such kits or systems may, in embodiments, be used particularly for ocular treatment, such as intrachoroidal or intravitreal injection. Depending on the treatment and the injection site involved, the target gel time in situ intravitreal or blood (IV) may be fast, as defined herein, while a relatively slow gel time is preferred in the case of intrachoroidal or intrabody tissue.

[0262] In certain embodiments, DBCO / azidohydrogel may have a gel time that is too fast for in situ injection, but it can be suitably used for gel formation outside the body to create implants or inserts such as plugs.

[0263] In other embodiments, norbornene / tetrazine-based systems may be good for in situ injection due to their relatively long gel time; however, in the absence of a dibasic acid linker, these systems, such as those illustrated in Example 8, are hardly degradable, and therefore the inclusion of a degradable linker is necessary for sustained-release systems.

[0264] In other embodiments, DBCO / tetrazine systems having a gelation time of 4–9 minutes are preferred for in-situ applications, although their gelation time is largely independent of temperature and pH. In vitro data show that DBCO / tetrazine PEG hydrogels can achieve degradation times or IgG protein release of up to 2 months; see Figure 10 and Example 9.

[0265] In other embodiments, the DBCO / azide / tetrazine tricomponent system allows for control and adjustment of gel time by varying the azide precursor concentration; see Example 10.

[0266] In certain embodiments, a drug delivery system or kit for in situ applications has injectability characterized by a glide force at 2 minutes after mixing of less than 10 N, such as 9 N, 8 N, 7 N, less than 6 N, or less than 5 Newtons, and less than 10 N, such as 9 N, 8 N, 7 N, 6 N, or 5 N or less, as described in Example 11.

[0267] Treatment methods and administration According to a particular embodiment of the present invention, a sustained-release drug delivery system comprising a hydrogel is configured for use as a pharmaceutical agent, such as in the treatment of a patient's disease or medical condition.

[0268] In one embodiment, a method for treating a patient's disease or condition includes administering a hydrogel to the patient to release an active agent over a long period of time.

[0269] The therapeutic methods of embodiments of the present invention include the treatment of the eye. In such a treatment, a hydrogel is used to release an active agent in the eye over a long period of time. In these embodiments, the disease or condition to be treated is an eye disease such as any posterior segment eye disease that affects the blood vessels and structures of the retina, macula, or choroid and causes visual impairment, vision loss, or blindness, in particular posterior segment eye conditions resulting from aging, trauma, or surgical intervention, such as age-related macular degeneration (AMD), cystoid macular edema (CME), diabetic macular edema (DME), posterior uveitis, and diabetic retinopathy.

[0270] Treatment methods may also involve the treatment of glaucoma, ocular hypertension, anterior chamber hemorrhage, presbyopia, cataracts, retinal vein occlusion, inflammation, miosis, mydriasis, conjunctivitis, intraocular infection, choroidal neovascularization (CNV), intraocular tumors, and retinal neuritis.

[0271] Furthermore, eye diseases include retinal neovascularization, choroidal neovascularization, exudative AMD, atrophic 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 multiple macular pigment epitheliopathy, Behçet's disease, and birdshot nettle. Choroidopathy, posterior uveitis, posterior scleritis, tortuosic choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusive disease, central retinal vein occlusion, disseminated intravascular coagulation, branching retinal vein occlusion, hypertensive fundus changes, ischemic syndrome, retinal artery microaneurysms, Coats' disease, parafoveal telangiectasia, hemiretinal vein occlusion, papillary phlebitis, carotid artery disease (CAD), dendritic vasculitis, sickle vein It may be one of the following: erythrocytic retinopathy, retinal pigment striata, familial exudative vitreoretinopathy, Eels disease, proliferative vitreoretinopathy, diabetic retinopathy, tumor-associated retinal diseases, congenital hypertrophy of the retinal pigment epithelium (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, complex hamartoma of the retina and retinal pigment epithelium, retinoblastoma, fundus vascular proliferative neoplasm, retinal astrocytoma, ocular lymphoid neoplasm, myopic retinal degeneration, acute retinitis pigmentosa, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal cancer, retinitis pigmentosa, Leber congenital amaurosis, choroidal atrophy, X-linked retinitis pigmentosa, Best vitiligo macular dystrophy, X-linked retinoschisis, color blindness CNGA3, color blindness CNGB3, LHON, Stargardt disease, Usher syndrome, Norrie disease, Valday-Beedl syndrome, and red-green color blindness.

[0272] The methods described in this section may also include administering a hydrogel drug delivery system in combination with another drug, also known as “combination therapy.”

[0273] In one embodiment, the combination therapy comprises administering the hydrogel in combination with one or more additional agents on either the same day or on different days. In one embodiment, the additional agents to be administered in the combination therapy may be liquid formulations of the agents or may be contained in oral dosage forms. Thus, the additional agents may be any small molecules, large molecules, proteins, nanoparticles, or any other activators described herein.

[0274] A particular embodiment of an ocular treatment method involving the administration of a hydrogel may include one of the following injections: intravitreous, intrachoracic, subconjunctival, retrobulbar, subtenon's capsule, subretinal, and suprachoroidal. The administration of the hydrogel or drug delivery system in a particular embodiment may also be topical, intradermal, or oral.

[0275] In certain embodiments, the drug delivery system is formulated for direct injection at the treatment site of the patient and may be administered by injection into the eye, such as parenteral administration, intratumoral injection, intravitreal injection, anterior chamber injection, subconjunctival injection, retrobulbar injection, sub-Tenon's capsule injection, subretinal injection, or suprachoroidal injection. In those embodiments, the drug delivery system is administered by direct injection, oral administration, or incorporated into an implant. In certain embodiments, direct injection involves positioning a depot and delivering the drug for local or systemic administration. Direct injection may involve injecting or implanting a pre-formed hydrogel at the treatment site and may further involve injecting a precursor of the hydrogel or a mixture thereof to form an in situ activator-releasing gel depot for delivering the drug for local or systemic administration.

[0276] In certain embodiments, the hydrogel or drug delivery system may be implanted or administered into the patient's body space by any one of the following methods: subcutaneous, intramuscular, intrathecal, epidural, intraperitoneal, intradermal, subcutaneous, intercostal, intra-articular, intrasynovial, intravertebral, oral, nasal, transrectal, intratumoral, or vaginal administration. Intra-articular administration is to a joint selected from the knee, elbow, hip, sternoclavicular, temporomandibular joint, carpal, tarsal, wrist, ankle, intervertebral disc, or ligamentum flavum.

[0277] The active or additional agents to be administered in combination therapy may be diagnostic agents. Diagnostic agents may be substances used to examine the body to detect impairments in normal bodily functions, as described above. In some cases, diagnostic agents may be agents with a functional purpose, such as being used to detect eye deformities, diseases, and pathophysiological aspects.

[0278] In certain embodiments of the present invention, a sustained-release drug delivery system is administered such that a bioorthogonal hydrogel is formed in situ at the treatment site of a patient. Gelation by click chemical crosslinking as described herein can occur under mild physiological conditions, and the functional groups do not interfere with physiological processes, so the gelation can occur within the patient's body. In those embodiments, the hydrogel is formed in situ at the treatment site of a patient by combining a first formulation comprising a first mixture containing a first precursor and an activator with a second formulation comprising a second mixture containing a second precursor, either at the treatment site or immediately before the treatment site, thereby enabling the combined formulation mixture to gel in situ at the treatment site by a click chemical crosslinking reaction.

[0279] In one embodiment, the first and second formulations are combined immediately before administering the formulation mixture at the treatment site. In a particular embodiment for treatment by in-situ gelation of a hydrogel, the first formulation is in a first syringe and the second formulation is in a second syringe, and the two formulations are combined in a Y-mixer before direct injection at the treatment site. Alternatively, the first and second formulations are each administered at the treatment site, for example, by separate injections, thereby allowing the formulations to be combined at the treatment site and the combined formulation to gel in situ at the treatment site.

[0280] Administration for in-situ gel formation can be performed by any of the following methods: subcutaneous, intramuscular, intrathecal, epidural, intraperitoneal, intradermal, subcutaneous, intercostal, intra-articular, synovial, intravertebral, oral, nasal, transrectal, intratumoral, or vaginal administration. In certain embodiments thereof, administration for in-situ gel formation includes injections into the eye, such as parenteral administration, intratumoral injection, intravitreal injection, anterior chamber injection, subconjunctival injection, retrobulbar injection, subtenon's capsule injection, subretinal injection, or suprachoroidal injection.

[0281] Kit for In-Situ Gel Formation Embodiments of the present invention provide a kit for forming a bio-orthogonal hydrogel containing an activator. The kit comprises at least two separate containers, such as vials or pre-filled syringes. The first container comprises a first precursor formulation comprising at least one first multi-arm precursor having a first functional group suitable for forming links or crosslinks by click chemistry with a complementary second functional group, and optionally at least one activator and / or at least one solvent. The second container comprises a second precursor formulation comprising at least one second multi-arm precursor having a first functional group for mixing and gel formation with a second functional group suitable for forming links by click chemistry, and optionally at least one activator and / or at least one solvent. At least one of the first multi-arm precursors, such as both of the second multi-arm precursors, is a polymer multi-arm precursor having polymer arms comprising polymer units, and at least one of the first multi-arm precursors or the second multi-arm precursors comprises linker units L comprising hydrolyzable ester bonds.

[0282] In another embodiment, a third container is provided, in which the activator is contained separately from the hydrogel precursor, and optionally, at least one solvent and / or the activator is contained in at least one or both of the first and second containers.

[0283] Each container may contain additional components such as a solvent, a pharmaceutical excipient, a buffer, an additional therapeutic or diagnostic agent, a thickener, or an adjuvant. In one embodiment of the kit, the first formulation is provided in a first vial or syringe, and the second formulation is provided in a second vial or syringe. The two syringes are formulated for simultaneous or sequential injection at the patient's treatment site. At least one solvent in the first, second, or third container is independently selected from a buffered aqueous medium such as water, PBS buffer, TBS buffer, or physiological saline, or any mixture thereof, and may optionally contain at least one additive selected from pharmaceutical excipients, sugars, salts, additional therapeutic or diagnostic agents, thickeners, viscosity enhancers, stabilizers, or adjuvant.

[0284] The kit is suitable for use in methods for preparing activators that release a hydrogel in situ at the treatment site within a patient's body by simultaneous or sequential injection.

[0285] In a further embodiment, the two syringes of the kit can be connected via a Y-mixer to combine two formulations. The kit is suitable for use in a method for preparing an activator that releases a hydrogel in situ at a treatment site within a patient's body by a single injection. The kit can also be used to prepare the hydrogel in the form of extruded strands that can be cut into individual dosage forms outside the body and to form implants or plugs, such as drug delivery plugs for insertion into the lacrimal duct of the eye.

[0286] In another embodiment, the first precursor mixture and / or the second precursor mixture may include a hydrophobic organic liquid such as an oil. The first precursor mixture and / or the second precursor mixture may be in the form of a suspension or emulsion, which may contain an activator dissolved or suspended therein.

[0287] In one embodiment, each of the first, second, third, or more containers may contain a solution of a precursor and / or activator in a suitable solvent such as water or a buffered water system. In another embodiment, at least one of the first, second, third, or more containers contains a solid that can be reconstituted by adding a solvent for use. Such embodiments may provide better storage stability. For example, in one embodiment of a kit comprising three vials or containers, the activator, such as a peptide or protein, may be a lyophilized API optionally containing a sugar buffer such as trehalose. This lyophilized API is first reconstituted into a solution by adding water, buffer, or solvent when the kit is used, and the solution may then be added to one or both hydrogel precursor solutions, which are then combined to initiate the gelation process.

[0288] Before or after freeze-drying, a glucose buffer may be added as needed to improve the solubility and stability of activators such as peptides or proteins. Even with non-peptide activators, the addition of a glucose buffer can improve stability and solubility. Exemplary glucose buffer formulations for use with embodiments of the present invention may include a solution of sugars such as trehalose, monophosphates, and diphosphates in water at a pH of about 6.4 at a suitable concentration, such as 3% (mg / mL).

[0289] In one embodiment of the kit, at least one first precursor formulation comprises a mixture of two or more first precursors, each having different first functional groups that are suitable for forming a link with a complementary second functional group by click chemistry but do not react with each other. In another embodiment, at least one second precursor formulation comprises a mixture of two or more second precursors, each having different second functional groups that are suitable for forming a link with a complementary first functional group by click chemistry but do not react with each other.

[0290] In such embodiments, different first or second functional groups are selected to have different kinetic rate constants or reaction rates in their click reactions with the corresponding second or first functional group counterparts. This can be used to adjust or modulate (i.e., delay or accelerate) the gel time and to adjust the viscosity (i.e., improve injectability), thereby optimizing in situ administration to different target sites.

[0291] Therefore, the use of two different second precursors provides control over solution viscosity and gel time, which is preferable for different therapeutic applications or administration modes. Very fast gel times (e.g., about 10 seconds to 2 minutes) can be suitable for in-situ gel-forming injections into liquid tissues such as vitreous fluid or blood, without dilution of the gel in a liquid environment. Shorter to moderate gel times (e.g., about 1 to 3 minutes) are preferred for injections into soft tissues, while slower gelation (e.g., about 2 to 8 minutes or more) may be more preferable for in-situ gel-forming injections into harder tissues.

[0292] In some embodiments, the first and second functional groups are selected to provide a gel time from combining the first and second precursor formulations of at least about 1 minute, for example, about 2 minutes or more, or about 3 minutes or more, for example, about 1 to 15 minutes, for example, about 2 to 10 minutes, 2 to 8 minutes, or 2 to 6 minutes, or 2 to 4 minutes.

[0293] In one embodiment, the kit comprises first and second functional groups selected from pairs including a strained alkyne moiety and azide such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonine (BCN), or norbornene or transcyclooctene (TCO) and tetrazine (Tz) moiety, or a strained alkyne moiety and tetrazine (Tz) moiety such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonine (BCN), or any combination thereof.

[0294] In another embodiment, the kit comprises a first precursor which is a dibenzocyclooctin (DBCO) group-functionalized multi-arm PEG, and a second precursor which is a combination of a tetrazine group-functionalized multi-arm PEG and an azide group-functionalized multi-arm PEG, or the first precursor is a combination of a dibenzocyclooctin (DBCO) group-functionalized multi-arm PEG and a norbornene group-functionalized multi-arm PEG, and the second precursor is a tetrazine group-functionalized multi-arm PEG.

[0295] The first and second formulations may be solutions or suspensions of the precursor and other optional components, each having a polymer precursor concentration of about 2.5% to about 30% by weight, such as about 5 to 20% by weight or about 10% by weight, based on the total weight of the formulation. In embodiments, the combined first and second formulations each have a polymer precursor concentration of about 5% to about 30% by weight, such as about 5 to 25% by weight, based on the total weight of the formulation. These concentration ranges can be suitably adapted as desired to achieve sufficient injectability and viscosity, depending on the processing means, such as the size of the needle used.

