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 the delivery of protein therapeutics (e.g., intravitreal delivery of protein therapeutics to the eye). In particular, the present disclosure describes drug delivery devices for injection into the eye of a subject in need thereof, comprising a capsule having a bilayer wall and a therapeutic agent, the therapeutic agent initially present within the lumenal space of the capsule. Methods of making and using these drug delivery devices are also described.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of 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.

[0002] The present disclosure relates to drug delivery compositions, and more particularly to compositions containing one or more multi-layered drug delivery capsules for the delivery of therapeutic agents to the eye. [Background technology]

[0003] Age-related macular degeneration (AMD) is the fourth most common cause of blindness worldwide after cataracts, premature birth, and glaucoma. Over 11 million people in the United States are diagnosed with wet AMD. It is estimated that this number will double in 30 years. Thus, much research has been conducted to understand the disease pathogenesis and develop treatment methods. It is widely known that age-related overexpression of vascular endothelial growth factor (VEGF) stimulates angiogenesis in the choroid, which leads to irreversible damage to the retina during bleeding and scarring of the newly formed blood vessels. The current gold standard treatment for wet AMD is monthly intravitreal injections of anti-VEGF (such as bevacizumab or ranibizumab) to inhibit VEGF and prevent angiogenesis. However, frequent injections often lead to infection, elevated intraocular pressure, and rhegmatogenous retinal detachment, as well as problems with patient compliance.

[0004] Recently, there have been reports of novel devices such as implants and microparticles / nanoparticles for long-term drug delivery in the eye. Unfortunately, such implants require surgical procedures for implantation and removal. Furthermore, currently known implantable devices tend to be off-target and reduce drug efficacy. Although the microparticles or nanoparticles have a relatively small size suitable for injection into the eye through a 30-gauge needle, the currently described microparticles or nanoparticles release therapeutic agents (such as anti-VEGF therapeutics) over a rapid release window due to the biodegradation of known particle compositions in the first three months.

[0005] Thus, despite the significant efforts directed towards the treatment of AMD or other ophthalmic disorders, there remains a lack of methods and compositions that minimize the adverse side effects of currently available treatment regimens.Furthermore, there remains a need for drug delivery systems and compositions that are biodegradable and can control drug release for up to 9 months or more after intravitreal injection.There remains a need for improved therapeutic approaches for the treatment of AMD and other ocular diseases that require the direct delivery of therapeutic agents to the eye.These and other needs are met by the present disclosure. Summary of the Invention

[0006] In accordance with the object(s) of the disclosure, as embodied and broadly described herein, the disclosure relates in one aspect to compositions, devices, and processes for the delivery of protein therapeutics, such as intravitreal delivery of protein therapeutics to the eye. The disclosed drug delivery compositions include a capsule having a bilayer wall and a therapeutic agent contained therein. In a further aspect, the disclosure relates to a method of treating an ophthalmic disease or disorder.

[0007] Thus, in one embodiment, one or more capsules each having a tubular shape with two closed ends, each of the one or more capsules independently comprising a multi-layered wall and at least one lumen compartment; and one or more therapeutic agents, each initially present within one or more of the at least one luminal compartment; Includes; Each multi-layer wall independently includes at least an inner layer and an outer layer; each inner layer comprises a first polymer having a net positive charge under physiological conditions; each outer layer independently comprises a second polymer different from the first polymer; A drug delivery composition is provided.

[0008] In some embodiments, the drug delivery composition may include two or more capsules. In some embodiments, a different therapeutic agent is 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.

[0009] In some embodiments, at least one or more of the capsules comprises two or more lumen compartments. In some embodiments, a different therapeutic agent is 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.

[0010] In some embodiments, the first polymer may comprise chitosan, polyethyleneimine, protamine, polypropylimine, 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 having an average diameter of about 50 nm to about 1000 nm.

[0011] In some embodiments, the second polymer may comprise a biodegradable polymer. In some embodiments, the second polymer comprises poly(ε-caprolactone) (PCL), poly-lactic acid (PLA), poly-glycolic acid (PGA), poly-lactide-co-glycolide (PLGA), polyester, poly(orthoester), poly(phosphazine), poly(phosphate ester), 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 having an average diameter of about 100 nm to about 2000 nm.

[0012] In some embodiments, the one or more capsules each independently have a length of about 0.1 cm to about 5 cm. In some embodiments, the one or more capsules each independently have an inner diameter of about 100 μm to about 2000 μm. In some embodiments, the one or more capsules each independently have an outer diameter of about 50 μm to about 300 μm greater than the inner diameter of the same capsule. In some embodiments, each multi-layered wall has a wall thickness of about 25 μm to about 150 μm. In some embodiments, each outer layer may further comprise pores having an average pore diameter of about 100 nm to about 10,000 nm.

[0013] In some embodiments, the one or more drug delivery capsules each have a surface charge, measured as a zeta potential at pH 7.4, of about -25 mV to about 25 mV. In some embodiments, the one or more therapeutic agents each have a net negative charge within a pH range of about 6.0 to about 7.4.

[0014] 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 (e.g., bevacizumab, ranibizumab, IBI305, or a combination thereof). In some embodiments, the anti-VEGF agent is a VEGF decoy receptor (e.g., aflibercept). In some embodiments, the anti-VEGF agent is a tyrosine kinase inhibitor (e.g., lapatinib, sunitinib, axitinib, pazopanib, or a combination thereof).

[0015] In some embodiments, the one or more therapeutic agents may include an anti-inflammatory agent (such as cyclosporine, a steroid or a nonsteroidal anti-inflammatory drug), an antimicrobial agent, an immunomodulatory agent, an ocular hypotensive agent, a neuroprotective agent, a gene therapy, a viral vector therapy, an alpha-adrenergic agonist, a beta-adrenergic agonist, or a combination thereof.

[0016] In another aspect, a method of treating an ophthalmic disorder in a subject is provided, comprising injecting a therapeutically effective amount of the drug delivery composition described herein into the eye of a subject in need thereof. In some embodiments, the ophthalmic disorder is selected from the group consisting of acute macular neuroretinopathy; Behcet's disease; angiogenesis (including choroidal neovascularization); diabetic uveitis; histoplasmosis; infection (such as 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 site or location in the posterior segment of the eye; ocular tumors; retinal disorders (such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinal disease, and the like); sympathetic ophthalmia; Vogt's disease; The ophthalmic disorder may include Koyanagi-Harada (VKH) syndrome; uveal exudation; posterior ocular pathologies caused or affected by ocular laser treatment; posterior ocular pathologies caused or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, non-retinopathy diabetic retinal dysfunction, retinitis pigmentosa, cancer, and glaucoma. In some embodiments, the ophthalmic disorder includes wet age-related macular degeneration (wet AMD), neovascularization, or macular edema. In some embodiments, injecting the described compositions into the subject's eye includes injection into the vitreous chamber of the eye. In other embodiments, injecting the described compositions into the subject's eye includes intravitreal injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, or suprachoroidal injection.

[0017] 1. A method of producing one or more capsules used in a drug delivery composition described herein, comprising: forming a first layer of a first polymer on the conductive rod, the forming of the first layer comprising electrospinning using a first solution comprising the first polymer in at least one organic solvent, the electrospinning being accomplished using a voltage difference of about 10 kV to about 30 kV; and forming a second layer of a second polymer on the first layer, the formation of the second layer comprising electrospinning a second solution comprising the first polymer and optionally a porogen onto the formed first layer, the electrospinning being accomplished using a voltage difference of about 10 kV to about 30 kV; The method also includes:

[0018] Also disclosed are kits that include one of: (a) the disclosed drug delivery composition; (b) the disclosed drug delivery composition in a sterile package; 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 disorder described herein.

[0019] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to those skilled in the art upon review of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this detailed description, be within the scope of the disclosure, and be protected by the accompanying claims. In addition, all optional preferred features and modifications of the described embodiments can be used in all aspects of the present disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional preferred features and modifications of the described embodiments, can be combined and exchanged with each other.

[0020] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, like reference numerals in the drawings designate corresponding parts throughout the several schematic views. [Brief description of the drawings]

[0021] [Figure 1A]A representative disclosed process is shown, which includes the steps of: a) collecting two layers of chitosan nanofibers and PCL nanofibers on a rotating rod using electrospinning; b) sintering the bilayer-coated rod in a vacuum oven at 100° C. for about 3 hours; c) removing the rod to generate a hollow cylinder at the center; d) generating a porous structure in the PCL layer by salt leaching; and e) loading a therapeutic substance into the capsules followed by sealing the ends. The prepared bilayer capsules can then be utilized in studies such as assessing drug release into an appropriate buffer (e.g., PBS) at 37° C. (e.g., step f); or can be used for delivery of the loaded drug to a suitable target (e.g., eye) via intravitreal injection (e.g., step g). [Figure 1B] 1 shows a schematic cross-sectional representation of a disclosed bilayer capsule, and a schematic representation of intravitreal injection of a disclosed bilayer capsule. [Diagram 2] Representative schematic representations of chitosan and PCL fiber mats formed using the disclosed techniques are shown (see Panel A). The following figures also show representative scanning electron micrograph (SEM) images: (Panel B) a representative SEM image of a cross-section of a bilayer chitosan-PCL fiber mat; and (Panel C) a representative SEM image of a PCL nanofiber layer and a chitosan nanofiber layer with diameters of 932.57±399.42 nm and 331.61±186.19 nm, respectively. [Diagram 3] Representative photographic images of the disclosed bilayer capsules are shown. The left box of the figure shows photographic images of two capsules, one with a diameter of 1.645 mm and the other with a diameter of 260 μm. The center box of the figure shows a representative SEM image of a PCL single-layer capsule with an inner diameter of 260 μm. The right box shows a representative SEM image of a chitosan-PCL bilayer capsule with a thickness of 89.85±4.27 μm. The image in the right box shows that in this representative example, a layer of chitosan fiber mat is attached to the outer PCL layer, with the chitosan layer occupying approximately 25% of the total wall thickness. [Figure 4]Representative images of disclosed PCL membranes prepared using the indicated concentrations of HEPES salt are shown, with the images showing the surface or cross-section of the disclosed PCL membrane as shown. The images show that increasing the ratio of HEPES sodium salt resulted in larger pores for the PCL membrane. Pore interconnections may be oversupplied in the interior of the membrane with salt concentrations above 5.0%. Arrows: characteristic interconnecting pores in the interior of the PCL film after salt leaching. Each image has a scalar bar shown in the lower left corner of the image. [Diagram 5] Figure 1 shows images and data related to the characterization of the disclosed bilayer capsules. Panel a shows a representative scheme of the bilayer structure after salt leaching and washing. Panel b shows representative SEM images of the disclosed bilayer membrane before and after salt leaching. As shown, a porous structure was generated by the chitosan fiber structure that was lost after salt leaching and washing with saturated sodium bicarbonate solution. The porous bilayer structure was observed in the cross section. Panel c shows representative FTIR spectra of the chitosan layer and PCL layer after salt leaching. As shown, the significant peak at 1752 cm-1 was assigned to carbonyl groups in PCL. The broad group at 3478 cm-1 was hydroxyl groups in chitosan. [Figure 6]1 shows data relating to the effect of porous bilayer structure on protein release from the disclosed bilayer capsules. Data were obtained from representative disclosed chitosan-PCL bilayer capsules (labeled Ch-PCL in the graph legend) and PCL monolayer capsules (labeled PCL in the graph legend) encapsulating BSA or bevacizumab, as determined as described herein from incubation in PBS, as described herein below. The percentage values ​​shown with the "Ch-PCL" or "PCL" labels in the graph legend indicate the w / v % used to prepare the bilayer or monolayer capsules. Panel a shows a representative BSA release profile from a 1.645 mm inner diameter bilayer capsule and a representative BSA release profile from a 260 μm inner diameter bilayer capsule. Panel b shows a representative bevacizumab release profile from a 1.645 mm inner diameter bilayer capsule and a representative bevacizumab release profile from a 260 μm inner diameter bilayer capsule. The drug delivery composition shows a lower cumulative release than the single-layer capsule at each time point (#=p≦0.05). The data also show that increasing salt concentration correlated with an increase in cumulative release at each time point (*=p≦0.05). [Figure 7]FIG. 1 shows data relating to the effect of porous bilayer structure on protein release from the disclosed bilayer capsules. In this figure, trend lines are fitted to the data with fitting parameters as indicated. Data were obtained as described herein below from representative disclosed chitosan-PCL bilayer capsules (labeled Ch-PCL in the graph legend) and PCL monolayer capsules (labeled PCL in the graph legend) encapsulating BSA or bevacizumab, determined as described herein from incubation in PBS. The percentage values ​​shown with the "Ch-PCL" or "PCL" labels in the graph legend indicate the w / v % used to prepare the bilayer or monolayer capsules. Panel a shows a representative BSA release profile from a 1.645 mm inner diameter bilayer capsule and a representative BSA release profile from a 260 μm inner diameter bilayer capsule. Panel b shows a representative bevacizumab release profile from a bi-layer capsule with an inner diameter of 1.645 mm and a representative bevacizumab release profile from a bi-layer capsule with an inner diameter of 260 μm. The data show that the disclosed bi-layer capsules can achieve near zero-order release kinetics. [Figure 8]1 shows an assay scheme for assessing potential toxicity of the disclosed capsules and data obtained from the assay. Panel a shows an assay scheme for assessing in vitro cytotoxicity using ARPE-19 cells by direct contact method. Panel b shows in vitro toxicity data for capsules prepared with 10.0% HEPES salt, 7.5% HEPES salt, and 5.0% HEPES salt by direct contact method. Panel c shows an assay scheme for assessing in vitro cytotoxicity using ARPE-19 cells by extract exposure method. Panel d shows in vitro cytotoxicity of capsule extracts prepared by different conditions. Each bar at different time points and salt concentrations represents the average measurement of three independent samples. Error bars indicate standard deviation. As above, data was obtained using a representative disclosed chitosan-PCL bilayer capsule (labeled Ch-PCL in the graph legend) and PCL monolayer capsule (labeled PCL in the graph legend). The data showed no significant differences in cell viability between cells treated with PCL extract or chitosan-PCL extract (p>0.05), and no significant differences were observed over time (p>0.05). [Figure 9]Representative fluorescence micrograph images and data relating to inhibition of cell-tubule length in VEGF-treated HUVEC cells exposed to bevacizumab delivered using PCL monolayer capsules or the disclosed bilayer capsules. Calcien AM was used to label the cells. Panel a shows representative fluorescence images showing HUVECs treated with 5 ng VEGF in the absence (left) and presence (right) of 10 mg original bevacizumab in cell culture medium. The data show significant disruption of cell tubules in the presence of bevacizumab compared to the control group. Panel b shows representative fluorescence images showing inhibition of cell tubules in cells exposed to 10 mg of bevacizumab released from 260 μm diameter PCL monolayer drug delivery devices and chitosan-PCL drug delivery devices at 1 week, 1 month, 3 months, and 9 months of exposure as indicated. Panel c shows the mean tube length inhibition of the indicated groups quantitatively analyzed using ImageJ software. Data are presented as mean ± standard deviation, n=3. Data show that significant differences in HUVEC tube length were observed in the groups treated with eluted bevacizumab from single-layer capsules compared to those from bilayer capsules (*=p≦0.05). Data also show that significant differences in the inhibition ability of HUVEC tubes of eluted bevacizumab compared to that of free parent bevacizumab were observed over time (#=p≦0.05). [Figure 10] 1 shows results from a study assessing the injection of a representative disclosed bilayer capsule into an ex vivo pig eye model. Panel a shows a schematic representation of the injection of a bilayer capsule into the vitreous humor via a hypodermic needle. Panel b shows a capsule preloaded into a 21-gauge needle that was injected 3 mm posterior to the limbus in the ex vivo pig eye (see center box). Following injection, the ex vivo pig eye was dissected and an intact capsule was observed to be intact in the vitreous humor of the ex vivo pig eye (see right box). [Figure 11]Figure 1 shows a comparison of biodegradation over one year of incubation of PCL single-layer capsules and bilayer capsules described herein. Panel a shows representative scanning electron micrograph (SEM) images prepared with different salt concentrations. An increase in the size of the pores on the PCL membrane was observed in all samples after 9 months. The cross-sectional images (see box on the right) show that the capsules remained intact over a one-year period. Panel b shows a representative SEM image of bilayer capsules. A fibrous framework could be observed in the porous chitosan layer. An intact bilayer structure is shown from the cross-sectional images (see box on the right). [Figure 12] Figure 1 shows the UV-visible absorption spectra of bevacizumab diluted in PBS at different concentrations. Panel a shows the absorbance of diluted bevacizumab measured by UV-visible spectroscopy. Panel b shows the standard curve of bevacizumab measured by a plate reader. The minimum concentration detectable by UV-visible spectroscopy and a plate reader is 5 μg / mL. [Figure 13] Figure 1 shows the effect of the porous bilayer structure of the capsules described herein on bevacizumab release, assessed by ELISA. The bilayer capsules described herein encapsulating bevacizumab and PCL single-layer capsules were incubated in PBS. The bevacizumab release profile was obtained for capsules with an inner diameter of 260 μm. The bilayer capsules disclosed effectively retain the protein inside the capsule for at least 9 months, with a lower cumulative release at each time point than the single-layer capsules (#=p≦0.05). Increasing the salt concentration also increases the cumulative release at each time point (*=p≦0.05). The release profile obtained by ELISA is consistent with the results determined by UV-visible spectroscopy. [Figure 14]Figure 1 shows the stability of free parent bevacizumab before and after lyophilization, and of bevacizumab eluted from the single-layer capsule and the bilayer capsule described herein over the first 3 months. Panel a shows the SEC-HPLC chromatograms of free parent bevacizumab, lyophilized bevacizumab, and bevacizumab in the device. Panel b shows the SEC-HPLC chromatograms of bevacizumab eluted from the single-layer capsule and the bilayer capsule incubated at physiological temperature for 1 month and 3 months. [Figure 15] Figure 1 shows the biodegradation of chitosan-PCL bilayer capsules exposed to PBS for 3 weeks. Representative SEM images of 260 μm inner diameter bilayer capsules prepared with 10% HEPES salt, which represents the most porous structure, are provided. Cross-section and internal images show that the capsules lost the inner chitosan layer after 3 weeks when exposed to PBS, but biodegradation was not significant when the chitosan layer was coated with PCL. The thickness of the bilayer capsules was 73.23 ± 3.62 μm.

[0022] Additional advantages of the present disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present disclosure. The advantages of the present disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure as claimed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Many modifications and other embodiments of the disclosed compositions and methods will occur to those skilled in the art having the benefit of the teachings presented in the foregoing description and the accompanying drawings. It should therefore be understood that the 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. Those skilled in the art will recognize many variations and adaptations of the aspects described herein. These variations and adaptations are intended to be included within the teachings of the disclosure and covered by the claims of this specification.

[0024] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0025] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and characteristics which may be readily separated from or combined with the characteristics of any of the other embodiments without departing from the scope or spirit of the disclosure.

[0026] Any recited method may be carried out in the order of events recited or in any other order that is logically possible. That is, unless expressly stated otherwise, it is not intended that any method or aspect described herein be interpreted as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically states in the claim or description that the steps are to be limited to a particular order, no order is intended to be implied in any respect. This is true for all potentially unclear criteria for interpretation, including questions of logic regarding the arrangement of steps or operational flow, the plain meaning derived from grammatical construction or punctuation, or the number or type of aspects described in the specification.

[0027] All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided herein may be different from the actual publication dates, which may require independent confirmation.

[0028] Although aspects of the present disclosure may be described and claimed in particular statutory classifications (such as systems statutory classifications), this is for convenience only and those skilled in the art will understand that each aspect of the present disclosure may be described and claimed in any statutory classification.

[0029] It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the disclosed compositions and methods belong.It will be further understood that terms (such as those defined in commonly used dictionaries) should be interpreted to have a meaning consistent with their meaning in the context of the specification and related art, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0030] Prior to description of the various aspects of this disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in this disclosure.

[0031] definition As used herein, "comprising" is to be construed as specifying the presence of the specified feature, integer, step, or component as referenced, but does not exclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Additionally, the terms "by," "comprising," "comprises," "comprised of," "including," "includes," "included," "involving," "involves," "involved," and "such as" are each used in their open, non-limiting sense and may be used interchangeably. Additionally, the term "comprising" is intended to include examples and embodiments covered by the terms "consisting essentially of" and "consisting of." Similarly, the term "consisting essentially of" is intended to include examples covered by the term "consisting of."

[0032] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "drug delivery composition," a "therapeutic agent," or a "clinical condition" includes, but is not limited to, two or more such drug delivery compositions, therapeutic agents, or clinical conditions, and the like.

[0033] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It will be further understood that each endpoint of a range is significant in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular 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 "about" one particular value and / or to "about" another particular value. Similarly, when values ​​are expressed as approximations by use of the antecedent "about," it will be understood that the particular value forms a further aspect. For example, if the value "about 10" is disclosed, then "10" is also disclosed.

[0034] When a range is expressed, a further embodiment includes from the one particular value and / or to the other particular value. For example, where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed in the disclosure (e.g., the phrase "from x to y" includes ranges greater than "x" and less than "y", as well as ranges from "x" to "y"). Ranges may also be expressed as upper limits (e.g., "about x, y, z, or less") and should be interpreted to include ranges "less than x", "less than y", and "less than z", as well as the specific ranges of "about x", "about y", and "about z". Similarly, the phrase "about x, y, z, or more" should be interpreted to include ranges "greater than x", "greater than y", and "greater than z", as well as the specific ranges of "about x", "about y", and "about z". In addition, the phrase "from 'x' to 'y'" includes "about 'x' to about 'y'", where "x" and "y" are numerical values.

[0035] It should be understood that such range formats are used for convenience and brevity and should therefore be interpreted in a flexible manner to include not only the numerical values ​​expressly recited as the limits of the range, but also all individual numerical values ​​or subranges encompassed within that range, as if each numerical value and subrange were expressly recited. By way of illustration, a numerical range of "about 0.1% to 5%" should be interpreted not only to include the explicitly recited values ​​of about 0.1% to about 5%, but also to include the individual values ​​(e.g., about 1%, about 2%, about 3%, 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%, as well as other possible subranges) within the indicated range.

