Drug delivery compositions for ocular administration of therapeutic agents and methods of use thereof

Abstract

The present disclosure relates to compositions, devices, and methods for delivering a protein therapeutic agent to the eye, for example, intravitreal delivery of a protein therapeutic agent. In particular, the present disclosure describes a drug delivery device for injection into the eye of a subject in need thereof comprising a capsule having a double-walled and a therapeutic agent, wherein the therapeutic agent is initially present within a lumen space of the capsule. Methods of making and using these drug delivery devices are also described.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 803,388, filed February 8, 2019, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to drug delivery compositions, and more particularly to compositions comprising one or more multilayer drug delivery capsules for delivering a therapeutic agent to the eye. Background Technology

[0004] Age-related macular degeneration (AMD) is the fourth leading cause of blindness worldwide after cataracts, premature birth, and glaucoma. In the United States, more than 11 million people are diagnosed with wet AMD. This number is estimated to double within 30 years. Therefore, much work has been done to understand the pathogenesis of the disease and to develop treatments. It is widely noted that overexpression of vascular endothelial growth factor (VEGF), along with aging, stimulates the formation of new blood vessels in the choroid, leading to irreversible damage to the retina during the bleeding and scarring of these newly formed vessels. The current gold standard treatment for wet AMD is monthly intravitreal injections of anti-VEGF drugs (such as bevacizumab or ranibizumab) to inhibit VEGF and prevent angiogenesis. However, frequent injections often lead to infections, increased intraocular pressure, and rhegmatogenous retinal detachment, as well as problems with patient compliance.

[0005] Recently, novel devices for long-term drug delivery in the eye, such as implants and micro / nanoparticles, have been reported. Unfortunately, such implants require surgical procedures for implantation and removal. Furthermore, known implants tend to be off-target and reduce drug efficacy. While microparticles or nanoparticles have a relatively small size suitable for injection into the eye using a 30-gauge needle, currently described microparticles or nanoparticles undergo rapid release of therapeutic agents, such as anti-VEGF agents, due to the biodegradation of known particle compositions during the first three months.

[0006] Therefore, despite significant efforts in the treatment of AMD and other ophthalmic conditions, methods and compositions that minimize the harmful side effects of currently available treatments remain lacking. Furthermore, there is a need for biodegradable drug delivery systems and compositions that allow for controlled drug release for up to nine months or longer after intravitreal injection. Improved treatment methods for AMD and other ophthalmic diseases requiring direct delivery of therapeutic agents to the eye are still needed. These and other needs are met by this disclosure. Summary of the Invention

[0007] In accordance with one or more purposes of this disclosure, as embodied and broadly described herein, in one aspect this disclosure relates to compositions, apparatus, and methods for delivering protein therapeutic agents to the eye, such as intravitreal delivery of protein therapeutic agents. The disclosed pharmaceutical delivery compositions comprise a capsule having a double-walled structure and containing a therapeutic agent therein. In another aspect, this disclosure relates to methods for treating ophthalmic diseases or conditions.

[0008] Therefore, in one aspect, a drug delivery composition comprising the following is provided:

[0009] One or more capsules, each having a tubular shape with two closed ends, wherein each of the one or more capsules independently comprises multiple layers of walls and at least one tubular compartment; and

[0010] One or more therapeutic agents, each of which is initially present in one or more of the at least one luminal compartment;

[0011] Each multi-layered wall independently contains at least an inner layer and an outer layer;

[0012] Each inner layer contains a first polymer that has a net positive charge under physiological conditions; and

[0013] Each outer layer independently contains a second polymer that is different from the first polymer.

[0014] In some embodiments, the drug delivery composition may comprise two or more capsules. In some embodiments, different therapeutic agents are initially present in each of the two or more capsules. In other embodiments, the same therapeutic agent is initially present in each of the two or more capsules.

[0015] In some embodiments, at least one or more capsules comprise two or more lumen compartments. In some embodiments, different therapeutic agents are initially present in each of the two or more lumen compartments. In other embodiments, the same therapeutic agent is initially present in each of the two or more lumen compartments.

[0016] In some aspects, the first polymer may comprise chitosan, polyethyleneimine, protamine, polypropyleneimine, poly-L-lysine, poly-L-arginine, poly-D-lysine, poly-D-arginine, cellulose, dextran, poly(amidoamine), poly(2-(dimethylamino)ethyl methacrylate), derivatives thereof, or combinations thereof. In some embodiments, the first polymer may comprise chitosan or a derivative thereof. In some embodiments, the first polymer comprises fibers with an average diameter of about 50 nm to about 1000 nm.

[0017] In some aspects, the second polymer may comprise a biodegradable polymer. In some embodiments, the second polymer comprises poly(ε-caprolactone) (PCL), polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolic acid (PLGA), polyester, poly(orthoester), poly(phosphonazine), poly(phosphate), gelatin, collagen, polyethylene glycol (PEG), derivatives thereof, or combinations thereof. In some embodiments, the second polymer comprises PCL. In other embodiments, the second polymer comprises PLA. In some embodiments, the second polymer comprises fibers with an average diameter of about 100 nm to about 2000 nm.

[0018] In some aspects, one or more capsules each independently have a length of about 0.1 cm to about 5 cm. In some embodiments, one or more capsules each independently have an inner diameter of about 100 μm to about 2000 μm. In some embodiments, one or more capsules each independently have an outer diameter of about 50 μm to about 300 μm larger than the inner diameter of an identical capsule. In some embodiments, each multilayer wall has a wall thickness of about 25 μm to about 150 μm. In some embodiments, each outer layer may further comprise pores with an average pore diameter of about 100 nm to about 10000 nm.

[0019] In some embodiments, each of the one or more drug delivery capsules has a surface charge measured at a zeta potential of about -25 mV to about 25 mV at pH 7.4. In some embodiments, one or more therapeutic agents each have a net negative charge in a pH range of about 6.0 to about 7.4.

[0020] In some embodiments, at least one of the one or more therapeutic agents is an anti-VEGF agent. In some embodiments, the anti-VEGF agent is a therapeutic antibody, such as bevacizumab, ranibizumab, IBI305, or a combination thereof. In some embodiments, the anti-VEGF agent is a VEGF decoy receptor, such as aflibercept. In some embodiments, the anti-VEGF agent is a tyrosine kinase inhibitor, such as lapatinib, sunitinib, axitinib, pazopanib, or a combination thereof.

[0021] In some implementations, one or more therapeutic agents may comprise anti-inflammatory agents, such as cyclosporine, steroid or non-steroidal anti-inflammatory drugs, antimicrobial agents, immunomodulatory drugs, ocular antihypertensive agents, neuroprotective agents, gene therapy, viral vector therapy, alpha-adrenergic agonists, beta-adrenergic agonists, or combinations thereof.

[0022] In another aspect, a method is provided for treating an ophthalmic condition in a subject of need, the method comprising injecting a therapeutically effective amount of the pharmaceutical delivery composition described herein into the subject's eye. In some embodiments, the ophthalmic condition may include acute macular neuroretinopathy; Behcet's disease; neovascularization, including choroidal neovascularization; diabetic uveitis; histoplasmosis; infection, such as an infection caused by fungi or viruses; macular degeneration, such as acute macular degeneration (AMD), including wet AMD, non-exudative AMD, and exudative AMD; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting a point or location in the posterior part of the eye; ocular tumors; retinal conditions, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), and proliferative vitreous. Retinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitis; sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; uveitis; posterior ocular conditions caused or affected by ocular laser treatment; posterior ocular conditions caused or affected by photodynamic therapy or photocoagulation, radiation retinopathy, epiretinal membrane disease, retinal branch vein occlusion, anterior ischemic optic neuropathy, non-retinopathy diabetic retinopathy, retinitis pigmentosa, cancer, and glaucoma. In some embodiments, the ocular condition includes wet age-related macular degeneration (wet AMD), neovascularization, or macular edema. In some embodiments, injecting the described composition into the subject's eye includes injection into the vitreous cavity of the eye. In other embodiments, injecting the described composition into the subject's eye includes intravitreal injection, subconjunctival injection, subtenon injection, retroocular injection, or suprachoroidal injection.

[0023] A method for forming one or more capsules used in a drug delivery composition as described herein is also provided, the method comprising:

[0024] A first polymer is formed into a first layer on a conductive rod, wherein forming the first layer includes electrospinning using a first solution containing at least one organic solvent containing the first polymer, and wherein electrospinning is performed using a voltage difference of about 10 kV to about 30 kV; and

[0025] A second polymer is formed on a first layer to form a second layer, wherein forming the second layer includes electrospinning a second solution comprising the first polymer and optionally a pore-forming agent onto the formed first layer, and wherein electrospinning is performed using a voltage difference of about 10 kV to about 30 kV.

[0026] Also disclosed are a medicine box comprising one of: (a) the disclosed drug delivery composition; (b) a sterile package containing the disclosed drug delivery composition; or (c) a pre-filled syringe or needle containing the disclosed drug delivery composition, and instructions for administering the drug delivery composition to treat an ophthalmic disease or condition as described herein.

[0027] Upon examination of the following figures and detailed embodiments, other systems, methods, features, and advantages of this disclosure will be apparent to or become apparent to those skilled in the art. All such other systems, methods, features, and advantages are intended to be included within this specification, within the scope of this disclosure, and protected by the appended claims. Furthermore, all optional and preferred features and modifications of the described embodiments are available in all aspects of this disclosure taught herein. Moreover, the individual features of the appended claims and all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with each other. Attached Figure Description

[0028] Many aspects of this disclosure can be better understood by referring to the following figures. The components in the figures are not necessarily drawn to scale, but are emphasized to clearly illustrate the principles of this disclosure. Furthermore, in the figures, similar reference numerals designate corresponding parts in several views.

[0029] Figure 1A A representative disclosed method is illustrated, comprising the steps of: a) collecting two layers of chitosan and PCL nanofibers on a rotating rod using electrospinning; b) sintering the bilayer coated rod in a vacuum furnace at 100°C for approximately 3 hours; c) removing the rod to form a centrally hollow cylinder; d) creating a porous structure in the PCL layers by salt leaching; and e) loading a therapeutic agent into the capsule, followed by end sealing. The prepared bilayer capsule can then be used in studies, such as (e.g., step f) to evaluate drug release into a suitable buffer (e.g., PBS at 37°C); or for delivering the loaded drug to a suitable target, such as via (e.g., step g) intravitreal injection into the eye.

[0030] Figure 1B A schematic cross-sectional view of the disclosed bilayer capsule and a schematic diagram of intravitreal injection of the disclosed bilayer capsule are shown.

[0031] Figure 2A representative schematic diagram of chitosan and PCL fiber mats formed using the disclosed technique is shown (see Figure A). The figure also shows representative scanning electron micrographs (SEM) images as follows: (Figure B) a representative SEM image of a cross-section of the bilayer chitosan-PCL fiber mat; and (Figure C) representative SEM images of PCL and chitosan nanofiber layers with diameters of 932.57 ± 399.42 nm and 331.61 ± 186.19 nm, respectively.

[0032] Figure 3 Representative photographic images of the disclosed bilayer capsules are shown. The left square of the figure shows photographic images of two types of capsules, one with a diameter of 1.645 mm and the other with a diameter of 260 μm. The middle square of the figure shows a representative SEM image of a PCL monolayer capsule with an inner diameter of 260 μm. The right square shows a representative SEM image of a chitosan-PCL bilayer capsule with a membrane thickness of 89.85 ± 4.27 μm. The image in the right square shows that in this representative example, the chitosan fiber pad layer is attached to the outer PCL layer, and the chitosan layer accounts for approximately 25% of the total wall thickness.

[0033] Figure 4 Representative images of the disclosed PCL membranes prepared using HEPES salts of indicated concentrations are shown, illustrating surface or cross-sectional views of the disclosed PCL membranes as indicated. The images demonstrate that increasing the ratio of sodium HEPES salt results in greater porosity in the PCL membrane. Excessive interconnecting pores can be provided within the membrane at salt concentrations exceeding 5.0%. Arrows: Characteristic interconnecting pores within the PCL film after salt leaching. Each image has a scale bar as shown in the lower left corner.

[0034] Figure 5 Images and data related to the characterization of the disclosed bilayer capsule are shown. Figure a shows a representative image of the bilayer structure after salt leaching and washing. Figure b shows representative SEM images of the disclosed bilayer membrane before and after salt leaching. As shown, a porous structure is generated by salt leaching and the chitosan fibrous structure is lost after washing with a saturated sodium bicarbonate solution. The porous bilayer structure is observed in cross-section. Figure c shows representative FTIR spectra of the chitosan layer and PCL layer after salt leaching. As shown, at 1752 cm⁻¹... -1 The significant peak at 3478 cm⁻¹ was assigned to the carbonyl group in PCL. -1 The broad group at that location is a hydroxyl group in chitosan.

[0035] Figure 6Data relating to the effects of porous and bilayer structures on protein release from the disclosed bilayer capsules are presented. As described below, data were obtained from representative disclosed chitosan-PCL bilayer capsules (labeled Ch-PCL in the legend) and PCL monolayer capsules (labeled PCL in the legend) encapsulating BSA or bevacizumab as described herein, as determined by incubation in PBS. The percentage values ​​shown in the legend along with the “Ch-PCL” or “PCL” label indicate the w / v% used to prepare the bilayer or monolayer capsules. Figure a shows representative BSA release patterns from a 1.645 mm inner diameter bilayer capsule and a 260 μm inner diameter bilayer capsule. Figure b shows representative bevacizumab release patterns from a 1.645 mm inner diameter bilayer capsule and a 260 μm inner diameter bilayer capsule. At each time point, the drug delivery composition showed lower cumulative release compared to the monolayer capsule (# = p ≤ 0.05). The data also showed that increasing salt concentration was associated with an increase in cumulative release at each time point (*=p≤0.05).

[0036] Figure 7 Data relating to the effects of porous and bilayer structures on protein release from the disclosed bilayer capsules are shown. In this figure, trend lines are fitted to the data using the fitting parameters shown. As described below, data were obtained from representative disclosed chitosan-PCL bilayer capsules (labeled Ch-PCL in the legend) and PCL monolayer capsules (labeled PCL in the legend) encapsulating BSA or bevacizumab as described herein, determined by incubation in PBS. The percentage values ​​shown in the legend along with the “Ch-PCL” or “PCL” label indicate the w / v% used to prepare the bilayer or monolayer capsules. Figure a shows representative BSA release patterns from a 1.645 mm inner diameter bilayer capsule and a 260 μm inner diameter bilayer capsule. Figure b shows representative bevacizumab release patterns from a 1.645 mm inner diameter bilayer capsule and a 260 μm inner diameter bilayer capsule. Data indicate that the disclosed bilayer capsules can achieve near-zero-order release kinetics.

[0037] Figure 8The analytical protocol used to assess the potential toxicity of the disclosed capsules, and the data obtained from the analysis, are shown. Figure a shows the analytical protocol used to assess in vitro cytotoxicity using ARPE-19 cells via the direct contact method. Figure b shows the in vitro toxicity data for capsules prepared via the direct contact method using 10.0% HEPES salt, 7.5% HEPES salt, and 5.0% HEPES salt. Figure c shows the analytical protocol used to assess in vitro cytotoxicity using ARPE-19 cells via the extraction-exposure method. Figure d shows the in vitro cytotoxicity of extracts from capsules prepared under different conditions. Each bar and salt concentration at different time points represents the average measurement of three independent samples. Error bars indicate the standard deviation. As described above, data were obtained using representative disclosed chitosan-PCL bilayer capsules (labeled Ch-PCL in the legend) and PCL monolayer capsules (labeled PCL in the legend). Data showed no significant difference in cell viability between cells treated with PCL or chitosan-PCL extract (p > 0.05), and no significant observed difference over time (p > 0.05).

[0038] Figure 9 Representative fluorescence micrographs and data relating to inhibition of cell tubule length in HUVEC cells treated with VEGF delivered using a PCL monolayer capsule or the disclosed dual-layer capsule are shown. Cells were labeled using calcein AM. Figure a shows representative fluorescence images of HUVECs treated with 5 ng VEGF in the absence of (left) and presence of (right) 10 mg of native bevacizumab in cell culture medium. The data show significant disruption of cell tubules in the cells in the presence of bevacizumab compared to the control group. Figure b shows representative fluorescence images of inhibition of cell tubules in cells exposed to 10 mg bevacizumab released by a 260 μm diameter PCL monolayer and a chitosan-PCL drug delivery device, as indicated, for 1 week, 1 month, 3 months, and 9 months. Figure c shows the mean tubule length inhibition in the indicated groups, quantitatively analyzed using ImageJ software. Data are presented as mean ± SD, n = 3. Data showed that the HUVEC tube length treated with bevacizumab eluted from monolayer capsules was significantly different from that of bilayer capsules (* = p ≤ 0.05). Data also showed that the HUVEC tube inhibition capacity of eluted bevacizumab was significantly different over time compared to free native bevacizumab (# = p ≤ 0.05).

[0039] Figure 10Results from a study evaluating the injection of representative, disclosed bilayer capsules into an isolated pig eye model are presented. Figure a shows a schematic diagram of the injection of the bilayer capsule into the vitreous fluid via a subcutaneous needle. Figure b shows the pre-loaded capsule in a 21-gauge needle, injected 3 mm posterior to the limbus in the isolated pig eye (see middle square). Following injection, the isolated pig eye was dissected, and the intact capsule was observed to be intact in the vitreous fluid of the isolated pig eye (see right square).

[0040] Figure 11 This paper compares the biodegradation of PCL monolayer and bilayer capsules after one year of incubation as described herein. Figure a shows representative scanning electron micrographs (SEM) of the capsules prepared at different salt concentrations. An increase in pore size was observed on the PCL membranes in all samples after nine months. Cross-sectional images (see right-hand squares) show that the capsules remained intact after one year. Figure b shows a representative SEM image of the bilayer capsules. A fibrous framework was observed in the porous chitosan layer. The complete bilayer structure is shown in the cross-sectional image (see right-hand squares).

[0041] Figure 12 The UV-Vis absorption spectra of bevacizumab diluted in PBS at different concentrations are shown. Figure a shows the absorbance of diluted bevacizumab measured by UV-Vis spectroscopy. Figure b shows the standard curve of bevacizumab measured by a plate reader. The minimum concentration detectable by UV-Vis spectroscopy and a plate reader is 5 μg / mL.

[0042] Figure 13 The effect of the porous and bilayer structure of the capsules described herein on bevacizumab release, as assessed by ELISA, is illustrated. The bilayer capsules and PCL monolayer capsules encapsulating bevacizumab described herein were incubated in PBS. Bevacizumab release patterns were obtained with capsules having an inner diameter of 260 μm. The disclosed bilayer capsules efficiently retained the protein within the capsule for at least nine months and exhibited lower cumulative release at each time point than the monolayer capsules (# = p ≤ 0.05). Increasing the salt concentration also increased the cumulative release at each time point (* = p ≤ 0.05). The release patterns obtained by ELISA were consistent with those determined by UV-Vis spectroscopy.

[0043] Figure 14 The stability of free, native bevacizumab before and after lyophilization, as well as that of bevacizumab eluted from the monolayer and bilayer capsules described herein during the first three months, is shown. Figure a shows SEC-HPLC chromatograms of free, native bevacizumab, lyophilized bevacizumab, and bevacizumab in the device. Figure b shows SEC-HPLC chromatograms of bevacizumab eluted from monolayer and bilayer capsules incubated at physiological temperatures for one and three months.

[0044] Figure 15 Biodegradation of chitosan-PCL bilayer capsules exposed to PBS for more than three weeks is shown. Representative SEM images of the 260 μm inner diameter bilayer capsules prepared with 10% HEPES salt are provided, revealing the predominantly porous structure. Cross-sectional and internal images indicate that the capsules lost their inner chitosan layer after three weeks of guided exposure to PBS; however, biodegradation was not significant when the chitosan layer was coated with PCL. The thickness of the bilayer capsules was 73.23 ± 3.62 μm.

[0045] Other advantages of this disclosure will be set forth in part in the description which follows, and will be apparent from the description or may be learned from practice of this disclosure. The advantages of this disclosure will be realized and achieved by means of the elements and combinations particularly pointed out in the appended claims. It should be understood that the foregoing general description and the following detailed description are merely exemplary and illustrative and do not limit the scope of the disclosure as claimed. Detailed Implementation

[0046] With the aid of the teachings and related drawings presented in the foregoing description, those skilled in the art will conceive of numerous modifications and other embodiments of the disclosed compositions and methods. Therefore, it should be understood that this disclosure is not limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. A skilled craftsman will recognize numerous variations and modifications of the aspects described herein. These variations and modifications are intended to be included in the teachings of this disclosure and are covered by the claims herein.

[0047] Although specific terms are used in this article, they are used only in a general and descriptive sense and are not intended for limiting purposes.

[0048] As will be apparent to those skilled in the art upon reading this disclosure, the discrete components and features of each of the individual embodiments described and illustrated herein may be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of this disclosure.

[0049] Any method described may be performed in the order of the described events or in any other logically possible order. In other words, unless expressly stated otherwise, no method or aspect set forth herein is intended to be construed as requiring its steps to be performed in a particular order. Therefore, unless a method claim is specifically stated in the claims or description as having steps limited to a particular order, no order is intended to be inferred in any respect. This applies to any possible non-representational basis used for interpretation, including logical elements concerning the arrangement of steps or the flow of operations, the general meaning derived from grammatical organization or punctuation, or the number or type of aspects described in this specification.

[0050] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials cited in connection with said publications. The publications discussed herein are provided solely by virtue of their disclosure prior to the date of this application. Nothing herein should be construed as an admission that the invention is not entitled to precedence over such publications due to prior art. Furthermore, the publication dates provided herein may differ from the actual publication dates, which may require independent verification.

[0051] While aspects of this disclosure may be described and required in specific statutory categories (such as the systems statutory category), this is for convenience only, and those skilled in the art will understand that each aspect of this disclosure may be described and required in any statutory category.

[0052] It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be restrictive. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods pertain. It will also be understood that terms such as those defined in common dictionaries should be interpreted as having the same meaning as they have in the context of this specification and related fields, and should not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0053] Before describing various aspects of this disclosure, the following definitions are provided and, unless otherwise indicated, should be used. Other terms may be defined elsewhere in this disclosure.

[0054] definition

[0055] As used herein, “comprising” should be interpreted as indicating the presence of the stated feature, whole, step, or component at the time of reference, but does not preclude the presence or addition of one or more features, wholes, steps, or components, or groups thereof. Furthermore, each of the terms “through,” “comprising / comprises / comprised of,” “including / includes / included,” “involving / involves / involved,” and “such as” is used in its open, non-restrictive sense and is interchangeable. Additionally, the term “comprising” is intended to include instances and aspects covered by the terms “substantially constitutes” and “consistent with.” Similarly, the term “substantially constitutes” is intended to include instances covered by the term “consistent with.”

[0056] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “a (or an)” and “the” include plural indicators. Thus, by way of example, references to “drug delivery composition,” “therapeutic agent,” or “clinical condition” include, but are not limited to, two or more such drug delivery compositions, therapeutic agents, or clinical conditions.

[0057] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed in range patterns herein. It will also be understood that the endpoints of various ranges are clearly both related to and independent of another endpoint. It should also be understood that there are many values ​​disclosed herein, and each value is disclosed herein as “about” the specific value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges may be expressed herein as from “about” one specific value and / or to “about” another specific value. Similarly, when a value is expressed as an approximation using the antecedent “about”, it should be understood that the specific value forms another aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0058] When describing a range, another aspect includes from one specific value and / or to another specific value. For example, where the stated range includes one or both of the limit values, the range excluding any or both of those ranges that include the limit values ​​is also included in this disclosure. For instance, the phrase "x to y" includes the range from 'x' to 'y' as well as the range greater than 'x' and less than 'y'. A range can also be expressed with an upper limit, such as 'about x, y, z or less' and should be interpreted as including the specific range of 'about x', 'about y', and 'about z' as well as the range of 'less than x', 'less than y', and 'less than z'. Similarly, the phrase 'about x, y, z or greater' should be interpreted as including the specific range of 'about x', 'about y', and 'about z' as well as the range of 'greater than x', 'greater than y', and 'greater than z'. Furthermore, the phrase "about 'x' to 'y'" (where 'x' and 'y' are numerical values) includes "about 'x' to about 'y'".

[0059] It should be understood that such range patterns are used for convenience and brevity, and therefore should be interpreted flexibly to include not only the numerical values ​​explicitly stated as the limits of the range, but also all individual numerical values ​​or subranges covered within the range, as if each numerical value and subrange were explicitly stated. For the purposes of this explanation, the numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly stated values ​​of about 0.1% to about 5%, but also the individual values ​​(e.g., about 1%, about 2%, about 3%, and about 4%) and subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%; and about 0.5% to about 4.4%, and other possible subranges) within the indicated range.

[0060] As used herein, the terms “about,” “approximately,” “equal to or about,” and “substantially” mean that the quantity or value in question may be an exact value or a value that provides an equivalent result or effect to that described in the claims or taught herein. In other words, it should be understood that quantities, dimensions, expressions, parameters, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller as needed, reflecting tolerances, conversion factors, rounding, measurement errors, and other factors known to those skilled in the art that result in an equivalent result or effect. In some cases, the value providing an equivalent result or effect cannot be reasonably determined. In such cases, it should generally be understood that, unless otherwise indicated or deduced, as used herein, “about” and “equal to or about” mean a nominal value indicating a variation of ±10%. Generally, quantities, dimensions, expressions, parameters, or other quantities or characteristics are “about,” “approximately,” or “equal to or about”, whether or not explicitly stated as such. It should be understood that, unless otherwise specifically stated, when “about,” “approximately,” or “equal to or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself.