[0296] In certain embodiments, the kit comprises three vials: one containing a lyophilized bevacizumab (or other activator) solid; a second vial containing a solution of a multi-arm DBCO-functionalized PEG precursor such as 8a15kPEG-DG; and a third vial containing a solution of a mixture of a tetrazine-functionalized PEG precursor such as 8a20kPEG-TET and an azide-functionalized PEG precursor such as 8a20kPEG-N3. A fourth vial containing a solvent for reconstituting the activator or dissolving / diluting the precursor formulation may also be included in the kit.

[0297] Certain aspects of further embodiments of the present invention are described in the following set of embodiments, which can be combined with each other in all embodiments. First set of embodiments 1. A sustained-release drug delivery system comprising a bioorthogonal hydrogel and at least one activator, wherein the bioorthogonal hydrogel comprises a covalently crosslinked polymer network and contains the at least one activator, and the polymer network comprises a) Multiple polymer units, b) A chemical bond that can be cleaved by hydrolysis, wherein at least one of the hydrolyzable bonds is an ester bond, c) The sustained-release drug delivery system comprising a crosslink formed by a click chemical reaction. 2. The system according to embodiment 1, wherein the polymer network comprises a multi-arm unit, each having a core unit X and three or more arms attached to the core unit, preferably in the range of 3 to 10, 4 to 8, or 4 to 6 arms, and crosslinks formed by a click chemical reaction are located on each arm. 3. The system according to embodiment 2, wherein the crosslinks formed by the click chemical reaction are located at the end of each arm of the multi-arm unit. 4. The system according to embodiment 2 or 3, wherein the polymer network comprises a first, second, and optionally more multi-arm units, and the crosslinks formed by the click chemical reaction are between the arms of the first multi-arm unit and the arms of the second or more multi-arm units. 5. The system according to embodiment 4, wherein the first multi-arm unit and the second multi-arm unit have the same or different number of arms and / or the same or different core unit X. 6. The system according to embodiments 2 to 5, wherein at least one of the multi-arm units is a polymer multi-arm unit having a polymer arm containing a polymer unit. 7. The system according to embodiment 6, wherein the polymer multi-arm unit has an average molecular weight of 1,000 to 100,000 daltons. 8. The system according to any of the prior embodiments, wherein the polymer units are selected from polyethylene glycol (PEG), polyethylene oxide, polypropylene oxide, polypropylene glycol (PPG), poly(ethylene glycol)-block-poly(propylene glycol)-block copolymer, polyvinyl alcohol (PVA), polyvinyl acetate, poly(vinylpyrrolidinone) (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-coglycolic acid (PLGA), p-dioxanone, trimethylene carbonate, caprolactone, random or block copolymers, or any combination or mixture thereof, or at least one of polyamino acids, glycosaminoglycans, polysaccharides, or proteins. 9. The system according to any of the prior embodiments, wherein the polymer unit is selected from at least one of polyethylene glycol (PEG) units, polypropylene glycol (PPG) units, polyglycolic acid (PGA), polylactic acid (PLA), or polylactic acid-coglycolic acid (PLGA), random or block copolymers, and / or combinations or mixtures thereof, preferably polyethylene glycol units. 10. The system according to any of the prior embodiments, wherein the covalently crosslinked polymer network comprises a plurality of hydrophobic polymer units and / or hydrophilic polymer units. 11. The system according to embodiment 10, wherein the hydrophobic polymer unit is selected from at least one of polylactic acid (PLA) and polylactic acid-coglycolic acid (PLGA) units. 12. The system according to any one of embodiment 10 or 11, wherein the hydrophilic polymer unit is selected from at least one of polyethylene glycol unit, polypropylene glycol unit, or polyglycolic acid (PGA), and is preferably polyethylene glycol unit. 13. The system according to any one of the prior embodiments, wherein each of the polymer units has an average molecular weight (Mn) in the range of about 1,000 to about 100,000 daltons, or about 2,000 to about 50,000 daltons, or about 3,000 to about 30,000 daltons. 14. The system according to any one of embodiments 4 to 13, wherein the average molecular weight (Mn) of the arms of the first multi-arm unit and the arms of the second multi-arm unit are the same or different. 15. The system according to any one of embodiments 4 to 14, wherein one of the first or second multi-arm units has a molecular weight of less than 1,000 daltons (and is thus a small molecule multi-arm unit), and the other multi-arm unit is a polymer multi-arm unit having an average molecular weight (Mn) of more than 5,000 daltons. 16. The system according to any one of embodiments 2 to 15, wherein the hydrolyzable ester bond is part of a linker unit L located between the polymer unit end of the arm of the multi-arm unit and the crosslink formed by the click chemical reaction. 17. The system according to any one of embodiments 4 to 16, wherein the first multi-arm unit and / or the second multi-arm unit includes a linker unit L containing the hydrolyzable ester bond, and the linker unit L may be the same or different in the first multi-arm unit and the second multi-arm unit. 18. The system according to any one of embodiment 16 or 17, wherein the linker unit L comprises a dibasic acid, or an acid and amide group such as succinic acid, glutaric acid, adipic acid, azelaic acid dibasic acid, or acid amide. 19. The linker unit L is given by equation (i): [ka] It includes a structure represented by the formula, where U 1 and U 2 However, independently, NH or O can be the same or different, U 1 and U 2The system according to any one of embodiments 16 to 18, wherein at least one of the elements is O and t is an integer between 0 and 10. 20. The polymer network is given by formula (ii): [ka] A system according to any one of embodiments 1 to 19, comprising a multi-arm unit represented by the formula, wherein X is the core unit of the multi-arm unit, Y is a polymer unit, L is a linker unit containing the hydrolyzable ester bond, A is a crosslink formed by a click chemical reaction, p is either 0 or 1, and q is an integer from 3 to 10. 21. The system according to any one of embodiments 2 to 15, wherein the core unit X is derived from a polyol having at least three hydroxyl groups, and the arm is connected to the hydroxyl groups, or the core unit X is derived from a polyamine having at least two amine groups, and the arm is connected to the amine groups. 22. The system according to embodiment 16, wherein the core unit X is derived from glycerol, pentaerythritol, xylitol, dipentaerythritol, tripentaerythritol, or hexaglycerol, isomalt, lactitol, maltitol, mannitol, or sorbitol, preferably pentaerythritol, or from a polyamine such as methylenediamine, ethylenediamine, 1,3-diaminopropane, putrescine, cadaverine, hexamethylenediamine, or 1,2-diaminopropane, or from a triamine such as diethylenetriamine, propane-1,2,3-triamine, propane-1,1,3-triamine, pentane-1,3,5-triamine, or from another polyamine such as triethylenetramine, butane-1,2,3,4-tetramine, or butane-1,1,4,4-tetramine. 23. The system according to any one of the prior embodiments, wherein the crosslink is formed by a click chemical reaction such as a copper-free SPAAC or IEDDA reaction, the first functional group of the first multi-arm precursor or unit reacts with at least one complementary second functional group of the second multi-arm precursor or unit, the second complementary functional group can react with the first functional group in a click chemical reaction, and the first and second functional groups are selected from a pair comprising an alkyne moiety and an azide such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonine (BCN), or norbornene or transcyclocten (TCO) and a tetrazine (Tz) moiety. 24. The system according to any one of the prior embodiments, wherein the crosslinking bond formed by the click chemical reaction comprises a chemical group selected from triazole and dihydropyridazine. 25. The system according to any one of the prior embodiments, wherein the activator is selected from the group consisting of therapeutic or diagnostic activators. 26. The system according to any one of embodiments 1 to 25, wherein the activator is a small molecule activator or a biomolecule activator, preferably a small molecule activator, a peptide, a protein, or a virus. 27. The activator is a steroid; nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, meclofename, mefanamic A, salsalate, sulindac, tolmetin, ketoprofen, diflunisal, piroxicam, naproxen, etodrug, fluviprofen, fenoprofen C, indomethacin, celecoxib, ketrolac, nepafenac; intraocular pressure lowering agents; antibiotics such as ciprofloxacin; analgesics such as bupivacaine; calcium channel blockers such as nifedipine; cell cycle inhibitors such as simvastatin; proteins such as insulin; small molecule hydrophilic drugs including carboxylates and amine salts; small molecule hydrophobic drugs, A system according to any one of the prior art embodiments, selected from thrin, hydrophilic peptides and protein drugs such as single-chain antibody fragments, Fab fragments, IgG antibodies, and fusion antibodies; aptamers; in particular bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, antibiotics, analgesics, calcium channel blockers, cell cycle inhibitors, chemotherapeutic agents, antiviral agents, anesthetics, hormones, anticancer agents, antitumor agents, viruses, genetically modified viruses such as AAV, nanobodies, aphibodies, ankyrin, DARPins, or any combination thereof. 28. The system according to any one of the prior art embodiments, wherein the activator is a peptide selected from the group consisting of Compstatin, APL-1, and Fc-III-4C, Beob 15 (brolucizumab), Zimura (abacincaptado pegol), pegcetacoplan, abisipal pegol, lamparizumab, Fovista, listiganib, AXT107, elamipretide, THR149, ALM201, VGB3, and Largazole. 29. The system according to any one of embodiments 1 to 28, wherein the activator is bevacizumab. 30. The system according to any one of embodiments 1 to 29, wherein the activator is contained in the hydrogel without being covalently conjugated to the hydrogel, or the activator is contained in the hydrogel and covalently conjugated to the hydrogel. 31. The system according to any one of embodiments 1 to 30, wherein the activator is contained in the hydrogel without being covalently conjugated to the hydrogel, and preferably, the activator is contained in the hydrogel without being covalently conjugated to the hydrogel via an oligonucleotide. 32. The system according to any one of embodiments 1 to 31, wherein the polymer network of the bio-orthogonal hydrogel is formed by reacting a first multi-arm precursor having a functional group containing an azide (N3) moiety with a second multi-arm precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety. 33. The system according to embodiment 32, wherein the polymer network of the bio-orthogonal hydrogel is formed by simply reacting a first multi-arm precursor having a functional group containing an azide (N3) moiety with a second multi-arm precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety. 34. The system according to any one of embodiments 1 to 33, wherein the polymer network of the bio-orthogonal hydrogel is formed by reacting a first multi-arm precursor having a functional group containing an azide (N3) moiety with a second multi-arm precursor having a functional group containing dibenzocyclooctin (DBCO), and the activator is a small molecule activator, a peptide, a protein, or a virus. 35. The system according to any one of embodiments 1 to 33, wherein the polymer network of the bio-orthogonal hydrogel is formed by reacting a first multi-arm precursor having a functional group containing an azide (N3) moiety with a second multi-arm precursor having a functional group containing dibenzocyclooctin (DBCO), and the activator is contained in the hydrogel without being covalently conjugated to the hydrogel, preferably the activator is contained in the hydrogel without being covalently conjugated to the hydrogel via an oligonucleotide. 36. The system according to any one of embodiments 1 to 33, wherein the polymer network of the hydrogel is formed by reacting a first multi-arm precursor having a functional group including an azide (N3) moiety with a second multi-arm precursor having a functional group including a dibenzocyclooctin (DBCO) moiety, and the activator is contained in the hydrogel without being covalently conjugated to the hydrogel via an oligonucleotide. 37. The system according to any one of embodiments 1 to 33, wherein the polymer network of the hydrogel is formed by reacting at least one first multi-arm precursor having a functional group comprising an azide (N3) moiety and / or a tetrazine moiety with a second multi-arm precursor having a functional group comprising a dibenzocyclooctin (DBCO) moiety, wherein the first multi-arm precursor having a functional group comprising an azide moiety is not derived from pentaerythritol tetrakis (2-bromoisobutyrate), the activator is a small molecule activator, a peptide, a protein, or a virus, and the activator is contained in the hydrogel without being covalently conjugated to the hydrogel via an oligonucleotide. 38. The first multi-arm precursor having a functional group including the azide (N3) moiety, [ka] A system according to any one of embodiments 32 to 37, selected from the group consisting of, where n is 1 to 2300, t is an integer from 0 to 10, or the corresponding 8-arm structure. 39. The system according to any one of embodiments 32 to 37, wherein the first multi-arm precursor having a functional group containing the azide (N3) moiety is selected from the group consisting of 8a5kPEG-N3, 8a10kPEG-N3, 8a15kPEG-N3, 8a20kPEG-N3, 4a20kPEG-N3, 4a20kPEG-PEG2N3S, 4a20kPEG-PEG2N3G, 4a20kPEG-PEG2N3AP, and 4aPEG-PEG2N3AZ. 40. The second multi-arm precursor having a functional group containing the dibenzocyclooctin (DBCO) moiety, [ka] A system according to any one of embodiments 32 to 37, selected from the group consisting of, where n is 1 to 2300, t is an integer from 0 to 10, or the corresponding 8-arm structure. 41. The second multi-arm precursor having a functional group containing the dibenzocyclooctin (DBCO) moiety is 4a20kPEG-D, 6a20kPEG-D, 8a20kPEG-D, 8a15kPEG-DG, 4a20kPEG-DS, 8a10kPEG-DG, 4a20kPEG-DG, 4a20kPEG-DAP, 4a20kPEG-DAZ, 4a20kPEG-DPEG 23 A system according to any one of embodiments 32 to 37 or 40, selected from the group consisting of AZ and 4a20kPEG-sDG. 42. The polymer network of the bio-orthogonal hydrogel is formed by reacting at least one, preferably one, of the following first multi-arm precursors having a functional group containing an azide (N3) moiety with at least one, preferably one, of the following second multi-arm precursors having a functional group containing a dibenzocyclooctin (DBCO) moiety: [ka] [ka] The system according to any one of embodiments 1 to 37, wherein n is between 1 and 2300, t is an integer between 0 and 10, or the above eight-arm version or the corresponding eight-arm structure. 43. The polymer network of the bio-orthogonal hydrogel is 8a10kPEG-N 3、At least one, preferably one, first multi-arm precursor selected from the group consisting of 4a20kPEG-N3, 4a20kPEG-PEG2N3S, 4a20kPEG-PEG2N3G, 4a20kPEG-PEG2N3AP, and 4aPEG-PEG2N3AZ, 8a20kPEG-Tetrazine, and 8a20kPEG-N3 is used to create a first multi-arm precursor consisting of 4a20kPEG-D, 6a20kPEG-D, 8a20kPEG-D, 4a20kPEG-DS, 8a10kPEG-DG, 4a20kPEG-DG, 8a15kPEG-DG, 4a20kPEG-DAP, 4a20kPEG-DAZ, and 4a20kPEG-DPEG 23 The system according to any one of embodiments 1 to 37 or 42, formed by reacting with at least one, preferably one, second multi-arm precursor selected from the group consisting of AZ, 4a20kPEG-sDG, and 4a20kPEG-PEG2BG. 44. The polymer network of the bio-orthogonal hydrogel is formed by reacting at least one, preferably one, of the following first multi-arm precursors having a functional group containing an azide (N3) moiety with at least one, preferably one, of the following second multi-arm precursors having a functional group containing a dibenzocyclooctin (DBCO) moiety: [ka] [ka] The system according to any one of embodiments 1 to 37, wherein x and y define the number of PPG, PEG, PLA, or PGA units, and t is an integer from 0 to 10, or the corresponding 8-arm structure. 45. The system according to any one of embodiments 1 to 37 or 44, wherein the first multi-arm precursor having a functional group containing the azide (N3) moiety is selected from the group consisting of 4a18Tetronic1307-PEG2N3, 4a20kPLA-PEG2N3, 8a20k50:50PLGA-PEG2N3, and 4a20kPLA-PEG2N3S. 46. ​​The system according to any one of embodiments 1 to 37 or 44, wherein the second multi-arm precursor having a functional group containing the dibenzocyclooctin (DBCO) moiety is selected from the group consisting of 4a20kPEG-DAZ, 8a20kPEG-D, and 4a18Tetronic1307-DAZ. 47. The system according to any one of embodiments 1 to 37 or 44, wherein the polymer network of the bio-orthogonal hydrogel is formed by reacting at least one, preferably one, first multi-arm precursor having a functional group containing an azide (N3) moiety selected from the group consisting of 4a18Tetronic1307-PEG2N3, 4a20kPLA-PEG2N3, 8a20k50:50PLGA-PEG2N3, and 4a20kPLA-PEG2N3S with at least one, preferably one, second multi-arm precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety selected from the group consisting of 4a20kPEG-DAZ, 8a20kPEG-D, and 4a18Tetronic1307-DAZ. 48. A method for producing a sustained-release drug delivery system defined in any one of embodiments 1 to 47, comprising a bio-orthogonal hydrogel and at least one activator described in any one of the prior embodiments, wherein the method is a) A step of preparing a first formulation comprising a first multi-arm precursor having a first functional group suitable for forming a link with a second functional group by click chemistry, b) Providing a second formulation comprising a second multi-arm precursor having a second functional group suitable for forming a link with a first functional group by a click chemical reaction, c) The step of combining the first and second formulations, d) Adding at least one activator to the first, second, or combined first and second formulations, thereby forming a combined reaction mixture; e) The step of enabling the combined reaction mixture to gel by forming a click chemical link between the first functional group and the second functional group, thereby forming a hydrogel containing the at least one activator, At least one of the first multi-arm precursor or the second multi-arm precursor is a polymer multi-arm precursor having polymer arms containing polymer units, The method wherein at least one of the first multi-arm precursor or the second multi-arm precursor comprises a linker unit L containing a hydrolyzable ester bond. 49. The method according to embodiment 48, wherein at least one third multi-arm precursor having a first or second functional group suitable for forming a click chemical bond with either the first multi-arm precursor or the second multi-arm precursor is added in one of steps a) to c). 50. The method according to embodiment 48 or 49, wherein the first and / or second formulation, preferably both, comprises at least one solvent. 51. The method according to embodiment 50, wherein at least one solvent is selected from acetone, acetonitrile, benzyl alcohol, chloroform, dichloromethane (DCM), dioxane, dimethyl carbonate (DMC), dimethyl sulfoxide (DMSO), methanol, ethanol or propanol, ethyl acetate, ethyl formate, ethyl propionate, glycoflor, hexafluoroisopropanol, isorpide dimethyl ether, isopropanol, methyl chloride, methylene chloride, methyl ethyl ketone, N-methylpyrrolidone, propylene carbonate, triethylamine, or tetrahydrofuran; a buffered aqueous medium such as water, PBS buffer or physiological saline; a sugar buffer; or any mixture thereof. 52. The method according to embodiment 51, wherein the at least one solvent is selected from water, a buffered aqueous medium such as PBS buffer or physiological saline, or a sugar buffer, or any mixture thereof. 53. The method according to any one of embodiments 48 to 52, further comprising the step of removing the solvent or solvent mixture. 54. The method according to embodiment 53, wherein the removal of the solvent is carried out by any of the following: nitrogen or other inert gas convection, hot air convection or direct drying, indirect or contact drying, spray drying, dielectric drying, vacuum drying, freeze drying, freeze-drying, supercritical or superheated steam drying, or any combination thereof. 55. The method according to any one of embodiments 48 to 54, wherein the multi-arm precursors, their cores and polymer units, linker L, crosslinking agent, and activator are as defined in system embodiments 2 to 47. 56. A sustained-release drug delivery system according to any one of embodiments 1 to 47, for use as a pharmaceutical product. 57. A method of treatment, comprising treating a disease or medical condition in a patient using a sustained-release drug delivery system described in any one of embodiments 1 to 47. 58. A sustained-release drug delivery system or method of treatment for use according to embodiment 56 or 57, wherein the sustained-release drug delivery system is used for the treatment of the eye. 59. A sustained-release drug delivery system or method of treatment for use according to any one of embodiments 56 to 58, used for the treatment of ocular diseases such as posterior segment diseases, in particular age-related macular degeneration (AMD), cystoid macular edema (CME), diabetic macular edema (DME), posterior uveitis, and diabetic retinopathy, which are caused by aging, trauma, or surgical intervention. 60. The drug delivery system is effective in treating retinal neovascularization, choroidal neovascularization, exudative AMD, atrophic 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, and macular edema. Acute multiple maculoplasty, Behçet's disease, birdshot choroidopathy, posterior uveitis, posterior scleritis, serpentine choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusive disease, central retinal vein occlusion, disseminated intravascular coagulation, branched retinal vein occlusion, hypertensive fundus changes, ischemic syndrome, retinal artery microaneurysms, Coats' disease, parafoveal telangiectasia, hemiretinal vein occlusion, papillary phlebitis, carotid artery disease (CAD), dendritic vasculitis, sickle vein Erythrocytic retinopathy, retinal pigment streaks, familial exudative vitreoretinopathy, Eels' disease, proliferative vitreoretinopathy, diabetic retinopathy, tumor-related retinal diseases, congenital hypertrophy of the retinal pigment epithelium (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, complex hamartoma of the retina and retinal pigment epithelium, retinoblastoma, fundus vascular proliferative neoplasm, retinal astrocytoma, ocular lymphoid neoplasm, myopic retinal degeneration, acute retinitis pigmentosa, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal cancer. A sustained-release drug delivery system or method of treatment for use according to any one of embodiments 56 to 59, used for the treatment of an ocular disease selected from the group consisting of retinitis pigmentosa, Leber congenital amaurosis, choroidal atrophy, X-linked 106 retinitis pigmentosa, Best vitiligo macular dystrophy, X-linked retinoschisis, CNGA3 color blindness, CNGB3 color blindness, LHON, Stargardt disease, Usher syndrome, Norrie disease, Valday-Beedl syndrome, and red-green color blindness. 61. A sustained-release drug delivery system or method of treatment for use according to any one of embodiments 56 to 60, wherein the drug delivery system is formulated for direct injection at the treatment site of the patient. 62. A sustained-release drug delivery system or method of treatment for use according to embodiment 61, wherein the drug delivery system is formulated for injection into the eye, such as parenteral administration, intratumoral injection, intravitreal injection, anterior chamber injection, subconjunctival injection, retrobulbar injection, subtenon's capsule injection, subretinal injection, or choroidal injection. 63. A sustained-release drug delivery system or method of treatment for use according to any one of embodiments 56 to 62, wherein the drug delivery system is administered by direct injection, by oral administration, or incorporated into an implant. 64. A sustained-release drug delivery system or method of treatment for use according to any one of embodiments 56 to 63, wherein the drug delivery system is administered into the patient's body space by any one of the following means: subcutaneous, intramuscular, intrathecal, epidural, intraperitoneal, intradermal, subcutaneous, intercostal, intra-articular, intrasynovial, intravertebral, oral, nasal, transrectal, intratumoral, or vaginal administration. 65. A sustained-release drug delivery system or method of treatment for use according to embodiment 64, wherein the intra-articular administration is to a joint selected from the knee, elbow, hip, sternoclavicular, temporomandibular joint, carpal, tarsal, wrist, ankle, intervertebral disc, or ligamentum flavum. 66. A sustained-release drug delivery system for use or method of treatment according to any one of embodiments 56 to 65, wherein the drug delivery system comprises two or more different active agents. 67. A sustained-release drug delivery system or method of treatment for use in combination therapy involving the administration of two or more activators, as described in embodiment 66. 68. A sustained-release drug delivery system or method of treatment for use according to any one of embodiments 56 to 67, wherein the hydrogel of the drug delivery system is formed in situ at the treatment site of the patient. 69. The sustained-release drug delivery system or method of treatment for use according to embodiment 68, wherein the hydrogel is formed in situ at the treatment site of a patient by combining a first formulation comprising a first mixture according to step a) of any one of embodiments 48 to 55 and a second formulation comprising a second mixture according to step b) of any one of embodiments 48 to 55, thereby enabling the combined formulation mixture to gel in situ at the treatment site. 70. A sustained-release drug delivery system or method of treatment for use according to embodiment 69, wherein the first formulation and the second formulation are combined immediately before administering the formulation mixture at the treatment site. 71. The sustained-release drug delivery system or method of treatment for use according to embodiment 70, wherein the first formulation is in a first syringe, the second formulation is in a second syringe, and the two formulations are combined in a Y-mixer before direct injection at the treatment site. 72. A sustained-release drug delivery system or method of treatment for use according to embodiment 69, wherein the first formulation and the second formulation are each administered at the treatment site, thereby enabling the formulations to be combined at the treatment site, and the combined formulation to gel in situ at the treatment site. 73. The sustained-release drug delivery system or method of treatment for use according to embodiment 72, wherein the first formulation is in a first syringe, the second formulation is in a second syringe, and the two formulations are injected simultaneously or sequentially at the treatment site. 74. A sustained-release drug delivery system or method of treatment for use according to embodiment 68, wherein the hydrogel is formed in situ at the treatment site of a patient by administering a suspension comprising a first mixture according to step a) of any embodiment 48 to 55, a second mixture according to step b) of any embodiment 48 to 55, and a hydrophobic organic liquid to the treatment site of the patient. 75. The sustained-release drug delivery system or method of treatment for use according to embodiment 74, wherein the hydrophobic organic liquid is contained in at least one of the first or second mixtures. 76. The hydrophobic organic liquid is triethyl citrate, acetyl triethyl citrate (ATEC), acetyl tributyl citrate (ATBC), α-tocopherol (vitamin E), α-tocopherol acetate; plant or vegetable oils such as sesame oil, olive oil, soybean oil, sunflower oil, coconut oil, canola oil, and rapeseed oil; nut oils such as hazelnut, walnut, pecan, and almond oil; cottonseed oil, corn oil, linseed oil, ethyl oleate, castor oil and its derivatives (Cremofor®); saturated or unsaturated fatty acids; mono A sustained-release drug delivery system or method of treatment for use according to embodiment 74 or 75, comprising lipids that are liquid at 37°C or below, such as glycerides, diglycerides, triglycerides (Myglyols®), phospholipids, glycerophospholipids, sphingolipids, sterols, prenols, and polyketides; a biocompatible oil selected from the group consisting of hydrophobic biodegradable liquid polymers (such as low molecular weight PLGA, PGA, or PLA), low-melting-point waxes such as plant, animal, or synthetic waxes, lanolin, jojoba oil, or combinations thereof. 77. A sustained-release drug delivery system or method of treatment for use according to any one of embodiments 74 to 76, wherein the activator is dissolved or dispersed in the hydrophobic organic liquid, or the activator is the hydrophobic organic liquid or forms at least a part thereof. 78. A kit for forming a hydrogel as defined in any one of the prior embodiments, comprising an activator as defined in any one of the prior embodiments, wherein the kit is in a separate container, a) A first precursor formulation comprising at least one first multi-arm precursor as defined in any one of the preceding embodiments, having a first functional group suitable for forming a link with a complementary second functional group by click chemistry, and optionally, at least one activator as defined in any one of the preceding embodiments, b) A second precursor formulation comprising at least one second multi-arm precursor as defined in any one of the preceding embodiments, having the first functional group for mixing and gel formation and a second functional group suitable for forming a linkage by click chemistry, c) Optionally, if not included in a), comprising at least one activator as defined in any of the prior art embodiments, The kit wherein at least one of the first multi-arm precursor or the second multi-arm precursor is a polymer multi-arm precursor having a polymer arm comprising a polymer unit, and at least one of the first multi-arm precursor or the second multi-arm precursor comprises a linker unit L comprising a hydrolyzable ester bond. 79. The kit according to embodiment 78, wherein the first formulation is provided in a first syringe, the second formulation is provided in a second syringe, and the syringes are connected via a Y-type mixer to combine the two formulations. 80. The kit according to embodiment 79 for use in a method for preparing an activator that releases a hydrogel in situ at a treatment site within a patient's body.