[0036] As used herein, the terms "about," "approximately," "is or is about," and "substantially" mean that the amount or value in question may be an exact value or a value that provides the same results or effects as those recited in the claims or taught herein. That is, it is understood that the amounts, sizes, formulations, parameters, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, as desired, to reflect tolerances, conversion factors, rounding, measurement error, and the like, and other factors known to those skilled in the art such that equivalent results or effects are obtained. In some circumstances, values ​​that provide the same results or effects cannot be logically determined. In such cases, generally, when used herein, unless otherwise indicated or implied, "about" and "is or is about" are understood to mean a variation of ±10% of the nominal value indicated. In general, an amount, size, formulation, parameter, or other quantity or characteristic is such, whether or not it is expressly stated to be "about," "approximately," or "is or is about." When "about," "approximately," or "is or is about" is used in front of a quantitative value, it is understood that the parameter also includes the particular quantitative value itself, unless specifically stated otherwise.

[0037] As used herein, "effective amount" may refer to an amount of a disclosed compound or pharmaceutical composition provided herein sufficient to achieve a beneficial or desired biological, emotional, medical, or clinical response in a cell, tissue, system, animal, or human. An effective amount may be administered in one or more administrations, applications, or dosages. The term may also include within its scope an amount effective to promote or restore substantially normal physiological function.

[0038] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to achieve a desired therapeutic result or have an effect on undesired symptoms, but generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the specific composition used; the age, weight, general health, sex, and diet of the patient; the time of administration; the route of administration; the excretion rate of the specific compound used; the duration of treatment; drugs used in conjunction with or simultaneously with the specific compound used, as well as similar factors within the knowledge and expertise of medical practitioners, and those that may be well known in the medical field. In the case of treating a particular disease or condition, in some instances, the desired response may be to inhibit the progression of the disease or condition. This may include only slowing the progression of the disease temporarily. However, in other instances, it may be desired to permanently halt the progression of the disease. This may be monitored by routine diagnostic methods known to those skilled in the art for any particular disease. The desired response to treatment of a disease or condition may be to delay or, in some cases, prevent the onset of the disease or condition.

[0039] For example, it is well within the skill of the artisan to start the dose of the compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for the purpose of administration. Thus, a single dose composition can contain such an amount or a submultiple thereof to constitute a daily dose. The dosage can be adjusted by the individual physician in the event of any contraindication. It is generally preferred that the maximum dose of the pharmacological agent of the present invention (alone or in combination with other therapeutic agents), i.e., the highest safe dose according to sound medical judgment, is used. However, it will be understood by those skilled in the art that a patient may insist on a lower dose or a tolerable dose for medical reasons, psychological reasons, or other reasons in practice.

[0040] Response to a therapeutically effective dose of the disclosed drug delivery composition can be measured by determining the physiological effect of the treatment or medicine (such as a reduction or absence of disease symptoms following administration of a treatment or pharmacological agent). Other assays are known to those skilled in the art and can be used to measure the level of response. The amount of treatment can be varied, for example, by increasing or decreasing the amount of the disclosed compounds and / or pharmaceutical compositions, by changing the disclosed compounds and / or pharmaceutical compositions administered, by changing the route of administration, by changing the timing of dosing, etc. Dosage can vary and can be administered daily for one or more days in one or more dose administrations. Guidance can be found in the literature for appropriate dosages for a given class of pharmaceutical products.

[0041] As used herein, the term "prophylactically effective amount" refers to an amount effective for preventing the development or onset of a disease or condition.

[0042] As used herein, the term "prevent" or "prevention" refers to eliminating, avoiding, forestalling, preempting, stopping, or impeding something from happening, especially by prior action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0043] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0044] As used herein, a "therapeutic agent" may refer to any substance, compound, molecule, and the like that may be biologically active or otherwise induce a pharmacological, immunogenic, biological, and / or physiological effect on the subject to which it is administered with local and / or systemic action. A therapeutic agent may be a primary active agent, or in other words, a component(s) of a composition to which all or part of the composition's effect is attributable. A therapeutic agent may be a secondary therapeutic agent, or in other words, a component(s) of a composition to which additional parts of the composition and / or other effects are attributable. Thus, the term encompasses compounds or chemicals that are traditionally considered to be drugs, vaccines, and biopharmaceuticals (including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, and the like). Examples of therapeutic agents are described in well-known literature references such as the Merck Index (14th edition), the Physicians' Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition), and include, but are not limited to, medicines; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure, or ameliorate a disease or illness; 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, but is not limited to, adjuvants; anti-infectives (such as antibiotics and antivirals); analgesics and analgesic combinations, anorectics, anti-inflammatory agents, anti-epileptic agents, local and general anesthetic agents, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergics and cholinomimetics, antimuscarinic and muscarinic agents, antiadrenergic agents, antiarrhythmic agents, antihypertensive agents, hormonal and nutritional agents, antiarthritic agents, antiasthmatic agents, anticonvulsants, antihistamines, antiemetic agents, antineoplastic agents, antipruritic agents, antipyretics; antiseizure agents, cardiovascular agents (including calcium channel blockers, beta blockers, beta agonists, and antiarrhythmic agents), antihypertensive agents, diuretics, vasodilators; central nervous system stimulants; cold remedies; and decongestants. The term encompasses compounds or compositions for use in all of the major therapeutic areas, including, but not limited to, substances; diagnostic substances; hormones; bone growth stimulators and bone resorption inhibitors; immunosuppressants; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized, or recombinantly produced); as well as nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA), including double-stranded and single-stranded molecules, genetic constructs, expression vectors, antisense molecules, and the like), small molecules (e.g., doxorubicin), and other biologically active macromolecules (e.g., proteins and enzymes). Drugs can be biologically active agents used in medical applications, including veterinary medicine, agriculture (such as by plants), and other areas. The term therapeutic agent also includes, but is not limited to, a medicine; a vitamin; a mineral supplement; a substance used to treat, prevent, diagnose, cure, or mitigate a disease or illness; or a substance that affects the structure or function of the body; or a prodrug that becomes biologically active or more active after being placed in a given physiological environment.

[0045] It is understood that the disclosure herein of a therapeutic agent also discloses pharma- ceutically acceptable salts, pharma- ceutically acceptable esters, pharma- ceutically acceptable amides, prodrug forms, and derivatives of the therapeutic agent.

[0046] The term "pharmaceutical acceptable salt" as used herein means a salt of an active agent prepared with an acid or base that is tolerated by biological systems or tolerated by subjects when administered in a therapeutically effective amount, or that is tolerated by biological systems and tolerated by subjects.When a compound of the present disclosure contains a relatively acidic functional group, a base addition salt can be obtained by contacting a neutral form of such a compound with a sufficient amount of a desired base, either without adding liquid or in a suitable inert solvent.Examples of pharmaceutical acceptable base addition salts include, but are not limited to, sodium salt, potassium salt, calcium salt, ammonium salt, organic amino salt, magnesium salt, lithium salt, strontium salt, or similar salts.When a compound of the present disclosure contains a relatively basic functional group, an acid addition salt can be obtained by contacting a neutral form of such a compound with a sufficient amount of a desired acid, either without adding liquid or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include, but are not limited to, those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydroiodic, or phosphorous acids, and the like, and those derived from relatively non-toxic organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids such as glucuronic or galactunoric acids, and the like.

[0047] The term "pharmaceutically acceptable ester" refers to esters of the compounds of the present disclosure, including those that hydrolyze in vivo and degrade readily in the human body to leave the parent compound or a salt thereof. Examples of pharmaceutically acceptable non-toxic esters of the present disclosure include C1-C6 alkyl esters and C5-C7 cycloalkyl esters, with C1-C4 alkyl esters being preferred. Esters of the disclosed compounds can be prepared according to conventional methods. Pharmaceutically acceptable esters can be added to a hydroxy group by reacting the compound containing a hydroxy group with an acid and an alkyl carboxylic acid (such as acetic acid), or an acid and an aryl carboxylic acid (such as benzoic acid). In the case of a compound containing a carboxyl acid group, a pharmaceutically acceptable ester is prepared from the compound containing a carboxyl acid group by reacting the compound with triethylamine and a base such as an alkyl halide (e.g., methyl iodide, benzyl iodide, cyclopentyl iodide, or an alkyl triflate). Esters can also be prepared by reacting the compound with an acid (such as hydrochloric acid) and an alcohol (such as ethanol or methanol).

[0048] The term "pharmaceutically acceptable amides" refers to the non-toxic amides of the present disclosure derived from ammonia (primary C1-C6 alkylamines and secondary C1-C6 dialkylamines). In the case of secondary amines, the amines can also be in the form of 5- or 6-membered heterocycles containing one nitrogen atom. Amides derived from ammonia (C1-C3 alkyl primary amides and C1-C2 dialkyl secondary amides) are preferred. 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 the compounds containing amino groups with alkyl anhydrides, aryl anhydrides, acyl halides, or aroyl halides. In the case of compounds containing a carboxylic acid group, pharma- ceutically acceptable amides are prepared from compounds containing a carboxylic acid group by reacting the compound with a base (such as triethylamine), a dehydrating agent (such as dicyclohexylcarbodiimide or carbonyldiimidazole), and an alkylamine (dialkylamine) (e.g., with methylamine, diethylamine, and piperidine). Amides can also be prepared by reacting the compound with an acid (such as sulfuric acid) and an alkyl carboxylic acid (such as acetic acid), or an acid and an aryl carboxylic acid (such as benzoic acid under dehydrating conditions, such as adding molecular sieves). The compositions can contain the compounds of the present disclosure in the form of pharma- ceutically acceptable prodrugs.

[0049] The term "pharmacologically acceptable prodrug" or "prodrug" refers to prodrugs of the disclosed compounds that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio, and effective for their intended use. The disclosed prodrugs can be rapidly transformed in vivo, for example by hydrolysis in blood, to the parent compound having the structure of the disclosed compound. A detailed discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V.14 of the ACS Symposium Series, and Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).

[0050] As used herein, "kit" refers to a collection of at least two components that make up the kit. The components together constitute a functional unit for a given purpose. The individual member components can be physically packaged together or separately. For example, a kit that includes instructions for using the kit may or may not physically include the instructions with other individual member components. Instead, the instructions can be provided as a separate member component, either in paper form, or on a computer-readable memory device, or downloaded from an internet website, or in electronic form as a recorded form.

[0051] As used herein, "instructions(s)" refers to documents describing the relevant materials or methodology associated with the kit. These materials may include any combination of the following: background information, a list of components and their availability (such as purchasing information), a brief or detailed protocol for use of the kit, troubleshooting, references, technical support, and other related documentation. The instructions may be provided in paper form, or on a computer readable memory device, or downloaded from an Internet website, or in electronic form as a recorded presentation, either with the kit or as a separate member component. The instructions may include one or more documents and are intended to encompass future revisions.

[0052] As used interchangeably herein, a "subject," "individual," or "patient" can refer to a vertebrate organism, such as a mammal (e.g., a human). A "subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably a human and components thereof.

[0053] As used herein, the terms "treating" and "treatment" may generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be, but is not necessarily, prophylactic, in terms of preventing or partially preventing a disease, condition or pathology (such as an ophthalmic disorder). The effect may be therapeutic, in terms of partially or completely curing a disease, condition, symptom, or adverse effects caused by a disease, disorder, or pathology. The term "treatment" as used herein may include any treatment of an ophthalmic disorder in a subject (especially a human), and may include any one or more of the following: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., preventing its development; and (c) relieving the disease, i.e., alleviating or ameliorating the disease and / or symptoms or pathology. The term "treatment" as used herein may refer to therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) may include those already with a disorder and / or those in whom a disorder is to be prevented. As used herein, the term "treating" may include inhibiting a disease, disorder, or condition (e.g., preventing its progression); and alleviating a disease, disorder, or condition (e.g., causing regression of a disease, disorder, and / or condition). Treating a disease, disorder, or condition may include ameliorating at least one symptom of a particular disease, disorder, or condition, even if the underlying pathophysiology is not affected (e.g., treating a subject's pain by administering an analgesic, even if such an agent does not treat the cause of the pain).

[0054] As used herein, "dose," "unit dose," or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or pharmaceutical composition thereof calculated to produce the desired response(s) associated with its administration.

[0055] As used herein, "therapeutic" can refer to treating, curing, and / or ameliorating a disease, disorder, condition, or side effect, or reducing the rate of progression of a disease, disorder, condition, or side effect.

[0056] As used herein, nomenclature for compounds (including organic compounds) may be given using common names, IUPAC, IUBMB, or CAS recommendations for nomenclature. When one or more stereochemical features are present, the Cahn-Ingold-Prelog rules for stereochemistry may be used to indicate stereochemical priority, E / Z designations, and the like. Given a name, one of skill in the art can readily ascertain the structure of a compound by systematic reduction of the compound structure using the naming rules, or by commercially available software such as CHEMDRAW™ (Cambridgesoft Corporation, USA).

[0057] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (ie, 1 atmosphere).

[0058] Drug delivery compositions having therapeutic or clinical utility are described herein. Methods of preparing or making the disclosed drug delivery compositions are also described herein. Methods of administering the disclosed drug delivery compositions to a subject in need thereof are also described herein. In some embodiments, the subject may have a clinical condition or pathology, such as an ophthalmic disorder. Other compositions, compounds, methods, properties, and advantages of the present disclosure will be or become apparent to one of ordinary skill in the art upon review of the following figures, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, properties, and advantages are encompassed within this detailed description and are within the scope of the present disclosure.

[0059] Drug Delivery Compositions Vascular endothelial growth factor (VEGF) is an essential regulator involved in abnormal angiogenesis, which supports 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):p.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):p.1097-1105; and Ferrara, N., Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney International, 1999.56(3):p.794-814). Anti-angiogenic strategies have been proposed to slow 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):p.1000-1017).Humanized monoclonal antibodies (anti-VEGF) are 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):p.4593-4599).

[0060] Treatment of this age-related retinal disease currently relies on the use of antiangiogenic agents to slow or halt progression. Intravitreal injection of anti-VEGF therapeutics (such as bevacizumab and ranibizumab) constitutes the current gold standard treatment for wet AMD, preventing irreversible retinal damage caused by VEGF-induced initiation of subretinal choroidal neovascularization (CNV) and hemorrhage and scarring of the newly formed blood vessels (see Delplace, V., S. Payne, and MJ JoCR Shoichet, Delivery strategies for treatment of age-related ocular diseases: From a biological understanding to biomaterial solutions. 2015. 219: p. 652-668; and Ohr, M. and PK J Eoop Kaiser, Intravitreal aflibercept injection for neovascular (wet) age-related macular degeneration. 2012. 13(4): p. 585-591). Bevacizumab, for example, is widely used to treat wet AMD because of its relatively low cost.However, these protein therapeutics have short half-lives in the vitreous humor and often require frequent (up to monthly) intravitreal injections to maintain efficacy in the eye (see Hard, AL and AJAH Hellstrom, On safety, pharmacokinetics and dosage of bevacizumab in ROP treatment-a review. 2011. 100(12): p. 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): p. 434-457). Unfortunately, this modality frequently results in side effects including pain, infection, endophthalmitis, elevated intraocular pressure, inflammation, retinal detachment, and cataract formation (see Sampat, KM and SJJCoioGarg, Complications of intravitreal injections. 2010. 21(3): p. 178-183). The main barrier to treatment is the high cost associated with each injection, imposing on patients and families the burden of undergoing monthly treatments (see Heimes, B., et al., Compliance von Patienten mit altersabhangiger Makuladegeneration unter Anti-VEGF-Therapie. Der Ophthalmologe, 2016. 113(11): p. 925-932). Thus, there is a clear need for easier and more efficient treatments for wet AMD.

[0061] Conventional delivery systems in the form of implants and particles have been developed to achieve controlled release for possible AMD treatments (see Delplace, V., S. Payne, and MJJoCRShoichet, Delivery strategies for treatment of age-related ocular diseases: From a biological understanding to biomaterial solutions. 2015. 219: p. 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): p. 416-423; Imperiale, JC, GBAcosta, and AJJoCRSosnik, 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): p. 2043-2053). Compared to microparticle / nanoparticle-based systems, implants have higher stability and drug payload due to their larger size (see Kim, YC, et al., Ocular delivery of macromolecules. Journal of Controlled Release, 2014. 190: p. 172-181). However, most implant-based treatments are associated with injection difficulties, and non-biodegradable intraocular lenses require additional surgical procedures 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: p. 585-595).These are accompanied by postoperative complications and increased costs. In addition, long-term sustained release from either particles or implants has been difficult due to poor 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 hydrolysis, often leading to therapeutic release for 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). Additional drawbacks include the formation of acidic by-products that can induce inflammation and exacerbate the foreign body response (see Lu, L., MJ Yaszemski, and AGB Mikos, Retinal pigment epithelium engineering using synthetic biodegradable polymers. 2001. 22(24): p. 3345-3355).

[0062] Disclosed herein is a drug delivery composition comprising an injectable, biodegradable, multi-layer capsule loaded with a therapeutic agent (e.g., bevacizumab) for achieving higher drug loading and longer drug release duration than previously available injectable drug delivery devices. Disclosed herein is a drug delivery composition comprising, for example, a nanoporous PCL outer shell and a chitosan inner layer to achieve physical trapping and electrostatic-based chemical absorption, respectively, to achieve highly sustainable and controllable drug release. A hollow structure encapsulated by a bilayer hybrid shell was utilized to load sufficient therapeutic substance for at least one year of long-term drug release. More specifically, the entire drug delivery composition is prepared by combining material processing techniques including electrospinning, sintering, and salt leaching. The disclosed method provides hollow-centered cylindrical microrods with high aspect ratios to enable injection feasibility through 21-gauge or smaller needles for intravitreal implant delivery. By optimizing the chemical and physical structure of the capsule using the disclosed methods, the disclosed drug delivery composition can be used to obtain a stable and controlled release of protein therapeutics for more than 10 months.By reducing the frequency of injections through small gauge needles, the disclosed drug delivery composition may improve the quality of life of patients with wet AMD.

[0063] Thus, in one embodiment, one or more capsules each having a tubular shape with two closed ends, each of the one or more capsules independently comprising a multi-layered wall and at least one lumen compartment; and one or more therapeutic agents, each initially present within one or more of the at least one luminal compartment; Including, Each multi-layer wall independently includes at least an inner layer and an outer layer; each inner layer independently comprises a first polymer having a net positive charge under physiological conditions; each outer layer independently comprises a second polymer different from the first polymer; A drug delivery composition is provided.

[0064] In some embodiments, the drug delivery composition may include two or more capsules (e.g., two capsules, three capsules, four capsules, five capsules, six capsules, seven capsules, eight capsules, nine capsules, ten capsules, or more). In such embodiments, the two or more capsules may include the same composition for the multi-wall of each capsule, or may differ in their composition. In some embodiments, the same or different therapeutic agents may be initially present in each of the two or more capsules.

[0065] In some embodiments, each capsule in the drug delivery composition may independently comprise two or more lumen compartments (e.g., two lumen compartments, three lumen compartments, four lumen compartments, or more). In some embodiments, the same or different therapeutic agents may be present initially in each of two or more lumen compartments within a single capsule.

[0066] In some embodiments, each capsule independently has a length of about 0.1 cm to about 5 cm (e.g., 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 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 to 1 cm, or 0.1 to 0.5 cm.

[0067] In some embodiments, the multi-layered wall has a wall thickness of about 25 μm to about 150 μm (eg, 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 is about 50 μm to 150 μm, about 55 μm to 150 μm, about 60 μm to about 150 μm, about 65 μm to about 150 μm, about 70 μm to about 150 μm, about 75 μm to about 150 μm, about 80 μm to about 150 μm, about 90 μm to about 150 μm, about 95 μm to about 150 μm, about 100 μm to about 150 μm, about 110 μm to about 150 μm, about 125 μm to about 150 μm, about 140 μm to about 150 μm, about 50 μm to 140 μm, about 55 μm to 140 μm, about 60 μm to about 140 μm, Approximately 65μm to approximately 140μm, 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, about 50μm to 125μm, about 55μm to 125μm, about 60μm to about 125μm, about 65μm to about 125μm, about 70μm to about 125μm, about 75μm to about 125μm, about 80μm to about 125μm, about 90μm to about 125μm, about 95μm Approximately 125 μm, approximately 100 μm to 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 8 0 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 75μm to approximately 100μm, approximately 80μm to approximately 100μm, approximately 90μm to approximately 100μm, approximately 95μm to approximately 100μm, approximately 50μm to 95μm, approximately 55μm to 95μm, approximately 60μm to approximately 95μm, approximately 65μm to approximately 95μm, approximately 70μm to approximately 95μm, approximately 75μm m ~ about 95μm, about 80μm - about 95μm, about 90μm - about 95μm, about 50μm - 90μm, about 55μm - 90μm, about 60μm - about 90μm, about 65μm - about 90μm, about 70μm - about 90μm, about 75μm - about 90μm, about 80μm - about 90μm,Approximately 50μm to 80μm, approximately 55μm to 80μm, approximately 60μm to approximately 80μm, approximately 65μm to approximately 80μm, approximately 70μm to approximately 80μm, approximately 75μm ~ Approx. 80 μm, Approx. 50 μm ~ 75 μm, Approx. 55 μm ~ 75 μm, Approx. 60 μm ~ Approx. 75 μm, Approx. 65 μm ~ Approx. 75 μm, Approx. 70 μm ~ Approx. 75 μm m, about 50 μm to 70 μm, about 55 μm to 70 μm, about 60 μm to about 70 μm, about 65 μm to about 70 μm, about 50 μm to 65 μm, about 55 μm to 65 μm, about 60 μm to about 65 μm, about 50 μm to 60 μm, about 55 μm to 60 μm, and about 50 μm to about 55 μm.

[0068] In some embodiments, the thickness of the inner layer can range from about 1 μm to about 100 μm. In some embodiments, the thickness of the inner layer can range from about 100 nm to about 990 nm (e.g., about 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 990 nm).

[0069] In some embodiments, the thickness of the outer layer can range from about 1 μm to about 100 μm. In some embodiments, the thickness of the outer layer can range from about 100 nm to about 990 nm (e.g., about 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 990 nm).

