Extended release drug delivery systems for ophthalmic drugs and methods of use - Patents.com

Abstract

Described herein are therapeutic compositions and methods of use for delivery of various drug substances into and around the eye, comprising a drug substance that non-covalently interacts with one or more complexing agent microparticles to form drug substance-complex microparticles, which are mixed in a hydrophobic dispersion medium, collectively forming a stable multi-phase colloidal suspension, which serves as a long-term release drug delivery system for ophthalmic drug delivery. The formulation of drug substance in a multi-phase colloidal suspension can be administered into and around the eye to produce sustained release of therapeutic levels of drug substance in ocular tissues for one or more months without the need for retreatment.

Description

[Technical field]

[0001] Claiming priority This patent application claims priority to U.S. Provisional Patent Application No. 63 / 195,697, filed June 1, 2021, entitled "INTRAVITREAL MITOCHONDRIAL-TARGETED PEPTIDE PRODRUGS AND METHODS OF USE," and U.S. Provisional Patent Application No. 63 / 281,052, filed November 18, 2021, entitled "INTRAVITREAL CORTICOSTEROID EXTENDED RELEASE IMPLANT AND METHODS OF USE," each of which is incorporated by reference in its entirety herein.

[0002] Incorporation by Reference All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. [Background technology]

[0003] Extended release drug delivery systems (XRDDS) include devices, compositions, formulations or other systems used in the design, manufacture and administration of specific drug substances in a manner that modulates drug release kinetics optimized to specific therapeutic goals for specific routes of administration.

[0004] Although several XRDDS have been developed and used for intraocular and periocular drug delivery, these systems have limitations including limited compatibility with only one type of drug substance, sudden and excessive release of drug payload (i.e., "dumping"), suboptimal rate or amount (i.e., insufficient or subtherapeutic drug release), or suboptimal duration of drug release (i.e., too short or too long duration).

[0005] Currently available ocular XRDDS have limited ability to customize specific kinetic drug release profiles. The majority exhibit zero-order (i.e. linear) release kinetics, but some may exhibit a short linear release followed by a short excessive recessive release or a "dumping" of all remaining drug substance (specifically an abrupt excess release). However, there is a demand for XRDDS that can be customized and equipped with different release kinetics, including biphasic and triphasic release kinetics. For example, many ocular diseases, such as wet age-related macular degeneration (AMD) and diabetic macular edema (DME), require a larger initial dose of drug to treat and reverse existing disease manifestations, followed by a reduced dose of drug to prevent disease recurrence, making these diseases ideal for biphasic release kinetics.

[0006] Several diseases of the retina, such as dry age-related macular degeneration (AMD), as well as several eye diseases, including diseases of the optic nerve, uvea, and anterior segment, have limited or no effective treatments, in part due to the lack of effective and versatile technologies for sustained drug delivery to the eye.

[0007] In diseases such as wet AMD, diabetic retinopathy, diabetic macular edema (DME) and retinal vein occlusion (RVO), effective treatments are available in the form of intravitreal anti-VEGF agents and corticosteroids, but these agents must be administered every 1-2 months to achieve optimal treatment outcomes. This creates a significant treatment burden for affected patients and their caregivers, as well as the treating physician. This ultimately leads to under- or inadequate treatment, and consequently suboptimal vision (i.e., limited vision gain or vision loss) in a high percentage of affected patients, regardless of the efficacy of available treatments.

[0008] In pathological conditions such as glaucoma, topical eyedrop drugs are effective in reducing intraocular pressure and the associated risk of vision loss, but poor compliance with self-administration and intermittent dosing lead to undertreatment and suboptimal outcomes for affected patients. Summary of the Invention

[0009] It is therefore highly desirable to provide a new, effective and versatile technology for sustained drug delivery that is compatible with a wide variety of drug types, including small molecules, antibodies, biologics, large proteins, peptides and other drugs that can be customized to achieve the desired drug release kinetics and duration to achieve therapeutic benefit.

[0010] Extended release drug delivery systems (XRDDS) include compositions, formulations, devices and / or systems used in the design, manufacture and administration of specific drug substances in a manner that tailors drug release kinetics to optimize specific therapeutic goals and for specific routes of administration.

[0011] Described herein are compositions, and methods of making and using, for a multi-purpose extended release drug delivery system (XRDDS) that delivers a variety of drug substances into and around the eye, comprising a drug substance that non-covalently interacts with one or more complexing agent microparticles to form drug substance-complex microparticles, which are miscible in a hydrophobic dispersion medium, thereby collectively forming a stable multi-phase colloidal suspension (Figure 1).

[0012] The drug substances herein may include various small polypeptides, proteins, aptamers, other nucleic acid drugs, hydrophobic chemicals, hydrophilic chemicals and other compounds used for therapeutic purposes that are capable of forming non-covalent complexes directly to one of six classes of complexing agents: fatty acids, organic compounds capable of forming keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids and polysaccharides; and to a prodrug of any active ingredient (API) that is linked via a cleavable covalent bond to a conjugate moiety that forms a complex with one of six classes of complexing agents: fatty acids, organic compounds capable of forming keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids and polysaccharides.

[0013] A conjugate moiety can be any chemical entity capable of covalently binding to an API. Certain conjugate moieties can be selected for their ability to provide properties not exhibited by the native API, particularly their ability to form reversible non-covalent complexes with complexing agents.

[0014] A complex is defined as a non-covalent interaction between a drug substance and a complexing agent. A complexing agent is defined as a chemical compound formulated as irregularly shaped microparticles ranging in size from 1 nanometer (nm) to 1000 micrometers (μm); that demonstrates a measurable binding capacity of a selected drug substance, defined as the amount of drug substance that binds to a known amount of complexing agent; that demonstrates a measurable unbound-bound ratio, or reversibility of drug binding, defined as Kd, in a particular dispersion medium; and that is not previously known or expected to form a complex with a selected drug substance. Binding of the drug substance to the complexing agent, either directly or, in the case of prodrugs, via a conjugate moiety, results in the formation of drug substance-complex microparticles. Certain well-known chemical compounds, including additives and excipients utilized in the pharmaceutical industry, when formulated as irregular microparticles, demonstrate the previously unknown and unexpected property of acting as complexing agents for various drug substances. These include six previously unknown classes of chemicals that, when formulated as irregularly shaped microparticles, act as complexing agents for a variety of drug substances: fatty acids, organic compounds capable of forming keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids and polysaccharides.

[0015] As used herein, specific complexing agents for drug substances include irregular particulate formulations such as magnesium stearate, lecithin, albumin, cyclodextrins, and the like, which exhibit previously unknown or unexpected properties in contrast to dissolved individual molecules.

[0016] A dispersion medium is a vehicle utilized in a colloidal mixture. As used herein, a dispersion medium is defined as a hydrophobic viscous oil selected from the four classes, saturated fatty acid methyl esters, unsaturated fatty acid methyl esters, saturated fatty acid ethyl esters, or unsaturated fatty acid ethyl esters, which when mixed with drug substance-complex microparticles can form a drug substance multi-phase colloidal suspension, and which has not previously been known to form a multi-phase colloidal suspension with the selected drug substance and selected complexing agent.

[0017] Colloidal suspensions include formulations that are viscous, flowable, pourable liquids (ie, colloidal mixtures) capable of forming a stable dispersion of particulates without migration or settling of the particulates. Containing a multi-phase colloidal suspension refers to a colloidal suspension in which the drug substance is present in at least two phases: free unbound drug substance and drug substance bound to a complexing agent (and, to a lesser extent, drug-drug aggregates). Drug substance-complex microparticles act as a reservoir for the drug substance when the microparticles are miscible in the dispersion medium.

[0018] Thus, the drug substance multi-phase colloidal suspension may comprise a viscous, flowable, injectable liquid that results in stably dispersed drug substance-complex microparticles without migration or settling, which may allow the free drug substance to dissociate from the drug substance-complex microparticles to create a free drug substance concentrate in the dispersion medium; the drug substance is free to diffuse out of the implant through the multi-phase colloidal suspension system and into the physiological environment of the adjacent eye. If the drug substance is a prodrug, when the prodrug is exposed to the physiological environment of the eye, the covalent bond linking the conjugate moiety is cleaved, releasing the free API.

[0019] API multi-phase colloidal suspensions enable drug delivery systems because the microparticles are reservoirs of bound API, each with a unique binding capacity and Kd (unbound-bound ratio), which in turn determines the amount of free API complexed in the dispersion medium. Using knowledge of the Kd and binding capacity of each API-complex microparticle, the total amount of free API in the system can be calculated, which in turn determines the rate and amount of release (Figure 2). The relative ratios and amounts of the various API-complex microparticles can be adjusted in a manner that creates calculable unbound free API in the system. The dynamic changes in the unbound free API in the system over the life of the implant are determined by the binding capacity and Kd of the API-complex microparticles in the API multi-phase colloidal suspension.

[0020] In the methods and compositions described herein, the bulk multi-phase colloidal suspension is injectable through a 20-gauge to 30-gauge needle (depending on the application) to provide a stable dispersion of microparticles without migration or settling when exposed to the physiological environment of the eye for the duration of the implant's life (1 to 12 months). The physiological environment of the eye is defined as an in vitro condition using phosphate buffered saline (or similar aqueous solvent) at 37° C. containing enzymes and proteins normally found in the vitreous (representing injection into the vitreous) or using phosphate buffered saline at 37° C. containing plasma (representing injection into various periocular tissues). Alternatively, the physiological environment of the eye may represent injection of the implant in vivo into the vitreous or periocular tissues.

[0021] The pharmaceutical multi-phase colloidal suspension also exhibits the property of biodegradability when exposed to the physiological environment of the eye, where the biodegradability occurs due to dissolution of the dispersion medium. The rate of biodegradation is proportional to the solubility of the dispersion medium in the physiological environment of the eye. Dispersion media with higher solubility allow faster biodegradation of the multi-phase colloidal suspension when exposed to the physiological environment of the eye, while dispersion media with lower solubility allow slower biodegradation of the multi-phase colloidal suspension when exposed to the physiological environment of the eye. This property of the pharmaceutical multi-phase colloidal suspension can be used together with the volume of implant injected to determine the durability of the implant in the physiological environment of the eye.

[0022] The formulation of drug substance in a multi-phase colloidal suspension (Figure 1), referred to as an implant, can be administered in and around the eye, i.e., into the vitreous humor, aqueous humor, suprachoroidal space, subretinal, subconjunctival, sub-Tenon's capsule, or orbital tissues, to produce sustained therapeutic release levels of drug substance for the desired duration (1 to 12 months) for the treatment of various diseases and disorders, and for the desired kinetics of release in the ocular tissues (Figure 3).

[0023] The extended release drug delivery systems (XRDDS) described herein consist of a drug substance that is admixed with one or more microparticle complexing agents to form "drug-complex" microparticles, which are combined and dispersed in a selected dispersion medium to form a stable multiphase colloidal suspension (Figure 1).

[0024] A colloid is a mixture in which particulate material is stably dispersed in a vehicle called the dispersion medium, but does not settle or migrate. This distinguishes colloids from suspensions in which particles settle in the suspension vehicle due to gravity. Typical particle sizes for colloids are in the nanometer range. In colloids, the defining property of the mixture is that the particulates remain stably dispersed with minimal settling or migration. Colloidal mixtures in which particulates are dispersed in a liquid are called "sols." Colloidal mixtures in which particulates are dispersed in a solid or semi-solid are called "solid colloids." Colloidal mixtures in which particulates are stably dispersed in a viscous semi-solid or solid dispersion medium are not given a defined name. Herein, we refer to stably dispersed particulates as "colloidal suspensions." In the methods and compositions described herein, the dispersion medium can be a hydrophobic dispersion medium that promotes stable colloidal suspensions. Drug substance multi-phase colloidal suspensions are suspensions in which the drug substance is present in more than one phase including free drug, drug-drug aggregates, and most importantly, drug non-covalently bound to complexing agent microparticles.

[0025] Complex formation occurs in two physicochemical situations. In one case, complex formation occurs through non-covalent interactions between individual molecules (e.g., receptor-ligand interactions). This type of complex formation is called molecular complexation and is not contemplated in the present composition.

[0026] The second situation involves molecules of a chemical, in this case a drug, that non-covalently bind or adsorb to the surface of a particulate, in this case the complexing agent. This type of complexing is referred to as particulate complexing. Different particulate adsorbents or complexing agents have different adsorptivities based on the size and shape of the particulate, the functional groups present on the surface of the particulate, and the roughness and porosity of the surface. The utility of particulate complexing has been realized in other disciplines, including soil science, where chemical adsorbents (e.g., alumina, silica gel, activated carbon) interact with certain chemicals (often foreign matter) in the soil; the hydrocarbon industry, where adsorbents (e.g., polypropylene, vermiculite, perlite, polyethylene, etc.) are used to clean up oil spills or remove residual oil from drilling and fracking equipment; and industrial coatings (e.g., zeolites, silica gel, aluminum phosphate), where adsorbents are used to bind chemicals for various purposes (i.e., lubrication, surface cooling).

[0027] In medical applications, sorbents are used to treat acute poisoning by ingestion (e.g., activated charcoal, calcium polystyrene sulfate, aluminum silicate), where they bind toxins and limit their adsorption from the intestine into the systemic circulation. In the pharmaceutical industry, the principles of adsorption complex formation are used to understand the chemistry of drug binding to plasma proteins in the blood, drug coatings on solid scaffolds for in situ drug release (e.g., drug-eluting stents), and the addition of excipients to insoluble drugs to improve oral bioavailability and intestinal absorption.

[0028] The methods and compositions described herein may utilize microparticle complexation, where the complexing agent is thus a chemical that is compatible with ocular tissue when formulated as irregularly shaped microparticles and has the ability to non-covalently bind to the drug substance and form drug substance-complex microparticles. One or more drug substance-complex microparticles are incorporated and mixed in a hydrophobic dispersion medium to form a stable multi-phase colloidal suspension that is safely delivered in and around the eye, resulting in a predictable, therapeutic level of the drug substance being continuously exposed in the ocular tissue for the desired duration of treatment. The complexing agent is selected from one of six classes of chemicals, including fatty acids, organic compounds that can form keto-enol tautomers, charged phospholipids, charged proteins, nucleic acids, and polysaccharides.

[0029] When the drug substance is a prodrug, the conjugate moiety of the prodrug is specifically selected for its ability to complex with or form a non-covalent interaction with one or more microparticle complexing agents to form a prodrug-complex microparticle. The one or more prodrug substance-complex microparticles are incorporated and mixed in a hydrophobic dispersion medium to form a stable multi-phase colloidal suspension that is safely delivered in and around the eye, resulting in a predictable, continuous exposure of the drug substance to therapeutic levels in ocular tissues over the desired duration of treatment. The complexing agent is selected from one of six classes of chemicals, including fatty acids, organic compounds that can form keto-enol tautomers, charged phospholipids, charged proteins, nucleic acids, and polysaccharides.

[0030] The methods and compositions described herein disclose a new and previously unappreciated property of these six classes of chemicals, fatty acids, organic compounds capable of forming keto-enol tautomers, charged phospholipids, charged proteins, nucleic acids and polysaccharides, that when in the form of irregularly shaped microparticles with irregular surfaces, they can act as effective complexing agents for drug substances. Criteria for a complexing agent include four characteristics: (1) the drug substance binds to the microparticle complexing agent, demonstrable by imaging with microscopy (see Figures 4-7); (2) when microparticles of a substance are added to a solution of the drug substance, upon centrifugation and pulling down of the microparticles, a pharmacologically significant amount of the drug substance is observed to complex to the microparticles, providing a quantitative measure of the binding capacity of the complexing agent (see Figures 15, 18, 20, 24); (3) when the drug substance-complexed microparticles are resuspended in an appropriate dispersion medium, they demonstrate partial release of drug, allowing the determination of the Kd or unbound-bound fraction of drug for a given drug substance-complexing agent pair in a particular dispersion medium (see Figure 25); and (4) when the drug substance-complexed microparticles are admixed in a dispersion medium, they exhibit a useful pharmacokinetic release profile to form a drug substance multiphase colloidal suspension (see Figure 9). Collectively, these four properties define a complexing agent and enable the complexation-based XRDDS described herein.

[0031] In contrast, spherical microparticles with a spherical smooth surface and a non-reactive coating, including, for example, silicone beads, latex beads, and certain polymeric microparticles, can be excluded because they fail to form stable complexes with the drug substance.

[0032] One class of complexing agents is fatty acids, which are carboxylic acids with an aliphatic chain that can be saturated or unsaturated and can be in the form of a salt or an ester (see example in Figure 4). For example, fatty acids are CH3(CH2) nCOOH, where n is equal to 4 to 30. Specific examples of fatty acids in salt form include magnesium stearate, magnesium palmitate, calcium stearate, calcium palmitate, and the like.

[0033] One class of complex formers are organic compounds that can form keto-enol tautomers. Tautomers refer to molecules that are capable of undergoing chemical equilibrium between a keto form (ketone or aldehyde) and an enol form (alcohol). Typically, compounds capable of undergoing keto-enol tautomerization contain a carbonyl group (C=O) that is in equilibrium with the enol tautomerization containing the pair C=C-OH of the double-bonded carbon atom adjacent to the hydroxyl (-OH) group, as depicted herein:

[0034] [ka] The relative concentrations of the keto and enol forms are determined by the chemistry of the particular molecule and the chemical microenvironment, including equilibrium, temperature, or redox state. Organic compounds capable of keto-enol tautomerization include, but are not limited to, phenols, tocopherols, quinones, ribonucleic acids, etc.

[0035] One class of complexing agents is charged phospholipids (see example in Figure 7). Generally, phospholipids consist of a glycerol molecule, two fatty acids, and a phosphate group modified by an alcohol, and the polar head of the phospholipid is typically negatively charged. Examples include lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, different phospholipids in oil, and many others, which can be used individually or in combination to act as complexing agents. Anionic phospholipids can include one of phosphatidic acid, phophatidyl serine, sphingomyelin, or phophatidyl inositol. In some examples, ionizable synthetic phospholipids with positive charges can be produced, including, but not limited to, DLin-MC3-DMA. The additional cationic phospholipid may include one of the following cationic triesters of phosphatidylcholine: 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC); 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC); 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP); 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE); 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC); 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (EDOPC); 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (EDMPC); 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (EDPPC). In pharmaceutical science, phospholipids are used in drug formulation and delivery applications to improve bioavailability, reduce toxicity, and improve cell permeability.However, in the methods and compositions described herein, phospholipids can be used as complexing agent microparticles that non-covalently bind to drug substance, and form drug substance-complex microparticles for the purpose of adjusting the free drug substance in the dispersion medium of the stable multi-phase colloidal suspension in which drug substance-complex microparticles are incorporated and dispersed.

[0036] One class of complexing agents is charged proteins. Proteins are large biomolecules and macromolecules that contain one or more long chains of amino acid residues. The amino acids that make up a protein can be positively charged, negatively charged, neutral or polar in nature, and collectively, the amino acids that comprise a protein give the protein its overall charge. A wide variety of proteins can act as complexing agents based on their size, molecular weight, ability to easily form microparticles, and compatibility with ocular tissues (see example in Figure 5). The charge of a protein determines its compatibility with a particular drug substance, such that negatively charged proteins easily complex with positively charged drug substances, while positively charged proteins (e.g., positively charged Arg-Gln-Ile-Arg-Arg-Ile-Ile-Gln-Arg-NH2 and synthetic peptides) easily complex with negatively charged drug substances. Examples of proteins that can act as complexing agents include albumin and collagen.

[0037] One class of complexing agents is nucleic acids, which are biopolymer macromolecules composed of nucleotides consisting of a 5-carbon sugar, a phosphate group, and a nitrogenous base. The importance of nucleic acids for biological functions and encoding genetic information is well established. However, nucleic acids also have versatile applications including nucleic acid enzymes (e.g., carbon nanomaterials), aptamers (e.g., for forming nucleic acid nanostructures and therapeutic molecules that act in an antibody-like fashion), and aptazymes (e.g., that can be used for in vivo imaging). In pharmaceutical science, specially designed nucleic acids have been considered and applied for use in carrier-based systems in which the nucleic acid serves as a carrier system for various types of drugs. However, in the methods and compositions described herein, the nucleic acid is not considered a carrier system, but rather a complexing agent, as it is highly negatively charged and thus formulated as a microparticle, which can then act as a complexing agent for a positively charged drug substance.

[0038] One class of complexing agents is polysaccharides, which are long chain polymeric carbohydrates composed of monosaccharide units linked together by glycosidic bonds. Often, they are quite heterogeneous and contain some variation of the repeating monosaccharide units. Depending on the structure, they may be insoluble in water. Complexation of polysaccharide particulate complexing agents to drug substances may occur through a variety of electrostatic interactions and may be influenced by the charge density of the drug substance and polysaccharide, the ratio of polysaccharide complexing agent to drug substance, ionic strength and other properties (see examples in Figure 6). Examples of polysaccharides that may serve as complexing agents include cyclic polysaccharide molecules, cyclodextrins, inclusion compounds, cellulose, pectin, or acid polysaccharides, which are polysaccharides that contain carboxyl, phosphate or other similarly charged groups.

[0039] In the methods and compositions described herein, a selected drug substance has a particular affinity for a given complexing agent and complexes with it to form a drug substance-complex microparticle, which affinity can be measured as the Kd, which is the unbound-bound fraction of the drug substance for a given drug substance-complex microparticle in a selected dispersion medium.

[0040] Another property of drug substance-complex microparticles is binding capacity, which is defined as the amount of drug substance that is bound to a known amount of complexing agent. The affinity and binding capacity of a drug substance for a particular complexing agent therefore serves to limit the free drug available for release from the drug substance-complex microparticles in a given dispersion medium.

[0041] The drug substances formulated in the multi-phase colloidal suspensions of the present extended release drug delivery systems (XRDDS) may include a variety of small polypeptides, proteins, aptamers, other nucleic acid drugs, hydrophobic chemicals, hydrophilic chemicals, and other compounds used for therapeutic purposes that are capable of forming non-covalent complexes directly to one of six classes of complexing agents: fatty acids, organic compounds that can form keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids, and polysaccharides.

[0042] The drug substance forms non-covalent high affinity interactions (or bonds) directly with one of six different classes of substances that are formulated as irregularly shaped microparticles: fatty acids, organic molecules capable of forming keto-enol tautomers, charged phospholipids, charged proteins, nucleic acids, and polysaccharides. Mixed in a dispersion medium, the resulting drug substance-complex microparticles modulate the release of free, unbound drug within a multiphase colloidal suspension, allowing for controlled, extended release from the formulated implant when administered into the physiological environment of the eye (see Figures 1-3).

[0043] The drug substance formulated in the multi-phase colloidal suspension can also be a prodrug of any active ingredient (API) linked via a cleavable covalent bond (see Figure 1) to a conjugate moiety that forms complexes with one of six classes of complexing agents: fatty acids, organic compounds capable of forming keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids, and polysaccharides.

[0044] The prodrug has the formula (I): R'-R (I) where R' is any active ingredient (API) covalently linked via a cleavable bond to R, a conjugate moiety that forms a non-covalent complex with one of five classes of complexing agents, and the covalent bond connecting R' and R can be removed by enzymatic cleavage, catalysis, hydrolysis or other reaction to provide the free API R' and the conjugate moiety R, and R has a C4-C30 lipid moiety (fatty acid or fatty alcohol), a C4-C30 straight chain or branched aliphatic moiety, a 2-mer to 30-mer peptide moiety, a PEGylated moiety, or a carbohydrate moiety.

[0045] The covalently linked conjugate moiety of the drug substance forms non-covalent high affinity interactions that bind to one of six different classes of substances that are formulated as irregularly shaped microparticles: fatty acids, organic molecules that can form keto-enol tautomers, charged phospholipids, charged proteins, nucleic acids, and polysaccharides (see Figures 4C, 5C, 6C, 7C). The formation of prodrug-complex microparticles optimizes the physicochemical properties of the API that are compatible with multiphase colloidal suspensions, and the prodrug-complex microparticles are miscible in a carrier medium that regulates the release of free, unbound prodrug within the multiphase colloidal suspension, allowing for controlled, extended release from the formulated implant when administered into the physiological environment of the eye.