[0298] Second set of embodiments In one embodiment, the present invention relates to a kit suitable for use in forming hydrogels in situ for sustained-release drug delivery, as described below. 1. A kit suitable for use in forming a hydrogel in situ for sustained-release drug delivery containing an activator, wherein the kit is in a separate container: a) A first precursor formulation comprising at least one first multi-arm precursor having a complementary second functional group and a first functional group suitable for forming a linkage by click chemistry, and a first container optionally comprising at least one solvent, b) A second precursor formulation comprising at least one second multi-arm precursor having a second functional group suitable for forming a link with the first functional group by a click chemical reaction, and a second container optionally comprising at least one solvent, c) comprising at least one activator contained in a separate third container and / or contained in at least one or both of the first and second containers, and optionally at least one solvent, The kit wherein at least one of the first multi-arm precursor and / or the at least one second multi-arm precursor is a polymer multi-arm precursor having a polymer arm comprising a polymer unit, and at least one of the first multi-arm precursor or the second multi-arm precursor comprises a linker unit L comprising a hydrolyzable ester bond. 2. The kit according to embodiment 1, wherein the at least one solvent in the first, second, or third container is independently selected from water, PBS buffer, TBS buffer, or a buffered aqueous medium such as physiological saline, or any mixture thereof, and optionally comprises at least one additive selected from pharmaceutical excipients, sugars, salts, additional therapeutic or diagnostic agents, thickeners, viscosity enhancers, stabilizers, or auxiliaries. 3. The kit according to any one of embodiment 1 or 2, wherein the at least one first precursor formulation comprises a mixture of two or more first precursors, each having different first functional groups that are suitable for forming a link with a complementary second functional group by click chemistry but do not react with each other. 4. The kit according to any one of embodiment 1 or 2, wherein the at least one second precursor formulation comprises a mixture of two or more second precursors, each having different second functional groups that are suitable for forming a link with a complementary first functional group by click chemistry but do not react with each other. 5. The kit according to any one of embodiment 3 or 4, wherein the different first or second functional groups are selected to have different kinetic rate constants or reaction rates in their click reactions with the corresponding second or first functional group counterparts. 6. The kit according to any one of embodiments 1 to 5, wherein the first and second functional groups are selected to provide a gel time from combining the first and second precursor formulations of at least about 1 minute, for example, about 2 minutes or more, or about 3 minutes or more, for example, about 1 to 15 minutes, for example, about 2 to 10 minutes, 2 to 8 minutes, or 2 to 6 minutes, or 2 to 4 minutes. 7. A kit according to any one of embodiments 1 to 5, wherein the first formulation is provided in a first syringe, the second formulation is provided in a second syringe, and the syringes are connected via a Y-type mixer to combine the two formulations. 8. The kit according to any one of embodiments 1 to 6, wherein the activator exists as a solid, such as obtained from a freeze-dried mixture that is suitable for reconstitution in an aqueous solution before use. 9. The kit according to any one of embodiments 1 to 8, wherein the at least one first precursor and the at least one second precursor are selected independently of each other or from multi-arm polyethylene glycol (PEG) precursors having 3 to 10 PEG arms, such as 4 to 8 PEG arms. 10. The kit according to any one of embodiments 1 to 8, wherein the at least one first precursor and the at least one second precursor are independently selected from each other to be 3 to 10 arm precursors of polymer units selected from polypropylene glycol (PPG), poly(ethylene glycol)-block-poly(propylene glycol) copolymer, poloxamers such as Tetronic®, polyvinyl alcohol (PVA), polyvinyl acetate, poly(vinylpyrrolidinone) (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-coglycolic acid (PLGA), p-dioxanone, trimethylene carbonate, caprolactone, random or block copolymers, or any combination or mixture thereof, or one or more units of polyamino acids, glycosaminoglycans, polysaccharides, or proteins. 11. The first and second multi-arm precursors independently have an average molecular weight (M) of 5,000 to 100,000 daltons, such as 5,000 to 40,000 daltons and 10,000 to 40,000 daltons. n A kit according to any one of embodiments 1 to 10, having ) 12. The kit according to any one of the prior art, wherein the linker unit L comprises a dibasic acid, or an acid and amide group such as succinic acid, glutaric acid, adipic acid, azelaic acid, or an acid amide thereof such as glutaramide. 13. The linker unit L is given by equation (i): [ka] It includes a structure represented by the formula, where U 1 and U 2 However, independently, it is NH or O, and U 1 and U 2 A kit according to any one of the prior embodiments, wherein at least one of the is O, preferably both are O, and t is an integer from 0 to 10. 14. The kit according to any one of the prior embodiments, wherein the first and second formulations each have a concentration of the polymer precursor of about 2.5% to about 30% by weight, such as about 5% to 20% by weight or about 10% by weight, based on the total weight of the formulation. 15. The kit according to any one of the prior embodiments, wherein the combined first and second formulations each have a concentration of the polymer precursor of about 5% to about 30% by weight, such as about 5% to 25% by weight, based on the total weight of the formulations. 16. The kit according to any one of the prior embodiments, wherein the first and second functional groups are selected from pairs comprising a strained alkyne moiety and azide such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonine (BCN), or norbornene or transcyclooctene (TCO) and tetrazine (Tz) moiety, or a strained alkyne moiety and tetrazine (Tz) moiety such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonine (BCN), or any combination thereof. 17. The system according to embodiment 16, wherein the first precursor is a dibenzocyclooctin (DBCO) group-functionalized multi-arm PEG, and the second precursor is a combination of a tetrazine group-functionalized multi-arm PEG and an azide group-functionalized multi-arm PEG, or the first precursor is a combination of a dibenzocyclooctin (DBCO) group-functionalized multi-arm PEG and a norbornene group-functionalized multi-arm PEG, and the second precursor is a tetrazine group-functionalized multi-arm PEG. 18. The kit according to any one of the prior embodiments, wherein the activator is contained in the hydrogel without being covalently conjugated to the hydrogel, or the activator is contained in the hydrogel and at least partially covalently conjugated to the hydrogel. 19. The kit according to any one of the prior embodiments, wherein the activator is selected from the group consisting of therapeutic or diagnostic activators such as those defined above, or the therapeutic activator is a small molecule activator or biomolecule activator, preferably a small molecule activator, peptide, protein, or virus, preferably a peptide selected from the group consisting of Compstatin, APL-1, and Fc-III-4C, Beob 15 (brolucizumab), bevacizumab (avastin), Zimura (abacincaptado pegol), pegcetacoplan, avisical pegol, lamparizumab, Fovista, listiganib, AXT107, elamipretide, THR149, ALM201, VGB3, and largazole, preferably bevacizumab. 20. A kit according to any one of embodiments 1 to 19 for use in a method of forming an activator that releases a hydrogel in situ at a treatment site within a patient's body. 21. A sustained-release drug delivery hydrogel available from a kit described in any one of embodiments 1 to 19.

