composition

Abstract

To provide a pharmaceutical composition comprising a peroxisome proliferator activated receptor (PPAR) modulator and a polymeric nanocarrier component.SOLUTION: A polymeric nanocarrier component according to the present invention is capable of solubilizing the PPAR modulator in an aqueous medium. In the polymeric nanocarrier component, a micelle is to form a non-ionic activator. Uses of the same in therapy are also provided.SELECTED DRAWING: Figure 18

Description

[Technical field]

[0001] The present invention relates to novel formulations of peroxisome proliferator-activated receptor (PPAR) modulators and their use in therapy. The present invention also relates to the use of acyl homoserine lactones (PPAR modulators) to enhance drug delivery by non-injection or topical routes of administration. [Background technology]

[0002] In one aspect, the present invention relates to the treatment or prevention of pulmonary arterial hypertension, cancer, and anti-fibrotic diseases, as well as neurodegenerative conditions, retinal diseases, and brain disorders. Neurodegenerative conditions affect various parts of the central and peripheral nervous system, and include Parkinson's disease, Alzheimer's disease, and Huntington's chorea. Retinal diseases can include retinal degenerative conditions, such as glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, and optic neuritis. Brain disorders can include traumatic brain injury, stroke, cerebral palsy (e.g., as caused by neonatal hypoxia), and cancer (including brain tumors). Summary of the Invention [Problem to be solved by the invention]

[0003] Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. These receptors were identified in rodent hepatocytes in 1990 and were named for their ability to induce peroxisome proliferation. PPARs directly interact with PPAR gamma coactivators 1-α (PGC-1α) and 1-β (PGC-1β) in regulating mitochondrial biogenesis by sensing and controlling lipid homeostasis. These receptors also regulate the expression of genes encoding uncoupling proteins (UCPs). UCPs are transporters of the inner mitochondrial membrane responsible for controlling heat production, ROS production, and oxidative function. By binding to specific sequences in the promoter regions of target genes, PPARs can regulate gene transcription. Activated PPARs can also directly inhibit transcription factors. These functions allow PPARs to promote lipid consumption in the production of ATP when there is a cellular demand for energy. Many PPAR modulators have poor aqueous solubility and / or poor biological activity, which can limit their therapeutic use.

[0004] Rosiglitazone is an exogenous PPAR-γ agonist that belongs to the thiazolidinedione class and acts as an insulin sensitizer. Initially used to combat insulin resistance in type 2 diabetes, rosiglitazone has recently shown promise as a treatment for animal models of PD (Normando et al., 2016). Rosiglitazone therapy has been reported to promote anti-inflammatory responses by attenuating microglial activation, proinflammatory cytokine release, oxidative stress, astrogliosis, and reversible inhibition of monoamine oxidase (a key enzyme for dopamine metabolism). Clinical trials of thiazolidinedione therapy for the treatment of PD have so far shown mixed outcomes, and administration of these drugs has not been reported to slow disease progression.

[0005] Recently, curcumin, resveratrol, and acyl homoserine lactones (AHLs) have each been reported to bind PPARs, and this interaction has been found to be involved in their biological activities and / or therapeutic effects, including attenuating microglial activation, release of proinflammatory cytokines, and oxidative stress, and promoting anti-inflammatory responses. AHLs have been reported to compete with RSG for binding to the same site of PPAR-γ (Jahoor et al., 2008; Cooley et al., 2010). Like other PPAR modulators, these compounds have poor aqueous solubility and / or bioavailability.

[0006] Thus, there is a need to provide improved formulations for PPAR modulators to improve bioavailability and / or bioactivity. [Means for solving the problem]

[0007] In a first aspect, the present invention provides a pharmaceutical composition comprising a peroxisome proliferator-activated receptor (PPAR) modulator incorporated within a polymeric nanocarrier component. The polymeric nanocarrier component is capable of solubilizing the PPAR modulator in an aqueous medium. PPAR modulators are known to have low solubility in water, requiring the use of a solvent such as dimethyl sulfoxide (DMSO). However, the inventors of the present invention have found that polymeric nanocarriers can be used to solubilize PPAR modulators at physiological pH (about pH 4 to about pH 8). Thus, the compositions of the present invention can be prepared without the need to use potentially harmful solvents, such as dimethylsulfoxide (DMSO). Surprisingly, the compositions of the present invention have been found to have a neuroprotective effect in an in vivo central nervous system injury model. Furthermore, systemic administration of this type of composition has been found to have a neuroprotective effect in the retina and central nervous system (CNS). The compositions of the present invention have been shown to exhibit a neuroprotective effect superior to administration of the PPAR modulator alone.

[0008] PPAR modulators may be endogenous or exogenous molecules and include compounds that modulate the activity of PPARs such that the actual receptor activity is altered, i.e., the compound can act as a PPAR agonist or inhibitor. The activity of PPARs can be modulated, for example, by PPAR agonists that bind to the receptor or by PPAR agonists that act on downstream components of the pathway activated by PPARs to induce similar activity. PPAR agonists may be superagonists, partial agonists or full agonists. PPAR modulators may have a binding affinity to PPARs of 100 μM or less (preferably 10 μM or less, 5 μM or less, or more preferably 2 μM or less or 1 μM or less). In an embodiment of the present invention, PPAR modulators may have a binding affinity to PPARs of 100 nM or less or 10 nM or less.

[0009] As used herein, "polymeric nanocarrier component" refers to a component that comprises a polymer. For example, the polymeric nanocarrier component may comprise a propylene glycol (PEG) group and / or a polymer-based component (e.g., a poloxamer). The polymeric nanocarrier component is preferably a surfactant or a synthetic derivative thereof.

[0010] The polymeric nanocarrier components may be non-ionic surfactants and / or micelle-forming surfactants. Non-ionic micelle-forming surfactants are advantageous because they form relatively soft and flexible micelles, which can enhance transport across non-injectable routes of administration, for example across mucous or biological membranes. Non-ionic surfactants include polysorbates (Tweens), Triton X-100, polyethoxylated castor oil, Solutol HS, and the like. In embodiments of the present invention, the micelle-forming surfactant may be selected from one or more of D-α-tocopherol·polyethylene glycol 1000 succinate (vitamin E TPGS), pegylated phospholipid derivatives (e.g., DSPE-PEG, DSPS-PEG, etc.), poloxamers (e.g., Lutrol F68, Lutrol F127, etc.), polylactic-co-glycolic acid (PLGA), or chitosan derivatives. Optionally, the PEG derivatives may be further functionalized by adding "click chemistry" reactive groups, such as maleimide-PEG for covalent attachment to thiol groups, or azide-PEG / alkyne-PEG for covalent attachment to alkyne / azide functional targeting moieties (e.g., TPGS-PEG-MAL, DSPE-PEG-AZIDE, etc.). The functional targeting moiety may comprise a protein or peptide, and may include a phosphatidylserine binding protein, such as an annexin, in particular annexin V, or a functional fragment or derivative thereof, preferably comprising annexin repeats. Alternatively, a His-tagged protein or peptide may be non-covalently attached to the particle surface using nickel-functionalized lipids (e.g. 18:1 DGS-NTA(Ni)).

[0011] In a preferred embodiment of the present invention, the polymeric nanocarrier component comprises Vitamin E TPGS, optionally in combination with Lutrol F127, Solutol HS, chitosan or DSPE-PEG. Vitamin E TPGS is a non-ionic surfactant that forms stable micelles at concentrations above 0.02% w / w and has a low critical micelle concentration. The α-tocopherol component has intrinsic and antioxidant properties as well as P-glycoprotein antagonism, which can improve the barrier penetration ability of formulations containing this agent. The polymeric nanocarrier composition of the present invention may contain Vitamin E TPGS at a concentration of about 0.02 mg / ml to about 100 mg / ml, preferably about 10 mg / ml to about 65 mg / ml, more preferably about 20 mg / ml to about 55 mg / ml.

[0012] Lutrol F127 is a bifunctional block copolymer surfactant consisting of a central hydrophobic polyoxypropylene group adjacent to a hydrophilic polyoxyethylene group, which can conformationally stabilize nanocarriers to prevent aggregation. The polymeric nanocarrier composition of the present invention may contain Lutrol F127 at a concentration of about 0.2% w / v to about 30% w / v, preferably about 5% w / v to about 20% w / v, more preferably about 10% w / v to about 20% w / v. Lutrol F127 may be used alone or in combination with Vitamin E TPGS.

[0013] Solutol HS (2-hydroxyethyl 12-methyl hydroxystearate) is a non-ionic solubilizer and emulsifier with low toxicity. The polymeric nanocarrier composition of the present invention may contain Solutol HS at a concentration of about 100 mg / ml to about 200 mg / ml. Solutol HS is preferably used in combination with Vitamin E TPGS.

[0014] The polymeric nanocarrier component may be up to 100% micelle-forming surfactant, for example, the polymeric nanocarrier component may be up to 100% Vitamin E TPGS or up to 100% Lutrol F 127. Alternatively, the micelle-forming surfactant may include a combination of surfactants, for example, Vitamin E TPGS in combination with a pegylated phospholipid derivative (e.g., DSPE-PEG), a non-ionic surfactant (e.g., Solutol HS), or a poloxamer (e.g., Lutrol F 127).

[0015] The composition is preferably in the form of a capsule formulation, and most preferably in the form of a micelle.Without being bound by theory, the inventors believe that encapsulation of the PPAR modulator provides sustained release of the PPAR modulator, improves the bioavailability of the component, and protects the PPAR modulator from hydrolysis.The composition of the present invention has been found to be more neuroprotective than PPAR modulators administered in non-encapsulated form.

[0016] The diameter of the micellar nanocarriers of the present invention is about 100 nm or less, preferably about 70 nm or less or about 50 nm or less. In a preferred embodiment of the present invention, the diameter of the micellar nanocarriers is about 30 nm or less. The minimum diameter of the micellar nanocarriers may be about 10 nm. The diameter of the micellar nanocarriers is preferably about 20 nm. The size of liposomes that are not subjected to sizing (e.g., extrusion process) is non-uniform, and their diameters are 100 nm to 1000 nm. In contrast, the diameter of the micellar nanocarriers of the present invention is substantially uniform. For example, in the composition of the present invention, at least 70%, or at least 80%, or at least 90% of the micellar nanocarriers may have a diameter of about 10 nm to about 30 nm.

[0017] Solubilization of the PPAR modulator preferably means encapsulation of the PPAR modulator, which can be quantified by encapsulation capacity. When performed at physiological pH, the encapsulation capacity is preferably at least 5%, at least 10%, or at least 15%. In embodiments of the present invention, the encapsulation capacity can be at least 20% or at least 25%. In embodiments of the present invention, the encapsulation capacity can be 50% or more, or 70% or more, or 80% or more, and can be up to 100%.