[0046] When the drug substance is a prodrug, an important feature of the prodrug is that the bond linking the API to the conjugate moiety is easily cleaved by enzymatic reaction, catalysis, hydrolysis or other chemical reaction. Upon cleavage of this bond in the prodrug, the released API retains sufficient biological activity for its mechanism of action (see examples in Figures 29A-29C).

[0047] The cleavable covalent bond may comprise one of an ester bond, a hydrazone bond, an imine bond, a disulfide bond, a thioester bond, a thioether bond, a phosphate ester bond, a phosphonate ester bond, a boronate ester bond, an amide bond, a carbamate ester bond, a carboxylate ester bond and a carbonate ester bond.

[0048] Generally, the conjugate moiety R to which the API is covalently linked is not selected based on its biological activity or mechanism of action on a target. Although not a preferred embodiment, disclosed herein are drug substances that can serve as functionally cleavable conjugate moieties and consist of homo- or hetero-dimers, trimers, multimers of any drug substance linked together, directly or indirectly, to chemical entities that serve as linker moieties.

[0049] As described herein, the API, R', may be covalently linked to a conjugate moiety, R, selected from one of the following five classes of chemicals: a C4-C30 lipid moiety, a C4-C30 straight chain or branched aliphatic moiety, a 2-mer to 30-mer peptide moiety, a PEGylated moiety, or a carbohydrate moiety.

[0050] One class of conjugated moieties is a C4-C30 lipid moiety, with or without a preceding linker moiety that connects the lipid moiety to an API (see example in Figure 27A). Lipids are defined herein as organic compounds that are insoluble in water but soluble in organic solvents. Lipids include fatty acids, fatty alcohols, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, prenol lipids (derived from the condensation of isoprene subunits), phospholipids, oils, waxes, and steroids.

[0051] One class of conjugated moieties is the C4-C30 straight chain or branched aliphatic moieties, with or without a preceding linker moiety that connects the aliphatic hydrocarbon to the API. This class includes alkanes, alkenes and alkynes and other hydrocarbon moieties composed of 4 to about 30 carbons.

[0052] One class of conjugate moieties are peptide moieties, with or without a preceding linker moiety that connects the peptide to the API (see examples in Figures 27B-27C), which include natural or synthetic amino acid polymers or polypeptide chains having lengths from 2-mer to 30-mer, which can be anionic, cationic or neutral in charge and contain homogeneous or heterogeneous amino acid repeats.

[0053] The anionic peptide portion may include at least one of poly-glutamate, poly-aspartate, or a combination of glutamate and aspartate. The cationic peptide portion may comprise at least one of poly-arginine, poly-lysine, poly-histidine, a combination of arginine and lysine, a combination of arginine and histidine, a combination of histidine and lysine, a combination of arginine, histidine and lysine, or a combination of arginine, histidine and lysine.

[0054] The peptide portion may have one or more PEGylation moieties for the addition of polyethylene glycol (PEG) groups, or may have one or more sites for modification by the addition of sugar or carbohydrate molecules, including glycosylation.

[0055] One class of conjugate moieties is a PEGylated compound moiety that includes a polyethylene glycol (PEG) polymer in a linear, branched, Y-shaped or multi-arm geometry, a PEGylated peptide or protein, or a PEGylated succinate, e.g., succinimidyl succinate, with or without a preceding linker moiety that connects the PEGylated compound to an API (see example in Figure 27D).

[0056] One class of conjugate moieties are carbohydrate molecule moieties including, but not limited to, monosaccharides or oligosaccharides of 2 to 20 sugars, with or without a preceding linker moiety that connects the carbohydrate to the API. The carbohydrate molecule may include one or more of glucose, galactose, lactose, mannose, ribose, fucose, N-acetylgalactosamine, N-acetylglucosamine, N-acetyleneuraminic acid, or an epimer or derivative of any of these.

[0057] One example of how a prodrug can be incorporated into a multi-phase colloidal suspension is represented by the formula (II): Hd-Arg-DMT-Lys-Phe(-O)-R, designated EY005-R(II)

[0058] [ka] where R is one of a class of mitochondria-targeting tetrapeptides (MTT) that can be used to form prodrugs that are the product of a condensation or esterification reaction of one of the following five classes of chemicals: a C4-C30 lipid moiety, a C4-C30 linear or branched aliphatic moiety, a 2-mer to 30-mer peptide moiety, a PEGylated moiety, or a carbohydrate moiety (FIG. 26).

[0059] One specific example of an EY005-prodrug includes EY005-stearyl (depicted in FIG. 27A), in which EY005 is linked via an ester bond to stearyl alcohol, a member from the group of long-chain saturated fatty alcohols. Upon cleavage of the ester bond, the prodrug EY005-stearyl releases EY005 MTT. To experimentally demonstrate this (FIG. 28), EY005-stearyl was incubated in vitro with carboxylesterase (0.1 μg / mL) at 37° C. to mimic the physiological environment of the eye and the type of esterase that tends to be abundant within the vitreous body therein. Incubation of EY005-stearyl with carboxylesterase produced abrupt cleavage of the ester bond of the prodrug, releasing EY005, as evidenced by high performance liquid chromatography (HPLC) analysis and quantification of the EY005 MTT and EY005-stearyl prodrugs in solution (FIG. 28B). When the EY005-stearyl prodrug is added to a phosphate buffered saline solution without esterase at 37° C., the ester bond of the EY005-stearyl prodrug is hydrolytically cleaved more slowly (about 36 hours) ( FIG. 28C ). Thus, in the physiological system of the eye, the covalent bond of the prodrug linking MTT to the inactive conjugate is readily cleaved by enzymatic cleavage or more slowly by hydrolysis, releasing active MTT.

[0060] Furthermore, upon cleavage of the covalent bond of the drug substance, the API, native MTT peptide, retains its biological activity for treating mitochondrial dysfunction. For example, as depicted in Figures 29A-29C, in an in vitro cell culture model of dry AMD, EY005-stearyl (5 μM) was added to RPE cells (possessing endogenous esterase) exhibiting mitochondrial dysfunction induced by exposure to hydroquinone (HQ). EY005-stearyl efficiently abolished HQ-induced mitochondrial dysfunction in RPE cells (as depicted by cellular flavoprotein-autofluorescence) with an efficacy equivalent to treatment with EY005 native peptide (5 μM). EY005-stearyl was also pre-incubated with carboxylesterase (0.1 μg / mL) in separate media. The harvested media containing cleaved EY005 (5 μM) was added to this RPE cell model of mitochondrial dysfunction and was similarly effective, with equipotency to the EY005 native peptide in reversing RPE mitochondrial dysfunction. Thus, these studies confirm that active MTT cleaved from the drug substance retains its essential and unmodified biological activity for treating mitochondrial dysfunction.

[0061] In some instances, conjugate moieties that can combine elements from two or more of these classes can serve as multimeric linker moieties that covalently link multiple molecules of the API to form dimers and / or multimers. Such linkers capable of generating dimers or multimers of mitochondrial targeting peptides can be referred to as "multimerization domains."

[0062] The prodrug having a multimerization domain has the formula (III): (R') n -R (III) where R is a linker or multimerization domain that is covalently linked to multiple APIs R' to form API dimers or multimers, and n is equal to 2 to about 100. Examples include PEG polymers (FIG. 27D), polyvinyl alcohol (PVA) polymers, or polypeptides, where the linker conjugate moiety R is covalently linked to two or more molecules of API R' to form dimers, trimers, multimers, etc. In some cases, the multimerization domain has an alcohol, i.e., multiple "-OH" groups, to which the API units R' are attached. In this setting, multiple APIs covalently linked (e.g., via an ester or another dynamic covalent bond) to a multimerization domain can be referred to as an API multimer.

[0063] One example of such a prodrug multimer has the formula:

[0064] [ka] where "n" is a number including the PVA polymer.

[0065] The dispersion medium of a pharmaceutical agent multi-phase colloidal suspension is defined herein as a hydrophobic liquid in which the pharmaceutical agent and particulate complexing agent are mixed to form a stable multi-phase colloidal suspension. The criteria that define a stable multi-phase colloidal suspension include a homogeneous mixture and distribution of drug substance-complex microparticles without settling, separation or dissociation of the microparticles after exposure to the physiological environment of the eye in vitro (i.e., buffered saline at 37° C., vitreous enzymes, dilute serum) or in vivo when injected into the eye, over a pre-specified duration of the implant's life. Stability also depends on the relative percentage of drug substance-complex microparticles to oil (weight to weight), as well as the size and mass of the microparticles.

[0066] The methods and compositions described herein describe previously unappreciated properties of certain oils that enable them to act as effective dispersion media (see examples in Figures 12A-12F). These include hydrophobicity, high initial viscosity, and other properties that enable them to form stable multi-phase colloidal suspensions when mixed with drug substance-complex microparticles.

[0067] Four classes of oils that meet these criteria for a dispersion medium include saturated fatty acid methyl esters, unsaturated fatty acid methyl esters, saturated fatty acid ethyl esters, or unsaturated fatty acid ethyl esters. The dispersion medium can be an individual oil from one of these classes, or it can be designed as a mixture of oils with different viscosity values ​​that are specifically designed and blended to achieve the desired goal of a stable colloidal suspension.

[0068] Saturated fatty acid methyl esters which may serve as dispersion media include methyl acetate, methyl propionate, methyl butyrate, methyl pentanoate, methyl hexanoate, methyl heptanoate, methyl octanoate, methyl nonanoate, methyl decanoate, methyl undecanoate, methyl dodecanoate (methyl laurate), methyl tridecanoate, methyl tetradecanoate, methyl 9(Z)-tetradecenoate, methyl pentadecanoate, methyl hexadecanoate, methyl heptadecanoate, methyl octadecenoate, methyl nonadecanoate, methyl eicosanoate, methyl heneicosanoate, methyl docosanoate, methyl tricosanoate, and the like.

[0069] Unsaturated fatty acid methyl esters which may serve as dispersion media include methyl 10-undecenoate, methyl 11-dodecenoate, methyl 12-tridecenoate, methyl 9(E)-tetradecenoate, methyl 10(Z)-pentadecenoate, methyl 10(E)-pentadecenoate, methyl 14-pentadecenoate, methyl 9(Z)-hexadecenoate, methyl 9(E)-hexadecenoate, methyl 6(Z)-hexadecenoate, methyl 7(Z))-hexadecenoate, methyl 11(Z)-hexadecenoate.

[0070] Saturated fatty acid ethyl esters which may serve as dispersion media include ethyl acetate, ethyl propionate, ethyl butyrate, ethyl pentanoate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl undecanoate, ethyl dodecanoate (ethyl laurate), ethyl tridecanoate, ethyl tetradecanoate, ethyl 9(Z)-tetradecenoate, ethyl pentadecanoate, ethyl hexadecanoate, ethyl heptadecanoate, ethyl octadecenoate, ethyl nonadecanoate, ethyl eicosanoate, ethyl heneicosanoate, ethyl docosanoate, and ethyl tricosanoate.

[0071] Unsaturated fatty acid ethyl esters which may serve as dispersion media include ethyl 10-undecenoate, ethyl 11-dodecenoate, ethyl 12-tridecenoate, ethyl 9(E)-tetradecenoate, ethyl 10(Z)-pentadecenoate, ethyl 10(E)-pentadecenoate, ethyl 14-pentadecenoate, ethyl 9(Z)-hexadecenoate, ethyl 9(E)-hexadecenoate, ethyl 6(Z)-hexadecenoate, ethyl 7(Z)-hexadecenoate, ethyl 11(Z)-hexadecenoate.

[0072] In contrast, certain other oils and viscous substances including water, silicone oil, viscous gelatin and viscous proteoglycans fail to form stable multi-phase colloidal suspensions or rapidly decompensate when exposed to the physiological ocular microenvironment (e.g., 37° C., buffered saline, vitreous enzymes, dilute serum) or in vivo when injected into the eye (see examples in Figures 10, 11, 13, 14).

[0073] Complexation of the drug substance to the particulate complexing agent within the dispersion medium serves to limit the release of free drug substance into the dispersion medium: the dispersion medium restricts water access to the drug substance-complex particulates while free, unbound drug substance is free to diffuse within the dispersion medium; the dispersion medium does not retain free, unbound drug, which may diffuse out of the multi-phase colloidal suspension.

[0074] Drug substance multi-phase colloidal suspensions can be designed by a specific process to meet pre-specified drug substance release rates and amounts by varying the ratios and amounts of different drug substance-complex microparticles with different Kd and binding capacities (see Figures 2 and 16). The Kd property is a measure of the affinity of the drug substance for a given complexing agent and is defined as the unbound-bound fraction of drug substance to drug substance-complex microparticles in a given dispersion medium. Specific Kd values ​​can be measured by a specified release assay as described herein. The binding capacity property is defined as the maximum amount of drug that will bind to a known amount of complexing agent.

[0075] The release of drug substance from the implant is determined in part by the unbound fraction in the dispersion medium, which is in turn determined in part by the Kd and binding capacity values ​​for the various drug substance-complexed microparticles. Knowledge of the Kd and binding capacity (see Figures 15, 18, 20, 24) allows for the selection of specific combinations of different prodrug-complexing agent microparticles to modulate the unbound fraction of drug in the dispersion medium over time, thus achieving a pre-specified release kinetic profile (see Figures 2, 3, and 16).

[0076] For example, the addition of drug substance-complex microparticles with high binding capacity and high Kd, indicating low affinity of the drug substance for the complex microparticles, can be used to create a short-term increase in the rate of release, or an initial burst. The addition of drug substance-complex microparticles with high binding capacity and moderate Kd, indicating moderate affinity of the drug substance for the complex microparticles, can be used to create a lower release rate over time, extending the duration of drug substance release from the implant. A combination of these two types of drug substance microparticles can be selected and mixed in the desired ratio and concentration to achieve creating an implant with a biphasic release kinetics of the drug substance from the implant (Figures 2 and 3). An implant with this release kinetic profile can be useful for diseases that require a "loading" phase to treat and reverse established disease symptoms, while a second "steady-state" phase can be effective in preventing the onset of new or recurrent disease.

[0077] In another example, the addition of drug substance-conjugate microparticles with high binding capacity and high Kd, indicating low affinity of the drug substance to the conjugate microparticles, can be used to create a short-term increase in the rate of release, or an initial burst. The addition of drug substance-conjugate microparticles with high binding capacity and moderate Kd, indicating moderate affinity of the drug substance to the conjugate microparticles, can be used to create a slower release rate over time, extending the duration of drug substance release from the implant. The addition of drug substance-conjugate microparticles with high binding capacity and low Kd, indicating high affinity of the drug substance to the conjugate microparticles, can slow the release and create a later burst in the life of the implant. A combination of these three types of drug substance microparticles can be selected and mixed in the desired ratios and concentrations to achieve the creation of an implant with a three-phase release kinetics of the drug substance from the implant (Figure 3). Implants with this release kinetic profile may be useful for diseases requiring a "loading" phase to treat and reverse established disease pathology, while a second "steady state" phase may be effective in preventing the onset of new or recurrent disease, and a third phase "late burst" may be useful for diseases where reduced target response to the drug occurs later in the life of the implant due to tachyphylaxis or other mechanisms mediating downregulation of the drug target or reduced responsiveness to the drug substance.

[0078] In such instances, the combined effect of a combination of two or more drug substance-complex microparticles incorporated into a selected dispersion medium is the release of the drug substance in two or more phases based on the integral of the release rates from the individual drug-complexant microparticle components incorporated and dispersed in the drug substance multi-phase colloidal suspension (see Figure 2).

[0079] The actual release kinetics achieved by the drug substance multi-phase colloidal suspension was measured at in vivo vitreous concentrations below EC 50 EC 50reflects the concentration of a drug substance that achieves 50% of the therapeutic effect of the maximum response for a given mechanism of action of the drug substance.

[0080] In formulations of drug substance multiphasic colloidal suspensions with biphasic release kinetics, the concentration of drug substance in the vitreous is increased during the initial burst phase by EC 50 (i.e., the drug concentration required to achieve 50% of the maximal effect), followed by a second (steady-state) phase where the EC 50 and the pre-specified release kinetics and desired duration of drug release have been achieved through the specific design and use of various drug substance-complex microparticles in the multi-phase colloidal suspensions described herein.

[0081] The pharmaceutical multi-phase colloidal suspension formulations can be delivered as one of three different implant formats including a flowable bolus implant, an erodible or non-bioerodible tube implant filled with the pharmaceutical multi-phase colloidal suspension, or a solid mold of the pharmaceutical multi-phase colloidal suspension made to a specific size and shape, dried and hardened, and configured for implant therapy (Figure 33). Any of these formulations can be injected into and around the eye, i.e., into the vitreous humor, aqueous humor, suprachoroidal space, subretinal, subconjunctival, sub-Tenon's capsule, or orbital tissues to produce sustained release of therapeutic levels of pharmaceutical in ocular tissues for the duration (1 to 12 months) desired for the treatment of various diseases and disorders.

[0082] The multi-phase colloidal suspensions described herein may incorporate a wide variety of drug substances that directly form non-covalent complexes with the microparticle complexing agent, as well as a wide variety of prodrugs consisting of an active ingredient (API) linked via a cleavable covalent bond to a conjugate moiety, which in turn forms a non-covalent complex with the microparticle complexing agent (Figures 1, 2, 3). Specifically, the multi-phase colloidal suspensions may incorporate a variety of hydrophobic and hydrophilic chemicals, small polypeptides, proteins, aptamers, other nucleic acid drugs, and other compounds.

[0083] Several examples are discussed herein to demonstrate the principles of complex formation for drug delivery. For example, fluorescently labeled cationic small molecules were mixed with known amounts of selected individual complexing agents (see Figures 4F, 5F, 6F, 7F). The various fluorescently labeled small molecule-complexed microparticles were then mixed in appropriate dispersion media and visualized under fluorescence microscopy. Using this approach, fluorescently labeled small molecules were observed to form fluorescently labeled small molecule-complexed microparticles with several different complexing agents, but did not adsorb to silica microbeads (Figure 8F, not the complexing agents).

[0084] In another example, fluorescently labeled, C12 (i.e., 12-carbon) lipid molecules were mixed with known amounts of selected individual complexing agents (see Figures 4E, 5E, 6E, 7E). The different fluorescently labeled lipid molecule-complex microparticles were then mixed in appropriate dispersion media and visualized under fluorescence microscopy. Using this approach, fluorescently labeled lipid molecules were observed to form fluorescently labeled lipid molecule-complex microparticles with several different complexing agents, but did not adsorb to silica microbeads (Figure 8E, not the complexing agents).

[0085] In another example, the tetrapeptide Hd-Arg-DMT-Lys-Phe was fluorescently labeled with fluorescein isothiocyanate (FITC) and mixed with known amounts of selected individual complexing agents (see Figures 4B, 5B, 6B, 7B). The various fluorescently labeled small molecule-complexed microparticles were then mixed in appropriate dispersion media and visualized under direct fluorescence microscopy. Using this approach, FITC-labeled Hd-Arg-DMT-Lys-Phe did not produce visible drug-complexed microparticles when mixed with different complexing agents (e.g., magnesium stearate, albumin, cyclodextrin, lecithin). FITC-labeled Hd-Arg-DMT-Lys-Phe was adsorbed to silica microbeads (but not the complexing agents, Figure 8B). This was not indicative of stable complex formation, but rather due to a weak affinity interaction of the microparticles resulting in fluorescence that disappeared when a colloidal suspension of Hd-Arg-DMT-Lys-Phe / microbeads was added to the physiological environment of the eye in vitro (see FIG. 25A).

[0086] In another example, the same tetrapeptide Hd-Arg-DMT-Lys-Phe was linked to stearyl alcohol by an ester bond to form the prodrug Hd-Arg-DMT-Lys-Phe(O)-stearyl. The prodrug Hd-Arg-DMT-Lys-Phe(O)-stearyl was fluorescently labeled with FITC and mixed with different complexing agents (see Figures 4C, 5C, 6C, 7C). The resulting mixture was then visualized under direct fluorescence microscopy. Using this approach, we observed that FITC-labeled Hd-Arg-DMT-Lys-Phe(O)-stearyl (the tetrapeptide is labeled with FITC) formed drug-complex microparticles with several different complexing agents: magnesium stearate (as previously described and expected); the large charged carrier protein albumin; and the large cyclic carbohydrate molecule cyclodextran, as well as the charged phospholipid lecithin. In contrast, FITC-labeled Hd-Arg-DMT-Lys-Phe(O)-stearyl did not adsorb to silica microbeads (FIG. 8C, not a complex former).

[0087] Since only the Hd-Arg-DMT-Lys-Phe(O)-stearyl prodrug with the conjugated moiety formed drug-conjugated microparticles, it is speculated that the conjugation was mediated by the conjugated moiety of the prodrug. To assess this, FITC-labeled Hd-Arg-DMT-Lys-Phe(O)-stearyl (the tetrapeptide is labeled with FITC) mixed with the complexing agent was treated with an aqueous solution of carboxylesterase (0.1 μg / mL) to hydrolyze the ester bond of the prodrug and release the fluorescent peptide. The conjugated microparticles were no longer fluorescently labeled by microscopy (see Figures 4D, 5D, 6D, 7D), confirming that the conjugation of the prodrug was specifically mediated by its conjugated moiety and validating the concept of using a prodrug with a compatible conjugated moiety to mediate the conjugation.

[0088] Furthermore, as described herein, the formation of drug-complex microparticles in which the complexing agent has a high affinity for the drug can be experimentally quantified and verified. For example, the prodrug Hd-Arg-DMT-Lys-Phe(O)-stearyl was mixed with a known amount of a selected individual complexing agent (see Figures 24 and 25). The Hd-Arg-DMT-Lys-Phe(O)-stearyl-complexing agent mixture was then added to an appropriate carrier medium (in this case methyl laurate) and centrifuged to "pull down" or separate the Hd-Arg-DMT-Lys-Phe(O)-stearyl bound to the complexing agent from the unbound prodrug present in the carrier medium. HPLC analysis of the microparticles and dispersion medium pulled down from the Hd-Arg-DMT-Lys-Phe(O)-stearyl content determined the fraction of prodrug bound to the complexing agent, as well as calculation of Kd values, which are the coefficient of binding versus non-binding, and the binding capacity of the prodrug-complexing agent microparticles (Figure 24). Using this type of assay, Kd values ​​and binding capacities can be generated for a particular prodrug-complexing agent pair in a selected dispersion medium.

[0089] Furthermore, the addition of conjugated moieties to the Hd-Arg-DMT-Lys-Phe native peptide (designated EY005) enabled non-covalent complexation to various complexing agents in a manner that dramatically altered the kinetic release profile from multiphasic colloidal suspensions added to the physiological environment of the eye in vitro (Figure 25). In various formulations of EY005 mixed with individual complexing agents in the dispersion medium, by day 4, the EY005 native peptide was rapidly "dumped" from all formulations, i.e., 100% of the EY005 peptide drug was completely released from all formulations (Figure 25A). In contrast, various formulations of Hd-Arg-DMT-Lys-Phe(O)-stearyl (designated EY005-stearyl) complexed to individual complexing agents in a dispersion medium (i.e., multi-colloidal suspension) produced sustained release of the EY005-stearyl prodrug over extended periods of time when added to the physiological environment of the eye in vitro, with variable release kinetics based on the Kd and binding capacity of the individual complexing agents (Figure 25B).

[0090] In some embodiments, the formulation of a drug substance (in this case EY005-prodrug) in a multi-phase colloidal suspension, consisting of two distinct prodrug-complex microparticles, in this example magnesium stearate and albumin, produces a unique kinetic release profile that reflects the integral of the ratio and concentration of the distinct prodrug-complex microparticles with their own unique Kd and binding capacity properties (Figure 25B).