[0070] In some embodiments, the tubular shape of the drug delivery capsule has an inner diameter of about 100 μm to about 1000 μm (eg, about 100 μm to about 1000 μm, about 100 μm to about 500 μm, or 100 μm to about 300 μm).In some embodiments, the tube shape of the drug delivery capsule is 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, about 400 μm m ~ approx. 1500 μm, approx. 500 μm ~ approx. 1500 μm, approx. 600 μm ~ approx. 1500 μm, approx. 700 μm ~ approx. 1500 μm, approx. 800 μm ~1500μm, 900μm~1500μm, 1000μm~1500μm, 100μm~1000μm, 200μm~approx. 1000μm, about 300μm to about 1000μm, about 400μm to about 1000μm, about 500μm to about 1000μm, about 600μm to about 10 00μm, about 700μm to about 1000μm, about 800μm to about 1000μm, about 900μm to about 1000μm, about 100μm to about 900μm m, 200μm to approx. 900μm, approx. 300μm to approx. 900μm, approx. 400μm to approx. 900μm, approx. 500μm to approx. 900μm, approx. 600 μm ~ approx. 900 μm, approx. 700 μm ~ approx. 900 μm, approx. 800 μm ~ approx. 900 μm, approx. 100 μm ~ approx. 800 μm, 200 μm ~ approx. 800 μm, approximately 300 μm to approximately 800 μm, approximately 400 μm to approximately 800 μm, approximately 500 μm to approximately 800 μm, approximately 600 μm to approximately 800 μm, approximately 70 0μm~about 800μm, about 100μm~about 700μm, 200μm~about 700μm, about 300μm~about 700μm, about 400μm~about 70 0 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 about 100 μm to about 300 μm larger than the inner diameter (e.g., 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 larger than the inner diameter).

[0071] In some embodiments, the first polymer may include chitosan, polyethyleneimine, protamine, polypropylenimine, 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.

[0072] 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%, at least about 80%.

[0073] In some embodiments, the first polymer has a molecular weight of about 50 kDa to about 500 kDa (e.g., 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 more (e.g., about 15 kDa or more, about 20 kDa or more, about 30 kDa or more, about 40 kDa or more, about 50 kDa or more, about 60 kDa or more, about 70 kDa or more, about 90 kDa or more, about 90 kDa or more, or about 100 kDa or more).

[0074] In some embodiments, the first polymer used in the inner layer comprises fibers. In some embodiments, the fibers can have a diameter of about 50 nm to about 1000 nm (e.g., about 100 nm to about 400 nm). In some embodiments, the fibers can 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.

[0075] In some embodiments, the second polymer may comprise poly(ε-caprolactone) (PCL), poly-lactic acid (PLA), poly-glycolic acid (PGA), poly-lactide-co-glycolide (PLGA), polyesters, poly(other esters), poly(phosphazines), poly(phosphate esters), gelatin, collagen, polyethylene glycol (PEG), derivatives thereof, and combinations thereof. In other embodiments, the second polymer may comprise PLGA, PCL, PLA, PGA, PEG, polysorbates, poly(ε-caprolactone-thioethylethylenephosphate) (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.

[0076] In some embodiments, the second polymer has a molecular weight of about 50 kDa to about 500 kDa (e.g., 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 more (e.g., about 15 kDa or more, about 20 kDa or more, about 30 kDa or more, about 40 kDa or more, about 50 kDa or more, about 60 kDa or more, about 70 kDa or more, about 90 kDa or more, about 90 kDa or more, or about 100 kDa or more).

[0077] In some embodiments, the second polymer is biodegradable in vivo and is well tolerated throughout the duration of the composition's presence and degradation. In some embodiments, under physiological conditions, the second polymer degrades by random chain scission, which results in a biphasic degradation. Initially, the physical structure is not significantly affected as the molecular weight decreases. Degradation occurs throughout the polymeric material and proceeds until a critical molecular weight is reached where the degradation products become small enough to be solubilized. At this point, the structure begins to become significantly porous and hydrated. In some embodiments, the second polymer has a molecular weight of about 90 kDa or more and does not degrade in the subject's eye until after six months or more. In some embodiments, the molecular weight of the biodegradable polymer is selected to tailor the degradation time of the material in vivo.

[0078] In some embodiments, the second polymer can comprise a blend of a high molecular weight polymer and a low molecular weight polymer. In some embodiments, the high molecular weight polymer can be about 25 kDa or more (e.g., about 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more) and the low molecular weight polymer can be about 20 kDa or less (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 high molecular weight polymer to lower molecular weight polymer is between about 1:9 and about 9:1 (e.g., 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).

[0079] In some embodiments, the outer layer of the second polymer used in the outer layer comprises fibers. In some embodiments, the fibers can have a diameter of about 100 nm to about 2000 nm (e.g., about 500 nm to about 1000 nm). In some embodiments, the fibers can have a diameter 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, about 750 nm to about 1500 nm, The diameter may be from about 1000 nm to about 1500 nm, from about 100 nm to about 1000 nm, from about 250 nm to about 1000 nm, from about 500 nm to about 1000 nm, from about 750 nm to about 1000 nm, from about 100 nm to about 750 nm, from about 250 nm to about 750 nm, from about 500 nm to about 750 nm, from about 100 nm to about 500 nm, from about 250 nm to about 500 nm, or from about 100 nm to about 250 nm.

[0080] In some embodiments, the outer layer may further comprise pores. In other embodiments, the outer layer does not comprise pores. In some embodiments, the outer layer comprises pores having an average pore diameter of about 1 nm to about 990 nm (e.g., 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 comprises pores having an average pore diameter of about 100 nm to 1000 nm (e.g., 350 nm to 650 nm). In some embodiments, the outer layer may have a thickness 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 100 0nm, about 100nm to about 900nm, 200nm to about 900nm, 300nm to about 900nm, about 400nm to about 900nm, about 450nm to about 900nm, about 500nm to about 900nm, about 550nm to about 900 nm, about 600nm to about 900nm, about 650nm to about 900nm, about 700nm to about 900nm, about 800nm ​​to about 900nm, about 100nm to about 800nm, 200nm to about 800nm, 300nm to about 800nm m, about 400nm to about 800nm, about 450nm to about 800nm, about 500nm to about 800nm, about 550nm to about 800nm, about 600nm to about 800nm, about 650nm to about 800nm, about 700nm to about 800 nm, about 100nm to about 700nm, 200nm to about 700nm, 300nm to about 700nm, about 400nm to about 700nm, about 450nm to about 700nm, about 500nm to about 700nm, about 550nm to about 700nm m, about 600nm to about 700nm, about 650nm to about 700nm, about 100nm to about 650nm, 200nm to about 650nm, 300nm to about 650nm, about 400nm to about 650nm, about 450nm to about 650nm , about 500nm to about 650nm, about 550nm to about 650nm, about 600nm to about 650nm, about 100nm to about 600nm, 200nm to about 600nm, 300nm to about 600nm, about 400nm to about 600nm,Approximately 450nm to approximately 600nm, approximately 500nm to approximately 600nm, approximately 550nm to approximately 600nm, approximately 100nm to approximately 550nm, 200nm to approximately 550nm, 300nm to approximately 550nm, approximately 40 0nm~about 550nm, about 450nm~about 550nm, about 500nm~about 550nm, about 100nm~about 500nm, 200nm~about 500nm, 300nm~about 500nm, about 400nm~ The nanopores may include pores having an average pore diameter of about 500 nm, about 450 nm to about 500 nm, about 100 nm to about 450 nm, 200 nm to about 450 nm, 300 nm to about 450 nm, about 400 nm to about 450 nm, about 100 nm to about 400 nm, 200 nm to about 400 nm, 300 nm to about 400 nm, about 100 nm to about 300 nm, about 200 nm to about 300 nm, or about 100 nm to about 200 nm. In some embodiments, the average pore size is similar to the size of the therapeutic agent, such that one or more therapeutic agents diffuse through the nanopore via single-file diffusion or hindered diffusion. Pores may not be necessary if the desired therapeutic agent is of a sufficiently small size (e.g., having a molecular weight of less than 500) that it can easily diffuse through the outer layer of the capsule.

[0081] In some embodiments, the first polymer or second polymer composition may provide a melt temperature between about 50° C. and about 70° C. In some embodiments, the first polymer or second polymer composition may provide a glass transition temperature (T g ) is provided to provide.

[0082] In some embodiments, each of the one or more capsules independently has a surface charge measurement, as a zeta potential at pH 7.5, of about -25 mV to about 25 mV (e.g., 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).

[0083] In some embodiments, the compositions of the first and second polymers are selected such that when exposed to physiological conditions, 50% of the mass of one or more of the layers remains after at least 3 months. If desired, the degradation rate of any one or more of the layers can be accelerated by adjusting such aspects of the capsule's manufacture (such as layer thickness or porosity) or by increasing the hydrophilicity of the polymer composition used to manufacture one or more layers.

[0084] Therapeutic Agents In a further aspect, the present disclosure also provides one or more therapeutic agents that can be used in the compositions disclosed herein.

[0085] In some embodiments, the one or more therapeutic agents each have a net negative charge within a pH range of about 6.0 to about 7.4.

[0086] As used herein, "therapeutic agent" refers to one or more therapeutic agents, active ingredients, or substances that can be used to treat ocular medical conditions or cancer.The therapeutic agent is typically provided in a form that is ophthalmologically acceptable and does not cause adverse reactions when the compositions disclosed herein are placed in the eye.As discussed herein, the therapeutic agent can be released from the compositions disclosed in a biologically active form.For example, when released from the system into the eye, the therapeutic agent can retain their three-dimensional structure.

[0087] As used herein, the term "therapeutic agent" is further understood to include any synthetic or naturally occurring biologically active compound or composition of matter that induces a desired pharmacological, immunogenic, and / or physiological effect by local and / or systemic action when administered to an organism (human or non-human animal). The term thus encompasses compounds or chemicals traditionally considered to be drugs, vaccines, and biopharmaceuticals (including molecules such as proteins, peptides, hormones, nucleic acids, genetic constructs, and the like). Examples of therapeutic agents are described in well-known literature references such as the Merck Index (14th edition), the Physicians' Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition), and include, but are not limited to, pharmaceuticals; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure, or mitigate diseases or conditions; 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, but is not limited to, adjuvants; anti-infectives (such as antibiotics and antivirals); analgesics and analgesic combinations, anorectics, anti-inflammatory agents, anti-epileptic agents, local and general anesthetic agents, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergics and cholinomimetics, antimuscarinic and muscarinic agents, antiadrenergic agents, antiarrhythmic agents, antihypertensive agents, hormonal and nutritional agents, anti-arthritic agents, anti-asthmatic agents, anticonvulsants, antihistamines, antiemetic agents, antineoplastic agents, antipruritic agents, antipyretics; anti-seizure agents, cardiovascular agents (including calcium channel blockers, beta blockers, beta agonists, and antiarrhythmic agents), antihypertensive agents, diuretics, vasodilators; central nervous system stimulants; cold remedies; and decongestants. The present invention encompasses compounds or compositions for use in all of the major therapeutic areas, including, but not limited to, excretory agents; diagnostic agents; hormones; bone growth stimulators and bone resorption inhibitors; immunosuppressants; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized, or recombinantly produced); as well as nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA), including double-stranded and single-stranded molecules, genetic constructs, expression vectors, antisense molecules, and the like), small molecules (e.g., doxorubicin), and other biologically active macromolecules (e.g., proteins and enzymes). Drugs can be biologically active agents used in medical applications, including veterinary medicine, agriculture (such as by plants), and other areas. The term therapeutic agent also includes, but is not limited to, a medicine; a vitamin; a mineral supplement; a substance used to treat, prevent, diagnose, cure, or mitigate a disease or illness; or a substance that affects the structure or function of the body; or a prodrug that becomes biologically active or more active after being placed in a given physiological environment.

[0088] In some embodiments, the therapeutic agent is a beta blocker (including timolol, betaxolol, levobetaxolol, and carteolol); miotics (including pilocarpine); carbonic anhydrase inhibitors; serotonergic agents; muscarinic agents; dopamine agonists; adrenergic agonists (including apraclonidine and brimonidine); anti-angiogenic agents; anti-infectives (quinolones, such as ciprofloxacin, and aminoglycosides, such as tobramycin and and gentamicin); nonsteroidal and steroidal anti-inflammatory agents (such as suprofen, diclofenac, ketorolac, rimexolone, and tetrahydrocortisol); growth factors (such as EGF); immunosuppressants; and anti-allergy agents (including olopatadine); prostaglandins (such as latanoprost); 15-ketolatanoprost; travoprost; and isopropyl unoprostone.

[0089] 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 can be cyclosporine. In some embodiments, the calcineurin inhibitor can be voclosporin. In some embodiments, the antibiotic can be amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, teicoplanin, vancomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, penicillin, piperacillin, ticarcin, cyclosporine ... The anti-lymphangiogenic agent may be selected from the group consisting of cyclosporine, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, mafenide, sulfacetamide, sulfamethizole, sulfasalazine, sulfisoxazole, trimethoprim, cotrimoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline. In some embodiments, the nicotinic acetylcholine receptor agonist may be any of pilocarpine, atropine, nicotine, epibatidine, 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.

[0090] In some embodiments, the therapeutic agent is a beta blocker (including levobunolol (BETAGAN), timolol (BETIMOL, TIMOPTIC), betaxolol (BETOPTIC), and metipranolol (OPTIPRANOLOL)); an alpha agonist (such as apraclonidine (IOPIDINE) and brimonidine (ALPHAGAN)); a carbonic anhydrase inhibitor (such as acetazolamide, methazolamide, dorzolamide (TRUSOPT), and brinzolamide (AZOPT)); a prostaglandin or prostaglandin analog (such as latanoprost (XALATAN), bimatoprost (LUMIGAN), and travoprost (TRAVATAN)); a miotic or cholinergic agent (such as pilocarpine (ISOPTO CARPINE, PILOPINE) and carbachol (ISOPTO CARPINE, PILOPINE)); CARBACHOL, etc.); epinephrine compounds (such as dipivefrin (PROPINE, etc.); forskolin; or neuroprotective compounds (such as brimonidine and memantine); steroid derivatives (such as 2-methoxyestradiol or an analogue or derivative thereof); or antibiotics.

[0091] The term "VEGF" refers to a vascular endothelial growth factor that induces angiogenesis or angiogenic processes, including but not limited to increased permeability. As used herein, the term "VEGF" encompasses the various subtypes of VEGF (also known as vascular permeability factor (VPF) and VEGF-A), which arise, for example, by alternative splicing of the VEGF-A / VPF gene, and includes VEGF121, VEGF165, and VEGF189. As further used herein, the term "VEGF" encompasses VEGF-related angiogenic factors, such as PIGF (placental growth factor), VEGF-B, VEGF-C, and VEGF-D, and VEGF-E, which act through their cognate VEGF receptors (i.e., VEGFRs) to induce angiogenesis or angiogenic processes. The term "VEGF" includes any member of a class of growth factors that bind to a VEGF receptor (VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flk-1), or VEGFR-3 (FLT-4)). The term "VEGF" may be used to refer to a "VEGF" polypeptide or a "VEGF"-encoding gene or nucleic acid.

[0092] The term "anti-VEGF agent" refers to an agent that reduces or inhibits the activity or production of VEGF, either partially or completely. Anti-VEGF agents can directly or indirectly reduce or inhibit the activity or production of a specific VEGF (such as VEGF165). In addition, "anti-VEGF agents" encompass agents that act on either the VEGF ligand or its cognate receptor to reduce or inhibit the receptor signal associated with VEGF. Non-limiting examples of "anti-VEGF agents" include antisense molecules, ribozymes, or RNAi that target VEGF nucleic acids; anti-VEGF aptamers, anti-VEGF antibodies to VEGF itself or its receptor, or soluble VEGF receptor decoys that prevent VEGF from binding to its cognate receptor; antisense molecules, ribozymes, or RNAi that target cognate VEGF receptor (VEGFR) nucleic acids; anti-VEGFR aptamers or anti-VEGFR antibodies that bind to cognate VEGFR receptors; and VEGFR tyrosine kinase inhibitors.

[0093] In some embodiments, the therapeutic agent may include an anti-VEGF agent. Representative examples of anti-VEGF agents include ranibizumab, bevacizumab, aflibercept, and KH902. VEGF receptor-Fc, fusion proteins, 2C3 antibody, ORA102, pegaptanib, bevasiranib, SIRNA-027, decursin, decursinol, picropodophyllin, guggulsterone, PLG101, eicosanoid LXA4, PTK787, pazopanib, axitinib, 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 proteins, sFLT01, sFLT02, peptide B3, TG100801, sorafenib, G6-31 antibody, fusion antibodies, and antibodies that bind to an epitope of VEGF. Additional non-limiting examples of anti-VEGF agents useful in the present methods 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), as well as antibodies that bind to an epitope of VEGF.

[0094] In various embodiments, 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 pegaptanib or a pharmaceutically acceptable salt thereof. Pegaptinib 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 an epitope of VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E). In some embodiments, the VEGF antagonist binds to an epitope of VEGF such that binding of VEGF to VEGFR is inhibited. In one embodiment, the epitope encompasses a component of the three-dimensional structure of VEGF that is displayed 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 from VEGF.

[0095] In various embodiments, the therapeutic agent may include an agent that blocks or inhibits VEGF-mediated activity (e.g., one or more VEGF antisense nucleic acids). The present disclosure provides therapeutic or prophylactic use of a nucleic acid that includes at least six nucleotides that is 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 that is capable of hybridizing by some sequence complementarity to a portion of an RNA (preferably an mRNA) encoding VEGF. 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 utility as compounds that prevent VEGF expression and may be used in the treatment of diabetes. The antisense nucleic acids of the present disclosure are double-stranded or single-stranded oligonucleotides, RNA, or DNA, or modifications or derivatives thereof, and may be administered directly to cells or produced intracellularly by transcription of an exogenously introduced sequence.

[0096] VEGF antisense nucleic acids are at least 6 nucleotides, preferably ranging from 6 to about 50 oligonucleotides. In certain embodiments, 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 versions thereof, and can be single-stranded or double-stranded. In addition, the antisense molecules can be polymers that are nucleic acid mimics (such as PNA, morpholino oligos, and LNA). Other types of antisense molecules include short double-stranded RNAs known as siRNAs, and short hairpin RNAs, and long dsRNAs (>50 bp, but usually ≧500 bp).

[0097] In various embodiments, the therapeutic agent may comprise one or more ribozyme molecules designed to catalytically cleave the transcript of the gene mRNA encoding VEGF, preventing translation of the target gene mRNA and thus expression of the gene product.

[0098] 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 complementary target RNA, followed by an endonucleolytic cleavage event. The composition of the ribozyme molecule must contain one or more sequences that are complementary to the target gene mRNA and must contain the well-known catalytic sequence responsible for cleaving the mRNA. For this sequence, see, for example, U.S. Pat. No. 5,093,246. Although ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy the mRNA encoding VEGF, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNA at positions defined by adjacent regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA has the following two-base sequence: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art. The ribozymes of the present disclosure also include RNA endoribonucleases (hereinafter "Cech-type ribozymes"), such as those naturally occurring in Tetrahymena thermophila (known as IVS or L-19 IVS RNA). Cech-type ribozymes have eight base-pair active sites that hybridize to a target RNA sequence, after which cleavage of the target RNA occurs. The present disclosure encompasses Cech-type ribozymes that target the eight base-pair active site sequence present in the gene encoding VEGF.

[0099] In further embodiments, the therapeutic agent may include an antibody that inhibits VEGF (such as bevacizumab or ranibizumab). In still further embodiments, the therapeutic agent includes an agent that inhibits VEGF activity (such as tyrosine kinase stimulated by VEGF), examples of which include, but are not limited to, lapatinib, sunitinib, sorafenib, axitinib, and pazopanib.

[0100] The term "anti-RAS agent" or "anti-renin angiotensin system agent" refers to an agent that reduces or inhibits, either partially or completely, the activity or production of a molecule of the renin angiotensin system (RAS). Non-limiting examples of "anti-RAS" or "anti-renin angiotensin system" molecules are one or more of angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, and renin inhibitors.

[0101] In some embodiments, the therapeutic agent may include a renin angiotensin system (RAS) inhibitor, which in some embodiments is one or more of an angiotensin converting enzyme (ACE) inhibitor, an angiotensin receptor blocker, and a renin inhibitor.

[0102] Non-limiting examples of angiotensin-converting enzyme (ACE) inhibitors useful in the present invention include alacepril, alatriopryl, althiopril calcium, ancovenin, benazepril, benazepril hydrochloride, benazeprilat, benzazepril, benzoylcaptopril, captopril, captopril cysteine, captopril glutathione, seranapril, seranopril, ceronapril, cilazapril, cilazapril. lat, conversta-tin, delapril, delapril diacid, enalapril, enalaprilat, enalkiren, enapril, epicaptopril, holoxymitine, fosphenopril, fosenopril, fosenopril sodium, fosinopril, fosinopril sodium, fosinoprilat, fosinoprilic acid, glycopril, hemorphin-4, idapril , imidapril, indolapril, indolaprilat, libenzapril, lisinopril, lyciumin A, lyciumin B, mixampril, moexipril, moexipril, moveltipril, muracein A, muracein B, muracein C, pentopril, perindopril, perindoprilat, pivalopril, pivopril, quinapril, quinapril hydrochloride, quinaprilat, ramipril, ramipril tetra, spirapril, spirapril hydrochloride, spiraprilat, spiropril, spirapril hydrochloride, temocapril, temocapril hydrochloride, teprotide, trandolapril, trandolaprilat, utibapril, zabicipril, zabiciprilat, zofenopril, zofenoprilat, pharmaceutically acceptable salts thereof, and mixtures thereof.

[0103] Non-limiting examples of angiotensin receptor blockers useful in the present invention include, but are not limited to, irbesartan (U.S. Pat. No. 5,270,317, incorporated herein by reference in its entirety), candesartan (U.S. Pat. Nos. 5,196,444 and 5,705,517, incorporated herein by reference in their entireties), valsartan (U.S. Pat. No. 5,399,578, incorporated herein by reference in its entirety), and losartan (U.S. Pat. No. 5,138,069, incorporated herein by reference in its entirety).

[0104] Non-limiting examples of renin inhibitors that may be used as therapeutic agents include, but are not limited to, aliskiren, ditekiren, enalkiren, remikiren, terlaquiren, ciprokiren, and zankiren, pharma- ceutically acceptable salts thereof, and mixtures thereof.

[0105] The term "steroid" refers to compounds belonging to or related to the following exemplary families of compounds: corticosteroids, mineralicosteroids, and sex steroids (including, for example, potentially androgenic or estrogenic, or antiandrogenic and antiestrogenic molecules). Among these are, for example, prednisone, prednisolone, methyl-prednisolone, triamcinolone, fluocinolone, aldosterone, spironolactone, danazol (otherwise known as OPTINA), and others. In some embodiments, the therapeutic agent may include a steroid.

[0106] The term "peroxisome proliferator-activated receptor gamma agent" or "PPAR-γ agent" or "PPARG agent" or "PPAR-γ agent" refers to an agent that acts directly or indirectly on peroxisome proliferator-activated receptors. The agent may also affect PPAR-α ("PPARA") activity.