[0299] Third set of embodiments In another embodiment, the present invention relates to a method of treating a disease or condition in a patient by forming a hydrogel in situ for sustained-release drug delivery at a treatment site within the patient's body, as defined below: 1. A method for treating a disease or condition in a patient by forming a hydrogel in situ for sustained-release drug delivery at a treatment site within the patient's body, wherein the method is a) Forming a reaction mixture by combining a first precursor formulation comprising at least one solvent and at least one first multi-arm precursor having a first functional group suitable for forming a link with a complementary second functional group by click chemistry, in the presence of an activator, and optionally a second precursor formulation comprising at least one solvent and at least one second multi-arm precursor having a second functional group suitable for forming a link with the first functional group by click chemistry, b) Injecting the reaction mixture into the treatment site of the patient, c) enabling the reaction mixture to gel at the treatment site, The method wherein at least one of the first multi-arm precursor and / or the at least one second multi-arm precursor is a polymer multi-arm precursor having a polymer arm comprising a polymer unit, and at least one of the first multi-arm precursor or the second multi-arm precursor comprises a linker unit L comprising a hydrolyzable ester bond. 2. The method according to Embodiment 1, wherein the first and second precursors, the first and second functional groups, the activator, the solvent, and the linker L are as defined in the first or second set of embodiments. 3. The aforementioned diseases or conditions are non-ocular diseases, or retinal neovascularization, choroidal neovascularization, exudative AMD, atrophic 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 tumor, retinal neuritis, inflammation, autoimmune uveitis, uveitis, proliferative vitreoretinopathy, and corneal degeneration, acute and chronic macular neuroretinopathy, central Serous chorioretinopathy, macular edema, acute multiple macular pigment epitheliopathy, Behçet's disease, birdshot chorioretinopathy, posterior uveitis, posterior scleritis, serpentine choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusive disease, central retinal vein occlusion, disseminated intravascular coagulation, branched retinal vein occlusion, hypertensive fundus changes, ischemic syndrome, retinal artery microaneurysms, Coats' disease, parafoveal telangiectasia, hemiretinal vein occlusion, papillary phlebitis Carotid artery disease (CAD), dendritic vasculitis, sickle cell retinopathy, retinal pigment streaks, familial exudative vitreoretinopathy, Eels' disease, proliferative vitreoretinopathy, diabetic retinopathy, tumor-related retinal diseases, congenital hypertrophy of the retinal pigment epithelium (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, complex hamartoma of the retina and retinal pigment epithelium, retinoblastoma, vascular proliferative neoplasm of the fundus, retinal astrocytoma, ocular lymphoid neoplasm, myopic retinal degeneration, acute retinal pigment epithelium The method according to embodiment 1 or 2, wherein the eye disease is selected from the group consisting of dermatitis, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal cancer, retinitis pigmentosa, Leber congenital amaurosis, choroidal atrophy, X-linked 106 retinitis pigmentosa, Best vitiligo macular dystrophy, X-linked retinoschisis, color blindness CNGA3, color blindness CNGB3, LHON, Stargardt disease, Usher syndrome, Norrie disease, Valday-Beedl syndrome, and red-green color blindness. 4. The method according to any one of the preceding embodiments, wherein the combined reaction mixture is administered into the body space of a patient for the treatment of an ocular disease by one of the following injections: intravitreous, intrachorium, subconjunctival, retrobulbar, subtenon's capsule, subretinal, or suprachoroidal, or for the treatment of a non-ocular disease by one of the following injections: subcutaneous, intramuscular, intrathecal, epidural, intraperitoneal, intradermal, subcutaneous, intercostal, intra-articular, synovial, intraspinal, or intratumor. 5. The method according to embodiment 4, wherein the intra-articular administration is to a joint selected from the knee, elbow, hip, sternoclavicular, temporomandibular joint, carpal, tarsal, wrist, ankle, intervertebral disc, or ligamentum flavum. 6. The method according to any one of the prior embodiments, wherein the kit described in any one of the first set of embodiments 1 to 19 is used. 7. The method according to any one of the preceding embodiments, wherein the first formulation is provided in a first syringe, the second formulation is provided in a second syringe, and the syringes are connected via a Y-mixer for combining the two formulations and directly injecting the reaction mixture. 8. The method according to embodiment 7, wherein the lyophilized activator is reconstituted by adding a solvent, and the resulting solution is added to the first syringe, the second syringe, or both syringes. 9. The method according to any one of the prior embodiments, wherein the gel time is controlled by using a first precursor formulation comprising a mixture of two or more first precursors, each having different first functional groups that are suitable for forming a linkage by a click chemical reaction with a complementary second functional group but do not react with each other, or by using a single second precursor formulation comprising a mixture of two or more second precursors, each having different second functional groups that are suitable for forming a linkage by a click chemical reaction with a complementary first functional group but do not react with each other, wherein the different first or second functional groups are selected to have different kinetic rate constants or reaction rates in their click reactions with the corresponding second or first functional group counterparts.

[0300] Fourth set of embodiments In another embodiment, the present invention relates to a hydrogel for sustained-release drug delivery obtained by simultaneously gelling two chemically reactive multi-arm polymer precursors, namely a precursor having a functional group reactive by click chemistry and a precursor having a functional group crosslinked by an electrophilic-nucleophilic reaction, as defined below: 1. A hydrogel for sustained-release drug delivery comprising an activator, wherein the hydrogel comprises a cross-penetrating covalently crosslinked dual polymer network, and contains at least one activator, and the dual polymer network comprises a) Multiple polymer units, b) A chemical bond that can be cleaved by hydrolysis, wherein at least one of the hydrolyzable bonds is an ester bond, c) A crosslink formed by a click chemical reaction between a first multi-arm precursor having a reactive functional group and a second multi-arm precursor, d) The hydrogel comprising a crosslink formed by an electrophilic-nucleophilic reaction between a third multi-arm precursor having a reactive functional group and a fourth multi-arm precursor. 2. The hydrogel according to Embodiment 1, wherein the reactive functional group is selected by click chemistry from a pair comprising a strained alkyne moiety such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonane (BCN) and an azide, norbornene, or transcyclooctene (TCO) and a tetrazine (Tz) moiety, or a strained alkyne moiety such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonane (BCN) and a tetrazine (Tz) moiety, or any combination thereof. 3. The hydrogel according to embodiment 1 or 2, wherein the functional group that is reactive by electrophilic-nucleophilic reactions is selected from activated ester groups such as succinimidyl esters and succinimidyl carbonates; electrophiles such as nitrophenyl carbonate, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, epoxides, mesylates, tosylates, tresyl, cyanurates, orthopyridyl disulfide or halogens; and nucleophiles such as amines such as primary amines, hydroxyls, alcohols, thiols, azido anions, and carboxyl groups; preferably selected from amines and N-hydroxysuccinimide groups. 4. The hydrogel according to any one of the prior embodiments, wherein the dual polymer network is formed by reacting a first multi-arm precursor having a functional group containing an azide (N3) and / or tetrazine (Tz) moiety with a second multi-arm precursor having a functional group containing dibenzocyclooctin (DBCO) or norbornene (Nor) moiety in the presence of a third precursor having a terminal amine (NH2) group and a fourth multi-arm precursor having a terminal N-hydroxysuccinimide (NHS) group. 5. The hydrogel according to any one of the prior embodiments, wherein the first to fourth precursors are selected independently of each other or from multi-arm polyethylene glycol (PEG) precursors having 3 to 10 PEG arms, such as 4 to 8 PEG arms. 6. The hydrogel according to any one of the prior art, wherein the first to fourth precursors are independently selected from polypropylene glycol (PPG), poly(ethylene glycol)-block-poly(propylene glycol) copolymer, poloxamers such as Tetronic®, polyvinyl alcohol (PVA), polyvinyl acetate, poly(vinylpyrrolidinone) (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-coglycolic acid (PLGA), p-dioxanone, trimethylene carbonate, caprolactone, random or block copolymers, or any combination or mixture thereof, comprising 3 to 10 arm precursors of polymer units, or one or more units of polyamino acids, glycosaminoglycans, polysaccharides, or proteins. 7. The first to fourth multi-arm precursors independently have an average molecular weight (M) of 5,000 to 100,000 daltons, such as 5,000 to 40,000 daltons and 10,000 to 40,000 daltons. n A hydrogel according to any one of the prior art embodiments. 8. The hydrogel according to any one of the prior art, wherein the dual polymer network comprises a linker unit L containing a dibasic acid, or an acid amide such as succinic acid, glutaric acid, adipic acid, azelaic acid, or glutaramide, and an amide thereof, and an amide. 9. The dual polymer network is given by formula (i): [ka] The formula includes a linker unit L which contains a structure represented by U 1 and U 2 However, independently, it is NH or O, and U 1 and U 2 A hydrogel according to any one of the prior art, wherein at least one of the elements is O, preferably both are O, and t is an integer from 0 to 10. 10. A hydrogel according to any one of the prior embodiments, wherein the activator is contained in the hydrogel without being covalently conjugated to the hydrogel, or the activator is contained in the hydrogel and at least partially covalently conjugated to the hydrogel. 11. The hydrogel according to any one of the prior embodiments, wherein the activator is selected from the group consisting of therapeutic or diagnostic activators such as those defined above, or the therapeutic activator is a small molecule activator or biomolecule activator, preferably a small molecule activator, peptide, protein, or virus, preferably a peptide selected from the group consisting of Compstatin, APL-1, and Fc-III-4C, Beob 15 (brolucizumab), bevacizumab (avastin), Zimura (abacincaptado pegol), pegcetacoplan, avisical pegol, lamparizumab, Fovista, listiganib, AXT107, elamipretide, THR149, ALM201, VGB3, and largazole, preferably bevacizumab. 12. A hydrogel according to any one of embodiments 1 to 11, formed in situ at a treatment site within the patient's body. 13. A method for forming a hydrogel according to any one of embodiments 1 to 11, wherein the method is a) A step of preparing a first formulation comprising a first multi-arm precursor having a first functional group suitable for forming a link with a second functional group by click chemistry, and at least one activator as defined in any of the preceding embodiments, b) Providing a second formulation comprising a second multi-arm precursor having a second functional group suitable for forming a link with the first functional group by a click chemical reaction, c) A step of preparing a third formulation comprising a third multi-arm precursor having a fourth functional group and a third functional group suitable for forming a linkage by electrophilic-nucleophilic reaction, d) Providing a fourth formulation comprising a fourth multi-arm precursor having a fourth functional group suitable for forming a linkage by an electrophilic-nucleophilic reaction with the third functional group, e) The step of combining the first to fourth formulations in the presence of at least one activator, thereby forming a combined reaction mixture, f) The step of forming a click chemical crosslink between the first functional group and the second functional group, and forming a crosslink between the third functional group and the fourth functional group by an electrophilic-nucleophilic reaction, thereby enabling the combined reaction mixture to gel and thereby form a hydrogel containing the at least one activator, The first to fourth multi-arm precursors each have polymer arms containing polymer units, The method wherein at least one of the first to fourth multi-arm precursors comprises a linker unit L containing a hydrolyzable ester bond. 14. The method according to embodiment 13, wherein the first to fourth functional groups are selected such that the first and second functional groups do not react with either the third or fourth functional group. 15. The method according to embodiment 13 or 14, wherein the first to fourth formulation further comprises acetone, acetonitrile, benzyl alcohol, chloroform, dichloromethane (DCM), dioxane, dimethyl carbonate (DMC), dimethyl sulfoxide (DMSO), methanol, ethanol or propanol, ethyl acetate, ethyl formate, ethyl propionate, glycoflor, hexafluoroisopropanol, isorpidodimethyl ether, isopropanol, methyl chloride, methylene chloride, methyl ethyl ketone, N-methylpyrrolidone, propylene carbonate, triethylamine, or tetrahydrofuran; a buffered aqueous medium such as water, PBS buffer or physiological saline; a sugar buffer; or any mixture thereof, the method according to embodiment 13 or 14. 16. The method according to embodiment 15, wherein the at least one solvent is selected from water, PBS buffer, TBS buffer, or a buffered aqueous medium such as physiological saline, or any mixture thereof, and optionally comprises at least one additive selected from pharmaceutical excipients, sugars, salts, additional therapeutic or diagnostic agents, thickeners, viscosity enhancers, stabilizers, or auxiliaries. 17. The method according to any one of embodiments 1 to 16, wherein the combined reaction mixture is injected into the treatment site of the patient before gelation is complete. 18. A sustained-release drug delivery system comprising a hydrogel according to any one of embodiments 1 to 12. 19. A hydrogel for sustained-release drug delivery, obtainable from the method described in any one of embodiments 13 to 17. 20. A method for adjusting the gel time of a combined reaction mixture produced in any one of embodiments 13 to 17, wherein the functional groups that are reactive in click chemistry and functional groups that are reactive in electrophilic-nucleophilic reactions are selected to have different rate constants or reaction rates. 21. The method according to embodiment 20, wherein the reaction rate of the electrophilic nucleophilic reaction is changed by adjusting the pH of the combined reaction mixture.