[0061] As used herein, "effective amount" can mean an amount of the disclosed compound or pharmaceutical composition provided herein sufficient to achieve a beneficial or desired biological, emotional, medical, or clinical response in cells, tissues, systems, animals, or humans. An effective amount may be administered, applied, or dosed once or multiple times. The term may also include, within its scope, amounts that effectively enhance or restore substantially normal physiological function.

[0062] As used herein, the term "therapeutic effective dose" refers to an amount sufficient to achieve the desired therapeutic outcome or to affect undesirable symptoms but generally insufficient to cause harmful side effects. The specific therapeutic effective dose level for any particular patient will depend on a variety of factors, including the condition being treated and its severity; the specific composition used; the patient's age, weight, general health, sex, and diet; the time of administration; the route of administration; the excretion rate of the specific compound used; the duration of treatment; the drugs used in combination with or concurrently with the specific compound used; and similar factors known and understood by a healthcare professional and likely familiar in the medical field. In the treatment of a particular disease or ailment, in some cases, the desired response may be to suppress the progression of the disease or ailment. This may involve only temporarily slowing the progression of the disease. However, in other cases, it may be necessary to permanently stop the progression of the disease. This can be monitored using routine diagnostic methods known to those skilled in the art for any particular disease. The desired response to the treatment of a disease or ailment may also be to delay the onset of the disease or ailment or even prevent its onset.

[0063] For example, starting the compound dose at a level below that required to achieve the desired therapeutic effect and gradually increasing the dose until the desired effect is achieved is entirely within the scope of the art. If necessary, the effective daily dose can be divided into multiple doses for administration purposes. Thus, a single-dose composition may contain such amounts or approximations to constitute the daily dose. If there are any contraindications, the dose can be adjusted by an individual physician. Generally, it is preferred to use the maximum dose of the pharmacological agent of the present invention (alone or in combination with other therapeutic agents), said maximum dose being the highest safe dose based on reasonable medical judgment. However, those skilled in the art will understand that patients may adhere to lower or tolerable doses for medical, psychological, or virtually any other reason.

[0064] The response to a therapeutically effective dose of the disclosed pharmaceutical delivery composition can be measured by determining the physiological effects of the treatment or the drug (such as a reduction or absence of disease symptoms after administration of the treatment or pharmacological agent). Other analyses will be known to those skilled in the art and may be used to measure the level of response. The amount of treatment can be altered, for example, by increasing or decreasing the amount of the disclosed compound and / or pharmaceutical composition, by changing the administered disclosed compound and / or pharmaceutical composition, by changing the route of administration, by changing the dosing schedule, and so on. The dosage can be varied and can be administered once or multiple times daily for one or more days. Guidance regarding appropriate dosages for a given class of pharmaceutical products can be found in the literature.

[0065] As used in this article, the term "preventive effective dose" refers to the amount that is effective in preventing the onset or onset of a disease or illness.

[0066] As used herein, the term "prevent / preventing" means, in particular, hindering, avoiding, eliminating, obstructing, preventing, or preventing something from happening by taking proactive measures. It should be understood that in the context of the use of reduction, suppression, or prevention, the use of two other terms is explicitly disclosed unless otherwise specified.

[0067] As used herein, the terms “optional” or “optionally” mean that the event or situation described below may or may not occur, and the description includes both the scenario in which the event or situation occurs and the scenario in which it does not occur.

[0068] As used herein, "therapeutic agent" can mean any substance, compound, molecule, etc., that may have biological activity or otherwise induce pharmacological, immunogenic, biological, and / or physiological effects in a subject administered via local and / or systemic action. A therapeutic agent can be a primary active agent, or in other words, one or more components of a composition that cause all or part of the effect of the composition. A therapeutic agent can be a secondary therapeutic agent, or in other words, one or more components of a composition that cause another part and / or other effects of the composition. Therefore, the term encompasses those compounds or chemicals conventionally considered as drugs, vaccines, and biological drugs, including molecules such as proteins, peptides, hormones, nucleic acids, and gene constructs. Examples of therapeutic agents are described in well-known references such as the Merck Index (14th edition), Physicians' Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition), and these include, but are not limited to, pharmaceutical preparations; vitamins; mineral supplements; substances intended to treat, prevent, diagnose, cure, or alleviate a disease or ailment; substances that affect the structure or function of the body, or prodrugs that become biologically active or more active after being placed in a physiological environment. For example, the term "therapeutic agent" includes compounds or compositions for use in all major therapeutic areas, including but not limited to adjuvants; anti-infective agents such as antibiotics and antiviral agents; analgesics and combinations of analgesics, appetite suppressants, anti-inflammatory agents, antiepileptic agents, local and general anesthetics, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, anxiolytics, antagonists, neuronal blocking agents, anticholinergics and cholinergic agents, antimuscarinic agents and muscarines, antiadrenergics, antiarrhythmics, antihypertensives, hormones and nutrients, antiarthritis drugs, bronchodilators, anticonvulsants, antihistamines, antinausea drugs, antitumor drugs, antipruritics, antipyretics; antispasmodics, cardiovascular agents (including calcium channel blockers, beta-blockers, beta-blockers) Agonists and antiarrhythmics, antihypertensives, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostic agents; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressants; muscle relaxants; psychotropic agents; sedatives; tranquilizers; proteins, peptides, and fragments thereof (naturally occurring, chemically synthesized, or recombinant); and nucleic acid molecules (polymeric forms of two or more nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA)), including double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules (e.g., doxorubicin), and other bioactive macromolecules such as proteins and enzymes. These agents may be bioactive agents for medical (including veterinary) applications and agricultural (e.g., plant) and other fields.The term therapeutic agent also includes, but is not limited to, pharmaceutical preparations; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure, or alleviate a disease or ailment; substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after being placed in a predetermined physiological environment.

[0069] It should be understood that the disclosure of the therapeutic agent herein also discloses pharmaceutically acceptable salts, pharmaceutically acceptable esters, pharmaceutically acceptable amides, prodrug forms, and derivatives of the therapeutic agent.

[0070] As used herein, the term "pharmaceutically acceptable salt" means a salt of the active principal agent, said salt being prepared with an acid or base and being tolerated by the biological system or by the subject when administered in a therapeutically effective amount, or both. When the compounds of this disclosure contain relatively acidic functional groups, a base addition salt can be obtained by contacting a neutral form of such a compound with an adequate amount of the desired base, either pure or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, calcium, ammonium, organic amino, magnesium, lithium, strontium, or similar salts. When the compounds of this disclosure contain relatively basic functional groups, an acid addition salt can be obtained by contacting a neutral form of such a compound with an adequate amount of the desired acid, either pure or in a suitable inert solvent. Pharmaceutically acceptable examples of acid addition salts include, but are not limited to, those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrocarbonic acid, phosphoric acid, monohydrophosphoric acid, dihydrophosphoric acid, sulfuric acid, monohydrosulfuric acid, hydroiodic acid, or phosphorous acid; and salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutylene, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid. Also included are salts of amino acids, such as arginine salts, and salts of organic acids such as glucuronic acid or galacturonic acid.

[0071] The term "pharmaceutically acceptable ester" refers to the ester of the disclosed compounds that is hydrolyzed in vivo, and includes those esters that readily decompose in the human body, leaving behind the parent compound or its salts. Examples of pharmaceutically acceptable, non-toxic esters of the disclosed invention include C1- to C6 alkyl esters and C5- to C7 cycloalkyl esters, although C1- to C4 alkyl esters are preferred. Esters of the disclosed compounds can be prepared according to conventional methods. Pharmaceutically acceptable esters can be attached to the hydroxyl group by reacting a hydroxyl-containing compound with an acid and an alkyl carboxylic acid (such as acetic acid) or with an acid and an aryl carboxylic acid (such as benzoic acid). In the case of compounds containing a carboxylic acid group, pharmaceutically acceptable esters are prepared from compounds containing a carboxylic acid group by reacting the compound with a base (such as triethylamine and alkyl halides, for example with methyl iodine, benzyl iodine, cyclopentyl iodine, or alkyl trifluoromethanesulfonate). They can also be prepared by reacting the compound with an acid (such as hydrochloric acid) and an alcohol (such as ethanol or methanol).

[0072] The term "pharmaceutically acceptable amide" refers to the non-toxic amides of this disclosure derived from ammonia, primary C1- to C6 alkylamines, and secondary C1- to C6 dialkylamines. In the case of secondary amines, the amine may also be in the form of a 5- or 6-membered heterocycle containing one nitrogen atom. Amides derived from ammonia, C1- to C3 alkyl primary amides, and C1- to C2 dialkyl secondary amides are preferred. The amides of the disclosed compounds can be prepared according to conventional methods. Pharmaceutically acceptable amides can be prepared from compounds containing primary or secondary amine groups by reacting an amino-containing compound with an alkyl anhydride, aryl anhydride, acyl halide, or aromatic acyl halide. In the case of compounds containing carboxylic acid groups, pharmaceutically acceptable amides are prepared from compounds containing carboxylic acid groups by reacting the compound with a base (such as triethylamine), a dehydrating agent (such as dicyclohexylcarbodiimide or carbonyl diimidazole), and alkylamines, dialkylamines, such as with methylamine, diethylamine, and piperidine. They can also be prepared by reacting the compounds with acids (such as sulfuric acid) and alkyl carboxylic acids (such as acetic acid) or with acids and aryl carboxylic acids (such as benzoic acid) under dehydration conditions (such as the addition of molecular sieves). The compositions may contain compounds of this disclosure in a pharmaceutically acceptable prodrug form.

[0073] The term "pharmaceutically acceptable prodrug" or "prodrug" refers to those prodrugs of the disclosed compounds that, within reasonable medical judgment, are suitable for contact with tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., meet a reasonable benefit / risk ratio, and are effective for their intended use. The prodrugs of the disclosed compounds can, for example, be rapidly converted in vivo into a parent compound having the structure of the disclosed compound by hydrolysis in the blood. Detailed discussions are provided in T. Higuchi and V. Stella, *Pro-drugs as Novel Delivery Systems*, ACSSymposium Series, Volume 14, and Edward B. Roche (ed.), *Bioreversible Carriers in Drug Design*, American Pharmaceutical Association and Pergamon Press (1987).

[0074] As used herein, "medicine box" means a collection of at least two components that constitute a medicine box. Together, the components form a functional unit for a given purpose. Individual component members may be physically packaged together or separately. For example, a medicine box containing instructions on how to use the medicine box may or may not physically include the instructions and other individual component members. Alternatively, the instructions may be supplied in paper form or in electronic form available on a computer-readable storage device or downloadable from an Internet website, or as a recorded report as individual component members.

[0075] As used herein, one or more “instruction manuals” refer to documents describing the relevant materials or methods relating to the medicine box. These materials may include any combination of the following: background information, a list of ingredients and information on their availability (purchase information, etc.), brief or detailed instructions for using the medicine box, troubleshooting, references, technical support, and any other relevant documentation. Instruction manuals may be supplied in paper form or electronic form available on a computer-readable storage device or downloadable from an internet website, or as a record report accompanying the medicine box or as a separate component. Instruction manuals may contain one or more documents and are intended to include future updates.

[0076] As used interchangeably in this document, “subject,” “individual,” or “patient” may refer to a vertebrate organism, such as a mammal (e.g., a human). “Subject” may also refer to a cell, cell population, tissue, organ, or organism, preferably a human or its constituent parts.

[0077] As used herein, the term "treating" generally refers to achieving the desired pharmacological and / or physiological effect. The effect may be, but does not necessarily have to be, preventative in the prevention or partial prevention of a disease, its symptoms, or ailment (such as an ophthalmic condition). The effect may be therapeutic in the partial or complete cure of a disease, ailment, symptom, or adverse effect attributable to a disease, condition, or symptom. As used herein, the term "treatment" may include any treatment of an ophthalmic condition in a subject (particularly a human) and may include any one or more of the following: (a) prevention of the occurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with it; (b) suppression of the disease, i.e., inhibition of its development; and (c) relief of the disease, i.e., mitigation or improvement of the disease and / or its symptoms or ailment. As used herein, the term "treatment" may refer to therapeutic treatment alone, preventative treatment alone, or both therapeutic and preventative treatment. Those subjects who require treatment (subjects in need) may include those who already have the condition and / or those who wish to prevent the condition. As used herein, the term "treatment" can include suppressing a disease, symptom, or disorder, such as hindering its progression; and alleviating a disease, symptom, or disorder, such as causing the disease, symptom, and / or disorder to subside. Treating a disease, symptom, or disorder can include improving at least one symptom of a particular disease, symptom, or disorder, even if the underlying pathophysiology is not affected, such as treating a subject's pain by administering an analgesic, although such an agent does not treat the cause of the pain.

[0078] As used herein, “dose / dosage” or “unit dose” may refer to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of the disclosed compound and / or its pharmaceutical composition calculated to produce one or more desired responses in relation to its administration.

[0079] As used in this article, "therapeutic" can mean treating, curing, and / or improving a disease, symptom, illness, or side effect, or reducing the rate of progression of a disease, symptom, illness, or side effect.

[0080] As used herein, the naming of compounds including organic compounds may use common names, IUPAC, IUBMB, or CAS nomenclature recommendations. When one or more stereochemical features are present, the Cahn-Ingold-Prelog rule for stereochemistry can be used to specify stereochemical priority, E / Z specifications, etc. If the compound structure is systematically simplified using nomenclature rules or commercially available software (such as CHEMDRAW) is used... TM (Cambridgesoft Corporation, USA) If a name is given, then those skilled in the art can easily determine the structure of the compound.

[0081] Unless otherwise specified, temperature in this document refers to atmospheric pressure (i.e., one atmosphere).

[0082] This document describes pharmaceutical delivery compositions having therapeutic or clinical efficacy. Methods for generating or preparing the disclosed pharmaceutical delivery compositions are also described. Methods for administering the disclosed pharmaceutical delivery compositions to a subject in need are further described. In some aspects, the subject may suffer from a clinical condition or lesion, such as an ophthalmic condition. Other compositions, compounds, methods, features, and advantages of this disclosure will be apparent or become apparent to those skilled in the art upon examination of the following figures, detailed descriptions, and examples. All such other compositions, compounds, methods, features, and advantages are intended to be included within the scope of this specification and disclosure.

[0083] Drug delivery composition

[0084] Vascular endothelial growth factor (VEGF) is an essential regulator of abnormal angiogenesis; it aids in rapid tumor growth and the formation of wet age-related macular degeneration (AMD) (see Holmes, DIR and I. Zachary, The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease. Genome biology, 2005.6(2): 209-209; Shibuya, M., Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis: A Crucial Target for Anti-and Pro-Angiogenic Therapies. Genes & Cancer, 2011.2(12): 1097-1105; and Ferrara, N., Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney International, 1999.56(3): 794-814). Anti-angiogenic strategies have been proposed to slow down wet AMD (see Ferrara, N. et al., Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. 2004.3(5): p.391; and Niu, G. and X. Chen, Vascular endothelial growth factor as an anti-angiogenic target for cancer therapy. Current drug targets, 2010.11(8): pp.1000-1017).Humanized monoclonal antibodies against VEGF have been used in ophthalmology for the off-label treatment of wet AMD (see Ferrara, N. et al., Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. 2004.3(5): p.391; and Presta, LG et al., Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. 1997.57(20): pp.4593-4599).

[0085] Treatment of this age-related retinal disease currently relies on the use of anti-angiogenic agents to slow or halt its progression. Intravitreal injection of anti-VEGF agents (such as bevacizumab and ranibizumab) constitutes the current gold standard therapy for wet AMD and prevents VEGF-initiated subretinal choroidal neovascularization (CNV) and irreversible retinal damage caused by hemorrhage and scarring of newly formed vessels (see Delplace, V., S. Payne and MJ Jo C.Shoichet, Delivery strategies for treatment of age-related ocular diseases: From abiological understanding to biomaterial solutions. 2015. 219: 652-668; and Ohr, M. and PKJ Eoop Kaiser, Intravitreal aflibercept injection for neovascular (wet) age-related macular degeneration. 2012. 13(4): 585-591). For example, bevacizumab has been widely used to treat wet AMD due to its relatively low cost. However, the short half-life of these protein therapeutics in the vitreous fluid often necessitates frequent, even monthly, intravitreal injections to maintain their effectiveness in the eye (see [link]). AL and AJAp On safety, pharmacokinetics and dosage of bevacizumab in ROP treatment – ​​a review. 2011. 100(12): 1523-1527; and Stewart, MW et al., Pharmacokinetic rationale for dosing every 2 weeks versus 4 weeks with intravitreal ranibizumab, bevacizumab, and aflibercept (vascular endothelial growth factor Trap-eye). Retina, 2012. 32(3): 434-457. Unfortunately, this regimen often produces side effects, including pain, infection, endophthalmitis, increased intraocular pressure, inflammation, retinal detachment, and cataract formation (see Sampat, KM and SJJ Coio Garg, Complications of intravitreal injections. 2010. 21(3): 178-183). The main barrier to treatment is the high cost associated with each injection, which places a burden on patients and families receiving monthly treatment (see Heimes, B. et al., Compliance von Patienten mit). Makuladegeneration under Anti-VEGF-Therapie. Der Ophthalmologe, 2016. 113(11): 925-932). Therefore, there is a clear need for simpler and more effective treatments for wet AMD.

[0086] Conventional delivery systems in the form of implants and particles have been developed to achieve controlled release for potential AMD treatment (see Delplace, V., S. Payne and MJ Jo C.Shoichet, Delivery strategies for treatment of age-related ocular diseases: From a biological understanding to biomaterial solutions. 2015. 219: 652-668; Radhakrishnan, K. et al., Protein delivery to the back of the eye: barriers, carriers and stability of anti-VEGF proteins. 2017. 22(2): 416-423; Imperiale, JC, GBAcosta and AJ Jo C.Shosnik, Polymer-based carriers for ophthalmic drug delivery. 2018; and Lee, SS et al., Biodegradable implants for sustained drug release in the eye. 2010. 27(10): 2043-2053). Compared to micro / nanoparticle-based systems, implants offer greater stability and drug delivery capacity due to their larger size (see Kim, YC, et al., Ocular delivery of macromolecules. Journal of Controlled Release, 2014, 190: 172-181). However, most implant-based therapies are accompanied by injection difficulties; non-biodegradable intraocular implants require additional surgery for implantation and removal (see Silva, GRd, et al., Implants as drug delivery devices for the treatment of eye diseases. Brazilian Journal of Pharmaceutical Sciences, 2010, 46: 585-595). These are associated with postoperative complications and increased costs. Furthermore, sustained long-term release from particles or implants remains challenging due to insufficient physical and chemical drug retention.For example, poly(lactic-co-glycolic acid) (PLGA), one of the most commonly used polymers for drug delivery, is characterized by rapid hydrolytic degradation, often resulting in therapeutic release over a period of up to 90 days (see Li, F. et al., Controlled release of bevacizumab through nanospheres for extended treatment of age-related macular degeneration. 2012.6: p. 54; and Sousa, F. et al., A new paradigm for antiangiogenic therapy through controlled release of bevacizumab from PLGA nanoparticles. 2017.7(1): p. 3736). Other disadvantages include the formation of acidic byproducts, which can induce inflammation and exacerbate foreign body reactions (see Lu, L., MJ Yaszemski and AGJ BMikos, Retinal pigment epithelium engineering using synthetic biodegradable polymers. 2001.22(24): pp. 3345-3355).

[0087] This paper discloses drug delivery compositions comprising injectable, biodegradable, and multilayered capsules loaded with therapeutic agents (e.g., bevacizumab), for achieving higher drug loading rates and longer drug release durations compared to conventionally available injectable drug delivery devices. To achieve highly sustained and controlled drug release, this paper discloses drug delivery compositions, for example, comprising a nanoporous PCL shell and a chitosan inner layer to achieve physical trapping and electrostatic-based chemisorption, respectively. To load sufficient therapeutic agents for long-term drug release up to at least one year, a hollow structure encapsulated by a bilayer hybrid shell is used. More specifically, the entire drug delivery composition is prepared by combining material processing techniques including electrospinning, sintering, and salt leaching. The disclosed methods provide centrally hollow cylindrical microrods with high aspect ratios to enable injection feasibility via 21-gauge or smaller needles for intravitreal implant delivery. By optimizing the chemical and physical structure of the capsule using the disclosed methods, stable and controlled protein therapeutic agent release lasting for more than ten months can be obtained using the disclosed drug delivery compositions. By reducing the frequency of injections via small needles, the disclosed drug delivery composition may potentially improve the quality of life for patients with wet AMD.

[0088] Therefore, in one aspect, a drug delivery composition comprising the following is provided:

[0089] One or more capsules, each having a tubular shape with two closed ends, wherein each of the one or more capsules independently comprises multiple layers of walls and at least one tubular compartment; and

[0090] One or more therapeutic agents, each of which is initially present in one or more of the at least one luminal compartment;

[0091] Each multi-layered wall independently contains at least an inner layer and an outer layer;

[0092] Each inner layer independently contains a first polymer that has a net positive charge under physiological conditions; and

[0093] Each outer layer independently contains a second polymer that is different from the first polymer.

[0094] In some embodiments, the drug delivery composition may comprise two or more capsules, such as two capsules, three capsules, four capsules, five capsules, six capsules, seven capsules, eight capsules, nine capsules, ten capsules, or more capsules. In such embodiments, the two or more capsules may contain the same composition or their composition may differ in the multilayered walls of each capsule. In some embodiments, the same therapeutic agent or different therapeutic agents may initially be present in each of the two or more capsules.

[0095] In some embodiments, each capsule in the drug delivery composition may independently contain two or more lumen compartments, such as two lumen compartments, three lumen compartments, four lumen compartments, or more lumen compartments. In some embodiments, the same or different therapeutic agents may initially be present in each of the two or more lumen compartments within a single capsule.

[0096] In some embodiments, each capsule independently has a length of about 0.1 cm to about 5 cm, for example, 0.5 cm to about 3 cm or 1 cm to about 3 cm. In some embodiments, each capsule independently has a length of about 0.1 cm to 5 cm, 0.5 cm to 5 cm, 1 cm to 5 cm, 2 cm to 5 cm, 3 cm to 5 cm, 4 cm to 5 cm, 0.1 cm to 4 cm, 0.5 cm to 4 cm, 1 cm to 4 cm, 2 cm to 4 cm, 3 cm to 4 cm, 0.1 cm to 3 cm, 0.5 cm to 3 cm, 1 cm to 3 cm, 2 cm to 3 cm, 0.1 cm to 2 cm, 0.5 cm to 2 cm, 1 cm to 2 cm, 0.1 cm to 1 cm, 0.5 cm to 1 cm, or 0.1 to 0.5 cm.

[0097] In some embodiments, the multilayer wall has a wall thickness of about 25 μm to about 150 μm, for example, about 70 μm to about 100 μm, about 75 μm to about 95 μm, or about 80 μm to about 90 μm. In some embodiments, the multilayer wall has a diameter of about 50 μm to 150 μm, about 55 μm to 150 μm, about 60 μm to 150 μm, about 65 μm to 150 μm, about 70 μm to 150 μm, about 75 μm to 150 μm, about 80 μm to 150 μm, about 90 μm to 150 μm, about 95 μm to 150 μm, about 100 μm to 150 μm, about 110 μm to 150 μm, about 125 μm to 150 μm, about 140 μm to 150 μm, about 50 μm to 140 μm, about 55 μm to 140 μm, about 60 μm to 140 μm, or about 65 μm to 140 μm. 0μm, approximately 70μm to approximately 140μm, approximately 75μm to approximately 140μm, approximately 80μm to approximately 140μm, approximately 90μm to approximately 140μm, approximately 95μm to approximately 140μm, approximately 100μm to approximately 140μm, approximately 110μm to approximately 140μm, approximately 125μm to approximately 140μm, approximately 50μm to 125μm, approximately 55μm to 125μm, approximately 60μm to approximately 125μm, approximately 65μm to approximately 125μm, approximately 70μm to approximately 125μm, approximately 75μm to approximately 125μm, approximately 80μm to approximately 125μm, approximately 90μm to approximately 125μm, approximately 95μm to approximately 125μm, approximately 100μm Approximately 125 μm, approximately 110 μm to approximately 125 μm, approximately 50 μm to 110 μm, approximately 55 μm to 110 μm, approximately 60 μm to approximately 110 μm, approximately 65 μm to approximately 110 μm, approximately 70 μm to approximately 110 μm, approximately 75 μm to approximately 110 μm, approximately 80 μm to approximately 110 μm, approximately 90 μm to approximately 110 μm, approximately 95 μm to approximately 110 μm, approximately 100 μm to approximately 110 μm, approximately 50 μm to 100 μm, approximately 55 μm to 100 μm, approximately 60 μm to approximately 100 μm, approximately 65 μm to approximately 100 μm, approximately 70 μm to approximately 100 μm, approximately 75 μm to approximately 100 μm, approximately 80 μm From to approximately 100 μm, from approximately 90 μm to approximately 100 μm, from approximately 95 μm to approximately 100 μm, from approximately 50 μm to 95 μm, from approximately 55 μm to 95 μm, from approximately 60 μm to approximately 95 μm, from approximately 65 μm to approximately 95 μm, from approximately 70 μm to approximately 95 μm, from approximately 75 μm to approximately 95 μm, from approximately 80 μm to approximately 95 μm, from approximately 90 μm to approximately 95 μm, from approximately 50 μm to 90 μm, from approximately 55 μm to approximately 90 μm, from approximately 60 μm to approximately 90 μm, from approximately 65 μm to approximately 90 μm, from approximately 70 μm to approximately 90 μm, from approximately 75 μm to approximately 90 μm, from approximately 80 μm to approximately 90 μm, from approximately 50 μm to 80 μm, from approximately 55 μm to 80 μmWall thicknesses ranging from approximately 60 μm to approximately 80 μm, approximately 65 μm to approximately 80 μm, approximately 70 μm to approximately 80 μm, approximately 75 μm to approximately 80 μm, approximately 50 μm to 75 μm, approximately 55 μm to 75 μm, approximately 60 μm to approximately 75 μm, approximately 65 μm to approximately 75 μm, approximately 70 μm to approximately 75 μm, approximately 50 μm to 70 μm, approximately 55 μm to 70 μm, approximately 60 μm to approximately 70 μm, approximately 65 μm to approximately 70 μm, approximately 50 μm to approximately 65 μm, approximately 55 μm to approximately 65 μm, approximately 60 μm to approximately 65 μm, approximately 50 μm to 60 μm, approximately 55 μm to 60 μm, and approximately 50 μm to approximately 55 μm.

