CN117651549A

CN117651549A - Anti-angiogenic agents and related methods

Info

Publication number: CN117651549A
Application number: CN202280049658.4A
Authority: CN
Inventors: 苏心怡; 赵欣欣; 思恩·奎妮·陈; 沃尔特·亨齐克; 刘增平; 罗贤俊; 薛琨; V·A·巴拉蒂
Original assignee: Agency for Science Technology and Research Singapore; Singapore Health Services Pte Ltd
Current assignee: Agency for Science Technology and Research Singapore; Singapore Health Services Pte Ltd
Priority date: 2021-07-26
Filing date: 2022-07-07
Publication date: 2024-03-05

Abstract

An anti-angiogenic agent is provided comprising: a multiblock copolymer in the form of one or more micelles, wherein the copolymer comprises a first poly (alkylene glycol) block, a second poly (alkylene glycol) block, and a polyester block. Also provided are methods of preparing the anti-angiogenic agents and pharmaceutical uses of the anti-angiogenic agents.

Description

Anti-angiogenic agents and related methods

Technical Field

The present disclosure relates generally to anti-angiogenic agents. The disclosure also relates to methods of preparing anti-angiogenic agents and related uses.

Background

Retinal diseases, such as neovascular age-related macular degeneration (nAMD) and diabetic macular edema (DMO), account for a significant proportion of visual impairments worldwide. Current first line treatment for these vision-threatening retinal diseases is intravitreal injection (IVT) anti-vascular endothelial growth factor (anti-VEGF) compounds to prevent abnormal vascular growth and leakage of fluids from these vessels. anti-VEGF compounds approved by the Food and Drug Administration (FDA) include ranibizumab @Novartis) and albesieadditional (>Bayer). Bevacizumab (+)>Novartis) are also not indicated for the treatment of such diseases and have similar efficacy. These anti-VEGF compounds are protein-based biologicals.

This places a significant burden on individuals and healthcare systems because patients require multiple intravitreal injections to control the disease. Furthermore, the invasiveness of intravitreal injections can lead to vision-threatening complications such as endophthalmitis. Because of the low compliance rate of long-term treatment regimens, there is a great interest in creating more tolerable treatment strategies. In recent years, pharmaceutical companies have developed compounds or sustained drug delivery systems with inherently more durable anti-angiogenic effects in order to reduce the number of intravitreal injections that a patient must undergo. In 2019, FDA approved Buxigroup monoclonal antibody @Novartis), which is able to maintain a longer effect than the previously described compounds. However, this strategy still does not completely avoid intravitreal injections, but only reduces the number of intravitreal injections that a patient must undergo.

Local delivery of anti-VEGF to the posterior segment of the eye will avoid the complications described above. However, multiple static and dynamic ocular barriers between the cornea and retina prevent the drug from reaching therapeutic concentrations on the retina sufficient to control the disease. While topical delivery systems for small molecule drugs have met with initial success, the development of such systems for FDA-approved protein-based anti-VEGF compounds has been challenging. Thus, different teams have attempted to locally deliver small molecules with anti-angiogenic effects, but have limited anti-disease efficacy. These successful topical delivery systems are mostly used to deliver small molecules to the anterior segment of the eye rather than the retina.

To date, the only anti-angiogenic eye drop in clinical trials is PAN-90806 (PanOptica), a small molecule Tyrosine Kinase Inhibitor (TKI) against VEGF-receptor 2 for nAMD treatment. PAN-90806 is a product under development. Other preclinical studies have demonstrated the use of hydrogel-based drug delivery systems to extend corneal residence time for delivery of hydrophobic small molecules to the retina, but have not demonstrated their use to enhance the permeability of hydrophilic protein-based drugs across the ocular barrier for drug delivery.

Few studies report the use of drug delivery platforms to locally deliver hydrophilic macromolecular biologics for retinal treatment. For example, liposomes with additional anionic phospholipid-binding protein annexin A5 (Anx 5) have been used to enhance delivery of bevacizumab to rabbit vitreous. The addition of AnxA5 significantly increased bevacizumab concentration in the vitreous of rat and rabbit eyes, but no therapeutic effect was demonstrated in this study. In addition, cell penetrating peptides composed of oligoarginines have also been used to successfully deliver bevacizumab to porcine vitreous, exhibiting therapeutic effects in Choroidal Neovascularization (CNV). Although cell penetrating peptides have been widely used for intracellular delivery, they have not been approved by the FDA due to persistent concerns about stability and immunogenicity.

In view of the above, there is a need to solve or at least ameliorate the problems described above. In particular, there is a need for anti-angiogenic agents, methods of preparing anti-angiogenic agents and related uses of anti-angiogenic agents that can solve or at least ameliorate the above problems.

Disclosure of Invention

In one aspect, an anti-angiogenic agent is provided, comprising:

multiblock copolymers in the form of one or more micelles,

wherein the copolymer comprises a first poly (alkylene glycol) block, a second poly (alkylene glycol) block, and a polyester block.

In one embodiment, the copolymer comprises at least urethane linkage groups (urethane/carbamate linkage) and/or allophanate linkage groups.

In one embodiment, the mole ratio of the first poly (alkylene glycol) block, the second poly (alkylene glycol) block, and the polyester block in the copolymer is from about 1 to 10:1:0.01 to 1.5.

In one embodiment, the first poly (alkylene glycol) and the second poly (alkylene glycol) are selected from the group consisting of poly (ethylene glycol) (PEG), poly (propylene glycol) (PPG), poly (butylene glycol), and combinations thereof; the polyester is selected from the group consisting of Polycaprolactone (PCL), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polyhydroxyalkanoate (PHA), and combinations thereof.

In one embodiment, the total polymer concentration of the copolymer is from 0.01 wt% to 6 wt%.

In one embodiment, the anti-angiogenic agent comprises at least 90 weight percent water content.

In one embodiment, the hydrodynamic size of the one or more micelles is 1nm to 100nm.

In one embodiment, the anti-angiogenic agent further comprises one or more bioactive substances complexed with or encapsulated by the copolymer micelle.

In one embodiment, the one or more bioactive substances include anti-vascular endothelial growth factor (anti-VEGF).

In one embodiment, the anti-VEGF is selected from bevacizumab, albespride, ranibizumab, and busitumumab.

In one embodiment, one or more bioactive substances are encapsulated by the copolymer micelle at an encapsulation rate of greater than 25%.

In one embodiment, the anti-angiogenic agent is formulated as a topical ophthalmic formulation.

In one aspect, there is provided a method of preparing an anti-angiogenic agent disclosed herein, the method comprising:

adding the copolymer to an aqueous medium at a concentration not lower than the critical micelle concentration of the copolymer but not higher than the sol-gel transition concentration of the copolymer to form micelles,

In one embodiment, the copolymer is present in the aqueous medium at a concentration of 0.01 wt% to 6 wt%.

In one embodiment, the method further comprises complexing or encapsulating the one or more bioactive substances with micelles.

In one embodiment, the method further comprises coupling the first poly (alkylene glycol) block, the second poly (alkylene glycol) block, and the polyester block together via at least a urethane linkage group and/or an allophanate linkage group.

In one embodiment, the coupling step is performed in the presence of a coupling agent comprising an isocyanate monomer containing two isocyanate functional groups.

In one embodiment, the coupling step is carried out in the presence of a catalyst selected from the group consisting of an alkyl tin compound, an aryl tin compound, and a dialkyl tin diester, such as dibutyl tin dilaurate, dibutyl tin diacetate, dibutyl tin dioctoate, and dibutyl tin distearate.

In one embodiment, the coupling step is performed in the presence of a solvent selected from toluene, benzene, xylene, halogenated organic solvents, halogenated alkane solvents, chlorinated solvents, methylene chloride, ethylene dichloride, carbon tetrachloride and chloroform (or chloroform).

In one aspect, there is provided an anti-angiogenic agent as disclosed herein for use in medicine.

In one aspect, there is provided an anti-angiogenic agent as disclosed herein for use in the prevention or treatment of ocular disease.

In one aspect, there is provided an anti-angiogenic agent disclosed herein for use in the prevention or treatment of cancer.

In one aspect, there is provided the use of an anti-angiogenic agent disclosed herein in the manufacture of a medicament for preventing or treating an ocular disease.

In one aspect, there is provided the use of an anti-angiogenic agent disclosed herein in the manufacture of a medicament for preventing or treating cancer.

In one aspect, there is provided a method of preventing or treating an ocular disease, the method comprising administering to a subject in need thereof an anti-angiogenic agent disclosed herein.

In one aspect, there is provided a method of preventing or treating cancer, the method comprising administering to a subject in need thereof an anti-angiogenic agent disclosed herein.

In one embodiment, the ocular disease is selected from angiogenic ocular disease, ocular disease of the anterior segment of the eye, ocular disease of the posterior segment of the eye, neovascular related ocular posterior segment disease, retinal disease, neovascular age-related macular degeneration (AMD) such as neovascular AMD, diabetic retinopathy, diabetic macular edema (DMO), choroidal Neovascularization (CNV), central Retinal Vein Occlusion (CRVO), corneal neovascularization, and retinal neovascularization.

In one embodiment, the anti-angiogenic agent is intended for topical administration to a subject in need thereof.

In one embodiment, the anti-angiogenic agent is formulated as an eye drop.

Definition of the definition

The term "polymer" as used herein refers to a compound comprising repeating units that is produced by a polymerization process. The units constituting the polymer are generally derived from monomers and/or macromers. The polymer typically comprises a number of repeats of the structural unit.

The term "monomer" or "macromer" as used herein refers to a chemical entity that can be covalently linked to one or more than one such entity to form a polymer.

The term "bond" refers to a connection between atoms in a compound or molecule. The bond may be a single bond, a double bond or a triple bond.

In the following definitions of many substituents, it is noted that "the group may be a terminal group or a bridging group". This is intended to indicate that the use of this term is intended to include the case where the group is a terminal group/moiety and the case where the group is a junction between two other moieties of a molecule. Using the term "alkyl" having 1 carbon atom as an example, it should be understood that when present as a terminal group, the term "alkyl" having 1 carbon atom may refer to the-CH ₃ When present as a bridging group, the term "alkyl" having 1 carbon atom may refer to the group-CH ₂ -and the like.

The term "alkyl" as a group or part of a group refers to a straight or branched chain aliphatic hydrocarbon group having 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. Examples of suitable straight and branched chain alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, hexyl, pentyl, 1, 2-dimethylpropyl, 1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2-dimethylbutyl, 3-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl 1, 2-trimethylpropyl, 1, 2-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2-dimethylpentyl, 3-dimethylpentyl, 4-dimethylpentyl, 1, 2-dimethylpentyl, 1, 3-dimethylpentyl, 1, 4-dimethylpentyl, 1,2, 3-trimethylbutyl, 1, 2-trimethylbutyl, 1, 3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl and the like. The group may be a terminal group or a bridging group.

The term "alkenyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, which may be straight or branched, having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms in the chain. The group may contain multiple double bonds, each of which may independently be in the configuration E or Z. Exemplary alkenyl groups include, but are not limited to, vinyl (ethenyl), vinyl (vinyl), allyl, 1-methylethenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1, 3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1, 3-pentadienyl, 2, 4-pentadienyl, 1, 4-pentadienyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1, 3-hexadienyl, 1, 4-hexadienyl, 2-methylpentanenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, and the like. The group may be a terminal group or a bridging group.

The term "alkynyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond, which may be straight or branched, having 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms in the chain. The group may contain multiple triple bonds. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 9-decynyl and the like. The group may be a terminal group or a bridging group.

The term "alkylene" as used herein is intended to broadly refer to a divalent aliphatic hydrocarbon group (e.g., alkyl, alkenyl, or alkynyl as defined herein). The alkylene groups may be linear, branched, saturated, unsaturated, cyclic, acyclic, substituted and/or unsubstituted. Examples of alkylene groups include methylene (i.e. -CH ₂ Or "alkylene" having 1 carbon atom, ethylene (i.e. -CH) ₂ CH ₂ Or "alkylene" having 2 carbon atoms), propylene (i.e. "alkylene" having 3 carbon atoms), and the like.

The term "poly (alkylene glycol)" as used herein is intended to broadly refer to polymers containing ether groups (i.e., -O-R-, where R is alkylene as defined herein) in the repeating units. In various embodiments, the term poly (alkylene glycol) may be used interchangeably with the terms "polyglycol", "polyether" or "poly (alkylene oxide)". Examples of the poly (alkylene glycol) include poly (ethylene glycol) (PEG) (or polyethylene oxide), poly (propylene glycol) (PPG) (or polypropylene oxide), poly (butylene glycol) (or polybutylene oxide), and the like.

The term "polyester" as used herein is intended to broadly refer to polymers containing ester groups (i.e. -O-C (=o) -) in the repeat unit. Examples of polyesters include Polycaprolactone (PCL), poly (lactic acid) or Polylactide (PLA), polyglycolic acid (PGA), polyethylene adipate (PEA), polyethylene terephthalate (PET), polyhydroxyalkanoates (PHA) (e.g., polyhydroxybutyrate (PHB)), polylactic acid-glycolic acid copolymers (PLGA), and the like.

The term "urethane linkage group" (urethane linkage/carbamate linkage) as used herein is intended to broadly refer to a group containing-O-C (=o) -N (R), wherein R is hydrogen or an organic group (e.g., a hydrocarbon group). For example, a "urethane linkage" may be-O- (c=o) -N (H) -. In various embodiments, a "urethane linkage group" may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 urethane groups. For example, the "urethane linkage" may also be-O- (c=o) -N (R) -R ' -N (R) - (c=o) -O-, or-O- (c=o) -N (H) -R ' -N (H) - (c=o) -O-, wherein R and R ' are each independently hydrogen or an organic group (e.g., a hydrocarbon group).

The term "allophanate linkage" as used herein is intended to broadly refer to groups containing [ -N (R) -C (=o) ] -N (R ') -C (=o) -O-, wherein R and R' are each independently hydrogen or an organic group (e.g., a hydrocarbon group). In various embodiments, an "allophanate linkage group" refers to a group formed by the reaction between an isocyanate group and a urethane group, wherein R and R' are derived from an isocyanate and a urethane, respectively. It should be appreciated that in various embodiments, allophanate formation is reversible. In various embodiments, an "allophanate linkage group" may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 allophanate groups.

The term "carbonate linkage" as used herein is intended to broadly refer to a group containing-O-C (=o) -O-. In various embodiments, a "carbonate linkage group" may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 carbonate groups.

The term "ester linkage" as used herein is intended to broadly refer to a group containing-O-C (=o) -. In various embodiments, an "ester linkage" may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 ester groups.

The term "urea linkage" as used herein is intended to broadly refer to groups containing-N (R) -C (=o) -N (R ') -, wherein R and R' are each independently hydrogen or an organic group (e.g., a hydrocarbon group). In various embodiments, a "urea linkage group" may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 urea groups.

When used in reference to a chemical structure or moiety, the term "substituted" means that one or more hydrogen atoms in the chemical structure or moiety is replaced by a chemical moiety or functional group, e.g., by an alcohol, alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g., methyl, ethyl, propyl, t-butyl), alkynyl, alkylcarbonyloxy (-O)C (O) alkyl), amide (-C (O) NH-alkyl-or-alkyl NHC (O) alkyl), amine (e.g., alkylamino, arylamino, arylalkylamino), aryl, aryloxy, azo, carbamate (carbamoyl) (-NHC (O) O-alkyl-or-OC (O) NH-alkyl), carbamoyl (carbamyl) (e.g., CONH) ₂ And CONH-alkyl, CONH-aryl and CONH-arylalkyl), carboxyl, carboxylic acid, cyano, ester, ether (e.g., methoxy, ethoxy), halogen, haloalkyl (e.g., -CCl) ₃ 、-CF ₃ 、-C(CF ₃ ) ₃ ) Heteroalkyl, isocyanate, isothiocyanate, nitrile, nitro, phosphodiester, sulfide, sulfonamide (e.g., SO) ₂ NH ₂ ) Sulfone, sulfonyl (including alkylsulfonyl, arylsulfonyl, and arylalkylsulfonyl), sulfoxide, thiol (e.g., mercapto, thioether), or urea (-NHCONH-alkyl-).

The term "micron" as used herein should be construed broadly to include dimensions of about 1 micron to about 1000 microns.

The term "nano" as used herein should be construed broadly to include dimensions of less than about 1000 nanometers, less than about 500 nanometers, less than about 100 nanometers, or less than about 50 nanometers.

The terms "treatment", "treatment" and "therapy" and their synonyms as used herein refer to therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a medical condition, including but not limited to diseases, symptoms and disorders. Medical conditions also include physical reactions to diseases or conditions, such as inflammation. Subjects in need of such treatment include subjects already with a medical condition, as well as subjects prone to develop a medical condition or subjects in need of prophylaxis of such a medical condition.

As used herein, the term "therapeutically effective amount" of a compound is an amount of an active agent that is capable of preventing or at least slowing (reducing) a medical condition, such as cancer, angiogenic eye disease, eye disease of the anterior segment of the eye, eye disease of the posterior segment of the eye, neovascular related eye posterior segment disease, retinal disease, neovascular age-related macular degeneration (AMD), such as neovascular AMD, diabetic retinopathy, diabetic macular edema (DMO), choroidal Neovascularization (CNV), central Retinal Vein Occlusion (CRVO), corneal neovascularization, and retinal neovascularization. The dosage and administration of the compounds, compositions and formulations of the present disclosure can be determined by one of ordinary skill in the clinical pharmacology or pharmacokinetic arts. See, e.g., mordinti and Rescino, (1992) Pharmaceutical research.9:17-25; morenti et al, (1991) Pharmaceutical research.8:1351-1359; and Mordinti and Chappell, "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development, yacobi et al (edit) (Pergamon Press: NY, 1989), pages 42-96. The effective amount of the active agents of the present disclosure used in therapy will depend, for example, on the purpose of the treatment, the route of administration, and the condition of the patient. Thus, it may be necessary for a therapist to adjust the dosage and alter the route of administration as required to obtain the best therapeutic effect.