[0301] It should be understood that the various embodiments described herein are merely examples and are not intended to limit the scope of the invention. For example, many of the materials and structures described herein can be replaced with other materials and structures without departing from the spirit of the invention. Thus, the invention as described in the claims may include variations from the specific examples and preferred embodiments described herein, as will be apparent to those skilled in the art. It should be understood that the various theories regarding why the invention works are not intended to limit it. [Examples]

[0302] Materials and methods Non-degradable 4a20kPEG-N 3、4 a20kPEG-D and Tetronic1307-N3 were purchased from Creative PEGworks. DBCO-NH2, sDBCO-NH2, BCN-PEG2-NH2, norbornene-NHS, tetrazine-NHS, tetrazine-PEG5-NHS, and N3-PEG2-NH2 (click chemical linker) were purchased from Broadpharm and are also commercially available from Sigma-Aldrich, Sichem, Sinopeg, TCI, and other vendors. All degradable (including dibasic acid or carboxamide linkers) NHS and NH2-terminated PEGs, PLAs, and PLGAs were purchased from Jenkem USA. [Table 1]

[0303] Buffer 7.4 of phosphate-buffered (PBS) is prepared by diluting 200 mL of PBS 10 times in RODI to a final volume of 2 mL, adding 200 mg of NaF and 600 μL of Tween 20, and then stirring for 24 hours.

[0304] Tris-HCl buffer (TBS) 8.5 buffer was prepared by diluting 100 mL of 1 M Tris-HCl solution to 50 mM and then storing it overnight at 37°C. Once the temperature had equilibrium, 200 mg of NaF was added, and the mixture was returned to a 37°C incubator for 1 hour to equilibrium. 600 μL of Tween was added, and the container was again allowed to equilibrium at 37°C for 1 hour. The pH was adjusted to 8.5 with 6N NaOH.

[0305] NHS conversion to DBCO and Azid 1 H-NMR analysis: The degree of NHS substitution (conversion %) is determined using the chemical shifts of different protons within the PEG structure. The degree of NHS substitution in multi-arm PEG is: 1 It is determined by the ratio of molecular weight determined by MALDI to molecular weight determined by 1H-NMR. The MALDI molecular weight is reported on the certificate of analysis of the purchased PEG material. Converted DBCO and azide 1 Using 1HNMR spectra, the conversion percentage can be calculated based on the amide peak and the free NHS peak.

[0306] HPLC analysis of DBCO-converted PEG: To further understand the purity of the converted DBCO material, an HPLC method was developed, and the amount of DBCO in the system was quantified using DBCO-amine as a standard. The degree of conversion can be calculated based on the residual concentration of unreacted DBCO-amine, and the purity of the material can be calculated by using the area of ​​different peaks corresponding to different substituted PEGs. A Waters UPLC system equipped with a PDA (photodiode array) detector was used for this analysis.

[0307] Example 1 Preparation of biodegradable polymer multi-arm precursors The preparation of the degradable polymer multi-arm precursors was carried out by the following general method. To create a DBCO-terminated degradable PEG multi-arm precursor, multi-arm PEGs having an NHS-terminated group, such as NHSS (succinimidyl succinate), NHSG (succinimidyl glutarate), NHSAP (succinimidyl adipate), or NHSAZ (succinimidyl azelaic acid), are converted to DS (dibenzocyclooctinamide succinate), DG (dibenzocyclooctinamide glutarate), DAP (dibenzocyclooctinamide adipic acid), or DAZ (dibenzocyclooctinamide azelaic acid) groups by reacting the NHS group with a DBCO-amine click chemical linker according to the following exemplary reaction scheme: [ka]

[0308] 5.5 mg of DBCO-amine (commercially available from Sigma-Aldrich, TCI, and other vendors) is required to ensure a 1:1 reaction with all NHS groups in 100 mg of 4a20kPEG-NHSS. Both solids are added to a 20 mL vial, and 15 mL of dichloromethane or other suitable solvent is added. The mixture is vortexed for 1 hour and dried overnight under nitrogen. The stoichiometry (for a 20k PEG precursor) is calculated as follows:

number

[0309] Similarly, to create a degradable PEG multi-arm precursor terminated with an azide group, the reaction of 4a20kPEG-NHSS with N3-PEG2NH2 (commercially available from Sigma-Aldrich, TCI, and other vendors) is carried out by weighing 100 mg of 4a20kPEG-NHSS and 3.5 mg of N3-PEG2-NH2 for a 1:1 reaction between the amine group of the linker and the NHS group that terminates the PEG, according to the following exemplary reaction scheme: [ka]

[0310] For the 4a20kPEG-NHSS starting material, the stoichiometry is calculated as follows:

number

[0311] Similarly, other multi-armed PEGs with different molecular weights, arm numbers, and other NHS- or NH2 terminal degradable linker groups can be converted to have terminal DBCO or azide groups. Characterization and purification of click chemically functionalized precursors are performed. 1 This can be performed by 1H-NMR and HPLC. Figure 1 shows the HPLC analysis of the product distribution for two reactions with 4a20kPEGSAP and DBCO-NH2 molar ratios of 1:1 and 1:2. The 1:1 stoichiometry yields significantly fewer byproducts.

[0312] NHS-functionalized magnetic beads can be used to remove residual unreacted DBCO-NH2 from the product. The corresponding HPLC analysis is shown in Figure 2. Unreacted DBCO-NH2 is efficiently removed by adding NHS-functionalized magnetic beads for 24 hours and extracting the beads with a magnet. 1 Based on H-NMR data, conversion of 80-90% or more of functional groups can be achieved with reactions that have not yet been optimized. Alternatively or in addition, purification of the click functionalization precursor may be carried out, if necessary, by centrifugation, filtration / sterilization filtration, and / or dialysis steps known to those skilled in the art to remove insoluble impurities. The precursor and / or activator, or combinations thereof, may optionally be dried and / or lyophilized by adding a sugar such as trehalose to a buffer such as PBS7.4.

[0313] To prepare the hydrogel, a pair of click chemically functionalized precursors is combined with a mixture from two separate syringes via, for example, the Y-type mixer described herein.

[0314] Example 2 Gel formation and characterization Non-degradable 4a20kPEG-N3 and 4a20kPEG-D were used to study the gel formation of click chemical polymer precursors. The following section describes the processes related to hydrogel formation and characterization.

[0315] A) Effect of buffer pH on gel formation and swelling Solutions of 10% by mass (50 mg precursor + 450 mg buffer) of non-degradable 4a20kPEG-N3 and 4a20kPEG-D in buffers with pH values ​​summarized in Table 1 were mixed via a syringe mixing process, cast into tubes, and dried overnight under nitrogen. The gel time and 24-hour swelling rate in buffers with pH 7.4–8.5 did not appear to show significant differences, suggesting that buffer pH does not affect the gelation process or gel quality. [Table 2]

[0316] The expansion rate is calculated by dividing the mass gain after 24 hours in the buffer solution by the original mass (%) of the gel. (Swelling % = 100 * (Mass after 24 hours - Original mass) / Original mass).

[0317] B) Effect of precursor concentration on gel formation and swelling The effect of precursor concentrations in the mixtures combined for gel formation was evaluated by syringe mixing a first precursor solution containing 5% by weight, 10% by weight, and 30% by weight of 4a20kPEG-N3 with a second precursor solution containing 5% by weight, 10% by weight, and 30% by weight of 4a20kPEG-D, and casting the gel into a tube. The results show that the initiation of gelation and gel quality are strongly dependent on the precursor concentration. Higher precursor concentrations result in much faster gel times and less swelling, suggesting a much denser polymer network. These findings are summarized in Table 2. [Table 3]

[0318] C) Effect of solvent polarity on gel formation and swelling Due to the nonpolar nature of the DBCO group, the effect of solvent polarity was investigated to determine whether solubility-solubility interactions influence the gelation process. The results shown in Table 3 indicate a tendency for gel time to increase as solvent polarity decreases (low dielectric constant). [Table 4]

[0319] D) Effect of arm number (functionality) on gel formation and swelling The effect of functional properties was evaluated by investigating the gelation process of 4a20kPEG-PEG2N3 with 4-arm, 6-arm, and 8-arm 20kPEG-D. The critical crosslinking value (a c )[(a c When the gel time and swelling rate obtained for a 10 wt% precursor system obtained by the syringe mixing process (where f is defined as the average number of arms) are plotted against (=1 / (f-1)), a strong linear dependence of the gel time and swelling rate inversely proportional to the number of arms is shown. See Table 4 and Figure 3. [Table 5]

[0320] E) Effect of degradable groups on the gelation process The conversion of NHS PEG to the corresponding DBCO and azide groups was performed according to the method described above. The effects of degradable DBCO versus degradable azide versus double-degradable gel were investigated to understand the effects on gel time, swelling, and persistence. Table 5 summarizes the gel time and swelling rate of different gels made from polymers with different degradation groups. The results in Table 6 below show that gels formed using non-degradable azide and degradable DBCO persisted longer than gels formed using non-degradable DBCO and degradable azide, as well as double-degradable gels. Interestingly, the persistence of the NHS-NH2 gel used as a control in this study was the same as that of the double-degradable DBCO-azide gel and the non-degradable azide / degradable DBCO gel. Swelling measurements were not performed on gels containing DS and AS because they degraded within 24 hours. [Table 6] [Table 7] 1 [ka] Gel still exists, 2 [ka] The gel exists and begins to decompose, 3 [ka] The gel has completely broken down.

[0321] Further examination of the chemical structures of these biodegradable polymers reveals that using biodegradable DBCOs such as DG, DAP, or DAZ further removes the DBCO group from the biodegradable ester bond to the PEG arm. This may explain the extended persistence compared to using AS, AG, AAP, and AAZ, which have non-biodegradable DBCOs. This also explains the similar persistence between NHS-NH2 chemistry and biodegradable azide / non-biodegradable DBCO samples.

[0322] F) Effects of click chemical linkers on gel duration, swelling, and degradation persistence Hydrogels were prepared using different click chemical linkers, as described in Examples 1 and 2. Approximately equimolar amounts of precursors were used. It can be observed that longer gel time chemistry provides a lower swelling rate. Using a more hydrophilic sulfo-DBCO(sD) functional group instead of DBCO, or using BCN-PEG2 instead of DBCO, may be used to increase persistence at TBS8.5 and modulate degradation kinetics. DBCO-PEG as the click chemical linker. 23 Using -NH2 to further detach the DBCO group from the linker L(AZ) of precursor 1 increases gel time and decreases persistence compared to the same DBCO-amine functionalized polymer. Table 7 summarizes the results. [Table 8]

[0323] Example 3 gels containing polymer units other than PEG In addition to hydrogels containing only PEG precursors, hydrogels have been prepared using DBCO-azidocrick chemistry for multi-arm precursors containing polymer units derived from polylactic acid (PLA), poly(lactic acid-coglycolic acid) PLGA, and poly(ethylene glycol)-blocked poly(propylene glycol)-blocked copolymers (poloxamer® or Tetronic® copolymer).

[0324] The exemplary multi-arm precursor used in this embodiment is as follows: [ka] [ka]

[0325] x and y define the number of PPG, PEG, PLA, or PGA units, and t is as previously defined herein.

[0326] General procedure for preparing gels: Approximately 100 mg of the first polymer precursor (e.g., 4a20kPEG-DAZ) was added to a 1 ml syringe, and the solvent (approximately 150 microliters) was added to the same syringe so that the final concentration of polymer in the gel conformed to Table 8. The mixture was vortexed at room temperature (20-25°C). The weight of the second polymer precursor (e.g., Tetronic 1307-N3) was adjusted to the molar equivalent required for crosslinking (90.5 mg). Similarly, the required amount of solvent was added to the syringe and vortexed. After complete dissolution, the two syringes were connected by a Luer lock, and the polymer solution was mixed for several seconds to initiate the gelation process. The solution was extruded through one syringe, and the gelation time was measured. The gel was dried under a nitrogen gas stream at room temperature for at least 2 days. The swelling of the gel was analyzed by measuring the weight of the gel pieces before and after immersion in 1×PBS (pH 7.4) at room temperature for 24 hours. The persistence of the gel was measured by immersing gel samples in TBS pH 8.5 buffer (Tris-buffered saline) at 37°C and visually inspecting the gel for degradation daily. The solvent used was dimethyl carbonate (DMC):acetone (80:20). The final concentration (mg / ml) of the polymer in the solvent after mixing the two pre-gelling solutions is given in %. Table 8 shows the combination of precursors used, gel time, swelling rate, and persistence of the resulting gel. [Table 9]

[0327] Example 4 Peptide compatibility of precursors To investigate the compatibility of DBCO-azide chemistry as an activator with peptides contained in the gel, precursors 8a20kPEG-NHSG, 8a10kPEG-NH2, 8a20kPEG-DG, and 8a10kPEG-PEG2N3 were dissolved at a 30% by mass concentration in a solution containing 9% by mass lysozyme in PBS 7.4 and peptides having a mass of 14 kDa. The solutions of 8a10kPEG-NH2, 8a10kPEG-PEG2N3, and 8a20kPEG-DG showed clear solutions when the precursors were dissolved in the lysozyme solution. However, small solid gel-like particles were formed while attempting to dissolve 8a20kPEG-NHSG in the lysozyme solution. This suggests that, under aqueous conditions, the primary amine group on lysozyme undergoes a nucleophilic reaction with the NHS group of 8a20kPEG-NHSG, resulting in pegylation and crosslinking with lysozyme. Precursors functionalized with click chemical functional groups do not interact with peptides, thus allowing the peptides to be incorporated into the gel formed from them.