[0018] The capsule formulation can contain a PAR modulator at a concentration of about 0.1 mg / mL to about 100 mg / mL, preferably about 0.5 mg / mL to about 50 mg / mL, and more preferably about 1 mg / mL to about 10 mg / mL.

[0019] In a preferred embodiment of the invention, the composition is in the form of a ternary system consisting of an aqueous continuous phase, and the PPAR modulator and polymeric nanocarrier components are primarily present in a dispersed phase distributed therein.

[0020] The PPAR modulator may be a PPAR-α modulator, a PPAR-γ modulator, a PPAR-δ modulator, a dual PPAR modulator, or a panPPAR modulator. In a preferred embodiment of the invention, the PPAR modulator is a PPARγ agonist or a compound having PPARγ agonist activity. Without wishing to be bound by theory, the inventors of the present invention believe that PPARγ agonists (or compounds having this type of activity) act on neurons and retinal ganglion cells (RGCs) to alleviate oxidative stress, reduce microglial activation and proinflammatory cytokine release, and promote mitochondrial biogenesis. These pathways have been implicated in the pathogenesis of certain CNS diseases, including glaucoma and Parkinson's disease, suggesting a mechanism of action in the treatment of such diseases as described herein.

[0021] The PPAR agonist may be a thiazolidinedione. In embodiments of the present invention, the PPAR agonist may be selected from one or more of pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, the phytocannabinoid Δ9-THCA, and troglitazone.

[0022] Alternatively, as described above, the PPAR modulator may be a compound that modulates PPAR activity such that the receptor activity itself is increased, i.e., a compound that has the same effect as an agonist. Compounds of this type include curcumin and resveratrol, which are known to have PPAR-γ activity.

[0023] Alternatively, the PPAR modulator may be an acyl homoserine lactone (AHSL) compound. This class of compounds has been shown to have PPAR modulation capabilities (Jahoor et al.). AHSL may have, for example, an acyl group with 4 to 20 carbon atoms. Tetramic and tetronic acid derivatives of AHSL may include 3-oxo and 3-hydroxy derivatives. Exemplary AHSL compounds include 3-hydroxydodecanoyl homoserine lactone and 3-oxododecanoyl homoserine lactone. AHSL compounds are further advantageous because they engage tight junctions to increase the permeability of biological barriers (Carlson et al.). This may enhance delivery of the composition to the intended tissue in vivo.

[0024] For example, a suitable polymeric nanocarrier composition of the present invention may be the following micellar formulation: Vitamin E TPGS at approximately 10mg / ml Approximately 20mg / ml of Lutrol F127 Approximately 5mg / ml of curcumin or Vitamin E TPGS at approximately 25mg / ml Solutol HS at approximately 150mg / ml About 5 mg / ml curcumin or about 15 mg / ml resveratrol or Vitamin E TPGS at approximately 50mg / ml Solutol HS at approximately 150mg / ml About 5 mg / ml curcumin or about 15 mg / ml resveratrol

[0025] The composition of the present invention may comprise a combination of two or more PPAR modulators. At least one of the PPAR modulators is preferably encapsulated as described above. Alternatively, both PPAR modulators may be encapsulated. When two or more PPAR modulators are encapsulated, the type of encapsulation may be the same or different. For example, both PPAR modulators may be encapsulated in a single polymeric nanocarrier, or each PPAR modulator may be encapsulated in a separate polymeric nanocarrier that is combined prior to administration. The composition of the present invention may comprise a combination of resveratrol and curcumin.

[0026] The composition may be sterile and may contain one or more pharmacologically acceptable carriers or excipients. Suitable carriers and excipients are well known to those skilled in the art and may be optimized for the intended delivery route. For example, the composition of the present invention may contain buffers, binders, preservatives, thickeners, or antioxidants (e.g., trehalose).

[0027] The composition is preferably suitable for local delivery, such as intraocular and nasal delivery, oral delivery, or dermal delivery. Local delivery of the composition can be advantageous because it reduces systemic exposure of the PPAR modulator. Systemic exposure is associated with adverse side effects, such as increased risk of myocardial infarction and death.

[0028] Topical preparations are preferably in the form of a solution or suspension in an aqueous medium (e.g. solutions, lotions, gels, creams, ointments, gels or foams). Oral preparations may be in the form of solutions, suspensions, tablets, capsules, powders or granules. Parenteral administration is intravenous, subcutaneous or intraperitoneal administration etc. Parenteral preparations may in particular be provided in the form of a solution or suspension in an aqueous medium or as a lyophilized powder.

[0029] In embodiments of the present invention, the compositions are suitable for intranasal delivery. Such formulations may be in the form of a solution, suspension, or dry powder suitable for inhalation.

[0030] Usually, and especially when the composition is administered in liquid form (via any of the above routes), the composition of the present invention can be provided as a lyophilized powder. The composition of the present invention is specified to be stable upon lyophilization, and can be reconstituted, for example, using saline or other (preferably aqueous) vehicles. When lyophilized, it is preferable to include a cryoprotectant material, such as trehalose, in the composition.

[0031] The compositions of the present invention can be used for therapy, in particular for the treatment or prevention of CNS disorders, such as neurodegenerative disorders, retinal disorders, or brain disorders.

[0032] In a further aspect, the invention provides a method of treating a CNS disorder (e.g. a neurodegenerative disorder, a retinal disorder, or a brain disorder) comprising administering a composition of the invention to a patient. The patient is preferably a mammal, including a human, and may be pediatric or elderly.

[0033] The neurodegenerative condition may be Parkinson's disease, Alzheimer's disease, or Huntington's chorea. The retinal disease may include retinal degenerative conditions, such as glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, and optic neuritis. The brain disorder may include traumatic brain injury, stroke, cerebral palsy, such as caused by neonatal hypoxia, and cancer, including brain tumors. In embodiments of the present invention, the composition may be intended for the treatment of pre-symptomatic Parkinson's disease.

[0034] The compositions of the invention may be administered topically, for example, intraocularly or intranasally, as described above. In one embodiment, the compositions of the invention may be administered in combination with one or more additional therapeutic agents, such as, for example, insulin, metformin, dipeptidyl peptidase-4 (DPP-4) inhibitors (e.g., alogliptin), glucagon-like peptide-1 (GLP-1) receptor agonists, antioxidants (e.g., resveratrol, coenzyme Q10, idebenone, quercetin, etc.), vitamin D compounds or derivatives thereof, vascular endothelial growth factor (VEGF) antagonists (e.g., ranibizumab, bevacizumab, or functional fragments thereof), N-methyl-D-aspartate (NMDA) receptor antagonists, glutamate antagonists, or memantine. The additional therapeutic agents may be administered simultaneously with the compositions of the invention or sequentially. When the additional therapeutic agents are administered simultaneously with the compositions of the invention, they may be included in the compositions of the invention in the same phase as the PPAR or in a consecutive phase of a ternary composition. In certain embodiments, the additional therapeutic agent has a hydrophobic nature such that it is present in the dispersed phase (i.e., co-encapsulated) with the PPAR.In certain embodiments, the composition of the present invention comprises curcumin and an antioxidant (e.g., resveratrol).

[0035] In a further aspect, the present invention provides a method for preparing a PPAR modulator micelle composition as described above, the method comprising: (1) Dissolving one or more polymeric nanocarrier components in a first solvent mixture (2) dissolving the PPAR modulator in a second solvent mixture; (3) Combining the dissolved polymeric nanocarrier components and the dissolved PPAR modulator and drying the combination to form a membrane. (4) Rehydrate the film with a buffer to form a micellar solution. (5) Filtration of the suspension to remove unencapsulated PPAR modulator. The first solvent mixture is preferably a short chain primary alcohol (e.g., ethanol). Also, ethanol has low toxicity compared to other solvents such as chloroform / methanol. This means that residual solvents that may be present in the composition are unlikely to cause problems, although they may. The first and second solvent mixtures in embodiments of the present invention may be the same. In a preferred embodiment of the method of the present invention, the PPAR modulator is curcumin, resveratrol, or AHSL, which have been shown to be poorly soluble in other solvent mixtures. The suspension is preferably filtered through a membrane filter having a pore size of about 0.22 μm, which can further remove any potential biological contaminants.

[0036] In a further aspect, the present invention provides a pharmaceutical composition comprising an active pharmaceutical ingredient (API) and an AHSL compound. The AHSL may, for example, have an acyl group having from 4 to 20 carbon atoms. Tetramic acid and tetronic acid derivatives of AHSL may include 3-oxo and 3-hydroxy derivatives. Exemplary AHSL compounds include 3-hydroxydodecanoyl homoserine lactone and 3-oxododecanoyl homoserine lactone.

[0037] As mentioned above, AHSL compounds engage tight junctions to increase the permeability of biological barriers. These compounds are known to be used by certain bacteria as part of the tissue invasion process during the establishment of infection in the host. However, the potential of these compounds as a means to deliver APIs to target tissues has not been recognized until now. The inventors of the present invention have determined that this type of approach can be used with a variety of APIs, including the PPAR modulators mentioned above (curcumin, resveratrol, etc.) and those with larger molecular weights, such as peptides or antibodies.

[0038] In a preferred embodiment, the composition of this aspect further comprises a polymeric nanocarrier component that encapsulates the AHSL and / or API in a liposome or micelle. The polymeric nanocarrier component is preferably in the form of a liposome, which comprises one or more phospholipids and may include a sterol (e.g., cholesterol) and / or a vitamin E derivative (e.g., TPGS). Suitable phospholipids include, for example, those based on phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. More particularly, the phospholipids used in the compositions of the present invention may comprise natural phospholipid derivatives or synthetic phospholipid derivatives. Natural phospholipid derivatives may include one or more of the following: egg phosphatidylcholine, hydrogenated egg phosphatidylcholine, soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, or sphingomyelin (e.g., 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine, 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn -glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine). The synthetic phospholipid derivatives may include one or more of the following: 1,2-didodecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dilauroyl-sn-glycero-3-phosphoserine (DLP), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOP), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn -glycero-3-phosphoethanolamine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and 1,2-distearoyl-sn-glycero-3-phosphoserine (DSP). In an embodiment of the invention, the surfactant or lipid may be combined with polyethylene glycol (PEG) (e.g. PLGA-PEG) and the polymeric nanocarrier component may be as defined in the first aspect of the invention.

[0039] Some AHSLs can form micelle-like structures in aqueous media and therefore can act as delivery systems for APIs without additional components, however the stability of the composition can be improved by inclusion of polymeric nanocarrier components, for example as described above.

[0040] The API contained in the composition of this embodiment is not particularly limited, and those skilled in the art can readily identify which APIs may potentially be used, but as an example, the various types of APIs described in the first embodiment may be considered.