The term "subject" as used herein includes both patients and non-patients. The term "patient" refers to an individual who has or is likely to have a medical condition, such as cancer, while "non-patient" refers to an individual who does not have, and is likely not to have, a medical condition. "non-patient" includes healthy individuals, non-diseased individuals, and/or individuals without a medical condition. The term "subject" includes humans and animals. Animals include mice and the like. "murine" refers to any mammal of the murine family, such as mice, rats, and the like.

The term "coupled" or "connected" as used in this specification is intended to encompass a direct connection or a connection through one or more intermediate means, unless otherwise indicated.

When referring to two elements, the term "associated" as used herein refers to a broad association between the two elements. The association includes, but is not limited to, a physical, chemical, or biological association. For example, when element a is associated with element B, elements a and B may be directly or indirectly attached to each other, or element a may contain element B, and vice versa.

When referring to two elements, the term "adjacent" as used herein means that one element is in close proximity to another element and may be, but is not limited to, elements in contact with each other or may also include elements separated by one or more additional elements disposed therebetween.

The term "and/or", e.g. "X and/or Y", is understood to mean "X and Y" or "X or Y", and should be used to provide explicit support for both meanings or for either meaning.

Furthermore, in the description herein, the word "substantially" wherever used is understood to include, but is not limited to, "all" or "completely" and the like. Furthermore, wherever terms such as "include," "comprising," and the like are used, they are intended to be non-limiting descriptive language, they broadly encompass the elements/components set forth after such terms, as well as other components not explicitly set forth. For example, when "comprising" is used, reference to "a" feature is also intended to reference "at least one" of the feature. Terms such as "comprising," "including," and the like, may be considered a subset of terms such as "comprising," "including," and the like, in the appropriate context. Thus, in the embodiments disclosed herein that use terms such as "comprising," "including," and the like, it is understood that these embodiments provide teachings for the use of corresponding embodiments that use terms such as "consisting," "consisting," and the like. Furthermore, terms such as "about," "approximately," and the like, are generally intended to cover a reasonable variation, such as a variation of +/-5% of the disclosed value, or a variation of 4% of the disclosed value, or a variation of 3% of the disclosed value, a variation of 2% of the disclosed value, or a variation of 1% of the disclosed value.

Furthermore, in the description herein, certain values may be disclosed within ranges. The values showing the endpoints of the ranges are intended to be illustrative of the preferred range. Wherever a range is described, it is intended that the range covers and teaches all possible sub-ranges as well as individual values within the range. That is, the endpoints of the range are not to be construed as limitations on the variation. For example, a description of a range of 1% to 5% is intended to specifically disclose sub-ranges of 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, etc., as well as individual values within the range, such as 1%, 2%, 3%, 4%, and 5%. The intent of the specific disclosure above is to be applicable to any range of depths/widths.

Additionally, when describing some embodiments, the present disclosure may have disclosed the methods and/or processes as a particular sequence of steps. However, unless otherwise required, it should be understood that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps are also possible. The particular sequence of steps disclosed herein should not be construed as undue limitations. Unless otherwise required, the methods and/or processes disclosed herein should not be limited to steps performed in the order recited. The order of the steps may be altered and still remain within the scope of the disclosure.

Furthermore, it should be understood that while the present disclosure provides embodiments having one or more of the features/characteristics discussed herein, in other alternative embodiments one or more of the features/characteristics may be disclaimed, and the present disclosure provides support for these disclaimers and these related alternative embodiments.

Detailed Description

Exemplary, non-limiting embodiments of anti-angiogenic agents, methods of making anti-angiogenic agents, and related methods/uses are disclosed below.

In various embodiments, polymeric particles, more particularly, multiblock copolymers in the form of one or more micelles, are provided, wherein the copolymer comprises/consists essentially of/consists of a first poly (alkylene glycol) block, a second poly (alkylene glycol) block, and a polyester block. Advantageously, in various embodiments, the multiblock copolymer micelle has intrinsic/inherent anti-angiogenic properties. Thus, in various embodiments, micelles may also be categorized as anti-angiogenic agents, as the micelles themselves are anti-angiogenic. Advantageously, embodiments of micelles may directly reduce proliferation, migration and tube formation of angiogenic cells in the absence of other anti-angiogenic factors. Even more advantageously, embodiments of the polymeric micelles (e.g., nanomicelles) can impart unexpectedly high anti-angiogenic effects compared to the corresponding hydrogels or free polymeric forms. In fact, experiments performed by the inventors have shown that the hydrogel form or the free polymer form does not achieve an anti-angiogenic effect.

Thus, in various embodiments, there is also provided an anti-angiogenic agent comprising a multiblock copolymer micelle.

In various embodiments, the multi-block copolymer is a three-component multi-block polymer. For example, a multi-block polymer consists of three different polymer blocks. Thus, in various embodiments, the first poly (alkylene glycol) block and the second poly (alkylene glycol) block are different from each other. In some embodiments, the multi-block copolymer comprises more than three polymer blocks.

In various embodiments, the multi-block copolymer is a polymer that is not chemically crosslinked, or is a non-crosslinked/uncrosslinked/uncrosslinkable polymer. The multiblock copolymer may form micelles by/due to physical interactions. Advantageously, in various embodiments, the preparation/formation of the copolymer micelles disclosed herein does not require the use of any additional chemical crosslinking agents or crosslinking agents.

The multi-block copolymer may have at least one unit of the following structural sequence a-B-C, wherein a comprises a first poly (alkylene glycol), B comprises a second poly (alkylene glycol), and C comprises a polyester. In various embodiments, a is different from B, and the positions of A, B and C can be interchanged. In various embodiments, the multi-block polymer may comprise a plurality of polymer blocks of a first poly (alkylene glycol), a plurality of polymer blocks of a second poly (alkylene glycol), and a plurality of polyester polymer blocks. In various embodiments, the multi-block copolymer comprises more than 3 polymer blocks. The blocks may be randomly distributed/arranged in the copolymer.

In various embodiments, the copolymer comprises at least one of a urethane, allophanate, carbonate, ester, urea linking group, and/or combinations thereof. The first poly (alkylene glycol) polymer block, the second poly (alkylene glycol) polymer block, and the polyester polymer block can be chemically coupled together by at least one of a urethane, allophanate, carbonate, ester, urea linkage, or a combination thereof. In some embodiments, the first poly (alkylene glycol) polymer block, the second poly (alkylene glycol) polymer block, and the polyester polymer block are chemically coupled together by at least one urethane or allophanate linking group, optionally further coupled by one of carbonate, ester, urea linking groups, or a combination thereof. For example, each of the first poly (alkylene glycol) polymer block, the second poly (alkylene glycol) polymer block, and the polyester polymer block may be linked to their respective adjacent blocks by at least one urethane, allophanate, carbonate, ester, urea linkage group, or combination thereof. In various embodiments, the copolymer is a poly (ether ester) urethane polymer.

In various embodiments, the first poly (alkylene glycol) and the second poly (alkylene glycol) are independently selected from the group consisting of poly (ethylene glycol) (PEG), poly (propylene glycol) (PPG), poly (butylene glycol), and combinations thereof; the polyester is selected from the group consisting of Polycaprolactone (PCL) (e.g., poly (ε -caprolactone), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polyhydroxyalkanoate (PHA) (e.g., polyhydroxybutyrate (PHB), poly-3-hydroxybutyrate (P3 HB), poly-4-hydroxybutyrate (P4 HB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO)) and combinations thereof.

In various embodiments, the polyester is selected from the group consisting of Polycaprolactone (PCL) (e.g., poly (ε -caprolactone), polylactic acid (PLA), and combinations thereof, in one embodiment, the polyester comprises PCL. In one embodiment, the first poly (alkylene glycol) comprises PEG and the second poly (alkylene glycol) comprises PPG, and the polyester comprises PCL. In various embodiments, the multiblock copolymer is entirely different from the poly (ethylene glycol) -poly (propylene glycol) (PEG-PPG), poly (propylene glycol) -poly (ε -caprolactone) (PPG-PCL), or poly (ethylene glycol) -poly (ε -caprolactone) (PEG-PCL) polymer.

In various embodiments, the molar ratio of the first poly (alkylene glycol) to the second poly (alkylene glycol) is from about 1:1 to about 10:1. The molar ratio of the first poly (alkylene glycol) to the second poly (alkylene glycol) can be about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In various embodiments, the amount/concentration of polyester is from about 1% to about 10% by weight of the multiblock copolymer. The amount/concentration of polyester may be about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, or about 10 wt% of the multi-block copolymer. In some embodiments, the multi-block polymer comprises about 1 wt% poly (caprolactone). In various embodiments, the molar ratio of the first poly (alkylene glycol) block, the second poly (alkylene glycol) block, and the polyester block in the copolymer is from about 1 to 10:1:0.01 to 1.5.

In various embodiments, the copolymer is amphiphilic and comprises hydrophilic and hydrophobic portions.

In various embodiments, the anti-angiogenic agent comprises/consists essentially of/consists of the multiblock copolymers disclosed herein, optionally with one or more than one bioactive agent and water/aqueous medium/aqueous buffer (e.g., aqueous solution). Thus, the anti-angiogenic agent may be present as a composition or formulation.

In various embodiments, the copolymer is not in the form of a hydrogel or free polymer because it exists as one or more micelles. Advantageously, in various embodiments, the micelles are suitable for non-invasive intraocular penetration. It will be appreciated that when embodiments of the copolymers disclosed herein are dissolved in an aqueous solvent, they can undergo three different forms of variation, depending on, for example, polymer concentration and temperature. For example, when the concentration is low (< Critical Micelle Concentration (CMC)), the copolymer is typically present in the form of a free polymer solution, on the other hand, when the concentration is increased to a concentration above CMC, micelles, such as nano-micelles, are typically formed. Finally, if the concentration is further increased, the nano-micelles may crosslink together to form a hydrogel. It should also be understood that although the polymer types may be the same, the three corresponding forms and their properties are significantly characterized and differ from each other. Without being bound by theory, it is believed that the hydrogel form does not spontaneously penetrate the cornea and migrate into the intraocular space. On the other hand, embodiments of the micelle are capable of spontaneously migrating (e.g., by diffusion) across one or more barriers (e.g., one or more barriers separating the intraocular space from the external environment, such as the sclera and/or cornea) into the intraocular space (e.g., the posterior segment of the eye, the retina, the vitreous cavity, the vitreous humor, etc.).

In various embodiments, the copolymer micelle is self-assembled/formed/created in the presence of water, buffer, or other aqueous medium (e.g., aqueous solution). In various embodiments, the copolymer micelles self-assemble/form/generate at a concentration above the Critical Micelle Concentration (CMC) but below the sol-gel transition concentration of the copolymer. In an example, the polymer/copolymer micelle comprises poly (ethylene glycol), poly (propylene glycol), poly (epsilon-caprolactone) (referred to herein as "EPC") self-assembled into a micelle (e.g., a nano-micelle) in an aqueous solution at an appropriate concentration in the presence of anti-VEGF. Advantageously, the inventors have unexpectedly found that EPC polymer micelles complexed with an anti-VEGF drug can exhibit ocular barrier penetrating activity by penetrating the cornea and reaching the retina. For example, the inventors have found that by complexing an anti-VEGF such as aflibercept with EPC (e.g., nano EPC or nzpc), penetration of the cornea and sclera can be enhanced, which results in a 4-fold increase in detection of aflibercept in mouse vitreous after a single local instillation compared to aflibercept alone. Without being bound by theory, it is believed that when anti-VEGF complexes with EPC micelles, enhancement of permeation is associated with enhancement of intracellular uptake.

In various embodiments, the Critical Micelle Concentration (CMC) of the copolymer is about 0.01 wt% to about 2.00 wt%, about 0.05 wt% to about 1.95 wt%, about 0.10 wt% to about 1.90 wt%, about 0.15 wt% to about 1.85 wt%, about 0.20 wt% to about 1.80 wt%, about 0.25 wt% to about 1.75 wt%, about 0.30 wt% to about 1.70 wt%, about 0.35 wt% to about 1.65 wt%, about 0.40 wt% to about 1.60 wt%, about 0.45 wt% to about 1.55 wt%, about 0.50 wt% to about 1.50 wt%, about 0.55 wt% to about 1.45 wt%, about 0.60 wt% to about 1.40 wt%, about 0.65 wt% to about 1.35 wt%, about 0.70 wt% to about 1.30 wt%, about 0.75 wt% to about 1.75 wt%, about 0.40 wt% to about 1.60 wt%, about 0.45 wt% to about 1.55 wt%, about 0.50 wt% to about 1.95 wt%, about 0.0.15 wt% to about 1.80 wt%, about 1.95 wt% to about 1.15 wt%. In various embodiments, the CMC is about 0.105 wt% when measured at 25 ℃, and about 0.046 wt% when measured at 37 ℃. The CMC may be greater than about 0.105 wt% when the temperature is reduced to 20 ℃.

In the context of a variety of embodiments of the present invention, the total polymer concentration of the copolymer in the composition or formulation is from about 0.01 wt% to about 6.00 wt%, from about 0.02 wt% to about 5.50 wt%, from about 0.03 wt% to about 5.00 wt%, from about 0.04 wt% to about 4.50 wt%, from about 0.05 wt% to about 4.00 wt%, from about 0.06 wt% to about 3.50 wt%, from about 0.07 wt% to about 3.00 wt%, from about 0.08 wt% to about 2.50 wt%, from about 0.09 wt% to about 2.00 wt%, from about 0.10 wt% to about 1.90 wt%, from about 0.15 wt% to about 1.85 wt%, from about 0.20 wt% to about 1.80 wt%, from about 0.25 wt% to about 1.75 wt%, from about 0.30 wt% to about 1.70 wt%, from about 0.35 wt% to about 1.65 wt%, from about 0.40 wt% to about 1.60 wt%, from about 0.45 wt% to about 3.00 wt%, from about 0.08 wt% to about 2.50 wt%, from about 0.09 wt% to about 2.00 wt%, from about 0.10 wt% to about 1.90 wt%, from about 0.15 wt% to about 1.85 wt%, from about 0.20 wt% to about 1.80 wt%, from about 0.25 wt% to about 1.75 wt%, from about 0.30 wt% to about 1.70 wt%, from about 0.35 wt% to about 1.35 wt%, from about 0.35 wt% to about 1.60 wt%, from about 0.0.35 wt% to about 1.0.0.75 wt%, from about 1.0.05 wt% to about 1.0.0.0 wt%, from about 1.0.0 wt% to about 1.0.0.0 wt% to about 0.0 wt% or from about 0.0. In various embodiments, the total polymer concentration is less than the sol-gel transition concentration or the concentration at which the polymer forms/converts to gel form. For example, the sol-gel transition concentration may be from about 2.0 wt% to about 10.0 wt%, so the total polymer concentration may be no more than about 10.0 wt%, no more than about 9.0 wt%, no more than about 8.0 wt%, no more than about 7.0 wt%, no more than about 6.0 wt%, no more than about 5.0 wt%, no more than about 4.0 wt%, no more than about 3.0 wt%, or no more than about 2.0 wt%.

In various embodiments, the composition or formulation comprises an aqueous medium or aqueous buffer. The aqueous medium may be a balanced salt solution. In various embodiments, the balanced salt solution is a solution having a physiological pH and an isotonic salt concentration. In various embodiments, the balanced salt solution comprises at least one of a sodium salt, a potassium salt, a calcium salt, and a magnesium salt, such as calcium chloride, potassium chloride, magnesium chloride, sodium acetate, sodium citrate, and sodium chloride.

In various embodiments, the anti-angiogenic agent has a high water content in excess of about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, about 96 wt%, about 97 wt%, about 98 wt%, about 99 wt%, about 99.5 wt%, or about 99.9 wt%.

In various embodiments, the hydrodynamic size of the micelle is from about 1.0nm to about 100.0nm, from about 2.0nm to about 99.0nm, from about 5.0nm to about 95.0nm, from about 10.0nm to about 90.0nm, from about 15.0nm to about 85.0nm, from about 20.0nm to about 80.0nm, from about 25.0nm to about 75.0nm, from about 30.0nm to about 70.0nm, from about 35.0nm to about 65.0nm, from about 40.0nm to about 60.0nm, from about 45.0nm to about 55.0nm, or from about 50.0nm. Thus, in various embodiments, the micelle is a nano-micelle, i.e., a nano-sized micelle. In various embodiments where the micelles are nano-sized, the material properties may differ significantly from the bulk material in the manner of interacting with the cells. Thus, the advantageous properties of nanomicelles (e.g., anti-angiogenic effect and ability to penetrate the ocular structural anatomy, such as the cornea, to reach the intraocular space) are unexpected and cannot be readily understood from the properties of the bulk material of the copolymer (e.g., hydrogel form or free form).

In various embodiments, the copolymer micelle comprises a hydrophobic core. Advantageously, the hydrophobic drug or bioactive substance may be supported within a hydrophobic core. Examples of hydrophobic drugs include, but are not limited to, paclitaxel, doxorubicin, teniposide, etoposide, daunorubicin, methotrexate, mitomycin C, indomethacin, ibuprofen, cyclosporine, and dimethyl phthalate (DDB).

In various embodiments, the copolymer micelle further comprises one or more than one bioactive substance that is complexed with, encapsulated by, or incorporated into the copolymer/micelle. Thus, in various embodiments, the copolymer micelle may exist as a complex. Advantageously, various embodiments of the copolymer micelle may be used as a drug delivery system or drug carrier/nanocarrier. For example, drug delivery systems comprising EPC polymer micelles complexed with an anti-VEGF drug may be used for topical application to the eye and still reach the retina.