[0098] In some embodiments, the thickness of the inner layer can vary from about 1 μm to about 100 μm. In some embodiments, the thickness of the inner layer can vary from about 100 nm to about 990 nm, for example, about 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm or 990 nm.

[0099] In some embodiments, the thickness of the outer layer may vary from about 1 μm to about 100 μm. In some embodiments, the thickness of the outer layer may vary from about 100 nm to about 990 nm, for example, about 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm or 990 nm.

[0100] In some embodiments, the tubular shape of the drug delivery capsule has an inner diameter of about 100 μm to about 1000 μm, such as about 100 μm to about 1000 μm, about 100 μm to about 500 μm, or about 100 μm to about 300 μm.In some embodiments, the tubular shape of the drug delivery capsule has a diameter of about 100 μm to about 2000 μm, 200 μm to about 2000 μm, about 300 μm to about 2000 μm, about 400 μm to about 2000 μm, about 500 μm to about 2000 μm, about 600 μm to about 2000 μm, about 700 μm to about 2000 μm, about 800 μm to about 2000 μm, about 900 μm to about 2000 μm, about 1000 μm to about 2000 μm, about 1500 μm to about 2000 μm, about 100 μm to about 1500 μm, 200 μm to about 1500 μm, about 300 μm to about 1500 μm, and about 400 μm to about 1500 μm. 0 μm, approximately 500 μm to approximately 1500 μm, approximately 600 μm to approximately 1500 μm, approximately 700 μm to approximately 1500 μm, approximately 800 μm to approximately 1500 μm, approximately 900 μm to approximately 1500 μm, approximately 1000 μm to approximately 1500 μm, approximately 100 μm to approximately 1000 μm, 200 μm to approximately 1000 μm, approximately 300 μm to approximately 1000 μm, approximately 400 μm to approximately 1000 μm, approximately 500 μm to approximately 1000 μm, approximately 600 μm to approximately 1000 μm, approximately 700 μm to approximately 1000 μm, approximately 800 μm to approximately 1000 μm, approximately 900 μm to approximately 1000 μm, approximately 100 μm to approximately 900 μm 200μm to about 900μm, about 300μm to about 900μm, about 400μm to about 900μm, about 500μm to about 900μm, about 600μm to about 900μm, about 700μm to about 900μm, about 800μm to about 900μm, about 100μm to about 800μm, 200μm to about 800μm, about 300μm to about 800μm, about 400μm to about 800μm, about 500μm to about 800μm, about 600μm to about 800μm, about 700μm to about 800μm, about 100μm to about 700μm, 200μm to about 700μm, about 300μm to about 700μm, about 400μm to about Inner diameters of 700 μm, about 500 μm to about 700 μm, about 600 μm to about 700 μm, about 100 μm to about 600 μm, 200 μm to about 600 μm, about 300 μm to about 600 μm, about 400 μm to about 600 μm, about 500 μm to about 600 μm, about 100 μm to about 500 μm, 200 μm to about 500 μm, about 300 μm to about 500 μm, about 400 μm to about 500 μm, about 100 μm to about 400 μm, 200 μm to about 400 μm, about 300 μm to about 400 μm, about 100 μm to about 300 μm, 200 μm to about 300 μm, and about 100 μm to about 200 μm.In some embodiments, the tubular shape has an outer diameter that is 100 μm to about 300 μm larger than the inner diameter, for example, about 100 μm to about 300 μm, 150 μm to about 300 μm, 200 μm to about 300 μm, about 250 μm to about 300 μm, about 100 μm to about 250 μm, about 150 μm to about 250 μm, about 200 μm to about 250 μm, about 100 μm to about 200 μm, about 150 μm to about 200 μm, or about 100 μm to about 150 μm.

[0101] In some embodiments, the first polymer may comprise chitosan, polyethyleneimine, protamine, polypropyleneimine, poly-L-lysine, poly-L-arginine, poly-D-lysine, poly-D-arginine, cellulose, dextran, poly(amidoamine), poly(2-(dimethylamino)ethyl methacrylate), derivatives thereof, or combinations thereof.

[0102] In some embodiments, the first polymer comprises chitosan or a derivative thereof. The chitosan may have a degree of deacetylation of about 60% to about 90%; at least about 70%, at least about 75%, or at least about 80%.

[0103] In some embodiments, the first polymer has a molecular weight of about 50 kDa to about 500 kDa, such as about 100 kDa to about 500 kDa, about 100 kDa to about 400 kDa, about 200 kDa to about 400 kDa, about 300 kDa to about 400 kDa, or about 310 kDa to about 375 kDa. In some embodiments, the first polymer has a molecular weight of about 10 kDa or greater, such as about 15 kDa or greater, about 20 kDa or greater, about 30 kDa or greater, about 40 kDa or greater, about 50 kDa or greater, about 60 kDa or greater, about 70 kDa or greater, about 90 kDa or greater, about 90 kDa or greater, or about 100 kDa or greater.

[0104] In some embodiments, such as the first polymer used in the inner layer, the fibers comprise fibers. In some embodiments, the fibers may have a diameter of about 50 nm to about 1000 nm, for example, about 100 nm to about 400 nm. In some embodiments, the fiber may have a diameter of about 50 nm to about 1000 nm, about 100 nm to about 1000 nm, about 200 nm to about 1000 nm, about 400 nm to about 1000 nm, about 600 nm to about 1000 nm, about 800 nm to about 1000 nm, about 50 nm to about 800 nm, about 100 nm to about 800 nm, about 200 nm to about 800 nm, about 400 nm to about 800 nm, about 600 nm to about 800 nm, about 50 nm to about 600 nm, about 100 nm to about 600 nm, about 200 nm to about 600 nm, about 400 nm to about 600 nm, about 50 nm to about 400 nm, about 100 nm to about 400 nm, about 200 nm to about 400 nm, about 50 nm to about 200 nm, about 100 nm to about 200 nm, or about 50 nm to about 100 nm.

[0105] In some embodiments, the second polymer may comprise poly(ε-caprolactone) (PCL), polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolic acid (PLGA), polyester, poly(orthoester), poly(phosphonazine), poly(phosphate), gelatin, collagen, polyethylene glycol (PEG), derivatives thereof, and combinations thereof. In other embodiments, the second polymer may comprise PLGA, PCL, PLA, PGA, PEG, polysorbate, poly(ε-caprolactone-thioethyl ethylidene phosphate) (PCLEEP), polyvinyl alcohol (PVA), or combinations thereof. In some embodiments, the second polymer comprises PLGA, PCL, PLA, PGA, or combinations thereof. In some embodiments, the second polymer may comprise PLGA, PCK, PLA, or combinations thereof. In some embodiments, the second polymer comprises PLGA. In some embodiments, the second polymer comprises PCL. In some embodiments, the second polymer comprises PLA.

[0106] In some embodiments, the second polymer has a molecular weight of about 50 kDa to about 500 kDa, such as about 100 kDa to about 500 kDa, about 100 kDa to about 400 kDa, about 200 kDa to about 400 kDa, about 300 kDa to about 400 kDa, or about 310 kDa to about 375 kDa. In some embodiments, the second polymer has a molecular weight of about 10 kDa or greater, such as about 15 kDa or greater, about 20 kDa or greater, about 30 kDa or greater, about 40 kDa or greater, about 50 kDa or greater, about 60 kDa or greater, about 70 kDa or greater, about 90 kDa or greater, about 90 kDa or greater, or about 100 kDa or greater.

[0107] In some embodiments, the second polymer is biodegradable in vivo and is fully tolerated throughout the presence of the composition and the duration of degradation. In some embodiments, under physiological conditions, the second polymer degrades via random chain scission, which causes a two-stage degradation process. Initially, the physical structure is not significantly affected as the molecular weight decreases. Degradation occurs in the polymer material and proceeds until a critical molecular weight is reached, at which point the degradation products become small enough to dissolve. At this point, the structure begins to become significantly more porous and hydration occurs. In some embodiments, the second polymer has a molecular weight of about 90 kDa or greater and remains undegraded from the subject's perspective until 6 months or longer. In some embodiments, the molecular weight of the biodegradable polymer is selected to adjust the degradation time of the material in vivo.

[0108] In some embodiments, the second polymer may comprise a blend of a high molecular weight polymer and a low molecular weight polymer. In some embodiments, the high molecular weight polymer may be about 25 kDa or greater (e.g., about 30 kDa or greater, 40 kDa or greater, 50 kDa or greater, 60 kDa or greater, 70 kDa or greater, 80 kDa or greater, 90 kDa or greater, or 100 kDa or greater), and the low molecular weight polymer may be about 20 kDa or greater (e.g., 15 kDa or less, 10 kDa or less, 8 kDa or less, 6 kDa or less, or 4 kDa or less). In some embodiments, the ratio of the high molecular weight polymer to the low molecular weight polymer is between about 1:9 and about 9:1, for example, between about 2:8 and about 8:2, between about 2:8 and about 6:4, or between about 2:8 and about 1:1.

[0109] In some embodiments, the outer layer of the second polymer used in the outer layer comprises fibers. In some embodiments, the fibers may have a diameter of about 100 nm to about 2000 nm, for example, about 500 nm to about 1000 nm. In some embodiments, the fibers may have diameters of about 100 nm to about 2000 nm, about 250 nm to about 2000 nm, about 500 nm to about 2000 nm, about 750 nm to about 2000 nm, about 1000 nm to about 2000 nm, about 1500 nm to about 2000 nm, about 100 nm to about 1500 nm, about 250 nm to about 1500 nm, about 500 nm to about 1500 nm, and about 750 nm to about 1500 nm. The diameter of approximately 1000 nm to approximately 1500 nm, approximately 100 nm to approximately 1000 nm, approximately 250 nm to approximately 1000 nm, approximately 500 nm to approximately 1000 nm, approximately 750 nm to approximately 1000 nm, approximately 100 nm to approximately 750 nm, approximately 250 nm to approximately 750 nm, approximately 500 nm to approximately 750 nm, approximately 100 nm to approximately 500 nm, approximately 250 nm to approximately 500 nm, or approximately 100 nm to approximately 250 nm.

[0110] In some embodiments, the outer layer may further include pores. In other embodiments, the outer layer does not include pores. In some embodiments, the outer layer includes pores with an average pore diameter of about 1 nm to about 990 nm, such as about 1 nm to about 100 nm, about 2 nm to about 700 nm, about 3 nm to about 400 nm, about 5 nm to about 200 nm, or about 7 nm to about 50 nm. In some embodiments, the outer layer includes pores with an average pore diameter of about 100 nm to 1000 nm, such as 350 nm to 650 nm. In some embodiments, the outer layer comprises an average pore diameter of about 100 nm to about 1000 nm, 200 nm to about 1000 nm, 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 450 nm to about 1000 nm, about 500 nm to about 1000 nm, about 550 nm to about 1000 nm, about 600 nm to about 1000 nm, about 650 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, about 900 nm to about 1000 nm, or about 100 nm. Approximately 900nm, 200nm to approximately 900nm, 300nm to approximately 900nm, approximately 400nm to approximately 900nm, approximately 450nm to approximately 900nm, approximately 500nm to approximately 900nm, approximately 550nm to approximately 900nm, approximately 600nm to approximately 900nm, approximately 650nm to approximately 900nm, approximately 700nm to approximately 900nm, approximately 800nm ​​to approximately 900nm, approximately 100nm to approximately 800nm, 200nm to approximately 800nm, 300nm to approximately 800nm, approximately 400nm to approximately 800nm, approximately 450nm m to approximately 800nm, approximately 500nm to approximately 800nm, approximately 550nm to approximately 800nm, approximately 600nm to approximately 800nm, approximately 650nm to approximately 800nm, approximately 700nm to approximately 800nm, approximately 100nm to approximately 700nm, 200nm to approximately 700nm, 300nm to approximately 700nm, approximately 400nm to approximately 700nm, approximately 450nm to approximately 700nm, approximately 500nm to approximately 700nm, approximately 550nm to approximately 700nm, approximately 600nm to approximately 700nm, approximately 650nm to approximately 700nm, approximately 100nm to approximately 650nm, 200nm to approximately 650nm, 300nm to approximately 650nm, approximately 400nm to approximately 650nm, approximately 450nm to approximately 650nm, approximately 500nm to approximately 650nm, approximately 550nm to approximately 650nm, approximately 600nm to approximately 650nm, approximately 100nm to approximately 600nm, 200nm to approximately 600nm, 300nm to approximately 600nm, approximately 400nm to approximately 600nm, approximately 450nm to approximately 600nm, approximately 500nm to approximately 600nm, approximately 550nm to approximately 600nm,Pores of approximately 100 nm to approximately 550 nm, 200 nm to approximately 550 nm, 300 nm to approximately 550 nm, approximately 400 nm to approximately 550 nm, approximately 450 nm to approximately 550 nm, approximately 500 nm to approximately 550 nm, approximately 100 nm to approximately 500 nm, 200 nm to approximately 500 nm, 300 nm to approximately 500 nm, approximately 400 nm to approximately 500 nm, approximately 450 nm to approximately 500 nm, approximately 100 nm to approximately 450 nm, 200 nm to approximately 450 nm, 300 nm to approximately 450 nm, approximately 400 nm to approximately 450 nm, approximately 100 nm to approximately 400 nm, 200 nm to approximately 400 nm, 300 nm to approximately 400 nm, approximately 100 nm to approximately 300 nm, approximately 200 nm to approximately 300 nm, and approximately 100 nm to approximately 200 nm. In some embodiments, the average pore size is similar to the size of the therapeutic agent, allowing one or more therapeutic agents to diffuse via single-row diffusion or hindered diffusion (through nanopores). Porosity may not be necessary when the desired therapeutic agent has a sufficiently small size (e.g., a molecular weight less than 500) to allow it to diffuse easily through the outer layer of the capsule.

[0111] In some embodiments, the composition of the first or second polymer can provide a melting temperature between about 50°C and about 70°C. In some embodiments, the composition of the first or second polymer is selected to provide a glass transition temperature (Tg) between about -50°C and about -80°C. g ).

[0112] In some embodiments, each of one or more capsules may independently have a surface charge that is measured to be about -25 mV to about 25 mV at pH 7.5, such as about -20 mV to about 20 mV, about -15 mV to about 15 mV, about -10 mV to about 10 mV, about -5 mV to about 5 mV, about -1 mV to about 1 mV, about -0.5 mV to about 0.5 mV, or about -0.1 mV to about 0.1 mV.

[0113] In some embodiments, the composition of the first and second polymers is selected such that, upon exposure to physiological conditions, 50% of the mass of one or more of the layers remains after at least three months. Ideally, the degradation rate of one or more of the layers can be accelerated by adjusting aspects such as layer thickness or porosity during capsule manufacturing or by increasing the hydrophilicity of the polymer composition used to manufacture one or more layers.

[0114] Therapeutic agents

[0115] In another aspect, this disclosure also provides one or more therapeutic agents that can be used in the compositions disclosed herein.

[0116] In some implementations, one or more therapeutic agents each have a net negative charge in a pH range of about 6.0 to about 7.4.

[0117] As used herein, "therapeutic agent" means one or more therapeutic agents, active ingredients, or substances that can be used to treat medical conditions or cancers of the eye. Therapeutic agents are typically ophthalmologically acceptable and provided in a form that does not cause adverse reactions when the compositions disclosed herein are placed in the eye. As discussed herein, therapeutic agents can be released from the disclosed compositions in a biologically active form. For example, a therapeutic agent may retain its three-dimensional structure when released from a system into the eye.

[0118] It should also be understood that, as used herein, the term "therapeutic agent" includes any synthetic or naturally occurring bioactive compound or composition of substances that, when administered to an organism (human or non-human animal), induce desired pharmacological, immunogenic, and / or physiological effects through local and / or systemic action. Therefore, the term encompasses those compounds or chemicals traditionally considered as pharmaceuticals, vaccines, and biological drugs, including molecules such as proteins, peptides, hormones, nucleic acids, and gene constructs. Examples of therapeutic agents are described in well-known references such as the Merck Index (14th edition), Physicians' Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition), and these include, but are not limited to, pharmaceutical preparations; vitamins; mineral supplements; substances intended to treat, prevent, diagnose, cure, or alleviate diseases or ailments; substances affecting the structure or function of the body; or prodrugs that become more bioactive or more active after being placed in a physiological environment. For example, the term "therapeutic agent" includes compounds or compositions for use in all major therapeutic areas, including but not limited to adjuvants; anti-infective agents such as antibiotics and antiviral agents; analgesics and combinations of analgesics, appetite suppressants, anti-inflammatory agents, antiepileptic agents, local and general anesthetics, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, anxiolytics, antagonists, neuronal blocking agents, anticholinergics and cholinergic-like agents, antimuscarinic agents and muscarines, antiadrenergics, antiarrhythmics, antihypertensives, hormones and nutrients, antiarthritis drugs, bronchodilators, anticonvulsants, antihistamines, antinausea drugs, antitumor drugs, antipruritics, antipyretics; antispasmodics, cardiovascular agents (including calcium channel blockers, beta-blockers, etc.) - Blockers, beta-agonists, and antiarrhythmics; antihypertensives, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostic agents; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressants; muscle relaxants; psychotropic agents; sedatives; tranquilizers; proteins, peptides, and fragments thereof (naturally occurring, chemically synthesized, or recombinant); and nucleic acid molecules (polymers of two or more nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA)), including double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules (e.g., doxorubicin) and other bioactive macromolecules, such as proteins and enzymes. These agents may be bioactive agents for medical (including veterinary) applications and agricultural (e.g., plant) and other fields. The term therapeutic agent also includes, but is not limited to, pharmaceutical preparations; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure, or alleviate a disease or ailment; substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after being placed in a predetermined physiological environment.

[0119] In some implementations, the therapeutic agent may comprise agents that can be used to treat ophthalmic conditions or eye diseases, such as: beta-blockers, including timolol, betaxolol, levobetalolol, and carteolol; miotics, including pilocarpine; carbonic anhydrase inhibitors; serotonergic agents; muscarines; dopaminergic agonists; adrenergic agonists, including araclonidine and brimonidine; anti-angiogenic agents; and anti-infective agents, including quinolones (such as ciprofloxacin) and aminoglycosides (such as...). Examples of anti-inflammatory agents include tobramycin and gentamicin; nonsteroidal and steroidal anti-inflammatory agents such as suprofen, diclofenac, ketorolac, rimexolone, and tetrahydrocortisol; growth factors such as EGF; immunosuppressants; and anti-allergic agents, including olopatadine; prostaglandins such as latanoprost, 15-ketolatanoprost, travoprost, and unoprostone isopropyl.

[0120] In some embodiments, the therapeutic agent is selected from the group consisting of: anti-inflammatory agents, calcineurin inhibitors, antibiotics, nicotinic acetylcholine receptor agonists, and anti-lymphangiogenic agents. In some embodiments, the anti-inflammatory agent may be cyclosporine. In some embodiments, the calcineurin inhibitor may be voclosporin. In some implementations, the antibiotic may be selected from the group consisting of: amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, teicoplanin, vancomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, acetylenolmycin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, penicillin (p Piperacillin, ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin ), sulfamethizole, sulfamethizole, sulfasalazine, sulfisoxazole, trimethoprim, cotrimoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline.In some embodiments, the nicotinic acetylcholine receptor agonist may be any one of pilucarpine, atropine, nicotine, scutellarin, lobeline, or imidacloprid. In some embodiments, the anti-lymphangiogenic agent may be a vascular endothelial growth factor C (VEGF-C) antibody, a VEGF-D antibody, or a VEGF-3 antibody.

[0121] In some respects, the therapeutic agents may be selected from: beta-blockers, including levobunolol (BETAGAN), timolol (BETIMOL, TIMOPTIC), betoprolol (BETOPTIC), and metipranolol (OPTIPRANOLOL); alpha-agonists, such as iodine and brimonidine (ALPHAGAN); carbonic anhydrase inhibitors, such as acetazolamide, methazolamide, dorzolamide (TRUSOPT), and brinzolamide (AZOPT); prostaglandins or prostaglandin analogs, such as latanoprost (XALATAN), bimatoprost (LUMIGAN), and travoprost (TRAVATAN); miotics or cholinergic agents, such as pilocrine (ISOPTO CARPINE, PILOPINE) and carbachol (ISOPTO CARPINE, PILOPINE). CARBACHOL; adrenaline compounds such as dipivefrin (PROPINE); forskolin; or neuroprotective compounds such as brimonidine and memantine; steroid derivatives such as 2-methoxyestradiol or its analogues or derivatives; or antibiotics.

[0122] The term “VEGF” refers to vascular endothelial growth factor that induces angiogenesis or the angiogenic process, including but not limited to increased permeability. As used herein, the term “VEGF” includes various isoforms of VEGF (also known as vascular permeability factor (VPF) and VEGF-A) generated through alternative splicing of genes such as VEGF-A / VPF, including VEGF121, VEGF165, and VEGF189. Additionally, as used herein, the term “VEGF” includes VEGF-related angiogenic factors that act through a homologous VEFG receptor (i.e., VEGFR) to induce angiogenesis or the angiogenic process, such as PIGF (placental growth factor), VEGF-B, VEGF-C, VEGF-D, and VEGF-E. The term “VEGF” includes any member of a growth factor class that binds to VEGF receptors such as VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flk-1), or VEGFR-3 (FLT-4)). The term “VEGF” can be used to refer to the “VEGF” polypeptide or the “VEGF”-encoded gene or nucleic acid.

[0123] The term "anti-VEGF agent" refers to an agent that reduces or partially or completely inhibits the activity or production of VEGF. Anti-VEGF agents can directly or indirectly reduce or inhibit the activity or production of specific VEGFs (such as VEGF165). Furthermore, "anti-VEGF agent" includes agents that act on VEGF ligands or their homologous receptors to reduce or inhibit VEGF-related receptor signaling. Non-limiting examples of "anti-VEGF agents" include antisense molecules, ribozymes, or RNAi targeting VEGF nucleic acids; anti-VEGF aptamers, anti-VEGF antibodies against VEGF itself or its receptors, or soluble VEGF receptor decoys that prevent VEGF from binding to its homologous receptors; antisense molecules, ribozymes, or RNAi targeting homologous VEGF receptor (VEGFR) nucleic acids; anti-VEGFR aptamers or anti-VEGFR antibodies that bind to homologous VEGFR receptors; and VEGFR tyrosine kinase inhibitors.

[0124] In some implementations, the therapeutic agent may comprise an anti-VEGF agent. Representative examples of anti-VEGF agents include ranibizumab, bevacizumab, aflibercept, KH902 VEGF receptor-Fc, fusion protein, 2C3 antibody, ORA102, pegaptanib, bevasiranib, siRNA-027, decursin, decursinol, picropodophyllin, guggulsterone, PLG101, arachidic acid LXA4, PTK787, pazopanib, and axiti. The following are examples of anti-VEGF agents that can be used in the methods of this invention: sorafenib, CDDO-Me, CDDO-Imm, shikonin, β-hydroxyisovalerylshikonin, ganglioside GM3, DC101 antibody, Mab25 antibody, Mab73 antibody, 4A5 antibody, 4E10 antibody, 5F12 antibody, VA01 antibody, BL2 antibody, VEGF-related protein, sFLT01, sFLT02, peptide B3, TG100801, sorafenib, G6-31 antibody, fusion antibodies, and antibodies that bind to epitopes of VEGF. Other non-limiting examples of anti-VEGF agents that can be used in the methods of this invention include substances that specifically bind to one or more of human vascular endothelial growth factor-A (VEGF-A), human vascular endothelial growth factor-B (VEGF-B), human vascular endothelial growth factor-C (VEGF-C), human vascular endothelial growth factor-D (VEGF-D), and human vascular endothelial growth factor-E (VEGF-E), and antibodies that bind to epitopes of VEGF.

[0125] In each embodiment, the anti-VEGF agent is the antibody ranibizumab or a pharmaceutically acceptable salt thereof. Ranibizumab is commercially available under the trademark LUCENTIS. In another embodiment, the anti-VEGF agent is the antibody bevacizumab or a pharmaceutically acceptable salt thereof. Bevacizumab is commercially available under the trademark AVASTIN. In another embodiment, the anti-VEGF agent is aflibercept or a pharmaceutically acceptable salt thereof. Aflibercept is commercially available under the trademark EYLEA. In one embodiment, the anti-VEGF agent is piperacanib or a pharmaceutically acceptable salt thereof. Piperacanib is commercially available under the trademark MACUGEN. In another embodiment, the anti-VEGF agent is an antibody or antibody fragment that binds to an epitope of VEGF, such as VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E. In some embodiments, the VEGF antagonist binds to the epitope of VEGF such that the binding of VEGF and VEGFR is inhibited. In one embodiment, the epitope encompasses components of the three-dimensional structure of the VEGF, such that the epitope is exposed on the surface of the folded VEGF molecule. In one embodiment, the epitope is a linear amino acid sequence derived from VEGF.