[0041] In a related aspect, the present invention provides the use of an AHSL to enhance delivery of an API, in particular across biological barriers (e.g., the blood-brain barrier, the blood-retinal barrier, etc.).

[0042] The invention will now be described in detail, by way of example only, with reference to the following figures: [Brief description of the drawings]

[0043] [Figure 1] Absorbance spectra and molar absorptivities of curcumin in DMSO and of curcumin stabilized and degraded in DMSO. [A] Calibration curve of known 0.005 mg / mL curcumin concentration dissolved in DMSO with peak optical density (OD) at 435 nm. [B] Slope of curcumin concentration versus absorbance at 435 nm shows molar absorptivity (ε). [C] Degraded curcumin in 5 mg / mL sodium hydroxide (NaOH) solution (left) is darker than the solubilized stabilized micellar curcumin formulation (right). [D] Molar absorptivity of DMSO-diluted curcumin solution degraded in NaOH compared to 5 mg / mL DMSO-diluted curcumin solution in ethanol. Over 72 hours of degradation, the molar absorptivity decreased from 22,462 L.mol-1.cm-1 to 2,477 L.mol-1.cm-1. This corresponds to 0.04% of the molar extinction coefficient of curcumin diluted in DMSO (58,547 L.mol-1.cm-1). [Diagram 2]A thin film hydration technique using vacuum-assisted rotary evaporation is presented, which involves adding dissolved material to a round-bottom flask [Ai] and evaporating ethanol (EtOH) under vacuum and elevated temperature [A.ii], leaving a dry thin film [A.iii], which is then resuspended in an aqueous buffer to generate curcumin micelles [A.iv]. Photographic images of the rotary evaporation [Bi] and the remaining thin film [B.ii]. [Diagram 3] Solubilization of curcumin in micellar formulations is shown. 2 mg / mL curcumin does not dissolve very well in distilled water [Ai] and forms a precipitate. This precipitate is removed by filtration using a 0.22 μm pore membrane, leaving the water behind [A.ii] [Bi]. A 2 mg / mL solution of curcumin-encapsulated micelles is passed through a 0.22 μm pore membrane filter [B.ii] [A.iii], showing good retention of curcumin. [Figure 4] Curcumin stability evaluation is shown, measuring encapsulation ability in different environments. [A] Comparison between 25°C and 4°C samples. Compared to baseline, the EE of 4°C samples declined from week 3 onwards. [B] Comparison between 25°C and freeze-dried (25°C) samples. The EE of freeze-dried samples declined significantly over the entire period. Data are presented as mean with SEM and analyzed using 2-way ANOVA with Bonferroni post-hoc analysis, ***p<0.001. [Diagram 5] Z-diameter and PDI of curcumin formulations over time are shown. [A] Micelle size (Z-diameter) remained without significant visible change over time. [B] Micelle spread (PDI) increased over time, but this change was not significant. Data are presented as mean with SEM and analyzed using one-way ANOVA with Bonferroni post-hoc analysis. [Figure 6]The discoloration of a resveratrol formulation is shown over a period of two weeks (configuration B). Images [A]-[E] are of the formulations stored on days 0, 7, 8, 9, and 14. [A] The initial formulation was clear of any precipitate formation. This formulation was stored at room temperature in a dry, dark environment for two weeks. [B] Regarding discoloration, within one week, the formulation was observed to gradually darken. [E] At two weeks, the formulation was at its darkest, but no precipitate formed. [Figure 7] Resveratrol formulations were stored in different environments over a period of 9 days (configuration C). Figures [A]-[E] show the stored formulations on days 0, 2, 3, 8, and 9. [i] Stored at room temperature (25°C), [ii] Stored at 4°C. During this period, the formulations were stored in a dry and dark environment. No discoloration or deposition was observed over the 9 days at 25°C and 4°C. [Figure 8] Resveratrol stability evaluation is shown, measuring encapsulation ability in different environments. [A] Comparison between 25°C and 4°C samples. [B] Comparison between 25°C and freeze-dried (25°C) samples. All samples showed significant differences at each time point compared to baseline. [B] also shows that there was a significant difference between the two samples at 6 and 9 weeks. Data are presented as mean with SEM and analyzed using 2-way ANOVA with Bonferroni post-hoc analysis, *p<0.05 **p<0.01. [Figure 9] Z-diameter and PDI of resveratrol formulations over time. [A] Micelle size (Z-diameter) remained without significant visible change over time. [B] Micelle spread (PDI) remained without significant visible change over time. Data are presented as mean with SEM and analyzed using 2-way ANOVA with Bonferroni post-hoc analysis. [Figure 10]In vitro pretreatment of primary RGCs (murine) with 50 μM curcumin micelle formulation or vehicle alone at equivalent concentration prior to paraquat treatment to induce oxidative stress insult. Cell viability was assessed using alamarBlue assay. [A] Dose-response curve showing decreased cell viability with increasing paraquat concentration. Cell viability was significantly increased in curcumin pretreated cells. [B] IC50 of curcumin treated RGCs is 2-fold higher than vehicle treated RGCs. [Figure 11] Overall RGC density is shown for treatment groups across retinas. pOINT = partial optic nerve section, Curc = curcumin, Resv = resveratrol. RGC density was significantly higher in treatment vs. control groups. There was a significant improvement in RGC density in the curcumin only and combined treatment groups compared to the pOINT only group. Data are presented as mean with SEM and analyzed using one-way ANOVA and post-hoc Tukey analysis, *p<0.05 **p<0.01. [Figure 12] [A] Topical administration of curcumin micelles (CN) eye drops (twice daily for 3 weeks) in an ocular hypertension model (Morrison model) showed significant neuroprotective effects compared to the OHT-only control group. The unformulated curcumin (FC) control group did not show significant protective effects on the RGC population in this model. This model is often used as a model of retinal ganglion cell degeneration in glaucoma, but the mechanism of neurodegeneration is increasingly recognized as sharing significant similarities with other diseases of the CNS, including Alzheimer's disease, PD, etc. RGC density was identified histologically using Brn3a assessment as described by Davis et al. in 2016. [B] Curcumin treatment showed no effect on IOP, suggesting that the neuroprotective effect was independent of IOP. [Figure 13]Spectrophotometric determination of curcumin content in nanocarrier formulations. [A] Keto and enol forms of curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione). [B] Suspension of 4.5 mg / mL curcumin in PBS (left), PBS after 0.2 μm filtration to remove insoluble material (middle), and in TPGS / Pluronic® F127 nanoparticles after 0.2 μm filtration to remove insoluble material (right). [C] Dissolved in DMSO, 22 μM curcumin has an absorbance peak at 435 nm. [D] Identification of the molar absorptivity of curcumin in DMSO solvent (58,547 L.mol-1.cm-1) and demonstration that accelerated oxidative degradation of curcumin at low pH 61 reduces the molar absorptivity (2133 L.mol-1.cm-1 after 72 h incubation in 1 M sodium hydroxide solution), suggesting that spectroscopic assessment can be used to monitor both the encapsulation and degradation of curcumin. [E] Dissolving 5 mg / mL curcumin in 1 M sodium hydroxide solution (right) resulted in a rapid color change compared to curcumin-loaded nanocarriers (left). [F] Calibration curve of known concentrations of curcumin measured by HPLC. [Figure 14A] Characterization and stability evaluation of curcumin-loaded nanoparticles over time. [A] Transmission electron microscopy images of curcumin-loaded nanoparticles (CN) negatively stained with 1% uranyl acetate. Scale bar = 50 nm. Dynamic light scattering revealed a homogenous particle population that did not change significantly when stored at 25°C or when lyophilized and stored at 25°C and resuspended after 9 weeks [B][C]. [Figure 14B] [D] Photograph of 1 mL of lyophilized CN in 10 mM HEPES and 50 mg / mL trehalose buffer showing good cake structure. [E] Illustrates the change in encapsulation ability over time in a stability study when CN was lyophilized and rehydrated versus stored at 25°C (solution). [F] Mean particle size and [G] dispersity index were recorded in both cases. Mean ± 95% CI. [Figure 15A] X-ray diffraction and FT-IR characterization of curcumin-loaded nanocarrier formulations. [Figure 15B] [A] X-ray diffraction patterns of naive curcumin (blue), empty nanocarriers (black), and curcumin nanocarriers (red). [B] FTIR analysis of (1) trehalose, (2) curcumin-loaded nanoparticles, (3) free curcumin, and (4) hollow nanoparticles. [Figure 16] In vitro release of curcumin. In vitro release of 4.5 mg / mL curcumin [A] from 95% ethanol solution, [B] from curcumin-loaded nanocarriers in PBS at 37°C (mean ± SE, n = 3). As curcumin is poorly soluble in aqueous buffers, the release of curcumin from ethanoic acid solution was limited by the formation of a visible precipitate from the 0.5 h time point. This type of aggregation was not observed in experiments using nanocarrier curcumin. [Figure 17] Curcumin nanoparticle treatment is neuroprotective against hypoxia mimicking cobalt chloride in immortalized retinal cells. Co-incubation of R28 cells with varying concentrations of CN using Alamar Blue cell viability assay significantly protected cells from glutamate-induced injury [A, B] or cobalt chloride-induced injury [C, D] (one-way ANOVA with Tukey post-hoc test, ***p < 0.001). Hollow nanoparticles containing TPGS were found to be neuroprotective against glutamate-induced toxicity [B] but not cobalt chloride-induced toxicity [D]. [Figure 18]Topical curcumin nanoparticles protect RGC somata from OHT-induced cell loss in vivo. [A] Schematic in vivo experimental design. OHT rats were randomly assigned to be untreated or to receive once-daily curcumin nanoparticle (CN) or free curcumin (FC) eye drops starting 2 days before induction of elevated IOP. Three weeks after surgery, animals were sacrificed moribund and retinal planes were flat-mounted prior to labeling with Brn3a. RGC populations were counted as previously described. Representative retinal images of corresponding Brn3a-labeled areas in the upper hemitina are shown. [B] Naive control animals, [C] animals without OHT treatment, [D] animals with OHT+CN treatment. [E] All OHT animals had significantly elevated IOP (mean ± SE) compared to baseline up to 21 days after surgery (Student's t test vs. contralateral eye, **p<0.01). There were no significant differences in IOP between OHT-treated groups at any time point, suggesting that the observed neuroprotective activity was independent of IOP. Higher IOP in [F]OHT-only eyes was associated with a significant decrease in RGC density (~23%), consistent with previous findings. CN, but not FC, treatment significantly reduced RGC loss (Kruskal-Wallis test with Dunns post-hoc test, **p < 0.01). [Figure 19] Topical curcumin nanoparticles protect RGC somata from optic nerve damage. Representative Brn3a-labeled hemiretinal sections were taken from similar regions of naive retina [A], pOINT retina [B], and pOINT model retina [C] after daily topical CN treatment for 21 days. [D] Whole retinal RGC density measurements showed that pOINT caused a significant decrease in RGC density, which was reduced by daily CN treatment (one-way ANOVA with Tukey post-hoc test, **p<0.01 and ***p < 0.0001). [E] Further division of each retina into superior and inferior quadrants using methods previously described (Davis et al., 2016) demonstrated that local CN somewhat prevented RGC density loss in both superior and inferior retinas (two-way ANOVA with Tukey post-hoc test, ***p<0.001). [Figure 20]Results of a cytotoxicity study with the acyl homoserine derivative 3-HO-C12 HSL (selected for its higher hydrolytic resistance than 3-oxo-C12 HSL) are shown (Davis et al., 2011). This molecule was found to be better tolerated in an immortalized human corneal epithelial cell line (HCE-S) than 3-oxo-C12 HSL [A and B] and common eye drops and the permeability enhancer benzalkonium chloride (BAK) [C]. [Figure 21] Negative stained TEM micrographs of TPGS micelles encapsulating 3-OH-C12 HSL after filtration [A]. 3-OH-C12 HSL-encapsulated micelles were prepared containing 1 mg / mL 3-OH-C12 HSL with TPGS alone [A&D], TPGS and 5 kaDa chitosan [B], and DSPE-PEG / cholesterol micelles [D]. Unencapsulated material was removed by 0.22 μm filtration due to the low water solubility of 3-OH-C12 HSL. After 2 h exposure to micelles containing up to 300 μM 3-OH-C12 HSL, encapsulation of 3-OH-C12 HSL was assessed by the ability of the molecule to reversibly disrupt HCE-S tight junctions. Because vehicle alone was unable to induce a reversible decrease in HCE-S transepithelial resistance, the micelles must contain 3-OH-C12 HSL (TER is expressed as a percentage of the control group, which is micelles without HSL). [Figure 22A] We demonstrate that acyl homoserine lactones (3-OH-C12 HSLs, abbreviated as QS in this figure) mediate reversible disruption of tight junctions and can therefore be used for the delivery of therapeutic molecules across biological barriers. [Figure 22B]Using an in vitro HCE-S transwell barrier model as described by Davis et al. in 2014, the transcytosis of [Ai-Aiii] 40 kDa FITC-dextran and [Bi-Biii] 149 kDa Avastin (antibody bevacizumab) was significantly enhanced after pretreatment of HCE-S with 300 μM 3-OH-C12HSL for 2 h. The control group was the equivalent formulation without AHSL (black circles in the graph). This demonstrates the potential of AHSL for enhancing drug delivery. [Diagram 23] It has been shown that coadministration of a model drug molecule with 3-OH-C12 HSL-containing micelles (containing 40 kDa Anx-FITC (annexin V conjugated to the dye fluorescein isothiocyanate) was sufficient to promote delivery of the drug across the HCE-S transwell model of biological barrier. [Figure 24] AHL-containing formulations (3-OH-C12 HSL) are shown to be well tolerated in in vitro HETCAM assays [A] and in in vivo Drazie assays [B] at concentrations of 0.3 mM (F1) and 0.6 mM (F2) versus vehicle control (TPGS micelles). These results are in sharp contrast to other permeability enhancers, such as benzalkonium chloride (BAK), which is known to induce potent ocular irritation in rabbits and humans. [Diagram 25] Figure 1 shows the results of a stability evaluation of a formulation of 4.5 mg / ml curcumin encapsulated in micelles formed from 25 mg / ml TPGS and 150 mg / ml Solutol, measuring the encapsulation ability (A), particle size (B), and polydispersity index (PDI) (C) over time. [Figure 26] FIG. 26 shows the results of stability evaluation of encapsulation ability (A), particle size (B), and polydispersity index (PDI) (C) of the formulations in FIG. 25 after lyophilization and subsequent rehydration. [Figure 27] Figure 2 shows that intranasal administration of curcumin micelles reduced DARC counts in the retina of a 3xTg-AD mouse model (B, C) compared to vehicle alone (A). [Figure 28]Figure 2 shows that intranasal administration of curcumin micelles prevented retinal ganglion cell (RGC) loss in the 3xTg-AD mouse model (B, C) compared to vehicle alone (A). [Figure 29] Figure 2 shows that intranasal administration of curcumin micelles reduced amyloid-β deposition in the hippocampus of 3xTg-AD mice (B, C) compared to vehicle alone (A). [Diagram 30] Figure 1 shows the results of a stability evaluation of a formulation of 15 mg / ml resveratrol encapsulated in micelles formed from 25 mg / ml TPGS and 150 mg / ml solutol, measuring the encapsulation ability (A), particle size (B), and polydispersity index (PDI) (C) over time. [Diagram 31] FIG. 31 shows the results of stability evaluation of encapsulation ability (A), particle size (B), and polydispersity index (PDI) (C) of the formulations in FIG. 30 after lyophilization and subsequent rehydration. [Diagram 32] Resveratrol nanoparticle treatment is neuroprotective against glutamate excitotoxicity in immortalized retinal cells. Co-incubation of R28 cells with RN significantly protected the cells from glutamate-induced damage using the Alamar Blue cell viability assay (A) (one-way ANOVA with Tukey post-hoc test, ***p < 0.001). Glutamate toxicity (B). Empty nanoparticles containing TPGS were found to be neuroprotective against glutamate-induced toxicity (A, C). Protective effect of RN (A, D). [Diagram 33] Resveratrol nanoparticle treatment has no neuroprotective effect against cobalt chloride-induced hypoxia in immortalized retinal cells (A), control (B), hollow nanoparticles (C), and RN (D). [Diagram 34] Figure 1. Intranasal administration of resveratrol micelles reduced DARC counts in the retina of the 3xTg-AD mouse model when compared to vehicle alone. [Diagram 35] Intranasal administration of resveratrol micelles reduced amyloid-β deposition in the hippocampus of 3xTg-AD mice (B, C) when compared to vehicle alone (A). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] experiment <Example 1> Micelles containing TPGS and Lutrol F127 were prepared with up to 5 mg / mL of curcumin or resveratrol and stability studies were performed. Micelles were also prepared with PEG-cholesterol and Lutrol F127.