In various embodiments, the copolymer micelle has an encapsulation efficiency/loading of at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60%. In various embodiments, the loading of the bioactive substance increases in a manner that is dependent on the micelle concentration.

In various embodiments, the bioactive substance includes small molecules, macromolecules (e.g., molecular weights greater than 50 kDa), biological macromolecules (e.g., carbohydrates, lipids, proteins, and nucleic acids), therapeutic agents, and/or drug molecules (e.g., anti-tumor drugs) capable of providing a biological effect, a therapeutic effect, a prophylactic effect, or a combination thereof. In various embodiments, the bioactive substance includes a macromolecular anti-VEGF drug/agent.

In various embodiments, copolymer micelles with intrinsic anti-angiogenic properties are capable of cooperating with biologically active substances to provide a combined and enhanced therapeutic effect. For example, the bioactive substance may be a substance having an anti-angiogenic effect. Advantageously, this enhances or synergistically enhances the anti-angiogenic effect already inherently or inherently present in embodiments of the copolymer micelles disclosed herein. For example, in embodiments where the biologically active substance comprises anti-VEGF, the inherent anti-angiogenic properties of the copolymer micelle act synergistically with the anti-angiogenic properties of the anti-VEGF to provide an overall enhanced anti-angiogenic effect. Advantageously, the copolymer micelles may be in the form of local micelles or nanomicelles having enhanced anti-vascular endothelial growth factor (anti-VEGF) penetration and intrinsic anti-angiogenic effects for use in the synergistic treatment of diseases responsive to anti-angiogenic inhibition, such as neovascular retinal diseases. In various embodiments, the polymer/copolymer micelle comprises poly (ethylene glycol), poly (propylene glycol), poly (epsilon-caprolactone) (i.e., EPC), and forms EPC-drug complexes with anti-VEGF drugs to treat retinal neovascular diseases, for example, as topical eye drops (e.g., in aqueous solution).

Thus, in various embodiments, the bioactive substance includes an anti-vascular endothelial growth factor (anti-VEGF). Examples of anti-VEGF include, but are not limited to, bevacizumab, albespride, ranibizumab, and the like.

In various embodiments, the bioactive substance includes a Tyrosine Kinase Inhibitor (TKI). Examples of TKIs include, but are not limited to, brinib, ceritinib, duo Wei Tini, sunitinib, sorafenib, vanadinib, and the like.

In various embodiments, the bioactive substance includes an anticancer drug. Examples of anticancer drugs include, but are not limited to, docetaxel, mitoxantrone, gemcitabine, capecitabine, oxaliplatin, interferon, sunitinib, sorafenib, carboplatin, doxorubicin, methotrexate, vincristine, vinorelbine, pemetrexed, gefitinib, etoposide, irinotecan, cyclophosphamide, topotecan, cyclophosphamide, paclitaxel, mitomycin, bevacizumab, trastuzumab, cetuximab, temozolomide, procarbazine, and the like.

While in some embodiments the copolymer micelles may be capable of acting as a carrier for genes, small molecular weight drugs such as TKIs (e.g., sunitinib), anticancer drugs (e.g., avastin), etc., it should be understood that in various embodiments the bioactive substances used do not include genes, small molecular weight drugs such as TKIs (e.g., sunitinib), anticancer drugs (e.g., avastin), etc. For example, in various embodiments, the anti-VEGF used as the bioactive substance is completely different in structure and function from the substances described above.

Furthermore, it should be understood that while the delivery means may be topical for the various embodiments disclosed herein, the delivery mechanism may still be different from those topical applications used in the art. For example, embodiments of the present disclosure differ from the prior art of topical delivery of avastin to the posterior segment of the eye using Cell Penetrating Peptide (CPP) facilitated penetration or annexin A5 related liposomes. It will be appreciated that in this approach, the permeation enhancement is from a peptide, such as annexin A5, which is a protein rather than a copolymer micelle or nanomicelle as disclosed herein. The embodiments of copolymer micelles disclosed herein are not peptides, nor protein-based carriers. It will be appreciated that cell penetrating peptides are protein based and have limitations in practical human use. Furthermore, embodiments of the copolymer micelles disclosed herein are also not liposomes.

In various embodiments, the copolymer micelle enhances/increases intraocular penetration of the bioactive substance through a barrier layer, such as the sclera and/or cornea layer, and enhances/increases intracellular uptake of the bioactive substance.

The bioactive material may be present at a concentration of about 0.1mg/mL to about 100.0 mg/mL. In various embodiments, the bioactive agent is present at a concentration of about 0.1mg/mL, about 0.2mg/mL, about 0.5mg/mL, about 1.0mg/mL, about 2.0mg/mL, about 5.0mg/mL, about 10.0mg/mL, about 15.0mg/mL, about 20.0mg/mL, about 25.0mg/mL, about 30.0mg/mL, about 35.0mg/mL, about 40.0mg/mL, about 45.0mg/mL, about 50.0mg/mL, about 55.0mg/mL, about 60.0mg/mL, about 65.0mg/mL, about 70.0mg/mL, about 75.0mg/mL, about 80.0mg/mL, about 85.0mg/mL, about 90.0mg/mL, about 95.0mg/mL, about 98.0mg/mL, about 99.0mg/mL, or about 100.0 mg/mL.

In various embodiments, the copolymer micelle is biocompatible and/or non-toxic, and/or does not elicit an inflammatory or adverse immune response in an animal or human, particularly in an eye of an animal or human (e.g., a corneal epithelial cell or corneal barrier).

In various embodiments, the copolymer is substantially free of heavy metals and/or contaminants. For example, the copolymer may be substantially free of antimony and/or arsenic and/or cadmium and/or cobalt and/or copper and/or lead and/or lithium and/or mercury and/or nickel and/or vanadium.

In various embodiments, the copolymer is substantially free of solvent contaminants. For example, the copolymer may be substantially free of benzene and/or carbon tetrachloride and/or 1, 2-dichloroethane and/or 1, 1-trichloroethane and/or acetonitrile and/or chlorobenzene and/or chloroform and/or cyclohexane and/or 1, 2-dichloroethylene and/or dichloromethane and/or 1, 2-dimethoxyethane and/or N, N-dimethylacetamide and/or N, N-dimethylformamide and/or 1, 4-dioxane and/or 2-ethoxyethanol and/or ethylene glycol and/or formamide and/or hexane and/or methanol and/or 2-methoxyethanol and/or methylbutyl ketone and/or methylcyclohexane and/or N-methylpyrrolidone and/or nitromethane and/or pyridine and/or sulfolane and/or tetrahydrofuran and/or tetrahydronaphthalene and/or toluene and/or 1, 2-trichloroethylene and/or xylene (m-, p-, m-phenylene, ortho-isomer) and/or acetic acid and/or acetone and/or anisole and/or 1-butanol and/or 2-butanol and/or butyl acetate and/or tert-butyl methyl ether and/or cumene and/or dimethyl sulfoxide and/or ethanol and/or ethyl acetate and/or ethyl ether and/or ethyl formate and/or formic acid and/or heptane and/or isobutyl acetate and/or isopropyl acetate and/or methyl acetate and/or 3-methyl-1-butanol and/or methyl ethyl ketone and/or methyl isobutyl ketone and/or 2-methyl-1-propanol and/or pentane and/or 1-pentanol and/or 1-propanol and/or 2-propanol and/or propyl acetate.

In various embodiments, the copolymer has a short residence time and/or is capable of natural degradation in an animal within about 6 months, about 5 months, about 4 months, about 3 months, or about 2 months. In some embodiments, the copolymer has a short residence time and/or is capable of natural degradation in an animal in a time period of about 2 months to about 6 months.

In various embodiments, the copolymer is biocompatible and/or non-toxic and/or does not elicit an inflammatory or adverse immune response in an animal or human, particularly in the eyes of an animal or human.

In various embodiments, the copolymer or at least one or more blocks of the copolymer are biodegradable and/or naturally decomposable. In some embodiments, all of the polymer blocks are biodegradable.

Advantageously, in various embodiments, the copolymer micelle can be repeatedly/continuously/consistently administered/delivered to an animal or human over a period of at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 14 days, or about 21 days without causing a change in the morphology, organization, structure, and/or function of the eye (e.g., refractive components such as cornea, lens, corneal epithelium, and endothelial cells). For example, repeated administration/delivery does not result in accelerated cataract formation. The route of administration or delivery may be topical and/or non-invasive. Thus, in various embodiments, the anti-angiogenic agent is formulated as a topical ophthalmic formulation, such as in the form of eye drops.

In various embodiments, methods of preparing the anti-angiogenic agents disclosed herein are also provided, the methods comprising adding a copolymer to an aqueous medium to form micelles at a concentration that is not less than the critical micelle concentration of the copolymer but not greater than the sol-gel transition concentration of the copolymer, wherein the copolymer comprises a first poly (alkylene glycol) block, a second poly (alkylene glycol) block, and a polyester block. The copolymer may be in powder form or in dry form prior to addition to the aqueous medium. It will be appreciated that the anti-angiogenic agent, copolymer, aqueous medium, micelle, first poly (alkylene glycol) block, second poly (alkylene glycol) block, polyester block, and the like may have one or more of the properties or characteristics as previously described.

In various embodiments, the copolymer is added to an aqueous medium, such that the total polymer concentration is from about 0.01 wt% to about 6.00 wt%, from about 0.02 wt% to about 5.50 wt%, from about 0.03 wt% to about 5.00 wt%, from about 0.04 wt% to about 4.50 wt%, from about 0.05 wt% to about 4.00 wt%, from about 0.06 wt% to about 3.50 wt%, from about 0.07 wt% to about 3.00 wt%, from about 0.08 wt% to about 2.50 wt%, from about 0.09 wt% to about 2.00 wt%, from about 0.10 wt% to about 1.90 wt%, from about 0.15 wt% to about 1.85 wt%, from about 0.20 wt% to about 1.80 wt%, from about 0.25 wt% to about 1.75 wt%, from about 0.30 wt% to about 1.70 wt%, from about 0.35 wt% to about 1.65 wt%, from about 0.40 wt% to about 1.60 wt%, from about 0.45 wt% to about 1.55 wt%, from about 0.0.0 wt% to about 1.80 wt%, from about 0.0.0 wt% to about 1.25 wt% to about 1.80 wt%, from about 0.25 wt% to about 1.75 wt%, from about 0.0.0 wt% to about 1.80 wt%, from about 0.0.35 wt% to about 1.35 wt%, from about 0.0.0 wt% to about 1.95 wt%, from about 0.0.0 wt% to about 1.0 wt% and. In various embodiments, the total polymer concentration is less than the sol-gel transition concentration or the concentration at which the polymer forms/converts to gel form. For example, the sol-gel transition concentration may be from about 2.0 wt% to about 10.0 wt%, so the total polymer concentration may be no more than about 10.0 wt%, no more than about 9.0 wt%, no more than about 8.0 wt%, no more than about 7.0 wt%, no more than about 6.0 wt%, no more than about 5.0 wt%, no more than about 4.0 wt%, no more than about 3.0 wt%, or no more than about 2.0 wt%.

In various embodiments, the method further comprises coupling the first poly (alkylene glycol) block, the second poly (alkylene glycol) block, and the polyester block together via at least one urethane, allophanate, carbonate, ester, urea linkage group, and/or combinations thereof. For example, each of the first poly (alkylene glycol) polymer block, the second poly (alkylene glycol) polymer block, and the polyester polymer block may be attached to their respective adjacent blocks by urethane, allophanate, carbonate, ester, urea linking groups, or combinations thereof.

In some embodiments, the first poly (alkylene glycol) polymer block, the second poly (alkylene glycol) polymer block, and the polyester polymer block are chemically coupled through at least one urethane linkage or allophanate linkage, and optionally further coupled through one of carbonate, ester, urea linkages, or a combination thereof. In various embodiments, the copolymer is a poly (ether ester) urethane polymer.

In various embodiments of the method, the first poly (alkylene glycol) and the second poly (alkylene glycol) are independently selected from the group consisting of poly (ethylene glycol) (PEG), poly (propylene glycol) (PPG), poly (butylene glycol), and combinations thereof; the polyester is selected from the group consisting of Polycaprolactone (PCL) (e.g., poly (ε -caprolactone), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polyhydroxyalkanoate (PHA) (e.g., polyhydroxybutyrate (PHB), poly-3-hydroxybutyrate (P3 HB), poly-4-hydroxybutyrate (P4 HB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO)) and combinations thereof.

In various embodiments of the method, the polyester is selected from the group consisting of Polycaprolactone (PCL) (e.g., poly (. Epsilon. -caprolactone), polylactic acid (PLA), and combinations thereof.

In various embodiments, the method comprises coupling or mixing PEG, PPG, and PCL.

In various embodiments of the method, the first poly (alkylene glycol) and the second poly (alkylene glycol) are coupled or mixed in a molar ratio of about 1:1 to about 10:1. The molar ratio of the first poly (alkylene glycol) to the second poly (alkylene glycol) coupled or mixed together can be about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In various embodiments, the polyesters are coupled or mixed in an amount/concentration of from about 1% to about 10% by weight of the multiblock copolymer. The polyesters may be coupled or mixed in an amount/concentration of about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, or about 10 wt% of the multiblock copolymer. In some embodiments, after the coupling step, the multi-block polymer contains about 1 wt% poly (caprolactone). In various embodiments, the first poly (alkylene glycol) block, the second poly (alkylene glycol) block, and the polyester block in the copolymer are coupled in a molar ratio of about 1 to 10:1:0.01 to 1.5.

In various embodiments, the coupling and/or mixing step is performed at an elevated temperature of about 70 ℃ to about 150 ℃, about 72 ℃ to about 148 ℃, about 74 ℃ to about 146 ℃, about 76 ℃ to about 144 ℃, about 78 ℃ to about 142 ℃, about 80 ℃ to about 140 ℃, about 82 ℃ to about 138 ℃, about 84 ℃ to about 136 ℃, about 86 ℃ to about 134 ℃, about 88 ℃ to about 132 ℃, about 90 ℃ to about 130 ℃, about 92 ℃ to about 128 ℃, about 94 ℃ to about 126 ℃, or about 96 ℃ to about 124 ℃, about 98 ℃ to about 122 ℃, about 100 ℃ to about 120 ℃, about 102 ℃ to about 118 ℃, about 104 ℃ to about 116 ℃, about 106 ℃ to about 114 ℃, about 108 ℃ to about 112 ℃, or about 110 ℃.

In various embodiments, the coupling and/or mixing step is performed for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 26 hours, at least about 28 hours, at least about 30 hours, at least about 32 hours, at least about 34 hours, at least about 36 hours, at least about 38 hours, at least about 40 hours, at least about 42 hours, at least about 44 hours, at least about 46 hours, or at least about 48 hours.

In various embodiments, the coupling and/or mixing steps are performed in the absence of air and/or water/moisture and/or in the presence of an inert gas such as nitrogen.

In various embodiments, the coupling step is performed in the presence of a coupling agent. In various embodiments, the coupling agent includes an isocyanate monomer that includes at least two (two or more) isocyanate functional groups. The coupling agent may be a diisocyanate selected from the group consisting of hexamethylene diisocyanate, tetramethylene diisocyanate, cyclohexane diisocyanate, tetramethylxylene diisocyanate, dodecyl diisocyanate, 2, 4-toluene diisocyanate and 2, 6-toluene diisocyanate. In various embodiments, the linear polymer is formed by using an isocyanate monomer containing two isocyanate functional groups as a coupling agent. In various embodiments, the coupling agent comprises no more than 2 isocyanates, which may otherwise form branched polymers that do not impart the same cell penetrating effect as linear polymers.

In various embodiments, the coupling step is performed in the presence of a solvent. The solvent includes an anhydrous solvent selected from toluene, benzene, xylene, halogenated organic solvents, halogenated alkane solvents, chlorinated solvents, methylene chloride, ethylene dichloride, carbon tetrachloride and chloroform (or chloroform).

In various embodiments, the coupling step is performed in the presence of a catalyst. The catalyst may be a metal catalyst. In various embodiments, the metal catalyst comprises a tin catalyst selected from the group consisting of an alkyl tin compound, an aryl tin compound, and a dialkyl tin diester, such as dibutyltin dilaurate, dibutyltin diacetate, dibutyltin dioctoate, and dibutyltin distearate.

In various embodiments, the method further comprises complexing or encapsulating the one or more bioactive substances with micelles. In various embodiments, the bioactive substance may already be present in the aqueous medium (e.g., aqueous solution, water, buffer, etc.) prior to the step of adding the copolymer to the aqueous medium. Thus, in various embodiments, the bioactive substance may be added to the aqueous solution prior to formation of the copolymer micelle. In various embodiments, encapsulation of the bioactive substance in the micelle may occur during formation of the micelle itself (e.g., self-formation of the micelle during addition of the copolymer to the aqueous medium). One or more than one bioactive substance may have one or more than one property or feature as previously described. For example, the bioactive substance may be anti-vascular endothelial growth factor (anti-VEGF) and/or may be selected from bevacizumab, albesipine, ranibizumab and busiximab.

In various embodiments, the complex of copolymer micelle and anti-VEGF is formed by directly mixing the copolymer micelle (e.g., nfpc) with anti-VEGF. Increasing the concentration of copolymer (e.g., EPC) increases the encapsulation efficiency of anti-VEGF, thus increasing the barrier penetration efficiency of anti-VEGF. In one embodiment, when EPC micelles and anti-VEGF (e.g., afflibecept) complexes (referred to herein as "nfepc+a") are formed, one or more of the following properties are found to be enhanced: (i) corneal permeability against VEGF (in mice), (ii) amount of anti-VEGF in vitreous humor after a single topical administration, (iii) scleral and corneal permeability in vitro in pigs, (iv) intracellular uptake in vitro, (iv) anti-angiogenic effect due to the synergistic effect of the nfpc. Thus, in various embodiments, methods of preparing EPC anti-VEGF drug complexes are also provided, the methods comprising mixing EPC polymer micelles with an anti-VEGF drug.