[0328] Example 5 AAV delivery Gold nanoparticles are used as a model to study the solution behavior of adeno-associated virus (AAV). Hydrogels can maintain the release of gold nanoparticles that have the same size and properties as AAV. By modifying the formulation, fast-release and delayed-release hydrogel formulations can be created and then tested for AAV delivery.

[0329] To evaluate the suitability of the hydrogel formed by the click chemical reaction for AAV release, lyophilized gold nanoparticles were suspended in a DMC solution containing 6% each of PEG precursors of azide and DBCO prior to gel formation. 4a20kPEG-PEG2N3 was used as the azide component, and 4a20kPEG-DS and 4a40kPEG-DG were used separately as DBCO components, as well as in a 50 / 50 blend of 4a20kPEG-DS / 4a40kPEG-DG. Gels containing dispersed lyophilized gold nanoparticles were generated and analyzed.

[0330] The results indicate that when 100% 4a20kPEG-DG is used as the DBCO component, the gel lasts for more than 20 days, with a burst of 40% release in the first few days, followed by a 15-day plateau, and then another sustained-release region until 100% of the gold nanoparticles are recovered, demonstrating the existence of a two-stage delivery method. Interestingly, when a 50 / 50 blend of 4a20kPEG-DS / 4arm40kPEG-DG is used as the DBCO component, the gel lasts for 5 days and shows a nearly linear sustained-release profile of gold nanoparticles. On the other hand, when 100% 4armk20PEG-DS is used as the DBCO component, the gel degrades overnight. These results demonstrate that DBCO / azidocrick chemistry can be used to maintain the release of gold nanoparticles used in AAV. The results are summarized in Figure 4.

[0331] Example 6 Protein delivery To test the feasibility of DBCO / azide PEG hydrogels for protein delivery, bevacizumab (Avastin kit) was selected as the API. To test this, several PEG precursors containing DBCO, azide, NHS, and NH2 functional groups were added to an aqueous solution containing bevacizumab, and aggregates were examined using SDS-PAGE. The results shown in Figure 5 and Table 9 demonstrate that large aggregates corresponding to pegylated bevacizumab are present only when NHS-containing PEG is added to the bevacizumab solution. These results suggest that protein delivery via DBCO / azide-PEG hydrogels can be achieved under aqueous conditions. [Table 10] *m20kPEG-SC is a linear mPEG20k succinimidyl carboxymethyl ester. [ka] It is commercially available from Sigma Aldrich.

[0332] Furthermore, SEC-HPLC analysis of samples that were not pegylated under gel electrophoresis revealed the presence of low-MW and high-MW species, as well as the main bevacizumab and PEG peaks (Figure 6). The bevacizumab recovery rate was reasonable (80-110%), suggesting that there was no pegylation by azide (N3), NH2, and DBCO groups on PEG.

[0333] Furthermore, the bevacizumab concentration in the solution was further confirmed using ELISA. Figure 7 shows good recovery rates of bevacizumab from gels of different concentrations, with 900 ng / mL being superior.

[0334] SPR analysis was performed to evaluate protein binding to VEGF. The results summarized in Table 10 and Figure 8 show no clear loss of bevacizumab function due to SPR. Theoretical K D and k a The characteristics are similar, mAb capture levels are similar, and no binding is observed when the PEG control is injected. [Table 11]

[0335] Example 7 Effect of crosslinking density (MWc) on release dynamics Bevacizumab (Avastin Kit) was selected as the API to test the effect of inter-crosslink molecular weight (MWc) on the API release kinetics in different hydrogels formed according to the general procedure described in Example 1. Several multi-arm PEG precursors containing DBCO, azide, NHS, and NH2 functional groups were added to an aqueous solution containing bevacizumab at a weight ratio of 3:1 total PEG:Avastin and a total PEG precursor concentration in a 10 wt% aqueous solution. Different MWc were obtained by varying the PEG arm length and number of arms. The compositions are summarized in Table 11, and the release kinetics are shown in Figure 9 as bevacizumab release rate % normalized to the assay. [Table 12]

[0336] The release dynamics data in Figure 9 reveal the control of bursts at MWc above 4375 and sustained in vitro release of API at TBS 8.5. As expected, higher MWc delays API release to 4 days at room temperature at TBS 8.5 and approximately 2 months at PBS 7.4 at 37°C.

[0337] Example 8 Norbornene / Tetrazine Hydrogel Following the general procedure described in Example 1, IgG-containing hydrogels (with different hydrogel:IgG ratios (PEG:IgG)) were produced by mixing equimolar amounts (each at approximately 10% precursor concentration (PEG%)) of aqueous solutions containing 8a20kPEG-norbornene (obtained by reacting 8a20kPEG-Nor, 8a20kPEG-NH2 with norbornene-NHS), as well as (A, B) tetrazine precursors (8a20kPEG-TET) and (C) PEG5 extended tetrazine precursors (8a20kPEG-PEG5TET), in the presence of IgG.

[0338] For the tetrazine precursor, 8a20kPEG-NH2 is mixed with either tetrazine-NHS ester in DCM (see scheme 8 below) or tetrazine-PEG5-NHS for 8 hours. For 8a20kPEG-TET, 1 Regarding the 82% conversion by H-NMR, and 8a20kPEG-PEG5TET: 1 The reaction was carried out by 100% conversion by H-NMR, the solvent was evaporated under a nitrogen atmosphere, and the mixture was dissolved in PBS 7.4, centrifuged at 10,000 rpm for 30 minutes, decanted, filtered through a 0.25 μm filter, dialyzed in WFI 3.8 at 10 k MWC for 24 hours, and subsequently obtained by sterile filtration or lyophilization. IgG was mixed in equal volumes into two precursor solutions in two separate syringes, and the two formulations from the two syringes were combined in a Y-mixer to produce an IgG-containing hydrogel. Scheme 8: [ka] Tetrazine NHS-ester

[0339] The gel time was found to be 2–6 minutes at room temperature (20°C). Each sample had a calculated molecular weight between crosslinked MWc of approximately 5000 Da. For all samples, IgG release data from the hydrogel in PBS 7.4 buffer at 37°C showed a relatively low initial burst of IgG (IgG burst %) of less than 10% at different PEG:IgG ratios. Pegylation of the precursor to the protein could not be determined by SEC. In a 1-week accelerated persistence test in TBS pH 8.5 buffer, no degradation was observed, as expected, due to the absence of a degradable linker in the precursor. The results are summarized in Table 12. [Table 13]

[0340] Example 9 DBCO / Tetrazine Hydrogel Following the general procedure described in Example 1, an IgG-containing hydrogel was produced by mixing an aqueous solution containing equimolar amounts of 8a15kPEG-DG and 8a20kPEG-TET precursor (same as in Example 7 above) in the presence of IgG. 8a15kPEG-DG was prepared by reacting 8-arm 15kDa PEG-glutarate succinimimidyl (8a15kPEG-SG) with DBCO-NH2 in DCM for 8 hours, evaporating the solvent under a nitrogen atmosphere, dissolving in sterile water for injection (WFI3.8), centrifuging at 10000 rpm for 30 minutes, decanting, filtering through a 0.25 μm filter, dialyzing with WFI3.8 at 10 kMWC for 24 hours, followed by sterile filtration. 1 The result was obtained by converting 94% using 1H-NMR.

[0341] The gelation time was 4.5 minutes (30% wt% PEG in the precursor solution) to 8.7 minutes (10% PEG in the precursor solution) at room temperature (20°C), indicating suitability for administration by in-situ gel formation. Each sample had a calculated MWc of approximately 4375 Da. IgG release data from hydrogels in PBS7.4 and TBS8.5 buffers at 37°C showed a relatively low initial burst of IgG in PBS7.4 (IgG burst %) and complete IgG release under accelerated conditions in TBS8.5 at room temperature within 11 days, demonstrating the suitability of DBCO / tetrazine hydrogel for long-term drug delivery. Release data is shown in Figure 10.

[0342] Example 10 DBCO / Tetrazine / Azidohydrogel To vary the gel time for in-situ applications, hydrogels were generated using two different functionalized multi-arm PEG precursors, both reacting with DBCO-functionalized multi-arm precursors at different reaction rates. Hydrogels were generated by reacting 8a20kTET and 8a20kN3 with varying amounts of azide precursors and 8a15kPEG-DG (see Example 8) according to the general procedure described in Example 1.

[0343] The gel initiation and complete gel formation times were measured using the stirring rod method, and the results are shown in Figure 11. The stirring rod method involves mixing two gel precursors together for 10 seconds, then injecting them into a vial containing a stirring magnetic stirring rod, and recording the time until the stirring rod decelerates and exhibits higher viscosity. This point is defined as "gel initiation," and "gel time" is defined as the point at which the stirring rod finally stops stirring.

[0344] The graph shows that, in the concentration range of azide precursors up to approximately 20 wt%, the gel time varies significantly from approximately 7 minutes with 0 wt% azide precursor to approximately 1 minute with approximately 20 wt% azide precursor. For this system, the inflection point of the curve at a concentration of approximately 13% azide precursor results in a gel time of approximately 3–4 minutes, which can be used to adapt the processing period for in situ gel formation applications.

[0345] Therefore, the use of two different second precursors provides control over solution viscosity and gel time, which is preferable for different therapeutic applications or administration modes. Very fast gel times (e.g., about 10 seconds to 2 minutes) can be suitable for in-situ gel-forming injections into liquid tissues such as vitreous fluid or blood, without dilution of the gel in a liquid environment. Shorter to moderate gel times (e.g., about 1 to 3 minutes) are preferred for injections into soft tissues, while slower gelation (e.g., about 2 to 8 minutes or more) may be more preferable for in-situ gel-forming injections into harder tissues.

[0346] Furthermore, 24-hour swelling data in PBS or TBS appeared unaffected by azide concentration, showing a fully crosslinked structure in each gel. Time-dependent accelerated degradation / IgG in vitro release kinetics data in TBS8.5 buffer were obtained for gel samples with 0, 10, 13, 18, and 100 wt% azide precursors and are shown in Figure 12.

[0347] Hydrogels containing 100 wt% azide precursor (equimolar amounts of 8a20kPEG-N3 / 8a15kPEG-DG) exhibit a nearly complete burst by day 2. Hydrogels containing 0 wt% azide (equimolar amounts of 8a20kPEG-TET / 8a15kPEG-DG) show a high burst on day 1, followed by a constant release until day 11. Hydrogels containing 10 wt%, 13 wt%, and 18 wt% azide precursor (equimolar amounts of 8a15kPEG-DG and the sum of 8a20kPEG-TET and 8a20kPEG-N3, in wt% ratios of 90:10, 87:13, and 82:18) yield sustained release of the IgG drug over 11 days under accelerated conditions with a relatively small initial burst. The degradation of TBS8.5 is accelerated by approximately 16 times compared to PBS7.4 under in vivo conditions, allowing for the long-term sustained release of the activator in vivo for up to approximately 6 months.

[0348] Example 11 Injectability A key parameter for applications using in-situ gel-forming sustained-release hydrogels is the injectability of the combined precursor solution. This parameter can be obtained by determining the glide force applied to a filled syringe of an Instron Model 3342 device equipped with a 50N load cell. The settings used are as follows: In a 1 ml syringe with a diameter of 4.78 mm, 100 μl volume of premixed aqueous precursor solution (8a15kPEG-DG as in Example 8 in one syringe, and a mixture of 95 wt% 8a20kPEG-tetrazine / 5 wt% 8a20kPEG-N3 in the other syringe) is injected per minute for 1 to 7 minutes at an injection rate of 100 mm / min, with an injection distance of 100 mm per 50 μl injection, and the displacement in response to the applied force is recorded. To compare different gel samples, the glide force was measured 2 minutes after mixing. The results are shown in Table 13. [Table 14]

[0349] The results suggest that, in the system tested in this example, a dose of 2.5–4 mg of the n-activator in a 50 μl injection volume appears to be ideal for injection.

Claims

1. A sustained-release drug delivery system comprising a bioorthogonal hydrogel and at least one activator, wherein the bioorthogonal hydrogel comprises a covalently crosslinked polymer network and contains the at least one activator, and the polymer network comprises a) Multiple polymer units, b) A chemical bond that can be cleaved by hydrolysis, wherein at least one of the hydrolyzable bonds is an ester bond, c) The sustained-release drug delivery system comprising a crosslink formed by a click chemical reaction.

2. The system according to claim 1, wherein the polymer network comprises or is composed of multi-arm units, each having a core unit X and three or more arms attached to the core unit, preferably in the range of 3 to 10, 4 to 8, or 4 to 6 arms, and crosslinks formed by a click chemical reaction are located at the ends of each arm and connect them to another arm attached to another core of the multi-arm unit.

3. The system according to claim 1 or 2, wherein the polymer network comprises a first, second, and optionally more multi-arm units, and the crosslinks formed by the click chemical reaction are between the arms of the first multi-arm unit and the arms of the second or more multi-arm units, and the multi-arm units have the same or different amounts of arms and / or the same or different core units X.

4. The system according to claim 2 or 3, wherein at least one of the multi-arm units is a polymer multi-arm unit having a polymer arm containing a polymer unit, and the polymer multi-arm unit has an average molecular weight of 1,000 to 100,000 daltons.

5. The system according to any of the prior claims, wherein the polymer unit is selected from at least one of polyethylene glycol (PEG) units, polypropylene glycol (PPG) units, polyglycolic acid (PGA), polylactic acid (PLA), or polylactic acid-coglycolic acid (PLGA), random or block copolymers, and / or combinations or mixtures thereof, preferably polyethylene glycol units.

6. The system according to any of the prior claims, wherein the covalently crosslinked polymer network comprises a plurality of hydrophobic polymer units and / or hydrophilic polymer units.