[0045] In Figure 1, the concentration of curcumin in the formulations was determined by spectroscopy. Comparing hydrolyzed curcumin (which has no therapeutic value) to the intact drug, which has virtually no absorbance at 435 nm, it was possible to determine not only the curcumin concentration in each formulation but also the amount of active agent (forced curcumin degradation achieved by treatment with sodium hydroxide (Figure 1D, red line)).

[0046] Manufacturing method Curcumin micelles were formed by the thin film rehydration method (see Figure 2). Curcumin, P127, and TPGS were first dissolved in absolute ethanol by sonication to form a stock solution (Fisher Scientific, UK). These were added to a round-bottom flask in the desired molar ratio (Table 1) and vortexed to mix. The ethanol solvent was evaporated using a vacuum-assisted rotary evaporator (Rotavapor R-210 / vacuum controller V-850, Buchi, Switzerland) under 50 mbar vacuum and at the desired temperature (Table 1) until a thin film of dissolved material remained as dry material. The dried film was resuspended in various aqueous buffer solutions at elevated temperature using a rotary evaporator (1 bar). The resulting micelles were separated from unencapsulated (insoluble) curcumin by filtration through a 0.22 μm filter (33 mm Millexfilter, Merck Millipore, USA). The micellar formulations were then characterized to determine their curcumin-encapsulation capacity, particle size, and stability over time. Rehydration buffer: distilled water, phosphate buffered saline (PBS), Tris (20 mM) trehalose (50 mg / mL) buffered saline. Drug stability was investigated using PBS buffer, as a neutral pH is preferred for topical instillation.

[0047] Details of individual formulation protocols and compositions JPEG2025016677000002.jpg148147Description of the formulations produced in this study. dH20 (distilled water), PBS (phosphate buffer), EtOH (ethanol), Cur (curcumin), TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate), P127 (Pluronic® F-127)

[0048] Unencapsulated curcumin was removed by filtration as shown in Figure 3.

[0049] Characterization and stability results (Curcumin micelles) JPEG2025016677000003.jpg62154

[0050] Freeze-drying: Three steps JPEG2025016677000004.jpg26154The freeze-drying protocol includes three stages: 1° (primary), 2° (secondary); h (hours).

[0051] Long-term stability of freeze-drying and rehydration (preliminary results for PBS) JPEG2025016677000005.jpg61145

[0052] The cryoprotectant was later replaced with 10 mg / mL to 100 mg / mL trehalose (10 mM HEPES, pH 7.4), which is more stable as shown in the following formulations.

[0053] In the presence of trehalose cryoprotectant (10 mg / mL to 100 mg / mL trehalose), the stability of curcumin micelles was good both at room temperature and after lyophilization. Micelles at pH 4.5 and pH 7.4 were prepared. See Figures 4 and 5.

[0054] Resveratrol micellar formulations (later co-administered with curcumin micelles in vivo) showed high stability using the same micellar formulations (see Table 5).

[0055] Composition of the initial formulation generated in the study: Formulation composition code PBS: phosphate buffered saline, Trehalose: Hepes-trehalose (50 mg / mL), TPGS: D-α-tocopheryl polyethylene glycol 100 succinate, P127: Pluronic (registered trademark) F-127, Res: resveratrol JPEG2025016677000006.jpg98169

[0056] Table 6: Amount of encapsulated resveratrol produced by different initial concentrations of drugs JPEG2025016677000007.jpg40161

[0057] The buffer composition preferably contains trehalose (10 mg / mL to 100 mg / mL) as this antioxidant prevents degradation of the product over time during storage, as compared to PBS, which causes progressive discoloration of the product (see Figures 6 and 7).

[0058] The samples showed high stability for up to 10 weeks (see Figures 8 and 9).

[0059] Curcumin micelle formulations were found to be significantly neuroprotective in vitro (see FIG. 10).

[0060] Topical curcumin micellar eye drops (twice a day for 3 weeks) and curcumin / resveratrol micellar combination therapy (2 drops a day) were found to be significantly neuroprotective in a rat model of optic neuropathy (partial optic nerve transection) (Figure 11). This neurodegenerative model is primarily used to evaluate the efficacy of therapies against retinal diseases, but due to the similarities in generative processes between the retina and the brain, it may also be used to provide information on the therapeutic potential of other CNS diseases. These results suggest a synergistic effect from the PPAR-g activity modulator curcumin and the antioxidant resveratrol with our micellar formulation.