[0126] In various aspects, the therapeutic agent may comprise an agent that blocks or inhibits VEGF-mediated activity, such as one or more VEGF antisense nucleic acids. This disclosure provides therapeutic or prophylactic uses of nucleic acids comprising at least six nucleotides that are antisense to a gene or cDNA encoding VEGF or a portion thereof. As used herein, a VEGF “antisense” nucleic acid refers to a nucleic acid capable of hybridizing to a portion of RNA (preferably mRNA) encoding VEGF by means of sequence complementarity. The antisense nucleic acid may be complementary to the coding and / or non-coding regions of the mRNA encoding VEGF. Such antisense nucleic acids have the efficacy of compounds that prevent VEGF expression and can be used to treat diabetes. The antisense nucleic acids of this disclosure are double-stranded or single-stranded oligonucleotides, RNA or DNA, or modified forms or derivatives thereof, and can be administered directly to cells or produced intracellularly through transcription of an exogenously introduced sequence.

[0127] VEGF antisense nucleic acids have at least six nucleotides, and preferably oligonucleotides in the range of 6 to 50. In certain aspects, the oligonucleotides are at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified forms thereof, and can be single-stranded or double-stranded. Furthermore, antisense molecules can be polymers that serve as nucleic acid mimics, such as PNA, morpholino oligonucleotides, and LNA. Other types of antisense molecules include short double-stranded RNA (called siRNA) and short hairpin RNA, as well as long dsRNA (>50 bp, but typically ≥500 bp).

[0128] In various aspects, therapeutic agents may contain one or more ribozyme molecules designed to catalyze the cleavage of gene mRNA transcripts encoding VEGF, thereby preventing the translation of target gene mRNA and thus preventing the expression of gene products.

[0129] Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to a complementary target RNA, followed by an endonuclease cleavage event. The composition of the ribozyme molecule must include one or more sequences complementary to the target gene mRNA and must include a well-known catalytic sequence responsible for mRNA cleavage. For this sequence, see, for example, U.S. Patent No. 5,093,246. While ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy mRNA encoding VEGF, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNA at a location indicated by a flanking region that forms a complementary base pair with the target mRNA. The only requirement is that the target mRNA has the following two-base sequence: 5′-UG-3′. The construction and preparation of hammerhead ribozymes are well known in the art. The ribozymes disclosed herein also include RNA endonucleases (hereinafter referred to as "Cech-type ribozymes"), such as the RNA endonuclease (called IVS, or L-19IVS RNA) naturally found in Tetrahymena thermophila. Cech-type ribozymes have an eight-base-pair active site that hybridizes to the target RNA sequence after cleavage of the target RNA. This disclosure covers those Cech-type ribozymes that target the eight-base-pair active site sequence present in genes encoding VEGF.

[0130] In other respects, the therapeutic agent may contain antibodies that inhibit VEGF, such as bevacizumab or ranibizumab. In other respects, the therapeutic agent may contain agents that inhibit VEGF activity, such as VEGF-stimulated tyrosine kinases, examples of which include, but are not limited to, lapatinib, sunitinib, sorafenib, axitinib, and pazopanib.

[0131] The term "anti-RAS agent" or "anti-renin-angiotensin system agent" refers to an agent that reduces or partially or completely inhibits the activity or production of molecules of the renin-angiotensin system (RAS). Non-limiting examples of "anti-RAS" or "anti-renin-angiotensin system" molecules include one or more of angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, and renin inhibitors.

[0132] In some embodiments, the therapeutic agent may comprise a renin-angiotensin system (RAS) inhibitor. In some embodiments, the renin-angiotensin system (RAS) inhibitor is one or more of angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, and renin inhibitors.

[0133] Non-limiting examples of angiotensin-converting enzyme (ACE) inhibitors that can be used in this invention include, but are not limited to: alacepril, alatriopril, altiopril calcium, ancovenin, benazepril, and benazepril hydrochloride. hydrochloride, benazeprilat, benazepril, benzoylcaptopril, captopril, captopril, captopril cysteine, captopril glutathione, ceranapril, ceranopril, ceronapril, cilazapril, cilazaprilat, convergestatin, delapril, delapric acid, enalapril, enalaprilat, enalkiren, enalapril Naphthyl (enapril), Epicaptopril, Foroxymithine, Fosfenopril, Fosenopril, Fosenopril Sodium, Fosinopril, Fosinopril Sodium, Fosinoprilat, Fosinopril Acid, Glycopril, Hemorphin-4, Itrapril, Imidapril, Indolapril, Indolaprilat, Libenzapril, Lisinopril, Lyciumin A) Lycium barbarum cyclic octapeptide B, Mixanpril, Moexipril, Moexiprilat, Movepril, Muracein AA) Cell wall pigment B) Cell wall pigment C) Pentopril, Perindopril, Perindoprilat, Pivalopril, Pivopril, Quinapril, Quinapril hydrochloride, Quinaprilat, Ramipril, Ramiprilat, Spirapril, Spirapril hydrochloride, Spirapril rilat), spiropril, spiropril hydrochloride, temocapril, temopril hydrochloride, teprotide, trandolapril, trandolaprilat, utibapril, zabicipril, zabiciprilat, zofenopril, zofenoprilat, their pharmaceutically acceptable salts, and mixtures thereof.

[0134] Non-limiting examples of angiotensin receptor blockers that can be used in this invention include, but are not limited to: irbesartan (U.S. Patent No. 5,270,317, incorporated herein by reference in its entirety), candesartan (U.S. Patent Nos. 5,196,444 and 5,705,517, incorporated herein by reference in their entirety), valsartan (U.S. Patent No. 5,399,578, incorporated herein by reference in its entirety), and losartan (U.S. Patent No. 5,138,069, incorporated herein by reference in its entirety).

[0135] Non-limiting examples of renin inhibitors that can be used as therapeutic agents include, but are not limited to: aliskiren, deteskiren, etanerskiren, remikiren, terlakiren, ciprokiren, and zankiren, pharmaceutically acceptable salts thereof, and mixtures thereof.

[0136] The term "steroid" refers to compounds that belong to or are associated with the following illustrative compound families: corticosteroids, mineralosteroids, and sex steroids (including, for example, potentially androgenic or estrogenic or antiandrogenic and antiestrogenic molecules). These substances include, for example, prednisone, prednisolone, methylprednisolone, triamcinolone, fluocinolone, aldosterone, spironolactone, danazol (or OPTINA), and other substances. In some embodiments, the therapeutic agent may comprise a steroid.

[0137] The terms "peroxisome proliferator-activated receptor gamma agent," "PPAR-γ agent," "PPARG agent," or "PPAR-γ agent" refer to agents that act directly or indirectly on the peroxisome proliferator-activated receptor. These agents can also affect the activity of PPAR-α, or "PPARA."

[0138] In some embodiments, the therapeutic agent may comprise a regulator of macrophage polarization. Illustrative macrophage polarization regulators include peroxisome proliferator-activated receptor γ (PPAR-g) regulators, including, for example, agonists, partial agonists, antagonists, or combinations of PPAR-γ / α agonists. In some embodiments, the therapeutic agent may comprise a PPARγ regulator, including a PPARγ regulator as a full agonist or a partial agonist. In some embodiments, the PPARγ regulator is a member of the pharmaceutical class of thiazolidinediones (TZDs or glitazones). By non-limiting example, the PPARγ regulator may be one or more of rosiglitazone (AVANDIA), pioglitazone (ACTOS), troglitazone (REZULIN), napoglitazone, rivoglitazone, ciglitazone, and rhodanine. In some embodiments, the PPARγ modulator is one or more of ibassartan and telmesartan. In some embodiments, the PPARγ modulator is a nonsteroidal anti-inflammatory drug (NSAID, such as ibuprofen) or indole. Known inhibitors include the experimental agent GW-9662. Other examples of PPARγ modulators are described in WIPO publications WO / 1999 / 063983, WO / 2001 / 000579, Nat Rev Immunol. 25 Oct 2011; 11(11):750-61 or are agents identified using the method of WO / 2002 / 068386, the contents of which are incorporated herein by reference in their entirety.

[0139] In some embodiments, the PPARγ modulator is a "bi" or "balanced" or "full" PPAR modulator. In some embodiments, the PPARγ modulator is glitazar, which is combined with two or more PPAR isoforms, such as muraglitazar (Pargluva), tesaglitazar (Galida), and alglitazar.

[0140] In some implementations, the therapeutic agent may include semapimod (CNI-1493) as described in Bianchi et al. (March 1995). Molecular Medicine (Cambridge, Mass.) 1(3):254-266, the contents of which are incorporated herein by reference in their entirety.

[0141] In some embodiments, the therapeutic agent may comprise a migration inhibitory factor (MIF) inhibitor. Illustrative MIF inhibitors are described in WIPO Publications WO2003 / 104203, WO 2007 / 070961, WO 2009 / 117706 and U.S. Patents 7,732,146, 7,632,505, and 7,294,753, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the MIF inhibitor is (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazolyl acetate (ISO-1), isoxazoline, p425 (J. Biol. Chem., 287, 30653-30663), neem-3-oxadione, or vitamin E.

[0142] In some embodiments, the therapeutic agent may comprise, for example, an inhibitor of chemokine receptor 2 (CCR2) as described in U.S. Patent Nos. 7,799,824, 8,067,415, 2007 / 0197590, 2006 / 0069123, 2006 / 0058289, and 2007 / 0037794, the contents of which are incorporated herein by reference in their entirety. In some implementations, the CCR2 inhibitors are Maraviroc, Cenicriviroc, CD192, CCX872, CCX140, 2-((isopropylaminocarbonyl)amino)-N-(2-((cis-2-((4-(methylthio)benzoyl)amino)-cyclohexyl)amino)-2-oxoethyl)-5-(trifluoromethyl)-benzamide, vicriviroc, SCH351125, TAK779, Teijin, RS-504393, compound 2, compound 14, or compound 19 (Plos ONE 7(3): e32864).

[0143] In some embodiments, the therapeutic agent may comprise an agent that modulates autophagy, microautophagy, mitophagy, or other forms of autophagy. In some embodiments, the therapeutic agent may comprise sirolimus, tacrolimus, rapamycin, everolimus, bafilomycin, chloroquine, hydroxychloroquine, spautin-1, metformin, perifosine, resveratrol, trichostocin, valproic acid, Z-VAD-FMK, or other substances known to those skilled in the art. Not wishing to be limited by theory, the agent that modulates autophagy, microautophagy, mitophagy, or other forms of autophagy may alter the recycling of intracellular components (e.g., but not limited to organelles, mitochondria, endoplasmic reticulum, lipids, or other components). Furthermore, not wishing to be limited by theory, this agent may act via or may not act via microtubule-associated protein 1A / 1B-light chain 3 (LC3).

[0144] In some embodiments, the therapeutic agent may comprise an agent for treating cancer, i.e., a cancer drug or anticancer agent. Exemplary cancer drugs may be selected from antimetabolite anticancer agents and antimitotic anticancer agents, as well as combinations thereof, administered to a subject. A variety of antimetabolites and antimitotic anticancer agents (including single such agents or combinations thereof) may be used in the methods and compositions described herein.

[0145] Antimetabolites are typically structurally similar to natural metabolites involved in normal metabolic processes in cancer cells, such as nucleic acid and protein synthesis. However, antimetabolites differ from natural metabolites sufficiently to interfere with these processes. In cells, antimetabolites are mistaken for similar metabolites and processed by the cell in a manner similar to normal compounds. The presence of these "decoy" metabolites prevents the cell from performing vital functions, and the cell cannot grow or survive. For example, antimetabolites can disrupt cell division by replacing these deceptive nucleotides into cellular DNA, or exert cytotoxic activity by inhibiting key cellular enzymes that prevent DNA replication.

[0146] Therefore, in one aspect, antimetabolite anticancer agents are nucleotides or nucleotide analogs. In other aspects, for example, with or without a linked sugar moiety, antimetabolite agents may contain purines (e.g., guanine or adenosine) or analogs thereof, or pyrimidines (cytidine or thymidine) or analogs thereof.

[0147] Suitable antimetabolite anticancer agents for use in this disclosure can be broadly classified according to the metabolic processes they affect, and may include, but are not limited to, analogues and derivatives of folic acid, pyrimidine, purine, and cytidine. Thus, in one aspect, one or more antimetabolite agents are selected from the group consisting of cytidine analogues, folic acid analogues, purine analogues, pyrimidine analogues, and combinations thereof.

[0148] In one particular aspect, for example, the antimetabolite is a cytidine analogue. According to this aspect, for example, cytidine analogues can be selected from the group consisting of: cytarabine (cytosine arabinoside), azacitidine (5-azacytidine), and their salts, analogues, and derivatives.

[0149] In another specific aspect, for example, antimetabolites are folic acid analogs. Folic acid analogs, or anti-folate agents, typically function by inhibiting dihydrofolate reductase (DHFR), an enzyme involved in nucleotide formation; when this enzyme is blocked, nucleotides are not formed, thereby disrupting DNA replication and cell division. According to some aspects, for example, folic acid analogs can be selected from the group consisting of: folate, methotrexate, pemetrexed, pteroxate, raltitrexed, trimetrexate, and their salts, analogs, and derivatives.

[0150] In another specific aspect, for example, antimetabolites are purine analogues. Purine-based antimetabolites function by inhibiting DNA synthesis, for example by interfering with the preparation of purine-containing nucleotides, adenine, and guanine (which prevents DNA synthesis and thus cell division). Purine analogues can also be incorporated into the DNA molecule itself during DNA synthesis, which can hinder cell division. According to some aspects, for example, purine analogues can be selected from the group consisting of: acyclovir, allopurinol, 2-aminoadenosine, ara-A, azacitidine, azathioprine, 8-aza-adenosine, 8-fluoroadenosine, 8-methoxyadenosine, 8-oxoadenosine, cladribine, deoxycoformycin, fludarabine (… fludarabine), ganciclovir, 8-aza-guanosine, 8-fluoro-guanosine, 8-methoxy-guanosine, 8-oxo-guanosine, guanosine diphosphate, guanosine diphosphate-β-L-2-aminotrehalose, guanosine diphosphate-D-arabinose, guanosine diphosphate-2-fluorotrehalose, guanosine diphosphate trehalose, mercaptopurine (6-MP), pentostatin, thioimidapurine, thioguanine (6-TG), and their salts, analogues, and derivatives.

[0151] In another specific aspect, for example, antimetabolites are pyrimidine analogues. Similar to the purine analogues discussed above, pyrimidine-based antimetabolites block the synthesis of pyrimidine-containing nucleotides (cytosine and thymine in DNA; cytosine and uracil in RNA). By acting as "decoys," pyrimidine-based compounds can prevent the preparation of nucleotides and / or can be incorporated into the growing DNA strand and cause its termination. Depending on certain aspects, for example, pyrimidine analogues may be selected from the group consisting of: cyclocytidine, azacitidine, 6-azouridine, bromouracil (e.g., 5-bromouracil), capecitabine, carmofur, chlorouracil (e.g., 5-chlorouracil), cytarabine (cytosine arabinoside), cytosine, dideoxyuridine, 3′-azido-3′-deoxythymidine, 3′-dideoxycytidine-2′-ene, 3′-deoxy-3′-deoxythymidine-2′-ene, dihydrouracil, deoxyfluorouridine, and enoxaflutole. Enocitabine, fluorouridine, 5-fluorocytosine, 2-fluorodeoxycytosine, 3-fluoro-3′-deoxythymidine, fluorouracil (e.g., 5-fluorouracil (also known as 5-FU), gemcitabine), 5-methylcytosine, 5-propynylcytosine, 5-propynylthymidine, 5-propynyluracil, thymidine, uracil, uridine, and their salts, analogs, and derivatives. In one aspect, the pyrimidine analog is not 5-fluorouracil. In another aspect, the pyrimidine analog is gemcitabine or a salt thereof.

[0152] In some aspects, the antimetabolite is selected from the group consisting of: 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and their salts, analogs, derivatives, and combinations thereof. In other aspects, the antimetabolite is selected from the group consisting of: capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and their salts, analogs, derivatives, and combinations thereof. In a particular aspect, the antimetabolite is not 5-fluorouracil. In a particularly preferred aspect, the antimetabolite is gemcitabine or a salt thereof (e.g., gemcitabine hydrochloride). ).

[0153] Other antimetabolite anticancer agents may be selected from, but are not limited to, the group consisting of: acanthifolic acid, aminothiadiazole, brequinar sodium, Ciba-Geigy CGP-30694, cyclopentylcytosine, cytarabine phosphate stearate, cytarabine conjugates, Lilly DATHF, MerrelDow DDFC, dezaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, Wellcome EHNA, Merck & Co. EX-015, fazarabine, fludarabine phosphate, N-(2′-furanyl)-5-fluorouracil, Daiichi Seiyaku FO-152, 5-FU-fibrinogen, isopropylpyrrolizine, Lilly LY-188011; Lilly LY-264618, methobenzaprim, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, tiazofurin, Erbamont TIF, tyrosine kinase inhibitors, Taiho UFT, and uricytin, etc.

[0154] In one aspect, the antimitotic agent is a microtubule inhibitor or a microtubule stabilizer. Generally, microtubule stabilizers (such as taxanes and epothilones) bind to the inner surface of the β-microtubule chain and enhance microtubule assembly by promoting the nucleation and elongation phases of polymerization and by reducing the concentration of key tubulin subunits required for microtubule assembly. Unlike microtubule inhibitors (such as vinca alkaloids) that inhibit microtubule assembly, microtubule stabilizers (such as taxanes) reduce hysteresis time and significantly alter the dynamic balance toward polymerization between tubulin dimers and microtubule polymers. Therefore, in one aspect, the microtubule stabilizer is a taxane or epothilone. In another aspect, the microtubule inhibitor is a vinca alkaloid.

[0155] In some embodiments, the therapeutic agent may comprise taxane or its derivatives or analogs. Taxane may be a naturally derived compound or related form, or a chemically synthesized compound or its derivative possessing antitumor properties. Taxanes belong to the terpene family, including but not limited to paclitaxel. And docetaxel The terpenoid family is primarily derived from the Pacific yew and the shortleaf yew, and exhibits activity against certain tumors, particularly breast and ovarian tumors. In one aspect, the taxane is docetaxel or parcitaxel. Parcitaxel is the preferred taxane and is considered an antimitotic agent that promotes the assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization. This stability results in the inhibition of normal dynamic reorganization of the microtubule network structure, which is essential for important interfaces and mitotic cell function.

[0156] It also includes a variety of known taxane derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113; piperazine and other derivatives described in WO 99 / 14209; taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Patent No. 5,869,680; 6-sulfur derivatives described in WO 98 / 28288; sulfinamide derivatives described in U.S. Patent No. 5,821,263; deoxyparcitaxel compounds, such as those described in U.S. Patent No. 5,440,056; and paclitaxel derivatives described in U.S. Patent No. 5,415,869. As mentioned above, this also includes prodrugs of parcitabine, including but not limited to those described in WO 98 / 58927; WO 98 / 13059; and U.S. Patent No. 5,824,701. Taxanes can also be taxane conjugates, such as parcitabine-PEG, parcitabine-dextran, parcitabine-xylose, docetaxel-PEG, docetaxel-dextran, docetaxel-xylose, etc. Other derivatives are mentioned in “Synthesis and Anticancer Activity of Taxol Derivatives,” DGI Kingston et al., Studies in Organic Chemistry, Vol. 26, entitled “New Trends in Natural Products Chemistry” (1986), Atta-ur-Rabman, P.Wle Quesne (eds.) (Elsevier, Amsterdam 1986) and other references. Each of these references is incorporated herein by reference in its entirety.

[0157] Various taxanes can be readily prepared using techniques known to those skilled in the art (see also WO 94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076; U.S. Patent Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267) (each of which is incorporated herein by reference in its entirety), or obtained from a variety of commercial sources, including, for example, Sigma-Aldrich Co., St. Louis, Mo.

[0158] Alternatively, the antimitotic agent may be a microtubule inhibitor; in a preferred aspect, the microtubule inhibitor is vinca alkaloid. Generally, vinca alkaloids are nuclear spindle toxins. Vinca alkaloids act before cell separation, during mitosis, when chromosomes divide and begin to migrate along the tubules of the mitotic spindle towards one of its poles. Under the action of these spindle toxins, the spindle becomes disordered due to the dispersion of chromosomes during mitosis, thereby affecting cell proliferation. According to certain aspects, for example, vinca alkaloids are selected from the group consisting of: vinca alkaloids, vincristine, vindesine, vinorelbine, and their salts, analogues, and derivatives.

[0159] Antimitotic agents can also be epothilones. Generally, members of the epothilone class of compounds stabilize microtubule function through a mechanism similar to taxanes. Epothilones can also induce cell cycle inhibition at the G2-M transition phase, leading to cytotoxicity and ultimately apoptosis. Suitable epothilones include epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, and epothilone F, as well as their salts, analogues, and derivatives. A specific epothilone analogue is the epothilone B analogue ixabepilone (Ixempra). TM ).

[0160] In some aspects, the antimitotic anticancer agent is selected from the group consisting of: taxanes, epothilone, vinca alkaloids, and their salts and combinations. Thus, for example, in one aspect, the antimitotic agent is taxane. More preferably, in this aspect, the antimitotic agent is parcilactone or docetaxel, more preferably parcilactone. In another aspect, the antimitotic agent is epothilone (e.g., an epothilone B analog). In yet another aspect, the antimitotic agent is vinca alkaloids.

[0161] Examples of cancer drugs that may be used in this disclosure include, but are not limited to: thalidomide; platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin, and carboplatin; anthraquinones such as mitoxantrone; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); adrenocortical inhibitors such as mitotane (o,p′-DDD) and aminoglutethimide; RXR agonists such as bexarotene; and tyrosine kinase inhibitors such as sunitinib and imatinib. Other examples of cancer drugs include alkylating agents, antimetabolites, natural products, hormones, antagonists, and other agents. Alternative names are indicated in parentheses. Examples of alkylating agents include nitrogen mustard, such as dichloromethyldiethylamine, cyclophosphamide, ifosfamide, melphalan, sarcolysin, and nitrogen mustard phenylbutyric acid; ethyleneimine and methyl melamine, such as hexamethyl melamine and thiotepa; alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine (BCNU), semustine (methyl-CCNU), lomustine (CCNU), and streptozocin; DNA synthesis antagonists, such as estramustine phosphate; and triazines, such as dacarbazine (DTIC, dimethyl-triazinylimidazolamide) and temozolomide. Examples of antimetabolites include folic acid analogs such as methotrexate; pyrimidine analogs such as fluorouracil (5-fluorouracil, 5-FU, SFU), fluorouridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside), and gemcitabine; purine analogs such as mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG), pentostatin (2′-deoxycofromycin, deoxycofromycin), cladribine, and fludarabine; and topoisomerase inhibitors such as amsacrine.Examples of natural products include vinca alkaloids, such as vincristine (VLB) and vinblastine; taxanes, such as parcitaxel, protein-bound parcitaxel (Abraxane), and docetaxel; epipodophyllotoxins, such as etoposide and teniposide; camptothecins, such as topotecan and irinotecan; antibiotics, such as dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, mitomycin (mitomycin C), idarubicin, and epirubicin; enzymes, such as L-asparaginase; and biological response regulators, such as interferon-α and interleukin-2. Examples of hormones and antagonists include luteinizing hormone-releasing hormone agonists, such as buserelin; adrenal steroids, such as prednisone and related formulations; progesters, such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate; estrogens, such as diethylstilbestrol and ethinylestradiol and related formulations; estrogen antagonists, such as tamoxifen and anastrozole; androgens, such as testosterone propionate and flumethasone and related formulations; androgen antagonists, such as flutamide and bicalutamide; and gonadotropin-releasing hormone analogs, such as leuprolide. Alternative names and brand names of these and other examples of cancer drugs, as well as their methods of use (including administration and dosage regimens), will be known to those skilled in the art.

[0162] In some respects, anticancer agents may include chemotherapeutic agents. Suitable chemotherapeutic agents include, but are not limited to, alkylating agents, antibiotics, antimetabolites, hormones, plant-derived agents and their synthetic derivatives, antiangiogenic agents, differentiation inducers, cell growth inhibitors, apoptosis inducers, cytotoxic agents, agents that affect cellular bioenergetics (i.e., affect cellular ATP levels) and molecules / actions that regulate these levels, biological agents (e.g., monoclonal antibodies, kinase inhibitors, and inhibitors of growth factors and their receptors), gene therapy agents, cell therapies (e.g., stem cells), or any combination thereof.

[0163] Based on these aspects, the chemotherapeutic agents are selected from the group consisting of: cyclophosphamide, nitrogen mustard chlorpyrifos, melphalan, dichloromethyldiethylamine, ifosfamide, busulfan, lomustine, strepzocin, temozolomide, dacarbazine, cisplatin, carboplatin, oxaliplatin, procarbazine, uracil nitrogen mustard, methotrexate, pemetrexed, fludarabine, cytarabine, fluorouracil, fluorouridine, gemcitabine, capecitabine, vinblastine, vincristine, vinorelbine, etoposide, parcitabine, docetaxel, doxorubicin, darubicin, epirubicin, idarubicin, mitoxantrone, bleomycin, mitomycin, hydroxyurea, topotecan, irinotecan, acridine, teniposide, erlotinib hydrochloride, and combinations thereof. Each possibility represents a single aspect of the invention.