7. The system according to claim 6, wherein the hydrophobic polymer unit is selected from at least one of polylactic acid (PLA) and polylactic acid-coglycolic acid (PLGA) units, and the hydrophilic polymer unit is selected from at least one of polyethylene glycol units, polypropylene glycol units, or polyglycolic acid (PGA), preferably polyethylene glycol units.

8. The system according to any one of the prior claims, wherein each of the polymer units has an average molecular weight (Mn) in the range of about 1,000 to about 100,000 daltons, or about 2,000 to about 50,000 daltons, or about 3,000 to about 30,000 daltons, and the average molecular weight (Mn) of the arms of the first multi-arm unit and the arms of the second multi-arm unit are the same or different.

9. The system according to any one of claims 3 to 8, wherein one of the first or second multi-arm units is a polymer multi-arm unit having a molecular weight of less than 1,000 daltons, and the other multi-arm unit is a polymer multi-arm unit having an average molecular weight (Mn) of more than 5,000 daltons.

10. The system according to any one of claims 2 to 9, wherein the hydrolyzable ester bond is part of a linker unit L located between the polymer unit end of the arm of the multi-arm unit and the crosslink formed by the click chemical reaction.

11. The aforementioned linker unit L is given by equation (i): 【Chemistry 1】 It includes a structure represented by the formula, where U １ and U ２ However, independently, NH or O, can be the same or different, U １ and U ２ The system according to claim 10, wherein at least one, preferably both, of the elements is O, t is an integer from 0 to 10, and is a dibasic acid such as succinic acid, glutaric acid, adipic acid, or azelaic acid.

12. The polymer network is given by equation (ii): 【Chemistry 2】 The system according to any one of claims 1 to 11, comprising a multi-arm unit represented by the formula, wherein X is the core unit of the multi-arm unit, Y is a polymer unit, L is a linker unit containing the hydrolyzable ester bond, A is a crosslink formed by a click chemical reaction, p is either 0 or 1, and q is an integer from 3 to 10.

13. The system according to any one of the prior claims, wherein the crosslinks are formed by a click chemical reaction such as a copper-free SPAAC or IEDDA reaction, the first functional group of a first multi-arm precursor or unit reacts with at least one complementary second functional group of a second multi-arm precursor or unit, the second complementary functional group can react with the first functional group in a click chemical reaction, and the first and second functional groups are selected from a pair comprising an alkyne moiety and an azide such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonine (BCN), or norbornene or transcyclocten (TCO) and a tetrazine (Tz) moiety.

14. The system according to any one of the prior claims, wherein the activator is selected from the group consisting of therapeutic or diagnostic activators.

15. The system according to any one of the prior claims, wherein the activator is a small molecule activator or a biomolecule activator, preferably a small molecule activator, a peptide, a protein, or a virus.

16. The aforementioned activators include steroids; nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, meclofename, mefanamic A, sarsalate, sulindac, tolmetin, ketoprofen, diflunisal, piroxicam, naproxen, etodrug, fluviprofen, fenoprofen C, indomethacin, celecoxib, ketrolac, and nepafenac; intraocular pressure lowering agents; antibiotics such as ciprofloxacin; analgesics such as bupivacaine; calcium channel blockers such as nifedipine; cell cycle inhibitors such as simvastatin; proteins such as insulin; small molecule hydrophilic drugs including carboxylates and amine salts; small molecule hydrophobic drugs, insulin A system according to any one of the prior claims, selected from hydrophilic peptides and protein drugs such as single-chain antibody fragments, Fab fragments, IgG antibodies, and fusion antibodies; aptamers; particularly bupivacaine (BPV-HCl or base), ropivacaine (RPV), dexamethasone, travoprost, axitinib, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, antibiotics, analgesics, calcium channel blockers, cell cycle inhibitors, chemotherapeutic agents, antiviral agents, anesthetics, hormones, anticancer agents, antitumor agents, viruses, genetically modified viruses such as AAV, protein binders such as nanobodies, aphibodies, ankyrin, DARPins, or any combination thereof.

17. The system according to any one of the prior claims, wherein the activator is a peptide selected from the group consisting of Compstatin, APL-1, and Fc-III-4C, Beob 15 (brolucizumab), bevacizumab (avastin), Zimura (abasincaptado pegol), pegcetacoplan, avisipal pegol, lamparizumab, Fovista, listiganib, AXT107, elamipretide, THR149, ALM201, VGB3, and largazole.

18. The system according to any one of the prior claims, wherein the activator is contained in the hydrogel without being covalently conjugated to the hydrogel, or the activator is contained in the hydrogel and covalently conjugated to the hydrogel.

19. The system according to any one of the prior claims, wherein the activator is contained in the hydrogel without being covalently conjugated to the hydrogel, and preferably, the activator is contained in the hydrogel without being covalently conjugated to the hydrogel via an oligonucleotide.

20. The polymer network of the bio-orthogonal hydrogel is azide (N ３ The system according to any one of the prior claims, wherein the reaction product is a reaction between a first multi-arm precursor having a functional group containing a ) moiety and a second multi-arm precursor having a functional group containing a dibenzocyclooctin (DBCO) moiety.

21. The polymer network of the bio-orthogonal hydrogel is azide (N ３ The system according to any one of the prior claims, wherein the reaction product is a first multi-arm precursor having a functional group including a portion of and a second multi-arm precursor having a functional group including dibenzocyclooctin (DBCO), and the activator is a small molecule activator, a peptide, a protein, or a virus.

22. The system according to any one of the prior claims, wherein the polymer network of the bio-orthogonal hydrogel is a reaction product of a first precursor formulation comprising a mixture of two or more first precursors, each having different first functional groups that are suitable for forming links with complementary second functional groups by click chemistry but do not react with each other.

23. The system according to claim 1 or 21, wherein the polymer network of the bio-orthogonal hydrogel is a reaction product of a second precursor formulation comprising a mixture of two or more second precursors, each having different second functional groups that are suitable for forming links with complementary first functional groups by click chemistry but do not react with each other.

24. The system according to any one of claims 22 or 23, wherein the different first or second functional groups are selected to have different kinetic constants or reaction rates in their click reactions with the corresponding second or first functional group counterparts.

25. The system according to any one of claims 22 to 24, wherein the first and second functional groups are selected from a pair comprising a strain alkyne moiety and azide such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonine (BCN), or norbornene or transcyclooctene (TCO) and tetrazine (Tz) moiety, or a strain alkyne moiety and tetrazine (Tz) moiety such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonine (BCN), or any combination thereof.

26. The system according to claim 25, wherein the first precursor is a dibenzocyclooctin (DBCO) group-functionalized multi-arm PEG, and the second precursor is a combination of a tetrazine group-functionalized multi-arm PEG and an azide group-functionalized multi-arm PEG, or the first precursor is a combination of a dibenzocyclooctin (DBCO) group-functionalized multi-arm PEG and a norbornene group-functionalized multi-arm PEG, and the second precursor is a tetrazine group-functionalized multi-arm PEG.

27. The aforementioned azide (N ３ A first multi-arm precursor having a functional group including the ) portion, 【Transformation 3】 A system according to any one of claims 20 to 26, selected from the group consisting of, where n is 1 to 2300, t is an integer from 0 to 10, or the corresponding eight-arm version thereof.

28. The second multi-arm precursor having a functional group containing the dibenzocyclooctin (DBCO) moiety is 【Chemistry 4】 A system according to any one of claims 20 to 27, selected from the group consisting of, where n is 1 to 2300, t is an integer from 0 to 10, or the corresponding eight-arm versions thereof.

29. The polymer network of the bio-orthogonal hydrogel is azide (N ３ The reaction product is obtained by reacting at least one, preferably one, of the following first multi-arm precursors having a functional group containing a ) moiety with at least one, preferably one, of the following second multi-arm precursors having a functional group containing a dibenzocyclooctin (DBCO) moiety: 【Chemistry 5-1】 【Chemistry 5-2】 The system according to any one of the prior claims, wherein n is between 1 and 2300, t is an integer between 0 and 10, or the corresponding eight-arm version described above.

30. The polymer network of the bioorthogonal hydrogel reacts at least one of the following first multi-arm precursors having a functional group containing an azide (N ３ ), such as one of the following second multi-arm precursors having a functional group containing a dibenzocyclooctyne (DBCO) moiety, to form a reaction product: 【Chemistry 6-1】 【Chemistry 6-2】 The system according to any one of the prior claims, wherein x and y define the number of PPG, PEG, PLA, or PGA units, and t is an integer from 0 to 10, or the corresponding eight-arm version thereof.

31. A method for producing a sustained-release drug delivery system as defined in any one of claims 1 to 30, comprising a bio-orthogonal hydrogel and at least one activator as described in any one of the prior claims, wherein the method is a) A step of preparing a first formulation comprising at least one first multi-arm precursor having a first functional group suitable for forming a link with a second functional group by click chemistry, b) Providing a second formulation comprising at least one second multi-arm precursor having a second functional group suitable for forming a link with the first functional group by click chemistry, c) The step of combining the first and second formulations, d) Adding at least one activator to the first, second, or combined first and second formulations, thereby forming a combined reaction mixture; e) a step of enabling the combined reaction mixture to gel by forming a click chemical link between the first functional group and the second functional group, thereby forming a hydrogel containing the at least one activator, At least one or both of the first multi-arm precursor or the second multi-arm precursor is a polymer multi-arm precursor having polymer arms containing polymer units, The method wherein at least one of the first multi-arm precursor or the second multi-arm precursor comprises a linker unit L containing a hydrolyzable ester bond.

32. The method according to claim 31, wherein at least one third multi-arm precursor having a first or second functional group suitable for forming a click chemical bond with either the first multi-arm precursor or the second multi-arm precursor is added in one of steps a) to c).

33. The method according to claim 31 or 32, wherein the first and / or second formulation, preferably both, comprises at least one solvent.

34. The method according to claim 33, wherein the at least one solvent is selected from water, a buffered aqueous medium such as PBS buffer or physiological saline, a sugar buffer, or any mixture thereof.

35. The method according to any one of claims 31 to 34, further comprising the step of removing the solvent or solvent mixture.

36. A sustained-release drug delivery system comprising a bio-orthogonal hydrogel according to any one of claims 1 to 30 and an activator, wherein the sustained-release drug delivery system further comprises a hydrogel which is a reaction product of at least two multi-arm precursors crosslinked via an electrophilic-nucleophilic reaction, and the hydrogel forms an interpenetrating network with the bio-orthogonal click chemical crosslinking network according to claims 1 to 30.

37. A hydrogel for sustained-release drug delivery, wherein the hydrogel contains an activator, The hydrogel comprises a cross-penetrating covalently crosslinked dual polymer network, and contains the at least one activator, and the dual polymer network comprises a) Multiple polymer units, b) A chemical bond that can be cleaved by hydrolysis, wherein at least one of the hydrolyzable bonds is an ester bond, c) A crosslink formed by a click chemical reaction between a first multi-arm precursor having a reactive functional group by click chemistry and a second multi-arm precursor, d) The hydrogel comprising a crosslink formed by an electrophilic-nucleophilic reaction between a third multi-arm precursor having a reactive functional group and a fourth multi-arm precursor.

38. The hydrogel according to claim 37, wherein the reactive functional group is selected by click chemistry from a pair comprising a strain alkyne moiety such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonane (BCN) and an azide, norbornene or transcyclooctene (TCO) and a tetrazine (Tz) moiety, or a strain alkyne moiety such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonane (BCN) and a tetrazine (Tz) moiety, or any combination thereof.

39. The hydrogel according to claim 37 or 38, wherein the functional group that is reactive by electrophilic-nucleophilic reactions is selected from activated ester groups such as succinimidyl esters and succinimidyl carbonates; electrophiles such as nitrophenyl carbonate, aldehydes, ketones, acrylates, acrylamides, maleimides, vinyl sulfones, iodoacetamides, alkenes, epoxides, mesylates, tosylates, tresyl, cyanurates, orthopyridyl disulfide or halogens; and nucleophiles such as amines such as primary amines, hydroxyls, alcohols, thiols, azido anions, and carboxyl groups; preferably selected from amines and N-hydroxysuccinimide groups.

40. The dual polymer network has terminal amines (NH ２ In the presence of a third precursor having a ) group and a fourth multi-arm precursor having a terminal N-hydroxysuccinimide (NHS) group, azide (N ３ The hydrogel according to any one of claims 37 to 39, formed by reacting a first multi-arm precursor having a functional group containing a tetrazine (Tz) moiety with a second multi-arm precursor having a functional group containing dibenzocyclooctin (DBCO) or norbornene (Nor) moiety.

41. The hydrogel according to any one of claims 37 to 40, wherein the first to fourth precursors are selected independently of each other or from multi-arm polyethylene glycol (PEG) precursors having 3 to 10 PEG arms, such as 4 to 8 PEG arms.

42. The hydrogel according to any one of claims 37 to 41, wherein the first to fourth precursors are independently selected from polypropylene glycol (PPG), poly(ethylene glycol)-block-poly(propylene glycol) copolymer, poloxamers such as Tetronic®, polyvinyl alcohol (PVA), polyvinyl acetate, poly(vinylpyrrolidinone) (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-coglycolic acid (PLGA), p-dioxanone, trimethylene carbonate, caprolactone, random or block copolymers, or any combination or mixture thereof, comprising 3 to 10 arm precursors of polymer units, or one or more units of polyamino acids, glycosaminoglycans, polysaccharides, or proteins.

43. The first to fourth multi-arm precursors independently have an average molecular weight (M) of 5,000 to 100,000 daltons, such as 5,000 to 40,000 daltons and 10,000 to 40,000 daltons. ｎ The hydrogel according to any one of claims 37 to 42.

44. The hydrogel according to any one of claims 37 to 43, wherein the dual polymer network comprises a linker unit L containing an acid and an amide group, such as a dibasic acid, or an acid amide such as succinic acid, glutaric acid, adipic acid, azelaic acid, or glutaramide.

45. The dual polymer network is defined by formula (i): 【Transformation 7】 The formula includes a linker unit L which contains a structure represented by U １ and U ２ However, independently, it is NH or O, U １ and U ２ The hydrogel according to any one of claims 37 to 44, wherein at least one of the is O, preferably both are O, and t is an integer from 0 to 10.

46. The hydrogel according to any one of claims 37 to 45, wherein the activator is contained in the hydrogel without being covalently conjugated to the hydrogel, or the activator is contained in the hydrogel and is at least partially covalently conjugated to the hydrogel.

47. The hydrogel according to any one of claims 37 to 46, wherein the activator is selected from the group consisting of therapeutic or diagnostic activators such as those defined above, or the therapeutic activator is a small molecule activator or biomolecule activator, preferably a small molecule activator, peptide, protein, or virus, preferably a peptide selected from the group consisting of Compstatin, APL-1, and Fc-III-4C, Beob 15 (brolucizumab), bevacizumab (avastin), Zimura (abasincaptado pegol), pegcetacoplan, avisical pegol, lamparizumab, Fovista, listiganib, AXT107, elamipretide, THR149, ALM201, VGB3, and largazole, preferably bevacizumab.