[0061] Curcumin treatment did not affect IOP, suggesting that the neuroprotective effect was independent of IOP (see FIG. 12).

[0062] <Example 2> Methods and Materials Preparation of curcumin-loaded nanocarriers Curcumin, d-α-tocopherol polyethylene glycol 1000 succinate (TPGS), and Pluronic F127 were obtained from Sigma Aldrich (Kent, UK) at the highest purity available. Curcumin-loaded nanocarriers (CNs) were prepared by applying the thin film hydration technique previously described (Davis et al., 2014). Curcumin, TPGS, and Pluronic F127 were dissolved in ethanol to concentrations of 5 mg / mL, 10 mg / mL, and 20 mg / mL, respectively. They were gently heated for 10 min to clarify, followed by bath sonication. The solutions were aliquoted into round-bottom flasks at the desired molar ratios (22.55 mM, 12.22 mM, and 7.94 mM MTPGS, curcumin, and Pluronic F127, respectively) and mixed well. The solvent was removed by rotary evaporation (50 mbar, 65° C., 2 h) using a Rotavapor R210 with a V850 vacuum controller (Buchi, Switzerland) while protected from light. After this time, the film was rehydrated (50° C., 0.5 h) with the desired buffer (distilled water, phosphate buffered saline (pH 7.4), or HEPES trehalose buffer (10 mM HEPES, 50 mg / mL trehalose, pH 7.4)). Unencapsulated curcumin was then removed from the formulation by 0.22 μm filtration (33 mm Millex filter, Merck Millipore, USA) as shown in FIG. 13B. Free curcumin (FC) was prepared following the same protocol as above without the addition of TPGS or Pluronic F127.

[0063] Freeze-drying of curcumin-loaded nanocarriers Lyophilization of the CN formulations in HEPES-trehalose buffer was achieved by equilibrating 1 mL aliquots of the nanocarriers in 7 mL screw-top squat glass vials (CamLab, Cambridge, UK) at 25°C before freezing at 760 Torr and -60°C for 2 hours. Primary drying of the samples was completed at 200 mTorr and -38°C for 24 hours, followed by a secondary drying step at 200 mTorr and 25°C for 2 hours. Samples were protected from light until required and were covered immediately after secondary drying ceased before storage at 25°C. For stability assessment, 1 mL of 0.22 μm filtered distilled water was added, gently mixed and the samples were allowed to rehydrate for 30 minutes.

[0064] The water content of the formulations was determined using thermogravimetric analysis (TGA). Freeze-dried (lyophilized) samples were placed in aluminum pans and analyzed by a Discovery TGA (TA Instruments, USA). Samples were purged with nitrogen gas at a flow rate of 25 mL / min and heated from 30°C to 200°C at a rate of 10°C / min. The percent mass loss was calculated for water content by TA Instruments Trios software at 120°C. Three freeze-dried formulations were measured in triplicate for each sample.

[0065] Curcumin Encapsulation Efficiency The loading efficiency of CN was determined spectroscopically and the results were confirmed using HPLC. Spectroscopic quantification of curcumin loading was achieved at 435 nm (standard for emptying nanocarriers) diluted 1:500 in DMSO. The molar extinction coefficient of curcumin was then used to determine the concentration of curcumin in each formulation, which was determined by drawing a calibration curve that determined the absorbance of known curcumin concentrations (Figure 13). The encapsulation capacity of each formulation was calculated using Equation 1: JPEG2025016677000008.jpg15170 formula [C] s is the concentration of curcumin initially added to the formulation (usually 4.5 mg / mL), and [C] Eis the concentration of curcumin detected spectroscopically in the nanocarriers after 0.22 μm filtration to remove unencapsulated material. Results were confirmed applying established HPLC techniques (Guddadarangavvanahally et al.). Briefly, samples containing curcumin were diluted with methanol before injecting 20 μL volumes at 25°C onto a Phenomenex® Synergi (4 μm Polar-RP 80A with dimensions 250 × 4.60 mm) column in an acetonitrile:0.1% trifluoroacetic acid (50:50) solvent system at a flow rate of 1 mL / min connected to an Agilent Technology 1260Infinity HPLC system. Absorbance was recorded at 420 nm and area under the curcumin elution curve compared to a calibration curve of known curcumin concentrations.

[0066] Dynamic Light Scattering Particle size was determined using a Malvern Zetasizer. Particle diameter and polydispersity index measurements were recorded from a minimum of three formulations for each experimental condition or post-manufacturing time point. Nanocarriers were diluted 1 / 10 with the appropriate buffer prior to recording.

[0067] Transmission electron microscopy The nanocarrier suspension was stained with 1% uranyl acetate for 1 min and treated with a carbon grid to absorb particles from the suspension before drying. The specimens were observed using a Joel-1010 transmission electron microscope operating at 100 kV with images obtained with a Gatan Orius digital camera.

[0068] X-ray diffraction and FT-IR X-ray diffraction graphs of the drug alone, hollow nanoparticles, or CN were prepared using an X-ray diffractometer (Rigaku MiniFlex 600), with the 2-thea angle set from 5 to 65 degrees with an angle increment of 0.05° / sec. The measurements were performed at a voltage of 40 kV and 15 mA. ―1 and 650cm ―1 The FT-IR spectra of free curcumin, hollow nanoparticles, and CN were measured with four scans between 4 cm ―1The spectra were recorded using a PerkinElmer Spectrum100 FT-IR spectrometer at 37.0°C.

[0069] Curcumin release assay In vitro curcumin release was assessed using a previously described protocol (Wang et al., 2012). Briefly, free curcumin (dissolved in 95% ethanol) or CN containing 4.5 mg / mL curcumin was loaded into a 1 mL Spectra-Por Float-A-Lyzer dialysis cassette (Sigma-Aldrich) with a molecular weight cutoff of 3.5–5 kDa. Samples were dialyzed against 200 mL of PBS containing 10% Tween-80 to act as a sink for released curcumin, which was kept at 37 °C with stirring at 50 rpm. At the designated time points, samples were removed from the mixture and replaced with fresh buffer. The concentration of curcumin was determined as described above. Results from three replicate experiments were consistent with single-phase binding (Equation 2). JPEG2025016677000009.jpg11162 where Y0=0, Plateau is the maximum release, and K is the curcumin release (h ―1 ), which results in a half-life (t 1 / 2 ) was calculated (t 1 / 2 = ln2 / K).

[0070] cell culture R28 cell line (Kerafast, Boston, MA) was cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Paisley, UK) supplemented with 5% fetal bovine serum (Invitrogen, UK), 100 U / ml penicillin, 100 μg / ml streptomycin and 0.292 mg / mL glutamine (Gibco, UK), 7.5% sterile dH20, and 1.5 mM KCl (Sigma-Aldrich, UK). Medium was changed every other day and cultures were passaged at 90% confluence.

[0071] Cell viability assessment R28 cells were plated at 4,000 cells / well in 96-well plates for 24 h before treatment with varying concentrations of curcumin (0-20 μM) or equivalent concentrations of TPGS / Pluronic® F127 nanocarriers alone (vehicle control) in combination with varying concentrations of cobalt chloride or glutamate insults for an additional 24 h. Cell viability was assessed using the Alamarblue (Invitrogen, UK) assay according to the manufacturer's instructions. Briefly, Alamarblue solution was added to each well plate and incubated for 4 h before fluorescence was recorded using a Safire plate reader (excitation wavelength 530 nm, emission wavelength 590 nm91).

[0072] animal All animal experiments were performed in accordance with procedures approved by the UK Home Office and the ARVO Declaration for the Use of Animals in Vision and Ophthalmological Research. For the in vivo evaluation of the experiments, 48 ​​adult male Dark Agouti (DA) rats (Harlan Laboratories, UK), weighing a total of 150–200 g, were housed in an air-conditioned environment at 21°C with a 12-h light / dark cycle (140–260 lux), where food and water were available ad libitum. Thirteen rats served as naive controls, with no additional manipulations prior to immunohistochemical staining.

[0073] Ocular hypertension (OHT) model Intraocular hypertension was surgically induced in the left eyes of 18 Da rats (5: OHT only, 5: OHT+CN, 8: OHT+FC) as previously described (Morrison et al., 1997). Procedures were performed under general anesthesia using a mixture of 37.5% ketamine (Pfizer Animal Health, Exton, PA), 25% dormitol (Pfizer Animal Health, Exton, PA), and 37.5% sterile water, administered intraperitoneally at a volume of 2 mL / kg. Briefly, 50 μL of hypertonic saline (1.8 M) was infused into two episcleral veins using a syringe pump (50 μL / min; UMP2; World Precision Instruments, Sorasota, FL, USA). A propylene ring with a slit cut 1 mm from the circumference was placed around the equator to prevent the outflow of the injected saline from interfering with other aqueous humor veins. The IOP of both eyes of each rat was measured at regular intervals using a TonoLab tonometer (Tiolat Oy, Helsinki, Finland) under inhalation anesthesia (0.4% isoflurane in oxygen). Five Da rats, along with five OHT-only control rats, received daily topical CN treatment (35 μL infusion / day, twice daily at 10 am, 5 min apart). This began 2 days before model induction and continued until the end of the model (21 days after IOP elevation). Eight additional rats received free curcumin (FC), prepared using the same protocol as CN curcumin, without the addition of TPGS or Pluronic F127. FC was administered to OHT animals following the same dosing regimen as described for CN. Animals were sacrificed moribund 3 weeks after unilateral IOP elevation, and retinas were flat-mounted before Brn3a immunohistochemical staining.

[0074] Partial Optic Nerve Section (POINT) Model Partial optic nerve transections were performed in the left eyes of 17 Da rats using a technique previously described (Levokovitch-Verbin et al., 2003). Under general anesthesia, the superior conjunctiva was incised to expose the optic nerve sheath. A longitudinal incision was then made to expose the dural ON, and a 0.2 mm incision was made on the dorsal surface of the ON, 2 mm behind the eye, using an ophthalmic scalpel with a steel cutting guard. The surgery was completed and examined by ophthalmoscopy, avoiding damage to major ocular blood vessels. Nine Da rats received daily topical administration of CN after pOINT model induction, with the remaining eight rats serving as pOINT-only controls, following the same treatment regimen previously described.