In various embodiments, the bioactive material is added to, complexed with, or encapsulated by the copolymer micelle at a concentration of about 0.1mg/mL to about 100.0 mg/mL. In various embodiments, the bioactive agent is present at a concentration of about 0.1mg/mL, about 0.2mg/mL, about 0.5mg/mL, about 1.0mg/mL, about 2.0mg/mL, about 5.0mg/mL, about 10.0mg/mL, about 15.0mg/mL, about 20.0mg/mL, about 25.0mg/mL, about 30.0mg/mL, about 35.0mg/mL, about 40.0mg/mL, about 45.0mg/mL, about 50.0mg/mL, about 55.0mg/mL, about 60.0mg/mL, about 65.0mg/mL, about 70.0mg/mL, about 75.0mg/mL, about 80.0mg/mL, about 85.0mg/mL, about 90.0mg/mL, about 95.0mg/mL, about 98.0mg/mL, about 99.0mg/mL, or about 100.0 mg/mL.

In various embodiments, the method further comprises removing contaminants from the multi-block copolymer; and dissolving the multi-block copolymer in an aqueous medium to form multi-block micelles. The step of removing contaminants from the multi-block polymer may comprise purifying and/or washing the multi-block copolymer. The step of dissolving the multi-block copolymer in an aqueous medium may include redissolving the polymer (e.g., the final polymer powder) in an equilibrium salt solution (BSS). In various embodiments, the BSS is water-based.

In various embodiments, the step of removing contaminants from the multi-block copolymer includes dialysis to remove unreacted reactants, solvents, and catalysts (e.g., extensive dialysis to remove unreacted PEG, solvents, and metal catalysts, etc.).

It should be appreciated that while the copolymer micelle may be purified and/or washed, in various embodiments, the purification and/or washing of the copolymer micelle is optional. In various embodiments, the methods of preparing copolymer micelles disclosed herein are essentially simple mixing processes and can be applied without further purification, e.g., purification or washing after encapsulation may not be required. For example, EPC forms nano-micelles (nfpc) in aqueous solution and forms complexes with aflibercept by a simple mixing process, which can be used without further purification or washing. Advantageously, such direct mixing compounding methods minimize protein-drug denaturation by mechanical agitation, heating, or organic solvents. In addition, this direct embodiment of the composite manufacturing process simplifies the manufacturing process and enables in situ drug encapsulation. Even more advantageously, by eliminating the need for purification and/or washing, substantial drug losses can be prevented, allowing for maximization of drug loading.

The method may further comprise sterilizing the material used to prepare the anti-angiogenic agent, for example by autoclaving methods or techniques.

In various embodiments, there is also provided a method of preparing a copolymer micelle comprising (i) coupling one or more than one polymer block selected from the group consisting of poly (alkylene glycol), polyester, and combinations thereof to form a copolymer, optionally, the polymer blocks being chemically coupled/linked by at least a urethane linkage group; and (ii) dissolving the copolymer in water, buffer or other aqueous medium (e.g., aqueous solution) to form copolymer micelles. The coupling step may comprise mixing one or more polymers selected from the group consisting of poly (alkylene glycol), polyesters, and combinations thereof with a coupling agent in the presence of a catalyst and a suitable solvent to form a copolymer. The method may further comprise (iii) mixing one or more bioactive agents with the copolymer micelle. In various embodiments, the bioactive substance may already be present in the water, buffer or other aqueous medium (e.g., aqueous solution) of step (ii) prior to dissolving the copolymer.

In various embodiments, there is also provided an anti-angiogenic agent disclosed herein for use in preventing or treating an ocular disease, use of an anti-angiogenic agent disclosed herein in the manufacture of a medicament for preventing or treating an ocular disease, and/or a method of preventing or treating an ocular disease, comprising administering an anti-angiogenic agent disclosed herein (e.g., in a therapeutically effective amount) to a subject in need thereof. The ocular disease may be selected from angiogenic ocular disease, ocular disease of the anterior segment of the eye, ocular disease of the posterior segment of the eye, neovascular related ocular posterior segment disease, retinal disease, neovascular age-related macular degeneration (AMD) such as neovascular AMD, diabetic retinopathy, diabetic macular edema (DMO), choroidal Neovascularization (CNV), central Retinal Vein Occlusion (CRVO), corneal neovascularization, and retinal neovascularization.

In various embodiments, the anti-angiogenic agent is topically administered or applied to a subject in need thereof (e.g., the subject's eye). Advantageously, this is different from the prior art which involves the use of intravitreal Injections (IVT). For example, it is understood that the mainstay of treatment for age-related macular degeneration (AMD) is intravitreal injection. In contrast, embodiments of the present invention include less invasive topical applications, for example in the form of eye drops, because embodiments of the copolymer micelles can pass through the ocular barrier for delivery to the retinal region of the eye following ocular administration of the topical application. In fact, this represents a significant contribution to the field.

In various embodiments, there is also provided an ocular delivery system for delivering an anti-vascular endothelial growth factor (anti-VEGF) in an eye, the system comprising a copolymer micelle disclosed herein; and anti-VEGF encapsulated by copolymer micelles. In various embodiments, the ocular delivery system is a non-invasive ocular delivery system.

In various embodiments, there is also provided an anti-angiogenic system for delivering an anti-vascular endothelial growth factor (anti-VEGF) comprising a copolymer micelle disclosed herein; and anti-VEGF encapsulated by copolymer micelles.

In various embodiments, also provided are anti-angiogenic agents disclosed herein for use in preventing or treating a proliferative disease, such as cancer, use of the anti-angiogenic agents disclosed herein in the manufacture of a medicament for preventing or treating a proliferative disease, such as cancer, or a method of preventing and/or treating a proliferative disease, such as cancer, comprising administering (e.g., in a therapeutically effective amount) the anti-angiogenic agents disclosed herein to a subject in need thereof. Advantageously, since embodiments of the anti-angiogenic agent or copolymer micelle exhibit anti-angiogenic effects, they can be used as agents or carriers to block or reduce angiogenesis in undesired proliferating cells, such as tumor or cancer cells with uncontrolled/undesired proliferation, thereby blocking the nutrient and oxygen supply to these cells and effectively "starving" them.

In various embodiments, also provided are anti-angiogenic agents, copolymer particles/micelles, micellar drug delivery systems, drug carriers, ocular delivery systems, anti-angiogenic systems disclosed herein, for use in medicine (e.g., for treating one or more of the diseases or conditions mentioned herein) and/or for drug delivery (e.g., for the eye).

Drawings

Fig. 1 is a schematic illustration of a multi-block copolymer (e.g., EPC polymer 100) according to various embodiments disclosed herein. As shown in fig. 1, EPC polymer 100 may self-assemble into micelles (e.g., polymer nanomicelle (nfpc) 102) in buffer 104. The nEPC 102 is capable of inhibiting angiogenesis alone, both in vitro and ex vivo. As shown in fig. 1, aflibercept 108 may be encapsulated by the nsec by direct mixing to form nsec+aflibercept (nsec+a) complex 106. When topically administered on the mouse cornea, the nEPC acts as a drug carrier, delivering a common transcornea of A Bai Xi to achieve therapeutic concentrations of retina in a laser-induced disease model of Choroidal Neovascularization (CNV).

Figures 2-4 illustrate characterization of EPC nanomicelles (nfcs) and their interactions with aflibercept according to various embodiments disclosed herein.

FIG. 2 shows Critical Micelle Concentration (CMC) values for EPC determined using the dye solubilization method, wherein absorbance change of the hydrophobic dye 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) is monitored in accordance with various embodiments disclosed herein. The CMC forming the nano-micelles was found to be 0.046% at 37 ℃.

FIG. 3 shows the use of fluorescence emitted by rhodamine-labeled Abelmoschus (Rho-A) and the observation of fluorescence intensity changes when the same amount of Rho-A was added to different concentrations of EPC to study the interaction of nEPC with Abelmoschus, according to various embodiments disclosed herein. The lower graph shows the graph in EPC concentration (wt%) and the upper graph shows the graph in log of EPC concentration. The high fluorescence intensity measured indicates that free Rho-a is abundant at 0.05 wt% EPC. However, the fluorescence intensity subsequently drops sharply (labeled "2" in the following figures) at no more than about 0.5 wt%, indicating that Rho-a is incorporated into the micelle when the nfpc is formed. In the following figures, "1" means 0.05 wt% and "2" means 0.2 wt%.

Fig. 4 shows interactions between micellar polymer components and aflibercept analyzed using 1H NMR, showing differences in EPC peak resonances at 3.57ppm PEG (labeled a) and 1.03ppm PPG (labeled b) with and without aflibercept (spectral reference to residual solvent peak of water at 4.66 ppm), according to various embodiments disclosed herein.

Fig. 5 shows the absorption spectra of 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) added to EPC copolymers at different concentrations at 25 ℃. According to various embodiments disclosed herein, absorption peaks for DPH were observed at 344nm, 358nm, and 376nm, and absorbance increased with increasing concentration.

Fig. 6 shows absorption spectra of 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) added to EPC copolymers at different concentrations at 37 ℃ according to various embodiments disclosed herein. Absorption peaks for DPH were observed at 344nm, 358nm and 376nm, and absorbance increased with increasing concentration.

Fig. 7 illustrates critical micelle concentrations of nfpc according to various embodiments disclosed herein. The Critical Micelle Concentration (CMC) value of EPC was measured using a dye solubilization method, in which the absorbance change of the hydrophobic dye 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) was observed. The CMC forming the nano-micelles was found to be 0.070% at 25 ℃.

Figures 8-9 illustrate characterization of size and morphology of EPC nanomicelle (nfpc) according to various embodiments disclosed herein.

Fig. 8 illustrates hydrodynamic dimensions of EPC nanomicelle (nsec), aflibercept, and aflibercept-loaded nsec (nsec+a) determined using Dynamic Light Scattering (DLS) according to various embodiments disclosed herein. The maximum hydrodynamic sizes of nEPC and Abelmoschus were 57.9nm and 13.1nm, respectively, when present alone. When EPC was mixed with aflibercept, only one 64.5nm band was formed, indicating the formation of nfpc+a.

Fig. 9 shows TEM images of ultrastructures of nfpc (upstream) and nfpc+a (downstream) according to various embodiments disclosed herein. Micelle morphology was determined using Transmission Electron Microscopy (TEM), scale 50nm.

Figures 10-17 show that nfpc (2% by weight) in vitro studies according to various embodiments disclosed herein demonstrate intrinsic anti-angiogenic properties, which may be mediated by Vascular Endothelial Growth Factor (VEGF) and platelet-derived growth factor (PDGF) pathways.

Fig. 10 shows that nfpc inhibits migration of VEGF-dependent Human Umbilical Vein Endothelial Cells (HUVEC) in scratch experiments: HUVECs require basal VEGF to proliferate, thus including two controls with and without VEGF. According to various embodiments disclosed herein, HUVECs treated with nlpc+vegf require the longest time to heal, indicating that migration of HUVECs is maximally inhibited.

Figure 11 shows quantification of scratch assay (wound recovery% at different time points): according to various embodiments disclosed herein, cells treated with both aflibercept+vegf and nepc+vegf exhibited a significant decrease in wound recovery compared to control and +vegf alone.

Fig. 12 shows HUVEC tube formation assay: phase contrast photomicrographs (taken 5 hours after exposure to medium): according to various embodiments disclosed herein, nEPC+VEGF is capable of inhibiting capillary formation to a greater extent than Abelmoschus+VEGF and VEGF only.

Figure 13 shows a quantitative analysis of total branch length performed in HUVEC tube formation assay: according to various embodiments disclosed herein, nEPC+VEGF shows minimal branch lengths and spacing.

Figure 14 shows quantitative analysis of branching intervals performed in HUVEC tube formation assay: according to various embodiments disclosed herein, nEPC+VEGF shows minimal branch lengths and spacing.

FIG. 15 shows RNA expression of key genes involved in angiogenesis in HUVECs 24 hours after treatment, as measured by qPCR for VEGF A-C, according to various embodiments disclosed herein. HUVECs treated with nEPC+VEGF showed significant differences in expression of VEGF-C, VEGFR and PDGFR-beta compared to treatment with Abelmoschus+VEGF. HUVECs treated with nEPC+VEGF showed significant differences in expression of VEGF-C, VEGFR3, PDGFB and PDGFR-alpha compared to treatment with VEGF alone. The values are expressed as mean.+ -. SD, n.gtoreq.3. P <0.0001 compared to +vegf control; * P <0.0002; * P <0.002, < p <0.0332.

Figure 16 shows RNA expression of key genes involved in angiogenesis in HUVECs 24 hours after treatment, as measured by qPCR for VEGFR 1-3, according to various embodiments disclosed herein. HUVECs treated with nEPC+VEGF showed significant differences in expression of VEGF-C, VEGFR and PDGFR-beta compared to treatment with Abelmoschus+VEGF. HUVECs treated with nEPC+VEGF showed significant differences in expression of VEGF-C, VEGFR3, PDGFB and PDGFR-alpha compared to treatment with VEGF alone. The values are expressed as mean.+ -. SD, n.gtoreq.3. P <0.0001 compared to +vegf control; * P <0.0002; * P <0.002, < p <0.0332.

Figure 17 shows RNA expression of key genes involved in angiogenesis in HUVECs 24 hours after treatment, as measured by qPCR for PDGF signaling molecules, according to various embodiments disclosed herein. HUVECs treated with nEPC+VEGF showed significant differences in expression of VEGF-C, VEGFR and PDGFR-beta compared to treatment with Abelmoschus+VEGF. HUVECs treated with nEPC+VEGF showed significant differences in expression of VEGF-C, VEGFR3, PDGFB and PDGFR-alpha compared to treatment with VEGF alone. The values are expressed as mean.+ -. SD, n.gtoreq.3. P <0.0001 compared to +vegf control; * P <0.0002; * P <0.002, < p <0.0332.

Fig. 18 shows HUVEC proliferation assay: according to various embodiments disclosed herein, nEPC+VEGF exhibits a greater inhibition of HUVEC proliferation than Abelmoschus+VEGF and VEGF alone. The values are expressed as mean.+ -. SD, n.gtoreq.3. P <0.0001 compared to +vegf control; * P <0.0002; * P <0.002, < p <0.0332.

Figures 19-21 show that nfpc (2 wt%) exhibits anti-angiogenic effects on a 3D AIM chip according to various embodiments disclosed herein.

Fig. 19 shows a schematic diagram 1900 of an AIM chip that allows HUVECs to germinate in a 3D environment, in accordance with various embodiments disclosed herein. The device includes a left microchannel 1902, a right microchannel 1904, and a middle channel 1906. The left and right microchannels were coated with fibronectin. The lateral fibronectin-coated flow channel 1902 on the left was then seeded with HUVEC 1908. The lateral fluid channel 1904, which is right coated with fibronectin, is empty. An in vitro anti-angiogenesis assay was performed at the intermediate channel 1906 of the device using an AIM 3D chip with type I collagen gel 1910. The intermediate channel 1906 is filled with a type I collagen gel 1910.

Fig. 20 shows confocal microscopy images of HUVEC AIM chips: according to various embodiments disclosed herein, HUVEC treated with albesieadditional+vegf exhibited stronger inhibition of the branch than nrpc+vegf and VEGF alone. The scale bar represents 100 μm.

Figure 21 shows quantification of total branch length formed after 5 days of HUVEC culture (left axis) and total cell count (line (- -) plot) for one triangle area count (right axis) in AIM chip, according to various embodiments disclosed herein.

Fig. 22A, 22B, 23-26 illustrate the anti-angiogenic effect of nfpc (2 wt%) in an isolated murine choroidal assay according to various embodiments disclosed herein.

Figure 22A shows an experimental design of a choroidal explant germination and degeneration assay according to various embodiments disclosed herein. In the figure, 2D represents 2 days, 3D represents 3 days, and 4D represents 4 days.

Figure 22B shows a data analysis of a choroidal explant germination and degeneration assay according to various embodiments disclosed herein. Quantification of choroidal sprouting area using the SWIFT-choroidal method according to Shao, Z.et al, PLoS One 2013,8, e69552, previously published, the contents of which are incorporated herein by reference in their entirety, shows (1) raw bright field images; (2) a computer-generated image after removal of the central explant; and (3) final SWIFT-choroidal image. The scale bar represents 100 μm.

Fig. 23 shows a germination degradation test (vessel germination was established prior to treatment): according to various embodiments disclosed herein, nEPC+VEGF does not cause regression of germinated vessels, but is capable of inhibiting further vessel sprouting.

Fig. 24 shows germination inhibition assays using VEGF, albespri+vegf and nfpc+vegf alone (after 48 hours of incubation): according to various embodiments disclosed herein, explants exposed to nEPC+VEGF produce fewer shoots than those exposed to Abelmoschus+VEGF and nEPC+VEGF.

Fig. 25 shows quantification of germination area (percent of total choroidal area) for germination assays, demonstrating reduced germination area in explants treated with npcp as compared to aflibercept and VEGF alone (p <0.05; p <0.01; p < 0.001), according to various embodiments disclosed herein.

Fig. 26 shows quantification of germination area [ (total area-initial area before treatment)/(total area) ], showing that a similar reduction in germination area between nfpc+vegf and aflibercept+vegf after 72 hours (< p < 0.05) was demonstrated according to various embodiments disclosed herein; p <0.01, p < 0.001).

Figures 27-29 show intracellular uptake of nepc+rho-a by hCEC in vitro, according to various embodiments disclosed herein.

Figure 27 shows that nsec+rho-a promotes in a concentration-dependent manner (from 0 wt% to 2 wt%) in vitro cellular uptake of aflibercept by hCEC, according to various embodiments disclosed herein. Confocal images were taken after incubation for 24 hours at different concentrations of nfpc (0.05 wt%, 0.2 wt%, 1 wt% and 2 wt%). Scale bar = 10 μm.