48. A method for forming a hydrogel according to any one of claims 37 to 47, wherein the method is a) A step of preparing a first formulation comprising a first multi-arm precursor having a first functional group suitable for forming a link with a second functional group by click chemistry, and at least one activator as defined in any of the preceding embodiments, b) Providing a second formulation comprising a second multi-arm precursor having a second functional group suitable for forming a link with the first functional group by a click chemical reaction, c) A step of preparing a third formulation comprising a third multi-arm precursor having a third functional group suitable for forming a link with a fourth functional group by an electrophilic-nucleophilic reaction, d) Providing a fourth formulation comprising a fourth multi-arm precursor having a fourth functional group suitable for forming a link with the third functional group by an electrophilic-nucleophilic reaction, e) The step of combining the first to fourth formulations in the presence of at least one activator, thereby forming a combined reaction mixture, f) The step of forming a click chemical crosslink between the first functional group and the second functional group, and forming a crosslink between the third functional group and the fourth functional group by an electrophilic-nucleophilic reaction, thereby enabling the combined reaction mixture to gel, and thereby forming a hydrogel containing the at least one activator, g) The first to fourth multi-arm precursors have polymer arms containing polymer units, h) The method wherein at least one of the first to fourth multi-arm precursors comprises a linker unit L containing a hydrolyzable ester bond.

49. The method according to claim 48, wherein the first to fourth functional groups are selected such that the first and second functional groups do not react with either the third or fourth functional group.

50. A sustained-release drug delivery system according to any one of claims 1 to 47, or a hydrogel according to any one of claims 37 to 47, for use as a pharmaceutical product.

51. A method of treatment comprising treating a disease or medical condition in a patient with a sustained-release drug delivery system according to any one of claims 1 to 47, or with a hydrogel according to any one of claims 37 to 47.

52. The sustained-release drug delivery system or method of treatment according to claim 50 or 51, wherein the sustained-release drug delivery system is used for the treatment of the eye.

53. A sustained-release drug delivery system or method of treatment for use according to any one of claims 50 to 52, used for the treatment of ocular diseases such as posterior segment diseases, in particular age-related macular degeneration (AMD), cystoid macular edema (CME), diabetic macular edema (DME), posterior uveitis, and diabetic retinopathy, which are caused by aging, trauma, or surgical intervention.

54. The drug delivery system is used for retinal neovascularization, choroidal neovascularization, exudative AMD, atrophic 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 Multiple maculoplasty, Behçet's disease, Birdshot chorioretinopathy, posterior uveitis, posterior scleritis, serpentine choroiditis, subretinal fibrosis, uveitis syndrome, Vogt-Koyanagi-Harada syndrome, retinal artery occlusive disease, central retinal vein occlusion, disseminated intravascular coagulation, branched retinal vein occlusion, hypertensive fundus changes, ischemic syndrome, retinal artery microaneurysms, Coats' disease, parafoveal telangiectasia, hemiretinal vein occlusion, papillary phlebitis, carotid artery disease (CAD), dendritic vasculitis, sickle cell anemia Bulbar retinopathy, retinal pigment striata, familial exudative vitreoretinopathy, Eels disease, proliferative vitreoretinopathy, diabetic retinopathy, tumor-related retinal diseases, congenital hypertrophy of the retinal pigment epithelium (RPE), posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, complex hamartoma of the retina and retinal pigment epithelium, retinoblastoma, fundus vascular proliferative neoplasm, retinal astrocytoma, ocular lymphoid neoplasm, myopic retinal degeneration, acute retinitis pigmentosa, glaucoma, endophthalmitis, cytomegalovirus retinitis, retinal cancer, retinoblastoma A sustained-release drug delivery system or method of treatment for use according to any one of claims 50 to 53, used for the treatment of an eye disease selected from the group consisting of membranous pigmentary degeneration, Leber congenital amaurosis, choroidal atrophy, X-linked 106 retinitis pigmentosa, Best vitiligo macular dystrophy, X-linked retinoschisis, CNGA3 color blindness, CNGB3 color blindness, LHON, Stargardt disease, Usher syndrome, Norrie disease, Valday-Beedle syndrome, and red-green color blindness.

55. A sustained-release drug delivery system or method of treatment for use according to any one of claims 50 to 54, wherein the drug delivery system is formulated for direct injection at the treatment site of the patient.

56. The drug delivery system is formulated for injection into the eye, such as parenteral administration, intratumoral injection, intravitreal injection, anterior chamber injection, subconjunctival injection, retrobulbar injection, sub-Tenon's capsule injection, subretinal injection, or suprachoroidal injection, according to claim 55, a sustained-release drug delivery system for use or method of treatment.

57. A sustained-release drug delivery system or method of treatment for use according to any one of claims 50 to 56, wherein the drug delivery system is administered by direct injection, by oral administration, or incorporated into an implant.

58. A sustained-release drug delivery system or method of treatment for use according to any one of claims 50 to 57, wherein the drug delivery system is administered into the patient's body space by any one of the following means: subcutaneous, intramuscular, intrathecal, epidural, intraperitoneal, intradermal, subcutaneous, intercostal, intra-articular, intrasynovial, intravertebral, oral, nasal, transrectal, intratumoral, or vaginal administration.

59. A sustained-release drug delivery system or method of treatment for use according to claim 58, wherein the intra-articular administration is to a joint selected from the knee, elbow, hip, sternoclavicular, temporomandibular joint, carpal, tarsal, wrist, ankle, intervertebral disc, or ligamentum flavum.

60. A sustained-release drug delivery system for use or method of treatment according to any one of claims 50 to 59, wherein the drug delivery system comprises two or more different activators.

61. A sustained-release drug delivery system or method of treatment for use according to claim 60, for use in combination therapy involving the administration of two or more activators.

62. A sustained-release drug delivery system or method of treatment for use according to any one of claims 50 to 61, wherein the hydrogel of the drug delivery system is formed in situ at the treatment site of the patient.

63. The sustained-release drug delivery system or method of treatment for use according to claim 62, wherein the hydrogel is formed in situ at the treatment site of a patient by combining a first formulation comprising a first mixture according to step a) of any one of claims 31 to 35 and a second formulation comprising a second mixture according to step b) of any one of claims 31 to 35, thereby enabling the combined formulation mixture to gel in situ at the treatment site.

64. The sustained-release drug delivery system or method of treatment for use according to claim 63, wherein the first formulation and the second formulation are combined immediately before administering the formulation mixture at the treatment site.

65. The sustained-release drug delivery system or method of treatment for use according to claim 64, wherein the first formulation is in a first syringe, the second formulation is in a second syringe, and the two formulations are combined in a Y-mixer before direct injection at the treatment site.

66. A sustained-release drug delivery system or method of treatment for use according to claim 63, wherein the first formulation and the second formulation are each administered separately or sequentially at the treatment site, thereby enabling the formulations to be combined at the treatment site and the combined formulation to gel in situ at the treatment site.

67. The sustained-release drug delivery system or method of treatment for use according to claim 66, wherein the first formulation is in a first syringe, the second formulation is in a second syringe, and the two formulations are injected simultaneously or sequentially at the treatment site.

68. A sustained-release drug delivery system or method of treatment for use according to claim 62, wherein the hydrogel is formed in situ at the treatment site of a patient by administering a suspension comprising a first mixture according to any one of claims 31 to 35, a second mixture according to any one of claims 48 to 74, 77 to 80, 83, or 84, and a hydrophobic organic liquid to the treatment site of the patient.

69. The sustained-release drug delivery system or method of treatment for use according to claim 68, wherein the hydrophobic organic liquid is included in at least one of the first or second mixtures.

70. The hydrophobic organic liquid is triethyl citrate, acetyl triethyl citrate (ATEC), acetyl tributyl citrate (ATBC), α-tocopherol (vitamin E), α-tocopherol acetate; plant or vegetable oils such as sesame oil, olive oil, soybean oil, sunflower oil, coconut oil, canola oil, and rapeseed oil; nut oils such as hazelnut, walnut, pecan, and almond oil; cottonseed oil, corn oil, linseed oil, ethyl oleate, castor oil and their derivatives (Cremofor®); saturated or unsaturated fatty acids; monoglycerides. A sustained-release drug delivery system or method of treatment for use according to claim 68 or 69, comprising lipids that are liquid at 37°C or below, such as riserids, diglycerides, triglycerides (Myglyols®), phospholipids, glycerophospholipids, sphingolipids, sterols, prenols, and polyketides; a biocompatible oil selected from the group consisting of hydrophobic biodegradable liquid polymers (such as low molecular weight PLGA, PGA, or PLA), low-melting-point waxes such as plant, animal, or synthetic waxes, lanolin, jojoba oil, or combinations thereof.

71. A sustained-release drug delivery system or method of treatment for use according to any one of claims 68 to 70, wherein the activator is dissolved or dispersed in the hydrophobic organic liquid, or the activator is the hydrophobic organic liquid or forms at least a part thereof.

72. A kit suitable for use in forming in situ a sustained-release drug delivery system or a hydrogel for sustained-release drug delivery containing an activator as defined in any one of the prior claims, wherein the kit is provided in a separate container, a) A first precursor formulation comprising at least one first multi-arm precursor having a complementary second functional group and a first functional group suitable for forming a linkage by click chemistry, and a first container optionally comprising at least one solvent, b) A second precursor formulation comprising at least one second multi-arm precursor having a second functional group suitable for forming a link with the first functional group by a click chemical reaction, and a second container optionally comprising at least one solvent, c) comprising at least one activator contained in a separate third container and / or contained in at least one or both of the first and second containers, and optionally at least one solvent, d) The kit wherein at least one of the first multi-arm precursor and / or the at least one second multi-arm precursor is a polymer multi-arm precursor having a polymer arm comprising a polymer unit, and at least one of the first multi-arm precursor or the second multi-arm precursor comprises a linker unit L comprising a hydrolyzable ester bond.

73. The kit according to claim 72, wherein the at least one solvent in the first, second, or third container is independently selected from water, PBS buffer, TBS buffer, or a buffered aqueous medium such as physiological saline, or any mixture thereof, and optionally comprises at least one additive selected from pharmaceutical excipients, sugars, salts, additional therapeutic or diagnostic agents, thickeners, viscosity enhancers, stabilizers, or auxiliaries.

74. The kit according to any one of claims 72 or 73, wherein the at least one first precursor formulation comprises a mixture of two or more first precursors, each having different first functional groups that are suitable for forming a link with a complementary second functional group by click chemistry but do not react with each other.

75. The kit according to any one of claims 72 or 73, wherein the at least one second precursor formulation comprises a mixture of two or more second precursors, each having different second functional groups that are suitable for forming a link with a complementary first functional group by click chemistry but do not react with each other.

76. The kit according to any one of claims 74 or 75, wherein the different first or second functional groups are selected to have different kinetic rate constants or reaction rates in their click reactions with the corresponding second or first functional group counterparts.

77. The kit according to any one of claims 72 to 76, wherein the first and second functional groups are selected to provide a gel time from combining the first and second precursor formulations of at least about 1 minute, for example, about 2 minutes or more, or about 3 minutes or more, for example, about 1 to 15 minutes, for example, about 2 to 10 minutes, 2 to 8 minutes, or 2 to 6 minutes, or 2 to 4 minutes.

78. The kit according to any one of claims 72 to 77, wherein the first formulation is provided in a first syringe, the second formulation is provided in a second syringe, and the syringes are connected via a Y-type mixer to combine the two formulations.

79. The kit according to any one of claims 72 to 78, wherein the activator exists as a solid, such as being obtained from a freeze-dried mixture that is suitable for reconstitution in an aqueous solution before use.

80. The kit according to any one of claims 72 to 79, wherein the at least one first precursor and the at least one second precursor are selected independently of each other or from multi-arm polyethylene glycol (PEG) precursors having 3 to 10 PEG arms, such as 4 to 8 PEG arms.

81. The first and second multi-arm precursors independently have an average molecular weight (M) of 5,000 to 100,000 daltons, such as 5,000 to 40,000 daltons and 10,000 to 40,000 daltons. ｎ A kit according to any one of claims 71 to 80, comprising ).

82. The kit according to any one of claims 71 to 81, wherein the linker unit L comprises a dibasic acid, or an acid and amide group such as succinic acid, glutaric acid, adipic acid, azelaic acid, or an acid amide thereof such as glutaramide.

83. The aforementioned linker unit L is given by equation (i): 【Transformation 8】 It includes a structure represented by the formula, where U １ and U ２ However, independently, it is NH or O, U １ and U ２ The kit according to any one of claims 71 to 82, wherein at least one of them is O, preferably both are O, and t is an integer from 0 to 10.

84. The kit according to any one of claims 71 to 83, wherein the first and second formulations each have a concentration of the polymer precursor of about 2.5% to about 30% by weight, such as about 5% to 20% by weight or about 10% by weight, based on the total weight of the formulation.

85. The kit according to any one of claims 71 to 84, wherein the combined first and second formulations each have a concentration of the polymer precursor of about 5% to about 30% by weight, such as about 5% to 25% by weight, based on the total weight of the formulations.

86. The kit according to any one of claims 71 to 85, wherein the first and second functional groups are selected from a pair comprising a strain alkyne moiety such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonine (BCN) and an azide, or norbornene or transcyclooctene (TCO) and a tetrazine (Tz) moiety, or a strain alkyne moiety such as dibenzocyclooctin (DBCO) or bicyclo[6.1.0]nonine (BCN) and a tetrazine (Tz) moiety, or any combination thereof.

87. The kit according to claim 86, wherein the first precursor is a dibenzocyclooctin (DBCO) group-functionalized multi-arm PEG, and the second precursor is a combination of a tetrazine group-functionalized multi-arm PEG and an azide group-functionalized multi-arm PEG, or the first precursor is a combination of a dibenzocyclooctin (DBCO) group-functionalized multi-arm PEG and a norbornene group-functionalized multi-arm PEG, and the second precursor is a tetrazine group-functionalized multi-arm PEG.

88. The kit according to any one of claims 71 to 87, wherein the activator is contained in the hydrogel without being covalently conjugated to the hydrogel, or the activator is contained in the hydrogel and at least partially covalently conjugated to the hydrogel.

89. The kit according to any one of claims 71 to 88, wherein the activator is selected from the group consisting of therapeutic or diagnostic activators such as those defined above, or the therapeutic activator is a small molecule activator or biomolecule activator, preferably a small molecule activator, peptide, protein, or virus, preferably compstatin, APL-1, and Fc-III-4C, Beob 15 (brolucizumab), bevacizumab (avastin), Zimura (abasincaptado pegol), pegcetacoplan, avisical pegol, lamparizumab, Fovista, listeganib, AXT107, elamipretide, THR149, ALM201, VGB3, and largazole, preferably bevacizumab.

90. A kit according to any one of claims 71 to 89 for use in a method for forming an activator that releases a hydrogel in situ at a treatment site within a patient's body.

91. A sustained-release drug delivery hydrogel available from a kit according to any one of claims 71 to 90.

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