[0075] Brn-3a immunohistochemical staining and confocal microscopy Brn-3a labeling of RGCs in retinal whole mounts was performed as previously described (Davis et al., 2016). Briefly, rats were sacrificed moribund and eyes enucleated. Retinal whole mounts were then fixed overnight in 4% paraformaldehyde at 4°C before dissection. Whole mounts were stained for the RGC-specific nuclear-localized transcription factor Brn3a using mouse anti-mAb (1:500, Merck Millipore, Darmstadt, Germany) and examined using confocal microscopy (LSM710, Carl Zeiss MicroImaging GmbH, Germany). Whole tissues from each retina were imaged as tiled Z-STACKs at ×10 magnification, which was used to generate single-plane maximum projections of the RGC layer of each retina for subsequent analysis. For reference, each whole tissue image was manually oriented with the superior retina at the top of the image using in vivo cSLO imaging of the retinal vasculature. Retinal image acquisition settings were constant for all retinas imaged. This allowed for comparison of Brn3a expression between each experimental group as previously described.94 Automated quantification of Brn3a-labeled RGCs in whole retinal tissue was achieved as previously described (Davis et al., 2016).

[0076] statistical analysis All data were analyzed by Student's t test (ANOVA) or appropriate post-hoc tests using GraphPad Prism5 (GraphPad Software, La Joola, CA, USA), as appropriate. Data are presented as mean ± standard deviation, and p < 0.05 was considered significant. Molecular structures were drawn using ACD / ChemSketch2015, and all images were taken by the authors (BMD).

[0077] Results and Discussion Spectroscopic methods can be used to evaluate curcumin encapsulation capacity and oxidation state Upon dilution in dimethyl sulfoxide (DMSO), curcumin exhibited a peak absorbance at 435 nm (Figure 13C) and a concentration of 58547 L.mol ―1 .cm ―1 The molar extinction coefficient of 0.01 mg / mL (Figure 13D&E) was comparable to previously reported values. The absorbance of curcumin diluted in DMSO at 435 nm followed the Beer-Lambert law up to 42 μM, and TPGS / Pluronic F127 nanocarriers in the absence of curcumin had no measurable absorbance at this wavelength. After separation of the unencapsulated material by 0.22 μm filtration, spectroscopic evaluation was used to identify the encapsulation efficiency (EE%) of the formulations containing curcumin. Spectroscopic measurements of EE% were confirmed by the established HPLC technique (Figure 13F) with both techniques showing good agreement (4.31 ± 0.18 mg / mL vs. 4.32 ± 0.33 mg / mL, respectively).

[0078] Spectroscopic quantification of curcumin concentration in nanocarriers can also be used to indicate the extent of curcumin degradation. Curcumin undergoes keto-enol tautomerization (Figure 13A) and exists in a stable keto form under acidic or neutral conditions and a water-soluble enol form under alkaline conditions. Like other molecules that undergo keto-enol tautomerization, the enol form of curcumin is prone to hydrolysis. Acceleration of the curcumin degradation process by dissolution in alkaline buffers resulted in a dramatic decrease in the molar extinction coefficient of curcumin at 435 nm compared to as-prepared curcumin (Figure 13C-D, 2133 L.mol after 72 h incubation in the presence of 1 M sodium hydroxide solution). ―1 .cm ―1 ). Furthermore, incubation of CN in alkaline conditions induced a dramatic color change from orange to brown (Figure S13E). Spectroscopic assessment of curcumin concentration after dissolution of sodium hydroxide showed that the curcumin molar extinction coefficient decreased rapidly, suggesting that this technique can be used not only to assess the entrapment efficiency of curcumin but also to monitor the extent of degradation of curcumin-containing formulations.

[0079] TPGS / Pluronic F127 nanocarriers enhance curcumin solubility and stability Curcumin-loaded nanocarriers were first prepared by incorporation into TPGS nanocarriers. TPGS was selected for the following reasons: the low critical micelle concentration of this excipient (0.02% w / w), the intrinsic and antioxidant properties of its α-tocopherol moiety, and its P-glycoprotein antagonism, which enhances the barrier-crossing ability of formulations containing this drug. TPGS is present in existing ophthalmic formulations, and both curcumin and TPGS can be readily solubilized in ethanol. And the solvent present in the commercially available eye drop (Optrex ActiMixt 2-in-1 Eye Spray / Dry Irritated Eye) at a concentration of 0.8% reduces the risk associated with residual solvents from the manufacturing process. Furthermore, the use of TPGS to enhance the bioavailability of orally administered drugs is well documented. Thus, together with the recent interest in the use of Pluronic F127 in food research, this suggests that the novel curcumin formulation described herein may be suitable for oral administration.

[0080] Formulation of curcumin with TPGS micelles was found to produce nanocarriers with a diameter of 16 nm as determined by dynamic light scattering (data not shown). Unfortunately, these formulations rapidly aggregated at 25°C and formed a precipitate within hours of resuspension that may indicate Ostwald ripening. Stabilization of the curcumin-loaded TPGS nanocarriers was achieved by the addition of the polymeric stabilizer Pluronic F127 (a triblock copolymer of polyoxyethylene and polyoxypropylene), which has been used previously to conformationally stabilize nanocarriers against aggregation.

[0081] Following the described method, curcumin-loaded nanocarriers (CNs) were prepared with the specified encapsulation capacity and average particle size (Figure 14). Upon resuspension in PBS (pH 7.4) or HEPES-trehalose buffer (pH 7.4), the nanocarriers were found to encapsulate 96.0% ± 2.0% (4.32 mg / mL) and 94.2% ± 4.1% (4.31 mg / mL) of curcumin, respectively. Transmission electron microscopy revealed that the nanocarriers were uniformly sized, typically 20 nm in diameter (Figure 14A). These results were confirmed by dynamic light scattering (Figure 14B-C), which identified a homogenous particle dispersion with a mean diameter of 16-20 nm, suggestive of a micellar formulation.

[0082] After storage at 25°C protected from light, the encapsulation ability and particle size of the CN formulations were evaluated over time. The CN formulations were found to exhibit excellent stability for 9 weeks at 25°C, with the formulations either not decreasing in EE% (Figure 14E), exhibiting significant changes in particle size (Figure 14F), or dispersing over this period (Figure 14G). This stability study was repeated with lyophilized CN prepared in the same buffer prior to storage at 25°C protected from light. The calculated residual water content at 120°C was 1.085±0.050%, indicating that the lyophilized formulations were properly prepared. The formulations were resuspended before recording the dispersibility (Figures 14E-G). This dispersibility was found to be consistent and similar to the results reported for the liquid formulations (Table 7). The EE% was found to decrease by an average of 20% compared to baseline at each time point evaluated, suggesting that this may be a result of the lyophilization or rehydration process.

[0083] Several groups have attempted to prepare curcumin-loaded nanoparticle formulations, including PLGA-nanocarriers, solid lipid nanocarriers, liposomes, and exosomes. Existing nanoparticle formulations of curcumin have limited stability (none of the cited studies assessed beyond 72 hours), only modest curcumin loading has been achieved (<0.77 mg / mL), and most protocols are difficult to translate to the clinic due to complex, multi-step manufacturing protocols requiring organic solvents. The TPGS / Pluronic F127 curcumin formulation described herein compares favorably with existing literature.

[0084] Characteristics and stability over time of curcumin-loaded nanocarriers (n=3) JPEG2025016677000010.jpg40169

[0085] To confirm the nature of the curcumin incorporated into the nanocarriers, XRD and FT-IR spectra were obtained (Figure 15). The X-ray diffraction pattern of free curcumin exhibited characteristic peaks at 5°-30°, indicating a highly crystalline structure. This characteristic was lost upon inclusion of curcumin in the nanocarrier formulation, indicating that curcumin was well incorporated into the amorphous nanocarrier structure and not bound to the particle surface. The FT-IR spectrum showed a peak at 3509 cm ―1 , 1626cm ―1 , 1601cm ―1 , 1505cm ―1 , 1271cm ―1 , 1024cm ―1 , 948cm ―1 , and 713 cm ―1 Free curcumin exhibits a characteristic peak at 3509 cm, which matches closely with previously reported values. ―1 The characteristic curcumin peak (representing the free hydroxyl group) is at 3352 cm ―1 The broad OH peak of the TPGS / Pluronic F127 support was associated with the broad OH peak of the TPGS / Pluronic F127 support, which may suggest complex formation. Furthermore, the aromatic C=C peak (1601 cm ―1 ~1588cm ―1 ) and C=O stretching, 1505 cm ―1 From 1514cm ―1 Characteristic shifts in the in-plane bending angles δ(CCC) and δ(CCO) to δ(CCO) have previously been interpreted as evidence of successful incorporation of curcumin into the complex.

[0086] The preparation of curcumin into nanocarriers substantially reduced the drug release rate (t and t , respectively) compared to the free drug at 37°C. 1 / 2= 22.6 h vs. 0.15 h, Figure 16). This is attributed to the slow release rate of curcumin from the nanocarriers. Less than 10% of the drug was released after 5 h of incubation, suggesting that there was little burst release from the CN formulation. This observation supports the FT-IR and XRD observations, which show that curcumin is not merely associated with the nanocarrier surface but is localized within the hydrophobic interior of the amorphous or disordered crystalline phase, and is consistent with previous studies. Together, these results suggest that the curcumin-loaded nanocarrier formulation described in this study has sustained release capabilities.

[0087] Curcumin-loaded nanocarriers protect retinal cell lines from glutamate- and hypoxia-induced damage. Glutamate excitotoxicity represents a potential mechanism leading to the loss of RGCs in glaucoma. Co-incubation of immortalized R28 cells with both CN and hollow nanoparticles using the AlamarBlue cell viability assay demonstrated no significant difference in glutamate-induced toxicity (Figure 17A and B, EM and IC50 in the injury-only treated group, respectively). 50 Curcumin was found to be significantly protective against glutamate-induced toxicity (28.3 ± 3.4 mM vs. 5.9 ± 1.2 mM, one-way ANOVA with Tukey post-hoc test (p < 0.001)). Also, there was no additive effect observed with the addition of curcumin to the nanoparticles (24.5 ± 1.2 mM, CN containing 20 μM curcumin). This observation is in line with previous studies suggesting that α-tocopherol (here appearing in the form of TPGS) is protective against glutamate-induced toxicity, suggesting that it is the result of the antioxidant function of this molecule. As TPGS was also not protective against cobalt chloride-induced damage, this suggests that curcumin and TPGS may have an additive therapeutic effect.

[0088] Upregulation of hypoxia-related factors such as hypoxia-inducible factor 1α (HIF-1α) has been suggested to implicate hypoxia in glaucoma pathology. Cobalt chloride (CoCl2) is a hypoxia mimetic and inducer of HIF-1α75 used as an in vitro glaucoma model. The IC of R28 cells exposed to CoCl2 for 24 h was 50 (Figure 17C and D) CN morphology (296 ± 53 μM vs. 757 ± 51 μM, respectively, one-way ANOVA with Tukey post-hoc test (p < 0.001)) was found to be significantly increased by co-incubation with 20 μM curcumin. Treatment with equivalent concentrations of nanoparticles in the absence of curcumin had no significant effect (296 ± 53 μM vs. 354 ± 8 μM, one-way ANOVA with Tukey post-hoc test (p > 0.05)), suggesting that a protective effect was observed with curcumin. At curcumin concentrations < 20 μM, it was not possible to determine whether it had a neuroprotective effect in this model. Curcumin has previously been reported to inhibit HIF-1α in hepatocellular carcinoma cells and has recently been reported to suppress HIF-1α synthesis in pituitary adenomas. HIF-1α inhibitors have previously been proposed as potential glaucoma treatments that merit further investigation.