Figure 28 shows quantitative cellular uptake results in hCEC by flow cytometry after incubation with nsec+a for 24 hours at different nsec concentrations, according to various embodiments disclosed herein.

FIG. 29 shows the cellular distribution and co-localization of fluorescent EPC-containing polymer (FEPC) and Rho-A observed with a confocal laser microscope (100X) using tomography (z-stack) imaging to confirm the intracellular distribution of the complexes, according to various embodiments disclosed herein. Ocular penetration of aflibercept in the sclera and cornea of ex vivo pigs with and without the presence of nfepc (2 wt%).

Figures 30-31 illustrate that nfpc+rho-a enhances ocular penetration of Rho-a in an ex vivo pig sclera model, according to various embodiments disclosed herein.

FIG. 30 shows the use of an Uper perfusion chamber (n.gtoreq.3, mean.+ -. SD) to measure permeability of Abelmoschus in ex vivo porcine sclera according to various embodiments disclosed herein.

Fig. 31 shows a measurement of the concentration of aflibercept in porcine vitreous (n=3, mean ± SD) 45 minutes after a single eye drop (20 μl) administration directly on porcine cornea-scleral eye cup, according to various embodiments disclosed herein.

Figures 32-33 illustrate that nfpc+rho-a enhances ocular penetration of Rho-a in an in vivo mouse ocular model, according to various embodiments disclosed herein.

Fig. 32 shows in vivo ocular distribution of abatvipx and nfpc+rho-a 45 minutes after single eye drops administration to mice, according to various embodiments disclosed herein. When administered directly, rho-A is found only in the corneal epithelium, whereas nEPC+rho-A is permeable to the cornea. (white arrow indicates Rho-A).

Fig. 33 shows the amount of Rho-a successfully penetrating the cornea to reach the mouse vitreous, assessed 45 minutes after administration of a single eye drop, and a comparison with the nlpc+rho-a eye drop, according to various embodiments disclosed herein. (n=10, mean ± SD). P < 0.05, p < 0.01, p < 0.001).

Fig. 34A, 34B, 34C, 35-36 illustrate a comparison of corneal residence time between ncp+rhoa and RhoA according to various embodiments disclosed herein.

Fig. 34A shows an anterior ocular segment optical coherence tomography (ASOCT) image after administration of 1 drop of each solution (nlpc+rho-A, rho-a) on the eye of a mouse, according to various embodiments disclosed herein. Photographs were taken at time points of 30s, 60s and manually blinked at fixed time intervals.

Fig. 34B shows an anterior ocular segment optical coherence tomography (ASOCT) image after administration of 1 drop of each solution (nlpc+rho-A, rho-a) on the eye of a mouse, according to various embodiments disclosed herein. Photographs were taken at time points of 120s, 210s and manually blinked at fixed time intervals.

Fig. 34C shows an anterior ocular segment optical coherence tomography (ASOCT) image after administration of 1 drop of each solution (nlpc+rho-A, rho-a) on the eye of a mouse, according to various embodiments disclosed herein. Photographs were taken at time points of 285s, 300s and manually blinked at fixed time intervals.

Fig. 35 illustrates the area occupied by eye drops on a cornea quantified using an ASOCT image, according to various embodiments disclosed herein. nEPC+rho-A showed a significantly larger area and persisted after 8 blinks. P < 0.05, p < 0.01, p < 0.001).

Fig. 36 shows a photograph of the anterior segment of a mouse eye after 20 blinks delivering 1 drop of each eye drop (nlpc+rho-A, rho-a) according to various embodiments disclosed herein. The photographs show that, unlike Rho-a alone, the nfpc+rho-a eye drops remain on the corneal surface.

Fig. 37A and 37B illustrate in vitro biocompatibility of the nfpc according to various embodiments disclosed herein.

FIG. 37A shows cellular activity measured on hCEC and ARPE-19 cell lines by lactate dehydrogenase release (LDH) assay according to various embodiments disclosed herein.

FIG. 37B shows cell death measured on hCEC and ARPE-19 cell lines by lactate dehydrogenase release (LDH) assay according to various embodiments disclosed herein.

Fig. 38 shows the in vitro biocompatibility of the nfpc. To assess the effect of nEPC on porcine corneal tissue integrity, transepithelial electrical impedance (TEER) after prolonged exposure to Abelmoschus or nEPC+A according to various embodiments disclosed herein was measured. Quantitative analysis showed no significant decrease in TEER for both groups after 24 hours of exposure.

Fig. 39A, 39B, 39C, 39D, 39E illustrate the in vivo biocompatibility of the nfpc according to various embodiments disclosed herein. After 14 days of topical eye drops (5 μl each, three times a day), the in vivo biocompatibility of nfpc (2 wt%) and nfpc+a was monitored with the mouse model. In all treatment groups, slit lamp imaging (no mydriasis) did not show any corneal haze and (mydriasis) did not show cataract formation. Histological results indicated that the cornea structure was preserved, and ZO-1 immunofluorescent staining results indicated that the integrity of the tight junctions of the corneal epithelium was maintained, TUNEL staining did not show any increase in apoptosis. Scale bar = 50 μm. (Epi: corneal epithelium layer; endo: corneal epithelium layer).

Figures 40-42 illustrate CNV degradation in a laser-induced mouse model caused by topical application of nfpc+a according to various embodiments disclosed herein. Local infusion of nfpc+a significantly reduced the laser-induced mouse CNV leakage area (n=8).

Fig. 40 shows Fluorescein Fundus Angiography (FFA) images taken from representative eyes on days 3, 7, and 14 after model creation, according to various embodiments disclosed herein.

Fig. 41 shows the extent of fluorescence leakage (n=8) in the choroidal lesion region quantified by ImageJ based on the FFA image shown in fig. 40. Daily recovery rate was calculated using the following formula: (leakage area on day 3-leakage area on day 14)/(leakage area on day 14-3). P < 0.05 compared to ncp+a; * P < 0.01; * P < 0.001.

Fig. 42 shows isolectin B4 (red) staining of endothelial cells on choroidal mount (flat mount), indicating an overall reduction in size of CNV lesions (white arrow point lesions produced by laser) following treatment with epc+a according to various embodiments disclosed herein.

Fig. 43 illustrates retention times of EPC copolymers in Gel Permeation Chromatography (GPC) using Tetrahydrofuran (THF) as a solvent, according to various embodiments disclosed herein.

FIG. 44A illustrates EPC copolymers in CDCl according to various embodiments disclosed herein ₃ In (a) and (b) ¹ H NMR spectrum.

FIG. 44B shows the EPC copolymer of FIG. 44A ¹ The identity of the corresponding protons (a, b, c, d, e, f, g and H) in the chemical structure shown in the H NMR spectrum. The integral of the corresponding proton, PEG, PPG, PCL characteristic peak in the chemical structure was identified and is shown in table 2.

Figures 45-47 illustrate characterization of fluorescein-diol according to various embodiments disclosed herein.

FIG. 45 illustrates a fluorescein-diol in CDCl according to various embodiments disclosed herein ₃ In (a) and (b) ¹ H NMR spectrum.

FIG. 46 illustrates a fluorescein-diol in CDCl according to various embodiments disclosed herein ₃ In (a) and (b) ¹³ C NMR spectrum.

FIG. 47 illustrates a fluorescein-diol in CDCl according to various embodiments disclosed herein ₃ 2D of (3) ¹ H- ¹ HCOSY profile.

Fig. 48-50 illustrate characterization of FEPC according to various embodiments disclosed herein.

FIG. 48 illustrates an FEPC polyurethane in CDCl according to various embodiments disclosed herein ₃ In (a) and (b) ¹ H NMR spectrum. The inset shows the enlarged aromatic region with peaks corresponding to the fluorescein aromatic groups.

Fig. 49 shows GPC Traces (THF) of FEPC thermal gel polymers according to various embodiments disclosed herein.

Fig. 50 shows Critical Micelle Concentration (CMC) values of FEPC measured at 37 ℃ using a dye solubilization method, wherein absorbance change of the hydrophobic dye 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) is monitored, according to various embodiments disclosed herein.

Fig. 51-53 illustrate characterization and evaluation of commercial F127.

FIG. 51 shows the CMC values of F127 measured at 25℃and 37℃compared with EPC. In the figure, square (■) represents F127, and circle (+) represents EPC.

FIG. 52 shows quantitative cell uptake results in hCEC by flow cytometry after 24 hours of incubation compared to nEPC+FITC-A and F127+FITC-A according to various embodiments disclosed herein.

FIG. 53 shows that in vivo local infusion of Rho-A complex F127 (F127-Rho-A) demonstrated poor corneal penetration in the eyes of mice.

Examples

Exemplary embodiments of the present disclosure will be better understood and apparent to those of ordinary skill in the art from the following examples, tables, and if applicable, in connection with the accompanying drawings. It is to be understood that other modifications relating to structural, biological and/or chemical changes may be made without departing from the scope of the invention. The exemplary embodiments are not necessarily mutually exclusive, as some embodiments may be combined with one or more embodiments to form new exemplary embodiments. The exemplary embodiments should not be construed as limiting the scope of the present disclosure.

The following examples describe nanomicelle drug delivery systems made of copolymers that are capable of delivering biologically active substances/drugs locally to the posterior segment of the eye via the cornea-scleral route. In the following examples, EPC was used as a copolymer comprising polyethylene glycol (PEG), polypropylene glycol (PPG) and Polycaprolactone (PCL) fragments; and the biological activity/drug used is aflibercept.

Fig. 1 shows a schematic diagram of a multi-block copolymer (e.g., EPC polymer 100) according to various embodiments disclosed herein. As shown in fig. 1, EPC polymer 100 self-assembles into micelles (e.g., polymer nanomicelle (nfpc) 102). The nfpc 102 is prepared by concentrating the EPC polymer 100 to a concentration above the Critical Micelle Concentration (CMC) but below that required for sol-gel transition.

Advantageously, the nfpc 102 alone is capable of inhibiting angiogenesis in vitro and ex vivo. The nEPC has intrinsic anti-angiogenesis activity, and can be used for treating neovascular retina diseases in cooperation with the drug delivery capacity.

As shown in fig. 1, by encapsulating aflibercept in EPC copolymer solution, an aflibercept-loaded nanomicelle (i.e., nfpc+a106) can be formed. The aflibercept 104 is encapsulated by the nEPC by direct mixing to form a nEPC+aflibercept (nEPC+A) complex 106. Abelmoschus was chosen because of its relatively long duration of action (i.e., FDA approval of its dosing interval of up to 3 months) compared to ranibizumab or bevacizumab with a one month duration of efficacy. When topically applied to murine corneas, the nEPC acts as a drug carrier, delivering a common transcornea of A Bai Xi to achieve therapeutic retinal concentrations in laser-induced CNV disease models. nEPC+A is capable of delivering clinically significant amounts of Abelmoschus to the retina to control choroidal neovascularization in mice.

As shown in the examples below, the abamectin-loaded nfpc (nfpc+a) was able to penetrate the cornea in an ex vivo porcine eye model and deliver clinically significant amounts of abamectin to the retina in a laser-induced Choroidal Neovascularization (CNV) mouse model, resulting in CNV degeneration (see, e.g., fig. 34A, 34B, 34C and fig. 41). nEPC+A also exhibits biocompatibility in vitro and in vivo (see, e.g., FIGS. 20 and 23). The ability of nEPC to deliver anti-VEGF drugs and the inherent anti-angiogenic properties can work synergistically and can be used for effective treatment. nEPC has been shown to be a promising local anti-VEGF delivery platform for the treatment of retinal diseases.

Example 1: development and characterization of nEPC+A

The thermodynamic self-assembly process of EPC copolymers to form micelles can be described in terms of CMC, or in terms of the minimum polymer concentration required for micelle formation. CMC was measured by monitoring the sharp increase in absorbance of the hydrophobic dye 1, 6-diphenyl-1, 3, 5-hexatriene (DPH) upon micelle formation (fig. 2 and 5 to 7). CMC values that formed nfpc at 37 ℃ were found to be 0.046 wt% (fig. 2). It was also compared to CMC values of Pluronic F127 (0.09 wt%). Pluronic F127 is an FDA approved polymer that has been widely used for drug delivery and drug controlled release. In contrast, the CMC formed by the nfpc was lower (0.09 wt%) than Pluronic F127 (fig. 2, 51).

To determine the ability of nEPC as an Abelmoschus drug delivery system, nEPC+A was first generated by dissolving EPC in Abelmoschus stock solution at a concentration above CMC but below the sol-gel transition. Under this condition, the EPC copolymer self-assembles into nepc+a. The formation of nEPC+A was observed by monitoring the hydrodynamic dimensions of 0.2 wt% of nEPC and Abelmoschus. The maximum hydrodynamic sizes of nEPC and Abelmoschus were 57.9nm and 13.1nm, respectively, when present alone. When EPC was mixed with the aflibercept solution to reach CMC, 2 size distributed bands were combined into 1 band and shifted to 64.5nm, indicating the formation of nfpc+a (fig. 8). The formation of nEPC+A was studied using a fluorometry wherein fixed amounts of fluorescent rhodamine conjugated Abelmoschus (Rho-A) were added to EPC solutions of different concentrations (FIG. 3). The initial fluorescence intensity from free Rho-a was higher, whereas at 0.05 wt% EPC the fluorescence intensity was drastically decreased. This decrease in fluorescence intensity occurs at a polymer concentration (CMC) similar to the CMC of EPC _EPC =0.046 wt%) indicates that when the nrpc is formed, free Rho-a is incorporated into the nrpc structure, forming nrpc+a. Rhodamine shows higher fluorescence in an aqueous environment, so it seems reasonable to encapsulate Rho-a into micelles. To further investigate micelle-drug interactions, EPCs with and without Abelmoschus were compared ¹ H NMR spectrum (fig. 4). The presence of aflibercept induces a significantly high magnetic field shift of the PEG protons, indicating that its hydration environment is due to non-drug interactionsCovalent interactions are modulated. This is probably due to the ion dipole interaction between the lewis basic oxygen atom on PEG and aflibercept, which is cationic at physiological pH. Furthermore, aflibercept results in the generation of PEG and PPG fragments of EPC ¹ The H NMR resonance state broadens, which is consistent with the reduced chain movement caused by the binding of aflibercept to the polymer micelles.

The size of nEPC and nEPC+A was observed by monitoring the hydrodynamic size of 0.2 wt% of nEPC and Abelmoschus. The maximum hydrodynamic sizes of nEPC and Abelmoschus were 57.9nm and 13.1nm, respectively, when present alone. When EPC was mixed with the aflibercept solution to reach CMC, 2 size distributed bands were combined into 1 band and shifted to 64.5nm, indicating the formation of nfpc+a (fig. 8). Transmission Electron Microscopy (TEM) was also used to study the nsec and nsec+a, and it was found that the particles they formed were mostly spherical and of approximately the same size. The particles in TEM were smaller than in DLS, probably due to air drying and collapse of the PEG shell of the micelle (fig. 9).

To determine the maximum Encapsulation Efficiency (EE) of the nfpc, various concentrations of nfpc (0.5 wt%, 1.0 wt% and 2.0 wt%) were studied. As the concentration of nfpc increases, the EE of aflibercept is: 1.3.+ -. 0.4% at 0.5 wt%, 17.4.+ -. 0.3% at 1.0 wt% and 47.3.+ -. 0.8% at 2.0 wt%. Subsequent experiments were performed with nEPC+A with 2 wt% EPC to obtain maximum EE. For subsequent experiments using the nEPC, a concentration of 2 wt% of nEPC was used.

Example 2: nEPC inhibits VEGF-induced endothelial cell migration, proliferation and angiogenesis both in vitro and ex vivo

In order to investigate the anti-angiogenic properties of nEPC, in vitro and ex vivo evaluations were performed. In vitro methods include HUVEC migration, proliferation and angiogenesis assays. In the HUVEC migration test, it took 30 hours for the wound to heal completely in the control experiment. When VEGF was added, the wound healing time was accelerated to 20 hours. Addition of aflibercept to the VEGF-treated experiments showed inhibition of VEGF. By 25 hours, the wound closure was incomplete, with a closure rate of only 76.0±11.3% achieved. Surprisingly, the addition of nfpc in VEGF-treated experiments also showed a similar slower wound closure process. By 25 hours, a wound closure rate of only 67.7±18.4% was achieved. This result suggests that nfpc alone was able to inhibit VEGF-induced HUVEC migration (fig. 10 and 11).

In addition, addition of nEPC also reduced HUVEC proliferation in HUVEC proliferation assay. After 48 hours incubation, addition of aflibercept alone produced 88.2±4.4% of cells, while addition of nsecp alone also reduced HUVEC proliferation, yielding 79.8±3.1% of cells after 48 hours (fig. 18).

Furthermore, in the HUVEC angiogenesis assay, nfpc was also able to significantly inhibit angiogenesis in terms of branch length and branch spacing (fig. 12). The use of nEPC alone was able to reduce vascular length formation (62.8% + -9.0) and branching spacing (43.9% + -11.4), more effective than the use of Abelmoschus alone (i.e., vascular length formation of 81.0% + -10.4 and branching spacing of 77.4% + -26.4) (FIGS. 13 and 14).

To elucidate the anti-angiogenic mechanism of nfpc, RNA expression of angiogenic genes in HUVEC was assessed using qPCR (fig. 15, 16 and 17). RNA expression of various VEGF isoforms (fig. 15) and their receptors (VEGFR) (fig. 16) known to play an important role in retinal neovascularization was assessed. The nEPC alone was able to significantly reduce the expression of VEGF-C and VEGFR3 compared to Abelmoschus, which was mainly down-regulating the expression of VEGFR 1. Up-regulation of Platelet Derived Growth Factor (PDGF) is known to result in anti-VEGF resistance. Interestingly, nEPC alone was able to significantly reduce the expression of PDGFB, PDGFR-alpha, PDGFR-beta compared to Abelmoschus. These results indicate that the anti-angiogenic effect of nfpc occurs through both VEGF and non-VEGF mediated pathways. Importantly, they appear to be different from, but synergistic with, the route of aflibercept dependence.