[0107] In some embodiments, the therapeutic agent may include a regulator of macrophage polarization. Exemplary regulators of macrophage polarization include peroxisome proliferator-activated receptor gamma (PPAR-g) regulators, including agonists, partial agonists, antagonists, or combinations of PPAR-gamma / alpha agonists. In some embodiments, the therapeutic agent may include a PPARgamma regulator, including PPARgamma regulators that are full agonists or partial agonists. In some embodiments, the PPARgamma regulator is a member of the thiazolidinedione (TZD, or glitazone) drug class. As non-limiting examples, the PPARgamma regulator may be one or more of rosiglitazone (AVANDIA), pioglitazone (ACTOS), troglitazone (REZULIN), netoglitazone, rivoglitazone, ciglitazone, rhodanine. In some embodiments, the PPARγ modulator is one or more of irbesartan and telmesartan. In some embodiments, the PPARγ modulator is a nonsteroidal anti-inflammatory drug (e.g., an NSAID such as ibuprofen) or an indole. Known inhibitors include the experimental drug GW-9662. Further examples of PPARγ modulators are those described in WIPO Publication Nos. WO / 1999 / 063983, WO / 2001 / 000579, Nat Rev Immunol.2011 Oct.25;11(11):750-61, or agents identified using the methods of WO / 2002 / 068386, the contents of which are incorporated herein by reference in their entirety.

[0108] In some embodiments, the PPARγ modulator is a "dual" PPAR modulator or a "balanced" PPAR modulator or a "pan" PPAR modulator. In some embodiments, the PPARγ modulator is a glitazar that binds two or more PPAR isoforms (e.g., muraglitazar (Pargluva) and tesaglitazar (Galida) and aleglitazar).

[0109] In some embodiments, the therapeutic agent may include semapimod (CNI-1493), 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.

[0110] In some embodiments, the therapeutic agent may comprise a migration inhibitory factor (MIF) inhibitor.Exemplary MIF inhibitors are described in WIPO Publication Nos. WO2003 / 104203, WO2007 / 070961, WO2009 / 117706, and U.S. Patent Nos. 7,732,146, 7,632,505, 7,294,753, 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-isoxazole acetic acid methyl ester (ISO-1), isoxazoline, p425 (J.Biol.Chem.,287,30653-30663), epoxyazadiradione, or vitamin E.

[0111] In some embodiments, therapeutic agents may include chemokine receptor 2 (CCR2) inhibitors, e.g., as described in U.S. patents and patent publications: U.S. Pat. No. 7,799,824, U.S. Pat. No. 8,067,415, US2007 / 0197590, US2006 / 0069123, US2006 / 0058289, and US2007 / 0037794, the contents of which are incorporated herein by reference in their entireties. In some embodiments, the CCR2 inhibitor is 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).

[0112] In some embodiments, the therapeutic agent may include an agent that modulates autophagy, microautophagy, mitophagy, or other forms of autophagy. In some embodiments, the therapeutic agent may include sirolimus, tacrolimus, rapamycin, everolimus, bafilomycin, chloroquine, hydroxychloroquine, spautin-1, metformin, perifosine, resveratrol, trichostatin, valproic acid, Z-VAD-FMK, or others known in the art. Without wishing to be bound by theory, an agent that modulates autophagy, microautophagy, mitophagy, or other forms of autophagy may alter the recycling of intracellular components (such as, but not limited to, organelles, mitochondria, endoplasmic reticulum, lipids, or others). Without further wishing to be bound by theory, the agent may or may not act through microtubule-associated protein 1A / 1B light chain 3 (LC3).

[0113] In some embodiments, the therapeutic agent may include an agent used in the treatment of cancer (i.e., an anticancer drug or anticancer drug). Exemplary anticancer drugs may be selected for a subject from antimetabolic and antimitotic anticancer drugs, and combinations thereof. A variety of antimetabolic and antimitotic anticancer drugs, including a single such drug or a combination of such drugs, may be used in the methods and compositions described herein.

[0114] Antimetabolites are typically structurally similar to natural metabolites that are involved in the normal metabolic processes of cancer cells (such as the synthesis of nucleic acids and proteins). However, antimetabolites are sufficiently different from natural metabolites that they interfere with the metabolic processes of cancer cells. In cells, antimetabolites are mistaken for similar metabolites and processed by the cells in a manner similar to normal compounds. The presence of the "decoy" metabolites prevents the cells from performing vital functions, and the cells cannot grow and survive. For example, antimetabolites can exert cytotoxic activity by substituting these incorrect nucleotides into cellular DNA, thereby preventing cell division, or by inhibiting important cellular enzymes and preventing DNA replication.

[0115] Thus, in one embodiment, the antimetabolite anticancer agent is a nucleotide or nucleotide analog.In certain embodiments, for example, the antimetabolite may comprise a purine (e.g., guanine or adenosine) or its analog, or a pyrimidine (cytidine or thymidine) or its analog, with or without a sugar moiety attached.

[0116] Suitable antimetabolic anti-cancer agents for use in the present disclosure are generally classified according to the metabolic processes they affect, and may include, but are not limited to, analogs and derivatives of folic acid, pyrimidines, purines, and cytidine. Thus, in one embodiment, the antimetabolic agent(s) is selected from the group consisting of cytidine analogs, folic acid analogs, purine analogs, pyrimidine analogs, and combinations thereof.

[0117] In one particular embodiment, for example, the antimetabolite is a cytidine analog. According to this embodiment, for example, the cytidine analog can be selected from the group consisting of cytarabine (cytosine arabinoside), azacytidine (5-azacytidine), and salts, analogs, and derivatives thereof.

[0118] In another particular embodiment, for example, the antimetabolite is a folic acid analog. Folic acid analogs or antifolates generally function by inhibiting dihydrofolate reductase (DHFR), an enzyme involved in the formation of nucleotides. When this enzyme is blocked, nucleotides are not formed, disrupting DNA replication and cell division. In one particular embodiment, for example, the folic acid analog can be selected from the group consisting of denopterin, methotrexate (amethopterin), pemetrexed, pteropterin, raltitrexed, trimetrexate, and salts, analogs, and derivatives thereof.

[0119] In another particular embodiment, for example, the antimetabolite is a purine analog. Purine-based antimetabolites function by inhibiting DNA synthesis (e.g., by interfering with the production of purine-containing nucleotides (adenine and guanine), which interrupts DNA synthesis and cell division). Purine analogs can also be incorporated into the DNA molecule itself during DNA synthesis, which can interfere with cell division. According to a particular embodiment, for example, the purine analog is acyclovir, allopurinol, 2-aminoadenosine, arabinosyl adenine (araA), azacytidine, azathiprine, 8-aza-adenosine, 8-fluoro-adenosine, 8-methoxy-adenosine, 8-oxo-adenosine, cladribine, deoxycoformycin, fludarabine, gancylovir, 8-aza-guanosine, 8-amino-adeno ... -fluoro-guanosine, 8-methoxy-guanosine, 8-oxo-guanosine, guanosine diphosphate, guanosine diphosphate-β-L-2-aminofucose, guanosine diphosphate-D-arabinose, guanosine diphosphate-2-fluorofucose, guanosine diphosphate fucose, mercaptopurine (6-MP), pentostatin, thiamiprine, thioguanine (6-TG), and salts, analogs, and derivatives thereof.

[0120] In yet another specific embodiment, for example, the antimetabolite is a pyrimidine analogue.Similar to the purine analogue 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 a "decoy", pyrimidine-based compounds can prevent the production of nucleotides and / or can be incorporated into growing DNA chains, leading to termination. In certain embodiments, for example, the pyrimidine analog is ancitabine, azacitidine, 6-azauridine, bromouracil (e.g., 5-bromouracil), capecitabine, carmofur, chlorouracil (e.g., 5-chlorouracil), cytarabine (cytosine arabinoside), cytosine, dideoxyuridine, 3'-azido-3'-deoxythymidine, 3'-dideoxycytidin-2'-ene, 3'-deoxy-3'-deoxythymidin-2'-ene, dihydrouridine, The pyrimidine analog may be selected from the group consisting of uracil, doxifluridine, enocitabine, floxuridine, 5-fluorocytosine, 2-fluorodeoxycytidine, 3-fluoro-3'-deoxythymidine, fluorouracil (e.g., 5-fluorouracil (also known as 5-FU)), gemcitabine, 5-methylcytosine, 5-propynylcytosine, 5-propynylthymine, 5-propynyluracil, thymine, uracil, uridine, and salts, analogs, and derivatives thereof. In one embodiment, the pyrimidine analog is other than 5-fluorouracil. In another embodiment, the pyrimidine analog is gemcitabine or a salt thereof.

[0121] In certain embodiments, the antimetabolite is selected from the group consisting of 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof. In other embodiments, the antimetabolite is selected from the group consisting of capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof. In one particular embodiment, the antimetabolite is other than 5-fluorouracil. In a particularly preferred embodiment, the antimetabolite is gemcitabine or a salt thereof (e.g., gemcitabine HCl (Gemzar®)).

[0122] Other antimetabolic anticancer drugs include acantifolic acid, aminothiadiazole, brequinar sodium, Ciba-Geigy CGP-30694, cyclopentylcytosine, cytarabine phosphate stearate, cytarabine conjugate, Lilly DATHF, Merrel Dow DDFC, desaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, Wellcome EHNA, Merck & Co. EX-015, fazarabine, fludarabine phosphate, N-(2'-furanidyl)-5-fluorouracil, Daiichi Seiyaku FO-152, 5-FU-fibrinogen, isopropylpyrrolidine, Lilly LY-188011; among others, Lilly LY-264618, methobenzaprim, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI The inhibitor may be selected from the group consisting of, but is not limited to, 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.

[0123] In one embodiment, the antimitotic agent is a microtubule inhibitor or a microtubule stabilizer. In general, microtubule stabilizers (such as taxanes and epothilones) bind to the inner surface of β-microtubule strands and promote microtubule assembly by enhancing the nucleation and elongation phases of the polymerization reaction, as well as by reducing the critical concentration of tubulin subunits required for the assembly of microtubules. Unlike microtubule inhibitors (such as vinca alkaloids) that prevent microtubule assembly, microtubule stabilizers (such as taxanes) reduce the lag time and dramatically shift the dynamic equilibrium between tubulin dimers and microtubule polymers toward polymerization. Thus, in one embodiment, the microtubule stabilizer is a taxane or an epothilone. In another embodiment, the microtubule inhibitor is a vinca alkaloid.

[0124] In some embodiments, the therapeutic agent may include a taxane or a derivative or analog thereof. Taxanes may be naturally derived compounds or related forms, or may be chemically synthesized compounds or derivatives thereof with antineoplastic properties. Taxanes are a family of terpenes, including but not limited to paclitaxel (Taxol®) and docetaxel (Taxotere®), which are derived primarily from the Pacific yew tree (Taxus brevifolia), and have activity against certain tumors, particularly breast tumors and ovarian tumors. In one aspect, the taxane is docetaxel or paclitaxel. Paclitaxel 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 the normal dynamic reorganization of the microtubule network, which is essential for vital interphase and mitotic cell function.

[0125] Also included are various known taxane derivatives, including both hydrophilic and hydrophobic derivatives.Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application No. WO99 / 18113; the piperazino and other derivatives described in WO99 / 14209; the taxane derivatives described in WO99 / 09021, WO98 / 22451 and U.S. Patent No. 5,869,680; the 6-thio derivatives described in WO98 / 28288; the sulfenamide derivatives described in U.S. Patent No. 5,821,263; deoxygenated paclitaxel compounds (such as those described in U.S. Patent No. 5,440,056); and the taxol derivatives described in U.S. Patent No. 5,415,869. As noted above, it further includes prodrugs of paclitaxel, including, but not limited to, those described in WO 98 / 58927; WO 98 / 13059; and U.S. Patent No. 5,824,701. The taxane can also be a taxane conjugate, such as paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, docetaxel-PEG, docetaxel-dextran, docetaxel-xylose, and the like. Other derivatives are mentioned in, among other references, "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, PWle Quesne, Eds. (Elsevier, Amsterdam 1986). Each of these references is incorporated herein in its entirety by reference.

[0126] A variety of taxanes can be readily prepared utilizing techniques known to those of skill in the art (see also WO94 / 07882, WO94 / 07881, WO94 / 07880, WO94 / 07876, WO93 / 23555, WO93 / 10076; U.S. Pat. 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 can be obtained from a variety of commercial sources, including, for example, Sigma-Aldrich Co., St. Louis, Mo.

[0127] Alternatively, antimitotic agents can be microtubule inhibitors. In one preferred embodiment, microtubule inhibitors are vinca alkaloids. In general, vinca alkaloids are mitotic spindle poisons. Vinca alkaloids act during mitosis, when chromosomes are separated and begin to move along the mitotic spindle tubules toward one of the poles before cell separation. Under the effect of these spindle poisons, spindle becomes disordered by the dispersion of chromosomes during mitosis, affecting cell proliferation. According to a particular embodiment, for example, vinca alkaloids are selected from the group consisting of vinblastine, vincristine, vindesine, and vinorelbine, as well as their salts, analogs, and derivatives.

[0128] The antimitotic agent may also be an epothilone. In general, members of the epothilone class of compounds stabilize microtubule function following a mechanism similar to that of taxanes. Epothilones may also cause cell cycle arrest at the G2-M transition, leading to cytotoxicity and ultimately apoptosis. Suitable epithiolones include epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, and epothilone F, as well as salts, analogs, and derivatives thereof. One particular epothilone analog is ixabepilone (Ixempra™), an epothilone B analog.

[0129] In certain embodiments, the antimitotic anticancer agent is selected from the group consisting of taxanes, epothilones, vinca alkaloids, and salts and combinations thereof. Thus, for example, in one embodiment, the antimitotic agent is a taxane. More preferably, in this embodiment, the antimitotic agent is paclitaxel or docetaxel, even more preferably paclitaxel. In another embodiment, the antimitotic agent is an epothilone (e.g., an epothilone B analog). In another embodiment, the antimitotic agent is a vinca alkaloid.

[0130] Examples of cancer drugs that may be used in the present disclosure include, but are not limited to, thalidomide; platinum coordination complexes (such as cisplatin (cis-DDP), oxaliplatin, and carboplatin); anthracenediones (such as mitoxantrone); substituted ureas (such as hydroxyurea); methylhydrazine derivatives (such as procarbazine (N-methylhydrazine, MIH)); adrenal cortex suppressors (such as mitotane (o,p'-DDD) and aminoglutethimide); RXR agonists (such as bexarotene); and tyrosine kinase inhibitors (such as sunitimib and imatinib). Additional examples of cancer drugs include alkylating agents, antimetabolites, natural products, hormones and antagonists, and miscellaneous drugs. Alternative names are indicated in parentheses. Examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphainide, and ifosfamide and melphalan (sarcolysin) and chlorambucil); ethylenimines and methylmelamines (such as hexamethylmelamine and thiotepa); alkylsulfonates (such as busulfan); nitrosoureas (such as carmustine (BCNU), semustine (methyl-CCNU), lomustine (CCNU), and streptozotocin); DNA synthesis antagonists (such as estramustine phosphate); and triazines (such as dacarbazine (DTIC, dimethyl-triazenoimidazole carboxamide), and temozolomide). Examples of antimetabolites include folic acid analogs (such as methotrexate (amethopterin)); pyrimidine analogs (such as fluorouracin (5-fluorouracil, 5-FU, SFU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside), and gemcitabine); purine analogs (such as mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG), and pentostatin (2'-deoxycoformycin, deoxycoformycin), cladribine, and fludarabine); and topoisomerase inhibitors (such as amsacrine).Examples of natural products include vinca alkaloids (such as vinblastine (VLB) and vincristine); taxanes (such as paclitaxel, protein-bound paclitaxel (Abraxane), and docetaxel (Taxotere)); 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, epirubicin, etc.); enzymes (such as L-asparaginase); and biological response modifiers (such as interferon alpha and interlelukin 2). Examples of hormones and antagonists include luteinizing releasing hormone agonists (such as buserelin); corticosteroids (such as prednisone and related preparations); progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); estrogens (such as diethylstilbestrol and ethinyl estradiol and related preparations); estrogen antagonists (such as tamoxifen and anastrozole); androgens (such as testosterone propionate and fluoxymesterone and related preparations); androgen antagonists (such as flutamide and bicalutamide); and gonadotropin releasing hormone analogs (such as leuprolide). Alternative names and trade names of these and additional examples of anticancer drugs, as well as their methods of use, including dosage and administration regimens, will be known to those skilled in the art.

[0131] In some embodiments, the anti-cancer agent may include a chemotherapeutic agent. Suitable chemotherapeutic agents include, but are not limited to, alkylating agents, antibiotics, antimetabolites, hormonal agents, plant-derived agents and their synthetic derivatives, anti-angiogenic agents, differentiation inducers, cell growth arrest inducers, apoptosis inducers, cytotoxic agents, agents that affect cellular bioenergetics (i.e., affect cellular ATP levels and the molecules / activities that regulate these levels), biological agents (e.g., monoclonal antibodies), kinase inhibitors, and inhibitors of growth factors and their receptors, gene therapy agents, cell therapy (e.g., stem cells), or any combination thereof.

[0132] In accordance with these embodiments, the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, chlorambucil, melphalan, mechlorethamine, ifosfamide, busulfan, lomustine, streptozocin, temozolomide, dacarbazine, cisplatin, carboplatin, oxaliplatin, procarbazine, uramustine, methotrexate, pemetrexed, fludarabine, cytarabine, fluorouracil, floxuridine, gemcitabine, capecitabine, vinblastine, vincristine, vinorelbine, etoposide, paclitaxel, docetaxel, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, bleomycin, mitomycin, hydroxyurea, topotecan, irinotecan, amsacrine, teniposide, erlotinib hydrochloride, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0133] According to certain embodiments, the therapeutic agent may comprise a biological drug, in particular an antibody. According to some embodiments, the antibody is selected from the group consisting of cetuximab, anti-CD24 antibody, panitumumab, and bevacizumab.

[0134] Therapeutic agents, as used in this disclosure, can include peptides, proteins (such as hormones, enzymes, antibodies, monoclonal antibodies, antibody fragments, monoclonal antibody fragments, and the like), nucleic acids (such as aptamers, siRNA, DNA, RNA, antisense nucleic acids or the like, antisense nucleic acid analogs or the like), low or high molecular weight compounds, receptor agonists, receptor antagonists, partial receptor agonists, and partial receptor antagonists.

[0135] Additional exemplary therapeutic agents may include, but are not limited to, peptide drugs, protein drugs, desensitizing agents, antigens, factors, growth factors, anti-infective agents (such as antibiotics, antimicrobial agents, antiviral agents, antibacterial agents, antiparasitic agents, antifungal agents, and combinations thereof), antiallergic agents, steroids, androgenic steroids, decongestants, hypnotic agents, steroidal anti-inflammatory agents, anticholinergic agents, sympathomimetics, sedative agents, miotic agents, psychostimulants, tranquilizers, vaccines, estrogens, progestational agents, humoral agents, prostaglandins, analgesics, antiseizure agents, antimalarials, antihistamines, cardiac agents, nonsteroidal anti-inflammatory agents, antiparkinsonian agents, antialzheimer's agents, antihypertensive agents, beta-adrenergic blocking agents, alpha-adrenergic blocking agents, nutritional agents, and benzophenanthridine alkaloids. Therapeutic agents may also be substances capable of acting as stimulants, sedatives, hypnotics, analgesics, anticonvulsants, and the like.

[0136] Additional therapeutic agents include CNS active agents, neuroactive agents, inflammatory and anti-inflammatory agents, renal and cardiovascular agents, gastrointestinal agents, antineoplastic agents, immunomodulators, immunosuppressants, hematopoietic agents, growth factors, anticoagulants, thrombolytic agents, antiplatelet agents, hormones, hormone activators, hormone antagonists, vitamins, ophthalmic agents, anabolic agents, antacid agents, antiasthmatic agents, anticholesterol and antilipid agents, anticonvulsants, antidiarrheal agents, antiemetic agents, antimanic agents, antimetabolites, antiemetic agents, antiobesity agents, antipyretics and analgesics, anticonvulsants, These may include antithrombotic agents, antitussives, antiuricemic agents, antianginal agents, antihistamines, appetite suppressants, biological agents, cerebral dilators, coronary vasodilators, bronchodilators, cytotoxic agents, decongestants, diuretics, diagnostic agents, erythropoietic agents, expectorants, gastrointestinal sedatives, hyperglycemic agents, hypnotic agents, hypoglycemic agents, laxatives, mineral supplements, mucolytic agents, neuromuscular drugs, peripheral vasodilators, psychotropic agents, stimulants, thyroid and antithyroid agents, tissue growth agents, uterine relaxants, vitamins, antigenic materials, etc. Other classes of therapeutic agents include those cited in Goodman & Gilman's The Pharmacological Basis of Therapeutics (McGraw Hill) and those contained in the Merck Index and The Physicians' Desk Reference (Thompson Healthcare).

[0137] Other therapeutic agents include androgen inhibitors, polysaccharides, growth factors (e.g., 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, codeine phosphate, codeine sulfate, morphine, mineral supplements, cholestryramine, N-acetylprocainamide, acetaminophen, aspirin, ibuprofen, phenylpropanolamine hydrochloride, caffeine, guaifenesin, aluminum hydroxide, magnesium hydroxide, peptides, polypeptides, proteins, amino acids, hormones, interferons, cytokines, and vaccines.

[0138] Further examples of therapeutic agents include, but are not limited to, peptide drugs, protein drugs, desensitizing agents, antigens, anti-infective agents (such as antibiotics, antimicrobial agents, antiviral agents, antibacterial agents, antiparasitic agents, antifungal agents, and combinations thereof), antiallergic agents, androgenic steroids, decongestants, hypnotic agents, steroidal anti-inflammatory agents, anticholinergic agents, sympathomimetics, sedative agents, miotic agents, psychostimulants, tranquilizers, vaccines, estrogens, progesterone agents, humoral agents, prostaglandins, analgesics, antiseizure agents, antimalarials, antihistamines, antiproliferative agents, anti-VEGF agents, cardiac agents, nonsteroidal anti-inflammatory agents, anti-Parkinson's agents, anti-Alzheimer's agents, antihypertensive agents, beta-adrenergic blocking agents, nutritional agents, and benzophenanthridine alkaloids. Medicaments may also be substances capable of acting as stimulants, sedatives, hypnotics, analgesics, anticonvulsants, and the like.