[0164] Depending on certain aspects, therapeutic agents may comprise biological drugs, particularly antibodies. Antibodies are selected from the group consisting of: cetuximab, anti-CD24 antibodies, panitumumab, and bevacizumab.

[0165] Therapeutic agents used in this disclosure may include peptides, proteins (such as hormones, enzymes, antibodies, monoclonal antibodies, antibody fragments, monoclonal antibody fragments, etc.), nucleic acids (such as aptamers, siRNA, DNA, RNA, antisense nucleic acids, antisense nucleic acid analogs, etc.), low molecular weight compounds or high molecular weight compounds, receptor agonists, receptor antagonists, partial receptor agonists, and partial receptor antagonists.

[0166] Other representative therapeutic agents may include, but are not limited to, peptide drugs, protein drugs, desensitizing materials, antigens, factors, growth factors, anti-infective agents (such as antibiotics, antimicrobial agents, antiviral agents, antibacterial agents, antiparasitic agents, antifungal substances, and combinations thereof), anti-allergic agents, steroids, androgenic steroids, decongestants, hypnotics, steroid anti-inflammatory agents, anticholinergics, sympathomimetic agents, sedatives, miotics, psychostimulants, tranquilizers, vaccines, estrogens, fertility stimulants, humoral agents, prostaglandins, analgesics, antispasmodics, antimalarial drugs, antihistamines, cardiostimulants, nonsteroidal anti-inflammatory agents, antiparkinsonian agents, anti-Alzheimer's agents, antihypertensive agents, beta-adrenergic blockers, alpha-adrenergic blockers, nutritional supplements, and benzophenanthridine alkaloids. Therapeutic agents can also be substances that can act as stimulants, sedatives, hypnotics, analgesics, anticonvulsants, etc.

[0167] Other therapeutic agents may include CNS-active drugs, neuroactive drugs, inflammatory and anti-inflammatory drugs, renal and cardiovascular drugs, gastrointestinal drugs, anti-tumor agents, immunomodulators, immunosuppressants, hematopoietic agents, growth factors, anticoagulants, thrombolytics, antiplatelet agents, hormones, hormone-active agents, hormone antagonists, vitamins, ophthalmic agents, synthetic agents, antacids, anti-asthmatics, cholesterol-lowering and lipid-lowering agents, anticonvulsants, anti-dysentery agents, antiemetics, antimanic agents, antimetabolites, antiemetics, antiobesity agents, antipyretics and analgesics, anticonvulsants, and antispasmodics. Thrombolytic agents, cough suppressants, antiuric agents, antianginal agents, antihistamines, appetite suppressants, biologics, cerebral dilators, coronary artery dilators, bronchodilators, cytotoxic agents, decongestants, diuretics, diagnostic agents, erythropoietin-producing agents, expectorants, gastrointestinal sedatives, hyperglycemic agents, hypnotics, hypoglycemic agents, laxatives, mineral supplements, mucolytic agents, neuromuscular drugs, peripheral vasodilators, psychotropic agents, stimulants, thyroid and antithyroid agents, tissue growth promoters, uterine relaxants, vitamins, antigens, etc. Other categories of therapeutic agents include those listed in Goodman and Gilman's *The Pharmacological Basis of Therapeutics* (McGraw Hill) and those included in the Merck Index and *The Physicians' Desk Reference* (Thompson Healthcare).

[0168] Other therapeutic agents include androgen inhibitors, polysaccharides, growth factors (such as vascular endothelial growth factor-VEGF), hormones, anti-angiogenic factors, dextromethorphan, dextromethorphan hydrobromide, noscapine, carbetapentane citrate, chlophedianol hydrochloride, chlorpheniramine maleate, phenindamine tartrate, pyrilamine maleate, doxylamine succinate, phenyltoloxamine citrate, phenylephrine hydrochloride, phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride, ephedrine, and codeine phosphate. Phosphate), codeine sulfate, morphine, mineral supplements, cholestryramine, N-acetylprocainamide, acetaminophen, aspirin, ibuprofen, phenylpropanolamine hydrochloride, caffeine, guaiacol, aluminum hydroxide, magnesium hydroxide, peptides, polypeptides, proteins, amino acids, hormones, interferon, cytokines, and vaccines.

[0169] Other examples of therapeutic agents include, but are not limited to, peptide drugs, protein drugs, desensitizing materials, antigens, anti-infective agents (such as antibiotics, antimicrobial agents, antiviral agents, antibacterial agents, antiparasitic agents, antifungal substances, and combinations thereof), anti-allergic agents, androgenic steroids, decongestants, hypnotics, steroid anti-inflammatory agents, anticholinergic agents, sympathomimetic agents, sedatives, miotics, psychostimulants, tranquilizers, vaccines, estrogens, fertility stimulants, humoral agents, prostaglandins, analgesics, antispasmodics, antimalarial drugs, antihistamines, antiproliferative agents, antiVEGF agents, cardiostimulants, nonsteroidal anti-inflammatory agents, anti-Parkinson's agents, antihypertensive agents, β-adrenergic blockers, nutritional agents, and benzophenanthridine alkaloids. These agents may also be substances capable of acting as stimulants, sedatives, hypnotics, analgesics, anticonvulsants, etc.

[0170] Other representative therapeutic agents include, but are not limited to, analgesics such as acetaminophen and acetylsalicylic acid; anesthetics such as lidocaine and xylocaine; appetite suppressants such as dexthyroxine and benzodiazepine tartrate; anti-arthritis drugs such as methylprednisolone and ibuprofen; bronchodilators such as terbutaline sulfate, theophylline, and ephedrine; antibiotics such as sulfamethoxazole, penicillin G, ampicillin, cephalosporins, amikacin, gentamicin, tetracycline, chloramphenicol, erythromycin, clindamycin, isoniazid, and rifampin; antifungals such as amphotericin B, nystatin, and ketoconazole; and antiviral agents such as acyclovir and amanstatin. Anticancer agents, such as cyclophosphamide, methotrexate, etretinate, parcitaxel, paclitaxel, etc.; anticoagulants, such as heparin, warfarin, etc.; anticonvulsants, such as phenyloin sodium, diazepam, etc.; antidepressants, such as isocarboxazid, amoxapine, etc.; antihistamines, such as diphenhydramine hydrochloride, etc. Drugs containing: HCl, chlorpromazine maleate, etc.; hormones, such as insulin, progesterone, estrogen, corticosteroids, glucocorticoids, and androgens; sedatives, such as thorazine, diazepam, chlorpromazine HCl, reserpine, chlordiazepoxide HCl, etc.; antispasmodics, such as belladonna alkaloids, bicyclic amine hydrochloride, etc.; vitamins and minerals, such as essential amino acids, calcium, iron, potassium, zinc, vitamin B12, etc.; cardiovascular agents, such as prazosin HCl, nitroglycerin, propranolol hydrochloride, etc. HCl), hydrazine hydrochloride, pancreatic lipase, succinate dehydrogenase, etc.; peptides and proteins, such as LHRH, somatostatin, calcitonin, growth hormone, glucagon-like peptide, growth-releasing factor, angiotensin, FSH, EGF, bone morphogenetic protein (BMP), erythropoietin (EPO), interferon, interleukin, collagen, fibrinogen, insulin, factor VIII, factor IX, etc. α-glucosidase, Cerazyme / Vasopressin, ACTH, human serum albumin, gamma globulin, structural proteins, blood product proteins, complex proteins, enzymes, antibodies, monoclonal antibodies, etc.; prostaglandins; nucleic acids; carbohydrates; fats; anesthetics, such as morphine and codeine, psychotropic agents; antimalarial agents, L-dopa, diuretics, such as furosemide and spironolactone; antiulcer drugs, such as ranitidine hydrochloride and cimetidine hydrochloride, etc.

[0171] Therapeutic agents can also be immunomodulators, including cytokines, interleukins, interferons, community-stimulating factors, and tumor necrosis factor; immunosuppressants, such as rapamycin and tacrolimus; allergens, such as cat dander, birch pollen, house dust mites, and grass pollen; and antigens of bacterial organisms, such as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, and Yersinia pestis. (The text appears to be a list of bacteria and viruses, possibly related to polio, influenza, and rabies. A direct translation wouldn't be meaningful without further context.) Antigens for diseases such as fever, lymphocytic choroid plexus meningitis, and hepatitis B; antigens for fungi, protozoa, and parasitic organisms such as: Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni.These antigens can be in the form of whole inactivated organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.

[0172] In another specific aspect, the therapeutic agent may contain antibiotics. Antibiotics may be one or more of the following, for example: amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin, ansamycin, geraniac, carbapenem, chloramphenicol, carbapenem, ertapenem, doripenem, imipenem / cilastatin, meropenem, cephalosporins (first generation), cefadroxil, cefazolin, cefalothin / cephalosporin. falothin), cephalexin, cephalosporins (second generation), cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cephalosporins (third generation), cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftiz oxime), ceftriaxone, cephalosporins (fourth generation), cefepime, cephalosporins (fifth generation), ceftobiprole, glycopeptides, teicoplanin, vancomycin, macrolides, azithromycin, clarithromycin, erythromycin, roxithromycin, acetomycin, telithromycin, spectinomycin, monotypic acid, aztreonam, penicillin, amoxicillin, ampicillin, azoxicillin, carbapenem, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin llin), nafcillin, oxacillin, penicillin, piperacillin, ticarcillin, polypeptides, bacitracin, colistin, polymyxin B, quinolones, ciprofloxacin, enoxacin, gatifloxacin, lofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trifloxacin, sulfonamides, sulfamidone, prontosil (obsolete), sulfacetamide, sulfamethoxazole, sulfanilimide (obsolete), sulfasalazine, sulfisoxazole, trimeprine, trimeprine-sulfamethoxazole (compound sulfamethoxazole) (TMP-SMX), tetracyclines (including demecycline, doxycycline, minocycline),Oxytetracycline, tetracycline, and other substances; Arsphenamine, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, acanthoxycycline, pyrazinamide, quinupristin / dalfopristin, rifampin (USA), timidazole, or combinations thereof. In one aspect, the therapeutic agent may be a combination of rifampin (USA) and minocycline.

[0173] Suitable growth factors as therapeutic agents include, but are not limited to, transforming growth factor-α (“TGF-α”), transforming growth factor (“TGF-β”), platelet-derived growth factor (“PDGF”), fibroblast growth factor (“FGF”) (including FGF acidic isoforms 1 and 2, FGF basic form 2, and FGF 4, 8, 9, and 10), and nerve growth factor (“NGF”) (including NGF 2.5S, NGF 2.5S, and NGF 2.5S). 7.0s and βNGF) and neurotrophic factors, brain-derived neurotrophic factor, chondroitin-derived factor, bone growth factor (BGF), basic fibroblast growth factor, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), insulin-like growth factor (IGF) I and II, hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF), transforming growth factor (TGF) (including TGFα, β, β1, β2, β3), bone growth factor, bone matrix-derived growth factor and bone-derived growth factor and mixtures thereof.

[0174] Cytokines suitable as therapeutic agents include, but are not limited to, cardiotrophins, stromal cell-derived factors, macrophage-derived chemokines (MDCs), melanoma growth stimulating factor (MGSA), macrophage inflammatory protein 1α (MIP-1α), macrophage inflammatory protein 2, macrophage inflammatory protein 3α, macrophage inflammatory protein 3β, macrophage inflammatory protein 4, and macrophage inflammatory protein 5, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TNF-α, and TNF-β. Immunoglobulins that can be used in this disclosure include, but are not limited to, IgG, IgA, IgM, IgD, IgE, and mixtures thereof. Some preferred growth factors include VEGF (vascular endothelial growth factor), NGF (nerve growth factor), PDGF-AA, PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF.

[0175] Other molecules that can be used as therapeutic agents include, but are not limited to, growth hormone, leptin, leukemia inhibitory factor (LIF), tumor necrosis factor α and β, endostatin, thromboretin, osteoblast-1, bone morphogenetic protein 2 and 7, osteonectin, somatostatin-like peptide, osteocalcin, interferon α, interferon αA, interferon β, interferon γ, interferon 1α, and interleukins 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, and 18.

[0176] In some implementations, the therapeutic agent is in the form of approximately 10, approximately 20, approximately 30, approximately 40, approximately 45, approximately 50, approximately 55, approximately 60, approximately 65, approximately 70, approximately 75, approximately 80, approximately 85, approximately 90, approximately 95, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150, approximately 160, approximately 170, approximately 180, approximately 190, approximately 200, approximately 210, approximately 220, approximately 230, approximately 240, approximately 250, approximately 260, approximately 270, approximately 280, approximately 290, approximately 3 00, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, or about 500; or a range of therapeutic doses formed by any of the foregoing values; or an amount (therapeutic dose micrograms per milligram of the weight of the disclosed drug delivery composition) present in the disclosed drug delivery composition.

[0177] In some embodiments, the therapeutic agent exhibits near-zero order release kinetics for at least 30 days, such as 45 days, 60 days, 3 months, 6 months, 9 months, 1 year, or longer. The therapeutic agent may exhibit near-zero order release kinetics upon implantation of the drug delivery composition or after a subsequent period, for example, approximately 30 days after implantation of the drug delivery composition. In other embodiments, the drug delivery composition may exhibit kinetics deviating from zero order kinetics.

[0178] Method for preparing the disclosed drug delivery composition

[0179] In various aspects, the disclosed drug delivery device is prepared by the methods disclosed herein and described in particular aspects as in the following representative embodiments.

[0180] Therefore, in one aspect, a method for preparing the drug delivery device described herein is provided, the method comprising: forming a first layer comprising a first polymer on a conductive rod; and forming a second layer comprising a second polymer on the first layer.

[0181] In some embodiments, forming the first layer includes electrospinning using a solution of the first polymer and a voltage difference of about 10 kV to about 30 kV.

[0182] In some embodiments, the first polymer solution is a solution of about 1 w / v% to about 10 w / v% in at least one organic solvent. In some embodiments, the at least one organic solvent in the first polymer solution comprises trifluoroacetic acid, dichloromethane, hexafluoroisopropanol, or a combination thereof. In some embodiments, trifluoroacetic acid and dichloromethane are present in a ratio of about 1:10 to about 10:1, for example, in a ratio of about 5:3 to about 10:3. In some embodiments, trifluoroacetic acid and dichloromethane are present in a ratio of about 7:3.

[0183] In some embodiments, forming the second layer includes electrospinning a solution containing a second polymer and optionally a pore-forming agent onto the formed first layer, wherein the voltage difference for electrospinning is about 20 kV to about 30 kV.

[0184] In some embodiments, the solution comprising the second polymer and optionally the porogen is about 1 w / v% to about 10 w / v% by weight of the total weight of the second polymer and the porogen, for example, about 2.5 w / v% to about 10 w / v% or about 5 w / v% to about 10 w / v%. In some embodiments, the solution comprising the second polymer and the porogen is a 1,1,1,3,3,3-hexafluoroprop-2-ol solution.

[0185] In some embodiments, the weight ratio of the second polymer to the porogen is from about 90:100 to about 100:1, for example, from about 90:100 to 99.9:0.1, from 90:100 to about 95:5, or from 95:5 to about 99.9:0.1. In some embodiments, the weight ratio of the second polymer to the porogen is about 99:1, about 95:5, about 92.5:7.5, or about 90:10. In some embodiments, the weight ratio of the second polymer to the porogen varies from about 50:50 to about 100:0.

[0186] As used herein, "porogen" refers to any material that can be used to form porous materials, such as porous polycaprolactone as described herein. In some embodiments, the porogen comprises a water-soluble compound, so that the porogen is substantially removed from the outer layer after washing the drug delivery device with water. In some embodiments, the porogen comprises a compound selected from: ([tris(hydroxymethyl)methylamino]propanesulfonic acid) (TAPS), (2-(bis(2-hydroxyethyl)amino)acetic acid) (Bicine), (tris(hydroxymethyl)aminomethane) or (2-amino-2-(hydroxymethyl)propane-1,3-diol) (Tris), (N-[tris(hydroxymethyl)methyl]glycine) (Tricine), (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropane The porogen may contain sulfonic acid (TAPSO), (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) (HEPES), (2-[[1,3-dihydroxy-2-(hydroxymethyl)prop-2-yl]amino]ethanesulfonic acid) (TES), (3-(N-morpholino)propanesulfonic acid) (MOPS), (piperazine-N,N′-bis(2-ethanesulfonic acid)) (PIPES), dimethylarsenic acid, (2-(N-morpholino)ethanesulfonic acid) (MES), or salts thereof, such as sodium salts thereof. In some embodiments, the porogen comprises sodium salt of HEPES. In some embodiments, the porogen comprises a water-soluble polymer (such as polyethylene glycol), a polyoxyethylene copolymer, an acrylate copolymer including a quaternary ammonium group, polyacrylamide, polyvinyl alcohol, hyaluronic acid, and polyvinylpyrrolidone. In other embodiments, the porogen comprises gelatin, polyethylene glycol (PEG), chitosan, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or agarose.

[0187] In some embodiments, the method further includes sintering the drug delivery device after forming the outer layer. In some embodiments, sintering is performed at a temperature of about 50°C to about 150°C, for example, about 90°C to about 110°C. In some embodiments, sintering includes heating for about 1 minute to about 6 hours, for example, about 30 minutes to about 6 hours.

[0188] In some embodiments, the method further includes washing the drug delivery device after sintering. In some embodiments, the drug delivery device is washed sequentially with a saturated sodium bicarbonate solution and deionized water. In some embodiments, the pore-forming agent is substantially removed from the drug delivery device after washing with deionized water.

[0189] In some embodiments, the method further includes drying the drug delivery device after washing. In some embodiments, drying is carried out in a vacuum. In some embodiments, drying is carried out at a temperature of about 50°C to about 150°C, for example, about 90°C to 110°C. In some embodiments, drying is carried out for about 1 minute to about 6 hours, for example, about 30 minutes to about 6 hours.

[0190] In other embodiments, the disclosed capsule may be manufactured by any suitable method that will be readily understood by those skilled in the art. In some embodiments, the disclosed capsule may be manufactured by forming an asymmetric membrane; a representative example of such a method is provided in Yen, C. et al., “Synthesis and characterization of nanoporous polycaprolactone membranes via thermally- and nonsolvent-induced phase separations for biomedical device application”, Journal of Membrane Science 2009, 343:180-88, which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the disclosed capsule may be manufactured using three-dimensional printing. In some embodiments, the disclosed capsule may be manufactured around methylcellulose, which is then removed to form a luminal compartment. In some embodiments, the disclosed capsules can be manufactured using methods described by Envisia Therapeutics in WO 2015 / 085251, WO 2016 / 144832, WO 2016 / 196365, WO 2017 / 015604, WO 2017 / 015616, or WO 2017 / 015675, each of which is incorporated herein by reference in its entirety for all purposes. In other embodiments, the disclosed capsules can be manufactured using methods similar to those used in the manufacture of hollow fiber membranes, such as phase inversions, including non-solvent-induced phase inversions (NIPS), (solvent) evaporation-induced phase inversions (EIPS), vapor adsorption-induced phase inversions (VIPS), and thermally induced phase inversions (TIPS). In some embodiments, the disclosed capsules can be manufactured using methods similar to those described in US 2015 / 232506, which is incorporated herein by reference in its entirety for all purposes. In some implementations, pores can be formed alternatively by laser diffraction of the capsule.

[0191] In some embodiments, the two ends of the tubular shape of the capsule are closed. The ends can be closed by any number of sealing techniques appropriately selected by those skilled in the art. In some embodiments, high-frequency sealing is used to seal both ends. In this technique, a high frequency generates eddies in the walls, which at least heat the polymer layer. When the temperature reaches the melting point of the polymer, the clamps are closed and the molten polymer is cooled and shaped. In some embodiments, thermal clamp sealing is used to seal both ends, wherein heated jaws apply heat to the outside of the tubular shape to heat the interior for sealing. In some embodiments, ultrasonic sealing can be used to seal both ends. In this technique, the polymer composition of the inner layer is heated and melted by high-frequency friction introduced from an ultrasonic amplitude transformer. The clamps are then closed around the portion to be sealed, cooled, and shaped to seal the end. In some embodiments, hot air sealing is used to seal both ends, wherein the system heats the sealing area inside the capsule with hot air, and then the ends are subsequently pressed and frozen at a subsequent station.

[0192] Treatment methods using the disclosed drug delivery device

[0193] This document also provides a method for treating a clinical disorder by administering the disclosed pharmaceutical delivery composition. The clinical disorder may be a clinical condition, disease, functional impairment, or other ailment that can be improved by the therapeutic composition.

[0194] The term "administering / administration" as used to describe a drug delivery device includes any route by which the device is introduced or delivered to a subject to perform its intended function. Administration can be performed via any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), or topical administration. Administration includes self-administration and administration by another person. In some cases, administration is performed via injection into the eye (including intraocular injection). In other cases, for example, in the treatment of cancer, administration may be performed via injection of the disclosed drug delivery composition into, adjacent to, near, or proximal to a tumor or other mass of cancer cells.

[0195] It should also be understood that the treatment or prevention of various modes of medical diseases and disorders as described are intended to mean "basic," which includes both complete and subtotal treatment or prevention, in which some biologically or medically relevant outcome is achieved. Treatment can be continuous long-term therapy for chronic diseases or single or fewer applications in the treatment of acute illnesses.

[0196] The term “single” application refers to the simultaneous or substantially simultaneous application of at least two active ingredients through different routes.

[0197] The term "sequential" application refers to the application of at least two active ingredients at different times, via the same or different routes of administration. More specifically, sequential use means that the entire application of one active ingredient occurs before the application of another active ingredient or other ingredients. Therefore, one active ingredient may be applied several minutes, hours, or days before the application of another active ingredient or other ingredients. Thus, the term "sequential" is different from "simultaneous" application.

[0198] The term “simultaneous” application refers to the simultaneous or substantially simultaneous application of at least two active ingredients via the same route.

[0199] As used in this article, the term "therapeutic" refers to treatment and / or prevention. Therapeutic effects are achieved by suppressing, alleviating, or eradicating a disease state.

[0200] This disclosure also provides methods for treating ophthalmic diseases or conditions by administering a therapeutically effective amount of the composition described herein. In some embodiments, the disclosed methods relate to the treatment of an ophthalmic condition and include injecting a therapeutically effective amount of the disclosed composition into the eye of a subject. The subject may be a patient; and the patient may have been diagnosed with an ophthalmic condition. In some aspects, the method may further include diagnosing the subject with an ophthalmic condition.

[0201] Ophthalmic conditions may include: acute macular neuroretinopathy; Behcet's disease; neovascularization, including choroidal neovascularization; diabetic uveitis; histoplasmosis; infections, such as those caused by fungi or viruses; macular degeneration, such as acute macular degeneration (AMD), including wet AMD, non-exudative AMD, and exudative AMD; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting specific points or locations of the posterior eye; ocular tumors; and retinal conditions, such as central retinal vein occlusion and diabetic retinopathy (including proliferative diabetic retinopathy). Diseases include proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitis; sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; uveitis; posterior ocular conditions caused or affected by ocular laser treatment; posterior ocular conditions caused or affected by photodynamic therapy or photocoagulation; radiation retinopathy; epiretinal membrane disease; retinal branch vein occlusion; anterior ischemic optic neuropathy; non-retinal diabetic retinopathy; retinitis pigmentosa; cancer; and glaucoma. In some cases, the ophthalmic condition is wet age-related macular degeneration (wet AMD), cancer, neovascularization, macular edema, or edema. In another specific aspect, the ophthalmic condition is wet age-related macular degeneration (wet AMD).

[0202] In various applications, injections for the treatment of ophthalmic conditions can be administered into the vitreous cavity of the eye. In some cases, the injection may be intravitreal, subconjunctival, subcapsular, retrobulbar, or suprachoroidal.

[0203] The term "ocular region" or "ocular location" means any area of ​​the eyeball (eyeball), including the anterior and posterior segments of the eye, and generally includes, but is not limited to, any functional (e.g., for vision) or structural tissue present in the eyeball, or any layer of tissue or cells that partially or completely line the interior or exterior of the eyeball. Specific examples of regions of the eyeball include, but are not limited to, the anterior chamber, posterior chamber, vitreous cavity, choroid, suprachoroidal space, conjunctiva, subconjunctival space, suprascleral space, intracorneal space, subretinal space, subcapsular space of Attennon, suprascleral space, sclera, pars plana, surgically induced avascular area, macula, and retina.

[0204] "Ophthalmological conditions" can refer to any disease, ailment, or disorder that affects or involves the eye or a part or area of ​​the eye. In general, the eye includes the eyeball (including the cornea) and other tissues and fluids that make up the eyeball, the muscles around the eye (such as the oblique and rectus muscles), and part of the optic nerve inside or near the eyeball.

[0205] "Glaucoma" refers to primary, secondary, and / or congenital glaucoma. Primary glaucoma can include open-angle and angle-closure glaucoma. Secondary glaucoma can occur as a complication of a variety of other conditions, such as injury, inflammation, pigment dispersion, vascular disease, and diabetes. The increased pressure in glaucoma causes blindness because it damages the optic nerve at its entry point into the eye. Therefore, in a non-limiting embodiment, reducing reactive oxygen species STC-1 or MSCs representing increased levels of STC-1 can be used to treat glaucoma and prevent or delay the onset of blindness.

[0206] "Inflammation-mediated" in relation to ocular conditions means any ocular disease that can benefit from treatment with anti-inflammatory agents, and includes, but is not limited to, uveitis, macular edema, acute macular degeneration, retinal detachment, ocular tumors, fungal or viral infections, multifocal choroiditis, diabetic retinopathy, uveitis, proliferative vitreoretinopathy (PVR), sympathetic ophthalmia, Vogt-Koyanagi-Harada (VKH) syndrome, histoplasmosis, and uveitis.