[0091] Thus, in some examples, the formation of a prodrug substantially alters the physicochemical properties of the API, allowing for optimization of suitability for complexation and formulation in multi-phase colloidal suspensions. In the example of Figure 25, the API Hd-Arg-DMT-Lys-Phe is highly hydrophilic and, as described above, did not mix with the complexing agent to produce visible drug-complex microparticles. The Kd and binding capacity of the unmodified API Hd-Arg-DMT-Lys-Phe are substantially different. Linkage to stearyl alcohol via an ester bond produces the prodrug Hd-Arg-DMT-Lys-Phe(O)-stearyl, which is highly hydrophobic compared to the unmodified API (Figures 24 and 25). Furthermore, the high affinity interaction between the hydrophobic long chain aliphatic alcohol of the MTT-prodrug conjugate moiety and the microparticle complexing agent serves to bind the MTT-prodrug and limits the amount of free unbound MTT-prodrug available for release from the dispersion medium in which the MTT-prodrug-complexed microparticles are dispersed (Figure 25).

[0092] Another specific example of a prodrug comprises Hd-Arg-DMT-Lys-Phe(O)-tri-arginine (tri-Arg) (FIG. 27C), in which Hd-Arg-DMT-Lys-Phe is linked via an ester bond to a positively charged peptide conjugate moiety, an arginine trimer / tripeptide, which readily forms a non-covalent complex with a negatively charged microparticle complexing agent to form the MTT-prodrug-complexed microparticle. The high affinity interaction between the positive conjugate moiety of the MTT-prodrug and the negative charge of the microparticle complexing agent serves to bind the MTT-tri-Arg prodrug, limiting the amount of free, unbound MTT-prodrug available for release from the dispersion medium in which the MTT-prodrug-complexed microparticles are dispersed.

[0093] Another specific example of a prodrug includes Hd-Arg-DMT-Lys-Phe(O)-tri-glutamate (triGlu) (FIG. 27B), in which Hd-Arg-DMT-Lys-Phe is linked via an ester bond to a negatively charged peptide conjugate moiety that is a glutamate trimer / tripeptide that readily forms a non-covalent complex with a positively charged microparticle complexing agent to form the MTT-prodrug-complexed microparticle. The high affinity interaction between the negatively charged conjugate moiety of the MTT-prodrug and the positive charge of the microparticle complexing agent serves to bind the MTT-triGlu prodrug, limiting the amount of free unbound MTT-prodrug available for release from the dispersion medium in which the MTT-prodrug-complexed microparticles are dispersed.

[0094] Several examples are discussed herein to specifically identify and distinguish materials that can (and cannot) act as dispersion media. As used herein, the dispersion medium is defined as a hydrophobic, viscous oil that, when mixed with the drug substance-complex microparticles, can form a stable multiphase colloidal suspension formed in the implant for administration in or around the eye. As used herein, colloidal indicates that the microparticles are uniformly dispersed and stable, thereby indicating that the microparticles will remain dispersed without settling or migration for the duration of the intended life of the implant.

[0095] To better understand these properties and to identify liquid substances that may serve as dispersion media, fluorescent particulate beads of two different sizes, 3 μm (micrometer or micron) and 10 μm, were used as surrogates for drug substance-complex particulates (to facilitate visualization and imaging of the particulates). These fluorescent particulate beads were suspended in various liquids in small shallow cylindrical wells, which were then evaluated by confocal fluorescence microscopy to assess the distribution of the particulate beads and whether any consistent settling of the fluorescent bead particulates occurred, as confirmed by the confocal capability to evaluate the liquid at various depths.

[0096] For example, when fluorescent microparticle beads were mixed in water (FIGS. 10A-10F) and silicone oil (FIGS. 11A-11F), they demonstrated a substantially higher number and density of microparticle beads in the lower level fluid and relatively much fewer beads in the upper liquid. Thus, water and silicone oil did not uniformly disperse the microparticles and colloidal suspensions were not formed as the microparticles settled within the liquid medium.

[0097] In another example, fluorescent microparticle beads were mixed in methyl laurate, a fatty acid methyl ester (Figures 12A-12F). Confocal microscopy demonstrated uniform distribution of the microparticles regardless of the depth of the liquid, indicating that the uniformly dispersed microparticles of methyl laurate form a multiphase colloidal suspension. Examination of this suspension in contact with the physiological environment of the eye (containing enzymes and proteins typically contained in ocular tissues) demonstrated the stability of the uniform distribution of the microparticles over long periods of time, over days, weeks, and months, without migration within the colloidal suspension.

[0098] In another example, fluorescent microparticle beads were mixed in 2% gelatin (FIGS. 13A-13F). Confocal microscopy demonstrated uniform distribution of the microparticles regardless of the depth of the liquid, indicating that the microparticles uniformly dispersed in 2% gelatin formed a colloidal suspension. However, after placing the 2% gelatin colloidal suspension (containing collagenase) in the physiological environment of the eye in vitro, the distribution of the microparticles within the suspension did not remain stable over time; the microparticles migrated and settled, indicating that the gelatin-based medium was unable to maintain the stability of the colloidal suspension over time.

[0099] Several examples are discussed herein to demonstrate proof of concept for the formulation and sustained release of various drug substances in multi-phase colloidal suspensions. For example, a formulation of the hydrophobic small molecule fluocinolone acetonide (FA) in a multi-phase colloidal suspension was developed (Figures 15-17). FA was blended with different microparticle complexing agents to form various FA-complexed microparticle formulations. The Kd and binding capacity properties for each FA-complexed microparticle in the dispersion medium were calculated (Figure 15). A two-phase kinetic release profile was desired in this example. Based on this, magnesium stearate and lecithin complexing agents were selected for incorporation into the dispersion medium methyl laurate with FA at specific ratios and concentrations to achieve two-phase release in a bioerodible tube formulation of FA multi-phase colloidal suspension. The formulation was iteratively refined by adjusting the ratio of FA-complexing agent microparticles for a given payload of drug to achieve an initial burst phase release followed by a steady-state release with a duration of release of approximately six months. Figure 16B illustrates another formulation, where different ratios of drug-complexed microparticles changed the release kinetics as the ratio of the complexing agent (KET) with higher affinity (Kd) for a given payload of FA increased throughout the formulation. For different formulations of FA in multiphase colloidal suspensions (Figure 17), a strong correlation was observed between the in vitro drug release kinetics (curves) and the in vivo release kinetics in the eye (colored dots at specific time points reflecting retinal tissue levels).

[0100] In another example, formulations of the hydrophilic small molecule dexamethasone phosphate (DexPh) in multiphase colloidal suspensions were developed (Figures 18-19). Kd and binding capacity properties were calculated for each DexPh-complexed microparticle in the dispersion medium (Figure 18). To understand how the physicochemical properties of the drug substance affect their interaction with the properties of the complexation, DexPh and FA (with the same payload) were blended with the same microparticle complexing agents (magnesium stearate and tocopherol) and dispersion medium (methyl laurate), respectively (Figure 19, curves for DexPh, curves for FA). The formulations of DexPh demonstrated a rapid and excessive release, or "dumping," of DexPh. Addition of a different complexing agent, lecithin, and decreasing the ratio of the other complexing agent, at a given payload (orange curve), altered the kinetic release profile, minimizing the dumping of DexPh and resulting in a more desirable sustained release profile, demonstrating the importance of choosing a complexing agent based on favorable non-covalent complexation with the specific drug substance of interest.

[0101] In another example, formulations of the hydrophilic small molecule sunitinib malate in a multi-phase colloidal suspension were developed (Figures 20-22). Sunitinib was blended with different microparticle complexing agents to form various sunitinib-complexed microparticle formulations (Figure 20). Complexation of sunitinib to the selected complexing agents was visually confirmed by blending and pull-down of the sunitinib-complexed microparticles, which was confirmed by the yellowish orange microparticles (sunitinib has an orange coloration) (Figure 21). Formulations of sunitinib in a bioerodible tube formulation of sunitinib multi-phase colloidal suspension were designed and manufactured and produced desirable tissue levels and durability of release when implanted in rabbit eyes in vivo (Figure 22).

[0102] For example, we developed a formulation of the hydrophobic small molecule axitinib in a multi-phase colloidal suspension. A formulation with a single-phase kinetic release profile was desired. Based on this, axitinib was mixed with a complexing agent with high binding capacity and low Kd (indicating high affinity) in a selected dispersion medium formulated as a bolus implant, which produced a slow release formulation with detectable drug in tissues and a desirable durability of release (Figure 23).

[0103] In another example, a formulation of the prodrug Hd-Arg-DMT-Lys-Phe(O)-stearyl, designated EY005-stearyl, was developed in a multi-phase colloidal suspension (Figures 24-32). EY005-stearyl was mixed with different microparticle complexing agents to form various EY005-stearyl-complex microparticle formulations. The Kd and binding capacity properties were calculated for each Y005-stearyl-complex microparticle in the dispersion medium (Figure 24). The various EY005-prodrug complex microparticle formulations demonstrated sustained release of the EY005-prodrug in vitro compared to a comparable formulation of the EY005 native peptide in a rapidly releasing, damped formulation (Figure 25). In an in vitro kinetic study, a pilot formulation of the prodrug multiphasic colloidal suspension as a bolus implant achieved zero order (i.e., linear) kinetics of release and a desirable durability of drug release of 3 months, where after release of the prodrug from the multiphasic colloidal suspension, the prodrug was present in the dispersion medium and the free API was present in the in vitro physiological environment (Figure 30).

[0104] In an in vitro efficacy study, bolus implants of the prodrug multiphasic colloidal suspension were added to endogenous esterases and RPE cell culture models. Cell culture data demonstrated restoration of the cytoskeleton with approximately 80% improvement at 21 days with abrogation of cellular mitochondrial dysfunction (Figures 31A-31D). The data confirm that prodrugs mixed with complexing agents and incorporated into the dispersion medium to form stable multiphasic colloidal suspensions can produce sustained release of the prodrug at predictable therapeutic levels and that the API remains bioactive upon MTT-prodrug cleavage in the surrounding in vitro physiological environment.

[0105] In vivo kinetic studies used LC / MS analysis to sustain high retinal levels (>300ng / g) of MTT-prodrug upon intravitreal injection of a bolus implant of the prodrug multiphasic colloidal suspension (Hd-Arg-DMT-Lys-Phe(O)-stearyl payload 1mg) in rabbit eyes for 6 weeks (Figure 32, EY005-Stearyl release from IVT MitoXR), confirming good in vivo-in vitro correlation for prodrug release. The recovered bolus had approximately 50% residual payload, indicating that the prodrug bolus implant achieved desirable approximately 90-day implant release kinetics based on zero-order release kinetics.

[0106] Furthermore, incorporation of a bioactive tetrapeptide API (without a prodrug) with the same complexing agent and in the same dispersion medium produces excess release, or "dumping," of the bioactive API in vitro (Figure 25A, Figure 30). Furthermore, multiphase colloidal suspension bolus formulation of the native API administered into the vitreous does not produce detectable tissue levels for more than 21 days (Figure 32, EY005 peptide release from formulated bolus), also pointing to excess release of the native API in vivo. Furthermore, there is no residual drug in the recovered bolus, consistent with excess drug release, or "dumping." Thus, incorporation of the native unmodified API into a multiphase colloidal suspension is insufficient to produce sustained release and fails to meet the specifications of an extended release drug delivery system. Importantly, these data confirm and highlight the need for specific interactions between the prodrug constructs and the prodrug conjugate moieties and complexing agents that form the drug substance-complex microparticles for some APIs that are not otherwise complexed to achieve controlled and durable release of active API from multiphasic colloidal suspensions XRDDS.

[0107] Pharmaceutical multi-phase colloidal suspension formulations, referred to as implants, can be administered intraocularly and periocularly, i.e., into the vitreous humor, aqueous humor, suprachoroidal space, subretinal, subconjunctival, sub-Tenon's capsule, or orbital tissues to produce sustained release of therapeutic levels of pharmaceutical agent in ocular tissues for a desired duration (1 to 12 months).

[0108] The pharmaceutical multi-phase colloidal suspension formulations can be used to prevent the onset or slow the progression, modify the pathology of disease, prevent vision loss or improve vision, or prevent the onset or ameliorate other destructive or degenerative aspects of ocular conditions and diseases including dry age-related macular degeneration (AMD), wet AMD, diabetic macular edema (DME), retinal vein occlusion (RVO), and inherited retinal degenerations (IRD), retinal degeneration, traumatic injury, ischemic vasculopathy, acquired or hereditary optic neuropathies, glaucoma, endophthalmitis, retinitis, uveitis, inflammatory diseases of the retina and uvea, Fuchs' corneal dystrophy, corneal edema, ocular surface diseases, dry eye diseases, diseases of the conjunctiva, diseases of the periocular tissues, and diseases of the orbit.

[0109] The methods may be used in conjunction with other treatment modalities including inhibition of vascular endothelial growth factor, complement inhibition, or administration of anti-inflammatory drugs, such as corticosteroids. All of the methods and devices described herein, in any combination, may be used to achieve the benefits contemplated and described herein.

[0110] For example, described herein are compositions of multi-phase colloidal suspensions that include a drug substance and one or more complexing agents admixed in a dispersion medium. The one or more complexing agents can be chemicals that are capable of forming drug substance-complex microparticles by non-covalent reversible binding to the drug substance and are formulated as irregularly shaped microparticles that are one of fatty acids, organic compounds that can form keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids, and polysaccharides.

[0111] For example, the complexing agent is CH3(CH2) n It may be a fatty acid comprising a carboxylic acid having an aliphatic chain with a chemical formula of COOH, where n is equal to 4 to 30, which may be saturated or unsaturated, a salt or an ester, including one or more of magnesium palmitate, magnesium stearate, calcium palmitate, calcium stearate.

[0112] The particulate complexing agent may be an organic compound capable of forming keto-enol tautomers and undergoing chemical equilibrium between the keto form and the enol form, which are composed of ketones or aldehydes, and includes one of phenolic compounds, tocopherol compounds, quinone compounds, and ribonucleic acid compounds. In some examples, the particulate complexing agent is a charged phospholipid, which is one or more of anionic phospholipids, lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, synthetic phospholipids with positive charge, and DLin-MC3-DMA. The particulate complexing agent may be a positively or negatively charged protein, which is one or more of albumin, synthetic polypeptide, plasma protein, alpha2-macroglobulin, fibrin, and collagen. The particulate complexing agent may be a biopolymer macromolecule that includes ribonucleic acid, a nucleotide that includes a 5-carbon sugar, a phosphate group, and a nitrogenous base. The particulate complexing agent may be a polysaccharide comprising a long chain polymeric carbohydrate comprising monosaccharide units linked together by glycosidic bonds, including one or more of cyclic polysaccharide molecules, cyclodextrins and inclusion compounds.

[0113] The drug substance may form a non-covalent complex with a particulate complexing agent and may include one of a small molecule, a small polypeptide, a protein, an aptamer, a nucleic acid drug, a hydrophobic chemical, and a hydrophilic chemical. In some examples, the drug substance is a prodrug of formula (I): R'-R(I), where R' is any active ingredient (API) covalently linked via a cleavable bond to R, a conjugate moiety that forms a non-covalent complex with one of five classes of complexing agents, and the covalent bond connecting R' and R can be removed by enzymatic cleavage, catalysis, hydrolysis, or other reaction to provide the free API R' and the conjugate moiety R, where R is selected from a C4-C30 lipid moiety (fatty acid or fatty alcohol), a C4-C30 linear or branched aliphatic moiety, a 2-mer to 30-mer peptide moiety, a PEGylated moiety, or a carbohydrate moiety. The cleavable covalent bond may comprise one of an ester bond, a hydrazone bond, an imine bond, a disulfide bond, a thioester bond, a thioether bond, a phosphate ester bond, a phosphonate ester bond, a boronate ester bond, an amide bond, a carbamate ester bond, a carboxylate ester bond and a carbonate ester bond.

[0114] The conjugated moieties, with or without a preceding linker moiety, are tert-butyl alcohol, tert-amyl alcohol, 3-methyl-3-pentanol, 1-heptanol (enanthyl alcohol), 1-octanol (capryl alcohol), 1-nonanol (pelargonic alcohol), 1-decanol (decyl alcohol, capric alcohol), undecyl alcohol (1-undecanol, undecanol, hendecanol), dodecanol (1-dodecanol, lauryl alcohol), tridecyl alcohol (1-tridecanol, tridecanol, isotridecanol), 1-tetradecanol (myristyl alcohol), pentadecyl alcohol (1-pentadecanol, pentadecanol), 1-hexadecanol (cetyl alcohol), c is-9-hexadecen-1-ol (palmitoleyl alcohol), heptadecyl alcohol (1-n-heptadecanol, heptadecanol), 1-octadecanol (stearyl alcohol), 1-octadecenol (oleyl alcohol), 1-nonadecanol (nonadecyl alcohol), 1-eicosanol (arachidyl alcohol), 1-heneicosanol (heneicosyl alcohol), 1-docosanol (behenyl alcohol), cis-13-docosen-1-ol (erucyl alcohol), 1-tetracosanol (lignoceryl alcohol), 1-pentacosanol, 1-hexacosanol (ceryl alcohol), 1-heptacosanol, 1-octacosanol (montanyl alcohol, cluytyl alcohol The alcohol may be an aliphatic alcohol including one or more of: 1-nonacosanol, 1-triacontanol (myricyl alcohol, melissyl alcohol).

[0115] The conjugate moiety may be any of tetradecanoic acid, pentadecanoic acid, (9Z)-hexadecenoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, (9Z,12Z)-octadeca-9,12-dienoic acid, (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid, (6Z,9Z,12Z)-octadeca-6,9,12-trienoic acid, and tetradecanoic acid, with or without a preceding linker moiety. The fatty acid may include one or more of oleic acid, (5E,9E,12E)-octadeca-5,9,12-trienoic acid, (6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraenoic acid, (Z)-octadec-9-enoic acid, (11E)-octadec-11-enoic acid, (E)-octadec-9-enoic acid, nonadecanoic acid, and eicosanoic acid.

[0116] In some examples, R is anionic, cationic or neutral, with or without a preceding linker moiety, and is a 2-mer to about 30-mer peptide moiety containing natural or synthetic amino acids, including one or more of poly-glutamate, poly-aspartate, or a combination of glutamate and aspartate; poly-arginine, poly-lysine, poly-histidine, a combination of arginine and lysine, a combination of arginine and histidine, a combination of histidine and lysine, or a combination of arginine, histidine and lysine; the peptide moiety has one or more PEGylation moieties for adding polyethylene glycol (PEG) groups; the peptide moiety has one or more moieties for modification by addition of sugar or carbohydrate molecules, including glycosylation. In some examples, R is one of a polyethylene glycol (PEG) polymer, a PEGylated peptide, or a PEGylated succinate, including PEG polymers in linear, branched, Y-shaped or multi-armed geometry. In some examples, R is a carbohydrate moiety that includes 2 to 20 sugar carbohydrates, including one or more of glucose, galactose, lactose, mannose, ribose, fucose, N-acetylgalactosamine, N-acetylglucosamine, N-acetyleneuramic acid, or epimers or derivatives of glucose, galactose, lactose, mannose, ribose, fucose, N-acetylgalactosamine, N-acetylglucosamine, and N-acetyleneuramic acid, with or without a preceding linker moiety.

[0117] R' can be an API, R is a linker or multimerization domain that covalently links multiple APIs to form a prodrug dimer or multimer, n is equal to 2 to about 100, and R is one of PEG, a PEG polymer, polyvinyl alcohol (PVA), or a peptide.

[0118] In any of these compositions, the dispersion medium can be a liquid oil capable of forming a multiphase colloidal suspension comprising a hydrophobic oil comprising at least one of saturated fatty acid methyl esters, unsaturated fatty acid methyl esters, saturated fatty acid ethyl esters, and unsaturated fatty acid ethyl esters.

[0119] Saturated fatty acid methyl esters may include one or more of methyl acetate, methyl propionate, methyl butyrate, methyl pentanoate, methyl hexanoate, methyl heptanoate, methyl octanoate, methyl nonanoate, methyl decanoate, methyl undecanoate, methyl dodecanoate (methyl laurate), methyl tridecanoate, methyl tetradecanoate, methyl 9(Z)-tetradecenoate, methyl pentadecanoate, methyl hexadecanoate, methyl heptadecanoate, methyl octadecenoate, methyl nonadecanoate, methyl eicosanoate, methyl heneicosanoate, methyl docosanoate, methyl tricosanoate, and the like.

[0120] Unsaturated fatty acid methyl esters can include methyl 10-undecenoate, methyl 11-dodecenoate, methyl 12-tridecenoate, methyl 9(E)-tetradecenoate, methyl 10(Z)-pentadecenoate, methyl 10(E)-pentadecenoate, methyl 14-pentadecenoate, methyl 9(Z)-hexadecenoate, methyl 9(E)-hexadecenoate, methyl 6(Z)-hexadecenoate, methyl 7(Z))-hexadecenoate, methyl 11(Z)-hexadecenoate.

[0121] Saturated fatty acid ethyl esters may include ethyl acetate, ethyl propionate, ethyl butyrate, ethyl pentanoate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl undecanoate, ethyl dodecanoate (ethyl laurate), ethyl tridecanoate, ethyl tetradecanoate, ethyl 9(Z)-tetradecenoate, ethyl pentadecanoate, ethyl hexadecanoate, ethyl heptadecanoate, ethyl octadecenoate, ethyl nonadecanoate, ethyl eicosanoate, ethyl heneicosanoate, ethyl docosanoate, ethyl tricosanoate.

[0122] Unsaturated fatty acid ethyl esters can include ethyl 10-undecenoate, ethyl 11-dodecenoate, ethyl 12-tridecenoate, ethyl 9(E)-tetradecenoate, ethyl 10(Z)-pentadecenoate, ethyl 10(E)-pentadecenoate, ethyl 14-pentadecenoate, ethyl 9(Z)-hexadecenoate, ethyl 9(E)-hexadecenoate, ethyl 6(Z)-hexadecenoate, ethyl 7(Z))-hexadecenoate, ethyl 11(Z)-hexadecenoate.

[0123] Also described herein are compositions of multi-phase colloidal suspensions comprising a drug substance and one or more complexing agents admixed in a dispersion medium having a release profile of one or more phases of drug release, wherein the one or more complexing agents are formulated as irregularly shaped microparticles that form drug substance-complex microparticles by non-covalent reversible binding to the drug substance, and wherein the drug substance comprises one of a fatty acid, an organic compound capable of forming keto-enol tautomers, a charged phospholipid, a charged protein, a ribonucleic acid, and a polysaccharide; and further wherein the drug substance comprises one of a small molecule, a small polypeptide, a protein, an aptamer, a nucleic acid drug, a hydrophobic chemical, and a hydrophilic chemical; and further wherein the dispersion medium is a hydrophobic liquid oil comprising at least one of a saturated fatty acid methyl ester, an unsaturated fatty acid methyl ester, a saturated fatty acid ethyl ester, and an unsaturated fatty acid ethyl ester.

[0124] For example, a multi-phase colloidal suspension composition may include a drug substance and one or more complexing agents admixed in a dispersion medium having a release profile of one or more phases of drug release, the one or more complexing agents being one of fatty acids, organic compounds capable of forming keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids and polysaccharides, formulated as irregularly shaped microparticles that reversibly bind to the drug substance by non-covalent bonding to form drug substance-complex microparticles; and further, the dispersion medium is a hydrophobic liquid oil comprising at least one of saturated fatty acid methyl esters, unsaturated fatty acid methyl esters, saturated fatty acid ethyl esters, unsaturated fatty acid ethyl esters.