[0139] Further exemplary therapeutic agents include analgesics (such as acetaminophen, acetylsalicylic acid, and the like); anesthetics (such as lidocaine, xylocaine, and the like); appetite suppressants (such as dexadrine, phendimetrazine tartrate, and the like); anti-arthritic substances (such as methylprednisolone, ibuprofen, and the like); anti-asthmatic substances (such as terbutaline sulfate, theophylline, ephedrine, and the like); antibiotics (such as sulfisoxazole, penicillin G, ampicillin, and the like). antifungals (such as amphotericin B, nystatin, ketoconazole, and the like); antivirals (such as acyclovir, amantadine, and the like); anticancer drugs (such as cyclophosphamide, methotrexate, etretinate, paclitaxel, taxol, and the like); anticoagulants substances (such as heparin, warfarin, and the like); anticonvulsants (such as sodium phenyloin, diazepam, and the like); antidepressants (such as isocarboxazid, amoxapine, and the like); antihistamines (such as diphenhydramine HCl, chlorpheniramine maleate, and the like); hormones (such as insulin, progestins, estrogens, corticoids, glucocorticoids, androgens, and the like); tranquilizers (such as solanacearum, serotonin, and the like); anti-seizure substances (such as belladonna alkaloids, dicyclomine hydrochloride, and the like); vitamins and minerals (such as essential amino acids, calcium, iron, potassium, zinc, vitamin B12, and the like); cardiovascular agents (such as prazosin HCl, nitroglycerin, propranolol HCl, hydralazine HCl, pancrelipase, succinate dehydrogenase, and the like);Peptides and proteins (LHRH, somatostatin, calcitonin, growth hormone, glucagon-like peptides, growth hormone releasing factor, angiotensin, FSH, EGF, bone morphogenetic proteins (BMPs), erythopoeitin (EPO), interferons, interleukins, collagen, fibrinogen, insulin, factor VIII, factor IX, Enbrel®, Rituxam®, Herceptin®, α-glucosidase, Cerazyme / Ceredo se®, vasopressin, ACTH, human serum albumin, gamma globulin, structural proteins, blood product proteins, complex proteins, enzymes, antibodies, monoclonal antibodies, and the like); prostaglandins; nucleic acids; carbohydrates; fats; narcotics (morphine, codeine, and the like, psychotherapeutics, and the like); antimalarials, L-dopa, diuretics (furosemide, spironolactone, and the like); antiulcer drugs (rantidine HCl, cimetidine HCl, and the like);

[0140] Therapeutic agents include immunomodulators (e.g., cytokines, interleukins, interferons, colony stimulating factors, tumor necrosis factors, and the like); immunosuppressants (such as rapamycin, tacrolimus, and the like); allergens (such as cat dander, birch pollen, house dust mites, grass pollen, and the like); bacterial organisms (such as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, Corynebacterium diphteriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitides, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptspirosis interrogans, Borrelia burgddorferi, Campylobacter jejuni, and the like);Antigens of viruses (such as smallpox virus, influenza A and B viruses, respiratory syncytial virus, parainfluenza virus, measles virus, HIV virus, SARS virus, varicella-zoster virus, herpes simplex virus types 1 and 2, cytomeglavirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps virus, rabies virus, rubella virus, coxsackievirus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, lymphocytic choriomeningitis virus, hepatitis B virus, and the like); such fungal, protozoan, and parasitic organisms (such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroids, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma The antigens may also be antigens of bacteria such as Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanasoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and the like. These antigens may be in the form of whole dead organisms, peptides, proteins, glycoproteins, carbohydrates, or combinations thereof;

[0141] In further particular embodiments, the therapeutic agent may comprise an antibiotic, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, paromomycin, ansamycin, geldanamycin, herbimycin, carbacephem, loracarbef, carbapenem, ertapenem, doripenem, imipenem / cilastatin, meropenem, cephalosporin (first generation), cefadroxil, cefazolin, cefalotin, cefalothin, cephalexin, cephalosporin (second generation), ... Faclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cephalosporin (third generation), cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cephalosporin (fourth generation), cefepime, cephalosporin (fifth generation), ceftobiprole, glycopeptides, teicoplanin, vancomycin, macrolides, azithromycin, clarithromycin, dirithromycin , erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin, monobactam, aztreonam, penicillin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin, piperacillin, ticarcillin, polypeptide, bacitracin, colistin, polymyxin B, quinolones, ciprofloxacin, enoxacin, gatifloxacin Sacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, sulfonamides, mafenide, prontosil (classic), sulfacetamide, sulfamethizole, sulfanilimide (classic), sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX), tetracyclines (demeclocycline, doxycycline, minocycline,oxytetracycline, tetracycline, and others); arsphenamine, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide, quinupristin / dalfopristin, rifampicin (rifampin in the US), timidazole, or combinations thereof. In one embodiment, the therapeutic agent can be a combination of rifampicin (rifampin in the US) and minocycline.

[0142] Growth factors useful as therapeutic agents include transforming growth factor-alpha ("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), nerve growth factor ("NGF") (NGF 2.5, NGF 7.0 and βNGF and neurotrophins, including brain derived neurotrophic factor, cartilage derived factors, 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), skeletal growth factor, bone matrix derived growth factor, and bone derived growth factor, and mixtures thereof.

[0143] Cytokines useful as therapeutic agents include, but are not limited to, cardiotrophin, stromal cell-derived factor, macrophage-derived chemokine (MDC), melanoma growth stimulating activity (MGSA), macrophage inflammatory protein 1 alpha (MIP-1 alpha), 2, 3 alpha, 3 beta, 4, and 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-alpha, and TNF-beta. Immunoglobulins useful in the present 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.

[0144] Other molecules useful as therapeutic agents include, but are not limited to, growth hormone, leptin, leukemia inhibitory factor (LIF), tumor necrosis factors alpha and beta, endostatin, thrombospondin, bone morphogenetic protein-1, bone morphogenetic proteins 2 and 7, osteonectin, somatomedin-like peptide, osteocalcin, interferon alpha, interferon alpha A, interferon beta, interferon gamma, interferon 1 alpha, and interleukins 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, and 18.

[0145] In some embodiments, the therapeutic agent is present in the disclosed drug delivery compositions at a concentration of about 10, about 20, about 30, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, In some embodiments, the therapeutic agent is present in an amount of 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 (in μg therapeutic agent per mg body weight of the disclosed drug delivery compositions); or in a range of amounts of therapeutic agent bracketed by any of the foregoing values; or in any combination of the foregoing values.

[0146] In some embodiments, the therapeutic agent exhibits near zero order release kinetics over a period of at least 30 days (e.g., 45 days, 60 days, 3 months, 6 months, 9 months, 1 year, or more). The therapeutic agent may exhibit near zero order release kinetics at the time of implantation of the drug delivery composition or after a period thereafter (e.g., the therapeutic agent begins to exhibit near zero order release kinetics about 30 days after implantation of the drug delivery composition). In other embodiments, the drug delivery composition may exhibit kinetics that deviate from zero order kinetics.

[0147] Methods of Preparing the Disclosed Drug Delivery Compositions In various aspects, the disclosed drug delivery devices are prepared by the methods disclosed herein below and as described in specific aspects of the representative examples that follow.

[0148] Thus, in one aspect, a method of preparing a drug delivery device described herein is provided 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.

[0149] In some embodiments, forming the first layer comprises electrospinning using a solution of the first polymer and a voltage difference of about 10 kV to about 30 kV.

[0150] In some embodiments, the first polymer solution is 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, the trifluoroacetic acid and dichloromethane are present in a ratio of about 1:10 to about 10:1 (e.g., a ratio of about 5:3 to about 10:3). In some embodiments, the trifluoroacetic acid and dichloromethane are present in a ratio of about 7:3.

[0151] In some embodiments, forming the second layer comprises electrospinning a solution comprising a second polymer and optionally a porogen onto the formed first layer, and the voltage difference used for electrospinning is about 20 kV to about 30 kV.

[0152] In some embodiments, the solution comprising the second polymer and optionally the porogen is about 1% to about 10% w / v (e.g., about 2.5% to about 10% w / v or about 5% to about 10% w / v) of the second polymer and the porogen based on the total weight. In some embodiments, the solution comprising the second polymer and the porogen is a 1,1,1,3,3,3,-hexafluoropropan-2-ol solution.

[0153] In some embodiments, the weight ratio of the second polymer to the porogen is about 90:100 to about 100:1 (e.g., about 90:100 to 99.9:0.1, about 90:100 to 95:5, or about 95:5 to 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 ranges from about 50:50 to about 100:0.

[0154] "Porogen," as used herein, refers to any material that can be used to generate a porous material (e.g., porous polycaprolactone as described herein). In some embodiments, the porogen comprises a water-soluble compound, i.e., such that upon washing the drug delivery device with water, the porogen is substantially removed from the outer layer. In some embodiments, the porogen is selected from the group consisting of ([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-hydroxypropanesulfonic acid) (T In some embodiments, the porogen comprises a compound selected from (APSO), (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-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 a salt thereof, such as a sodium salt thereof. In some embodiments, the porogen comprises a HEPES sodium salt. In some embodiments, the porogen comprises a water soluble polymer, such as polyethylene glycol, polyoxyethylene copolymer, acrylate copolymer (including quaternary ammonium groups, polyacrylamide, polyvinyl alcohol, hyaluronan, and polyvinylpyrrolidone). In other embodiments, the porogen comprises gelatin, polyethylene glycol (PEG), chitosan, polyvinylpyrrolidone (PVP), polyvinyl alcohol, or agarose.

[0155] In some embodiments, the method further comprises sintering the drug delivery device following formation of the outer layer. In some embodiments, sintering comprises heating at a temperature of about 50° C. to about 150° C. (e.g., about 90° C. to about 110° C.). In some embodiments, sintering comprises heating for a period of about 1 minute to about 6 hours (e.g., about 30 minutes to about 6 hours).

[0156] In some embodiments, the method further comprises washing the drug delivery device following sintering. In some embodiments, the drug delivery device is washed with a saturated sodium bicarbonate solution followed by deionized water. In some embodiments, the porogen is substantially removed from the drug delivery device upon washing with deionized water.

[0157] In some embodiments, the method further comprises drying the drug delivery device following washing. In some embodiments, the drying is under vacuum. In some embodiments, the drying is at a temperature of about 50° C. to about 150° C. (e.g., about 90° C. to 110° C.). In some embodiments, the drying occurs for a period of about 1 minute to about 6 hours (e.g., about 30 minutes to about 6 hours).

[0158] In other embodiments, the disclosed capsules can be manufactured by any suitable method as would be readily understood by one of ordinary skill in the art. In some embodiments, the disclosed capsules can be manufactured by asymmetric membrane formation. 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 capsules can be manufactured using three-dimensional printing. In some embodiments, the disclosed capsules can be manufactured around methylcellulose, which can be subsequently removed to form the lumen compartment. In some embodiments, the disclosed capsules can be manufactured by methods described by Envisia Therapeutics in WO2015 / 085251, WO2016 / 144832, WO2016 / 196365, WO2017 / 015604, WO2017 / 015616, or WO2017 / 015675 (each of which is incorporated herein by reference in its entirety for all purposes). In still other embodiments, the disclosed capsules can be manufactured by methods similar to those used in the manufacture of hollow fiber membranes, such as phase inversion, including non-solvent induced phase inversion (NIPS), (solvent) evaporation induced phase inversion (EIPS), vapor sorption induced phase inversion (VIPS), and thermally induced phase inversion (TIPS). In some embodiments, the disclosed capsules can be manufactured using methods similar to those described in US2015 / 232506 (which is incorporated herein by reference in its entirety for all purposes). In some embodiments, the pores may instead be formed by laser diffraction of the capsule.

[0159] In some embodiments, the two ends of the tube shape of the capsule are closed. The ends can be closed by any sealing technique as appropriately selected by a person skilled in the art. In some embodiments, the two ends are sealed using a radio frequency tube sealing technique. In such a technique, radio frequency generates eddy currents in the wall, which heats at least the polymer layer. When the temperature reaches the melting point of the polymer, the clamp is closed and the molten polymer is cooled and formed. In some embodiments, the two ends are sealed using hot-jaw tube sealing, where a heated jaw applies heat to the outside of the tube shape and heats the inside for sealing. In some embodiments, the two ends can be sealed using ultrasonic tube sealing. In such a technique, the polymer composition of the inner layer is heated and melted by radio frequency frictional force introduced from an ultrasonic horn. The clamp is then closed around the section intended to be sealed, cooled, and formed to seal the ends. In some embodiments, the two ends are sealed using hot air sealing, where the system heats the interior seal area of ​​the capsule with hot air and then subsequently presses and cools the ends in the next station.

[0160] Methods of Treatment Using the Disclosed Drug Delivery Devices Also provided herein are methods of treating a clinical condition by administration of the disclosed drug delivery compositions. The clinical condition may be a clinical disorder, disease, dysfunction, or other condition that may be ameliorated by the therapeutic composition.

[0161] The term "administering" the disclosed drug delivery device to a subject or "administration" thereof encompasses any route of introducing or delivering the device to a subject to perform its intended function. Administration can be performed by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), or topical. Administration includes self-administration and administration by another. In some instances, administration is via injection into the eye (including intraocular injection). In other instances, for example, in the treatment of cancer, administration can be via injection of the disclosed drug delivery composition into, adjacent to, adjacent to, or proximal to a tumor or other mass of cancer cells.

[0162] It will also be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean "substantially," which encompasses total and less than total treatment or prevention, where some degree of biologically or medically significant results is achieved. Treatment may be continuous extended treatment for chronic conditions, or single or several administrations for treatment of acute conditions.

[0163] The term "separate" administration refers to the administration of at least two active ingredients at the same time or substantially the same time, but by different routes.

[0164] The term "sequential" administration refers to the administration of at least two active ingredients at different times, with the same or different routes of administration. More specifically, sequential use refers to the totality of administering one of the active ingredients before the administration of the other(s) begins. Thus, it is possible to administer one of the active ingredients for minutes, hours, or days before the administration of the other active ingredient(s). Thus, the term "sequential" is different from "simultaneous" administration.

[0165] The term "simultaneous" administration refers to the administration of at least two active ingredients at the same or substantially the same time, by the same route.

[0166] The term "therapeutic" as used herein means treatment and / or prophylaxis. A therapeutic effect is achieved by suppression, amelioration, or eradication of a disease state.

[0167] The present disclosure further provides a method of treating an ophthalmic disease or disorder by administering a therapeutically effective amount of a composition described herein. In some embodiments, the disclosed method involves treating an ophthalmic disorder, comprising 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 disorder. In some aspects, the method may further comprise diagnosing the subject with an ophthalmic disorder.

[0168] Ophthalmologic disorders include acute macular neuroretinopathy; Behcet's disease; angiogenesis (including choroidal neovascularization); diabetic uveitis; histoplasmosis; infection (such as infections caused by fungi or viruses); macular degeneration (such as acute macular degeneration (AMD) including wet AMD, non-exudative AMD, and wet AMD); edema (such as macular edema, cystoid macular edema, and diabetic macular edema); multifocal choroiditis; ocular trauma affecting a site or location in the posterior segment of the eye; ocular tumors; retinal disorders (such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreous retinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinal disease, etc.); sympathetic ophthalmia; Vogt's disease; The ophthalmological disorder may be Koyanagi-Harada (VKH) syndrome; uveal exudation; posterior ocular pathology caused or affected by ocular laser treatment; posterior ocular pathology caused or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, non-retinopathy diabetic retinal dysfunction, retinitis pigmentosa, cancer, and glaucoma.In certain instances, the ophthalmological disorder is wet age-related macular degeneration (wet AMD), cancer, neovascularization, macular edema, or edema.In further particular embodiments, the ophthalmological disorder is wet age-related macular degeneration (wet AMD).

[0169] In various embodiments, the injection for treating an ophthalmic disorder can be an injection into the vitreous chamber of the eye. In some cases, the injection is an intravitreal injection, a subconjunctival injection, a sub-Tenon injection, a retrobulbar injection, or a suprachoroidal injection.

[0170] "Ocular region" or "ocular site" means any section of the ocular globe (eyeball), including the anterior and posterior segments, and includes, but is not limited to, any functional (e.g., visual) or structural tissue generally found in the eyeball, or tissue or cell layer that partially or completely lines the interior or exterior of the eyeball. Specific examples of sections of the eyeball in an ocular region include, but are not limited to, the anterior chamber, the posterior chamber, the vitreous cavity, the choroid, the suprachoroidal space, the conjunctiva, the subconjunctival space, the extrascleral space, the intracorneal space, the subretinal space, the subtenon space, the epicorneal space, the sclera, the pars plana, the surgically induced avascular region, the macula, and the retina.

[0171] "Ophthalmic disorder" may mean a disease, illness, or condition that affects or involves the eye or one of the parts or regions of the eye. Broadly speaking, the eye includes the eyeball (including the cornea) and other tissues and fluids that make up the eyeball, the periocular muscles (such as the oblique and rectus muscles), and the portion of the optic nerve within or adjacent to the eyeball.

[0172] "Glaucoma" refers to primary glaucoma, secondary glaucoma, and / or congenital glaucoma. Primary glaucoma can include open-angle glaucoma and closed-angle glaucoma. Secondary glaucoma can occur as a complication of various other conditions, such as injury, inflammation, pigment scattering, vascular disease, and diabetes. Increased intraocular pressure in glaucoma damages the optic nerve that enters the eye, causing blindness. Thus, in one non-limiting embodiment, STC-1, or MSCs expressing increased amounts of STC-1, can be used in the treatment of glaucoma to prevent or delay the onset of blindness by reducing reactive oxygen species.

[0173] "Inflammation-mediated" in reference to ocular conditions means any condition of the eye that may benefit from treatment with an anti-inflammatory agent, and is intended to include, 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 uveal effusion.

[0174] With respect to ocular conditions, "injury" or "damage" are used interchangeably and refer to cellular and morphological signs and symptoms resulting from inflammatory-mediated conditions (e.g., inflammation), tissue injury caused by means other than inflammation (e.g., chemical injury, including chemical burns), and injury caused by infection (including, but not limited to, bacterial, viral, or fungal infection).

[0175] "Intraocular" means within or under the tissue of the eye. Intraocular administration of a drug delivery system includes administration of the drug delivery system to sub-Tenon, subconjunctival, suprachoroidal, subretinal, intravitreal, anterior chamber, and the like locations. Intraocular administration of a drug delivery system excludes administration of the drug delivery system to topical, systemic, intramuscular, subcutaneous, intraperitoneal, and the like locations.

[0176] "Macular degeneration" refers to any of a number of disorders and conditions in which the macula degenerates or loses functional activity. The degeneration or loss of functional activity may result, for example, from cell death, reduced cell proliferation, loss of normal biological function, or a combination of the above. Macular degeneration may lead to and / or manifest as alterations in the structural integrity of the cells and / or extracellular matrix of the macula, alterations in the normal cell-matrix architecture and / or extracellular matrix architecture, and / or loss of function of macular cells. The cells may be any cell type normally present in or near the macula, including RPE cells, photoreceptors, and capillary endothelial cells. Age-related macular degeneration (or ARMD) is the major macular degeneration-related condition, but numerous others are known, including, but not limited to, Best macular degeneration, Stargardt macular degeneration, 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 those with AMD and is characterized by abnormal blood vessels growing under or through the retinal pigment epithelium (RPE), resulting in bleeding, seepage, scarring, or serous retinal detachment. Eighty to ninety percent of AMD patients have the atrophic form, characterized by atrophy of the retinal pigment epithelium and loss of macular photoreceptors. Drusen may or may not be present in the macula. There may also be atrophy of areas of the retinal pigment epithelium in the macula that causes vision loss. There is currently no cure for any form of AMD, but some success in attenuating wet AMD has been achieved with photodynamic therapy and especially anti-VEGF therapy.

[0177] "Drusen" are debris-like material that accumulates under the RPE with age. Drusen are observed using fundus examination. A normal eye has a drusen-free macula, but drusen may be abundant in the retinal periphery. The presence of soft drusen in the macula, in the absence of any macular vision loss, is considered an early stage of AMD. Drusen contain a variety of lipids, polysaccharides, and glycosaminoglycans, along with multiple proteins, modified proteins, or protein adducts. There is no generally accepted treatment to address drusen formation and thereby manage the progressive nature of AMD.

[0178] "Ocular neovascularization" (ONV) is used herein to refer to choroidal or retinal neovascularization, or both.

[0179] "Retinal neovascularization" (RNV) refers to the abnormal development, proliferation, and / or growth of retinal blood vessels, for example, on the surface of the retina.

[0180] "Subretinal neovascularization" (SRNVM) refers to the abnormal development, proliferation, and / or growth of blood vessels beneath the surface of the retina.

[0181] "Cornea" refers to the transparent structure that forms the anterior part of the fibrous membrane of the eye. The cornea consists of five layers: 1) the anterior corneal epithelium (continuous with the conjunctiva); 2) the anterior limiting membrane (Bowman's layer); 3) the stroma propria or stromal cell layer; 4) the posterior limiting membrane (Descemet's membrane); and 5) the endothelium of the anterior chamber or corneal endothelium (keratoderma).

[0182] "Retina" refers to the innermost layer of the eyeball, which surrounds the vitreous body and is continuous posteriorly with the optic nerve. The retina is composed of layers including: 1) the internal limiting membrane; 2) the nerve fiber layer; 3) the ganglion cell layer; 4) the inner plexiform layer; 5) the inner nuclear layer; 6) the outer plexiform layer; 7) the outer nuclear layer; 8) the external limiting membrane; and 9) the rod-cone layer.

[0183] "Retinal degeneration" refers to any inherited or acquired degeneration of the retina and / or retinal pigment epithelium. Non-limiting examples include retinitis pigmentosa, Best's disease, RPE pattern dystrophy, and age-related macular degeneration.