[0207] "Damage" or "impairment" in relation to ocular conditions is interchangeable and refers to cellular and morphological manifestations and symptoms caused by inflammatory-mediated disorders (such as inflammation), tissue damage caused by means other than inflammation (such as chemical damage, including chemical burns), and damage caused by infection (including but not limited to bacterial, viral, or fungal infections).

[0208] "Intraocular" means within or beneath the ocular tissues. Intraocular drug delivery systems include those administered subcapsular, subconjunctival, suprachoroidal, subretinal, vitreous, anterior chamber, and similar locations. Intraocular drug delivery systems exclude those administered superficially, systemically, intramuscularly, subcutaneously, intraperitoneally, and similar locations.

[0209] "Macular degeneration" refers to any of a number of conditions and disorders in which the macula undergoes degeneration or loses its functional activity. Degeneration or loss of functional activity can result from, for example, cell death, decreased cell proliferation, loss of normal biological function, or a combination of the foregoing. Macular degeneration can lead to and / or manifest as alterations in the structural integrity of macular cells and / or the extracellular matrix, changes in the normal cellular and / or extracellular matrix architecture, and / or loss of function of macular cells. Cells can be any cell type normally present in or near the macula, including RPE cells, photoreceptor cells, and capillary endothelial cells. Age-related macular degeneration (AMD), or ARMD, is the primary macular degeneration-related disorder, but many other macular degeneration-related disorders are known, including but not limited to Best macular dystrophy, Stargardt macular dystrophy, Sorsby fundus dystrophy, Mallatia Leventinese, Doyne honeycomb retinal dystrophy, and RPE pattern dystrophy. Age-related macular degeneration (AMD) is described as either “dry” or “wet.” The wet exudative neovascular form of AMD affects approximately 10–20% of patients with AMD and is characterized by abnormal vascular growth beneath or within the retinal pigment epithelium (RPE), leading to hemorrhage, exudation, scarring, or serous retinal detachment. Eighty to ninety percent of AMD patients have the dry form, characterized by retinal pigment epithelial cell atrophy and loss of macular photoreceptors. Choroidal verrucous verrucous verrucous may or may not be present in the macula. Atrophy of the retinal pigment epithelium cells, which explains vision loss, may also be present in the macula. Currently, there is no treatment for any form of AMD, although some success has been achieved in alleviating wet AMD using photodynamic therapy and, in particular, anti-VEGF therapy.

[0210] "Choroid verrucous" are fibrous materials that accumulate beneath the retinas per the retina with age. They are observed using an ophthalmoscopy. A normal eye may have a macula without choroidal verrucous ...

[0211] "Ocular neovascularization" (ONV) is used in this article to refer to choroidal neovascularization or retinal neovascularization, or both.

[0212] "Retinal neovascularization" (RNV) refers to, for example, the abnormal development, proliferation, and / or growth of retinal vessels on the surface of the retina.

[0213] Subretinal neovascularization (SRNVM) refers to the abnormal development, proliferation, and / or growth of blood vessels beneath the surface of the retina.

[0214] The cornea is the transparent structure that forms the anterior part of the fibrous membrane of the eye. It consists of five layers: 1) the anterior corneal epithelium connected to the conjunctiva; 2) the anterior limiting layer (Bowman's layer); 3) the intrinsic matrix or stroma; 4) the posterior limiting layer (Descemet's membrane); and 5) the endothelium of the anterior chamber or the stratum corneum of the skin.

[0215] The retina is the innermost layer of the eyeball that surrounds the vitreous body and is connected to the optic nerve at the back. The retina consists of the following layers: 1) internal limiting membrane; 2) nerve fiber layer; 3) ganglion cell layer; 4) internal reticular layer; 5) inner nuclear layer; 6) outer reticular layer; 7) outer nuclear layer; 8) external retina; and 9) rod and cone layer.

[0216] "Retinal degeneration" refers to any hereditary or acquired degeneration of the retina and / or retinal pigment epithelial cells. Non-limiting examples include retinitis pigmentosa, Best's disease, RPE pattern malnutrition, and age-related macular degeneration.

[0217] In various aspects, treatment of ophthalmic conditions can include treating a variety of retinal diseases or disorders, including: macular degeneration / retinal degeneration: macular degeneration, including age-related macular degeneration (ARMD), such as non-exudative and exudative age-related macular degeneration; choroidal neovascularization; retinopathy, including diabetic retinopathy, acute and chronic macular neuroretinopathy, central serous chorioretinopathy; and macular edema, including cystoid macular edema and diabetic macular edema. Uveitis / Retinitis / Choroiditis: Acute multifocal squamous pigment epithelial lesions, Behcet's disease, shotgun retinochoroidal lesions, infectious diseases (syphilis, Lyme disease, tuberculosis, toxoplasmosis), uveitis (including intermediate uveitis (paris plana inflammation) and anterior uveitis), multifocal choroiditis, multiple transient white spot syndrome (MEWDS), ocular sarcoidosis, posterior scleritis, creeping choroiditis, subretinal fibrosis, uveitis syndrome, and Vogt-Koyanagi-Harada syndrome. Vascular / exudative diseases: retinal artery occlusive disease, central retinal vein occlusion, disseminated intravascular coagulation, retinal branch vein occlusion, hypertensive fundus changes, ocular ischemia syndrome, retinal microaneurysms, Coats' disease, parafoveal telangiectasia, hemilateral retinal vein occlusion, papillary phlebitis, central retinal artery occlusion, retinal branch artery occlusion, carotid artery disease (CAD), frost-like dendritic retinal vasculitis, sickle cell retinopathy and other hemoglobinopathies, vascular streaks, familial exudative vitreoretinopathy, Eales' disease; Traumatic / surgical diseases: sympathetic ophthalmia, uveitis-related retinopathy, retinal detachment, trauma, laser treatment, PDT, photocoagulation, inadequate perfusion during surgery, radiation retinopathy, retinopathy after bone marrow transplantation. Proliferative diseases: proliferative vitreoretinopathy and epiretinal membranes, proliferative diabetic retinopathy. Infectious diseases: ocular histoplasmosis, ocular toxocariasis, ocular histoplasmosis syndrome (OHS), endophthalmitis, toxoplasmosis, HIV-associated retinal disease, HIV-associated choroidal disease, HIV-associated uveitis, viral retinitis, acute retinal necrosis, progressive external retinal necrosis, fungal retinal disease, ocular syphilis, ocular tuberculosis, diffuse unilateral subacute optic neuritis, and myiasis.Hereditary disorders: Retinitis pigmentosa, systemic diseases with associated retinal dystrophy, congenital stationary night blindness, cone cell dystrophy, Staggart's disease and macular degeneration, Best's disease, pattern dystrophy of retinal pigment epithelial cells, X-linked retinoschisis, Sotheby's retinal dystrophy, benign concentric macular degeneration, Bietti's crystalline dystrophy, and fibroelastoma. Retinal tears / holes: Retinal detachment, macular hole, and giant retinal tears. Tumors: Tumor-related retinal diseases, congenital RPE hypertrophy, posterior uveal melanoma, choroidal hemangioma, choroidal osteoma, choroidal metastasis, retinal and retinal pigment epithelial cell hamartoma, retinoblastoma, fundus vascular proliferative tumors, retinal astrocytoma, and intraocular lymphoma. Others: Punctate inner choroidal lesions, acute posterior multifocal squamous pigment epithelial lesions, myopic retinal degeneration, and acute retinal pigment epitheliitis.

[0218] Anterior ocular diseases are conditions, ailments, or disorders affecting or involving the anterior (i.e., in front of the eye) ocular region or site, such as the periocular muscles, eyelids, or ocular tissues or fluids located anterior to the posterior wall of the lens capsule or ciliary muscle. Therefore, anterior ocular diseases primarily affect or involve the conjunctiva, cornea, anterior chamber, iris, posterior chamber (behind the iris but anterior to the posterior wall of the lens capsule), lens or lens capsule, and the blood vessels and nerves that vascularize or innervate the anterior ocular region or site.

[0219] Therefore, anterior ocular conditions can include diseases, ailments, or disorders such as: aphakia; pseudophakia; astigmatism; blepharospasm; cataracts; conjunctival diseases; conjunctivitis, including but not limited to atopic keratoconjunctivitis; corneal damage, including but not limited to damage to the corneal stroma; corneal diseases; corneal ulcers; dry eye syndrome; eyelid diseases; lacrimal apparatus diseases; lacrimal duct obstruction; myopia; presbyopia; pupillary disorders; refractive disorders; and strabismus. Glaucoma can also be considered an anterior ocular condition because the clinical goal of glaucoma treatment can be to reduce the hypertension of the aqueous fluid in the anterior chamber of the eye (i.e., reduce intraocular pressure).

[0220] Other eye diseases or conditions that can be treated according to the present invention include, but are not limited to, ocular cicatricial pemphigoid (OCP), Stevens Johnson syndrome, and cataracts.

[0221] Posterior ocular symptoms are diseases, ailments, or disorders that primarily affect or involve the posterior ocular region or site, such as the choroid or sclera (located posterior to the plane passing through the posterior wall of the lens capsule), vitreous body, vitreous cavity, retina, optic nerve (i.e., optic disc), and the blood vessels and nerves that vascularize or innervate the posterior ocular region or site. Therefore, posterior ocular conditions can include diseases, ailments, or disorders such as: acute macular neuroretinopathy; Behçet's disease; choroidal neovascularization; diabetic retinopathy; uveitis; ocular histoplasmosis; infections, such as those caused by fungi or viruses; macular degeneration, such as acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting the posterior ocular site or location; ocular tumors; and retinal diseases, such as central retinal vein occlusion and diabetic retinopathy. Diseases including proliferative diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery or vein occlusive disease, retinal detachment, uveitis; sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; uveitis; posterior ocular conditions caused or affected by ocular laser treatment; posterior ocular conditions caused or affected by photodynamic therapy or photocoagulation; radiation retinopathy, epiretinal membrane disease, retinal branch vein occlusion, anterior ischemic optic neuropathy, non-retinal diabetic retinopathy, retinitis pigmentosa, and glaucoma. Glaucoma can be considered a posterior ocular condition because the treatment goal is to prevent or reduce visual loss attributable to damage or loss of retinal ganglion cells or retinal nerve fibers (i.e., neuroprotection).

[0222] In some embodiments, the ocular condition is an ocular inflammation caused by, for example, iritis, conjunctivitis, seasonal allergic conjunctivitis, acute and chronic endophthalmitis, anterior uveitis, uveitis associated with systemic disease, posterior uveitis, chorioretinitis, pars plana inflammation, masquerade syndrome (including ocular lymphoma), pemphigoid, scleritis, keratitis, severe ocular allergic reactions, corneal abrasion, and disruption of the blood-aqueous barrier. In another embodiment, the ocular condition is a postoperative ocular inflammation caused by, for example, refractive keratotomy, cataract removal surgery, intraocular lens transplantation, vitrectomy, corneal transplantation, various forms of thin-slice keratotomy (DSEK, etc.), and radial keratotomy.

[0223] In various applications, injections for the treatment of ophthalmic conditions can be administered into the vitreous cavity of the eye. In some cases, the injection may be intravitreal, subconjunctival, subcapsular, retrobulbar, or suprachoroidal.

[0224] In various aspects, methods for treating ophthalmic conditions include the administration of a disclosed drug delivery device containing a therapeutic agent, for example, by injection of about 0.01 mg to about 25 mg; or about 1 mg to about 15 mg. In some embodiments, the drug delivery composition can release an amount of drug that maintains a concentration of about 10 picomoles to about 500 picomoles in the vitreous humor of the eye for about 10 days to about 12 months. The amount of therapeutic agent in the drug delivery composition will depend on the amount of therapeutic agent that may be present in one or more capsules and the amount necessary to achieve the desired therapeutic effect.

[0225] In some embodiments, the disclosed drug delivery can protect the bioactivity of the encapsulated therapeutic agent for up to 12 months. The level of protection of bioactivity will depend on both the therapeutic agent used and the selected composition of the disclosed capsule, but can be quantified by methods such as HPLC (for determining the amount and form of the drug present in the eye), cellular analysis of the activity against a positive control (such as using a single therapeutic agent), and ELISA for characterizing the form of other therapeutic agents or assessing changes in bioactivity (such as transcription factor expression).

[0226] medicine box

[0227] This disclosure also relates to a medicine box containing one of the following: (a) a drug delivery composition as described herein; (b) a sterile package containing a drug delivery composition as described herein; or (c) a pre-filled syringe or needle containing a drug delivery composition as described herein; and instructions for administering the drug delivery composition as described herein to treat a clinical condition or lesion.

[0228] In another aspect, the disclosed pillbox can be packaged for daily dosing regimens (e.g., on a card, in a dosing card, on a blister pack, or in blow-molded plastic). Such packaging enhances the product and increases ease of use for administration by healthcare professionals. It also reduces potential medical errors. The invention also provides such pillboxes that further include instructions for use.

[0229] In another aspect, this disclosure also provides a pill packet or box containing one or more packages containing the disclosed drug delivery composition. Associated with such packaging may be a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of a drug or biological product, reflecting agency approval for manufacture, use, or sale for human use.

[0230] In various aspects, the disclosed kit may also contain other therapeutic agents, compounds, and / or products co-packaged, co-formulated, and / or co-delivered with other components. For example, a drug manufacturer, drug reseller, physician, compounding shop, or pharmacist may provide a kit containing the disclosed drug delivery composition and another component for delivery to a patient.

[0231] It is anticipated that the disclosed kit can be used in conjunction with the disclosed preparation method, the disclosed use or treatment method and / or the disclosed composition.

[0232] As can be seen from the foregoing, the aspects of this paper are well suited to achieve all the purposes and objectives set forth above, along with other obvious advantages inherent to the structure.

[0233] While specific elements and steps are discussed in combination with each other, it should be understood that any element and / or step provided herein is to be considered as being able to be combined with any other element and / or step, regardless of whether such element and / or step is explicitly provided, while still remaining within the scope provided herein.

[0234] It should be understood that certain features and sub-combinations are effective and can be employed without mentioning other features and sub-combinations. This is covered by and within the scope of the claims.

[0235] Since many possible aspects can be carried out without departing from the scope, it should be understood that all subjects set forth or illustrated herein with the accompanying drawings and detailed embodiments should be interpreted as illustrative and not limiting.

[0236] It should also be understood that the terminology used herein is for descriptive purposes only and is not intended to be limiting. A skilled craftsman will recognize many variations and modifications of the aspects described herein. These variations and modifications are intended to be included in the teachings of this disclosure and are covered by the claims herein.

[0237] Having generally described aspects of this disclosure, the following embodiments illustrate some other aspects of this disclosure. While aspects of this disclosure have been described in conjunction with the following embodiments and corresponding text and figures, it is not intended to limit the aspects of this disclosure to this description. Rather, it is intended to cover all alternatives, modifications, and equivalents included within the spirit and scope of this disclosure.

[0238] Example

[0239] The following examples are provided to provide a complete disclosure and description, for those skilled in the art, of how to prepare and evaluate the compounds, compositions, articles, apparatus, and / or methods claimed herein, and are intended merely to illustrate the disclosure and not to limit the scope of what the inventors consider to be its disclosure. Efforts have been made to ensure accuracy regarding figures (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, portions are parts by weight, temperatures are °C or equal to ambient temperature, and pressures are equal to or close to atmospheric pressure.

[0240] Material

[0241] Chitosan (DD>75%, Mw 310,000-375,000 Da), polycaprolactone (Mn 80,000), trifluoroacetic acid (TFA), sodium 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), and Tween 20 were purchased from Sigma-Aldrich Inc. (St. Louis, MO). 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) was purchased from Oakwood Products Inc. (Estill, SC). Dichloromethane (DCM), chromatographically purified bovine serum albumin (BSA), and recombinant human VEGF protein were purchased from Fisher Scientific International Inc. (Hampton, NH). Bevacizumab (Avastin) was purchased from Genentech, Inc. (San Francisco, CA). The quinolinic acid (BCA) protein assay kit, colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetraazonium (MTS) analyte, horseradish peroxidase (HRP)-conjugated goat anti-human immunoglobulin G (IgG) fragment crystallizable (Fc) secondary antibody, and 3,3',5,5'-tetramethylbenzidine (TMB) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Human retinal pigment epithelial cell line (ARPE-19 cells, CRL2302) and DMEM:F-12 medium were purchased from the American Type Culture Collection (Rockville, MD). Human umbilical vein endothelial cells (HUVECs), 200 PRF of culture medium, low serum growth supplement, and low growth factor basement membrane matrix without lactose dehydrogenase-enhancing virus (LDEV) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA). All other reagents used were analytical grade.

[0242] Capsule making

[0243] In this study, capsules of two different sizes (260 μm and 1645 μm) were fabricated. The 1.645 μm capsule primarily served as a base model for the smaller capsules to optimize processing conditions. The 260 μm capsule was used in subsequent studies.

[0244] Figure 1A The capsule manufacturing method is illustrated in the figure. Based on previous research, chitosan fiber layers were prepared by electrospinning with modifications (see Gu, BK et al., Fabrication of sonicated chitosan nanofiber mat with enlarged porosity for use as hemostatic materials. 2013.97(1): 65-73). In short, a 5.0% (w / v) chitosan solution prepared in a mixture of TFA and DCM at a volume ratio of 7:3 was extruded through a No. 20 stainless steel needle connected to the cathode of a high-voltage DC generator. The ground was connected to a drum collector at a speed of 500 rpm, in which the electrospun fibers were deposited. To obtain capsules with two different inner diameters, the fibers were collected using 315 stainless steel rods with a diameter of 1.645 mm or 260 μm. The solution was continuously fed at a voltage of 25.0 kV to the 1.645 mm drum collector at a feed rate of 3.0 mL / h and to the 260 μm drum collector at a feed rate of 1.0 mL / h. Use a nitrogen-filled glove box to control humidity at 30% during electrospinning.

[0245] To prepare PCL nanoporous layers (see Cipitria, A. et al., Design, fabrication and characterization of PCL electrospun scaffolds—a review. 2011.21(26): 9419-9453; Chaparro, FJ et al., Sintered electrospun polycaprolactone for controlled model drug delivery. 2019; Nam, J. et al., Modulation of embryonic mesenchymal progenitor cell differentiation via control over pure mechanical modulus inelectrospun nanofibers. 2011.7(4): 1516-1524; and Chaparro, FJ et al., Sintered electrospun poly(ε-caprolactone)–poly(ethylene terephthalate) for drug delivery. Journal of Applied Polymer Science, 2019.0(0): Page 47731), 0.5 g of a combination of PCL and sodium HEPES salt was dissolved in 10.0 g of HFP, and the solution was continuously stirred overnight at 40 °C. Five mass ratios of PCL to sodium HEPES salt (100:0; 99:1; 95:5; 92.5:7.5; 90:10) were investigated to evaluate the effect of salt-induced porous structure of PCL films on drug release. PCL solution was continuously fed using an injection pump at a feed rate of 3.0 mL / h to a 1.645 mm drum collector and at a feed rate of 1.0 mL / h to a 260 μm drum collector. A high-voltage DC generator was set to 24.0 kV to generate PCL nanofibers, which were deposited on 1.645 mm and 260 μm diameter 315 stainless steel rods with or without a primary chitosan layer to form bilayer and monolayer films, respectively.

[0246] Electrospun capsules were sintered in a vacuum at 100°C for 3 hours using an AccuTemp digital vacuum furnace to remove surface porosity, and then the capsules were gently removed from the rods (see Chaparro, FJ et al., Sintered electrospun polycaprolactone for controlled model drug delivery. 2019). The samples were washed with a saturated sodium bicarbonate solution to neutralize TFA, followed by washing with deionized water to dissolve and remove HEPES sodium salt. The capsules were vacuum-dried overnight. The outer diameter of the capsules prepared using a 1.645 mm rod was measured using a digital micrometer (Keyence) before and after sintering. The film thickness was calculated in the form of [sintered outer diameter of capsule - 1.645 mm] / 2. Images of the capsules prepared using a 260 μm diameter rod were obtained using an optical microscope (Cole-Parmer). The images were analyzed using Motic Image Plus to determine the outer diameter of the capsules. The film thickness was calculated in the form of [sintered outer diameter of capsule - 260 μm] / 2.

[0247] Capsule characterization

[0248] The morphological characteristics of the capsules were examined using scanning electron microscopy (SEM) (FEI, Quanta 200). Cross-sections of the chitosan fiber layer, PCL fiber layer, and bilayer and monolayer films before and after salt leaching were attached to a carbon ribbon placed on an aluminum stub base and sputter-coated with a gold-palladium layer. The capsules were immersed in liquid nitrogen and broken up to obtain cross-sections for imaging. The average fiber size and pore size of the PCL and chitosan layers were characterized, and quantification was performed using ImageJ (NIH) from SEM images of three samples.

[0249] Surface chemical analysis of the electrospun samples was performed using a Fourier transform infrared (FTIR) spectrometer (Thermo Scientific, Nicolet Nexus 670) in attenuated total reflectance (ATR) mode. Germanium crystals were placed in contact with the samples at an 8 cm⁻¹ depth. -1 100 scans were collected at a specific resolution. The image was taken at 1727 cm⁻¹. -1 and 1590cm -1The standard peak positions were used to identify PCL (carbonyl peak) and chitosan (amine band) respectively (see Elzein, T. et al., FTIR study of polycaprolactone chainorganization at interfaces. Journal of Colloid and Interface Science, 2004, 273(2): 381-387; and Osman, Z. and AKArof, FTIR studies of chitosan acetate based polymer electrolytes. Electrochimica Acta, 2003, 48(8): 993-999).

[0250] Drug release profile and loading / encapsulation efficacy

[0251] Hollow bilayer capsules with two open ends were obtained by removing the drum collector. For capsules with an inner diameter of 1.645 mm, 2.0 mg of BSA powder (model protein) or 2.0 mg of lyophilized bevacizumab powder (Avastin, anti-VEGF) dissolved in phosphate-buffered saline (PBS) at a concentration of 0.1 mg / μL was loaded into capsules that were sealed at the ends using a sealing device (Doug Care Equipment, TTS-8C) (see Chaparro, FJ et al., Sintered electrospun polycaprolactone for controlled model drug delivery. 2019; and Bernards, DA et al., Nanostructured thin film polymer devices for constant-rate protein delivery. 2012.12(10): pp. 5355-5361). For capsules with an inner diameter of 260 μm, considering the limited internal volume of the capsule, a concentrated 1.0 mg BSA or 1.0 mg bevacizumab slurry at a concentration of 1.0 mg / μL is loaded into the capsule using a 31-gauge needle.

[0252] The in vitro BSA release patterns of PCL monolayer capsules and PCL-chitosan bilayer capsules were obtained as described in the following steps. The capsules were immersed in 1 mL of PBS in 1.5 mL weakly binding centrifuge tubes to reduce binding of the centrifuge tubes to the eluted proteins. The centrifuge tubes containing the immersed capsules were incubated at 37°C to simulate physiological conditions. The eluent was collected at 1 h, 3 h, 6 h, 12 h, 24 h, 3 days, 1 week, 2 weeks, 1 month and thereafter every month (see Sousa, F. et al., A new paradigm for antiangiogenic therapy through controlled release of bevacizumab from PLGA nanoparticles. 2017.7(1): 3736; Yandrapu, SK et al., Nanoparticles in Porous Microparticles Prepared by Supercritical Infusion and Pressure Quench Technology for Sustained Delivery of Bevacizumab. Molecular Pharmaceutics, 2013.10(12): 4676-4686; and Tyagi, P. et al., Light-activated, in situ forming gel for sustained suprachoroidal delivery of bevacizumab. Molecular pharmaceutics, 2013.10(8): 2858-2867). Then, 1.0 mL of fresh PBS was added and incubation was maintained. The BSA release pattern was obtained by determining the uptake of eluted BSA via BCA analysis and quantifying the concentration using a BSA protein-based standard curve. The same protocol was used to obtain the bevacizumab eluent for in vitro bevacizumab release from PCL monolayer capsules and PCL-chitosan bilayer capsules.The characteristic absorbance of bevacizumab was identified at 277 nm using UV-Vis spectroscopy (Agilent, Cary 100UV-Vis), and the release rate of bevacizumab from the capsule was determined at 277 nm using a microplate reader (BioTek, Synergy HT). Quantification was performed based on standard curves of stock bevacizumab solutions at different concentrations (see Li, F. et al., Controlled release of bevacizumab through nanospheres for extended treatment of age-related macular degeneration. 2012.6: p. 54). Experiments were performed in triplicate.

[0253] To determine the rate of release of reactive bevacizumab from a 260 μm inner diameter capsule, enzyme-linked immunosorbent assay (ELISA) was performed as reported (see Tyagi, P. et al., Light-activated, in situ forming gel for sustained suprachoroidal delivery of bevacizumab. Molecular pharmaceutics, 2013.10(8): 2858-2867; and Varshochian, R. et al., Albuminated PLGA nanoparticles containing bevacizumab intended for ocular neovascularization treatment. Journal of Biomedical Materials Research Part A, 2015.103(10): 3148-3156). In short, 100 μL of a pH 9.6 sodium carbonate buffer solution containing 1 μg / mL VEGF recombinant human protein was immobilized overnight at 4°C on a 96-well Nunc Maxisorp plate (Thermo Fisher Scientific). The plate was blocked for 2 h at room temperature with 200 μL of PBS / T containing 2% BSA (0.05% v / v Tween 20 / pH 7.4 PBS) and washed three times with 300 μL of PBS / T. Then, the bevacizumab eluted from the capsule was diluted to 0 ng / mL to 10 ng / mL in 0.1% BSA-PBS / T solution (as determined by a standard curve), and 100 μL of the sample was added to each well and incubated for another 2 h at room temperature. Subsequently, the plate was washed three times with PBS / T, and 100 μL of HRP goat anti-human IgG Fc secondary antibody PBS / T solution (1:1000) was added to each well. The entire plate was incubated in the dark at room temperature for 1 h and washed five times with PBS / T. Color was indicated by adding 100 μL of TMB and stopped with 100 μL of 1N sulfuric acid. The concentration of active bevacizumab in each test sample was determined by comparing the absorbance at 450 nm with a standard curve.