[0125] In some examples, the multi-phase colloidal suspension composition comprises a drug substance and one or more complexing agents admixed in a dispersion medium having a release profile of one or more phases of drug release, the one or more complexing agents are formulated as irregularly shaped microparticles that form drug substance-complex microparticles by non-covalent reversible binding to the drug substance, and the drug substance comprises one of a fatty acid, an organic compound capable of forming keto-enol tautomers, a charged phospholipid, a charged protein, a ribonucleic acid, and a polysaccharide, further, the drug substance comprises one of a small molecule, a small polypeptide, a protein, an aptamer, a nucleic acid drug, a hydrophobic chemical, and a hydrophilic chemical; and further, the dispersion medium is a hydrophobic liquid oil comprising at least one of a saturated fatty acid methyl ester, an unsaturated fatty acid methyl ester, a saturated fatty acid ethyl ester, and an unsaturated fatty acid ethyl ester.

[0126] Also described herein is a method for designing a composition of a multi-phase colloidal suspension that includes a drug substance and one or more complexing agents mixed in a dispersion medium to meet a pre-specified release rate and amount of the drug substance.For example, the method may include varying the ratio and amount of different drug substance-complex microparticles with different binding capacities and Kd.The method that may include varying the ratio and amount of different drug substance-complex microparticles with different binding capacities and Kd includes adding drug substance-complex microparticles with high binding capacity and high Kd, which indicates that the drug substance has a low affinity for the microparticle complexing agent, to create a short-term increase in the rate of release, or an initial burst.Any of these methods may include adding drug substance-complex microparticles with high binding capacity and low Kd, which indicates that the drug substance has a high affinity for the microparticle complexing agent, to extend the duration of release of the drug substance from the implant. Any of these methods may include formulating the drug substance-multiphase colloidal suspension for ocular injection as one of a flowable bolus implant, an erodible or non-bioerodible tube implant filled with the drug substance-multiphase colloidal suspension, or a drug substance-multiphase colloidal suspension made into a solid mold of a particular size and shape and configured for implant therapy.

[0127] Also described herein are methods of treatment using any of these compositions. For example, described herein are methods of treating ocular disorders and diseases, in which a drug substance-multiphase colloidal suspension is administered in and around the eye into one of the following tissue compartments: vitreous humor, aqueous humor, suprachoroidal space, subretinal, subconjunctival, subtenon's capsule, or orbital tissue to produce sustained release of therapeutic levels of drug substance in ocular tissue for one or more months.

[0128] For example, a method of treating vision loss in a subject by intravitreal or periocular injection of a formulation of an extended release drug delivery system that produces highly sustained retinal and retinal pigment epithelium (RPE) tissue levels of active drug may include the steps of delivering a prodrug drug substance in combination with the extended release drug delivery system into the subject's eye at the start of treatment; and cleaving the prodrug by the action of esterases or bioactive enzymes in the subject's eye to release the active ingredient (API) of the prodrug into the eye at a burst phase release rate during a first phase; and cleaving the prodrug by the action of esterases or bioactive enzymes to release the API into the eye at a steady state administration rate during a second phase, wherein the burst phase rate exceeds the steady state release rate, and wherein the first phase extends for about 2-6 weeks from the start of treatment and the subsequent phase extends for one or more months from the end of the first phase.

[0129] For example, a method of preventing the onset or slowing the progression of atrophy of the retinal neurosensory epithelium and / or retinal pigment epithelium (RPE) in a subject by intravitreal or periocular injection of a formulation of an extended release drug delivery system that produces highly sustained retinal and RPE tissue levels of active drug may include delivering into the subject's eye at the start of treatment an active ingredient (API) prodrug drug substance in combination with an extended release drug delivery system; and cleaving the prodrug by the action of esterases or bioactive enzymes in the subject's eye to release the API into the eye at a burst phase release rate during a first phase; and cleaving the prodrug by the action of esterases or bioactive enzymes to release the API into the eye at a steady state administration rate during a second phase, wherein the burst phase rate exceeds the steady state release rate, and wherein the first phase extends for about 2-6 weeks from the start of treatment and the subsequent phase extends for one or more months from the end of the first phase.

[0130] All of the methods and devices described herein, in any combination, may be used to achieve the benefits contemplated and described herein. A better understanding of the features and advantages of the methods and apparatus described herein can be obtained by reference to the following detailed description that sets forth illustrative embodiments and the accompanying drawings. [Brief description of the drawings]

[0131] [Figure 1] FIG. 1 illustrates the components of a multiphase colloidal suspension, in which a drug substance (100) (defined as a variety of small polypeptides, proteins, aptamers, other nucleic acid drugs, hydrophobic chemicals, hydrophilic chemicals, and other compounds used for therapeutic purposes) or prodrug (101) (e.g., any active ingredient (103) linked via a cleavable covalent bond to one of five classes of conjugate moieties (105)) is added and mixed with one or more complexing agents in a hydrophobic dispersion medium (107). These collectively form a multiphase colloidal suspension extended release drug delivery system (109) that can be administered intraocularly and periocularly in a variety of formulations to achieve durations of treatment of 1-12 months, for example, to treat a variety of diseases of the eye, periocular tissues, and orbit. [Diagram 2] 2A-2E illustrate an approach to mathematically custom design a formulation for a specific pharmacokinetic release profile using a multiphasic colloidal suspension extended release drug delivery system, including, in this specific example, one method of constructing a biphasic release profile (FIG. 2A) for the release of the drug substance described herein. [Diagram 3] 3A-3C illustrate three different potential release kinetic profiles of a drug substance from a multiphasic colloidal suspension, including (FIG. 3A) monophasic (zero order) release kinetics, (FIG. 3B) biphasic release kinetics, and (FIG. 3C) triphasic release kinetics. [Figure 4]Figures 4A-4F illustrate the complexation of magnesium stearate with various fluorescent small molecules assayed using fluorescence microscopy. Figure 4A shows magnesium stearate alone, which exhibits low intrinsic fluorescence. Figure 4B shows magnesium stearate incubated with FITC-labeled EY005 native peptide (Hd-Arg-DMT-Lys-Phe). FITC-labeled EY005 native peptide alone showed minimal complexation with magnesium stearate, which was reflected by minimal fluorescent labeling of the microparticles. Figure 4C shows magnesium stearate incubated with FITC-labeled EY005-stearate prodrug (Hd-Arg-DMT-Lys-Phe(O)-stearyl). Complexation of the EY005-stearyl prodrug with magnesium stearate was evident due to the moderate fluorescence of the imaged magnesium stearate microparticles. FIG. 4D shows that treatment of FITC-labeled EY005-stearyl prodrug (FITC-labeled peptide and unlabeled stearyl conjugate moiety) complexed with magnesium from sample C with carboxylesterase (0.1 μg / mL) reduced the level of fluorescence, demonstrating that complexation is specifically mediated by the stearyl conjugate moiety. FIG. 4E shows magnesium stearate incubated with fluorescent C12 lipid compound. Strong complexation of C12 lipid compound with magnesium stearate was evident due to the bright fluorescence of the imaged magnesium stearate microparticles. FIG. 4F shows magnesium stearate incubated with fluorescent cationic small molecule, which strongly complexed with magnesium stearate as seen by the bright fluorescence of the magnesium stearate microparticles. [Diagram 5]Figures 5A-5F illustrate the complexation of albumin with various fluorescent small molecules assayed using fluorescence microscopy. In Figure 5A, albumin alone exhibits low intrinsic fluorescence. Figure 5B shows albumin incubated with FITC-labeled EY005 native peptide (Hd-Arg-DMT-Lys-Phe). FITC-labeled EY005 native peptide alone showed minimal complexation with albumin as reflected by the negative staining of albumin crystals, which are surrounded by diffuse fluorescence from dissolved FITC-labeled EY005 native peptide. Figure 5C shows albumin incubated with FITC-labeled EY005-stearate prodrug (Hd-Arg-DMT-Lys-Phe(O)-stearyl). Strong complexation of the EY005-stearyl prodrug with albumin was evident as bright fluorescence of the imaged albumin crystals. In FIG. 5D, FITC-labeled EY005-stearyl prodrug (FITC-labeled peptide and unlabeled stearyl conjugate moiety) complexed with albumin from sample C was treated with carboxylesterase (0.1 μg / mL) resulting in a decrease in the level of fluorescence, demonstrating that the complexation was specifically mediated by the stearyl conjugate moiety. FIG. 5E shows albumin incubated with fluorescent C12 lipid compound. Strong complexation of the fluorescent C12 lipid compound with albumin was evident as bright fluorescence of the imaged albumin microparticles. FIG. 5F shows albumin incubated with fluorescent cationic small molecule, which was moderately complexed with albumin as seen by the moderate fluorescence of the albumin microparticles. [Figure 6]Figures 6A-6F illustrate the complexation of various fluorescent small molecules with cyclodextrin gamma as assayed using fluorescence microscopy. In Figure 6A, cyclodextrin alone exhibits low intrinsic fluorescence. Figure 6B shows cyclodextrin incubated with FITC-labeled EY005 native peptide (Hd-Arg-DMT-Lys-Phe). FITC-labeled EY005 native peptide alone exhibited minimal complexation with cyclodextrin, as reflected by the minimal increase in fluorescence over that of cyclodextrin alone. FIG. 6C shows cyclodextrin incubated with FITC-labeled EY005-stearate prodrug (Hd-Arg-DMT-Lys-Phe(O)-stearyl. Complexation of the EY005-stearyl prodrug with cyclodextrin was evident as moderate fluorescence of the imaged prodrug-cyclodextrin microparticles. FIG. 6D shows the FITC-labeled EY005-stearyl prodrug complexed with cyclodextrin from sample C (FITC-labeled peptide and unlabeled stearyl conjugate moiety) was incubated with carboxylesterase (0.1 μg / mL). Treatment with 100 µg / mL of cyclodextrin showed a decrease in the level of fluorescence, demonstrating that complexation is specifically mediated by the stearyl conjugate moiety. Figure 6E shows cyclodextrin incubated with a fluorescent C12 lipid compound. Complexation of the fluorescent C12 lipid compound with cyclodextrin was evident as moderate fluorescence of the imaged cyclodextrin microparticles. In Figure 6F, cyclodextrin was incubated with a fluorescent cationic small molecule that complexes more strongly with cyclodextrin, creating brightly fluorescent microparticles. [Figure 7]Figures 7A-7F illustrate the complexation of various fluorescent small molecules with lecithin assayed using fluorescence microscopy. In Figure 7A, lecithin alone exhibits low intrinsic fluorescence. Figure 7B shows lecithin incubated with FITC-labeled EY005 native peptide (Hd-Arg-DMT-Lys-Phe). FITC-labeled EY005 native peptide showed minimal complexation with lecithin as reflected by a minimal increase in fluorescence over that of lecithin alone. Figure 7C shows lecithin incubated with FITC-labeled EY005-stearate prodrug (Hd-Arg-DMT-Lys-Phe(O)-stearyl). Complexation of lecithin with EY005-stearyl prodrug was evident as bright fluorescence for all lecithin samples. In FIG. 7D, FITC-labeled EY005-stearyl prodrug (FITC-labeled peptide and unlabeled stearyl conjugate moiety) complexed with lecithin from sample C was treated with carboxylesterase (0.1 μg / mL) resulting in a decrease in the level of fluorescence, demonstrating that complexation is specifically mediated by the stearyl conjugate moiety. FIG. 7E shows lecithin incubated with fluorescent C12 lipid compound. Complexation of the fluorescent C12 lipid compound with lecithin was evident as bright fluorescence in all lecithin samples. FIG. 7F shows lecithin incubated with fluorescent cationic small molecule, which showed minimal complexation with lecithin as evidenced by only dim fluorescence in the lecithin sample. [Figure 8]Figures 8A-8F illustrate the complexation of various fluorescent small molecules assayed using fluorescence microscopy with silica microbeads, which are microparticles that do not act as complexing agents. In Figure 8A, silica microbeads alone were imaged as a negative control and showed minimal intrinsic fluorescence. Figure 8B shows silica microbeads incubated with FITC-labeled EY005 native peptide (Hd-Arg-DMT-Lys-Phe). FITC-labeled EY005 native peptide was shown complexed with silica microbeads that produced circular shapes of fluorescence and disperse upon addition of the multiphase colloidal suspension to the physiological environment of the eye, indicating low avidity of complexation. Figure 8C shows silica microbeads incubated with FITC-labeled EY005-stearyl prodrug (Hd-Arg-DMT-Lys-Phe(O)-stearyl). There was no evidence of complexation of the EY005-stearyl prodrug with the silica microbeads. In Figure 8D, FITC-labeled EY005-stearyl prodrug (FITC-labeled peptide and unlabeled stearyl conjugate moiety) complexed with silica microbeads from sample C was treated with carboxylesterase (0.1 μg / mL) without altering the very low level of fluorescence. Figure 8E shows silica microbeads incubated with fluorescent C12 lipid compounds. The dim fluorescence at the surface of the silica microbeads suggests minimal complexation with the fluorescent C12 lipid compounds. Figure 8F shows silica microbeads incubated with fluorescent cationic small molecules that complex extensively with the silica microbeads, producing brightly fluorescent circular shapes. [Figure 9] FIG. 9 demonstrates examples of release kinetics of fluocinolone acetonide (F17 FA), dexamethasone free base (F10 DEX) and dexamethasone phosphate (F1 DEX PHOS) with daily release rates for various formulations. [Figure 10]10A-10F illustrate fluorescent microbeads (3 μm and 10 μm) in water. Very rapid settling occurs in the upper regions of the mixture, showing very few microbeads, while the lower levels of the mixture show very densely packed microbeads. Thus, this mixture does not function as a multiphase colloidal suspension, as the particles are not uniformly dispersed. [Figure 11] 11A-11F illustrate fluorescent microbeads (3 μm and 10 μm) in silicone oil. A very rapid settling occurs in the upper region of the mixture, showing only a few microbeads, while the lower level of the mixture shows very densely packed microbeads. Thus, this mixture does not function as a multiphase colloidal suspension, as the particles are not uniformly dispersed. [Figure 12] Figures 12A-12F illustrate fluorescent microbeads (3 μm and 10 μm) in methyl laurate. The beads remain uniformly dispersed with no evidence of settling or migration. Thus, methyl laurate is an effective dispersion medium as it forms a stable multiphase colloidal suspension. [Figure 13] 13A-13F illustrate fluorescent microbeads (3 μm and 10 μm) in 2% gelatin. The beads remain uniformly dispersed with no evidence of settling or migration, indicating the formation of a colloidal suspension. [Figure 14] 14A-14F illustrate fluorescent microbeads (3 μm and 10 μm) in 2% gelatin treated with collagenase. After treatment, the beads settle rapidly, resulting in a higher ratio of beads in the lower wells, indicating that gelatin cannot serve as a dispersion medium in the physiological environment of the eye, which has a large amount of enzymes that degrade gelatin. Therefore, this does not represent a stable multiphase colloidal suspension, and 2% gelatin is not an effective dispersion medium. [Figure 15]Figure 15 shows Table 1 illustrating the binding capacity (μg complexed fluocinolone acetonide per mg complexing agent) and Kd (unbound-bound ratio) of fluocinolone acetonide added to methyl laurate and mixed with various complexing agents, centrifuged and pulled down microparticles after 1 hour of incubation. The amount of fluocinolone acetonide complexed with each complexing agent was determined by HPLC. These data demonstrate various degrees of complexation with each class of complexing agents. [Figure 16] FIG. 16A illustrates how different representative formulations of drug and complexing agent(s) produce specific and distinct release kinetics in vitro, with the drug release kinetics of each formulation being engineered and tailored in a predictable manner by varying the ratio of two different complexing agents in this example. Formulation 1 depicts a shorter duration release profile (i.e., 120 days), while formulation 2 depicts a biphasic release profile with a longer duration (i.e., 210 days). FIG. 16B illustrates the effect of various ratios of complexing agents on the time release kinetics described herein for a given drug payload. As described in FIG. 2, drug release kinetics measured from individual drug-complexes can be utilized to determine the predicted target release kinetics for a blend of two or more drug-complexes, which can be experimentally confirmed by in vitro release studies, as in FIG. 9. [Figure 17] 17A and 17B illustrate good in vitro to in vivo correlation for two different formulations of fluocinolone acetonide in multiphasic colloidal suspensions. The depicted curves reflect the in vitro release profiles, while the individual colored round dots represent the in vivo release data from rabbit eyes. [Figure 18]Figure 18 shows Table 2, illustrating the binding capacity (μg fluocinolone acetonide complexed per mg complexing agent) and Kd (unbound-bound ratio) of dexamethasone phosphate added to methyl laurate and mixed with various complexing agents, centrifuged and pulled down microparticles after 1 hour of incubation. The amount of dexamethasone phosphate complexed with each complexing agent was determined by HPLC. These data demonstrate various degrees of complexation with each class of complexing agents. [Figure 19] FIG. 19 illustrates which physicochemical properties of the drug substance affect its interaction with the complexation properties. The hydrophilic drug substance dexamethasone phosphate (DexPh) and the hydrophobic drug substance fluocinolone acetonide (FA) (at the same payload) were each miscible with the same particulate complexing agents (magnesium stearate and tocopherol) and dispersion medium (methyl laurate) (circles for DexPh, triangles on the lower curve for FA). Formulations of DexPh demonstrated abrupt and excessive release, or "dumping," of DexPh. The addition of a different complexing agent, lecithin, at a given payload with a lower ratio of the other complexing agent (triangles, middle curve), altered the kinetic release profile, minimizing the dumping of DexPh and resulting in a more desirable sustained release profile. [Figure 20] Figure 20 shows Table 3, illustrating the binding capacity (μg complexed fluocinolone acetonide per mg complexing agent) and Kd (unbound-bound ratio) of sunitinib malate added to the dispersion medium and mixed with various complexing agents, centrifuged and pulled down the microparticles after 1 hour of incubation. The amount of sunitinib malate complexed with each complexing agent was determined by HPLC. These data demonstrate various degrees of complexation with each class of complexing agents. [Figure 21]Figure 21 illustrates that complexation can also be assessed by colorimetric analysis of certain compounds. Sunitinib is a brightly colored yellow compound. To assess complexation, various complexing agents were incubated with sunitinib maleate solution for 1 hour. The complexing agents were then rinsed 5 times to remove any free sunitinib. After rinsing, the complexing agents were imaged to assess the relative extent of sunitinib complexation. As can be seen, colorimetric changes occur to different degrees with each complexing agent, suggesting different levels of complexation with sunitinib. [Figure 22] Figure 22 illustrates the formulation (as a multiphase colloidal suspension in a bioerodible tube) and drug substance release of low and high dose implants at multiple time points with detectable drug levels in rabbit retinal tissue, and the durability of release of each. Tissue levels at the high dose implants remained consistently above IC90 levels of sunitinib. [Figure 23] Figure 23 illustrates the formulation (as a multi-phase colloidal suspension as a flowable bolus) and drug substance release of axitinib at multiple time points with detectable drug levels in rabbit retinal tissue from implants, and durability of release. Tissue levels remained consistently above the IC90 levels of axitinib. [Figure 24] Figure 24 is Table 4, illustrating the binding capacity (μg fluocinolone acetonide complexed per mg complexing agent) and Kd (unbound-bound ratio) of EY005-stearyl prodrug added to dispersion medium and mixed with various complexing agents, centrifuged and pulled down microparticles after 1 hour of incubation. The amount of centrate complexed with each complexing agent was determined by HPLC. These data demonstrate various degrees of complexation with each class of complexing agent. [Diagram 25]FIG. 25A shows data from an accelerated in vitro release assay in which EY005 MTT was formulated with various complexing agents using methyl laurate as the carrier medium. In all cases, all EY005 was released rapidly, in some cases within hours, and in all cases by day 3. FIG. 25B shows data from an accelerated in vitro release assay in which EY005-stearyl prodrug was formulated with various complexing agents using methyl laurate as the carrier medium. Formulations without complexing agents show a rapid release of EY005 into the medium. Formulations with silica microbeads that are not complexed with EY005-stearyl prodrug also show a rapid release of EY005 into the medium. In contrast, formulations with other complexing agents demonstrate sustained release of EY005 at various rates. Of particular note, when magnesium stearate and albumin are both used as complexing agents in the same formulation, EY005 is released at a moderate rate between those of the formulations using either complexing agent alone. [Figure 26] Figure 26 illustrates generally one example of the mitochondria-targeting tetrapeptide EY005 (103), which when linked to one of several classes of conjugate moieties (105) constitutes a mitochondria-targeting peptide prodrug (101). This mitochondria-targeting peptide prodrug is mixed with a selected complexing agent in a dispersion medium to form a multi-phase colloidal suspension in a bolus formulation (107), which can be injected into the vitreous of the eye (109) as part of an intravitreal (IVT) extended release drug delivery system. [Figure 27]Figure 27A is an example of a prodrug of an EY005 mitochondrial targeting tetrapeptide that includes a stearyl alcohol or octadecyl moiety linked to the mitochondrial targeting tetrapeptide via an ester bond. Figure 27B is an example of a prodrug of an EY005 mitochondrial targeting tetrapeptide that includes a peptide motif (e.g., an anionic tri-Glu peptide) and a linker moiety linked to EY005 via an ester bond. Figure 27C is an example of a prodrug of an EY005 mitochondrial targeting tetrapeptide that includes a peptide motif (e.g., a cationic tri-Arg peptide) and a linker moiety linked to EY005 via an ester bond. Figure 27D is an example of a prodrug of an EY005 mitochondrial targeting tetrapeptide that includes a polyethylene glycol (PEG) linked to EY005 via an ester bond. [Figure 28] Figures 28A-28C demonstrate the cleavage of esters based on the EY005-stearyl prodrug by carboxylesterase and by spontaneous hydrolysis. Figure 28A shows baseline HPLC analysis of EY005-stearyl prodrug (top trace) and EY005 MTT (bottom trace). EY005-stearyl was incubated with carboxylesterase (0.1 μg / mL) in vitro at 37°C to mimic the physiological environment of the eye and the type of esterase that is prone to abundance in the vitreous. Incubation of EY005-stearyl with carboxylesterase produces abrupt cleavage of the ester bond of the prodrug, releasing EY005, as evidenced by the disappearance of the EY005-stearyl prodrug peak and the appearance of an EY005 peak in high performance liquid chromatography (Figure 28B). When EY005-stearyl prodrug is added to a phosphate buffered saline solution without esterase at 37° C., the ester bond of EY005-stearyl prodrug is cleaved more slowly by hydrolysis (FIG. 28C). After 6 hours, partial cleavage of EY005-stearyl prodrug to EY005 MTT is shown. [Figure 29]FIG. 29A shows an in vitro culture model of dry AMD, in which RPE cells possessing endogenous esterases are exposed to hydroquinone (HQ) to induce mitochondrial dysfunction. Mitochondrial dysfunction is manifested as increased flavoprotein autofluorescence (upper panel) and abnormal morphology of the actin cytoskeleton (lower panel). EY005-stearyl (5 μM) efficiently reversed HQ-induced mitochondrial dysfunction in RPE cells (portrayed by a reduction in cellular flavoprotein-autofluorescence and normalized actin cytoskeleton dysfunction) with an efficacy equivalent to treatment with EY005 native peptide (5 μM). EY005-stearyl was also pre-incubated with carboxylesterase (0.1 μg / mL) in separate media. The collected media containing cleaved EY005 (5 μM) was added to this RPE cell model of mitochondrial dysfunction and was similarly effective, with equal potency to the EY005 native peptide in abrogating RPE mitochondrial dysfunction. Figure 29B shows quantification of flavoprotein autofluorescence (FP-AF) from at least three replicates of each condition depicted in Figure 29A. Both EY005-stearyl and esterase-cleaved EY005-stearyl show equal potency to the native EY005 peptide. Figure 29C shows quantification of actin cytoskeleton abnormal morphology from at least three replicates of each condition depicted in Figure 29A. Both EY005-stearyl and esterase-cleaved EY005-stearyl show equal potency to the native EY005 peptide. [Diagram 30] Figure 30 shows the in vitro pharmacokinetics of the pilot formulation of Mito XR (triangles). Mito XR achieved zero-order (i.e. linear) kinetics of release of the EY005 bioactive tetrapeptide, which achieved the desired durability of drug release for 3 months, and the free bioactive MTT in the dispersion medium was released from the implant into the physiological environment of the eye. In contrast, the similarly formulated EY005 native peptide (circles) showed a very rapid release, which did not provide the desired durability of drug release. [Diagram 31]Figures 31A-31C depict an in vitro culture model of dry AMD, in which RPE cells, possessing endogenous esterases, are exposed to hydroquinone (HQ) to induce mitochondrial dysfunction. Mito dysfunction manifests as abnormal morphology of the actin cytoskeleton. In these efficacy studies, a bolus implant of Mito XR (formulated with EY005-stearyl in a multi-phase colloidal suspension) was added to an RPE cell culture model of dry AMD in which endogenous esterases are present. Treatment with Mito XR implants resulted in abrogation of mitochondrial dysfunction and concomitant restoration of actin cytoskeleton morphology. Figure 31D shows a graphical representation of the data from Figures 31A-31C. Cultured RPE cells were graded for severity of abnormal morphology of the actin cytoskeleton in control, cells exposed to HQ, and cells exposed to HQ and treated with Mito XR. Results from at least three replicates were quantified. Cultures treated with Mito XR demonstrate an 80% reduction in the severity of RPE cell actin cytoskeleton abnormalities compared to control, HQ-exposed cells. [Diagram 32]Figure 32 shows the superior in vivo pharmacokinetics of EY005-stearyl prodrug formulated as Mito XR. Rabbits were injected with intravitreal Mito XR implants containing formulated EY005-stearyl prodrug (EY005-stearyl release from IVT MitoXR) or identical bolus formulations containing EY005 native peptide (EY005 peptide release from formulated bolus). EY005-stearyl prodrug formulated as Mito XR showed retinal tissue concentrations above the EC50 for reversing mitochondrial dysfunction. These therapeutic drug levels were sustained until the 7-week time point, at which time Mito XR implants were recovered that still contained 50% payload, indicating that this formulation achieved the desired 90-day durability. In contrast, native EY005 peptide was rapidly released with nearly undetectable tissue concentrations by 3.5 weeks. The retrieved implants did not contain the EY005 native peptide, suggesting rapid dumping of the drug in vivo. [Diagram 33] Figures 33A-33C illustrate examples of delivery forms or modes for delivering implants of either a multi-phase colloidal suspension extended release drug delivery system that may contain one or more complexing agents that non-covalently complex with a prodrug of a mitochondria-targeting tetrapeptide. Figure 33A shows an example of a bolus injection, where the extended release drug delivery system material is formulated as an injectable liquid bolus. Figure 33B is an example where the MTT-prodrug multi-phase colloidal suspension is formulated as a tube implant with a biodegradable outer sleeve / tubing filled with a multi-phase colloidal suspension containing the prodrug and complexing agent. Figure 33C is an example where the extended release drug delivery system material is molded into a solid state specific shape for implant therapy. Figure 33D illustrates two methods of injecting the multi-phase colloidal suspension formulation into the eye, either as a bolus injection or as a tube implant. [Diagram 34]FIG. 34A-34B illustrate the effect of various inner diameters / radii of the open end of an injectable tube implant format on XRDDS based complex formation. The release rate predictably decreased in proportion to the radius / diameter of the tube end. FIG. 34A illustrates the dimensions of the end of the tube (depot). FIG. 34B is a graph showing the release rate over time (days) for two examples of a given extended release corticosteroid formulation, each released from a tube of different inner diameter / radius (r). As predicted, the formulation in the PE10 tube, which has a lower r value, has a lower release rate compared to the formulation in the PE50, which has a higher r value. FIG. 34C illustrates the use of an ultra-thin walled 25 gauge needle suitable for intravitreal injection, which has a bioerodible or non-bioerodible tube (depot) for releasing the extended release corticosteroid formulation out of the lumen of the 25 gauge needle. [Diagram 35] 35A-35D illustrate that the composition of the bioerodible tube may allow for a biphasic drug release profile (FIG. 35B), or a triphasic drug release profile, allowing for accelerated release later. In FIG. 35A, the bioerodible tube is a PLGA composition 82L / 18G tube, which shows the tube intact after all of the drug has been released. FIG. 35B is a graph showing the biphasic kinetic profile that occurred with the tube of FIG. 35A filled with an extended release corticosteroid formulation. FIG. 35C shows the bioerodible tube with PLGA composition 80L / 20G tube degrading before all of the drug is released, resulting in a triphasic release profile when releasing the extended release corticosteroid formulation, as shown in FIG. 35D. [Diagram 36]Figures 36A and 36B illustrate that irradiation of a corticosteroid drug in an XRDDS matrix can be used to tailor the release rate, particularly for the initial burst phase of release and the early period of the subsequent steady state of release, with more drug release seen from implants irradiated at higher doses. Figure 36A shows the release rate over an extended period of time of an example of a long-term release corticosteroid formulation (fluocinolone acetonide) that was not irradiated ("non-irradiated") compared to the same formulation that was irradiated ("irradiated to 40 kGy"). Figure 36B shows the release rate over an extended period of time of yet another example of a long-term release corticosteroid formulation (fluocinolone acetonide) that was not irradiated ("non-irradiated") compared to the same formulation that was irradiated ("irradiated to 40 kGy"). In both cases (Figures 36A and 36B), the irradiated implants show a higher release rate during the initial burst at one month, as well as a higher release rate at one month of the maintenance phase, compared to the non-irradiated implants. [Figure 37] Figures 37A and 37B illustrate tuning the duration of drug release by controlling the length of the implant, for example, using longer or shorter bioerodible or non-bioerodible tubes, with shorter tubes exhibiting a relatively shorter duration of release and longer tubes resulting in a longer duration of drug release. Figure 37A illustrates exemplary dimensions of a bioerodible tube. Figure 37B is a graph comparing the duration of in vivo release of an extended release corticosteroid formulation over time (days) for a 4mm vs. 6mm long implant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0132] Described herein are compositions and methods of use for a novel, multi-purpose extended release drug delivery system (XRDDS) for delivery of a variety of drug substances into and around the eye, comprising a drug substance that non-covalently interacts with one or more complexing agent microparticles to form drug substance-complex microparticles, which are miscible in a hydrophobic dispersion medium, thereby collectively forming a stable multi-phase colloidal suspension (Figure 1).