To further characterize the ability of nEPC to inhibit angiogenesis, 3D cell models were utilized to evaluate HUVEC migration and angiogenesis simultaneously. In this single assay, HUVECs germinated and migrated from pre-existing monolayers into the attached 3D collagen matrix, providing a concentration gradient of angiogenic stimulus (fig. 19). In the VEGF control, HUVECs migrate to the central collagen channel and form tubular structures after 5 days of culture. Migration and angiogenesis are significantly inhibited in the presence of aflibercept, minimizing migration of cells to collagen channels. Although the nfpc did not inhibit migration of HUVEC to the central collagen channel (fig. 20), the total branch length was reduced from 336.0 pixels (in +vegf control) to 106.7 pixels (p=0.0087) (fig. 21).

To further assess the ability of nEPC to inhibit angiogenesis, robust and quantifiable ex vivo experiments were performed on nEPC using murine choroidal explants. These explants allowed to study the sprouting and degeneration of murine vascular endothelial cells under the influence of exogenous angiogenic or anti-angiogenic factors (fig. 22A and 22B). Vascular germination inhibition assays were performed by exposing murine choroidal explants to aflibercept and nfpc, respectively, on experimental day 2 after initial vascular germination (fig. 24 and 25). After exposure to aflibercept and nEPC, the germination area was reduced from 16.2% + -4.0 to 10.0% + -0.9 and 1.5% + -0.38, respectively. Vascular regression testing was performed to rule out the possibility that the results were due to the nrpc-induced toxicity and cell death rather than its anti-angiogenic properties. In this test, aflibercept and nfpc were added only after 4 days of vessel sprouting. The area of vessel sprouting was then measured after 48 hours and 72 hours (figure 23). In contrast to toxicity, the nEPC treated explants did not induce regression of germinated vessels (FIG. 26), but the vessel sprouting was further reduced (FIG. 25) to a comparable extent to that observed in the Abelmoschus control (nEPC: 10.3.+ -. 2.7% at 48 hours, 11.7.+ -. 1.5% at 72 hours; abelmoschus: 9.9.+ -. 2.1% at 48 hours, 12.2.+ -. 1.8%) indicating anti-angiogenic effect but not cytotoxicity. These results provide pathological evidence of the anti-angiogenic effect of nEPC.

Example 3: nEPC as nano-carrier to promote in vitro intracellular uptake of Abelmoschus by human corneal epithelial cells (hCEC)

To determine whether nEPC was able to act as an in vitro drug carrier for Abelmoschus, rho-A was incubated with hCEC for 24 hours. Maximum intracellular uptake of Rho-a was observed at 2 wt% of the nfpc (fig. 27), indicating that internalization of aflibercept increased with increasing nfpc wt%. Flow cytometry was performed to quantify Rho-A concentration dependent uptake. When 2 wt% of nEPC+rho-A was administered, rho-A emitted fluorescence intensity approximately 2-fold higher than when Rho-A was administered alone (FIG. 28). The fluorescence intensity of internalized nEPC+A was higher compared to F127+ Abelmoschus (FIG. 52). Confocal tomography of hCEC, FEPC and Rho-a also showed that FEPC and Rho-a co-localize at the cytoplasm of hCEC rather than adhering to the cell surface, indicating intracellular uptake of nsec+a (fig. 29).

Example 4: nEPC enhanced ocular penetration of Abelmoschus in vitro pig models and in vivo murine models

To assess the ability of nEPC to enhance ex vivo penetration of Abelmoschus, pigs were sclerally resected and clamped on vertical Eus perfusion chambers, which allowed for measurement of drug transport in tissues. Pig sclera was continuously exposed to Rho-a or nsec+rho-a for 40 minutes. Throughout the process, PBS solutions at opposite sites were collected at multiple time points. At 40 minutes, the detected Rho-A concentration (541 ng/mL) in the presence of nEPC+rho-A was higher than in the case where Rho-A alone was present (70 ng/mL) (FIG. 30). Similarly, when tested using porcine cornea-scleral glasses, rho-a concentration (6 ng/mL) in the vitreous was 6-fold higher (0.09 ng/mL) when given with nrpc+rho-a than with Rho-a alone (fig. 31).

To further verify whether nEPC could promote corneal penetration in Abelmoschus, wild type mice were treated with a single local dose of Rho-A (40 mg/mL), nEPC+rho-A or F127-Rho-A (n=3). It was observed that nEPC+rho-A was able to penetrate the cornea and accumulate below the corneal endothelium, whereas Rho-A and F127-Rho-A alone accumulated above the corneal epithelium (FIGS. 32, 53). Consistent with this, the amount of aflibercept detected in the vitreous of mice treated with nrpc+rho-a (n=10) (2362±354.7 ng/mL) was 4-fold higher than that of Rho-a alone (633±133.9 ng/mL) (fig. 33).

Example 5: nEPC+rho-A increased corneal surface residence time compared to Rho-A alone

Comparison of corneal residence time of single drop of nEPC+RhoA versus single drop of Rho-A alone was performed on murine eyes (FIGS. 34A, 34B, 34C, 35 and 36). Anterior ocular segment optical coherence tomography (ASOCT) imaging shows that over time and repeated blinks, the eye drop volume at the corneal surface decreases. After 8 blinks of about 120 seconds, the area of nEPC+rho-A left on the corneal surface is significantly larger (FIG. 35). Anterior ocular segment photographs also showed that the nEPC+rho-A eye drops remained on the corneal surface after 20 blinks compared to Rho-A alone (FIG. 36).

Example 6: nEPC has biocompatibility with in vitro human cell line and in vivo mouse eye cell

Cytotoxicity of nEPC was assessed using an LDH assay on hCEC and ARPE-19 cells (human retinal pigment epithelial cell line) (FIG. 37A). Minimal cell death was observed 24 hours after co-culture with EPC polymers at concentrations of 0.01 wt% to 2 wt% (fig. 37B).

To evaluate the effect of nEPC on corneal barrier integrity, transepithelial electrical impedance (TEER) of porcine cornea was measured after prolonged exposure to Abelmoschus and nEPC+A, respectively. TEER is a powerful indicator of cellular barrier integrity. When comparing before and after exposure, no significant change in TEER was measured with exposure to both abacisand nfepc+a, indicating that the corneal barrier was not disrupted after exposure to nfepc+a (fig. 38).

To evaluate the biocompatibility of the locally applied nfpc, eyes of wild-type mice were treated with buffer, aflibercept, nfpc or nfpc+a solutions (n=8). Each eye was administered 3 times per day for a total duration of 14 days. An image of the anterior segment of the eye was taken using a slit lamp microscope on both the non-mydriatic and mydriatic eyes to evaluate the sharpness of the cornea and lens. On day 14, eyes treated with either nEPC and nEPC+A did not show any corneal or lens haze compared to eyes treated with the control buffer solution. Histological sections of the cornea showed that in the eyes treated with nEPC and nEPC+A, the cornea was histologically intact and not infiltrated with inflammatory cells. ZO-1 immunofluorescent staining is a marker of tight junctions of the corneal epithelium, indicating that tight junctions of the cornea remain unchanged in nEPC and nEPC+A treated eyes. Terminal deoxynucleotidyl transferase dUTP notch end marker (TUNEL) staining did not show an increase in apoptosis in the nupc and nupc+a treated eyes. This is similar to the buffer control group. In addition, all keratocytes exhibit a large polygonal, squamous cell shape and clear cell boundaries. These findings indicate that nEPC and nEPC+A have no significant adverse effect on corneal epithelial cells and retinal cells and are therefore biocompatible (FIG. 39A, FIG. 39B, FIG. 39C, FIG. 39D and FIG. 39E).

Example 7: local application of nEPC+A reduces vascular leakage in laser induced CNV mouse models

The biological activity of nEPC+A was determined using a murine model of laser induced CNV. CNV eyes were treated with buffer, aflibercept (40 mg/mL), nrpc or nrpc+a solution 3 times daily for 14 days. Fundus Fluorescein Angiography (FFA) was performed on days 3, 7 and 14, respectively, to monitor the resolution of vascular leakage response to treatment (fig. 40) and calculate recovery (see experimental method in example 10 below) (fig. 41). The leak area was not reduced when the buffer solution was administered, and recovery rates of 118.3.+ -. 48.7 pixels/day when Abelmoschus, nEPC and nEPC+A were administered to the eyes, respectively; 239.7 + -108.8 pixels/day; 568.1.+ -. 68.9 pixels/day. The recovery rate of nEPC+A was significantly faster compared to the buffer. At the end of day 14, the eyeballs were removed to obtain choroidal flatmount for endothelial cell lectin B4 staining (fig. 42). The choroidal paving tablet can display CNV lesions in choroidal tissues in a panoramic manner, and provides a pathological basis for comparing the sizes of CNV lesions. Consistent with FFA results, the nfpc+a treated eyes retained minimal CNV lesion area.

Example 8: discussion of the invention

IVT against VEGF compounds remains the primary method of treating retinal vascular disease. However, local delivery of anti-VEGF compounds via eye drops represents a more desirable and easier way to repeatedly deliver anti-VEGF to the retina due to the therapeutic burden associated with IVT and the potential vision threatening complications. Ideally, a topical anti-VEGF delivery system must be able to 1) deliver therapeutic concentrations of drugs to the posterior segment of the eye against the ocular barrier, 2) exhibit biocompatibility, particularly upon repeated use, 3) retain bioactivity and therapeutic effects at the retina. To date, published approaches have met with limited success in meeting these criteria. In particular, achieving therapeutic concentrations of anti-VEGF in the posterior segment of the eye to control disease has been challenging. In the previous examples, the results indicate that nEPC+A is able to overcome these obstacles that hamper the successful development of effective local anti-VEGF formulations.

It should be appreciated that the nEPC better meets the above criteria for an ideal local anti-VEGF delivery system compared to other nanoformulations. Importantly, as assessed in the validated disease model, nEPC+A was able to achieve therapeutic concentrations of aflibercept in the posterior ocular segment of mice. When a single drop of nEPC+A was administered to the eyes of the mice, the amount of aflibercept detected in the vitreous of mice treated with nEPC+A was four times higher than when aflibercept was topically administered alone. The concentration of nEPC+A in the vitreous was as high as 2362.5 ng/mL.+ -. 354.6. This is higher than IC reported for VEGF for Abelmoschus of about 1.8ng/ml ₅₀ . Consistent with the murine model, nrpc+a can reach an aflibercept concentration in the vitreous of up to 6ng/ml when administered topically on an ex vivo pig eye model, as compared to administration alone. These results demonstrate that nEPC+A is capable of significantly enhancing the delivery of topically applied Abelmoschus to the posterior segment of the eye. More importantly, nEPC+A can reach concentrations of Abelmoschus in murine vitreous that are higher than the clinically significant concentrations required to inhibit VEGF activity.

Without being bound by theory, it is believed that the ability of nEPC+A to reach therapeutic concentrations of Abelmoschus in the retina may be due to two reasons. First, nEPC+A has a high EE. In the experiment, nEPC+A was able to achieve 47.3% of Abelmoschus EE. Previous studies on local delivery of approved anti-VEGF compounds to the retina utilized liposomes to encapsulate bevacizumab. These liposomes contain AnxA5, which enhances absorption of the liposomal drug carrier across the corneal epithelial barrier. In contrast, the EE of AnxA 5-associated liposomes was 22% to 25%, almost half that achieved by nlpc+a. Since EE of nlpc+a is significantly higher, it is able to package and ultimately deliver a larger drug payload to the posterior segment of the eye. Second, nEPC+A is able to enhance the delivery of Abelmoschus across the corneal epithelium and scleral barrier. This was demonstrated using both ex vivo porcine cornea model and in vivo murine model tests of ewing perfusion chamber, where nepc+a was able to penetrate the cornea to the endothelial layer. Preservation of TEER indicated that the barrier function of the cornea was not significantly disrupted. While the clear mechanism by which nEPC promotes corneal penetration remains to be further investigated, it is noted that nEPC+A is taken up in vitro by corneal epithelial cells, suggesting that nEPC+A may pass through the cornea by endocytic transport. Corneal surface retention experiments also showed that nEPC+A was retained at the corneal surface for a longer period of time than the Abelmoschus solution. Longer residence times may allow better absorption of the nanomicelles by the corneal epithelial cells. Taken together, these results demonstrate that nEPC+A is capable of acting as a carrier to promote corneal penetration of Abelmoschus for posterior ocular segment drug delivery without disrupting the function or structure of the corneal barrier. The subsequent pathways that occur after corneal penetration of nEPC+A can be deduced from the experiments performed. Accumulation of aflibercept in the pig ex vivo model vitreous was significantly increased following administration of single drop of nepc+rhoa eye drops, and the reduction of CNV lesions following administration of nepc+a eye drops suggests that nepc+a can overcome vitreous arrival at the retina to achieve its intended activity.

To achieve safe drug delivery from the ocular surface to the posterior segment, the biocompatibility of the nfpc was demonstrated to be critical. When co-cultured with the cornea (hCEC) and retinal pigment epithelium (ARPE-19) cell lines, nEPC+A showed good biocompatibility. This was further confirmed in the in vivo mouse model, when the administration of nfpc+a was repeated on the ocular surface for 14 days, the cornea remained clear. Histological analysis showed no change in morphology and tissue structure of both corneal epithelial and endothelial cells. In particular, the tightly linked corneal epithelium marker ZO-1 did not rupture compared to the control experiment. Tight junctions are extremely important for the homeostasis of the cornea, as they constitute the primary barrier to passive movement of fluids, electrolytes, macromolecules and cells. Given that nEPC+A may pass through the cornea by endocytic transport, these results indicate that endocytic transport does not cause any toxicity to the cornea in the short term. Administration of nEPC+A also did not accelerate cataract formation. The results also indicate that nEPC+A is able to reach the posterior segment of the eye without affecting the structure or function of the cornea and lens. This is particularly important because the cornea and lens are the primary refractive components of the eye. Thus, any inflammation in these tissues may reduce the final vision.

nEPC+A also successfully retained the biological activity of Abelmoschus in the posterior segment of the eye. This is demonstrated by the results of administering nEPC+A on a laser-induced in vivo mouse CNV model. After 2 weeks of continuous treatment, CNV degradation rate was highest in the eyes treated with nsec+a compared to either abamectin alone or nsec alone. This suggests a synergistic effect between the anti-angiogenic effect of the aflibercept compound and the nlpc.

The consequence that nEPC alone can inhibit angiogenic activity in vitro and ex vivo was unexpected. In studies of HUVEC migration, proliferation and angiogenesis, it was noted that nfpc could also inhibit VEGF-driven processes. In vitro and ex vivo, the effect of nEPC alone was sometimes observed to be greater than that of aflibercept alone. RNA expression analysis of HUVECs treated with nEPC also showed that anti-angiogenic effects may be mediated by both VEGF and non-VEGF mediated pathways. In addition, nEPC and A Bai Xi down-regulate the respective angiogenic pathways. Abelmosil down regulates mainly VEGFR1, while nEPC down regulates VEGF-C and VEGFR3 pathways, suggesting that a dual-tubular mechanism is possible, helping to achieve better anti-angiogenic effects. To further understand the effect of nEPC on the angiogenic process, studies were performed using AIM chips. This makes it possible to study angiogenesis in 3D micro-networks, so that cell-cell and cell-extracellular matrix interactions can be studied simultaneously. In particular, different VEGF gradients were used to induce sprouting of HUVECs into centrally filled collagen channels. Observations of this 3D vascular micro-network experiment further elucidate the anti-angiogenic mechanism of the nlpc. While cell proliferation and angiogenesis continue to be inhibited, HUVEC migration is not significantly inhibited. Without being bound by theory, it is believed that this phenomenon may be due to the addition of collagen. It is well known that immobilized extracellular matrix components such as collagen drive endothelial cell migration independently of chemotactic cytokines-known as tactility. Thus, it is speculated that nEPC is capable of inhibiting VEGF-driven angiogenic pathways responsible for endothelial cell proliferation and angiogenesis, but not of inhibiting the chemotaxis driven by ECM components such as collagen.

Although the pharmacokinetics of the smaller murine eye is not the same as the human eye due to the apparent differences in size, this problem was partially solved by using an ex vivo pig model, where similar results were produced and the nEPC+A was able to achieve greater corneal penetration. The present disclosure may also serve as a platform for further research to further explore the mechanisms behind the anti-angiogenic properties of nfpc, as angiogenesis is a dynamic process regulated by various pro-angiogenic mediators and anti-angiogenic factors to enable endothelial cells to proliferate, migrate, adhere and angiogenesis. For example, proteomic analysis can be used to find the modulating effect of nEPC on angiogenic signaling pathways. In addition, additional studies can be performed on other possible delivery routes, such as the transscleral route, to elucidate the complete mechanism of nEPC+A.

Example 9: summary

In summary, the present study discusses novel topical formulations consisting of the anti-VEGF compound albesiput encapsulated by polymeric nanomicelles having intrinsic anti-angiogenic properties. In addition to being a drug carrier with high payload and corneal barrier penetration enhancer, the results of this study also demonstrate that the anti-angiogenic properties inherent to nEPC may enhance the anti-angiogenic effect of aflibercept. It is understood that this was the first report of the local application of polymeric micelles loaded with macromolecular biological agents and exhibiting therapeutic effects on the retina, and also the first report of the study of the intrinsic anti-angiogenic effect of the nEPC. The nfpc is able to meet the necessary features of an effective local anti-VEGF delivery system for retinal diseases. The ability of nEPC to deliver therapeutically significant concentrations of Abelmoschus to the retina, together with the inherent anti-angiogenic properties, demonstrate a synergistic effect useful for the effective local delivery of existing anti-VEGF compounds. This suggests that nEPC+A may be a promising topical pharmaceutical formulation for the treatment of retinal diseases.