[0254] Drug loading was determined by rupturing three different sizes of monolayer and bilayer capsules loaded with BSA and bevacizumab in PBS. In simple terms, the three types of monolayer and bilayer capsules loaded with BSA and bevacizumab were ruptured and immersed in 1 mL of PBS solution. The apparatus was vigorously washed five times with 1 mL of PBS using a vortex mixer. Each wash lasted at least ten minutes. The collected BSA and reactive bevacizumab eluents were determined by BCA analysis, UV-Vis spectroscopy, and ELISA. Drug encapsulation efficiency was calculated as free drug in eluent / total drug amount * 100%. Drug loading efficiency was calculated as drug loading / capsule weight * 100%. Cumulative release percentage was calculated as cumulative amount of drug eluted from the capsule / [drug loading * encapsulation efficiency] * 100%.

[0255] Capsule biodegradation

[0256] Long-term in vitro degradation and erosion of 260 μm inner diameter PCL monolayer capsules and PCL-chitosan bilayer capsules were determined by morphological changes. In short, capsules with two sealed ends and bilayer capsules with two open ends, incubated in PBS at physiological temperature for more than 9 months and 3 weeks respectively, were removed and vacuum-dried for characterization. Cross-sections of the PCL outer layer, chitosan inner layer, and monolayer and bilayer capsules were examined using SEM. Large cracks and pores were evaluated, and the average pore size of PCL layers prepared with different ratios of HEPES sodium salt was quantified by analyzing three different images using Image J. The pore size was compared with the initial pore size of the capsules before incubation using one-way ANOVA with a post-hoc Tukey test at a significance level of 0.05. Data are presented as mean ± standard deviation.

[0257] Cytotoxicity

[0258] The in vitro cytotoxicity of PCL monolayer capsules and PCL-chitosan bilayer capsules was evaluated by MTS analysis using human retinal pigment epithelial (ARPE-19) cells (see Sur, A. et al., Pharmacological protection of retinal pigmented epithelial cells by sulindac involves PPAR-α. 2014. 111(47): 16754-16759; Andrés-Guerrero, V. et al., Novel biodegradable polyesteramide microspheres for controlled drug delivery in ophthalmology. 2015. 211: 105-117; and Huhtala, A. et al., In vitro biocompatibility of degradable biopolymers in cell line cultures from various ocular tissues: extraction studies. Journal of Materials Science: Materials in Medicine, 2008. 19(2): 645-649). In all experiments, ARPE-19 cells were used at a rate of 4 × 10⁶ cells / year. 4 Cells were seeded at a density of 100 cells / well in 48-well plates. Cytotoxicity was analyzed using both the direct contact method and the extraction-exposure method. In the direct contact method, 1 cm PCL monolayer capsules or PCL-chitosan bilayer capsules were placed in the wells of the cell-seeded plates for 24 hours. In the extraction-exposure method, PCL monolayer capsules or PCL-chitosan bilayer capsules were immersed in 1 mL of fresh culture medium for 1 day, 3 days, 1 week, 2 weeks, and 1 month. At each time point, the capsule conditioned medium was transferred to ARPE-19 cell culture, and measurements were taken. Each sample was incubated with cells for 24 hours. For cytotoxicity analysis, cell culture medium was mixed with 20 μL of MTS reagent and then incubated at 37°C for 3 hours. The absorbance of the supernatant was measured at 490 nm using a microplate reader. Cell viability of the experimental groups was normalized relative to the control group (untreated). All experiments were repeated three times, and the data were analyzed using one-way ANOVA with a post-hoc graphical test at a significance level of 0.05. Data are presented as mean ± standard deviation.

[0259] Assessment of aggregation and anti-angiogenic activity

[0260] The stability of bevacizumab was determined using an ultra-high performance liquid chromatography (UHPLC) 3000 system (Thermo Fisher Scientific Inc., Waltham, MA) with a SEC-1000 column. To determine the stability of bevacizumab during the lyophilization process, 500 μL of 25 mg / mL bevacizumab (Avastin) was lyophilized using a Labconco lyophilizer, and the powder was then redissolved in 500 μL of PBS. The instability of concentrated bevacizumab was also assessed by diluting the bevacizumab slurry from the device to 25 mg / mL in PBS. Before injection, free native bevacizumab, concentrated bevacizumab, and bevacizumab eluted from monolayer and bilayer capsules at specific time points were filtered through a 0.2 μm Whatman SPARTAN HPLC syringe filter (VWR International, Radnor, PA) before and after lyophilization. The fractions of natural bevacizumab monomers, aggregates, and fragments were analyzed by deconvolving HPLC spectra to the separated individual elution peaks. The overall area of ​​the monomers, aggregates, and fragments was normalized to the total area of ​​the HPLC peaks to obtain the percentage of each component. The average molecular weight was then calculated from the fraction percentage (%) and the molecular weight of each component.

[0261] The anti-angiogenic activity of bevacizumab released from PCL monolayer capsules and PCL-chitosan bilayer capsules was further evaluated using capillary tube formation assay (see Elsaid, N. et al., PLGA microparticles entrapping chitosan-based nanoparticles for the ocular delivery of ranibizumab. 2016. 13(9): 2923-2940; Arnaoutova, I. and HKJNpKleinman, In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. 2010. 5(4): 628; and DeCicco-Skinner, KL et al., Endothelial cell tube formation assay for the in vitro study of angiogenesis. 2014(91)). More specifically, HUVECs were exposed to VEGF (5 ng / mL), an angiogenesis promoter, and mixed with: i) 10 μg / mL native bevacizumab; ii) 10 μg / mL bevacizumab released from a PCL monolayer capsule; and iii) 10 μg / mL bevacizumab released from a PCL-chitosan bilayer capsule (at 1 week, 2 weeks, 1 month, 3 months, 6 months, and 9 months). Six hours later, calcein AM was added to the cells, followed by incubation for 30 min. The cells were then directly observed visually using a fluorescence microscope (Nikon, Eclipse TS100) equipped with a digital camera (Qimaging). Three images were analyzed using ImageJ to quantify the length of the formed capillaries. For each sample, the total tubular length of the experimental group was normalized to the control group treated with VEGF, and all experiments were repeated three times. Data were analyzed by one-way ANOVA with a post-hoc graphical test, with a significance level of 0.05. Data are presented as mean ± standard deviation.

[0262] Injection feasibility

[0263] The feasibility of device injection was evaluated using fresh pig eyes obtained from a local slaughterhouse (Delaware Meats, Delaware, Ohio) (see Hoshi, S. et al., In Vivo and In Vitro Feasibility Studies of Intraocular Use of Polyethylene Glycol–Based Synthetic Sealant to Close Retinal Breaks in Porcine and Rabbit Eyes. 2015. 56(8): 4705-4711). The capsule was preloaded into a 21-gauge subcutaneous injection needle connected to a 1 mL syringe. The 21-gauge needle used in this study had an inner diameter similar to that of the commercial intraocular implant injector Ozurdex applicator (see Lee, SS et al., Biodegradable implants for sustained drug release in the eye. 2010. 27(10): 2043-2053). Intraocular injection was performed using the syringe needle at a depth of 3 mm posterior to the limbus. The capsule was injected into the porcine vitreous fluid using a small volume of PBS (100 μL) to mitigate the effects of increased IOP. After injection, the needle was removed, and the sclera was cut around the middle of the eye to verify the capsule's position in the vitreous fluid.

[0264] Electrospun chitosan and PCL nanofibers as building blocks for IBB capsules

[0265] The disclosed strategy for fabricating IBB capsules is based on a two-step coating of chitosan and PCL films onto a rod-shaped template, followed by removal from the template. To form a porous, centrally hollow bilayer structure, electrospinning is used. Electrospinning provides a high surface area to volume ratio for protein chemisorption and adjustable porosity for drug diffusion to achieve the desired functionality. Electrospinning as a method for nanofiber fabrication is based on using electricity to draw a charged polymer solution into nanoscale fibers. For the synthesis of chitosan nanofibers, processing parameters, including humidity and voltage, are optimized. For example, high humidity (above 30%) or low voltage (below 24 kV) causes significant charge loss at the spinneret and prevents the formation of fibers from the chitosan solution. Therefore, low humidity and a relatively high voltage are used. Simultaneously, the chitosan precursor is dissolved in TFA and DCM, as the addition of TFA better dissolves chitosan, while DCM allows for timely solvent evaporation, both of which are necessary for electrospinning. To place chitosan nanofibers onto a steel rod template, the chitosan nanofibers are directly collected on the rotating steel rod. Figure 2The SEM images shown indicate that the chitosan fibers have a diameter of 331.61 ± 186.19 nm and are highly interconnected, forming a highly porous structure that allows for efficient drug diffusion. However, the chitosan fiber pads were found to be fragile, consistent with reports of their low mechanical flexibility (see Jayakumar, R. et al., Biomedical applications of chitin and chitosan based nanomaterials—A short review. 2010. 82(2): pp. 227-232). To address this, a second layer of PCL was added, which not only provides physical encapsulation of the drug but also imparts improved flexibility. More specifically, PCL nanofibers with a diameter of 932.57 ± 399.42 nm were coated on top of the chitosan nanofibers (see Baker, SR et al., Determining the mechanical properties of electrospun poly-ε-caprolactone (PCL) nanofibers using AFM and a novel fiber anchoring technique. 2016. 59: pp. 203-212). To this end, nanofiber-based cylinders with high surface area, high mechanical flexibility, and strong adhesion between different layers were constructed as building blocks for IBB capsules.

[0266] Synthesis and characterization of injectable and bilayered microcapsules

[0267] Using two nanofiber layers as building blocks, a process is achieved by directly removing a steel rod template after electrospinning. Figure 3The hollow capsule structure is shown. While the bilayer PCL-chitosan nanofiber structure provides significant physical and electrostatic interactions with protein therapeutics, burst release can still occur due to the significantly larger size of the continuous porous structure of the nanofibers compared to the size of the protein (see Chaparro, FJ et al., Sintered electrospun polycaprolactone for controlled model drug delivery. 2019). Sintering is used to melt the PCL nanofiber layer to reduce its porosity and decrease burst release of the drug. On the other hand, directly coating the PCL layer without starting with the initial nanofiber structure makes it difficult to achieve a thin-layer structure on top of the chitosan, which is important for the preparation of injectable microcapsules. The mechanism for forming the bilayer structure based on sintering is based on the relatively low melting point of PCL (60°C) compared to the 220°C of chitosan nanofibers. Therefore, while PCL becomes mostly non-porous during the process of physically holding the drug, the chitosan remains porous for electrostatically binding the drug. Furthermore, this method also better integrates the bilayer during the melting process. Figure 3 As shown in the SEM images, the chitosan fiber layer adheres to the outer PCL layer, which stabilizes the bilayer structure because the molten PCL nanofibers increase the adhesion between the two layers. However, a framework composed of large fibers can still be observed on the PCL surface after sintering. During the sintering process, the film thickness decreases by 80% due to pressing and increased density; therefore, the capsule size can be controlled by adjusting the thickness of the chitosan and PCL fiber layers during the electrospinning process.

[0268] Following the sintering of chitosan and PCL fiber films, bilayer microcapsules with hollow structures were generated using a template strategy and by leveraging the mechanical robustness of the PCL outer layer. The size and structure of the capsules could be effectively controlled by manipulating the shape and size of the template rods. As a proof of concept, two sizes of monolayer PCL and bilayer chitosan-PCL capsules were prepared: one size with a larger inner diameter of 1.645 mm (pre-model), which could be used as a scaffold for implantation, and one size with a smaller inner diameter of 260 μm (final model), which could be injected via a 21-gauge needle. While the hollow structure of the steel rod template primarily allows for higher volumetric drug loading, the bilayer membrane provides both physical trapping and chemical non-covalent bonding for sustained, long-duration release.

[0269] In the device used for drug release studies, capsules with an inner diameter of 1.645 mm have an outer diameter of approximately 1.815 mm and a wall thickness of 89.36 ± 11.52 μm. Similarly, capsules with an inner diameter of 260 μm have an outer diameter of approximately 430 μm and a membrane thickness of 89.85 ± 4.27 μm, and these capsules are designed for injection via a 21-gauge needle. Increasing the capsule thickness enhances its mechanical properties, preventing breakage during injection. However, increasing the capsule size could potentially hinder intravitreal injection. Therefore, a wall thickness of 80–90 μm was determined to balance mechanical robustness and injection feasibility. Furthermore, membrane thickness is closely related to the drug diffusion rate; therefore, controlling and minimizing the thickness difference between monolayer and bilayer capsules reduces the impact of thickness on drug release.

[0270] Modulating the nanopore structure of bilayer membranes

[0271] After sintering, the PCL layer becomes non-porous and the drug release rate is significantly limited (see Chaparro, FJ et al., Sintered electrospun polycaprolactone for controlled model drug delivery. 2019). However, without sintering, the bilayer capsules become highly porous, resulting in undesirable high drug release rates. Therefore, a salt leaching method is used to precisely tune the 3D porous structure of the bilayer membrane to achieve long-term sustained drug release. More specifically, different amounts of water-soluble salts (HEPES) are mixed into the nanofibers during electrospinning. Before drug loading, the capsules are incubated in water to dissolve the HEPES inside the membrane, thereby reforming the porous structure on the bilayer membrane. By adjusting the concentration of HEPES in the PCL nanofibers, the porosity can be effectively controlled. Figure 4 As shown, the pore size and distribution height in the PCL sintered films depend on the mass ratio of salt to PCL. For example, at lower salt concentrations, the pores tend to be smaller and more dispersed across the entire surface of the film. However, low salt content also hinders the formation of interconnected pores for macromolecular diffusion and release. Table 1 shows the analytical pore size results for different ratios of PCL to HEPES sodium salt. For this purpose, PCL films prepared with salt concentrations above 5.0% were used for capsule fabrication and drug release studies because interconnected pore structures were observed in their cross-sections via SEM.

[0272] Table 1. Porosity and pore size of PCL membranes prepared with different ratios of PCL and sodium HEPES.

[0273] 0.0% HEPES salt none no 1.0% HEPES salt 237.26±96.93 no 5.0% HEPES salt 371.65±156.77 yes 7.5% HEPES salt 582.21±302.17 yes 10% HEPES salt 608.55±273.90 yes

[0274] To assess the changes in the bilayer structure before and after salt leaching, SEM imaging was used to observe the inner, outer, and cross-sectional surfaces of the bilayer capsules. Before salt leaching, the PCL sintered film was rough, containing embedded HEPES sodium salt crystals. After salt leaching, a porous structure appeared in the PCL layer, and the chitosan layer lost its fibrous structure and formed a porous layer. The average pore size of the chitosan layer was 802.47 ± 501.02 nm, which allowed for 3D diffusion of proteins and interactions with them to facilitate sustained release by maximizing their interaction with proteins through electrostatic interactions. The inner chitosan layer exhibited a more dense structure with nanopores up to 25 μm thick, which can be attributed to the relatively high melting point of chitosan. In contrast, the outer PCL layer had a denser structure with nanochannels running through it and a total thickness of 65 μm, supporting protein diffusion while physically capturing the drug. These results are consistent with the morphologies collected on the individual layers of PCL and chitosan.

[0275] To further confirm the chemical properties of the double-layer capsules after sintering and washing, the following was conducted: Figure 5 The final capsule shown was subjected to FTIR spectral analysis. In the spectrum shown, at 1727 cm⁻¹... -1 The significant peak at 2963 cm⁻¹ was assigned to the carbonyl group in PCL. -1 and 2995cm -1 The peak at 3478 cm⁻¹ represents CH stretching in the main chain of PCL. -1 Broad groups were observed at [location], which is attributed to the OH stretching vibration from hydroxyl groups, which are abundant in the chitosan backbone. Furthermore, at 1571 cm⁻¹... -1 The characteristic peaks of chitosan at the NH stretching point were assigned to the chitosan layer. These peaks provide strong evidence of the chemical properties of the chitosan and PCL layers, even after exposure to sintering. Therefore, the cationic chitosan remains active and can chemically and non-covalently bind the anionic protein bevacizumab. The feasibility of the disclosed bottom-up method for synthesizing hybrid nanostructured capsules with wide tunable pore size, high aspect ratio, and dimensionality, and providing optimal physical and chemical properties for drug loading and controlled release of protein therapeutics, was confirmed using SEM and FTIR.

[0276] Bevacizumab's high effective load and long-lasting drug release

[0277] To demonstrate the high effective loading of protein drugs achieved through the hollow structure of capsules, drug encapsulation efficacy was determined by rupturing drug-loaded capsules and assessing the amount of BSA and bevacizumab leakage from the capsules, respectively. BSA was used as the model protein drug, and bevacizumab is a clinically used anti-VEGF therapeutic agent for the treatment of AMD. Considering that both BSA and bevacizumab can adsorb to the chitosan layer of the bilayer capsule, both monolayer and bilayer capsules were used to assess drug effective loading. No significant difference in drug effective loading was found between the two types of capsules. Based on this study, the BSA encapsulation efficacy of the three large capsules and three small capsules was 100.39 ± 6.46% and 69.64 ± 7.15%, respectively. Lower encapsulation efficacy was observed when bevacizumab was loaded into both large and small capsules, assessed as 52.66 ± 6.47% by UV-Vis spectroscopy, a common instrument used to determine the concentration of proteins with characteristic absorbance of approximately 280 nm. Figure 12 As shown in the figure. However, a higher amount of reactive bevacizumab (729.02 ± 84.67 μg) was obtained by ELISA quantification, which gave about 70% bevacizumab encapsulation efficacy. The lower encapsulation efficiency can be attributed to the reduced sensitivity of UV-Vis spectroscopy to lower concentrations of bevacizumab, while the cumulative release can be effectively detected by ELISA. The loading capacity of the capsule was about 26.60 ± 1.90% w / w, which is higher than most reported loading capacities of 10-15% (see Li, F. et al., Controlled release of bevacizumab through nanospheres for extended treatment of age-related macular degeneration. 2012.6: p. 54; and Badiee, P. et al., Ocular implant containing bevacizumab-loaded chitosannanoparticles intended for choroidal neovascularization treatment. Journal of Biomedical Materials Research Part A, 2018.106(8): pp. 2261-2271).

[0278] like Figure 6 As shown, using the model drug BSA, the ability of the exemplary capsule to modulate drug release patterns by altering the capsule's surface morphology and porosity was demonstrated. As a proof of concept, single-layer and double-layer capsules with inner diameters of 1.645 mm and 260 μm were prepared, and samples prepared under salt leaching conditions of 5%, 7.5%, and 10% salt were also investigated. Figure 6BSA release morphologies for monolayer and bilayer capsules of two sizes are illustrated. As expected, burst release occurred in the 1.645 mm inner diameter PCL monolayer capsule during the first month, with over 75% of the loaded BSA eluting from the capsule, significantly limiting the duration of drug release. Burst release time in the 260 μm inner diameter capsule was shortened to two weeks, attributed to the increased surface area to volume ratio, and the cumulative release percentage was similar to that of the 1.645 mm inner diameter capsule during this period. Thus, the drug diffusion rate is accelerated in the smaller capsules. However, burst release followed stable release in both capsule sizes. The maximum drug release time for the 1.645 mm inner diameter monolayer capsule was approximately five months, while that for the 260 μm inner diameter capsule prepared with 10% salt was three months. The effect of HEPES sodium salt was also investigated. Higher salt concentrations resulted in faster release rates due to increased pore connectivity. In capsules with lower salt concentrations, burst release was slower but still uncontrollable.

[0279] In contrast, PCL-chitosan bilayer capsules showed no evidence of explosive release. The bilayer capsules significantly slowed BSA release. The release pattern of the bilayer capsules exhibited high linearity, which is summarized as follows: Figure 7 One month later, the 1.645 mm inner diameter bilayer capsules showed a greater ability to retain BSA within the device and released approximately 15% of the loaded BSA, which was 60% less than that released by monolayer PCL capsules over the same time period. Similarly, the 260 μm inner diameter bilayer capsules significantly reduced burst release. Only 25% of the BSA eluted from the 260 μm inner diameter bilayer capsules, which was higher than that from the 1.645 mm capsules because the area-to-volume ratio for diffusion was relatively larger. The chitosan layer effectively limited drug diffusion, and the porosity of the PCL shell did not play a significant role in controlling BSA release. There was no significant difference in diameter between monolayer and bilayer capsules (p>0.05), therefore the effect of thickness on drug release was negligible in these studies. Theoretically, based on the cumulative release data, the bilayer structure has the potential to control drug release for at least one year for both capsule sizes.

[0280] After a comprehensive study and optimization of the porous structure of the bilayer capsules, bevacizumab, a target drug for the clinical treatment of AMD, was loaded and released using capsules with a bilayer structure and leached with 10% HEPES. Consistent with BSA drug release experiments, sustained release patterns exceeding one year and nine months, respectively, were successfully achieved in 1.645 μm and 260 μm capsules, without significant initial burst release. Interestingly, during drug release based on the bilayer capsules, the burst release of bevacizumab was further reduced compared to BSA. This can be attributed to the increased molecular weight and lower effective charge of bevacizumab compared to BSA. In this case, pore size dominates the diffusion rate of bevacizumab. Bevacizumab with a higher molecular weight may be more difficult to elute from capsules with limited porous channels. This explains why the overall release of the monolayer capsules prepared with 5% HEPES salt had similar release kinetics to that of the bilayer capsules. Furthermore, the capsules prepared with 5% HEPES salt exhibited the lowest release rate compared to two other capsules with larger pores within the membrane. Notably, near-zero-order release kinetics were achieved after burst release in a 260 μm inner diameter bilayer capsule loaded with bevacizumab, which is significant. Figure 7 The result is shown in the figure (p<0.05).

[0281] Simple UV absorption was used to assess and quantify the amount of bevacizumab, but it could not indicate reactive bevacizumab or distinguish the background of broken polymers from the capsule over time. Therefore, bevacizumab eluted from the 260 μm capsule was re-determined by ELISA. Figure 13 The overall trend of bevacizumab release was consistent with previous results obtained by UV-Vis spectroscopy. For example, the long-term cumulative release of bevacizumab from monolayer capsules prepared with 5% HEPES salt was assessed to be approximately 160 μg by UV-Vis spectroscopy, which was the same as that characterized by ELISA over nine months. Therefore, by comparing the release results of UV-Vis and ELISA, the release pattern obtained by UV-Vis is reliable and can provide the overall trend of bevacizumab release from both monolayer and bilayer capsules. Similarly, over nine months, the capsules prepared with 5% salt had a relatively slower release rate compared to those prepared with 7.5% and 10% salt. Based on these considerations, bilayer capsules with higher (7.5% and 10%) HEPES salt concentrations were then identified for long-term release against VEGF. Furthermore, the high drug loading capacity and stable drug release pattern over nine months strongly indicate the potential of the exemplary bilayer capsule as a universal platform for delivering anti-VEGF therapeutics.

[0282] Capsule biodegradation

[0283] The in vitro degradation of PCL monolayer capsules and chitosan-PCL bilayer capsules was investigated. Capsules with two closed ends were placed in PBS at 37°C for over nine months, then removed and characterized by SEM. The mechanical integrity of the entire device was determined primarily by the slow hydrolysis of the PCL layer at the ester bonds (see Darwis, D. et al., Enzymatic degradation of radiation crosslinked poly(ε–caprolactone). Polymer Degradation and Stability, 1998, 62(2): 259-265). Therefore, assessing the erosion and degradation of the PCL layer over time is important. Figure 11 As shown in the characteristic SEM images, both monolayer and bilayer capsules remained intact after nine months of incubation. However, compared to the initial capsules, the pores on the PCL membrane surface became larger and more dispersed after nine months, indicating slow degradation of the PCL membrane. Table 2 summarizes the analytical pore sizes of the capsules under different HEPES salt conditions. The average diameter of the pores on the PCL surface increased significantly by approximately 180 nm (p ≤ 0.05), but the entire device remained intact without any obvious breakage or fragmentation. The bilayer capsules were also characterized. After nine months, the chitosan layer remained tightly adhered to the PCL layer, and the fibers remained well-defined and intact. The fibrous structure of the chitosan surface layer remained clear without any significant changes. Furthermore, the membrane thickness of both monolayer and bilayer capsules ranged from 80 μm to 90 μm, similar to its original thickness before incubation. However, as... Figure 15As shown, when capsules with two open ends were immersed in PBS at physiological temperature for more than three weeks, a significant reduction and loss of chitosan fiber thickness was observed. This may be due to the slow degradation of chitosan when exposed to direct water for a long period of time (see Kean, T. and M. Thaou, Biodegradation, biodistribution and toxicity of chitosan. Advanced Drug Delivery Reviews, 2010, 62(1): 3-11; and Onishi, H. and Y. Machida, Biodegradation and distribution of water-soluble chitosan in mice. Biomaterials, 1999, 20(2): 175-182). Furthermore, due to the weak mechanical properties of chitosan, the inner chitosan fiber membrane can become fragile under shear stress during long-term incubation in water, leading to significant chitosan loss (see Sangsanoh, P. and PJBSupaphol, Stability improvement of electrospun chitosan nanofibrous membranes in neutral or weak basic aqueous solutions. 2006.7(10): pp. 2710-2714; and Chen, Z. et al., Mechanical properties of electrospun collagen–chitosan complex single fibers and membrane. Materials Science and Engineering: C, 2009.29(8): pp. 2428-2435). Therefore, a hydrophobic PCL layer can prevent the inner chitosan layer from breaking down and further reduce the degradation of the entire device.