[0089] Topically administered curcumin nanocarrier therapy protects RGCs in a rodent model of ocular hypertension and optic nerve injury Having established the neuroprotective effect of CN in vivo with vehicle-only treatment, we next assessed the neuroprotective effect of this formulation on RGC integrity using an established in vivo rodent model (RGC loss). We expect that topically applied curcumin-loaded nanoparticles will reach the retina by a combination of local and systemic absorption. In support of this hypothesis, Sigurdsson et al. reported that their dexamethasone formulation (with a similar molecular weight to curcumin (392 vs. 368 Da, respectively)) was taken up by the retina by 60% via local penetration and 40% via systemic absorption. We expect that the well-documented P-gp inhibitory activity of tocopherol and curcumin, combined with the enhanced corneal penetration activity previously reported for PEGylated micelle formulations, will enhance curcumin delivery to the retina via the local absorption route.

[0090] The optimal time point post model induction (maximal RGC loss at shortest time after induction) was selected based on our previous studies characterizing the natural history of OHT and pOINT models, which were assessed at multiple time points after model induction. We recently reported that administration of TPGS-containing micelles had no neuroprotective effect in vivo. This, combined with our in vitro observations, suggests that any neuroprotective effects observed were the result of curcumin treatment. Rats received CN topically according to the drug injection method illustrated in Figure 18A. Briefly, rodents received CN dosing of 2 drops (35 μL each) per day at 5-minute intervals, starting 2 days prior to OHT model induction, for 3 weeks from the day of model induction. Topical administration of CN was found to be well tolerated by rats, with no signs of ocular irritation or inflammation reported in the unmedicated eye monitored by a qualified ophthalmologist. Rodent IOP profiles after IOP elevation by injection of hypertonic saline into two episcleral veins (Figure 18B) show that IOP remained significantly elevated for at least 7 days after model induction compared to unmedicated eyes. No significant differences in IOP profiles were observed between the CN and OHT-only groups, suggesting that any neuroprotective effects of curcumin are due to IOP-independent processes. RGC health was assessed histologically from whole retinal mounts by brn3a assessment (Figure 18C). This approach was chosen because Brn3a is a nuclear-restricted, RGC-specific transcription factor. This transcription factor exclusively labels 97% of the RGC population (excluding light-sensitive RGCs). We have also recently developed an algorithm to accurately and automatically quantify the total RGC population in rodent models of retinopathy, allowing reliable assessment of RGC health. Using this approach, we found that OHT induction produced a significant reduction in global RGC density of ~23% compared to the contralateral eye, which is comparable to previous studies using this model.CN administration significantly increased the RGC density ratio between OHT eyes versus contralateral untreated eyes (Kruskal-Wallis test with Dunns post-hoc test (**p < 0.01)), whereas administration of non-encapsulated curcumin solubilized in PBS (FC - free curcumin) did not have this effect (Figure 18D-F).

[0091] To further explore the neuroprotective potential of CN in topical application, a population assessment of whole retina brn3a-labeled RGCs was performed in the pOINT model (Figure 19A). In this model, twice-daily topical administration of CN was found to significantly protect RGCs (one-way ANOVA, ***p < 0.001). Sectioning of whole retina mounts into superior and inferior quadrants (Figure 19B) showed that treatment with CN protected the population of RGCs in both superior and inferior quadrants, but this effect was more pronounced in the superior hemitreatum (two-way ANOVA, ***p < 0.001). This may imply that the protective effect of curcumin therapy is exerted by anti-apoptotic as well as antioxidant mechanisms. Representative areas from the upper quadrants of Brn3a-labeled retinal whole mounts (Figure 19C-E) show that the RGC population was reduced in retinal pOINT subjects (Figure 19D) versus naive controls (Figure 19C). Three weeks of CN treatment were found to protect RGC somata from pOINT-induced damage (Figure 19E). Preservation of RGC somata was observed in both the upper and lower retinal quadrants, suggesting that curcumin may elicit neuroprotective activity through multiple pathways involving both primary and secondary neurodegenerative processes.

[0092] The possibility of neuroprotection with TPGS through inhibition of glutamate excitotoxicity is intriguing and may contribute to the neuroprotective effect of our formulation in vivo. In support of this hypothesis and our current in vitro findings, Nucci et al. previously reported that intraocular administration of a total of 10 μL of 0.5% (w / v) TPGS (equivalent to a total dose of 0.5 mg TPGS) provided neuroprotection against ischemia / reperfusion injury in rats. We have previously reported that topical administration of TPGS at the same concentration had no neuroprotective effect in vivo. This discrepancy is likely due to the lower concentration reaching the retina compared to intravenous application, which is usually estimated to be ~3% of the topically applied dose. Although our previous work with this model suggests that TPGS administration alone did not appear to have a neuroprotective effect in and of itself, it is highly likely that synergy between curcumin and TPGS enhances curcumin transport across the ocular barrier, possibly through TPGS modulation of P-gp activity, if not through the neuroprotective effects of TPGS alone.

[0093] The neuroprotective effect of curcumin-loaded nanocarriers observed in this study may be the result of treatment starting 2 days before model induction, suggesting that this therapy may be most effective for patients at risk of IOP spikes (e.g. following phacoemulsification surgery) or as a preventative agent for patients identified as at high risk of developing glaucoma (e.g. conditions with high intraocular pressure or other glaucoma risk factors). Furthermore, by developing new technologies such as DARC (detection of apoptotic retinal cells) that have the potential to diagnose glaucoma early in the disease progression (Cordeiro et al., 2017), therapies that slow or prevent the loss of RGCs at early disease stages could play a major role in managing glaucoma.

[0094] In conclusion, this study describes a novel nanocarrier formulation of curcumin in TPGS / Pluronic™ F127 that increases the solubility of this poorly soluble drug by approximately 400,000-fold. The formulation incorporates 4.3 mg / mL of curcumin with a consistent encapsulation capacity of >90% and excellent stability (characterized by HPLC and spectroscopic techniques) in liquid or lyophilized form for at least 2 months when stored at room temperature. The formulation was found to be neuroprotective against glutamate- and cobalt chloride-induced injury of in vitro retinal cultures, and significantly preserved RGC density in two ocular injury-established rodent models. In conclusion, we demonstrate that curcumin-loaded nanoparticles have an excellent potential to cross the ocular barrier, allowing easy translation of curcumin-based therapies into clinics treating ocular conditions, such as glaucoma.

[0095] Example 3: Acyl homoserine lactones (AHSLs) are enhancers of drug delivery The results shown in Figures 20-23 demonstrate the safety and efficacy of AHSL as a drug delivery enhancer of compositions according to further aspects of the invention described above. The results demonstrate enhanced transepithelial delivery of two structurally unrelated model substances, FITC-dextran and FITC-annexin V, and an antibody (bevacizumab) from AHSL-containing micelles. Micelles (or other ternary formulation systems) can be prepared by a variety of methods as will be appreciated by those skilled in the art. However, thin film hydration may refer to any warming, as it may be a simple mixture of the formulation components in aqueous media.

[0096] Exemplary Composition Formulations In more detail, and by way of example only, the compositions can be prepared as follows (Figure 21 reports formulations 1-3). Formulation 1. 3-OH-C using TPGS or DSPE-PEG and cholesterol micelles 12 HSL (a) 100 mg TPGS (or DSPE-PEG and cholesterol) and 5 mg.mL ―1 3-OH-C12 HSL was solubilized in 5:1 (chloroform:methanol). (b) a desired amount of TPSG (or DSPE-PEG and cholesterol) and 3-OH-C 12 Aliquots of HSL (usually 0.5% and 0.1% w / v, respectively) were taken and the solvent was evaporated by rotary evaporation (1 h at 65° C., 50 mbar). (c) The cake was resuspended in PBS buffer at 65° C. for 30 min before filtration (0.22 μm) to remove unencapsulated material. Formulation 2. 3-OH-C using TPGS micelles 12 HSL (a) TPGS powder was directly solubilized in PBS buffer by heating to 65°C for 2 h to a final concentration of 0.5% (v / v) to generate TPGS micelles. (b) Alternatively to (a), TPGS micelles were prepared by sonication of a solution of A. (c) 3―OH―C 12 HSL was solubilized in DMSO to a concentration of 10 mg / mL, before being mixed with the TPGS micelle solution at 0.1% (v / v) for one hour. (d) Unencapsulated material was removed by 0.22 μm filtration. Preparation 3. 3-OH-C using chitosan micelles of TPGS / 5k 12 HSL (a) 55 mg / mL of 5K chitosan was prepared by dissolving 110 mg of 5K chitosan in 2 mL of buffer (10 mM HEPES, 140 mM sodium chloride, pH 7.4) and heating to 37° C. for 1 hour. (b) TPGS solutions were prepared as described above (F1 or F2). (c) 25 mg of 5K chitosan was mixed with 2.75 mg of TPGS from the above solution and 0.1 mg of 3-OH-C 12 HSL was added and incubated at 37°C for 1 h with brief sonication to ensure complete dissolution. (d) Unencapsulated material was removed by filtration (0.22 μm pores). Preparation 4.3-OH-C12 HSL-encapsulated liposomes (1) PC with 3.25 mM QSSM was prepared by dissolving in chloroform:methanol (5:1). 50% P.S. 15% Cholesterol 30% QSSM 5% - The following formulations of PLVs (65 mM) are prepared: PC 70% (25mg / ml):1001μl P.S. 15% (25mg / ml):322μl Cholesterol (50mg / ml): 151μl 3―OH―C 12 HSL(5mg / ml):972μl (2) Place under argon and then evaporate under vacuum for 1 hour to produce a dry lipid cake. (3) Meanwhile, 1 ml of AnxV-776 (13-03, 0.475 mg / ml) is dialyzed against 10 mM HEPES, 140 mM NaCl, pH 6.5 for 3 hours. (4) Mix gently (150 rpm) and incubate at 37°C for 1 hour to rehydrate the lipid cake with 1 ml of AnxV-776 solution. (5) Freeze and thaw the mixture five times in liquid nitrogen. (6) Sequentially extrude through 400 nm, 200 nm, and finally 100 nm pore filters.