[0133] As used herein, drug substances may include 1) a variety of small polypeptides, proteins, aptamers, other nucleic acid drugs, hydrophobic chemicals, hydrophilic chemicals and other compounds used for therapeutic purposes that are capable of forming non-covalent complexes directly to one of six classes of complexing agents: fatty acids, organic compounds that can form keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids and polysaccharides, and 2) prodrugs of any active ingredient (API) linked via a cleavable covalent bond to a conjugate moiety that forms a complex with one of six classes of complexing agents: fatty acids, organic compounds that can form keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids and polysaccharides (Figure 1).

[0134] A conjugate moiety is any chemical entity that can be covalently attached to an API. Certain conjugate moieties may be selected for their ability to provide properties that the native API does not exhibit, particularly their ability to form reversible non-covalent complexes with complexing agents.

[0135] A complex is defined as a non-covalent interaction between a drug substance and a complexing agent. A complexing agent is defined as a chemical compound formulated as irregularly shaped microparticles ranging in size from 1 nanometer (nm) to 1000 micrometers (μm); that demonstrates a measurable binding capacity of a selected drug substance, defined as the amount of drug substance that binds to a known amount of complexing agent; that demonstrates a measurable unbound-bound ratio, or reversibility of drug binding, defined as Kd, in a particular dispersion medium; and that is not previously known or expected to form a complex with a selected drug substance. Binding of the drug substance to the complexing agent, either directly or, in the case of prodrugs, via a conjugate moiety, results in the formation of drug substance-complex microparticles. Certain well-known chemical compounds, including additives and excipients utilized in the pharmaceutical industry, when formulated as irregular microparticles, demonstrate the previously unknown and unexpected property of acting as complexing agents for various drug substances. These include six previously unknown classes of chemicals that, when formulated as irregularly shaped microparticles, act as complexing agents for a variety of drug substances: fatty acids, organic compounds capable of forming keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids and polysaccharides.

[0136] Irregular particulate formulations that are not dissolved into individual molecules, such as magnesium stearate, lecithin, albumin, and cyclodextrin, all meet the definition of a particulate complexing agent for a drug substance, a property that is previously unknown or unexpected (Figures 4-7).

[0137] A dispersion medium is a vehicle utilized in a colloidal mixture. As used herein, a dispersion medium is defined as a hydrophobic viscous oil selected from the four classes, saturated fatty acid methyl esters, unsaturated fatty acid methyl esters, saturated fatty acid ethyl esters, or unsaturated fatty acid ethyl esters, which when mixed with drug substance-complex microparticles can form a drug substance multi-phase colloidal suspension, and which has not previously been known to form a multi-phase colloidal suspension with the selected drug substance and selected complexing agent.

[0138] As used herein, a colloidal suspension is a formulation that is a viscous, flowable, pourable liquid (ie, a colloidal mixture) that forms a stable dispersion of fine particles without migration or settling of the particles.

[0139] Containing a multi-phase colloidal suspension refers to a colloidal suspension in which the drug substance is present in at least two phases: free unbound drug substance and drug substance bound to a complexing agent (and, to a lesser extent, drug-drug aggregates). Drug substance-complex microparticles act as a reservoir for the drug substance when the microparticles are miscible in the dispersion medium.

[0140] Thus, the drug substance multi-phase colloidal suspensions described herein can be viscous, flowable, injectable liquids that produce stably dispersed drug substance-complex microparticles without migration or settling, which can allow the free drug substance to dissociate from the drug substance-complex microparticles to create a free drug substance concentrate in the dispersion medium. The drug substance can freely diffuse out of the implant through the multi-phase colloidal suspension system and into the physiological environment of the adjacent eye. If the drug substance is a prodrug, the covalent bond linking the conjugate moiety is cleaved when the prodrug is exposed to the physiological environment of the eye, releasing the free API.

[0141] Formation of stably dispersed drug substance-complex microparticles in a drug substance multi-phase colloidal suspension, occurring by blending as defined herein, refers to the mixing and incorporation of a drug substance and one or more complexing agents in a dispersion medium by using a strategy incorporating a variety of mixing techniques, including stand paddle mixing, centrifugal shear mixing, high shear mixing, ribbon blenders, anchor mixers, static mixers, V-blenders, planetary mixers, kneading, kneading and folding, stirring, resonant acoustic mixers, Banbury mixers, dispersion mixers, vacuum mixers, high shear rotor mixers, and various other types of mixing techniques. The final blend may be homogeneously mixed (e.g., have a uniform or substantially uniform distribution). In some instances, the final blend may be non-homogeneously mixed (e.g., have a distribution or gradient of drug substance complex microparticles within the dispersion medium).

[0142] Drug substance multi-phase colloidal suspensions enable drug delivery systems because the microparticles are reservoirs of bound drug substance, each with a unique binding capacity and Kd (unbound-bound ratio), which in turn determines the amount of complexed free drug substance in the dispersion medium. Using knowledge of the Kd and binding capacity of each drug substance-complex microparticle, the total amount of free drug substance in the system can be calculated, which in turn determines the rate and amount of release. The relative ratios and amounts of the various drug substance-complex microparticles can be adjusted in a manner that creates calculable unbound free drug substance in the system (Figures 2A-2E). The dynamic changes in unbound free drug substance in the system over the life of the implant are determined by the binding capacity and Kd of the drug substance-complex microparticles in the drug substance multi-phase colloidal suspension.

[0143] In the methods and compositions described herein, the bulk multi-phase colloidal suspension is injectable through a 20-gauge to 30-gauge needle (depending on the application) to provide a stable dispersion of microparticles without migration or settling when exposed to the physiological environment of the eye for the duration of the implant's life (1 to 12 months). The physiological environment of the eye is defined as an in vitro condition using phosphate buffered saline (or similar aqueous solvent) at 37° C. containing enzymes and proteins normally found in the vitreous (representing injection into the vitreous) or using phosphate buffered saline at 37° C. containing plasma (representing injection into various periocular tissues). Alternatively, the physiological environment of the eye may represent injection of the implant in vivo into the vitreous or periocular tissues.

[0144] The pharmaceutical multi-phase colloidal suspension also exhibits the property of biodegradability when exposed to the physiological environment of the eye, where the biodegradability occurs due to dissolution of the dispersion medium. The rate of biodegradation is proportional to the solubility of the dispersion medium in the physiological environment of the eye. Dispersion media with higher solubility allow faster biodegradation of the multi-phase colloidal suspension when exposed to the physiological environment of the eye, while dispersion media with lower solubility allow slower biodegradation of the multi-phase colloidal suspension when exposed to the physiological environment of the eye. This property of the pharmaceutical multi-phase colloidal suspension can be used together with the volume of implant injected to determine the durability of the implant in the physiological environment of the eye.

[0145] The formulation of pharmaceutical active ingredients in a multi-phase colloidal suspension, referred to as an implant, can be administered intraocularly and periocularly, i.e., into the vitreous humor, aqueous humor, suprachoroidal space, subretinal, subconjunctival, sub-Tenon's capsule, or orbital tissues, to produce sustained release of therapeutic levels of pharmaceutical active ingredients in ocular tissues over desirable durations (1 to 12 months) for the treatment of various diseases and disorders.

[0146] The multi-phase colloidal suspension extended release compositions described herein (e.g., extended release drug delivery systems, XRDDS) may contain drug substances that are admixed with one or more microparticle complexing agents to form "drug-complex" microparticles, which are combined and dispersed in a selected dispersion medium to form a stable multi-phase colloidal suspension.

[0147] A colloid is a mixture in which particulate material is stably dispersed in a vehicle called the dispersion medium, but does not settle or migrate. This distinguishes colloids from suspensions in which particles settle in the suspension vehicle due to gravity. Typical particle sizes for colloids are in the nanometer range. In colloids, the defining property of the mixture is that the particulates remain stably dispersed with minimal settling or migration. Colloidal mixtures in which particulates are dispersed in a liquid are called "sols." Colloidal mixtures in which particulates are dispersed in a solid or semi-solid are called "solid colloids." Colloidal mixtures in which particulates are stably dispersed in a viscous semi-solid or solid dispersion medium are not given a defined name. Herein, we refer to stably dispersed particulates as "colloidal suspensions." In the methods and compositions described herein, the dispersion medium can be a hydrophobic dispersion medium that promotes stable colloidal suspensions. Drug substance multi-phase colloidal suspensions are suspensions in which the drug substance is present in more than one phase including free drug, drug-drug aggregates, and most importantly, drug non-covalently bound to complexing agent microparticles.

[0148] Complex formation occurs in two physicochemical situations. In one case, complex formation occurs through non-covalent interactions between individual molecules (e.g., receptor-ligand interactions). This type of complex formation is called molecular complex formation and is not contemplated in the present composition.

[0149] The second situation involves molecules of a chemical, in this case a drug, that non-covalently bind or adsorb to the surface of a particulate, in this case the complexing agent. This type of complexing is referred to as particulate complexing. Different particulate adsorbents or complexing agents have different adsorptivities based on the size and shape of the particulate, the functional groups present on the surface of the particulate, and the roughness and porosity of the surface. The utility of particulate complexing has been realized in other disciplines, including soil science, where chemical adsorbents (e.g., alumina, silica gel, activated carbon) interact with certain chemicals (often foreign matter) in the soil; the hydrocarbon industry, where adsorbents (e.g., polypropylene, vermiculite, perlite, polyethylene, etc.) are used to clean up oil spills or remove residual oil from drilling and fracking equipment; and industrial coatings (e.g., zeolites, silica gel, aluminum phosphate), where adsorbents are used to bind chemicals for various purposes (i.e., lubrication, surface cooling).

[0150] In medical applications, sorbents are used to treat acute poisoning by ingestion (e.g., activated charcoal, calcium polystyrene sulfate, aluminum silicate), where they bind toxins and limit their adsorption from the intestine into the systemic circulation. In the pharmaceutical industry, the principles of adsorption complex formation are used to understand the chemistry of drug binding to plasma proteins in the blood, drug coatings on solid scaffolds for in situ drug release (e.g., drug-eluting stents), and the addition of excipients to insoluble drugs to improve oral bioavailability and intestinal absorption.

[0151] The methods and compositions described herein may utilize microparticle complexation, where the complexing agent is thus a chemical that is compatible with ocular tissue when formulated as irregularly shaped microparticles and has the ability to non-covalently bind to the drug substance and form drug substance-complex microparticles. One or more drug substance-complex microparticles are incorporated and mixed in a hydrophobic dispersion medium to form a stable multi-phase colloidal suspension that is safely delivered in and around the eye, resulting in a predictable, therapeutic level of the drug substance being continuously exposed in the ocular tissue for the desired duration of treatment. The complexing agent is selected from one of six classes of chemicals, including fatty acids, organic compounds that can form keto-enol tautomers, charged phospholipids, charged proteins, nucleic acids, and polysaccharides.

[0152] When the drug substance is a prodrug, the conjugate moiety of the prodrug is specifically selected for its ability to complex with or form a non-covalent interaction with one or more microparticle complexing agents to form a prodrug-complex microparticle. The one or more prodrug substance-complex microparticles are incorporated and mixed in a hydrophobic dispersion medium to form a stable multi-phase colloidal suspension that is safely delivered in and around the eye, resulting in a predictable, continuous exposure of the drug substance to therapeutic levels in ocular tissues over the desired duration of treatment. The complexing agent is selected from one of six classes of chemicals, including fatty acids, organic compounds that can form keto-enol tautomers, charged phospholipids, charged proteins, nucleic acids, and polysaccharides.

[0153] The methods and compositions described herein disclose a new and previously unappreciated property of these six classes of chemicals, fatty acids, organic compounds capable of forming keto-enol tautomers, charged phospholipids, charged proteins, nucleic acids and polysaccharides, that when in the form of irregularly shaped microparticles with irregular surfaces, they can act as effective complexing agents for drug substances. The complexation agent criteria include four characteristics: (1) the drug substance binds to the microparticle complexation agent, demonstrable by imaging with microscopy (Figures 4A-4F, 5A-5F, 6A-6F, and 7A-7F); (2) when microparticles of the material are added to a solution of the drug substance, a pharmacologically significant amount of the drug substance is observed to be complexed to the microparticles upon centrifugation and pull-down of the microparticles, providing a quantitative measure of the binding capacity of the complexation agent (see Figures 15, 18, 20, 24); (3) when the drug substance-complexed microparticles are resuspended in an appropriate dispersion medium, they demonstrate partial release of drug, allowing the determination of the Kd or unbound-bound fraction of drug for a given drug substance-complexation agent pair in a particular dispersion medium (see Figures 25A-25B); and (4) when the drug substance-complexed microparticles are admixed in a dispersion medium, they exhibit a useful pharmacokinetic release profile to form a drug substance multi-phase colloidal suspension (see Figure 9). Collectively, these four properties define a complexing agent and enable the complexation-based XRDDS described herein.

[0154] In contrast, spherical microparticles with a spherical smooth surface and a non-reactive coating, including, for example, silicone beads, latex beads, and certain polymeric microparticles, fail to form complexes with drug substances (Figures 8A-8F) and are therefore excluded from the methods and compositions described herein.

[0155] One class of complexing agents is fatty acids, which are carboxylic acids with an aliphatic chain that may be saturated or unsaturated and may be in the form of a salt or an ester. For example, fatty acids are CH3(CH2) nIt may have a chemical formula of COOH, where n is equal to 4 to 30. The fatty acid may include one of tetradecanoic acid, pentadecanoic acid, (9Z)-hexadecenoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, (9Z,12Z)-octadeca-9,12-dienoic acid, (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid, (6Z,9Z,12Z)-octadeca-6,9,12-trienoic acid, (5E,9E,12E)-octadeca-5,9,12-trienoic acid, (6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraenoic acid, (Z)-octadec-9-enoic acid, (11E)-octadec-11-enoic acid, (E)-octadec-9-enoic acid, nonadecanoic acid, eicosanoic acid, and the like. The fatty acid can be an unbranched fatty acid between C14 and C20. The fatty acid can be a saturated fatty acid including one of myristic acid (tetradecanoic acid), palmitic acid (hexadecanoic acid), stearic acid (octadecanoic acid), arachidic acid (eicosanoic acid). Specific examples of fatty acids in salt form include magnesium stearate (Figures 4A-4F), magnesium palmitate, calcium stearate, calcium palmitate, and the like.

[0156] One class of complex formers are organic compounds that can form keto-enol tautomers. Tautomers refer to molecules that are capable of undergoing chemical equilibrium between a keto form (ketone or aldehyde) and an enol form (alcohol). Typically, compounds capable of undergoing keto-enol tautomerization contain a carbonyl group (C=O) in equilibrium with the enol tautomer, which contains a pair of C=C-OH double-bonded carbon atoms adjacent to a hydroxyl (-OH) group, as depicted herein:

[0157] [ka] The relative concentrations of the keto and enol forms are determined by the chemistry of the particular molecule and the chemical microenvironment, including equilibrium, temperature, or redox state. Organic compounds capable of keto-enol tautomerization include, but are not limited to, phenols, tocopherols, quinones, ribonucleic acids, etc.

[0158] One class of complexing agents is the charged phospholipids. Generally, phospholipids consist of a glycerol molecule, two fatty acids, and a phosphate group modified by an alcohol, with the polar head of the phospholipid typically being negatively charged. Examples include lecithin (Figures 7A-7F), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, different phospholipids in oil, and many others, which can be used individually or in combination to act as complexing agents. Anionic phospholipids can include one of phosphatidic acid, phosphatidylserine, sphingomyelin, or phosphatidylinositol. In some examples, ionizable synthetic phospholipids with positive charges can be produced, including, but not limited to, DLin-MC3-DMA. The additional cationic phospholipid may include one of the following cationic triesters of phosphatidylcholine: 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC); 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC); 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP); 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE); 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC); 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine (EDOPC); 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (EDMPC); 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine (EDPPC). In pharmaceutical science, phospholipids are used in drug formulation and delivery applications to improve bioavailability, reduce toxicity, and improve cell permeability.However, in the methods and compositions described herein, phospholipids are used as complexing agent microparticles that non-covalently bind to drug substance, forming drug substance-complex microparticles for the purpose of adjusting the free drug substance in the dispersion medium of the stable multi-phase colloidal suspension in which drug substance-complex microparticles are incorporated and dispersed.

[0159] In some instances, anionic phospholipids may form non-covalent complexes with the cationic conjugate portion of a prodrug. Cationic phospholipids may form non-covalent complexes with the anionic conjugate portion of a prodrug.

[0160] One class of complexing agents is charged proteins. Proteins are large biomolecules and macromolecules that contain one or more long changes of amino acid residues. The amino acids that make up a protein can be positively charged, negatively charged, neutral or polar in nature, and collectively, the amino acids that comprise a protein give the protein its overall charge. A wide variety of proteins can act as complexing agents based on their size, molecular weight, ability to readily form microparticles, and compatibility with ocular tissues. The charge of a protein determines its compatibility with a particular drug substance, such that negatively charged proteins readily complex with positively charged drug substances, while positively charged proteins (e.g., positively charged Arg-Gln-Ile-Arg-Arg-Ile-Ile-Gln-Arg-NH2 and synthetic peptides) readily complex with negatively charged drug substances. Examples of proteins that can act as complexing agents include albumin (Figures 5A-5F) and collagen.

[0161] One class of complexing agents is nucleic acids, which are biopolymer macromolecules composed of nucleotides consisting of a 5-carbon sugar, a phosphate group, and a nitrogenous base. The importance of nucleic acids for biological functions and encoding genetic information is well established. However, nucleic acids also have versatile applications including nucleic acid enzymes (e.g., carbon nanomaterials), aptamers (e.g., for forming nucleic acid nanostructures and therapeutic molecules that act in an antibody-like fashion), and aptazymes (e.g., that can be used for in vivo imaging). In pharmaceutical science, specially designed nucleic acids have been considered and applied for use in carrier-based systems in which the nucleic acid serves as a carrier system for various types of drugs. However, in the methods and compositions described herein, the nucleic acid is not considered a carrier system, but rather a complexing agent, as it is highly negatively charged and thus formulated as a microparticle, which can then act as a complexing agent for a positively charged drug substance.