[0184] In various embodiments, the methods of treating ophthalmic disorders include treating various diseases or conditions of the retina of the eye, including the following: maculopathy / retinal degeneration: macular degeneration (including age-related macular degeneration (ARMD) such as non-exudative age-related macular degeneration and exudative age-related macular degeneration); choroidal neovascularization; retinopathies (including diabetic retinopathy, acute macular neuroretinopathy and chronic macular neuroretinopathy, central serous chorioretinopathy); and macular edema (including cystoid macular edema and diabetic macular edema); uveitis / retinitis. / choroiditis: acute multifocal spotty pigment epitheliopathy, Behcet's disease, scattershot chorioretinopathy, infectious (syphilis, Lyme disease, tuberculosis, toxoplasmosis), uveitis (including intermediate uveitis (pars planitis) and anterior uveitis), multifocal choroiditis, multiple ephemeral white dot 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, Branch retinal vein occlusion, Hypertensive changes, Ocular ischemic syndrome, Retinal artery microaneurysms, Coats' disease, Parafoveal telangiectasia, Hemiretinal vein occlusion, Papillary vasculitis, Central retinal artery occlusion, Branch retinal artery occlusion, Carotid artery disease (CAD), Dendritic vasculitis, Sickle cell retinopathy and other hemoglobinopathies, Pigmented streaks, Familial exudative vitreoretinopathy, Eales' disease; Traumatic / surgical diseases: Sympathetic ophthalmia, Uveitic retinal disease, Retinal detachment, Trauma, Laser, PDT, Photocoagulation, Blood flow during surgery depression, radiation retinopathy, bone marrow transplant retinopathy;proliferative disorders: proliferative vitreoretinopathy and epiretinal membrane, proliferative diabetic retinopathy;infectious disorders: ocular histoplasmosis, ocular toxocariasis, ocular histoplasmosis syndrome (OHS), endophthalmitis, toxoplasmosis, retinal disease associated with HIV infection, choroidal disease associated with HIV infection, uveitis disease associated with HIV infection, viral retinitis, acute retinal necrosis, progressive outer retinal necrosis, fungal retinal disease, ocular syphilis, ocular tuberculosis, generalized unilateral subacute neuroretinitis, and myiasis;Genetic Disorders: Retinitis Pigmentosa, Systemic Disorders Associated with Retinal Dystrophies, Congenital Stationary Night Blindness, Cone Dystrophies, Stargardt Disease, Fundus Flavicolor, Best Disease, Pattern Dystrophy of the Retinal Pigment Epithelium, X-Linked Retinoschisis, Sorsby Fundus Dystrophy, Benign Central Maculopathy, Bietti Crystalline Dystrophy, Pseudoxanthoma Elasticum;Retinal Tears / Leaks: Retinal Detachment, Macular Hole, Giant Retinal Breaks;Tumors: Retinal Disease Associated with Tumors, Congenital Hypertrophy of the RPE, Posterior Uveal Melanoma, Choroidal Hemangioma, Choroidal Osteoma, Choroidal Metastases, Retinal and Retinal Pigment Epithelial Hamartoma, Retinoblastoma, Angioproliferative Tumor of the Fundus, Retinal Astrocytoma, Intraocular Lymphoid Tumor;Other: Punctate Inner Choroidopathy, Acute Posterior Multifocal Spotted Pigment Epitheliopathy, Myopic Retinal Degeneration, Acute Retinal Pigment Epitheliitis, and the like;

[0185] An anterior ocular condition is a disease, illness, or condition that affects or involves an anterior (i.e., front of the eye) region or site, such as the periocular muscles, eyelids, or tissues or fluids of the eye that are located behind the posterior wall of the lens capsule or anterior to the ciliary muscle. Thus, anterior ocular conditions primarily affect or involve the conjunctiva, cornea, anterior chamber, iris, posterior chamber (behind the iris but in front of 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.

[0186] Thus, anterior ocular conditions may include, for example, diseases, illnesses, or conditions such as aphakia; pseudophakia; astigmatism; blepharospasm; cataract; conjunctival disease; conjunctivitis (including but not limited to atopic keratoconjunctivitis); corneal injury (including but not limited to injury to the corneal stromal region); corneal disease; corneal ulcer; dry eye syndrome; eyelid disease; lacrimal system disease; lacrimal duct obstruction; myopia; presbyopia; pupillary disorder; refractive disorder, and strabismus. Glaucoma may be determined to be an anterior ocular condition because the clinical goal of glaucoma treatment may be to reduce the high pressure of aqueous humor in the anterior chamber (i.e., reduce intraocular pressure).

[0187] Other diseases or disorders of the eye that may be treated according to the present invention include, but are not limited to, ocular cicatricial pemphigoid (OCP), Stevens Johnson syndrome, and cataracts.

[0188] A posterior ocular condition is a disease, illness, or condition that primarily affects or involves a region or area of ​​the posterior segment of the eye, such as the choroid or sclera (in a posterior position relative to the plane through the posterior wall of the lens capsule), vitreous body, vitreous chamber, retina, optic nerve (i.e., optic disc), and the blood vessels and nerves that vascularize or innervate the posterior ocular region or area. Thus, posterior segment pathologies include, for example, acute macular neuroretinopathy; Behcet'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, nonexudative 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 a site or location in the posterior segment; ocular tumors; retinal disorders (such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreous retinopathy (PVR), retinal artery occlusion, and the like). The diseases, illnesses, or conditions may include retinal vein occlusive disease, retinal detachment, uveitis retinal disease, etc.); sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; uveal exudation; posterior segment pathologies caused or affected by ocular laser treatment; posterior segment pathologies caused or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, non-retinopathy diabetic retinal dysfunction, retinitis pigmentosa, and glaucoma. Glaucoma may be considered a posterior segment pathology because the treatment goal may be to prevent or reduce the occurrence of vision loss due to damage to or loss of retinal ganglion cells or retinal nerve fibers (i.e., neuroprotection).

[0189] In some embodiments, the ocular disorder is ocular inflammation resulting from, for example, iritis, conjunctivitis, seasonal allergic conjunctivitis, acute and chronic endophthalmitis, anterior uveitis, uveitis associated with systemic disease, posterior uveitis, chorioretinitis, pars planitis, masked syndrome (including ocular lymphoma), pemphigoid, scleritis, keratitis, severe ocular allergies, corneal abrasion, and blood-aqueous barrier disruption. In yet another embodiment, the ocular disorder is post-operative ocular inflammation resulting from, for example, laser photorefractive keratectomy, cataract removal surgery, intraocular lens insertion, vitrectomy, corneal transplantation, forms of superficial keratectomy (such as DSEK), and radial keratectomy.

[0190] In various embodiments, the injection for treating an ophthalmic disorder can be an injection into the vitreous chamber of the eye. In some cases, the injection is an intravitreal injection, a subconjunctival injection, a sub-Tenon injection, a retrobulbar injection, or a suprachoroidal injection.

[0191] In various aspects, the method for the treatment of an ophthalmic disorder includes administration, e.g., via injection, of the disclosed drug delivery device containing an amount of about 0.01 mg to about 25 mg of therapeutic agent; or about 1 mg to about 15 mg of therapeutic agent. In some embodiments, the drug delivery composition can release an amount of drug that maintains a concentration of about 10 picomolar to about 500 picomolar in the vitreous of the eye over a period of about 10 days to about 12 months. The amount of therapeutic substance in the drug delivery composition will depend on the amount of therapeutic agent that can be present in one or more capsules and the amount necessary to achieve the desired therapeutic effect.

[0192] In some embodiments, the disclosed drug delivery can protect the biological activity of the encapsulated therapeutic agent for periods of up to 12 months. The level of protection of biological activity will depend on both the therapeutic agent used and the selected composition of the disclosed capsules, but can be quantified by methods such as HPLC (to determine the amount and form of drug present in the eye), cellular assays of activity against a positive control (such as the use of a therapeutic agent alone), and ELISA to characterize other forms of the therapeutic agent or assess changes in biological activity (such as transcription factor expression).

[0193] kit The present disclosure also relates to a kit comprising (a) a drug delivery composition described herein; (b) a drug delivery composition described herein in a sterile package; or (c) one of a pre-filled syringe or needle containing a drug delivery composition described herein; and instructions for administering the drug delivery composition described herein to treat a clinical condition or pathology.

[0194] In further embodiments, the disclosed kits can be packaged with a daily dosing regimen (e.g., packaged on a card, packaged with a dosing card, packaged on a blister or blow molded plastic, etc.). Such packaging enhances the product and increases ease of use for administration by health care professionals. Such packaging can also reduce potential medical errors. The invention also features such kits that further contain instructions for use.

[0195] In a further aspect, the disclosure also provides a pharmaceutical pack or kit comprising one or more packages containing the disclosed drug delivery compositions. Associated with such packages may be a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, the notice reflecting approval by that agency of the manufacture, use, or sale for human administration.

[0196] In various embodiments, the disclosed kits can further include therapeutic agents, compounds, and / or products that are co-packaged, co-formulated, and / or co-delivered with other components. For example, a drug manufacturer, drug reseller, physician, compounding facility, or pharmacist can provide a kit that includes the disclosed drug delivery composition and another component for delivery to a patient.

[0197] It is contemplated that the disclosed kits can be used in connection with the disclosed methods of making the disclosed compositions, the disclosed methods of using or processing the disclosed compositions, and / or the disclosed compositions.

[0198] From the foregoing, it will be seen that the aspects herein are well adapted to attain all the ends and objectives set forth above, together with other advantages which are obvious and inherent in the structure.

[0199] Although certain elements and steps are discussed in relation to each other, it is understood that any element and / or step provided herein is contemplated as being combinable with other elements and / or steps, regardless of explicit provision of that same combination (while still being within the scope provided herein).

[0200] It will be understood that certain features and subcombinations are advantageous and may be used without reference to other features and subcombinations, which are contemplated by and within the scope of the appended claims.

[0201] Since many possible embodiments can be made without departing from the scope thereof, it will be understood that all matters herein described or shown in the accompanying drawings and detailed description are to be interpreted in an illustrative and not a limiting sense.

[0202] It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Those skilled in the art will recognize many variations and adaptations of the embodiments described herein. These variations and adaptations are intended to be included within the teachings of the present disclosure and covered by the claims of this specification.

[0203] Having described aspects of the present disclosure, the following examples generally describe some additional aspects of the present disclosure. Aspects of the present disclosure will be described in conjunction with the following examples and corresponding text and figures, but there is no intent to limit the aspects of the present disclosure to this description. On the contrary, it is intended to cover all alternatives, modifications, and equivalents that are encompassed within the spirit and scope of the present disclosure. EXAMPLES

[0204] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how the compounds, compositions, products, devices, and / or methods claimed herein are made and evaluated, are intended to be purely illustrative of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations will occur. Unless otherwise indicated, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric pressure.

[0205] material Chitosan (DD>75%, Mw 310,000-375,000 Da), polycaprolactone (Mn 80,000), trifluoroacetic acid (TFA), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) sodium salt, 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 VEGF recombinant human protein were purchased from Fisher Scientific International Inc. (Hampton, NH). Bevacizumab (Avastin) was purchased from Genentech, Inc. (San Francisco, CA). Bicinchoninic acid (BCA) protein assay kit and colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, horseradish peroxidase (HRP)-conjugated goat anti-human immunoglobulin G (IgG) crystallographic fragment (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 American Type Culture Collection (Rockville, MD). Human umbilical vein endothelial cells (HUVEC), medium 200PRF and low serum growth supplement and lactose dehydrogenase-elevating virus (LDEV)-free growth factor-reduced basement membrane matrix were purchased from Thermo Fisher Scientific Inc. (Waltha, MA). All other reagents used were of analytical grade.

[0206] Capsule Production Two sizes of capsules with different inner diameters (260 μm and 1645 μm) were fabricated in this study. The 1.645 mm size capsules mainly served as a preliminary model for smaller capsules to optimize the processing conditions. The 260 μm size capsules were used for subsequent studies.

[0207] The capsule fabrication process is shown in Figure 1A. Chitosan fiber layers were prepared via electrospinning based on a modified previous study (see Gu, BK, et al., Fabrication of sonicated chitosan nanofiber mat with enlarged porosity for use as hemostatic materials. 2013.97(1):p.65-73). Briefly, a 5.0% (w / v) chitosan solution prepared in a mixture of TFA and DCM with a volume ratio of 7:3 was extruded through a 20-gauge stainless steel needle connected to the cathode of a high-voltage direct current generator. The substrate was attached to a rotating drum collector at a speed of 500 rpm, on which the electrospun fibers were deposited. To obtain capsules with two different inner diameters, 315 stainless steel rods with diameters of either 1.645 mm or 260 μm were used for fiber collection. The solution was continuously fed at a voltage of 25.0 kV with a feed rate of 3.0 mL / h for the 1.645 mm drum collector and 1.0 mL / h for the 260 μm drum collector. Humidity during electrospinning was controlled at 30% using a nitrogen-filled glove box.

[0208] To prepare the PCL nanoporous layer (Cipitria, A., et al., Design, fabrication and characterization of PCL electrospun scaffolds-a review. 2011.21(26):p.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 in electrospun nanofibers. 2011.7(4):p.1516-1524; and Chaparro, FJ, et al., Sintered electrospun poly(ε-caprolactone)-poly(ethylene terephthalate) for drug delivery. Journal of Applied Polymer See Science, 2019.0(0):p.47731), a combination of 0.5 g of PCL and HEPES sodium 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 HEPES sodium salt (100:0; 99:1; 95:5; 92.5:7.5; 90:10) were studied to assess the effect of the salt-induced porous structure of the PCL film on drug release. A syringe pump was used to continuously feed the PCL solution at a feed rate of 3.0 mL / h for the 1.645 mm drum collector and 1.0 mL / h for the 260 μm drum collector. A high-voltage DC generator was set at 24.0 kV to produce PCL nanofibers that were deposited on 1.645 mm and 260 μm diameter 315 stainless steel rods with or without an unstretched chitosan layer to form bilayer and monolayer films, respectively.

[0209] The electrospun capsules were sintered under vacuum at 100 °C for 3 hours using an AccuTemp digital vacuum oven 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 saturated sodium bicarbonate solution to neutralize the TFA, then with deionized water to dissolve and remove the HEPES sodium salt. The capsules were vacuum dried overnight. The outer diameters of capsules prepared using a 1.645 mm rod before and after sintering were measured using a digital micrometer (Keyence). The thickness of the film was calculated as [outer diameter of sintered capsule - 1.645 mm] / 2. Images of capsules prepared using a 260 μm diameter rod were acquired using an optical microscope (Cole-Parmer). The images were analyzed by Motic Image Plus to determine the outer diameter of the capsules. The thickness of the film was calculated as [outer diameter of sintered capsule - 260 μm] / 2.

[0210] Capsule characterization The morphological characteristics of the capsules were investigated by 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 deposited on carbon tape placed on an aluminum stub mount and sputter-coated with a layer of gold-palladium. The capsules were immersed in liquid nitrogen and fractured to obtain cross sections for imaging. The average fiber and pore sizes of the PCL and chitosan layers were characterized and quantified from the SEM images of the three samples using ImageJ (NIH).

[0211] 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. A germanium crystal was placed in contact with the sample and 100 scans were performed at 8 cm -1 The data was collected at a resolution of 1727 cm -1 and 1590cm -1 Standard peak positions in the FTIR spectra were used to identify PCL (carbonyl peak) and chitosan (amine band), respectively (see Elzein, T., et al., FTIR study of polycaprolactone chain organization at interfaces. Journal of Colloid and Interface Science, 2004. 273(2): p. 381-387; and Osman, Z. and A. K. Arof, FTIR studies of chitosan acetate based polymer electrolytes. Electrochimica Acta, 2003. 48(8): p. 993-999).

[0212] Drug release profile and loading / encapsulation effectiveness Hollow bilayer capsules with two open ends were obtained by removal of 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 were loaded into the end-sealed capsules using a tube sealer (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):p.5355-5361). For capsules with an inner diameter of 260 μm, considering the limited internal volume of the capsule, a slurry of concentrated 1.0 mg BSA or 1.0 mg bevacizumab at a concentration of 1.0 mg / μL was loaded into the capsule using a 31-gauge needle.

[0213] In vitro BSA release profiles from 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 a 1.5 mL low-binding centrifuge tube to reduce the binding of the centrifuge tube to the eluted proteins. The centrifuge tube with the immersed capsules was incubated at 37° C. to mimic physiological conditions. Elution was collected at 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 3 days, 1 week, 2 weeks, 1 month, and monthly thereafter. (See Sousa, F., et al., A new paradigm for antiangiogenic therapy through controlled release of bevacizumab from PLGA nanoparticles. 2017.7(1):p.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):p.4676-4686; and Tyagi, P., et al., Light-activated, in situ forming gel for sustained suprachoroidal delivery of bevacizumab. Molecular pharmaceutics, 2013.10(8):p.2858-2867.) Then, 1.0 mL of fresh PBS was added and kept under incubation. The BSA release profile was obtained by determining the absorption of the eluted BSA by BCA assay and quantifying the concentration using a BSA protein-based standard curve. The same protocol was applied to obtain the bevacizumab eluate for in vitro bevacizumab release from PCL monolayer capsules and PCL-chitosan bilayer capsules.The characteristic absorbance of bevacizumab was identified at 277 nm by UV-visible spectroscopy (Agilent, Cary 100 UV-Vis), and the release rate of bevacizumab from the capsules was determined by a microplate reader (BioTek, Synergy HT) at 277 nm and quantified based on a standard curve 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.

[0214] To determine the release rate of reactive bevacizumab from capsules with an internal diameter of 260 μm, an 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):p.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):p.3148-3156). Briefly, 100 μL of VEGF recombinant human protein at 1 μg / mL in a pH 9.6 sodium carbonate buffer solution was immobilized on a 96-well Nunc maxisorp plate (Thermo Fisher Scientific) overnight at 4°C. The plate was blocked with 200 μL of 2% BSA solution in PBS / T (0.05% v / v Tween 20 in PBS pH 7.4) for 2 hours at room temperature and washed three times with 300 μL of PBS / T. The bevacizumab eluted from the capsules was then diluted between 0 ng / mL and 10 ng / mL (determined by a standard curve) in a solution of 0.1% BSA-PBS / T, and 100 μL of the sample was added to each well and further incubated for 2 hours at room temperature. After that, the plate was washed three times with PBS / T and 100 μL of HRP goat anti-human IgG Fc secondary antibody in PBS / T (1:1000) was added to each well. The whole plate was incubated in the dark for 1 hour at room temperature and washed five times with PBS / T. The color was developed 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 to a standard curve.

[0215] Drug payload was determined by disruption of three BSA and bevacizumab loaded monolayer and bilayer capsules of different sizes in PBS. Briefly, three BSA and bevacizumab loaded monolayer and bilayer capsules were disrupted and immersed in 1 mL of PBS solution. Using a vortex mixer, the devices were washed vigorously with 1 mL of PBS five times. Each wash took at least 10 minutes. The collected eluates of BSA and reactive bevacizumab were determined by BCA assay, UV-visible spectroscopy, and ELISA. Drug encapsulation efficiency was calculated as free drug in eluate / total amount of drug*100%. Drug loading efficiency was calculated as drug payload / capsule weight*100%. Cumulative release % was calculated as cumulative amount of drug dissolved from capsule / [drug payload*encapsulation efficiency]*100%.

[0216] Biodegradation of capsules In vitro degradation and erosion of 260 μm inner diameter PCL single-layer capsules and PCL-chitosan double-layer capsules over a long period of time was determined by morphological changes. Briefly, capsules with two sealed ends and double-layer capsules with two open ends incubated in PBS at physiological temperature for 9 months and 3 weeks, respectively, were collected and then vacuum-dried for characterization. The PCL outer layer, the chitosan inner layer, and cross-sections of both single-layer and double-layer capsules were examined using SEM. Gross fractures and fissures were assessed, and the average pore size of the PCL layers prepared with different ratios of HEPES sodium salt was quantified by analyzing three different images using Image J and compared with the initial pore size of the capsules before incubation by one-way ANOVA with post-hoc Tukey test at a significance level of 0.05. Data are presented as mean ± standard deviation.

[0217] cytotoxicity The in vitro cytotoxicity of PCL monolayer and PCL-chitosan bilayer capsules was assessed by MTS assay performed with human retinal pigmented epithelial (ARPE-19) cells (see Sur, A., et al., Pharmacological protection of retinal pigmented epithelial cells by sulindac involves PPAR-α. 2014. 111(47): p. 16754-16759; Andres-Guerrero, V., et al., Novel biodegradable polyesteramide microspheres for controlled drug delivery in ophthalmology. 2015. 211: p. 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): p. 645-649). ARPE-19 cells were cultured at 4 × 10 4Cells were seeded in 48-well plates at a density of 10 ... All experiments were repeated in triplicate and data were analyzed by one-way ANOVA with post-hoc Tukey test at a significance level of 0.05. Data are presented as mean ± standard deviation.

[0218] Assessment of aggregation and anti-angiogenic activity The stability of bevacizumab was determined by an ultra-high performance liquid chromatography (UHPLC) 3000 system (Thermo Fisher Scientific Inc., Waltham, MA) using a SEC-1000 column. To determine the bevacizumab stability during the lyophilization process, 500 μL of 25 mg / mL bevacizumab (Avastin) was freeze-dried by a freeze-drying apparatus (Labconco) and the powder was rediluted 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. Free parent bevacizumab before and after lyophilization, concentrated bevacizumab, and bevacizumab eluted from the single-layer and double-layer capsules at specific time points were filtered through a 0.2 μm Whatman SPARTAN HPLC Syringe Filter (VWR International, Radnor, PA) before injection. The original bevacizumab monomer, aggregate, and fragment fractions were analyzed by HPLC spectral deconvolution and separated into individual elution peaks. The integrated areas of the monomer, aggregate, and fragment were normalized to the total area of ​​the HPLC peak to obtain the percentage of each component. The average molecular weight was then calculated from the fraction percentage and molecular weight of each component.

[0219] The antiangiogenic activity of bevacizumab released from PCL monolayer and PCL-chitosan bilayer capsules was further assessed using a capillary-like tubule formation assay (see Elsaid, N., et al., PLGA microparticles entrapping chitosan-based nanoparticles for the ocular delivery of ranibizumab. 2016.13(9):p.2923-2940; Arnaoutova, I. and HKJNpKleinman, In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract. 2010.5(4):p.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 the angiogenesis promoter VEGF (5ng / mL) mixed with i) 10μg / mL original bevacizumab; ii) 10μg / mL bevacizumab released from PCL monolayer capsules; and iii) 10μg / mL bevacizumab released from PCL-chitosan bilayer capsules at 1 week, 2 weeks, 1 month, 3 months, 6 months, and 9 months. After 6 hours, calcein AM was added to the cells, followed by incubation for 30 minutes. The cells were then visualized directly using a fluorescent microscope (Nikon, Eclipse TS100) equipped with a digital camera (Qimaging). The three images were analyzed using Image J to quantify the length of the capillary structures formed. The total tube length from the experimental groups was normalized to the VEGF-treated control group for each sample, and all experiments were repeated three times. Data were analyzed by one-way ANOVA with post-hoc Tukey test at a significance level of 0.05. Data are presented as mean ± standard deviation.

[0220] Injection feasibility Fresh porcine eyes obtained from a local slaughterhouse (Delaware Meats, Delaware, Ohio) were used for assessment of the injectability feasibility of the device (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): p. 4705-4711). The capsules were preloaded into a 21-gauge hypodermic needle connected to a 1 mL syringe. The 21-gauge needle used in this study has an internal diameter similar to a commercialized intraocular lens injector (Ozurdex applicator) (see Lee, SS, et al., Biodegradable implants for sustained drug release in the eye. 2010. 27(10): p. 2043-2053). Intraocular injections were placed 3 mm posterior to the limbus using a syringe needle. A small volume (100 μL) of PBS was used to extrude the capsule into the porcine vitreous humor to reduce the effects of elevated intraocular pressure. After injection, the needle was removed and the sclera was cut around the center of the eye to check for capsule placement in the vitreous humor.