Example 10: experimental method

10.1.Materials and reagents

PEG, PPG and PCL were purchased from Sigma-Aldrich (Missouri, united States). Pluronic F127 (P2443) was purchased from Sigma. Abelmoschus is available from Bayer Healthcare (Berlin, germany). NHS-fluorescein and NHS-rhodamine were purchased from Thermo Fisher Scientific (Waltham, mass. USA). 3D cell culture chips were purchased from AIM BIOTECH (Singapore). Lactate dehydrogenase release (LDH) assay kit was purchased from DojinDo EU (Kumamoto, japan). Optimal Cleavage Temperature (OCT) compound (Tissue-) Purchased from Sakura Finetek (USA).

The chemical structures of exemplary copolymers designed according to the various embodiments disclosed herein are shown in scheme 1. The polymer is a three-component multi-block thermogel polymer consisting of hydrophilic poly (ethylene glycol) (PEG), thermosensitive poly (propylene glycol) (PPG), and hydrophobic biodegradable polyesters, such as, but not limited to, biodegradable poly (epsilon-caprolactone) (PCL) segments linked together by urethane linkages.

Synthesis of polymers

The general steps for preparing the multiblock copolymers according to various embodiments disclosed herein include: in the presence of a metal catalyst (in the examples below, dibutyltin dilaurate is used) and a suitable solvent (in the examples below, toluene is used), one or more hydrophilic polymers, one or more hydrophobic polymers and one or more thermosensitive polymers are mixed with a coupling agent (in the examples below, hexamethylene-1, 6-diisocyanate is used) as shown in scheme 1.

Examples of preparing polymers designed according to the various embodiments disclosed herein are described in detail below.

Poly (PEG/PPG/PCL carbamate) is synthesized from PEG, PPG and PCL-diol using hexamethylene-1, 6-diisocyanate as a coupling agent. Added hexamethyleneThe amount of 1, 6-diisocyanate corresponds to the amount of reactive hydroxyl groups in the solution. Typically, 0.15g of PCL-diol (mn= 2000,7.50 ×10 ^-5 Molar), 12g PEG (mn= 2050,5.85 ×10) ^-3 Molar) and 3g PPG (mn= 2000,1.50 ×10 ^-3 Moles) were dried overnight at 50 ℃ under high vacuum in a 250mL two-necked flask. Then, 100mL of anhydrous 1, 2-toluene was added to the flask, and any traces of water in the system were removed by two azeotropic distillation. 100mL of anhydrous 1, 2-toluene was added to the flask followed by two subsequent drops of dibutyltin dilaurate (-8X 10) ^-3 g) And 1.27g of hexamethylene-1, 6-diisocyanate (7.58X10) ^-3 Moles). The reaction mixture was stirred at 60 ℃ to 110 ℃ under nitrogen atmosphere for 24h. The resulting copolymer was precipitated from diethyl ether and further purified by redissolving in chloroform followed by precipitation in diethyl ether. The yield after isolation and purification was 85%.

10.2.Preparation of EPC copolymer and fluorescein-containing EPC Polymer (FEPC)

EPC copolymers were synthesized by ligating PEG, PPG and PCL. The PEG to PPG feed ratio was fixed at 4:1, with PCL (1%) added. To prepare FEPC, PEG (4.0 g, average MW 2050), PPG (1.0 g, average MW 2000) and PCL (50 mg, average MW 2000) were dried by azeotropic distillation using anhydrous toluene (2 x 20 mL) on a rotary evaporator and then heated at 110 ℃ in vacuo for 1 hour. Then, fluorescein-diol (75 mg) (scheme 2) was added to the mixture in multiple portions, followed by zinc diethyldithiocarbamate (12.4 mg) catalyst, anhydrous toluene (30 mL), and hexamethylene diisocyanate (0.44 mL). The reaction was stirred at 300RPM for 2 hours at 110 ℃. The bright yellow polymer was isolated by precipitation of a hot toluene solution in vigorously stirred diethyl ether (500 mL). The resulting polymer was purified by dialysis against a dialysis tube (MWCO 3500 Da) in distilled water for 3 days, followed by freeze-drying to give a yellow solid (yield = 4.5g, 81%). Fig. 43 shows the retention time of Gel Permeation Chromatography (GPC) of EPC copolymers, and table 1 shows the molecular weight details of EPC copolymers. Molecular weights of the constituent polymers (Table 2), EPC (FIG. 44 and Table 2), fluorescein-diol (FIGS. 45 to 47) and FEPC (FIGS. 48 to 50) were determined by Gel Permeation Chromatography (GPC) and were determined by ¹ H nuclear magnetic resonance (NM)R) chemical composition was evaluated. CMC values of FEPC were determined using dye solubilization method (fig. 48-50).

Scheme 2 shows a method for synthesizing a fluorescein-EPC polyurethane random block copolymer by (a) synthesizing fluorescein-diol from fluorescein and (B) through a polyaddition reaction between a diol reagent and hexamethylene diisocyanate under the catalysis of zinc diethyldithiocarbamate. Average M _n 2050g mol ^-1 PEG, average M of (v) _n 2000g mol ^-1 Is (1) PPG, average M _n 2000g mol ^-1 Is available from Sigma-Aldrich, hexamethylene-1, 6-diisocyanate (HMDI, 99%), dibutyltin dilaurate (DBTL, 95%), zinc diethyldithiocarbamate (ZDTC, 97%), potassium carbonate, potassium iodide, 3-bromo-1-propanol. Anhydrous toluene was purchased from Tedia, N-Dimethylformamide (DMF), ethyl acetate from VWR Chemicals, dichloromethane from Fisher Scientific, methanol (CMOS grade) from j.t.baker. All chemicals, reagents and solvents were used as received without further purification. Fluorescein (600 mg,1.81 mmol) was dissolved in anhydrous DMF (6 mL) under sonication and gentle heating. To the bright red solution were added potassium carbonate (525 mg,3.80 mmol), potassium iodide (60 mg,0.36 mmol) and 3-bromo-1-propanol (0.34 ml,3.80 mmol). The reaction was heated under Ar atmosphere with vigorous stirring at 80 ℃ overnight. Thereafter, the crude reaction was poured into water (100 mL) to form a bright orange suspension which was extracted with ethyl acetate (5×30 mL). The combined organics were washed with brine (2X 30 mL), dried over MgSO ₄ Dried and depressurized on a rotary evaporator to remove the solvent. Column chromatography (eluent: 10 v/v% methanol in dichloromethane) afforded the target product as a bright orange powder (yield 340mg, 42%).

¹ H NMR(500MHz，Chloroform-d)δ8.26(d，J＝7.6Hz，1H，H _d )，7.75(td，J＝7.5，1.4Hz，1H，H _b )，7.69(td，J＝7.7，1.4Hz，1H，H _c )，7.32(d，J＝7.6Hz，1H，H _a )，7.00(d，J＝2.4Hz，1H，H _h )，6.91(d，J＝9.0Hz，1H，H _i )，6.88(d，J＝9.6Hz，1H，H _f )，6.76(dd，J＝9.0，2.4Hz，1H，H _j )，6.56(dd，J＝9.6，2.0Hz，1H，H _e )，6.48(dd，J＝1.9，0.5Hz，1H，H _g )，4.26(t，J＝6.1Hz，2H，H _k )，4.21-4.07(m，2H，H _n )，3.88(t，J＝6.2Hz，2H，H _m )，3.39(td，J＝6.2，1.2Hz，2H，H _p )，2.10(quint.，J＝6.0Hz，2H，H _l )，1.59(quint.，J＝6.0Hz，2H，H _o ). ¹³ C NMR(125MHz，ChIoroform-d)δ185.5，165.9，163.7，159.1，154.5，150.5，134.3，132.9，131.5，130.7，130.6，130.5，130.0，129.9，129.1，117.7，115.0，114.0，105.9，101.1，66.1，62.6，59.6，58.9，31.9，31.4.λ _max (DMSO)/nm 437(ε/dm ³ mol ^-1 cm ^-1 39000)，460(47100)，488(30500).MS(ESI+ve)m/z 449.093([M+H] ⁺ ，C ₂₆ H ₂₄ O ₇ ，calc.449.160).

TABLE 1 summary of molecular weight details of EPC copolymers

TABLE 2 molar ratio of PEG (E), PPG (P) and PCL (C) added to each polymer from 3.60ppm to 3.70ppm, 1.10ppm to 1.15ppm and 4.05ppm, respectively ¹ H NMR resonance state determination

10.3.Preparation of nEPC, F127 nano micelle and rhodamine-labeled Abelmoschus nEPC or F127

CMC values for EPC and F127 were determined using the dye solubilization method. In vitro tests used either nEPC (2 wt%) or nEPC+rho-A. nEPC (2 wt%) was prepared by diluting EPC solution (10 wt.%% of the total weight of the composition). To prepare nEPC+rho-A, according to Pierce ^TM The NHS-rhodamine antibody labeling kit provides a protocol for chemically conjugating aflibercept to rhodamine. To ensure conjugation, a 5x excess of rhodamine was used. Unreacted excess rhodamine was then removed using a 50kDA filtration device. The filtration process was repeated until a clear filtrate was obtained. Rhodamine is a small molecule that passes through a filter and is separated from conjugated rhodamine. Because aflibercept is a macromolecule with a molecular weight greater than 50kDA, the aflibercept conjugated to rhodamine will be collected in the filter. To calculate the drug concentration, a standard curve of rhodamine conjugate concentration versus fluorescence intensity (Ex: 552nm, em:575 nm) was obtained using an enzyme labelling instrument (Infinite M200, tecan).

Different nEPC concentrations (0.05 wt%, 0.2 wt%, 1 wt%, 2 wt%) of nEPC+rho-A were prepared by dissolving a 10 wt% EPC solution in a Rho-A solution (0.5 mg/ml). In vivo testing used nEPC+A (nEPC 2 wt%, abelmoschus 40 mg/ml). nEPC+A was prepared by diluting a 10 wt% EPC solution in an Abelmoschus solution (0.5 mg/ml).

10.4.nEPC dissolution and NMR methods

Recording was performed at room temperature using a JEOL 500MHz NMR spectrometer (Tokyo, japan) ¹ H Nuclear Magnetic Resonance (NMR) spectra. EPC was dissolved in 0.3mL of aqueous Eylea buffer and further treated with 0.4mL of D ₂ O dilution. For samples containing Abelmoschus, an equal amount of EPC was dissolved in an Abelmoschus solution in aqueous Eylea buffer and 0.4mL of D was used ₂ O was further diluted. These samples were subjected to standard water inhibition and the spectra were analyzed using the MestReNova software (version 12.0.4) with reference to the residual peak of the solvent at 4.66 ppm.

10.5.Transmission electron microscope characterization of nEPC morphology

The EPC copolymer was dissolved in water or an aflibercept solution (0.05 wt%) at 1 wt% to form blank nlpc and nlpc+a solutions, respectively. Samples were prepared on a 400 mesh formvar-carbon EM grid (TeddPella 01754-F), stained negatively with 2% uranyl acetate and air dried. The grids were analyzed on a TALOS120c G transmission electron microscope (ThermoFisher Scientific, massachusetts, USA) with an operating voltage of 120 kV. Images were acquired using a CETA-16M camera at 120000 x magnification.

10.6.Characterization of nEPC and interaction with Abelmoschus

The average hydrodynamic nanomicelle sizes of nEPC (500. Mu.L, 0.2 wt% to avoid particle aggregation) and Abelmoschus (1000. Mu.L, 0.5 mg/mL) were measured by dynamic light scattering (DLS, zetasizer Nano-Malvern Panalytical, UK) at 25℃and pH7.2, respectively. The aflibercept and the nlpc solution were then mixed and the hydrodynamic size measured to monitor the encapsulation of aflibercept. The average of 3 micelle diameter measurements for 12 runs was calculated for all samples.

Fluorescence intensity was used to determine the EE of nEPC+rho-A. Briefly, rho-A (32. Mu.L, 1000 ng/mL) was added to a different wt% EPC solution (288. Mu.L) and homogenized. After homogenization, the solution was kept at room temperature for one hour and then ultracentrifuged with 100kDA filterFiltration was performed to collect free Rho-a at the bottom of the centrifuge tube. The solution containing free Rho-A was then transferred to a spectrophotometer (Ex/Em: 520.+ -. 20/590.+ -. 20 nm) for reading. EE is calculated using the following formula:

10.7.cell lines and culture media

Human umbilical vein endothelial cells (HUVEC, C2519A) were obtained from Lonza (Basel, switzerland) and stored in EGM in 25-T flasks ^TM -2 endothelial cell growth Medium-2 (EGM, CC-3162). Human corneal epithelial cells (hCECs, PCS-700-010 ^TM ) Corneal epithelial cell growth medium (=) obtained from ATCC (Manassas, virginia) and stored in T-25 flasks>PCS-700-040 ^TM ) Is a kind of medium. Immortalized adult retinal pigment epithelial cells (ARPE-19 cells,) and (E) a plant>CRL-2302) was obtained from ATCC (Manassas, virginia) and maintained in Dulcitol Modified Eagle Medium (DMEM) -F12 (1:1) supplemented with fetal bovine serum (FBS, 10%) and penicillin-streptomycin (1%).

10.8.In vitro anti-angiogenic assay

In the HUVEC proliferation experiments, HUVEC (passage 5) was seeded in EGM on 24-well plates at a density of 15000 per well. After overnight incubation, cell starvation was performed with Endothelial Basal Medium (EBM) +2% Fetal Bovine Serum (FBS) instead of EGM for 6h, then with appropriate medium according to the following 4 experimental arms: CTR (EGM), VEGF (50 ng/mL VEGF 165), abelmoschus+VEGF (50 ng/mL VEGF165+50 μg/mL Abelmoschus), and nEPC+VEGF (50 ng/mL VEGF165+2 wt% nEPC). Cell proliferation and death were assessed 24 hours and 72 hours later using the LDH assay according to kit instructions.

In the HUVEC migration assay, HUVEC (generation 5) was seeded onto 96-well plates at a density of 20000 per well. Once fused, use WoundMaker ^TM (ESSEN Bioscience 4379, UK) scratches were formed in each well. Culture medium (100 uL) based on the above 4 experimental arms was injected into the corresponding wells, respectively. Using a living cell analysis system (intucyte) Sartorius) takes phase difference images for each hole at each time point at the same position. The image was analyzed using MATLAB (MathWorks; version R2019 a), the boundary of the cell area was approximated using a method involving frequency filtering and mathematical morphology, which was adapted from an algorithm according to c.c. byes-aldaro, D.Biram, G.M.Tozer, C.Kanthou, electronics Letters2008,44,791. Wound recovery (%) [ (a) was used _t＝0h -A _t＝Δh )/A _t＝0h ]X 100 calculation, where A _t＝0h Is the wound area measured immediately after the scratch (time zero), A _t＝Δh Is the wound area measured at a selected point in time after the laceration.

In HUVEC tube formation assay, starved HUVEC (passage 5) was inoculated into matrigel @Growth For Reduced) coated 96-well plates with a density of 20000 cells/well. Solutions (100 μl) based on the above 4 experimental arms were injected into the corresponding wells, respectively. Bright field images were taken after 5 hours of exposure. The formation of blood vessels was analyzed with an angiogenesis analyzer in ImageJ software.

In vitro anti-angiogenic assays were performed using AIM 3D chips (AIM Biotech, singapore) using type I collagen solutions (2 mg/mL) in the intermediate channel of the device according to AIM biotechnology protocols. The left and right microchannels were then coated with fibronectin. HUVEC in EGM was then run at 3X 10 ⁶ The density of individual cells/mL was seeded in the left fibronectin-coated lateral fluid channel. After 24 hours, human VEGF in EGM was removed ₁₆₅ (40 ng/mL) and sphingosine-1-phosphate (S1P) (125 nM) were added to the left and right channels. This served as a positive control. Three groups were then prepared with VEGF and S1P: abelmosil (50. Mu.g/mL), nEPC (2 wt%) and nEPC+A, and applied to the cell channel and right side fluid channel. AIM chip (FIG. 19) at 37℃and 5% CO ₂ The culture medium was changed daily for 3 days. Angiogenesis sprouting of HUVEC in collagen hydrogels was monitored daily with a phase contrast microscope. Confocal imaging was performed on day 5 using a laser scanning microscope (Carl Zeiss LSM800 scanning head on an imager.Z2 microscope controlled by Zen2.1) using a Plan-Apochromat10x/0.45-NA objective.

10.9.HUVEC RNA expression analysis

HUVEC RNA expression was determined by qPCR. HUVECs were seeded at 100000 cells/well in 24-well plates. After 24 hours, 400 μl of solution based on 4 groups of experimental arms (buffer, VEGF, aflibercept+vegf, nfcp+vegf) was added. After 24 hours of incubation, total RNA was extracted and purified using RNasy Mini Kit (Qiagen, gmbH, hilden, germany) and converted to cDNA using iScript reverse transcription supermix for RT-qPCR (Bio-Rad Laboratories Inc, USA). The reaction mixture was added with RNase-free water to 20. Mu.L, and then the synthesis was performed in a thermal cycler. The cDNA was then amplified for real-time PCR analysis. Each real-time PCR reaction included 2. Mu.L of diluted cDNA solution, RNase-free water, the corresponding forward and reverse primer mix (10. Mu.M) and SYBR Green real-time PCR mix. Reactions were performed in triplicate in a real-time PCR system (Applied BioSystems QuantStudio 5). Each single reaction plate was GAPDH as an internal standard.