[0162] One class of complexing agents is polysaccharides, which are long chain polymeric carbohydrates composed of monosaccharide units linked together by glycosidic bonds. Often, they are quite heterogeneous and contain some variation of the repeating monosaccharide units. Depending on the structure, they may be insoluble in water. Complexation of polysaccharide particulate complexing agents to drug substances may occur through a variety of electrostatic interactions and may be influenced by the charge density of the drug substance and polysaccharide, the ratio of polysaccharide complexing agent to drug substance, ionic strength, and other properties. Examples of polysaccharides that may serve as complexing agents include cyclic polysaccharide molecules, cyclodextrins (Figures 6A-6F), inclusion compounds, cellulose, pectin, or acidic polysaccharides, which are polysaccharides that contain carboxyl, phosphate, or other similarly charged groups.

[0163] The complexing agent may be a compound that contains a metal ion. In any of these therapeutic compositions, ion coordination complex formation can occur around a central ion that forms extensive non-covalent interactions, which can be a central metal ion including one of copper, iron, zinc, platinum or lithium.

[0164] Ion coordination complexation is a chemical complexation process around a central ion, usually a metal, capable of forming extensive non-covalent electrostatic interactions with a wide range of chemicals. It is one of the most common chemical processes in nature. The affinity of binding varies among different coordinating ions, some of which are nearly irreversible, while others display relatively labile bonds. Central metal ions include copper, iron, zinc, platinum, lithium, etc. Three classes that can serve as complexation agents for drug delivery are chelating agents (EDTA), complexes to certain metals (platinum, lithium, lanthanum), and molecules with metalloprotein elements (hemoglobin, porphyrins, superoxide dismutase, etc., with zinc or copper binding domains).

[0165] The complexing agent may include a chelating agent configured for complexation with a metal, a metalloprotein, or a superoxide dismutase (SOD). The complexing agent may include a chelating agent configured for complexation with one or more of platinum, lithium, lanthanum, hemoglobin, porphyrin, zinc binding domain, or a superoxide dismutase (SOD).

[0166] In the methods and compositions described herein, a selected drug substance has a particular affinity for a given complexing agent and complexes with it to form a drug substance-complex microparticle, which affinity can be measured as the Kd, which is the unbound-bound fraction of the drug substance for a given drug substance-complex microparticle in a selected dispersion medium.

[0167] Another property of drug substance-complex microparticles is binding capacity, which is defined as the amount of drug substance that is bound to a known amount of complexing agent. The affinity and binding capacity of a drug substance for a particular complexing agent (Figures 15, 18, 20, 24) therefore serves to limit the free drug available for release from the drug substance-complex microparticles in a given dispersion medium.

[0168] Thus, in a multiphasic colloidal suspension consisting of one or more drug substance-complex microparticles incorporated in a hydrophobic dispersion medium, rather than the use of complexation that is the formulation of the multiphasic colloidal suspension to improve bioavailability, the use of complexation that limits the free unbound drug substance is available for release from a given dispersion medium of the multiphasic colloidal suspension.

[0169] The drug substances formulated in the multi-phase colloidal suspensions of the present extended release drug delivery systems (XRDDS) may include a variety of small polypeptides, proteins, aptamers, other nucleic acid drugs, hydrophobic chemicals, hydrophilic chemicals, and other compounds used for therapeutic purposes that are capable of forming non-covalent complexes directly to one of six classes of complexing agents: fatty acids, organic compounds that can form keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids, and polysaccharides.

[0170] The drug substance is one of six different classes of substances that are formulated as irregularly shaped microparticles: fatty acids, organic molecules that can form keto-enol tautomers, charged phospholipids, charged proteins, nucleic acids, and polysaccharides that form non-covalent high affinity interactions (or bonds) directly. Mixed in a dispersion medium, the resulting drug substance-complex microparticles regulate the release of free, unbound drug within a multiphase colloidal suspension, allowing for controlled, extended release from the formulated implant when administered into the physiological environment of the eye.

[0171] The drug substance formulated in the multi-phase colloidal suspension can also be a prodrug of any active ingredient (API) linked via a cleavable covalent bond to a conjugate moiety that forms complexes with one of six classes of complexing agents: fatty acids, organic compounds capable of forming keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids, and polysaccharides.

[0172] The prodrug has the formula (I): R'-R(I) where R' is any active ingredient (API) covalently linked via a cleavable bond to R, a conjugate moiety that forms a non-covalent complex with one of five classes of complexing agents, and the covalent bond connecting R' and R can be removed by enzymatic cleavage, catalysis, hydrolysis or other reaction to provide the free API R' and the conjugate moiety R, and R has a C4-C30 lipid moiety (fatty acid or fatty alcohol), a C4-C30 straight chain or branched aliphatic moiety, a 2-mer to 30-mer peptide moiety, a PEGylated moiety, or a carbohydrate moiety.

[0173] A prodrug can be the product of a condensation or esterification reaction between an API and a conjugate moiety. In pharmacology, a prodrug is a chemical modification of an API. Prodrugs are metabolized in the recipient by tissue enzymes or by hydrolysis to the free API and inactive conjugate moieties. Prodrugs are typically used to modify the physicochemical properties of an API to improve absorption, bioavailability or pharmacokinetics (PK). However, in the methods and compositions described herein, the goal of the prodrug strategy is to optimize the physicochemical properties of the drug for suitability for multiphasic colloidal suspension extended release drug delivery systems (XRDDS). In most cases, this results in a controlled release rate of the API that cannot be achieved otherwise using the non-prodrug native form of the API.

[0174] The covalently linked conjugate moiety of the drug substance forms non-covalent high affinity interactions that bind to one of six different classes of substances that are formulated as irregularly shaped microparticles: fatty acids, organic molecules that can form keto-enol tautomers, charged phospholipids, charged proteins, nucleic acids, and polysaccharides. The formation of prodrug-complex microparticles optimizes the physicochemical properties of the API that are compatible with multiphase colloidal suspensions, and the prodrug-complex microparticles are miscible in a carrier medium that regulates the release of free unbound prodrug within the multiphase colloidal suspension, allowing for controlled, extended release from the formulated implant when administered into the physiological environment of the eye.

[0175] When the drug substance is a prodrug, an important feature of the prodrug is that the bond linking the API to the conjugate moiety is easily cleaved by enzymatic reaction, catalysis, hydrolysis or other chemical reaction (Figures 28A-28C). Upon cleavage of this bond in the prodrug, the released API retains sufficient biological activity for its mechanism of action (Figures 29A-29C).

[0176] A number of metabolic enzymes have been detected in ocular tissues, including esterases, peptidases, phosphatases, oxime hydrolases, ketone reductases, etc. The linkage to the conjugate moiety in the prodrugs described herein can be configured to achieve specific cleavage by any of these metabolic enzymes.

[0177] The cleavable covalent bond may comprise an ester bond, a hydrazone bond, an imine bond, a disulfide bond, a thioester bond, a thioether bond, a phosphate ester bond, a phosphonate ester bond, a boronate ester bond, an amide bond, a carbamate ester bond, a carboxylate ester bond, a carbonate ester bond or one others known to one skilled in the art of medicinal chemistry.

[0178] Ester prodrugs may be desirable, particularly since ocular tissues contain abundant esterase activity. In some examples of prodrugs, cleavage and release of free API can be assessed in an in vitro release assay, where the prodrug is incubated in a solution containing a carboxylesterase (or other natural or synthetic esterase) and isolated vitreous collected from animals (e.g., pigs, rabbits, etc.) or vitreous collected from human donors at 37 degrees Celsius, 25 degrees Celsius, or other temperatures. Analytical methods, such as HPLC or mass spectrometry, can be used to calculate the amount of free API and unchanged prodrug at various time points after the start of incubation (Figure 28B).

[0179] In some examples, cleavage and release of free API can be assessed in an in vitro release assay, where the prodrug is incubated in media at 37 degrees Celsius, 25 degrees Celsius, or other temperatures. Analytical methods, such as HPLC or mass spectrometry, can be used to calculate the amount of free API and unchanged prodrug at various time points after the start of incubation (Figure 28C).

[0180] In some examples, cleavage and release of free API can be assessed following in vivo injection of the prodrug into the vitreous cavity or periocular tissues of preclinical animal models (e.g., mice, rats, rabbits, pigs, etc.), ocular tissues can be harvested, and analytical methods, such as HPLC or mass spectrometry, can be used to calculate the amount of free API and unchanged prodrug at various time points following in vivo injection (Figures 31A-31D).

[0181] Generally, the conjugate moiety R to which the API is covalently linked is not selected based on its biological activity or mechanism of action on a target. Although not a preferred embodiment, disclosed herein are drug substances that can serve as functionally cleavable conjugate moieties and consist of homo- or hetero-dimers, trimers, multimers of any drug substance linked together, directly or indirectly, to chemical entities that serve as linker moieties.

[0182] As described herein, the API, R', may be covalently linked to a conjugate moiety, R, selected from one of the following five classes of chemicals: a C4-C30 lipid moiety, a C4-C30 straight chain or branched aliphatic moiety, a 2-mer to 30-mer peptide moiety, a PEGylated moiety, or a carbohydrate moiety.

[0183] One class of conjugate moieties is a C4-C30 lipid moiety, with or without a preceding linker moiety that connects the lipid moiety to the API. Lipids are defined herein as organic compounds that are insoluble in water but soluble in organic solvents. Lipids include fatty acids, fatty alcohols, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, prenol lipids (derived from the condensation of isoprene subunits), phospholipids, oils, waxes, and steroids.

[0184] Aliphatic alcohols include tert-butyl alcohol, tert-amyl alcohol, 3-methyl-3-pentanol, 1-heptanol (enanthic alcohol), 1-octanol (caprylic alcohol), 1-nonanol (pelargonic alcohol), 1-decanol (decyl alcohol, capric alcohol), undecyl alcohol (1-undecanol, undecanol, hendecanol), dodecanol (1-dodecanol, lauryl alcohol), tridecyl alcohol (1-tridecanol, tridecanol, isotridecanol), 1-tetradecanol (myristyl alcohol), pentadecyl alcohol (1-pentadecanol, pentadecanol), 1-hexadecanol (cetyl alcohol), cis-9-hexadecen-1-ol (palmitoleic alcohol), It may include one or more of heptadecyl alcohol (1-n-heptadecanol, heptadecanol), 1-octadecanol (stearyl alcohol), 1-octadecenol (oleyl alcohol), 1-nonadecanol (nonadecyl alcohol), 1-eicosanol (arachidyl alcohol), 1-heneicosanol (heneicosyl alcohol), 1-docosanol (behenyl alcohol), cis-13-docosen-1-ol (erucyl alcohol), 1-tetracosanol (lignoceryl alcohol), 1-pentacosanol, 1-hexacosanol (seryl alcohol), 1-heptacosanol, 1-octacosanol (montanyl alcohol, cruityl alcohol), 1-nonacosanol, 1-triacontanol (myricyl alcohol, melissyl alcohol).

[0185] The fatty acids may include one or more of tetradecanoic acid, pentadecanoic acid, (9Z)-hexadecenoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, (9Z,12Z)-octadeca-9,12-dienoic acid, (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid, (6Z,9Z,12Z)-octadeca-6,9,12-trienoic acid, (5E,9E,12E)-octadeca-5,9,12-trienoic acid, (6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraenoic acid, (Z)-octadec-9-enoic acid, (11E)-octadec-11-enoic acid, (E)-octadec-9-enoic acid, nonadecanoic acid, and eicosanoic acid.

[0186] One class of conjugated moieties is the C4-C30 straight chain or branched aliphatic moieties, with or without a preceding linker moiety that connects the aliphatic hydrocarbon to the API. This class includes alkanes, alkenes and alkynes, as well as other hydrocarbon moieties composed of from 4 to about 30 carbons, and can include unbranched, branched and cyclic groups.

[0187] One class of conjugate moieties are peptide moieties, with or without a preceding linker moiety that connects the peptide to the API, which include natural or synthetic amino acid polymers or polypeptide chains having lengths from 2-mer to 30-mer, which are anionic, cationic or neutral in charge and contain homogeneous or heterogeneous amino acid repeats.

[0188] Examples of anionic peptide sequences that can serve as the conjugate moiety R include, but are not limited to, peptides consisting of polyaspartic acid (aspartate), polyglutamic acid (glutamate), poly-(aspartic acid-glutamic acid) or poly-(glutamic acid-aspartic acid) repeats.

[0189] Examples of cationic peptide sequences which may serve as the conjugate moiety R include, but are not limited to, poly-lysine, poly-arginine, poly-histidine, peptides consisting of poly-(lysine-arginine) (or arginine-lysine) repeats, peptides consisting of poly-(lysine-histidine) (or histidine-lysine) repeats, peptides consisting of poly-(arginine-histidine) (or histidine-arginine) repeats, peptides consisting of poly-(lysine-arginine-histidine) repeats, peptides consisting of poly-(lysine-histidine-arginine) repeats, peptides consisting of poly-(arginine-lysine-histidine) repeats, peptides consisting of poly-(arginine-histidine-lysine) repeats, peptides consisting of poly-(histidine-arginine-lysine) repeats, peptides consisting of poly-(histidine-arginine-lysine) repeats, peptides consisting of poly-(histidine-lysine-arginine) repeats.

[0190] The peptide moiety may have one or more pegylation moieties for the addition of polyethylene glycol (PEG) groups. The peptide portion may have one or more sites for modification by the addition of a sugar or carbohydrate molecule, including glycosylation.

[0191] One class of conjugate moieties is a PEGylated compound moiety that includes a polyethylene glycol (PEG) polymer in a linear, branched, Y-shaped or multi-arm geometry, a PEGylated peptide or protein, or a PEGylated succinate, e.g., succinimidyl succinate, with or without a preceding linker moiety that connects the PEGylated compound to an API.

[0192] One class of conjugate moieties are carbohydrate molecule moieties including, but not limited to, monosaccharides or oligosaccharides of 2 to 20 sugars, with or without a preceding linker moiety that connects the carbohydrate to the API. The carbohydrate molecule may include one or more of glucose, galactose, lactose, mannose, ribose, fucose, N-acetylgalactosamine, N-acetylglucosamine, N-acetyleneuraminic acid, or epimers or derivatives of any of these.

[0193] One example of how a prodrug can be incorporated into a multi-phase colloidal suspension is represented by the formula (II): Hd-Arg-DMT-Lys-Phe(-O)-R(II)

[0194] [ka] where R is one of a class of mitochondria-targeting tetrapeptides (MTT) that can be used to form prodrugs that are the product of a condensation or esterification reaction of one of the following five classes of chemicals: a C4-C30 lipid moiety, a C4-C30 straight or branched aliphatic moiety, a 2-mer to 30-mer peptide moiety, a PEGylated moiety, or a carbohydrate moiety, covalently linked via an ester bond to the hydroxyl group of the amino acid at position 4 of MTT.

[0195] In some examples, the prodrug Hd-Arg-DMT-Lys-Phe(-O)-R has the formula Hd-Arg-DMT-Lys-Phe(-O-)-non-polar lipid. The non-polar lipid may include one of several molecules including octadecyl (wherein -OR is derived from stearyl alcohol) (FIG. 27A) or hexadecyl (wherein -OR is derived from palmityl alcohol) or other similar molecules as the conjugated moiety. Prodrugs with non-polar lipids as the conjugated moiety are just one class of prodrugs described herein that may be suitable for lipid-based complexing agents, including complexing agents that are also non-polar lipids. Non-polar lipids are hydrophobic molecules that are solid at temperatures between 27° C. and 50° C., inclusively containing ketoacyl and isoprene groups, including but not limited to fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits).

[0196] One specific example of Hd-Arg-DMT-Lys-Phe(-O)-R includes Hd-Arg-DMT-Lys-Phe(-O)-stearyl (depicted in FIG. 27A), in which Hd-Arg-DMT-Lys-Phe is linked via an ester bond to stearyl alcohol, a member from the group of long-chain saturated fatty alcohols. Upon cleavage of the ester bond, the prodrug Hd-Arg-DMT-Lys-Phe(-O)-stearyl releases native MTT. To demonstrate this, experimentally Hd-Arg-DMT-Lys-Phe(-O)-stearyl was incubated in vitro at 37° C. with carboxylesterase (0.1 μg / mL) to mimic the physiological environment of the eye and the type of esterase that is likely to be abundant within the vitreous body therein. Incubation of Hd-Arg-DMT-Lys-Phe(-O)-stearyl with carboxylesterase produced rapid cleavage of the ester bond of the prodrug, releasing Hd-Arg-DMT-Lys-Phe as evidenced by high performance liquid chromatography (HPLC) analysis and quantification of the Hd-Arg-DMT-Lys-Phe and Hd-Arg-DMT-Lys-Phe(-O)-stearyl prodrugs in solution (Figure 28B). When the Hd-Arg-DMT-Lys-Phe(-O)-stearyl prodrug was added to a phosphate buffered saline solution at 37°C without esterase, the ester bond of the Hd-Arg-DMT-Lys-Phe(-O)-stearyl prodrug was hydrolytically cleaved more slowly (approximately 36 hours) (Figure 28C). Thus, in the physiological system of the eye, the covalent bond of the prodrug linking MTT to the inactive conjugate is easily cleaved by enzymatic cleavage or more slowly by hydrolysis, releasing the active MTT.

[0197] Furthermore, upon cleavage of the covalent bond of the drug substance, the API, the native MTT peptide, retains its biological activity for treating mitochondrial dysfunction. For example, as depicted in Figures 29A-29C, in an in vitro cell culture model of dry AMD, Hd-Arg-DMT-Lys-Phe(-O)-stearyl (5 μM) was added to RPE cells (possessing endogenous esterases) exhibiting mitochondrial dysfunction induced by exposure to hydroquinone (HQ). Hd-Arg-DMT-Lys-Phe(-O)-stearyl efficiently abolished HQ-induced mitochondrial dysfunction in RPE cells (as depicted by cellular flavoprotein-autofluorescence) with an efficacy equivalent to treatment with Hd-Arg-DMT-Lys-Phe native peptide (5 μM). Hd-Arg-DMT-Lys-Phe(-O)-stearyl was also pre-incubated with carboxylesterase (0.1 μg / mL) in another medium. The collected medium (5 μM) containing the cleaved Hd-Arg-DMT-Lys-Phe was added to this RPE cell model of mitochondrial dysfunction and was similarly effective, with equal potency to the Hd-Arg-DMT-Lys-Phe native peptide for reversing RPE mitochondrial dysfunction. Thus, these studies confirm that the active API cleaved from the prodrug retains its essential and unmodified biological activity for treating mitochondrial dysfunction.

[0198] In some instances, the conjugate moiety may combine elements from two or more of these classes and may serve as a multimeric linker moiety covalently linking multiple molecules of the API to form dimers and / or multimers. Such linkers capable of generating dimers or multimers of mitochondrial targeting peptides may be referred to as "multimerization domains."

[0199] The prodrug having a multimerization domain has the formula (III): (R') n -R(III) where R is a linker or multimerization domain that is covalently linked to multiple APIs R' to form API dimers or multimers, and n is equal to 2 to about 100. Examples include PEG polymers, polyvinyl alcohol (PVA) polymers, or polypeptides, where the linker conjugate moiety R is covalently linked to two or more molecules of API R' to form dimers, trimers, multimers, etc. In some cases, the multimerization domain has an alcohol, i.e., multiple "-OH" groups, to which the API units R' are attached. In this setting, multiple APIs covalently linked (e.g., via an ester or another dynamic covalent bond) to a multimerization domain can be referred to as an API multimer.

[0200] One example of such a prodrug multimer has the formula:

[0201] [ka] where "n" is a number including the PVA polymer.

[0202] The dispersion medium of a pharmaceutical agent multi-phase colloidal suspension is defined herein as a hydrophobic liquid in which the pharmaceutical agent and particulate complexing agent are mixed to form a stable multi-phase colloidal suspension. The criteria that define a stable multi-phase colloidal suspension include a homogeneous mixture and distribution of drug substance-complex microparticles without settling, separation or dissociation of the microparticles after exposure to the physiological environment of the eye in vitro (i.e., buffered saline at 37° C., vitreous enzymes, dilute serum) or in vivo when injected into the eye, over a pre-specified duration of the implant's life. Stability also depends on the relative percentage of drug substance-complex microparticles to oil (weight to weight), as well as the size and mass of the microparticles.

[0203] The methods and compositions described herein describe new and previously unappreciated properties of certain oils that enable them to act as effective dispersion media. These include hydrophobicity, high initial viscosity, and other properties that enable them to form stable multi-phase colloidal suspensions when combined with drug substance-complex microparticles.

[0204] Four classes of oils that meet these criteria for a dispersion medium include saturated fatty acid methyl esters, unsaturated fatty acid methyl esters, saturated fatty acid ethyl esters, or unsaturated fatty acid ethyl esters. The dispersion medium can be an individual oil from one of these classes, or it can be designed as a mixture of oils with different viscosity values ​​that are specifically designed and blended to achieve the desired goal of a stable colloidal suspension.

[0205] Saturated fatty acid methyl esters that may serve as dispersion media include methyl acetate, methyl propionate, methyl butyrate, methyl pentanoate, methyl hexanoate, methyl heptanoate, methyl octanoate, methyl nonanoate, methyl decanoate, methyl undecanoate, methyl dodecanoate (methyl laurate) (Figures 12A-12F), methyl tridecanoate, methyl tetradecanoate, methyl 9(Z)-tetradecenoate, methyl pentadecanoate, methyl hexadecanoate, methyl heptadecanoate, methyl octadecenoate, methyl nonadecanoate, methyl eicosanoate, methyl heneicosanoate, methyl docosanoate, methyl tricosanoate, and the like.

[0206] Unsaturated fatty acid methyl esters which may serve as dispersion media include methyl 10-undecenoate, methyl 11-dodecenoate, methyl 12-tridecenoate, methyl 9(E)-tetradecenoate, methyl 10(Z)-pentadecenoate, methyl 10(E)-pentadecenoate, methyl 14-pentadecenoate, methyl 9(Z)-hexadecenoate, methyl 9(E)-hexadecenoate, methyl 6(Z)-hexadecenoate, methyl 7(Z))-hexadecenoate, methyl 11(Z)-hexadecenoate.

[0207] Saturated fatty acid ethyl esters which may serve as dispersion media include ethyl acetate, ethyl propionate, ethyl butyrate, ethyl pentanoate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl undecanoate, ethyl dodecanoate (ethyl laurate), ethyl tridecanoate, ethyl tetradecanoate, ethyl 9(Z)-tetradecenoate, ethyl pentadecanoate, ethyl hexadecanoate, ethyl heptadecanoate, ethyl octadecenoate, ethyl nonadecanoate, ethyl eicosanoate, ethyl heneicosanoate, ethyl docosanoate, and ethyl tricosanoate.

[0208] Unsaturated fatty acid ethyl esters which may serve as dispersion media include ethyl 10-undecenoate, ethyl 11-dodecenoate, ethyl 12-tridecenoate, ethyl 9(E)-tetradecenoate, ethyl 10(Z)-pentadecenoate, ethyl 10(E)-pentadecenoate, ethyl 14-pentadecenoate, ethyl 9(Z)-hexadecenoate, ethyl 9(E)-hexadecenoate, ethyl 6(Z)-hexadecenoate, ethyl 7(Z)-hexadecenoate, ethyl 11(Z)-hexadecenoate.

[0209] In contrast, certain other oils, as well as viscous substances including silicone oils, viscous gelatins and viscous proteoglycans (Figures 10A-10F, 11A-11F, 13A-13F and 14A-14F), fail to form stable multiphase colloidal suspensions or rapidly decompensate when exposed to the physiological ocular microenvironment (e.g., 37°C, buffered saline, vitreous enzymes, dilute serum) or in vivo when injected into the eye.