[0221] Electrospinning of chitosan and PCL nanofibers as building blocks for IBB capsules The disclosed strategy to fabricate IBB capsules is based on a two-step coating of chitosan and PCL films on rod-shaped templates, followed by their removal from the templates. Electrospinning is used to generate bilayer structures with a hollow porous center, which can provide high surface area-to-volume ratios for protein chemisorption and tunable porosity for drug diffusion to obtain the desired functionality. Electrospinning as a method for nanofiber fabrication is based on drawing a charged polymer solution into nano-sized fibers using electrical forces. To synthesize chitosan nanofibers, processing parameters (including humidity and voltage) were optimized. For example, either high humidity (greater than 30%) or low voltage (less than 24 kV) caused significant loss of charge from the spinning head, preventing the chitosan solution threads from forming fibers. Therefore, low humidity and relatively high voltage were used. Meanwhile, chitosan precursor was dissolved in TFA and DCM because the addition of TFA could dissolve chitosan better and DCM could allow the evaporation of the solvent in a timely manner, both of which are required for electrospinning. To place chitosan nanofibers onto the steel rod mold, chitosan nanofibers were collected directly on the rotating steel rod. From the SEM image shown in Figure 2, the diameter of the chitosan fibers was 331.61 ± 186.19 nm, and these fibers were highly interconnected, forming a highly porous structure to allow efficient drug diffusion. However, the chitosan fiber mat was found to be brittle, which is consistent with the reports of low mechanical flexibility (see Jayakumar, R., et al., Biomedical applications of chitin and chitosan based nanomaterials-A short review. 2010.82(2):p.227-232). To this end, a second layer of PCL was added, which not only provided physical entrapment of the drug, but also imparted improved flexibility.More specifically, PCL nanofibers with a diameter of 932.57±399.42 nm were coated onto 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:p.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.

[0222] Synthesis and characterization of injectable double-layered microcapsules Utilizing two nanofiber layers as building blocks, hollow capsule structures were formed by directly removing the steel rod template after electrospinning as shown in Figure 3. Although the bilayer PCL-chitosan nanofiber structure can provide significant physical and electrostatic interactions with protein therapeutics, burst release may still occur considering the significant large size of the continuous porous structure of nanofibers compared to the size of the protein (see Chaparro, FJ, et al., Sintered electrospun polycaprolactone for controlled model drug delivery. 2019). Sintering was used to melt the PCL nanofiber layer to reduce the porosity and thus the burst release of the drug. Meanwhile, directly coating the PCL layer without starting with an initial nanofiber structure made it difficult to achieve a thin layer structure on top of the chitosan, which is important for the fabrication of injectable small capsules. The mechanism of sintering-based formation of the bilayer structure was based on the relatively low melting point of PCL at 60 °C compared to chitosan nanofibers at 220 °C. Thus, PCL became almost non-porous during the process to physically hold the drug, while chitosan remained porous to electrostatically bind the drug. In addition, this process also successfully integrated the two layers during the melting process. From the SEM images shown in Figure 3, the chitosan fiber layer adhered to the PCL outer layer, as the melting of the PCL nanofibers increased the adhesion between the two layers, which stabilized the bilayer structure. However, a framework composed of large fibers can still be observed on the surface of the PCL after sintering. During the sintering process, the thickness of the film was reduced by 80% due to the compaction and increased density, so the capsule size can be controlled by adjusting the thickness of the chitosan and PCL fiber layers during the electrospinning process.

[0223] After sintering the chitosan and PCL fiber films, bilayer microcapsules with hollow structures were generated by a template strategy and by taking advantage of the mechanical robustness of the PCL outer layer. The size and structure of the capsules could be effectively controlled by controlling the shape and size of the template rod. As a proof of concept, two sizes of single-layer PCL capsules and bilayer chitosan-PCL capsules were prepared: one with a larger inner diameter of 1.645 mm (previous model) that could be implanted as a scaffold, and one with a smaller inner diameter of 260 μm (final model) that could be injectable through a 21-gauge needle. The hollow structure, with the steel rod as the template, allowed for a high volume primarily for drug loading, while the bilayer membrane provided physical trapping and chemical non-covalent bond formation to achieve long-term sustainable release.

[0224] In the device used for drug release study, the outer diameter of the 1.645 mm inner diameter capsule was approximately 1.815 mm and the wall thickness was 89.36 ± 11.52 μm. Similarly, the outer diameter of the 260 μm inner diameter capsule was approximately 430 μm and the membrane thickness was 89.85 ± 4.27 μm, designed to be injectable via a 21-gauge needle. The increase in capsule thickness promoted the mechanical properties of the capsule, which prevented rupture during injection. However, the increase in capsule size may hinder intravitreal injection. Therefore, 80-90 μm was determined as the wall thickness that balanced mechanical robustness and injectability. Also, the membrane thickness is closely related to the diffusivity of the drug, so the difference in thickness between the single-layered and bilayered capsules was controlled and minimized to reduce the effect of thickness on drug release.

[0225] Tuning the nanoporous structure of bilayer membranes After sintering, the PCL layer became non-porous and the drug release rate was significantly limited (see Chaparro, FJ, et al., Sintered electrospun polycaprolactone for controlled model drug delivery. 2019). However, without sintering, the bilayer capsules would be highly porous, resulting in an undesirably high drug release rate. Therefore, the salt leaching method was used to precisely adjust the three-dimensional porous structure of the bilayer membrane to enable long-term sustainable release of the drug. More specifically, varying amounts of a water-soluble salt (HEPES) were mixed into the nanofibers during electrospinning. Incubation of the capsules in water before drug loading led to the elution of HEPES inside the film, thereby forming a porous structure on the bilayer membrane again. By adjusting the concentration of HEPES in the PCL nanofibers, the porosity could be effectively controlled. As shown in Figure 4, the pore size and distribution in the PCL sintered film were highly dependent on the mass ratio of salt to PCL. For example, the pores tended to be smaller and more dispersed across the entire surface of the film at lower salt concentrations. However, low amounts of salt also hindered the creation of interconnected pores for the diffusion and release of large molecules. Table 1 shows the analytical pore size results for different ratios of PCL to HEPES sodium salt. To this end, PCL films prepared with salt concentrations above 5.0% were used in capsule fabrication and drug release studies, since interconnected porous structures were observed in cross-section via SEM. TIFF0007636334000001.tif69150

[0226] To assess the changes in the bilayer structure before and after salt leaching, SEM imaging was used to observe the inner surface, outer surface, and cross-section of the bilayer capsule. Before salt leaching, the PCL sintered film was rough with some HEPES sodium salt crystals embedded therein. 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 three-dimensional diffusion of proteins for sustainable release and interaction with the proteins by maximizing the interaction with the proteins via electrostatic interactions. The inner chitosan layer showed a more nanoporous structure with a thickness of 25 μm, which may be due to the relatively high melting point of chitosan. In contrast, the outer PCL layer had a denser structure with a total thickness of 65 μm with nanochannels passing through it to physically trap the drug while supporting protein diffusion. These results are consistent with the morphology collected on separate layers of PCL and chitosan.

[0227] To further confirm the chemical nature of the bilayer capsules after sintering and washing, FTIR spectroscopy was performed on the final capsules shown in FIG. -1 A significant peak at 2963 cm was assigned to the carbonyl group in PCL. -1 and 2995 cm -1 The peak at 3478 cm was due to the CH stretching vibration in the backbone of PCL. The peak at 3478 cm was due to the OH stretching vibration from the abundant hydroxyl groups in the backbone of chitosan. -1 In addition, a wide range of groups of 1571 cm -1A characteristic peak for chitosan at 100 nm was assigned to NH stretching. Such peaks provide strong evidence of the chemical properties of the chitosan and PCL layers even after exposure to sintering. Thus, cationic chitosan remains active and capable of chemically non-covalently binding anionic protein (bevacizumab). SEM and FTIR demonstrated the feasibility of the disclosed bottom-up approach for the synthesis of hybrid nano-microstructured capsules with widely tunable pore sizes, aspect ratios, and dimensions, which can provide optimal physical and chemical properties for drug loading and controlled release of protein therapeutics.

[0228] High payload and long-term sustainable drug release of bevacizumab To confirm the high payload of protein drugs enabled by the hollow structure of the capsules, the drug encapsulation efficacy was determined by the destruction of the drug-loaded capsules and the assessment of the amount of BSA and bevacizumab leaking from the capsules, respectively. BSA was used as a model protein drug, and bevacizumab is a clinically used anti-VEGF therapeutic for the treatment of AMD. Considering that BSA and bevacizumab can be adsorbed to the chitosan layer of the bilayer capsules, both single-layer and bilayer capsules were used for the assessment of drug payload. No significant difference in drug payload was found between both capsules. Based on the study, the BSA encapsulation efficacy of the three large capsules and the three small capsules was 100.39±6.46% and 69.64±7.15%, respectively. Lower encapsulation efficiency was observed in both large and small capsules loading of bevacizumab, which was assessed as 52.66±6.47% by UV-visible spectroscopy (a commonly used instrument that determines the concentration of proteins with characteristic absorption around 280 nm), as shown in Figure 12. However, a higher amount of reactive bevacizumab (729.02±84.67 μg) was quantified by ELISA, which gave a bevacizumab encapsulation efficiency of approximately 70%. The lower encapsulation efficiency could be attributed to the decreased sensitivity of UV-visible spectroscopy to lower concentrations of bevacizumab and the fact that cumulative release can be effectively detected by ELISA.The loading capacity of the capsules is approximately 26.60±1.90% w / w, which is higher than most reported devices with 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 chitosan nanoparticles intended for choroidal neovascularization treatment. Journal of Biomedical Materials Research Part A, 2018.106(8):p.2261-2271).

[0229] Using the model drug BSA, we demonstrated the ability of the exemplary capsules to modulate the drug release profile by modifying the capsule surface morphology and porosity, as shown in Figure 6. 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 by salt leaching conditions of 5%, 7.5%, and 10% salt were also investigated. Figure 6 shows the BSA release profiles of single-layer and double-layer capsules for both sizes. As expected, a burst release occurred in the 1.645 mm inner diameter PCL single-layer capsules during the first month, with more than 75% of the loaded BSA eluting from the capsules, which significantly limited the duration of drug release. Due to the increase in the surface area to volume ratio, the period of burst release was shortened to 2 weeks in the 260 μm inner diameter capsules, and the cumulative release percentage is similar to that of the 1.645 mm inner diameter capsules during this period. Therefore, the drug diffusion rate of the small capsules was improved. However, in both capsule sizes, there was a steady release followed by a burst release. The maximum drug release period for the single-layer capsules was approximately 5 months for the 1.645 mm inner diameter capsules and 3 months for the 260 μm inner diameter capsules prepared with 10% salt. The effect of HEPES sodium salt was also investigated. Higher salt concentrations resulted in a more rapid release rate due to increased pore interconnectivity. In capsules with lower salt concentrations, the burst release was delayed but still uncontrollable.

[0230] In contrast, the PCL-chitosan bilayer capsules showed no obvious evidence of burst release. The bilayer capsules significantly delayed BSA release. The release profile of the bilayer capsules showed high linearity, which is summarized in Figure 7. After one month, the 1.645 mm inner diameter bilayer capsules released approximately 15% of the loaded BSA, which was 60% lower release compared to the single-layer PCL capsules in the same period, indicating a higher ability to retain BSA inside the device. Similarly, the 260 μm inner diameter bilayer capsules significantly reduced the burst release. Only 25% of the BSA was eluted from the 260 μm inner diameter bilayer capsules, which was higher than the 1.645 mm capsules due to the relatively larger area-to-volume for diffusion. The chitosan layer was effective in limiting drug diffusion, and the porosity of the PCL shell did not play a significant role in controlling the BSA release. There was no significant difference between the diameters of single-layer and bilayer capsules (p>0.05), so the effect of thickness on drug release was negligible in these studies. Theoretically, the bilayer structure has the ability to control drug release for at least one year for both capsule sizes based on cumulative release data.

[0231] After the porous structure of the bilayer capsules was comprehensively investigated and optimized, capsules with bilayer structure were used for loading and releasing bevacizumab (a targeted drug for clinical AMD treatment) and leaching with 10% HEPES. Consistent with the BSA drug release experiment, a sustainable release profile over 1 year and 9 months without obvious initial burst release was successfully achieved in both 1.645 mm capsules and 260 μm capsules, respectively. Interestingly, there was a further reduction in the burst release of bevacizumab during bilayer capsule-based drug release compared to BSA. This may be due to the increased molecular weight and lower effective charge of bevacizumab compared to BSA. In this condition, the pore size dominates the diffusion rate of bevacizumab. It would be difficult for higher molecular weight bevacizumab to elute from capsules with limited porous channels. This explains why the overall release of both single-layer and bilayer capsules prepared with 5% HEPES salt has similar release kinetics. Also, compared with the other two capsules with larger pores on the inside of the membrane, the capsule prepared with 5% HEPES salt has the lowest release rate. It is noteworthy that the bilayer capsules with inner diameter of 260 μm loaded with bevacizumab achieved near zero-order release kinetics after burst release, as shown in Figure 7 (P<0.05).

[0232] Although simple UV absorption was used to assess and quantify the amount of bevacizumab, it could not identify reactive bevacizumab and distinguish capsules from the background of destroyed polymer over time. Therefore, the bevacizumab eluted from the 260 μm capsules was determined again by ELISA. FIG. 13 shows that the general trend of releasing bevacizumab was consistent with the previous results determined by UV-visible light. For example, the long-term cumulative release of bevacizumab from the single-layer capsules made with 5% HEPES salt was assessed to be approximately 160 μg by UV-visible light, which was the same as that characterized by ELISA over 9 months. Thus, by comparing the release results from UV-visible light and ELISA, the release profile obtained by UV-visible light was reliable, which could provide the general trend of bevacizumab release from single-layer and double-layer capsules. Similarly, the capsules prepared with 5% salt had a relatively slower release rate over 9 months compared to those with 7.5% and 10% salt. Based on these considerations, bilayer capsules with higher (7.5% and 10%) HEPES salt concentrations were then identified and used for long-term release of anti-VEGF. At the same time, the high drug loading capacity and stable drug release profile over a 9-month period strongly indicate the potential of the exemplary bilayer capsules as a versatile platform for the delivery of anti-VEGF therapeutics.

[0233] Biodegradation of capsules The in vitro degradation of PCL single-layer capsules and chitosan-PCL double-layer capsules was studied. Capsules with two closed ends were placed in PBS for 9 months at 37°C, and the capsules were retrieved and characterized by SEM. The mechanical integrity of the entire device is mainly determined by the PCL layer, which slowly undergoes hydrolysis at the ester bonds (see Darwis, D., et al., Enzymatic degradation of radiation crosslinked poly(ε-caprolactone). Polymer Degradation and Stability, 1998. 62(2): p. 259-265). Therefore, it is important to assess the erosion and degradation of the PCL layer over time. From the characteristic SEM images shown in Figure 11, both single-layer capsules and double-layer capsules remained intact after 9 months of incubation. However, compared to the initial capsules, after 9 months the pores on the surface of the PCL film became larger and more dispersed, indicating a slow degradation of the PCL film. Table 2 summarizes the analytical pore size of capsules with different conditions of HEPES salt. The pores on the PCL surface significantly increased by approximately 180 nm in diameter on average (p≦0.05), but the entire device maintained its integrity without obvious cracks and fractures. The bilayer capsules were also characterized. After 9 months, the chitosan layer was still tightly adhered onto the PCL layer and the fibers were still well-defined and intact. The fiber framework of the chitosan surface layer was still evident without significant changes. Furthermore, the membrane thickness of both the single-layer capsules and the bilayer capsules was in the range of 80 μm to 90 μm, which is similar to its original thickness before incubation. However, as shown in FIG. 15, a significant thickness decrease and loss of chitosan fibers was observed when immersing capsules with two open ends in PBS at physiological temperature for 3 weeks.This is likely caused by the slow degradation of chitosan when exposed directly to water over time (see Kean, T. and M. Thanou, Biodegradation, biodistribution and toxicity of chitosan. Advanced Drug Delivery Reviews, 2010. 62(1): p. 3-11; and Onishi, H. and Y. Machida, Biodegradation and distribution of water-soluble chitosan in mice. Biomaterials, 1999. 20(2): p. 175-182). Furthermore, due to the weak mechanical properties of chitosan, the inner chitosan fibrous membrane may become fragile under shear forces during long-term incubation in water, resulting in significant loss of chitosan (see Sangsanoh, P. and PJB Supahol, Stability improvement of electrospun chitosan nanofibrous membranes in neutral or weak basic aqueous solutions. 2006.7(10):p.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):p.2428-2435). Therefore, the hydrophobic PCL layer can protect the inner chitosan layer from degradation and further reduce the degradation of the entire device. TIFF0007636334000002.tif61150

[0234] cytotoxicity One of the most important attributes for any drug delivery device is acceptable biocompatibility in the presence of target cells or tissues, both short-term and long-term. To this end, the cytotoxicity of the exemplary bilayer capsules was investigated using retinal pigment epithelial (ARPE-19) cells, since they are the most predominant cells in the retina and are highly sensitive to toxic and exogenous materials, both by direct contact and by extract exposure methods. The cell viability of RPE cells was measured with and without treatment of monolayer and bilayer capsules by using a standard mitochondrial activity measurement assay. From the results shown in Figure 8, both PCL monolayer capsules and PCL-chitosan bilayer capsules showed negligible toxicity to RPE cells during 24 hours of direct incubation. Both PCL and chitosan were reported to have 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: p. 45-51; and Wassmer, S., et al., Chitosan microparticles for delivery of proteins to the retina. Acta Biomaterialia, 2013. 9(8): p. 7855-7864). Similarly, extracts of both capsules did not affect cell viability over one month, as shown in FIG. 8. Even at the one-month time point, viability above 95% was found for the bilayer capsules. Collectively, these results indicate negligible cytotoxicity to retinal pigment epithelial cells and suggest the potential of the bilayer capsules for preclinical evaluation of ocular models in future studies.

[0235] Stability of released VEGF inhibitors and therapeutic efficacy on anti-angiogenesis One major issue that hampers the development of long-term sustained protein delivery systems is protein aggregation and degradation in aqueous environments. Bevacizumab is unstable under physiological conditions and prone to degradation and aggregation over time in the body (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):p.99-112; Oliva, A., M. Llabres, and JBFarina, 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:p.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): p. 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): p. 519-522.) The device is further loaded with highly concentrated bevacizumab obtained from freeze-drying.Aggregation and loss of activity can 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): p. 341-352). This becomes important because bevacizumab aggregates may not be released from the implant at the same rate as the monomers. Therefore, a bevacizumab stability study was required to assess the aggregation and fragmentation of bevacizumab during device fabrication and device incubation over time using HPLC. The analytical aggregation and fragmentation of bevacizumab is summarized in Table 3 and the HPLC spectrum is shown in Figure 14. To confirm the stability of bevacizumab during lyophilization, the HPLC spectrum of lyophilized bevacizumab was compared to that of commercially available bevacizumab (Avastin). 16% aggregates were formed in the free parent bevacizumab, and a slight increase in bevacizumab aggregates was observed during the lyophilization cycle. Aggregation in concentrated solutions was also assessed by dilution in PBS followed by immediate HPLC characterization. However, no change in bevacizumab aggregates was observed, indicating that the protein is fairly stable in concentrated solutions, further proving that the exemplary device can delay the release of bevacizumab monomers. The potency of bevacizumab released over time from single-layer and bilayer capsules was also assessed. The percentage of aggregates ranged between 11% and 16% for both capsules over three months, and it was also found that bevacizumab underwent fragmentation during long-term incubation. However, the potency of bevacizumab was still maintained at a high level over this period as shown in Table 3. Bevacizumab monomer eluted from 260 μm inner diameter PCL monolayer capsules accounted for 84% at 1 month, and this figure decreased slightly to 79% at 3 months.Similarly, the bevacizumab monomer released from the 260 μm inner diameter chitosan-PCL bilayer capsule was 82% over the first 3 months. This enhanced stability may be attributed to its adhesion to chitosan by ionic binding to glycoproteins and its increased bioavailability. Also, the hydrophobic PCL layer slowed down the process of fragmentation by reducing the liquid exchange across the capsule. Therefore, the efficacy of the long-term released bevacizumab was well maintained, suggesting the high potential of the exemplary capsule for the treatment of AMD without frequent injections. TIFF0007636334000003.tif135150

[0236] While HPLC provides clear information about the bevacizumab monomer and aggregation delivered by the exemplary capsules, ELISA characterizes the potency and amount of VEGF-reactive bevacizumab released over time to ensure its effect on angiogenesis. Therefore, bevacizumab ELISA was performed to determine the reactive bevacizumab released over time from capsules with an inner diameter of 260 μm. After one month, the release rate of active bevacizumab was maintained at about 20 μg / mL per month, which is similar to the amount of bevacizumab determined by UV-visible light. In addition, the bioactivity percentage of the eluted bevacizumab was also calculated by comparing the cumulative release percentage measured by ELISA to that determined by UV / visible light. From this result, bevacizumab released from the single-layer capsules can maintain more than 90% bioactivity during a period of nine months, which indicates the potential in protecting the protein. Fluctuations in bioactivity were observed in the bilayer capsules, which were maintained at around 80%. A background increase in UV-visible absorbance effect due to slow biodegradation of the inner layer over the long period of incubation as mentioned above may cause a lower bioactivity percentage. However, both results strongly support the high bioactivity of proteins protected by single-layer and bilayer capsules. In this regard, the exemplary hollow bilayer capsules that physically protect the drug have the potential to overcome this obstacle to sustained release.

[0237] Bevacizumab eluted from single-layer PCL and PCL-chitosan bilayer capsules was also assessed for its inhibitory effect on VEGF-induced tubule growth in a tube formation assay using HUVEC, as shown in Figure 9. At a concentration of 10 μg / mL, the positive control original bevacizumab resulted in 93.15±1.49% inhibition of tubule length. Bevacizumab eluted from single-layer and bilayer capsules after one month led to approximately 13.33±6.51% and 12.33±4.63% tube formation in 260 μm inner diameter capsules and 1.645 mm inner diameter capsules, respectively, which was more effective compared to conventional injection of original bevacizumab. A slight increase in tube length formation appeared after three months due to loss of bioactivity caused by long-term incubation at physiological temperature. However, there was no significant difference in the antiangiogenic properties of bevacizumab dissolved from bilayer and single-layer capsules (p>0.05). It is expected that the slow drug diffusion delayed the process of enzymatic degradation of bevacizumab, which significantly protected the proteins inside the capsule. Overall, the antiangiogenic bioactivity was well maintained at a high level over a period of 9 months, suggesting a possible protective effect of the capsules for long-term drug release.