10.10.Cytotoxicity study

The cytotoxic effects of nEPC (0.01 to 2 wt%) on hCEC and ARPE-19 were evaluated using LDH proliferation and cell leakage assays (cytotoxic LDH detection kit, dojindo, DOJD-CK12, japan) according to the manufacturer's protocol. Briefly, cells were seeded in 96-well plates at a density of 10 k/well. After overnight incubation, the cells were exposed to 100 μl of the nfpc solution (0 wt%, 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.1 wt%, 0.2 wt%, 0.5 wt%, 1 wt% and 2 wt%) for 24 hours. In the LDH proliferation assay, all groups were lysed and absorbance at 490nm was measured and absorbance at 650nm was subtracted. Proliferation rate (%) =100% × (a/B). For the test group, supernatants were collected. For positive controls, cells were lysed prior to collection of the supernatant. The absorbance of all groups was measured as described previously. Cell death rate (%) =100% × [ (a-C)/(B-C) ]. The same cytotoxicity study was also performed on HUVEC (fig. 8 and 9).

10.11.Transepithelial electrical impedance in porcine tissue

For porcine cornea tissue, tissue integrity was monitored by ewing perfusion chamber (WPI, u.s). Briefly, freshly resected porcine cornea was gently mounted in a sample holder and then inserted vertically between two halves of ews. The donor (corneal epithelial side) and the recipient (corneal endothelial side) were each filled with 7.5mL of Glutathione Bicarbonate Ringer's (GBR) solution and continuously vented with a Carbogen gas mixture (95% O) ₂ And 5% CO ₂ ) To maintain the activity of the cornea. The cornea was stimulated with 10mV continuous current pulses every 1 minute for 0.2s and electrical parameters of the cornea were monitored in real time by labgart 7 software control. When electrical parameters areAfter substantial stabilization, all liquid on both sides is removed. Immediately 0.2mL of sample solution was added to the donor chamber and 0.4mL GBR was added to the receiving chamber at 37 ℃. At pre-fixed 10 minute intervals, 0.2ml fbs was removed from the receiving chamber and an equal volume of GBR solution was replenished. Sample collection lasted 40 minutes. At the end of infiltration, GBR was refilled into the chamber, checked for tissue integrity and monitored for an additional 30 minutes. The permeated Rho-aflibercept was analyzed by an enzyme-labeled instrument to detect fluorescence intensity.

10.12.In vitro anti-angiogenic choroidal test

Choroidal germination tests were performed according to the protocol disclosed in Shao, z. Et al, PLoS One 2013,8, e69552, the contents of which are incorporated herein by reference in their entirety. Briefly, murine choroidal tissues were cut into small pieces and embedded in matrigel. Explants were cultured ex vivo in egm+5% fbs on 24 well plates. After 48 hours incubation at 37 ℃, the vascular growth of the explants was monitored. The medium per well was replaced with the appropriate medium (500 μl) based on the following 3 experimental groups: VEGF (EGM+5% FBS+50ng/mL VEGF 165), abelmoschus+VEGF (EGM+5% FBS+50ng/mL VEGF165+1mg/mL Abelmoschus), nEPC+VEGF (EGM+5% FBS+50ng/mLVEGF165+2 wt% nEPC). After 2 days, the explants were monitored with a bright field microscope (Nikon Eclipse Ti using Plan UW 2x/0.06-NA objective) and images were taken.

For the choroidal degeneration assay, the explants were cultured in egm+5% FBS for 4 days. Once vessel germination has been established, the medium of each well is replaced by the same experimental group used for the germination test. On days 3 and 4, the explants were monitored. The germination area was quantified by TRI2 software and standardized for explant size based on the protocol published according to Shao, z. Et al, PLoS One 2013,8, e69552, the contents of which are incorporated herein by reference in their entirety. The phase contrast image of the choroidal bud was analyzed with ImageJ 1.46r (NIH). The choroidal tissues of the center of germination were delineated and removed by adjusting the magic wand tool to 30%. The capillary sprouting and background and edges are defined by a threshold function. The total number of threshold contour pixels is then calculated for quantization. In the inhibition test, the germination area was measured directly in pixels. To correct for the differences in initial explant size, the germination area was calculated using the following formula:

10.13.in vitro assessment of hCEC uptake of Rho-A and nEPC+rho-A

hCEC were seeded onto 24-well plates on gelatin-coated slides at a cell density of 10000 cells/well in corneal epithelial cell growth medium (500 μl). At 37℃with 5% CO ₂ After 24 hours of incubation in humidified atmosphere, the medium is replaced with growth medium containing Rho-A (0.5 mg/mL) and a different nEPC solution (0 wt%, 0.05 wt%, 0.2 wt%, 1 wt%, 2 wt%). The cells were washed 4 times with PBS to remove any excess extracellular aflibercept. Finally, cells were observed under a confocal laser scanning microscope (Carl Zeiss LSM800 scanning head on an imager.Z2 microscope controlled by Zen 2.1). Images were acquired using a Plan-Apochromat 100x/1.4-NA oil DIC objective for the slides. The laser lines on the system were 405nm, 488nm, 561nm and 640nm.

10.14.Quantification of hCEC in vitro cellular uptake by FACT

hCEC were seeded onto 12-well plates at a density of 60000 cells/well. Cells were cultured and exposed to the same medium used for internalization. After 24 hours of exposure, the samples were aspirated, the cells were carefully washed four times with PBS and digested with trypsin. Cells were suspended in growth medium (400. Mu.L), and internalized Rho-A (0.5 mg/mL) or nEPC+rho-A (Rho-A: 0.5mg/mL, nEPC:2 wt%) were quantified by flow cytometry (BD Bioscience FACS Aria II, united States), excited at 564nm and monitored at 590 nm. The results were analyzed by FlowJo software. All evaluations of F127 strictly followed the same conditions.

10.15.Local permeability ex-vivo model of porcine cornea

The local penetration of nEPC+rho-A (nEPC: 2 wt%, rho-A:0.5 mg/ml) was evaluated using an ex vivo model of porcine cornea. Adult pig eyes were obtained within 3 hours after death of the animals. The eyes were rinsed with PBS and a drop of Rho-A (20 uL,40 mg/mL) and nEPC+rho-A were applied directly to the cornea. After incubation at 37 ℃ for 40 minutes, the eyes were washed with PBS. The glass bodies were then collected to determine the intensity of fluorescence emitted by Rho-a. This was done using a microplate reader (ex: 560nm/em:594 nm).

Rho-A (40 mg/mL) and nEPC+rho-A penetration through the pig sclera was evaluated using a vertical Eudraga electrode kit (World Precision Instruments, florida, U.S.). The sclera was placed vertically between the diffusing cells with the epithelium facing the donor cells. The setting was maintained at 37 ℃. Donor cells contained Rho-a (0.2 mL) and nrpc+rho-a solution, while recipient chambers contained PBS (0.4 mL). PBS was collected every 10 minutes and the chamber was filled with additional PBS (0.4 mL). The experiment was stopped at 40 minutes and the concentration of infiltrated aflibercept was calculated from the fluorescence intensity (sample size > 3).

10.16.Animal study

Male wild-type C57B/6J mice of 6 to 8 weeks of age were obtained from In vivo (Singapore) and used for all In vivo experiments. All animal procedures were performed in accordance with the ARVO statement for ophthalmic and vision study animals. Experiments were approved by the Institutional Animal Care and Use Committee (IACUC): #191 488, item name: testing of therapeutic agents for ocular delivery of drugs.

10.17.In vivo evaluation of corneal residence time

To evaluate the corneal residence time of nanomicelles complexed with Rho-A (nEPC+rho-A: 2 wt%, rho-A:0.5 mg/mL) and Rho-A (5. Mu.L, 40 mg/mL), a single eye drop was administered to the murine corneal surface, respectively. The eyes of the mice were manually blinked every 15 seconds. Anterior ocular segment optical coherence tomography (optvue, RTVue) was performed 30 seconds, 60 seconds, 120 seconds, 210 seconds, 285 seconds, 300 seconds after the first administration of the eye drops. For each aset image, imageJ was used to select and quantify the area on the corneal surface occupied by eye drops.

10.18.In vivo Abelmosipu corneal permeability study

To evaluate the local permeability of nanomicelles complexed with Rho-A (nEPC or F127:2% by weight, rho-A:0.5 mg/mL), single eye drops of Rho-A (5. Mu.L, 40 mg/mL) or nEPC+rho-A (F127+rho-A) were applied to murine corneal surfaces. Mice were sacrificed 40 minutes after application of eye drops and eyeballs were removed. The eyes were then embedded in OCT compound (Sakura Finetek, USA), followed by fixation with 4% paraformaldehyde, and then frozen sections were made 10 μm thick for viewing under confocal laser scanning microscopy (Olympus FV1000 confocal head on IX81 microscope controlled by Fluoview 4.2).

Rho-a permeability to vitreous cavities was assessed by taking a sample of vitreous humor. The glass body was removed by a thin glass capillary tube by performing edge penetration with a 30-gauge needle. Vitreous humor from 10 eyes was collected from each group, and the amount of Abelmoschus was evaluated by a spectrophotometer (ex: 560.+ -. 20nm/em: 590.+ -. 20 nm).

10.19.Evaluation of in vivo biological Activity of nEPC+A

The biological activity of nEPC+A was assessed using a laser-induced CNV murine model previously published in Nirmal, J. Et al, exp Eye Res 2020,199,108187, the contents of which are incorporated herein by reference in their entirety. Mice were anesthetized with intraperitoneal ketamine (150 mg/kg) and xylazine (10 mg/kg). In these eyes, photocoagulation was induced using an image guided laser system (Micron IV, phoenix Research Laboratories, plasasanton, CA). Mice were divided into 4 groups of 8 eyes each, including: buffer (PBS); abelmosipu (40 mg/mL); nEPC (2 wt%) and nEPC+A (Abelmoschus 40mg/mL, nEPC 2 wt%). Each solution was applied immediately after laser treatment, 3 times per day, 1 hour apart, for 14 days.

Mice were anesthetized as described above, and then subjected to Fundus Fluorescein Angiography (FFA) using a retinal imaging system (Micron IV, phoenix Research Laboratories) at days 3, 7 and 14 after laser photocoagulation. FFA images were taken 5 minutes and 10 minutes after fluorescein injection.

Mice were euthanized and the eyeballs were removed 14 days after laser treatment to make choroidal flatmount. Eyes were fixed overnight at 4 ℃ in 4% paraformaldehyde in PBS. Anterior ocular segments and retinas were embedded in paraffin for immunostaining. The eye cups were incubated with isolectin B4 at 4 ℃ for choroidal vascular staining and then washed 3 times with PBS. After four incisions were made radially into the optic nerve, the tissue was plated and a laminographic image of the CNV lesions was taken with a confocal microscope (LSM 700, zeiss, thornwood, NY).

Angiography and blanket imaging were imported into ImageJ (US National Institutes of Health, bethesda, MD, USA). The maximum boundary of CNV lesions on each image was manually delineated under magnification, the area quantified as pixels per 100 μm. Fluorescence intensity of CNV lesions was single blind graded by 2 independent graders using ImageJ (US National Institutes of Health, bethesda, MD, USA). CNV degradation rate was calculated using the following:

10.20.statistical analysis

All data are reported as mean ± s.d. Statistical significance differences between samples were determined by one-way analysis of variance (ANOVA) and then pair-wise tested using Tukey Honest Significant Differences (HSD) post-hoc test. P values below 0.05 in the 2-tailed test were considered significant (< 0.05, <0.002 and <0.0002, < 0.0001). All assays were performed using GraphPad Prism (version 8.1.1).

10.21.Characterization of materials

NMR spectra were recorded at room temperature on a JEOL ECA 500MHz NMR spectrometer running at 500MHz, with the sample dissolved in CDCl ₃ (NMR solvents purchased from Cambridge Isotopes Laboratory inc.). Chemical shifts are reported in parts per million (ppm) on the delta scale. Gel Permeation Chromatography (GPC) analysis was performed at 40 ℃ on a Waters GPC machine equipped with a 515HPLC pump, a Waters Styragel column, and a Waters 2414 refractive index detector. HPLC grade THF was used as the eluent at a flow rate of 1.0 ml/min. Calibration curves were generated using monodisperse polystyrene standards.

Those skilled in the art will appreciate that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, employed, modified, included, or the like, among the different exemplary embodiments in the present description. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

1. An anti-angiogenic agent comprising:

multiblock copolymers in the form of one or more micelles,

2. An anti-angiogenic agent according to claim 1 wherein the copolymer comprises at least urethane and/or allophanate linkages.

3. The anti-angiogenic agent according to claim 1 or 2, wherein the molar ratio of the first poly (alkylene glycol) block, the second poly (alkylene glycol) block to the polyester block in the copolymer is from about 1 to 10:1:0.01 to 1.5.

4. The anti-angiogenic agent according to any one of the preceding claims, wherein the first poly (alkylene glycol) and the second poly (alkylene glycol) are selected from the group consisting of poly (ethylene glycol) (PEG), poly (propylene glycol) (PPG), poly (butylene glycol), and combinations thereof; the polyester is selected from the group consisting of Polycaprolactone (PCL), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polyhydroxyalkanoate (PHA), and combinations thereof.

5. The anti-angiogenic agent according to any one of the preceding claims, wherein the total polymer concentration of the copolymer is 0.01 to 6 wt%.

6. The anti-angiogenic agent according to any one of the preceding claims, wherein the anti-angiogenic agent comprises at least 90 weight percent water content.

7. The anti-angiogenic agent according to any one of the preceding claims, wherein the hydrodynamic size of the one or more micelles is 1nm to 100nm.

8. The anti-angiogenic agent according to any one of the preceding claims, wherein the anti-angiogenic agent further comprises one or more than one bioactive substance complexed with or encapsulated by the copolymer micelle.

9. The anti-angiogenic agent according to claim 8, wherein the one or more bioactive substances include anti-vascular endothelial growth factor (anti-VEGF).

10. The anti-angiogenic agent according to claim 9, wherein the anti-VEGF is selected from bevacizumab, albespride, ranibizumab and busimumab.

11. An anti-angiogenic agent according to any one of claims 8 to 10 wherein one or more bioactive substances are encapsulated by the copolymer micelle at an encapsulation rate of greater than 25%.

12. The anti-angiogenic agent according to any one of the preceding claims, wherein the anti-angiogenic agent is formulated as a topical ophthalmic formulation.

13. A method of preparing an anti-angiogenic agent according to any one of claims 1 to 12, the method comprising:

14. The method of claim 13, wherein the concentration of copolymer in the aqueous medium is from 0.01 wt% to 6 wt%.

15. The method of claim 13 or 14, further comprising complexing or encapsulating one or more bioactive substances with micelles.

16. The method of any one of claims 13 to 15, further comprising coupling the first poly (alkylene glycol) block, the second poly (alkylene glycol) block, and the polyester block together through at least a urethane linkage group and/or an allophanate linkage group.

17. The method of any one of claims 13 to 16, wherein the first poly (alkylene glycol) and the second poly (alkylene glycol) are selected from the group consisting of poly (ethylene glycol) (PEG), poly (propylene glycol) (PPG), poly (butylene glycol), and combinations thereof; the polyester is selected from the group consisting of Polycaprolactone (PCL), polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polyhydroxyalkanoate (PHA), and combinations thereof.

18. The method of any one of claims 13 to 17, wherein the coupling step is performed in the presence of a coupling agent comprising an isocyanate monomer containing two isocyanate functional groups.

19. The process according to any one of claims 13 to 18, wherein the coupling step is carried out in the presence of a catalyst selected from the group consisting of an alkyl tin compound, an aryl tin compound and a dialkyl tin diester, such as dibutyltin dilaurate, dibutyltin diacetate, dibutyltin dioctoate and dibutyltin distearate.

20. The method according to any one of claims 13 to 19, wherein the coupling step is performed in the presence of a solvent selected from toluene, benzene, xylene, halogenated organic solvents, halogenated alkane solvents, chlorinated solvents, methylene chloride, dichloroethane, carbon tetrachloride and chloroform (or chloroform).

21. An anti-angiogenic agent according to any one of claims 1 to 12 for use in medicine.

22. An anti-angiogenic agent according to any one of claims 1 to 12 for use in the prevention or treatment of an ocular disease.

23. An anti-angiogenic agent according to any one of claims 1 to 12 for use in the prevention or treatment of cancer.

24. Use of an anti-angiogenic agent according to any one of claims 1 to 12 in the manufacture of a medicament for the prevention or treatment of an ocular disease.

25. Use of an anti-angiogenic agent according to any one of claims 1 to 12 in the manufacture of a medicament for the prevention or treatment of cancer.

26. A method of preventing or treating an ocular disease, the method comprising administering to a subject in need thereof an anti-angiogenic agent according to any one of claims 1 to 12.

27. A method of preventing or treating cancer, the method comprising administering to a subject in need thereof an anti-angiogenic agent according to any one of claims 1 to 12.

28. The anti-angiogenic agent according to claim 22, the use according to claim 24 or the method according to claim 26, wherein the ocular disease is selected from angiogenic ocular disease, ocular disease of the anterior segment of the eye, ocular disease of the posterior segment of the eye, neovascular related posterior segment disease, retinal disease, neovascular age-related macular degeneration (AMD) such as neovascular AMD, diabetic retinopathy, diabetic macular edema (DMO), choroidal Neovascularization (CNV), central Retinal Vein Occlusion (CRVO), corneal neovascularization and retinal neovascularization.

29. The anti-angiogenic agent according to claim 22, the use according to claim 24 or the method according to claim 26, wherein the anti-angiogenic agent is to be topically administered to a subject in need thereof.

30. The anti-angiogenic agent according to claim 22, the use according to claim 24 or the method according to claim 26, wherein the anti-angiogenic agent is formulated as an eye drop.