[0210] Complexation of the drug substance to the particulate complexing agent within the dispersion medium serves to limit the release of free drug substance into the dispersion medium: the dispersion medium restricts water access to the drug substance-complex particulates while free, unbound drug substance is free to diffuse within the dispersion medium; the dispersion medium does not retain free, unbound drug, which may diffuse out of the multi-phase colloidal suspension.

[0211] This complex formation-based XRDDS feature is clearly different from the established prior art of XRDDS for ophthalmic drug delivery. The support vehicle is a liquid or semi-solid material that is selected based on the physicochemical properties of the vehicle for interaction with the drug substance in a manner that restricts or limits the release from the support vehicle. Examples include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, viscous gelatin, hydrogels, and viscous chondroitin sulfate. Support vehicle-based XRDDS does not require any stable dispersion of the drug substance-complex microparticles, and drug release is determined by the interaction of the support vehicle with the drug substance, and the support vehicle retards or slows the diffusion from the vehicle into the physiological environment of the eye. These properties are different from the preferred embodiment of the drug substance in multi-phase colloidal suspension XRDDS, where the drug substance-complex microparticles are stably dispersed without settling or migration, and there is no need for a dispersion medium to retard or slow the diffusion of the drug substance from the implant.

[0212] Carrier-based XRDDS represents a strategy of passively releasing bioerodible formulations. Carrier-based XRDDS are designed to physically entrap the drug substance in a particular carrier, but then the system must degrade via interactions with tissue, rather than mechanisms inherent in XRDDS, to release the free drug substance. In some embodiments, the carrier formulation comprises a single device that compartmentalizes the drug substance from the tissue. Examples include, but are not limited to, polymer-based rods or other shapes (drugs entrapped in chemicals are extruded into rods or molded into different shapes), photopolymerized or photocrosslinked block polymers made of PLGA, and other crosslinkable substrates with drug substances entrapped within the polymer and injectable viscous polymers or polymer-based rods or other shapes, polymer-based microparticles (requiring chemical covalent crosslinking of small block polymers to entrap the drug), and liposomes (phospholipids in water emulsions) that have been sonicated to entrap the drug, all of which can be used to formulate the drug substance. A common feature of all carrier-based systems is that the drug substance is entrapped within the carrier material, and as the carrier degrades, dissolves, or otherwise disintegrates, the free drug substance is released into the tissue. This may require chemical or enzymatic reactions afforded by the tissue microenvironment. Furthermore, defects in the carrier system during degradation allow access to water from the microenvironment, which further enhances the release of the drug substance. Carrier-based systems differ from multiphase colloidal suspensions, which have a hydrophobic dispersion medium, preventing water from entering the system. Furthermore, in multiphase colloidal suspensions, the drug substance is released from the implant, eliminating the need for the system to degrade via interaction with the tissue. The release kinetics are not determined by the drug entrapped in the multiphase colloidal suspension.

[0213] Thus, the present multi-phase colloidal suspensions differ from previously conceived and designed systems, such as those based on retention vehicles and carriers, because they instead utilize a complexation system chemistry specific to sustained release drug delivery to the eye. The present systems use the complexation of drug onto one or more complexing agents in a bioerodible format or formulation as a method to limit the unbound free drug available for release and to modulate the kinetics of drug release in ocular tissues.

[0214] Drug substance multi-phase colloidal suspensions can be designed by a specific process to meet pre-specified drug substance release rates and amounts by varying the ratio and amount of different drug substance-complex microparticles with different Kd and binding capacities (Figures 2A-2E). The Kd property is a measure of the affinity of the drug substance for a given complexing agent and is defined as the unbound-bound fraction of drug substance to drug substance-complex microparticles in a given dispersion medium. Specific Kd values ​​can be measured by specified release assays as described herein (Figures 15, 18, 20, 24). The binding capacity property is defined as the maximum amount of drug that will bind to a known amount of complexing agent.

[0215] The release of drug substance from the implant is determined in part by the unbound fraction in the dispersion medium, which is in turn determined in part by the Kd and binding capacity values ​​for the various drug substance-complexed microparticles. Knowledge of the Kd and binding capacity allows the selection of specific combinations of different prodrug-complexing agent microparticles to modulate the unbound fraction of drug in the dispersion medium over time and thus achieve a pre-specified release kinetics profile (Figures 2A-2E and 9). The inclusion of more than one complexing agent in the multi-phase colloidal suspension can be used to modulate the unbound fraction of drug in the dispersion medium over time and thus the release kinetics of the system.

[0216] For example, the addition of drug substance-complex microparticles with high binding capacity and high Kd, indicating low affinity of the drug substance to the complex microparticles, can be used to create a short-term increase in the rate of release, or an initial burst. The addition of drug substance-complex microparticles with high binding capacity and moderate Kd, indicating moderate affinity of the drug substance to the complex microparticles, can be used to create a longer-term lower release rate, extending the duration of drug substance release from the implant. A combination of these two types of drug substance microparticles can be selected and mixed in the desired ratio and concentration to create an implant with a biphasic release kinetics of the drug substance from the implant (Figure 3B). An implant with this release kinetic profile can be useful for diseases that require a "loading" phase to treat and reverse established disease symptoms, while a second "steady-state" phase can be effective in preventing the onset of new or recurrent disease.

[0217] In another example, the addition of drug substance-conjugate microparticles with high binding capacity and high Kd, indicating low affinity of the drug substance to the conjugate microparticles, can be used to create a short-term increase in the rate of release, or an initial burst. The addition of drug substance-conjugate microparticles with high binding capacity and moderate Kd, indicating moderate affinity of the drug substance to the conjugate microparticles, can be used to create a longer-term lower release rate, extending the duration of drug substance release from the implant. The addition of drug substance-conjugate microparticles with high binding capacity and low Kd, indicating high affinity of the drug substance to the conjugate microparticles, can slow the release and create a later burst in the life of the implant. A combination of these three types of drug substance microparticles can be selected and mixed in the desired ratios and concentrations to achieve the creation of an implant with a three-phase release kinetics of the drug substance from the implant (Figure 3C). Implants with this release kinetic profile may be useful for diseases requiring a "loading" phase to treat and reverse established disease pathology, while a second "steady state" phase may be effective in preventing the onset of new or recurrent disease, and a third phase "late burst" may be useful for diseases where reduced efficacy of the drug or reduced target response to the drug occurs later in the life of the implant due to tachyphylaxis or other mechanisms mediating downregulation of the drug target or reduced responsiveness to the drug substance.

[0218] In such instances, the combined effect of a combination of two or more drug substance-complex microparticles incorporated in a selected dispersion medium is the release of drug substance in two or more phases based on the integral of the release rates from the individual drug-complexant microparticle components incorporated and dispersed in the drug substance multi-phase colloidal suspension.

[0219] For example, Figure 2 illustrates the theoretical basis for designing and constructing an extended release drug delivery system (XRDDS) implant that produces a desired drug release kinetic profile for a drug substance. First, a theoretical pharmacokinetic release curve (i.e., target release profile) is designed to represent a desired initial burst phase and a subsequent steady-state release phase, which in this depiction is linearized by log transformation, to obtain the desired daily release rate, total delivery duration, and drug payload in the final formulation. An iterative process is performed to identify specific member compounds from two or three different classes of complexing agents that are expected to form non-covalent interactions with the drug substance based on the physicochemical properties of the drug substance. Each drug substance-complex microparticle is first combined in an initial amount and ratio, and the drug-complex microparticles are then mixed and incorporated into the desired dispersion medium. The drug substance multi-phase colloidal suspension was used as the "sink" condition and two properties of the drug substance-conjugate microparticles were measured: Kd (fraction unbound - bound) at days 1, 3, 7, 14 and 21 (a good indicator of burst and general binding affinity); and release kinetics (% of initial payload of drug released over time), where Kd1 corresponds to drug substance-conjugate 1 and Kd2 corresponds to drug substance-conjugate 2.

[0220] Curve fitting is then applied to the release curve of each drug-conjugate and the linearized curves are then interpreted to determine the correct combination (of two or three specific drug-conjugate pairs) that results in release kinetics that fit the predetermined desired composite target product profile.

[0221] This "theoretically designed" formulation, containing a combination of two or three drug substance-complex microparticles, is then compounded and tested for actual release kinetics, as shown in Figure 2. If necessary, the ratio of the two to three selected drug substance-complex microparticles can be iteratively readjusted until the final release kinetics meets the predetermined target product release profile.

[0222] In some examples, when the drug substance is a prodrug, in the second or third drug substance-conjugate microparticle, the bioactive agent may be covalently linked to a different conjugate moiety to form a different prodrug structure, and the complexing agent may be different from the first, and the Kd values ​​are different, and the Kd1 and Kd2 of the drug substance-conjugate microparticle are based on both the different conjugate moieties and the different complexing agents between the pairs.

[0223] Alternatively, in some instances, where the drug substance is a prodrug, the conjugate moieties of the prodrug may be different between the first and second drug-conjugate pairs, but the complexing agents may be the same, with the Kd values ​​being different and the Kd1 and Kd2 of the drug-conjugate pairs being based on the different conjugate moieties between the pairs.

[0224] The composite extended release drug delivery system is designed and customized for the physicochemical properties of the drug substance that regulate the release of the free drug substance from the system into the tissue. The actual release kinetics achieved by the drug substance multi-phase colloidal suspension was measured at in vivo vitreous concentrations below EC 50 EC 50 reflects the concentration of a drug substance that achieves 50% of the therapeutic effect of the maximum response for a given mechanism of action of the drug substance.

[0225] In formulations of drug substance multiphasic colloidal suspensions with biphasic release kinetics, the concentration of drug substance in the vitreous is increased during the initial burst phase by EC 50 (i.e., the drug concentration required to achieve 50% of the maximal effect), followed by a second (steady-state) phase where the EC 50 and the pre-specified release kinetics and desired duration of drug release have been achieved through the specific design and use of various drug substance-complex microparticles in the multi-phase colloidal suspensions described herein.

[0226] The pharmaceutical multi-phase colloidal suspension formulation may be delivered as one of three different implant formats including a flowable bolus implant, an erodible or non-bioerodible tube implant filled with the pharmaceutical multi-phase colloidal suspension, or a solid mold of the pharmaceutical multi-phase colloidal suspension made to a specific size and shape, dried and hardened, and configured for implant therapy (Figures 33A-33D). In some instances, the tube may itself be formed of an extended release drug delivery system. In other instances, the tube may be made of a bioerodible polymer that is compatible with ocular tissue (e.g., poly(lactic-co-glycolic acid, PLGA). In some instances, the tube may have one or both ends open to release the mitochondrial targeted extended release compound.

[0227] Any of these formulations can be injected intraocularly and periocularly, i.e., into the vitreous humor, aqueous humor, suprachoroidal space, subretinal, subconjunctival, sub-Tenon's capsule or orbital tissues to produce sustained release of therapeutic levels of active pharmaceutical ingredient in ocular tissues for desired durations (1 to 12 months) for the treatment of various diseases and disorders.

[0228] As a versatile extended release drug delivery system (XRDDS), the multi-phase colloidal suspension described herein can incorporate a wide variety of drug substances that directly form non-covalent complexes with the particulate complexing agent, as well as a wide variety of prodrugs consisting of an active ingredient (API) linked via a cleavable covalent bond to a conjugate moiety, which in turn can form a non-covalent complex with the particulate complexing agent. Specifically, the multi-phase colloidal suspension can incorporate a variety of hydrophobic and hydrophilic chemicals, small polypeptides, proteins, aptamers, other nucleic acid drugs, and other compounds.

[0229] Several examples are discussed herein to demonstrate the principles of complex formation for drug delivery. For example, fluorescently labeled cationic small molecules were mixed with known amounts of selected individual complexing agents (Figures 4F, 5F, 6F, 7F). The various fluorescently labeled small molecule-complexed microparticles were then mixed in appropriate dispersion media and visualized under fluorescence microscopy. Using this approach, fluorescently labeled small molecules were observed to form fluorescently labeled small molecule-complexed microparticles with several different complexing agents.

[0230] In another example, the tetrapeptide Hd-Arg-DMT-Lys-Phe was fluorescently labeled with fluorescein isothiocyanate (FITC) and mixed with known amounts of selected individual complexing agents (Figures 4B, 5B, 6B, 7B). The various fluorescently labeled small molecule-complexed microparticles were then mixed in appropriate dispersion media and visualized under direct fluorescence microscopy. Using this approach, FITC-labeled Hd-Arg-DMT-Lys-Phe did not produce visible drug-complexed microparticles when mixed with different complexing agents (e.g., magnesium stearate, albumin).

[0231] In another example, the same tetrapeptide Hd-Arg-DMT-Lys-Phe was linked to stearyl alcohol by an ester bond to form the prodrug Hd-Arg-DMT-Lys-Phe(O)-stearyl. The prodrug Hd-Arg-DMT-Lys-Phe(O)-stearyl was fluorescently labeled with FITC and mixed with different complexing agents (Figures 4C, 5C, 6C, 7C). The resulting mixture was then visualized under direct fluorescence microscopy. Using this approach, we observed that FITC-labeled Hd-Arg-DMT-Lys-Phe(O)-stearyl (the tetrapeptide is labeled with FITC) formed drug-complex microparticles with several different complexing agents: magnesium stearate (as previously described and expected); the large charged carrier protein albumin; and the large cyclic carbohydrate molecule cyclodextran. In contrast, FITC-labeled Hd-Arg-DMT-Lys-Phe(O)-stearyl was not observed to consistently form drug-complexed microparticles with silica microbeads (Figure 8C), indicating that the process of complexation and drug-complexed microparticle formation is highly dependent on favorable noncovalent interactions between the drug and the complexing agent.

[0232] Since only the Hd-Arg-DMT-Lys-Phe(O)-stearyl prodrug with the conjugated moiety formed drug-conjugated microparticles, it is speculated that the conjugation was mediated by the conjugated moiety of the prodrug. To assess this, FITC-labeled Hd-Arg-DMT-Lys-Phe(O)-stearyl (the tetrapeptide is labeled with FITC) mixed with the complexing agent was treated with an aqueous solution of carboxylesterase (0.1 μg / mL) to hydrolyze the ester bond of the prodrug and release the fluorescent peptide (Figures 4D, 5D, 6D, 7D). The conjugated microparticles were no longer fluorescently labeled by microscopy, confirming that the conjugation of the prodrug was specifically mediated by its conjugated moiety, validating the concept of using a prodrug with a compatible conjugated moiety to mediate conjugation.

[0233] Furthermore, as described herein, the formation of drug-complex microparticles in which the complexing agent has a high affinity for the drug can be experimentally quantified and verified. For example, the prodrug Hd-Arg-DMT-Lys-Phe(O)-stearyl was mixed with a known amount of a selected individual complexing agent (FIG. 24). The Hd-Arg-DMT-Lys-Phe(O)-stearyl-complexing agent mixture was then added to an appropriate carrier medium (in this case methyl laurate) and centrifuged to "pull down" or separate the Hd-Arg-DMT-Lys-Phe(O)-stearyl bound to the complexing agent from the unbound prodrug present in the carrier medium. HPLC analysis of the microparticles and dispersion medium pulled down from the Hd-Arg-DMT-Lys-Phe(O)-stearyl content determined the fraction of prodrug bound to the complexing agent, as well as calculation of Kd values, which are coefficients of binding versus non-binding, and the binding capacity of the prodrug-complexing agent microparticles (Figure 24). Using this type of assay, Kd values ​​and binding capacities can be generated for a particular prodrug-complexing agent pair in a selected dispersion medium (see Figure 24).

[0234] Thus, in some instances, the formation of a prodrug substantially alters the physicochemical properties of the API, allowing for optimization of its suitability for complexation and formulation in multiphase colloidal suspensions. The API Hd-Arg-DMT-Lys-Phe is highly hydrophilic and, as noted above, was not miscible with the complexing agent to produce visible drug-complex microparticles. Linkage to stearyl alcohol via an ester bond produced the prodrug Hd-Arg-DMT-Lys-Phe(O)-stearyl, which is highly hydrophobic compared to the unmodified API. Furthermore, the high affinity interaction between the hydrophobic long chain aliphatic alcohol of the conjugate portion of this MTT-prodrug and the microparticle complexing agent serves to bind the MTT-prodrug, limiting the amount of free unbound MTT-prodrug available for release from the dispersion medium in which the MTT-prodrug-complex microparticles are dispersed (Figure 25B).

[0235] Another specific example of a prodrug comprises Hd-Arg-DMT-Lys-Phe(O)-tri-arginine (tri-Arg) (depicted in FIG. 27C), in which Hd-Arg-DMT-Lys-Phe is linked via an ester bond to a positively charged peptide conjugate moiety that is an arginine trimer / tripeptide that readily forms a non-covalent complex with a negatively charged microparticle complexing agent to form the MTT-prodrug-complexed microparticle. The high affinity interaction between the positive conjugate moiety of the MTT-prodrug and the negative charge of the microparticle complexing agent serves to bind the MTT-tri-Arg prodrug and limits the amount of free unbound MTT-prodrug available for release from the dispersion medium in which the MTT-prodrug-complexed microparticles are dispersed.

[0236] Another specific example of a prodrug includes Hd-Arg-DMT-Lys-Phe(O)-tri-glutamate (triGlu) (depicted in FIG. 27B), in which Hd-Arg-DMT-Lys-Phe is linked via an ester bond to a negatively charged peptide conjugate moiety that is a glutamate trimer / tripeptide that readily forms a non-covalent complex with a positively charged microparticle complexing agent to form the MTT-prodrug-complex microparticle. The high affinity interaction between the negatively charged conjugate moiety of the MTT-prodrug and the positive charge of the microparticle complexing agent serves to bind the MTT-triGlu prodrug, limiting the amount of free unbound MTT-prodrug available for release from the dispersion medium in which the MTT-prodrug-complex microparticles are dispersed.

[0237] In an example where the conjugate moiety of the EY005-prodrug is a pegylated peptide, e.g., EY005-polyethylene glycol (PEG) (Figure 27D), the complexing agent may form a non-covalent interaction with the PEG or PEGylated conjugate moiety based on its size and charge.

[0238] Several examples are discussed herein to specifically identify and distinguish materials that can (and cannot) act as dispersion media. As used herein, the dispersion medium is defined as a hydrophobic, viscous oil that, when mixed with the drug substance-complex microparticles, can form a stable multiphase colloidal suspension formed in the implant for administration in or around the eye. As used herein, colloidal indicates that the microparticles are uniformly dispersed and stable, thereby indicating that the microparticles will remain dispersed without settling or migration for the duration of the intended life of the implant.

[0239] To better understand these properties and to identify liquid substances that may serve as dispersion media, fluorescent particulate beads of two different sizes, 3 μm (micrometer or micron) and 10 μm, were used as surrogates for drug substance-complex particulates (to facilitate visualization and imaging of the particulates). These fluorescent particulate beads were suspended in various liquids in small shallow cylindrical wells, which were then evaluated by confocal fluorescence microscopy to assess the distribution of the particulate beads and whether any consistent settling of the fluorescent bead particulates occurred, as confirmed by the confocal capability to evaluate the liquid at various depths.

[0240] For example, when fluorescent microparticle beads were mixed in water (FIGS. 10A-10F) and silicone oil (FIGS. 11A-11F), they demonstrated a substantially higher number and density of microparticle beads in the lower level fluid and relatively much fewer beads in the upper liquid. Thus, water and silicone oil did not disperse the microparticles uniformly and colloidal suspensions were not formed.

[0241] In another example, fluorescent microparticle beads were mixed in fatty acid methyl ester (Figures 12A-12F). Confocal microscopy demonstrated uniform distribution of the microparticles regardless of the depth of the liquid, indicating that the uniformly dispersed microparticles of methyl laurate form a multiphase colloidal suspension. Examination of this suspension in contact with the physiological environment of the eye (containing enzymes and proteins typically contained in ocular tissues) demonstrated the stability of the uniform distribution of the microparticles over long periods of time, 1 day, 1 week, and 1 month, without migration within the colloidal suspension.

[0242] In another example, fluorescent microparticle beads were mixed in 2% gelatin (Figures 13A-13F). Confocal microscopy demonstrated uniform distribution of the microparticles regardless of the depth of the liquid, indicating that the microparticles uniformly dispersed in the 2% gelatin formed a multiphase colloidal suspension. However, after being placed in the physiological environment of the eye in vitro (which typically contains enzymes and proteins contained in ocular tissues) (Figures 14A-14F), the distribution of the microparticles within the suspension did not remain stable over time; the microparticles migrated and the 2% gelatin was eroded and destroyed.

[0243] Several examples are discussed herein to demonstrate proof of concept for the formulation and sustained release of various drug substances in multi-phase colloidal suspensions. For example, a formulation of the hydrophobic small molecule fluocinolone acetonide (FA) in a multi-phase colloidal suspension was developed. FA was blended with different microparticle complexing agents to form various FA-complexed microparticle formulations (Figures 15, 16A-16B and 17-17B). The Kd and binding capacity properties for each FA-complexed microparticle in different dispersion media were calculated (data shown for methyl laurate dispersion media). A two-phase kinetic release profile was desired in this example. Based on this, magnesium stearate and tocopherol complexing agents were selected for incorporation into the methyl laurate dispersion media along with FA at specific ratios and concentrations to achieve two-phase release in a bioerodible tube formulation of FA multi-phase colloidal suspension. The formulation was iteratively refined by adjusting the ratio of FA-complexing agent microparticles for a given payload of drug to achieve an initial burst phase release followed by a steady-state release with a duration of release of approximately 6 months.

[0244] In another example, a formulation of the hydrophilic small molecule dexamethasone phosphate (DexPh) in a multiphase colloidal suspension was developed (Figures 18-19). To understand how the physicochemical properties of the drug substance affect their interaction with the complexation properties, DexPh was miscible with the same particulate complexing agents, magnesium stearate and tocopherol, and the dispersion medium selected for the hydrophobic small molecule FA. The formulation of DexPh demonstrated abrupt and excessive release, or "damping," of DexPh. Adding different complexing agents and decreasing the ratio of other complexing agents, for a given payload, altered the kinetic release profile to minimize damping of DexPh and obtain a more desirable sustained release profile, demonstrating the importance of selecting a complexing agent based on favorable non-covalent complexation with the particular drug substance of interest.

[0245] In another example, a formulation of the hydrophilic small molecule sunitinib malate in a multi-phase colloidal suspension was developed. Sunitinib was blended with different microparticle complexing agents to form various sunitinib-complexed microparticle formulations (Figures 20-22). Complexation of sunitinib to the selected complexing agent was visually confirmed by blending and pulling down the sunitinib-complexed microparticles, which was confirmed by the yellowish orange microparticles (sunitinib has an orange coloration). A formulation of sunitinib in a bioerodible tube formulation of sunitinib multi-phase colloidal suspension was designed and manufactured that exhibited biphasic release kinetics. The pharmacokinetics demonstrated a biphasic release with a large initial burst followed by a steady-state release. Varying the ratio of sunitinib-complexed microparticles reduced the amount of drug released during the initial burst and decreased the steady-state release.

[0246] For example, a formulation of the hydrophobic small molecule axitinib in a multi-phase colloidal suspension was developed. Axitinib was mixed with different microparticle complexing agents to form various axitinib-complex microparticle formulations (Figure 23). A formulation with a single-phase kinetic release profile was desired in this example. Based on this, axitinib was mixed with a complexing agent with high binding capacity and low Kd (indicating high affinity) in a selected dispersion medium that was formulated as a bolus implant, which produced a slow release of the formulation with the drug detectable in tissues.

[0247] In another example, a formulation of the prodrug Hd-Arg-DMT-Lys-Phe(O)-Stearyl in a multiphasic colloidal suspension was developed (Figures 24-32). In an in vitro kinetic study, a pilot formulation of the prodrug multiphasic colloidal suspension as a bolus implant achieved zero order (i.e. linear) kinetics of release and a desirable durability of drug release of 3 months, where after release of the prodrug from the multiphasic colloidal suspension, the prodrug was present in the dispersion medium and the free API was present in the in vitro physiological environment (see Figure 30).