[0238] Injection feasibility In addition to high drug loading capacity, sustainable release of protein therapeutics, and maintenance of anti-VEGF bioactivity, these capsules can also be made injectable. To demonstrate this, an injection feasibility study was performed by transscleral delivery of 10 mm long capsules into ex vivo porcine vitreous humor via a 21-gauge needle, as shown in Figure 10. Capsules with an outer diameter of 430 μm were used in this study because they are similar in size to the commercialized intraocular lens (Ozurdex) with a diameter of 460 μm and a length of 6 mm. The Ozurdex applicator is fitted with a 22 gauge TSK needle. (Chan, A., L.-S. Leung, and MS Blumenkranz, Critical appraisal of the clinical utility of the dexamethasone intravitreal implant (Ozurdex®) for the treatment of macular edema related to branch retinal vein occlusion or central retinal vein occlusion. Clinical Ophthalmology (Auckland, NZ), 2011.5:p.1043; Arcinue, CA, OMCeron, and CS Foster, A comparison between the fluocinolone acetonide (Retisert) and dexamethasone (Ozurdex) intravitreal implants in uveitis. Journal of ocular pharmacology and therapeutics, 2013.29(5):p.501-507; and Querques, L., et al., Repeated intravitreal dexamethasone implant (Ozurdex®) for retinal vein occlusion. See Ophthalmologica, 2013. 229(1):p.21-25).The inner diameter of the needle is approximately 500 μm, which can fit 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):p.387-391). More specifically, in clinical applications, typically anti-VEGF-loaded capsules can be delivered intravitreally by a similar applicator, which can avoid invasive open surgery. Therefore, the advanced drug delivery system based on the exemplary bilayer capsule will be quite compatible with currently used clinical approaches.

[0239] In this study, to address the key challenge of long-term therapy for the treatment of wet AMD, a polymer-based microstructured delivery platform was designed and developed to achieve sustainable release of anti-VEGF in vitro. Sustainable protein release was achieved by designing and optimizing the structure of chitosan-PCL bilayer microcapsules through the use of composite chemistry and engineering methods. The key properties of these chitosan-PCL microcapsules include the size, porosity of the PCL shell, and hollow structure for simple and elegant drug loading.

[0240] PCL-chitosan microcapsules were synthesized by a novel combination of electrospinning, sintering, and salt leaching. Preliminary studies showed that chitosan fibers lost their structure after salt leaching. It was believed that the formation of trifluoroacetate during fiber preparation accelerated the process of chitosan elution while using TFA and DCM as solvents, and therefore a necessary step of neutralization with sodium bicarbonate solution was required during washing to reduce the effect of acid salts on the bioactivity of bevacizumab (see Sangsanoh, P. and PJB Supahol, Stability improvement of electrospun chitosan nanofibrous membranes in neutral or weak basic aqueous solutions. 2006.7(10):p.2710-2714).

[0241] The thickness of the membrane correlated with the drug release period. Theoretically, a thicker membrane resulted in a slower diffusion of the drug. Even though increasing the capsule size could help achieve slower drug release, increasing the size of the microcapsules would prevent injection through small gauge needles. Therefore, a thinner membrane was required to make the capsules injectable for clinical application. The chitosan layer was added to address this issue. In this study, all capsules had a thickness between 80 and 95 μm, which minimized the effect of thickness in exploring the relationship between drug release rate and chitosan-PCL composites.

[0242] After optimizing these key factors to control drug release, the performance of microcapsules was evaluated and optimized for the sustainable release of anti-VEGF. Bevacizumab has been used clinically in the treatment of 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): p. 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): p. 4807-4813). Its net charge calculated from the pI should be slightly positive at pH 7.4 as reported by numerous studies.However, protein aggregation in water and other organic solvents typically used during device fabrication has the potential to reduce biological activity and cause unwanted side effects (Varshochian, R., et al., Albuminated PLGA nanoparticles containing bevacizumab intended for ocular neovascularization treatment. Journal of Biomedical Materials Research Part A, 2015. 103(10): p. 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): p. 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 (See Sciences, 2013.50(3):p.341-352.) Therefore, PBS is widely used for suspending bevacizumab to maintain its stability and bioactivity.Bevacizumab has been reported to be net negatively charged in PBS at pH 7.4, suggesting binding to chitosan and may provide a more sustainable release from the exemplary capsules (see Li, S.K., et al., Effective electrophoretic mobilities and charges of anti-VEGF proteins determined by capillary zone electrophoresis. 2011. 55(3): p. 603-607; and Garcia-Quintanilla, L., et al., Pharmacokinetics of Intravitreal Anti-VEGF Drugs in Age-Related Macular Degeneration. Pharmaceutics, 2019. 11(8): p. 365). The binding of buffer ions in PBS to bevacizumab increases its hydrophilicity, which further increases its stability and causes a difference between the theoretical and experimental net charges 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): p. 603-607; and Chopra, P., J. Hao, and SKJIjopLi, Iontophoretic transport of charged macromolecules across human sclera. 2010. 388(1-2): p. 107-113). It is therefore hypothesized that bevacizumab carries a negative charge in the vitreous and capsule, and therefore, the protein can be retained by the positively charged chitosan via electrostatic attraction. Similarly, BSA is a negatively charged protein in water with an isoelectric point around 4.7. BSA was able to bind cationic ions and increase its surface charge under physiological conditions (in PBS).However, as previously reported, these ions have little effect on the charge of BSA, so BSA still remains negatively charged in PBS (see Li, SK, et al., Effective electrophoretic mobilities and charges of anti-VEGF proteins determined by capillary zone electrophoresis. 2011. 55(3): p. 603-607; and Alis, A., et al., Measurements and Theoretical Interpretation of Points of Zero Charge / Potential of BSA Protein. Langmuir, 2011. 27(18): p. 11597-11604). By providing a combined electrostatic interaction between bevacizumab or BSA and chitosan and a protective effect from the PCL shell, the desired sustained drug release profile could be achieved.

[0243] In this study, the release rate for a similar payload of bevacizumab was also significantly improved. The average payload of the reported devices ranged 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):p.3148-3156). However, these devices had limitations including not being injectable and not sustaining release over 3 months. The drug loading capacity of these devices was not as expected. Also, the bioactivity of anti-VEGF may be affected during the fabrication process in these devices due to the interaction of the therapeutic with the solvent or high temperature. However, the drug loading was processed after the device was fabricated, which avoided the drug loss and inactivation that commonly occurs using traditional preparation methods (such as emulsion). Thus, the capsule designed herein guaranteed a drug payload of 700 μg of bevacizumab due to the narrow space in the injectable capsule and the high molecular weight of bevacizumab. The template rod can be selectively increased to expand the inner space and promote drug loading. Also, in pharmaceutical applications, the dry powder of bevacizumab can be accurately replaced and loaded under a microscope, which can further promote drug loading efficiency and bevacizumab stability.In addition, other therapeutic agents with lower molecular weights comparable to the model drug BSA could be evaluated using the exemplary device and potentially further increase the drug payload significantly (see Rosenfeld, PJ, et al., Optical coherence tomography findings after an intravitreal injection of bevacizumab (Avastin®) for neovascular age-related macular degeneration. 2005. 36(4): p. 331-335).

[0244] The bilayer capsule can effectively control the drug release rate by utilizing electrostatic interactions between protein therapeutics and polymers, and can address many of the current problems associated with the clinical treatment of wet AMD. It also provides an alternative method for some diseases that require long-term treatment with protein therapeutics, such as colorectal and breast cancers, and some brain tumors. However, the device fabrication method still needs to be optimized for the requirements of different protein therapeutics, and there will be great potential for ophthalmic, cancer, and other biomedical applications.

[0245] In conclusion, a polymer-based delivery platform was developed for the controlled release of anti-VEGF, which is based on a bilayer microstructure that synergistically combines electrostatic binding between chitosan and anti-VEGF with a protective hydrophobic layer of PCL, providing an effective means of modulating polymer-protein interactions for controlled release of therapeutic agents. The bilayer structure was characterized in detail, and the capsule performance for protein delivery was further determined. Most importantly, the exemplary designed delivery platform significantly improved the in vitro long-term release of anti-VEGF compared to most current devices, supporting its potential to treat AMD. Future studies are required to evaluate and reoptimize the therapeutic efficacy of anti-VEGF-loaded devices in in vivo AMD models.

[0246] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims. The present invention includes the following aspects. [Item 1] One or more capsules each having a tubular shape with two closed ends, each of the one or more capsules independently comprising a bilayer wall and at least one lumen compartment; and one or more therapeutic agents, each present within one or more of said at least one luminal compartment; Includes; Each bi-ply wall includes an inner layer and an outer layer; the inner layer comprises a first polymer having a net positive charge under physiological conditions; the outer layer independently comprises a second polymer different from the first polymer; Drug delivery compositions. [Item 2] The drug delivery composition described in Item 1, wherein the composition is intended for injection into the eye of a subject. [Item 3] The drug delivery composition described in Item 2, wherein the injection is into the vitreous chamber of the eye. [Item 4] The drug delivery composition described in Item 2, wherein the injection is an intravitreal injection, a subconjunctival injection, a sub-Tenon injection, a retrobulbar injection, or a suprachoroidal injection. [Item 5] The drug delivery composition according to any one of items 1 to 4, wherein the first polymer comprises chitosan, polyethyleneimine, protamine, polypropylenimine, poly-L-lysine, poly-L-arginine, poly-D-lysine, poly-D-arginine, cellulose, dextran, poly(amidoamine), poly(2-(dimethylamino)ethyl methacrylate), a derivative thereof, or a combination thereof. [Item 6] The drug delivery composition described in any one of items 1 to 5, wherein the first polymer comprises chitosan or a derivative thereof. [Item 7] The drug delivery composition according to any one of items 1 to 6, wherein the second polymer comprises poly(ε-caprolactone) (PCL), polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polyester, poly(other ester), poly(phosphazine), poly(phosphate ester), gelatin, collagen, polyethylene glycol (PEG), derivatives thereof, and combinations thereof. [Item 8] The drug delivery composition according to any one of items 1 to 7, wherein the second polymer comprises PCL or a derivative thereof. [Item 9] The drug delivery composition described in any one of items 1 to 8, wherein each of the one or more capsules has a length of about 0.1 cm to about 5 cm. [Item 10] The drug delivery composition described in Item 9, wherein each of the one or more capsules has a length of about 0.5 cm to about 3 cm. [Item 11] The drug delivery composition described in any one of Items 9 or 10, wherein each of the one or more capsules has a length of about 1 cm to about 3 cm. [Item 12] The drug delivery composition according to any one of items 1 to 11, wherein each of the one or more capsules has an inner diameter of about 100 μm to about 2000 μm. [Item 13] The drug delivery composition according to Item 12, wherein the inner diameter is about 100 μm to about 500 μm. [Item 14] The drug delivery composition according to any one of items 12 and 13, wherein the inner diameter is about 100 μm to about 300 μm. [Item 15] The drug delivery composition according to any one of items 12 to 14, wherein each of the one or more capsules has an outer diameter that is about 50 μm to about 300 μm larger than the inner diameter. [Item 16] The drug delivery composition according to any one of items 1 to 15, wherein the bilayer wall has a wall thickness of about 25 μm to about 150 μm. [Item 17] The drug delivery composition described in Item 16, wherein the wall thickness is about 70 μm to about 100 μm. [Item 18] The drug delivery composition according to any one of items 16 or 17, wherein the wall thickness is about 75 μm to about 95 μm. [Item 19] The drug delivery composition according to any one of items 16 to 18, wherein the wall thickness is about 80 μm to about 90 μm. [Item 20] The drug delivery composition of any one of items 1 to 19, wherein the outer layer further comprises pores having a pore diameter of about 100 nm to about 10,000 nm. [Item 21] The drug delivery composition described in Item 20, wherein the pore diameter is about 350 nm to 650 nm. [Item 22] The drug delivery composition of any one of items 1 to 21, wherein the outer layer comprises fibers having a diameter of about 100 nm to about 2000 nm. [Item 23] The drug delivery composition described in Item 22, wherein the fibers have a diameter of about 500 nm to about 1000 nm. [Item 24] The drug delivery composition of any one of items 1 to 12, wherein the inner layer comprises fibers having a diameter of about 50 nm to about 1000 nm. [Item 25] The drug delivery composition described in Item 24, wherein the fibers have a diameter of about 100 nm to about 400 nm. [Item 26] The drug delivery composition according to any one of items 1 to 25, wherein the therapeutic agent has a net negative charge at any pH within the range of about pH 6.0 to about pH 7.4. [Item 27] ​​The drug delivery composition according to any one of Items 1 to 26, wherein the therapeutic agent is an anti-VEGF therapeutic agent. [Item 28] The drug delivery composition described in Item 27, wherein the anti-VEGF therapeutic agent is a therapeutic antibody, a therapeutic protein, or a combination thereof. [Item 29] The drug delivery composition of item 28, wherein the therapeutic antibody is bevacizumab, ranibizumab, IBI305, or a combination thereof. [Item 30] The drug delivery composition described in Item 28, wherein the therapeutic protein is a VEGF decoy receptor. [Item 31] The drug delivery composition described in Item 30, wherein the VEGF decoy receptor is aflibercept. [Item 32] The drug delivery composition described in Item 27, wherein the anti-VEGF therapeutic agent is a tyrosine kinase inhibitor. [Item 33] The drug delivery composition described in Item 32, wherein the tyrosine kinase inhibitor is lapatinib, sunitinib, sorafenib, axitinib, pazopanib, or a combination thereof. [Item 34] The drug delivery composition described in Item 27, wherein the anti-VEGF therapeutic agent is an antisense nucleic acid targeting VEGF or a VEGF receptor. [Item 35] The drug delivery composition according to any one of items 1 to 34, wherein the therapeutic agent is present in an amount of about 0.01 mg to about 3 mg. [Item 36] The drug delivery composition according to Item 35, wherein the therapeutic agent is present in an amount of about 0.5 mg to about 2 mg. [Item 37] The drug delivery composition of any one of Items 35 or 36, wherein the therapeutic agent is present in an amount of about 0.5 mg to about 1.5 mg. [Item 38] The drug delivery composition of any one of items 1 to 37, wherein the therapeutic agent exhibits near zero order release kinetics over a period of at least 30 days. [Item 39] The drug delivery composition of Item 38, wherein the therapeutic agent exhibits near zero order release kinetics over a period of at least three months. [Item 40] The drug delivery composition of item 38, wherein the therapeutic agent exhibits near zero order release kinetics over a period of at least six months. [Item 41] The drug delivery composition of item 38, wherein the therapeutic agent exhibits near zero order release kinetics over a period of at least 9 months. [Item 42] A method for treating an ophthalmic disorder in a subject in need thereof, comprising injecting a therapeutically effective amount of the drug delivery composition described in any one of items 1 to 41 into the eye of the subject. [Item 43] The ophthalmic disorder is selected from the group consisting of acute macular neuroretinopathy; Behcet's disease; angiogenesis (including choroidal neovascularization); diabetic uveitis; histoplasmosis; infection (such as 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 site or location of the posterior segment of the eye; ocular tumors; retinal disorders (such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreous retinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinal disease, etc.); sympathetic ophthalmia; Vogt's disease; Item 43. The method according to item 42, wherein the treatment is Koyanagi-Harada (VKH) syndrome; uveal effusion; a posterior ocular condition caused by or affected by ocular laser treatment; a posterior ocular condition caused by or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, non-retinopathy diabetic retinal dysfunction, retinitis pigmentosa, cancer, and glaucoma. [Item 44] The method of item 42, wherein the ophthalmic disorder is wet age-related macular degeneration (wet AMD), neovascularization, macular edema, or edema. [Item 45] The method of any one of items 42 to 44, wherein injecting into the eye of the subject comprises injecting into the vitreous chamber of the eye. [Item 46] The method according to any one of items 42 to 44, wherein injecting into the subject's eye includes intravitreal injection, subconjunctival injection, sub-Tenon injection, retrobulbar injection, or suprachoroidal injection. [Item 47] A method for preparing a drug delivery capsule for injection into an eye of a subject, comprising: forming an inner layer on the conductive rod comprising a first polymer having a net positive charge under physiological conditions; and forming an outer layer over the inner layer, the outer layer comprising a second polymer different from the first polymer; Includes; said forming said inner layer comprises electrospinning using a solution of said first polymer and a voltage differential of about 10 kV to about 30 kV; the solution of the first polymer is about 1% w / v to about 10% w / v in at least one organic solvent; said forming said outer layer comprises electrospinning onto said formed inner layer using a solution comprising said second polymer and optionally a porogen; said voltage differential used for electrospinning is between about 10 kV and about 30 kV; the solution comprising the second polymer and optionally the porogen is about 1 w / v % to about 10 w / v % of the second polymer and the porogen based on the total weight; the weight ratio of the second polymer to the optional porogen is from about 50:50 to about 100:0; The method. [Item 48] The method of item 47, wherein the first polymer comprises chitosan or a derivative thereof. [Item 49] The method of any one of items 47 or 48, wherein the second polymer comprises PCL or a derivative thereof. [Item 50] The method according to any one of items 47 to 49, wherein at least one organic solvent in the solution of the first polymer is a mixture of trifluoroacetic acid and dichloromethane; and the trifluoroacetic acid and the dichloromethane are present in a ratio of about 1:10 to about 10:1. [Item 51] The method according to Item 50, wherein the trifluoroacetic acid and the dichloromethane are present in a ratio of about 5:3 to about 10:3. [Item 52] The method of item 51, wherein the trifluoroacetic acid and the dichloromethane are present in a ratio of about 7:3. [Item 53] The method according to any one of Items 47 to 52, wherein a weight ratio of the second polymer to the porogen is from about 90:100 to about 99.9:0.1. [Item 54] The method according to Item 53, wherein the weight ratio of the second polymer to the porogen is about 90:100 to about 95:5. [Item 55] The method according to Item 53, wherein the weight ratio of the second polymer to the porogen is from about 95:5 to about 99.9:0.1. [Item 56] The method according to any one of Items 47 to 55, wherein the solution containing the second polymer and the porogen is about 2.5 w / v % to about 10 w / v % of the second polymer and the porogen, based on the total weight. [Item 57] The method according to Item 56, wherein the solution containing the second polymer and the porogen is about 5 w / v % to about 10 w / v % of the second polymer and the porogen, based on the total weight. [Item 58] The method of any one of items 47 to 57, further comprising sintering the drug delivery capsule following formation of the outer layer. [Item 59] The method of item 58, wherein sintering comprises heating at a temperature of about 50° C. to about 150° C. for a period of about 1 minute to about 6 hours. [Item 60] The method of item 58, wherein sintering comprises heating at a temperature of about 90°C to about 110°C for a period of about 30 minutes to about 6 hours. [Item 61] The method of any one of items 58 to 60, further comprising washing the drug delivery capsule following sintering. [Item 62] The method of item 61, wherein washing comprises washing the drug delivery capsule with a basic solution, an aqueous solution, or a combination thereof. [Item 63] The method of item 61, wherein washing comprises washing with saturated sodium bicarbonate solution, followed by washing with deionized water. [Item 64] The method of any one of items 61 to 63, further comprising drying the drug delivery capsule following washing. [Item 65] The method according to Item 64, wherein the drying is carried out under vacuum at a temperature of about 50°C to about 150°C for a period of about 1 minute to about 6 hours. [Item 66] The method according to Item 64, wherein the drying is carried out under vacuum at a temperature of about 90°C to about 110°C for a period of about 30 minutes to about 6 hours. [Item 67] A drug delivery capsule produced by the method according to any one of items 47 to 66. [Item 68] The drug delivery capsule described in Item 67, further comprising a therapeutic agent.

Claims

1. one or more capsules, each having a tubular shape with two closed ends, each of the one or more capsules independently comprising a bilayer wall and at least one lumen compartment; and one or more therapeutic agents, each present within one or more of said at least one luminal compartment; Including; Each bi-ply wall includes an inner layer and an outer layer; the inner layer comprises chitosan; the outer layer independently comprises poly(ε-caprolactone) (PCL); The therapeutic agent has a net negative charge at any pH between pH 6.0 and pH 7.4; Drug delivery compositions.

2. the composition is intended for injection into the eye of a subject; the injection is into the vitreous chamber of the eye; or 2. The drug delivery composition of claim 1, wherein the injection is an intravitreal injection, a subconjunctival injection, a subtenon injection, a retrobulbar injection, or a suprachoroidal injection.

3. 3. The drug delivery composition of claim 1 or 2, wherein each of the one or more capsules has a length of 0.1 cm to 5 cm, a length of 0.5 cm to 3 cm, or a length of 1 cm to 3 cm.

4. 4. The drug delivery composition of claim 1, wherein each of the one or more capsules has an inner diameter of 100 μm to 2000 μm, 100 μm to 500 μm, or 100 μm to 300 μm.

5. 5. The drug delivery composition of claim 1, wherein the bilayer wall has a wall thickness of 25 μm to 150 μm, 70 μm to 100 μm, 75 μm to 95 μm, or 80 μm to 90 μm.

6. The drug delivery composition of any one of claims 1 to 5, wherein the outer layer further comprises pores having a pore diameter of 100 nm to 10,000 nm or 350 nm to 650 nm.

7. The drug delivery composition of any one of claims 1 to 6, wherein the outer layer comprises fibers having a diameter of 100 nm to 2000 nm or 500 nm to 1000 nm.

8. The drug delivery composition of any one of claims 1 to 7, wherein the inner layer comprises fibers having a diameter of 50 nm to 1000 nm or 100 nm to 400 nm.

9. The drug delivery composition of any one of claims 1 to 8, wherein the therapeutic agent is an anti-VEGF therapeutic agent.

10. 10. The drug delivery composition of claim 9, wherein the anti-VEGF therapeutic agent is selected from bevacizumab, ranibizumab, IBI305, aflibercept, lapatinib, sunitinib, sorafenib, axitinib, pazopanib, an antisense nucleic acid targeting VEGF or a VEGF receptor, or a combination thereof.

11. The drug delivery composition of any one of claims 1 to 10, wherein the therapeutic agent is present in an amount of 0.01 mg to 3 mg, 0.5 mg to 2 mg, or 0.5 mg to 1.5 mg.

12. The drug delivery composition of any one of claims 1 to 11, for use in treating an ophthalmic disorder in a subject in need thereof.

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