[0284] Table 2. Porosity and pore size of PCL membranes prepared with different ratios of PCL and HEPES sodium salt after one year of incubation. After nine months of incubation, the pore size of PCL was found to increase significantly at different salt concentrations (p≤0.05).

[0285] 5.0% HEPES salt 550.83±243.33 179.81±16.71 7.5% HEPES salt 662.00±238.43 79.79±22.26 10% HEPES salt 935.17±331.41 326.62±24.82

[0286] Cytotoxicity

[0287] One of the most critical properties of any drug delivery device is acceptable short-term and long-term biocompatibility in the presence of target cells or tissues. To this end, cytotoxicity of an exemplary bilayer capsule was studied using retinal pigment epithelial (ARPE-19) cells, as they are the most prevalent cells in the retina and are highly sensitive to toxicity and exogenous materials by both direct contact and extraction exposure methods. Cell viability of RPE cells was measured with and without treatment of monolayer and bilayer capsules using standard mitochondrial activity assays. Figure 8 The results shown indicate that both PCL monolayer capsules and PCL-chitosan bilayer capsules exhibited negligible toxicity to RPE cells during a 24-hour direct incubation period. Both PCL and chitosan are known for their good biocompatibility in intraocular applications (see Kim, J. et al., Long-term intraocular pressure reduction with intracameral polycaprolactone glaucoma devices that deliver a novel anti-glaucoma agent. 2018. 269: 45-51; and Wassmer, S. et al., Chitosan microparticles for delivery of proteins to the retina. Acta Biomaterialia, 2013. 9(8): 7855-7864). Similarly, extracts from both capsules did not affect cell viability within one month, which is... Figure 8 The results show that the bilayer capsules retained over 95% of their activity even at a time point up to one month old. These results collectively indicate negligible cytotoxicity to retinal pigment epithelial cells and suggest the potential of the bilayer capsules for preclinical evaluation in ocular models in future studies.

[0288] The stability of the released VEGF inhibitors and their therapeutic effect against angiogenesis.

[0289] A major problem hindering the development of long-term sustained protein delivery systems is the aggregation and degradation of proteins in aqueous environments. Bevacizumab is physiologically unstable and tends to degrade and aggregate in vivo over time (see Courtois, F. et al., Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab. mAbs, 2016.8(1): 99-112; Oliva, A., M. Llabrés and JB). Capability measurement of size-exclusion chromatography with a light-scattering detection method in a stability study of bevacizumab using the process capability indices. Journal of Chromatography A, 2014, 1353: 89-98; Latypov, RF et al., Elucidation of acid-induced unfolding and aggregation of human immunoglobulin IgG1 and IgG2 Fc. Journal of Biological Chemistry, 2012, 287(2): 1381-1396; and Bakri, SJ et al., Six-month stability of bevacizumab (Avastin) binding to vascular endothelial growth factor after withdrawal into a syringe and refrigeration or freezing. Retina, 2006, 26(5): 519-522). In addition, the device was loaded with highly concentrated bevacizumab obtained by freeze-drying. Aggregation and loss of activity may occur at high concentrations or during the lyophilization process (see Varshochian, R. et al., The protective effect of albumin on bevacizumab activity and stability in PLGA nanoparticles intended for retinal and choroidal neovascularization treatments. European Journal of Pharmaceutical Sciences, 2013, 50(3): 341-352). This becomes important because bevacizumab aggregates may not be released from the implant at the same rate as monomers. Therefore, bevacizumab stability studies are required to assess the aggregation and fragmentation of bevacizumab over time during device fabrication and device incubation using HPLC. The analytical aggregation and fragmentation of bevacizumab are summarized in Table 3, and Figure 14The HPLC chromatograms are shown in Table 3. To confirm the stability of bevacizumab during lyophilization, the HPLC chromatograms of lyophilized bevacizumab were compared with those of the commercial bevacizumab Avastin. 16% aggregates were formed in free native bevacizumab, and a slight increase in bevacizumab aggregates was observed during lyophilization cycles. Additionally, aggregation in concentrated solutions was assessed by dilution in PBS followed immediately by HPLC characterization. However, no change in bevacizumab aggregates was observed, indicating that the protein is fairly stable in concentrated solutions and also confirming that the exemplary device slows the release of bevacizumab monomers. The potency of bevacizumab released over extended periods from monolayer and bilayer capsules was also evaluated. The percentage of aggregates varied between 11% and 16% for both types of capsules over three months, and fragmentation of bevacizumab was also observed during extended incubation. However, the potency of bevacizumab remained high during this period, as shown in Table 3. At one month, 84% of the bevacizumab monomers were eluted from the 260 μm inner diameter PCL monolayer capsule, and this figure decreased slightly to 79% at three months. Similarly, 82% of the bevacizumab monomers were released from the 260 μm inner diameter chitosan-PCL bilayer capsule during the first three months. This enhanced stability can be attributed to the attachment to chitosan via ionic binding to the glycoprotein and increased bioavailability. Additionally, the hydrophobic PCL layer slows the fragmentation process by reducing fluid exchange through the capsule. Consequently, the potency of the long-term released bevacizumab is well preserved, demonstrating the good potential of the exemplary capsule for the treatment of AMD without frequent injections.

[0290] Table 3. Analysis of aggregated and fragmented free natural bevacizumab before and after freeze-drying, and bevacizumab eluted from monolayer and bilayer capsules at 1 month and 3 months.

[0291]

[0292] While HPLC provides clear information about bevacizumab monomers and aggregates delivered by the exemplary capsules, ELISA characterizes the potency and quantity of VEGF-responsive bevacizumab for long-term release to ensure its effect on angiogenesis. Therefore, a bevacizumab ELISA was performed to determine the reactive bevacizumab released from a 260 μm inner diameter capsule over time. After one month, the release rate of active bevacizumab remained at approximately 20 μg / mL per month, similar to the amount of bevacizumab determined by UV-Vis. Furthermore, the percentage of eluted bevacizumab bioactivity was calculated by comparing the cumulative release percentage measured by ELISA with that measured by UV / Vis. The results show that bevacizumab released from the monolayer capsule maintained over 90% of its bioactivity over nine months, indicating its potential in protecting proteins. Fluctuations in bioactivity were observed in the bilayer capsule, where bioactivity remained at approximately 80%. The lower percentage of bioactivity could be caused by increased UV-VIS absorbance background resulting from the slow biodegradation of the inner layer during prolonged incubation, as mentioned above. However, both results strongly support the high bioactivity of proteins protected by both single-layer and double-layer capsules. In this regard, exemplary hollow double-layer capsules that physically protect drugs have the potential to overcome this barrier to sustained release.

[0293] In addition, such as Figure 9 As shown, the inhibitory effect of bevacizumab eluted from PCL monolayer and PCL-chitosan bilayer capsules on VEGF-induced tubule growth was evaluated in a tube formation analysis using HUVEC. At a concentration of 10 μg / mL, the positive control, native bevacizumab, induced a 93.15 ± 1.49% inhibition of tubule length. After one month, bevacizumab eluted from monolayer and bilayer capsules induced approximately 13.33 ± 6.51% and 12.33 ± 4.63% of tubule formation, respectively, for capsules with an inner diameter of 260 μm and 1.645 mm, respectively, which was more effective than routine injection of native bevacizumab. After three months, a slight increase in tubule length formation was observed, attributed to loss of bioactivity due to prolonged incubation at physiological temperatures. However, there was no significant difference in the anti-angiogenic properties between bevacizumab eluted from bilayer capsules and bevacizumab eluted from monolayer capsules (p > 0.05). The expected slow drug diffusion delays the enzymatic breakdown of bevacizumab, which significantly protects the proteins within the capsule. Overall, the anti-angiogenic bioactivity remained well maintained at a high level over nine months, suggesting the capsule's potential protective effect against long-term drug release.

[0294] Injection feasibility

[0295] In addition to high drug loading capacity, sustained release of protein therapeutic agents, and maintenance of anti-VEGF bioactivity, these capsules can also be formulated as injectable formulations. To demonstrate this, a feasibility test was conducted by delivering a 10mm long capsule through the sclera using a 21-gauge needle into isolated porcine vitreous fluid. Figure 10 As shown in the figure. A capsule with an outer diameter of 430 μm was used in this study because its size is similar to that of the commercially available intraocular implant Ozurdex, which has a diameter of 460 μm and a length of 6 mm. The Ozurdex applicator is equipped with a 22-gauge TSK needle (see Chan, A., L.-S. Leung and MS Blumenkranz, Critical appraisal of the clinical utility of the dexamethasone intravitreal implant). for the treatment of macularedema related to branch retinal vein occlusion or central retinal vein occlusion. Clinical Ophthalmology (Auckland, NZ), 2011.5: page 1043; Arcinue, CA, OMCerón and CSFoster, A comparison between the fluocinolone acetonide (Retisert) and dexamethasone (Ozurdex) intravitreal implants in uveitis. Journal of ocular pharmacology and therapeutics, 2013.29(5): pages 501-507; and Querques, L. et al., Repeated intravitreal dexamethasone implant For retinal veinocclusion. Ophthalmologica, 2013, 229(1): pp. 21-25). The inner diameter of the needle is approximately 500 μm, which can be adapted to the exemplary capsule (see Meyer, CH et al., Penetration force, geometry, and cutting profile of the novel and old Ozurdex needle: the MONO study. Journal of Ocular Pharmacology and Therapeutics, 2014, 30(5): pp. 387-391). More specifically, in clinical applications, anti-VEGF-loaded capsules can typically be delivered intravitreal via a similar applicator, avoiding invasive open surgery. Therefore, the advanced drug delivery system based on the exemplary bilayer capsule is fully compatible with currently used clinical methods.

[0296] In this study, to address a significant challenge in long-term therapies for wet AMD, a polymer-based microstructure delivery platform was designed and developed to achieve sustained in vitro release of anti-VEGF agents. Sustained protein release was achieved by designing and optimizing the structure of chitosan-PCL bilayer microcapsules using combinatorial materials chemistry and engineering methods. Key features of these chitosan-PCL microcapsules include the size and porosity of the PCL shell, and a hollow structure for simple and concise drug loading.

[0297] PCL-chitosan microcapsules were synthesized using a novel combination of electrospinning, sintering, and salt leaching. In the preliminary study, attention should be paid to the loss of structure of the chitosan fibers after salt leaching. It is believed that the formation of trifluoroacetate during fiber preparation accelerates the dissolution of chitosan when using TFA and DCM as solvents; therefore, a necessary step of neutralization with sodium bicarbonate solution during washing is required to reduce the effect of acidic salts on the bioactivity of bevacizumab (see Sangsanoh, P. and PJBSupaphol, Stability improvement of electrospun chitosan nanofibrous membranes in neutral or weak basic aqueous solutions. 2006.7(10): pp. 2710-2714).

[0298] Membrane thickness is related to drug release time. Theoretically, thicker membranes result in slower drug diffusion. While increasing capsule size could potentially help achieve slower drug release, the increased microcapsule size would hinder injection through small needles. Therefore, thinner membranes are needed to prepare injectable capsules for clinical applications. A chitosan layer was added to address this issue. In this study, all capsules had a thickness between 80 and 95 μm, which minimized the impact of thickness on exploring the relationship between drug release rate and the chitosan-PCL complex.

[0299] After optimizing these key factors to control drug release, the performance of the microcapsules was evaluated and optimized for sustained anti-VEGF release. Bevacizumab has been used clinically to treat wet AMD since 2004 (see Michels, S. et al., Systemic bevacizumab (Avastin) therapy for neovascular age-related macular degeneration: twelve-week results of an uncontrolled open-label clinical study. 2005. 112(6): 1035-1047.e9). Theoretically, the isoelectric point (pI) of bevacizumab is 7.8 (see Nomoto, H. et al., Pharmacokinetics of bevacizumab after topical, subconjunctival, and intravitreal administration in rabbits. 2009. 50(10): 4807-4813). At pH 7.4, its net charge calculated from the pI will be slightly positive, as reported by numerous studies.However, protein aggregates in water and other organic solvents typically used during device fabrication have the potential to reduce bioactivity and cause undesirable side effects (see Varshochian, R. et al., Albuminated PLGA nanoparticles containing bevacizumab intended for ocular neovascularization treatment. Journal of Biomedical Materials Research Part A, 2015. 103(10): 3148-3156; Courtois, F. et al., Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab. mAbs, 2016. 8(1): 99-112; and Varshochian, R. et al., The protective effect of albumin on bevacizumab activity and stability in PLGA nanoparticles intended for retinal and choroidal neovascularization treatments. European Journal of Pharmaceutical). Sciences, 2013, 50(3): 341-352. Therefore, PBS is widely used to suspend bevacizumab to maintain its stability and biological activity.Bevacizumab has been reported to carry a net negative charge in PBS at pH 7.4, which suggests binding to chitosan and the possibility of providing a more sustained release from the exemplary capsule (see Li, SK et al., Effective electrophoretic mobilities and charges of anti-VEGF proteins determined by capillary zone electrophoresis. 2011.55(3): 603-607; and García-Quintanilla, L. et al., Pharmacokinetics of Intravitreal Anti-VEGF Drugs in Age-Related Macular Degeneration. Pharmaceutics, 2019.11(8): 365). The binding of buffer ions in PBS to bevacizumab increases its hydrophilicity, which further increases its stability and causes a discrepancy between the theoretical and experimental net charge of the protein (see Li, SK et al., Effective electrophoretic mobilities and charges of anti-VEGF proteins determined by capillary zone electrophoresis. 2011. 55(3): 603-607; and Chopra, P., J. Hao and SKJIjopLi, Iontophoretic transport of charged macromolecules across human sclera. 2010. 388(1-2): 107-113). Therefore, bevacizumab is negatively charged in the vitreous body and capsule; thus, it is assumed that this protein can be trapped by positively charged chitosan via electrostatic attraction. Similarly, BSA is a negatively charged protein in water with an isoelectric point of approximately 4.7. Under physiological conditions (in PBS), BSA can bind to cations and increase its surface charge.However, as previously reported, BSA remains negatively charged in PBS because these ions have a smaller effect on the charge of BSA (see Li, SK et al., Effective electrophoretic mobilities and charges of anti-VEGF proteins determined by capillary zone electrophoresis. 2011. 55(3): 603-607; and Alis, A. et al., Measurements and Theoretical Interpretation of Points of Zero Charge / Potential of BSA Protein. Langmuir, 2011. 27(18): 11597-11604). The desired sustained drug release profile can be achieved by providing a combined electrostatic interaction between bevacizumab or BSA and chitosan, along with protection from the PCL shell.

[0300] In this study, the release rate of bevacizumab with similar effective loads was also significantly improved. The average effective load of the reported devices varied from 500 μg to 1000 μg (see Li, F. et al., Controlled release of bevacizumab through nanospheres for extended treatment of age-related macular degeneration. 2012.6: p. 54; and Varshochian, R. et al., Albuminated PLGA nanoparticles containing bevacizumab intended for ocular neovascularization treatment. Journal of Biomedical Materials Research Part A, 2015.103(10): 3148-3156). However, these devices have limitations, including not being injectable or providing non-sustained release for more than three months. The drug loading capacity of these devices differed from what was expected. In addition, the anti-VEGF bioactivity in these devices may be affected during the fabrication process due to interactions between the therapeutic agent and the solvent or high temperatures. However, after device fabrication, drug loading was processed to avoid drug loss and inactivation typically occurring with conventional preparation methods such as emulsions. Therefore, the capsule designed in this paper ensures a drug loading of 700 μg of bevacizumab due to the limited space within the injectable capsule and the high molecular weight of bevacizumab. The template rod can be selectively enlarged to expand the internal space and enhance drug loading. Additionally, the dried powder of bevacizumab can be precisely replaced and loaded under a microscope within the drug, further enhancing drug loading efficiency and bevacizumab stability. Furthermore, the exemplary device can be used to evaluate other therapeutic agents with lower molecular weights similar to the model drug BSA and has the potential to further significantly increase drug loading (see Rosenfeld, PJ, et al., Optical coherence tomography findings after an intravitreal injection of bevacizumab). for neovascular age-related maculardegeneration. 2005.36(4): pp. 331-335).

[0301] The bilayer capsule can efficiently control the drug release rate by utilizing the electrostatic interaction between the protein therapeutic agent and the polymer, which can solve many of the current problems associated with the clinical treatment of wet AMD. It also provides an alternative approach for some diseases requiring long-term treatment with protein therapeutic agents, such as colorectal cancer, breast cancer, and some brain tumors. However, further optimization of the device fabrication methods may still be needed to meet the needs for different protein therapeutic agents with great potential for ophthalmic, cancer, and other biomedical applications.

[0302] In summary, a polymer-based delivery platform for controlled release of anti-VEGF has been developed. This platform is based on a bilayer microstructure that synergistically combines the electrostatic binding between chitosan and anti-VEGF with a protective hydrophobic PCL layer to provide an efficient pathway for modulating polymer-protein interactions to achieve controlled therapeutic release. The bilayer structure was characterized in detail, and the performance of the capsule for protein delivery was further determined. Most importantly, the exemplary designed delivery platform significantly improves the long-term in vitro release of anti-VEGF compared to most current devices, supporting its potential for treating AMD. Future research should evaluate and further optimize the therapeutic effect of anti-VEGF-loaded devices in in vivo AMD models.

[0303] It will be apparent to those skilled in the art that various modifications and variations may be made to this disclosure without departing from its scope or spirit. Other embodiments of this disclosure will be apparent to those skilled in the art from the considerations of this specification and from the practice of this disclosure. This specification and embodiments are intended to be considered exemplary only, and the true scope and spirit of this disclosure are indicated by the following claims.

Claims

1. A drug delivery composition comprising: A capsule having a tubular shape with two closed ends, wherein the capsule comprises a double-walled structure and a tubular compartment; and Therapeutic agent, the therapeutic agent being present in the lumen compartment; The double-walled structure comprises an inner layer and an outer layer; The inner layer contains chitosan; The outer layer independently comprises poly(ε-caprolactone) (PCL); The therapeutic agent has a net negative charge at any pH value within the range of pH 6.9 to pH 7.4; and The therapeutic agent mentioned above is a therapeutic protein.

2. The drug delivery composition of claim 1, wherein the capsule has a length of 0.1 cm to 5 cm.

3. The drug delivery composition of claim 2, wherein the capsule has a length of 0.5 cm to 3 cm.

4. The drug delivery composition of claim 2, wherein the capsule has a length of 1 cm to 3 cm.

5. The drug delivery composition of claim 1, wherein the capsule has an inner diameter of 100 μm to 2000 μm.

6. The drug delivery composition of claim 5, wherein the inner diameter is from 100 μm to 500 μm.

7. The drug delivery composition of claim 5, wherein the inner diameter is from 100 μm to 300 μm.

8. The drug delivery composition of claim 5, wherein the capsule has an outer diameter that is 100 μm to 300 μm larger than the inner diameter.

9. The drug delivery composition of claim 1, wherein the double wall has a wall thickness of 50 μm to 150 μm.

10. The drug delivery composition of claim 9, wherein the wall thickness is 70 μm to 100 μm.

11. The drug delivery composition of claim 9, wherein the wall thickness is 75 μm to 95 μm.

12. The drug delivery composition of claim 9, wherein the wall thickness is 80 μm to 90 μm.

13. The drug delivery composition of claim 1, wherein the outer layer further comprises pores with a pore diameter of 100 nm to 10,000 nm.

14. The drug delivery composition of claim 13, wherein the pore diameter is 350 nm to 650 nm.

15. The drug delivery composition of claim 1, wherein the outer layer comprises fibers with a diameter of 100 nm to 2000 nm.

16. The drug delivery composition of claim 15, wherein the fiber has a diameter of 500 nm to 1000 nm.

17. The drug delivery composition of claim 1, wherein the inner layer comprises fibers with a diameter of 50 nm to 1000 nm.

18. The drug delivery composition of claim 17, wherein the fiber has a diameter of 100 nm to 400 nm.

19. The drug delivery composition of claim 1, wherein the therapeutic agent is an anti-VEGF therapeutic agent.

20. The drug delivery composition of claim 1, wherein the therapeutic protein is a therapeutic antibody selected from bevacizumab, ranibizumab, IBI305, or combinations thereof.

21. The drug delivery composition of claim 1, wherein the therapeutic protein is a VEGF decoy receptor.

22. The drug delivery composition of claim 21, wherein the VEGF decoy receptor is aflibercept.

23. The drug delivery composition of claim 19, wherein the anti-VEGF therapeutic agent is a tyrosine kinase inhibitor.

24. The drug delivery composition of claim 1, wherein the therapeutic agent is present in an amount of 0.1 mg to 3 mg.

25. The drug delivery composition of claim 24, wherein the therapeutic agent is present in an amount of 0.5 mg to 2 mg.

26. The drug delivery composition of claim 24, wherein the therapeutic agent is present in an amount of 0.5 mg to 1.5 mg.

27. The drug delivery composition of claim 1, wherein the therapeutic agent exhibits near-zero order release kinetics over a period of at least 30 days when measured using a PBS release assay.

28. The drug delivery composition of claim 27, wherein the therapeutic agent exhibits near-zero order release kinetics over a period of at least 45 days.

29. The drug delivery composition of claim 27, wherein the therapeutic agent exhibits near-zero order release kinetics over a period of at least 90 days.

30. Use of the drug delivery composition of any one of claims 1-18 and 24-29 for the preparation of a medicament for treating age-related macular degeneration in a subject of need, wherein the medicament is bevacizumab.

31. The use as described in claim 30, wherein the treatment comprises injection into the vitreous cavity of the eye.

32. The use as described in claim 30, wherein the treatment includes intravitreal injection, subconjunctival injection, subcapsular injection, retroocular injection, or suprachoroidal injection.

33. A method for preparing a drug delivery capsule for injection into the eye of a subject, the method comprising: An inner layer containing chitosan is formed on the conductive rod; and An outer layer is formed on the inner layer, wherein the outer layer comprises poly(ε-caprolactone) (PCL); The formation of the inner layer includes electrospinning using a solution of the chitosan and a voltage difference of 20 kV to 30 kV; The chitosan solution is a 1 w / v% to 10 w / v% solution in at least one organic solvent; The outer layer is formed by electrospinning a solution containing the poly(ε-caprolactone) (PCL) and optionally a pore-forming agent onto the inner layer; and the voltage difference used for electrospinning is 20 kV to 30 kV. The solution containing PCL and the optional porogen is a 1 w / v% to 10 w / v% solution based on the total weight of PCL and the porogen; and The weight ratio of PCL to the optional porogen is 90:100 to 100:

0.

34. The method of claim 33, wherein at least one organic solvent in the chitosan solution is a mixture of trifluoroacetic acid and dichloromethane; and wherein the trifluoroacetic acid and the dichloromethane are present in a ratio of 1:10 to 10:

1.

35. The method of claim 34, wherein the trifluoroacetic acid and the dichloromethane are present in a ratio of 5:3 to 10:

3.

36. The method of claim 35, wherein the trifluoroacetic acid and the dichloromethane are present in a ratio of 7:

3.

37. The method of claim 33, wherein the weight ratio of PCL to the porogen is from 90:100 to 99.9:0.

1.

38. The method of claim 37, wherein the weight ratio of PCL to the porogen is 90:100 to 95:

5.

39. The method of claim 37, wherein the weight ratio of PCL to the porogen is from 95:5 to 99.9:0.

1.

40. The method of claim 33, wherein the solution comprising PCL and the porogen is a solution of 2.5 w / v% to 10 w / v% based on the total weight of PCL and the porogen.

41. The method of claim 40, wherein the solution comprising PCL and the porogen is a solution of 5 w / v% to 10 w / v% based on the total weight of PCL and the porogen.

42. The method of claim 33, further comprising sintering the drug delivery capsule after forming the outer layer.

43. The method of claim 42, wherein sintering comprises heating at a temperature of 50°C to 150°C for a period of 1 minute to 6 hours.

44. The method of claim 42, wherein sintering comprises heating at a temperature of 90°C to 110°C for 30 minutes to 6 hours.

45. The method of claim 42, further comprising washing the drug delivery capsule after sintering.

46. ​​The method of claim 45, wherein washing comprises washing the drug delivery capsule with an alkaline solution, an aqueous solution, or a combination thereof.

47. The method of claim 45, wherein washing comprises washing with a saturated sodium bicarbonate solution, followed by washing with deionized water.

48. The method of claim 45, further comprising drying the drug delivery capsule after washing.

49. The method of claim 48, wherein drying is carried out in a vacuum at a temperature of 50°C to 150°C for a period of 1 minute to 6 hours.

50. The method of claim 48, wherein drying is carried out in a vacuum at a temperature of 90°C to 110°C for a period of 30 minutes to 6 hours.

51. A drug delivery capsule prepared by the method described in any one of claims 33-50.

52. The drug delivery capsule of claim 51, further comprising a therapeutic agent, wherein the therapeutic agent has a net negative charge at any pH value from pH 6.9 to pH 7.4, and wherein the therapeutic agent is a therapeutic protein.

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