[0248] In an in vitro efficacy study, bolus implants of the prodrug multiphasic colloidal suspension were added to endogenous esterases and RPE cell culture models (Figures 31A-31D). Cell culture data demonstrated restoration of the cytoskeleton with approximately 80% improvement at 21 days with abrogation of cellular mitochondrial dysfunction (Figure 31D). The data confirm that prodrugs mixed with complexing agents and incorporated into the dispersion medium to form stable multiphasic colloidal suspensions can produce sustained release of the prodrug at predictable therapeutic levels and that the API remains bioactive upon MTT-prodrug cleavage in the surrounding in vitro physiological environment.

[0249] In vivo kinetic studies using LC / MS analysis demonstrated that high retinal levels (>300ng / g) of MTT-prodrug were sustained for 6 weeks by intravitreal injection of a bolus implant of the prodrug multiphasic colloidal suspension (Hd-Arg-DMT-Lys-Phe(O)-stearyl payload 1mg) in rabbit eyes (Figure 32), confirming good in vivo-in vitro correlation for prodrug release. The recovered bolus had approximately 50% residual payload, indicating that the prodrug bolus implant achieved desirable approximately 90-day implant release kinetics based on zero-order release kinetics.

[0250] Relationship C ss = release rate / clearance and half-life (t 1 / 2 ) can be utilized to calculate the approximate desired daily release rate and drug payload of an extended release drug delivery system implant.

[0251] Furthermore, incorporation of a bioactive tetrapeptide API (without a prodrug) with the same complexing agent and in the same dispersion medium produces excess release, or "dumping," of the bioactive API in vitro (Figure 30). Furthermore, multiphase colloidal suspension bolus formulations of native API administered into the vitreous do not produce detectable tissue levels beyond 21 days (Figure 32), similarly pointing to excess release of the native API in vivo. Furthermore, there is no residual drug in the retrieved bolus, consistent with excess drug release, or "dumping." Thus, incorporation of native unmodified API into a multiphase colloidal suspension is insufficient to produce sustained release and fails to achieve the specifications of an extended release drug delivery system. Importantly, these data confirm and highlight the need for specific interactions between the prodrug construct and the prodrug conjugate moiety and complexing agent that form the drug substance-complex microparticles in some APIs that are not otherwise complexed to achieve controlled and durable release of the active API from the multiphase colloidal suspension XRDDS.

[0252] The pharmaceutical agent multi-phase colloidal suspension formulations, referred to as implants, can be administered intraocularly and periocularly, i.e., into the vitreous humor (Figure 33D), aqueous humor, suprachoroidal space, subretinal, subconjunctival, sub-Tenon's capsule, or orbital tissues to produce sustained release of therapeutic levels of pharmaceutical agent in ocular tissues for a desired duration (1 to 12 months).

[0253] The pharmaceutical multi-phase colloidal suspension formulations can be used to prevent the onset or slow the progression, modify the pathology of disease, prevent vision loss or improve vision, or prevent the onset or ameliorate other destructive or degenerative aspects of ocular conditions and diseases including dry age-related macular degeneration (AMD), wet AMD, diabetic macular edema (DME), retinal vein occlusion (RVO), and inherited retinal degenerations (IRD), retinal degeneration, traumatic injury, ischemic vasculopathy, acquired or hereditary optic neuropathies, glaucoma, endophthalmitis, retinitis, uveitis, inflammatory diseases of the retina and uvea, Fuchs' corneal dystrophy, corneal edema, ocular surface diseases, dry eye diseases, diseases of the conjunctiva, diseases of the periocular tissues, and diseases of the orbit.

[0254] The methods may be used in conjunction with other treatment modalities including inhibition of vascular endothelial growth factor, complement inhibition, or administration of anti-inflammatory drugs, such as corticosteroids. All of the methods and devices described herein, in any combination, may be used to achieve the benefits contemplated and described herein.

[0255] It should be recognized that all combinations of the foregoing concepts, and additional concepts discussed in more detail below, are contemplated as being part of the inventive subject matter disclosed herein (provided such concepts are not mutually inconsistent) and may be used to achieve the benefits described herein.

[0256] The process parameters and sequences of steps described and / or illustrated herein are given by way of example only and may be varied as desired. For example, although the steps illustrated and / or described herein may be shown or discussed in a particular order, these steps do not necessarily have to be performed in the order illustrated or described. The various example methods described and / or illustrated herein may omit one or more of the steps described or illustrated herein or may include additional steps in addition to those disclosed.

[0257] When a feature or element is referred to herein as "on" another feature or element, it may be directly on the other feature or element, or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as "directly on" another feature or element, there are no intervening features or elements. When a feature or element is referred to as "connected," "attached," or "connected" to another feature or element, it is understood that it is directly connected, attached, or connected to the other feature or element, or intervening features or elements may also be present. In contrast, when a feature or element is referred to as "directly connected," "directly attached," or "directly connected" to another feature or element, there are no intervening features or elements. Although described or illustrated with respect to one embodiment, features and elements so described or illustrated may be applied to other embodiments. It may also be recognized by those skilled in the art that a reference to a structure or feature that is located "adjacent" to another feature may have portions that overlap or underlie the adjacent feature.

[0258] The terminology used herein is merely for the purpose of describing the detailed embodiments and is not intended to limit the present invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It is further understood that the terms "comprise" and / or "comprising", as used herein, specify the presence of the stated features, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0259] Spatial relationship terms, such as "below," "below," "lower," "above," "above," and the like, may be used herein for ease of description to describe the relationship of one element or feature to another element(s) or feature(s) as illustrated in the figures. It is understood that spatial relationship terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is inverted, an element described as "below" or "directly below" the other element or feature would be oriented "above" the other element or feature. Thus, the exemplary term "below" may encompass both an upward and downward orientation. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the descriptive terms used herein to describe spatial relationships are to be interpreted accordingly. Similarly, terms such as "upward," "downward," "vertical," "horizontal," and the like are used herein for descriptive purposes only, unless specifically indicated otherwise.

[0260] The terms "first" and "second" may be used herein to describe various features / elements (including steps), but these features / elements should not be limited by these terms unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another. Thus, a first feature / element discussed below could be referred to as a second feature / element, and similarly, a second feature / element discussed below could be referred to as a first feature / element without departing from the teachings of the present invention.

[0261] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" refer to various components that may be jointly employed in methods and articles (e.g., devices and compositions and apparatuses that include methods). For example, the term "comprising" is understood to imply the inclusion of any stated element or step but not the exclusion of any other element or step.

[0262] In general, any of the devices and methods described herein should be understood to be inclusive, however, all or a subset of the components and / or steps may alternatively be excluded and may be expressed as "consisting of" or "consisting essentially of" various components, steps, subsets or substeps.

[0263] As used herein, in the specification and claims, including those used in the examples, and unless expressly specified otherwise, all numbers may be read as if they are preceded by the word "about" or "approximately", even if the term does not appear explicitly. The phrase "about" or "approximately" may be used to describe a magnitude and / or location that indicates that the described value and / or location is within a reasonable expected range of values ​​and / or locations. For example, a numerical value may have a value that is + / -0.1% of the stated value (or range of values), + / -1% of the stated value (or range of values), + / -2% of the stated value (or range of values), + / -5% of the stated value (or range of values), + / -10% of the stated value (or range of values), etc. Any numerical value provided herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Any numerical range recited herein is intended to include all subranges subsumed therein. When a value is disclosed, it is also understood that "less than or equal to" that value, "more than or equal to" that value, and possible ranges between the values ​​are also disclosed, as would be well understood by one of ordinary skill in the art. For example, if a value "X" is disclosed, "less than or equal to" and "more than or equal to X" (e.g., X is a number) are also disclosed. It is also understood that throughout this application, data is presented in several different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood that greater than, greater than, less than, less than, less than, and equal to 10 and 15 are considered to be disclosed, as well as between 10 and 15. It is also understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0264] Although various illustrative embodiments have been described above, any of a number of changes may be made to the various embodiments without departing from the scope of the invention as set forth in the claims. For example, the order in which the various described method steps are performed may often be varied in alternative embodiments, and in other alternative embodiments, one or more method steps may be skipped entirely. Optional features of the various device and system embodiments may be included in some embodiments and not in other embodiments. Thus, the foregoing description is provided primarily for exemplary purposes and should not be construed as limiting the scope of the invention as set forth in the claims.

[0265] The examples and illustrations contained herein are presented for the purpose of illustrating specific embodiments in which the subject matter may be practiced, and are not intended to be limiting. As mentioned, other embodiments are available and can be derived therefrom, such that structural and logical substitutions and changes can be made without departing from the scope of the present disclosure. Such embodiments of the subject matter of the present invention may be referred to herein, individually or collectively, simply by the term "invention", for convenience, and it is not intended to spontaneously limit the scope of this application to a single invention or inventive concept, if more than one is actually disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiment shown. The present disclosure is intended to cover any of the various embodiments, and all adaptations or variations. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those skilled in the art upon reviewing the above description.

Claims

1. A multi-phase colloidal suspension composition comprising a drug substance and one or more complexing agents admixed in a dispersion medium.

2. 1. A composition of a multi-phase colloidal suspension comprising a drug substance and one or more complexing agents admixed in a dispersion medium having a release profile of one or more phases of drug release, wherein the one or more complexing agents are formulated as irregularly shaped microparticles which form drug substance-complex microparticles by non-covalent reversible binding to the drug substance and are one of: fatty acids, organic compounds capable of forming keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids and polysaccharides, and further wherein the dispersion medium is a hydrophobic liquid oil comprising at least one of saturated fatty acid methyl esters, unsaturated fatty acid methyl esters, saturated fatty acid ethyl esters, unsaturated fatty acid ethyl esters.

3. A composition of a multi-phase colloidal suspension comprising a drug substance and one or more complexing agents admixed in a dispersion medium having a release profile of one or more phases of drug release, wherein the one or more complexing agents are formulated as irregularly shaped microparticles that form drug substance-complex microparticles by non-covalent reversible binding to the drug substance, and wherein the drug substance comprises one of a fatty acid, an organic compound capable of forming keto-enol tautomers, a charged phospholipid, a charged protein, a ribonucleic acid, and a polysaccharide, and further wherein the drug substance comprises one of a small molecule, a small polypeptide, a protein, an aptamer, a nucleic acid pharmaceutical, a hydrophobic chemical, and a hydrophilic chemical; and further wherein the dispersion medium is a hydrophobic liquid oil comprising at least one of a saturated fatty acid methyl ester, an unsaturated fatty acid methyl ester, a saturated fatty acid ethyl ester, and an unsaturated fatty acid ethyl ester.

4. 10. The composition of claim 1, wherein the one or more complexing agents are chemicals capable of forming drug substance-complex microparticles by non-covalent reversible binding to the drug substance and formulated as irregularly shaped microparticles that are one of fatty acids, organic compounds capable of forming keto-enol tautomers, charged phospholipids, charged proteins, ribonucleic acids and polysaccharides.

5. One or more complexing agents may be CH3(CH2) ｎ 4. The composition according to any one of claims 1 to 3, which is a fatty acid comprising a carboxylic acid having an aliphatic chain with the chemical formula COOH, where n is equal to 4 to 30, saturated or unsaturated, and a salt or ester, and which comprises one or more of magnesium palmitate, magnesium stearate, calcium palmitate, calcium stearate.

6. 4. The composition of any one of claims 1 to 3, wherein the one or more complexing agents comprises an organic compound capable of forming keto-enol tautomers and undergoing chemical equilibrium between the keto form and the enol form of a ketone or an aldehyde, and is a particulate complexing agent comprising one or more of a phenolic compound, a tocopherol compound, a quinone compound, a ribonucleic acid compound.

7. 4. The composition of any of claims 1 to 3, wherein the one or more complexing agents are charged phospholipids and are particulate complexing agents comprising one or more of anionic phospholipids, lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, positively charged synthetic phospholipids, and DLin-MC3-DMA.

8. 4. The composition of any of claims 1 to 3, wherein the one or more complexing agents are positively or negatively charged proteins and are particulate complexing agents comprising one or more of albumin, synthetic polypeptides, plasma proteins, alpha2-macroglobulin, fibrin and collagen.

9. 4. The composition of any of claims 1 to 3, wherein the one or more complexing agents are particulate complexing agents that are one or more of a biopolymer macromolecule comprising a ribonucleic acid, a nucleotide comprising a 5-carbon sugar, a phosphate group and a nitrogenous base.

10. 4. The composition of any of claims 1 to 3, wherein the one or more complexing agents are polysaccharides comprising long chain polymeric carbohydrates comprising monosaccharide units linked together by glycosidic bonds, and are particulate complexing agents comprising one or more of cyclic polysaccharide molecules, cyclodextrins and inclusion compounds.

11. The composition of any one of claims 1 to 4, wherein the active pharmaceutical ingredient forms a non-covalent complex with a microparticle complexing agent and includes one of a small molecule, a small polypeptide, a protein, an aptamer, a nucleic acid drug, a hydrophobic chemical, and a hydrophilic chemical.

12. 4. The pharmaceutical agent has the formula (I): R'-R(I) where R′ is any active ingredient (API) covalently linked via a cleavable bond to R, a conjugate moiety that forms a non-covalent complex with one of five classes of complexing agents, and the covalent bond linking R′ and R can be removed by enzymatic cleavage, catalysis, hydrolysis or other reaction to provide the free API R′ and the conjugate moiety R, where R is: C4-C30 lipid moiety (fatty acid or fatty alcohol); a C4 to C30 straight or branched aliphatic moiety; a 2-mer to 30-mer peptide portion, a pegylated moiety, or 5. The composition of claim 1, which is a prodrug of a cyclic amine (e.g., a cyclic amine ...

13. 13. The composition of claim 12, wherein the cleavable covalent bond comprises one of an ester bond, a hydrazone bond, an imine bond, a disulfide bond, a thioester bond, a thioether bond, a phosphate ester bond, a phosphonate ester bond, a boronate ester bond, an amide bond, a carbamate ester bond, a carboxylate ester bond, and a carbonate ester bond.

14. The conjugated moiety R may be selected from the group consisting of tert-butyl alcohol, tert-amyl alcohol, 3-methyl-3-pentanol, 1-heptanol (enanthyl alcohol), 1-octanol (capryl alcohol), 1-nonanol (pelargonic alcohol), 1-decanol (decyl alcohol, capric alcohol), undecyl alcohol (1-undecanol, undecanol, hendecanol), dodecanol (1-dodecanol, lauryl alcohol), tridecyl alcohol (1-tridecanol, tridecanol, isotridecanol), 1-tetradecanol (myristyl alcohol), pentadecyl alcohol (1-pentadecanol, pentadecanol), 1-hexadecanol (cetyl alcohol), cis-9-hexadecen-1-ol (palmitoleyl alcohol), hepta 13. The composition of claim 12, wherein the fatty alcohol comprises one or more of the following: decyl alcohol (1-n-heptadecanol, heptadecanol), 1-octadecanol (stearyl alcohol), 1-octadecenol (oleyl alcohol), 1-nonadecanol (nonadecyl alcohol), 1-eicosanol (arachidyl alcohol), 1-heneicosanol (heneicosyl alcohol), 1-docosanol (behenyl alcohol), cis-13-docosen-1-ol (erucyl alcohol), 1-tetracosanol (lignoceryl alcohol), 1-pentacosanol, 1-hexacosanol (seryl alcohol), 1-heptacosanol, 1-octacosanol (montanyl alcohol, cruityl alcohol), 1-nonacosanol, 1-triacontanol (myricyl alcohol, melissyl alcohol).

15. Conjugated moiety R may be selected from the group consisting of tetradecanoic acid, pentadecanoic acid, (9Z)-hexadecenoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, (9Z,12Z)-octadeca-9,12-dienoic acid, (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid, (6Z,9Z,12Z)-octadeca-6,9,12-trienoic acid, 13. The composition of claim 12, wherein the fatty acid comprises one or more of (5E,9E,12E)-octadeca-5,9,12-trienoic acid, (6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraenoic acid, (Z)-octadec-9-enoic acid, (11E)-octadec-11-enoic acid, (E)-octadec-9-enoic acid, nonadecanoic acid, and eicosanoic acid.

16. 13. The composition of claim 12, wherein R is one of anionic, cationic or neutral, with or without a preceding linker moiety, and is a 2-mer to about 30-mer peptide moiety comprising natural or synthetic amino acids including one or more of poly-glutamate, poly-aspartate or a combination of glutamate and aspartate; poly-arginine, poly-lysine, poly-histidine, a combination of arginine and lysine, a combination of arginine and histidine, a combination of histidine and lysine or a combination of arginine, histidine and lysine; the peptide moiety has one or more PEGylation moieties for the addition of polyethylene glycol (PEG) groups; and the peptide moiety has one or more moieties for modification by addition of sugar or carbohydrate molecules including glycosylation.

17. 13. The composition of claim 12, wherein R is one of a polyethylene glycol (PEG) polymer, including linear, branched, Y-shaped or multi-arm geometry PEG polymers, a PEGylated peptide, or a PEGylated succinate.

18. 13. The composition of claim 12, wherein R is a carbohydrate moiety comprising 2 to 20 sugar carbohydrates including one or more of glucose, galactose, lactose, mannose, ribose, fucose, N-acetylgalactosamine, N-acetylglucosamine, N-acetyleneuramic acid, or epimers or derivatives of glucose, galactose, lactose, mannose, ribose, fucose, N-acetylgalactosamine, N-acetylglucosamine, and N-acetyleneuramic acid, with or without a preceding linker moiety.

19. 13. The composition of claim 12, wherein R' is an API, R is a linker or multimerization domain that covalently links multiple APIs to form a prodrug dimer or multimer, n is equal to 2 to about 100, and R is one of PEG, a PEG polymer, polyvinyl alcohol (PVA), or a peptide.

20. 5. The composition of claim 1, wherein the dispersion medium is a liquid oil capable of forming a multi-phase colloidal suspension comprising a hydrophobic oil comprising at least one of saturated fatty acid methyl esters, unsaturated fatty acid methyl esters, saturated fatty acid ethyl esters, and unsaturated fatty acid ethyl esters.

21. 21. The composition of claim 20, wherein the dispersion medium comprises saturated fatty acid methyl esters including one or more of methyl acetate, methyl propionate, methyl butyrate, methyl pentanoate, methyl hexanoate, methyl heptanoate, methyl octanoate, methyl nonanoate, methyl decanoate, methyl undecanoate, methyl dodecanoate (methyl laurate), methyl tridecanoate, methyl tetradecanoate, methyl 9(Z)-tetradecenoate, methyl pentadecanoate, methyl hexadecanoate, methyl heptadecanoate, methyl octadecenoate, methyl nonadecanoate, methyl eicosanoate, methyl heneicosanoate, methyl docosanoate, methyl tricosanoate, and the like.

22. 21. The composition of claim 20, wherein the dispersion medium comprises an unsaturated fatty acid methyl ester comprising one or more of the methyl esters methyl 10-undecenoate, methyl 11-dodecenoate, methyl 12-tridecenoate, methyl 9(E)-tetradecenoate, methyl 10(Z)-pentadecenoate, methyl 10(E)-pentadecenoate, methyl 14-pentadecenoate, methyl 9(Z)-hexadecenoate, methyl 9(E)-hexadecenoate, methyl 6(Z)-hexadecenoate, methyl 7(Z))-hexadecenoate, methyl 11(Z)-hexadecenoate.

23. 21. The composition of claim 20, wherein the dispersion medium comprises a saturated fatty acid ethyl ester comprising one or more of ethyl acetate, ethyl propionate, ethyl butyrate, ethyl pentanoate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, ethyl undecanoate, ethyl dodecanoate (ethyl laurate), ethyl tridecanoate, ethyl tetradecanoate, ethyl 9(Z)-tetradecenoate, ethyl pentadecanoate, ethyl hexadecanoate, ethyl heptadecanoate, ethyl octadecenoate, ethyl nonadecanoate, ethyl eicosanoate, ethyl heneicosanoate, ethyl docosanoate, ethyl tricosanoate.

24. 21. The composition of claim 20, wherein the dispersion medium comprises an unsaturated fatty acid ethyl ester comprising one or more of ethyl 10-undecenoate, ethyl 11-dodecenoate, ethyl 12-tridecenoate, ethyl 9(E)-tetradecenoate, ethyl 10(Z)-pentadecenoate, ethyl 10(E)-pentadecenoate, ethyl 14-pentadecenoate, ethyl 9(Z)-hexadecenoate, ethyl 9(E)-hexadecenoate, ethyl 6(Z)-hexadecenoate, ethyl 7(Z))-hexadecenoate, ethyl 11(Z)-hexadecenoate.

25. A method for designing a composition of a multi-phase colloidal suspension comprising a drug substance and one or more complexing agents mixed in a dispersion medium to match a pre-specified drug substance release rate and amount, the method comprising the steps of varying the ratio and amount of different drug substance-complex microparticles having different binding capacities and Kds.

26. 26. The method of claim 25, wherein varying the ratio and amount of different drug substance-complex microparticles having different binding capacities and Kd comprises adding drug substance-complex microparticles with a high binding capacity and a high Kd, indicating a low affinity of the drug substance for the microparticle complexing agent, to create a short-term increase in the rate of release, or an initial burst.

27. 26. The method of claim 25, further comprising extending the duration of release of the drug substance from the implant by adding drug substance-complex microparticles that have a high binding capacity and a low Kd, indicating a high affinity of the drug substance for the microparticle complexing agent.

28. 26. The method of claim 25, further comprising formulating the drug substance-multiphase colloidal suspension for ocular injection as one of a flowable bolus implant, an erodible or non-bioerodible tube implant filled with the drug substance-multiphase colloidal suspension, or a drug substance-multiphase colloidal suspension made in a solid mold of a particular size and shape and configured for implant therapy.

29. A method of treating ocular disorders and diseases, wherein a drug substance-multiphasic colloidal suspension is administered in and around the eye into one of the following tissue compartments: vitreous humor, aqueous humor, suprachoroidal space, subretinal, subconjunctival, sub-Tenon's capsule, or orbital tissue to produce sustained release of therapeutic levels of drug substance in ocular tissues over one or more months.

30. 1. A method of treating vision loss in a subject by intravitreal or periocular injection of a formulation of an extended release drug delivery system that produces highly sustained retinal and retinal pigment epithelium (RPE) tissue levels of an active drug, comprising: delivering a prodrug drug substance in combination with a long-term release drug delivery system into the subject's eye at the start of treatment; cleaving the prodrug by the action of an esterase or bioactive enzyme in the subject's eye to release the active ingredient (API) of the prodrug into the eye at a burst phase release rate during a first phase; and cleaving the prodrug by the action of an esterase or bioactive enzyme to release the API into the eye at a steady state administration rate during a second phase, wherein the burst phase rate exceeds the steady state release rate, and wherein the first phase extends for about 2-6 weeks from the start of treatment and the subsequent phase extends for one or more months from the end of the first phase.

31. 1. A method of preventing the onset or slowing the progression of atrophy of the retinal neurosensory epithelium and / or retinal pigment epithelium (RPE) in a subject by intravitreal or periocular injection of a formulation of an extended release drug delivery system that produces highly sustained retinal and RPE tissue levels of active drug, comprising: delivering a drug substance, which is a prodrug of an active ingredient (API) in combination with a long-term release drug delivery system, into the eye of a subject at the start of treatment; cleaving the prodrug by the action of an esterase or bioactive enzyme in the subject's eye to release the API into the eye at a burst phase release rate during a first phase; and cleaving the prodrug by the action of an esterase or bioactive enzyme to release the API into the eye at a steady state administration rate during a second phase, wherein the burst phase rate exceeds the steady state release rate, and wherein the first phase extends for about 2-6 weeks from the start of treatment and the subsequent phase extends for one or more months from the end of the first phase.

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