[0097] In these typical compositions, 3-OH-C 12 HSL can be substituted for any AHSL. Transwell Assay Protocol (based in part on Davis et al., 2014) HCE-S cells (immortalized human corneal epithelial cells) were cultured as monolayers in 90% DMEM supplemented with 10% heat-inactivated FB and penicillin-streptomycin (100u / mL). Cells were cultured at 37°C in a humidified incubator with 5% CO2 and growth medium was replaced every 3 days. HCE-S populations were grown to 80% confluence before passage to 20% using 0.25% trypsin-EDTA. Cell populations were estimated using a hemocytometer and trypan blue exclusion assay before each experiment.

[0098] A previously described method (Reichl, 2008) was adapted to prepare the HCE-S barrier model for the transcytosis assay. HCE-S cells (5 × 10 4 The cells / well were placed in a Transwell insert (polycarbonate, 3 μm pore size, 1.13 cm 2 ) and kept for 7 days, replacing the medium in the apical and basal chambers every other day. After this time point, cells were cultured for an additional 10 days at the apical air interface to encourage the development of a multilayered barrier. Before each experiment, the medium was replaced with phenol red-free DMEM and the barrier integrity was measured via transepithelial resistance (TER). TER values ​​were measured between 300 and 600 Ω cm. 2 And, TER>300Ω 2 Only epithelial barriers of 0.1 μm (the threshold recognized for a tight epithelial barrier) were used in transcytosis experiments (Becker et al.).

[0099] Figures 21 and 22. 300 μM of 300 3-OH-C was added to micelles (considered 100% drug loading) or equivalent concentration of micelles alone (vehicle control) dissolved in phenol red-free medium for 2 h. 12Baseline TER was recorded before the addition of HSL. From this time on, TER was recorded before replacing the medium with fresh one. Model drug molecules (FITC-dextran or Avastin) were added to the apical chamber and the concentration of drug in the basolateral chamber was measured by monitoring a standard curve of FITC fluorescence (FITC-dextran) versus known concentrations or by using a commercially available Avastin sandwich ELISA kit as previously described (Davis et al., 2014). TER measurements at each time point were normalized to vehicle-treated controls, clearly demonstrating the ability of AHSL to reversibly disrupt biological barriers and, more importantly, the utilization of this concept for drug delivery applications.

[0100] Figure 23. To demonstrate that co-administration and pre-treatment of AHSL-containing formulations may be useful to improve drug delivery with therapeutic effects across biological barriers, FITC ANXV (e.g., a model protein drug) was co-administered with AHSL-loaded micelles for 3 hours. Protocol as described for Figures 21 and 22.

[0101] Although the HCE-S transwell model used here is a model of the corneal epithelium, the process of acyl homoserine lactone-mediated reversible barrier disruption is through interaction with IQGAP1 [Karlsson et al.], and thus similar effects are observed at other biological barriers (i.e. blood-brain barrier, blood-retinal barrier, gut and skin barrier).

[0102] To date, the ability of acyl homoserine lactones to disrupt biological barriers has been described solely as the ability of these agents (produced by bacteria and containing 3-OH-C) to allow bacteria to enter host tissues. 12 HSL is 3-oxo-C 12 Only HSL (synthetic analogs of HSL) has been of interest. This work presents the first demonstration of the use of these agents to facilitate delivery of therapeutics. This includes macromolecules such as polysaccharides and large proteins (Figures 25 and 26), although one of skill in the art will appreciate that the concept also applies to small molecule APIs crossing biological barriers.

[0103] Example 4: HET-CAM test Materials and Methods To study ocular tolerance in vitro, the HETCAM® test was developed as described in the INVITTOX no15 protocol (Warren, 1990).

[0104] The test is based on the observation of the irritant effect (bleeding, vasoconstriction and coagulation) of the chorioallantoic membrane (CAM) of 10-day-old larvae-formed eggs, induced by the application of 0.3 ml of the preparation to be studied (application lasting for the first 5 minutes).

[0105] These eggs (farm GALLSA, Tarragona, Spain) were kept at 12 ± 1°C for at least 24 h before being placed in an incubator where temperature (37.8°C) and humidity (50–60%) were controlled during incubation.

[0106] A series of controls were performed: SDS 1% (positive control for slow stimulation), 0.1 N NaOH (positive control for fast stimulation), NaCl 0.9% (negative control).

[0107] Data were analyzed as the mean ± SD of the time that injury occurred (n=3 / group). Irritation scores can be classified into four categories (Table 8).

[0108] HET-CAM calculation and classification JPEG2025016677000011.jpg56169

[0109] result The analyzed preparations were shown to be non-irritating in vitro (Table 9). Interestingly, the only phenomenon that emerged in all cases was a slight vasoconstriction effect (Figure 24). No bleeding or clotting was observed with any of the preparations.

[0110] Formulation evaluation results JPEG2025016677000012.jpg102169

[0111] In vivo Materials and Methods In vivo ocular tolerance was assessed using the Draize irritation test, which was performed using New Zealand white male rabbits of 2.5 kg (average weight) from San Bernardo firm (Bavarra). The test was performed in accordance with the Ethical Committee for Animal Experimentation of the UB and current legislation (Decret 214 / 97, Gencat).

[0112] The samples were placed in the conjunctival sac of the left eye and gently massaged to ensure adequate blood circulation (Nobraga et al., 2012). The appearance of irritation was observed at the time of administration and 1 hour later, with the right eye serving as a negative control (n=3 / group).

[0113] The evaluation was performed by direct observation of the anterior segment of the eye, noting possible damage to the conjunctiva (inflammation, chemosis, redness, or oozing), iris, and cornea (opacification and affected surface). The eye irritation index (OII) was assessed according to the damage observed (Tables 10 and 11).

[0114] Draize test calculations and classifications JPEG2025016677000013.jpg50169

[0115] Draize test evaluation score JPEG2025016677000014.jpg190141

[0116] result There was no irritation (OII=0) in any of the formulations. The animals did not show any signs of in vivo irritation at the time of application (eye drops) or after 1 hour (Figure 24). This correlates with the results of the in vitro evaluation showing that the product is non-irritating.

[0117] <Example 5> A formulation of 4.5 mg / ml curcumin encapsulated in micelles formed from 25 mg / ml TPGS and 150 mg / ml Solutol was prepared using the thin film rehydration technique previously described.

[0118] The formulation demonstrated high encapsulation capacity and stability over 90 days (Figures 25A-C). Encapsulation capacity remained high (65%) upon lyophilization and subsequent rehydration, while particle size and polydispersity also showed stability over 90 days (Figures 26A-C).

[0119] The formulation was then tested in the 3xTg-AD mouse model of Alzheimer's disease, administered intranasally, 5 days per week for 3 months. Compared to vehicle alone, curcumin nanoparticles reduced retinal DARC counts (see e.g. WO 2011 / 055121 for details regarding DARC counts), indicating reduced cell death (Figures 27A-C). Curcumin nanoparticles also protected retinal ganglion cells (RGCs) from loss (Figures 28A-C) and reduced amyloid-β deposition in the hippocampus (Figures 29A-C).

[0120] <Example 6> A formulation of 15 mg / ml resveratrol encapsulated in micelles formed from 25 mg / ml TPGS and 150 mg / ml Solutol was prepared using the thin film rehydration technique previously described.

[0121] The formulation demonstrated high encapsulation capacity (>70%) and stability over 90 days (Figures 27A-C). The encapsulation capacity remained high upon lyophilization and subsequent rehydration, while particle size and polydispersity also showed stability over 90 days (Figures 28A-C).

[0122] R28 cells were cultured as above prior to treatment with resveratrol (20 μ m )-containing micelles or equivalent concentrations of TPGS / Solutol-only micelles (i.e., empty) in combination with varying concentrations of cobalt chloride or glutamate insults.

[0123] Resveratrol-containing micelles provided neuroprotection against glutamate excitotoxicity (FIG. 29A-D), but had no neuroprotective effect against cobalt chloride-induced hypoxia (FIG. 30A-D).

[0124] The formulation was then tested in the 3xTg-AD mouse model of Alzheimer's disease, where the formulation was administered intranasally 5 days per week for 3 months. Compared to vehicle alone, resveratrol nanoparticles reduced DARC counts in the retina, indicating reduced cell death (Figure 31), as well as reduced amyloid-β deposits in the hippocampus (Figure 32A-C).

[0125] <Reference> Becker U, Ehrhardt C, Daum N, Baldes C, Schaefer UF, Ruprecht KW, Kim KJ, Lehr CM (2007) Expression of ABC-transporters in human corneal tissue and the transformed cell line, HCE-T. J Ocul Pharmacol Ther. 23(2):172-81 Biehlmaier O, Alam M, Schmidt WJ (2007) A rat model of Parkinsonism shows depletion of dopamine in the retina. Neurochemistry international 50: 189-195 Doi 10.1016 / j.neuint.2006.08.001 Bizrah M, Dakin SC, Guo L, Rahman F, Parnell M, Normando E, Nizari S, Davis B, Younis A, Cordeiro MF (2014) A semi-automated technique for labeling and counting of apoptosing retinal cells. BMC bioinformatics 15: 169 Doi 10.1186 / 1471-2105-15-169 Cooley MA, Whittall C, Rolph MS (2010) Pseudomonas signal molecule 3-oxo-C12-homoserine lactone interferes with binding of rosigliatzone to human PPARγ. Microbes and Infection 12 231-237 Cordeiro MF, Guo L, Luong V, Harding G, Wang W, Jones HE, Moss SE, Sillito AM, Fitzke FW (2004) Real-time imaging of single nerve cell apoptosis in retinal neurodegeneration. Proc Natl Acad Sci U S A 101: 13352-13356 Doi 10.1073 / pnas.0405479101 Cordeiro, M. F. et al. Real-time imaging of single neuronal cell apoptosis in patients with glaucoma. Brain 274, 61-5 (2017). Davis, B. M., Richens, J. L. & O’Shea, P. Label-free critical micelle concentration determination of bacterial quorum sensing molecules. Biophys. J. 101, 245-54 (2011). Davis BM, Normando EM, Guo L, Turner LA, Nizari S, O'Shea P, Moss SE, Somavarapu S, Cordeiro MF (2014) Topical delivery of Avastin to the posterior segment of the eye in vivo using annexin A5-associated liposomes. Small 10(8):1575-84. Davis BM, Guo L, Brenton J, Langley L, Normando EM, Cordeiro MF (2016) Automatic quantitative analysis of experimental primary and secondary retinal neurodegeneration: implications for optic neuropathies. Cell Death Discovery 2, Article number 16031 (2016) doi:10.1038 / cddiscovery.2016.31 Galvao J, Davis B, Tilley M, Normando E, Duchen MR, Cordeiro MF (2014) Unexpected low-dose toxicity of the universal solvent DMSO. FASEB Journal: official publication of the Federation of American Societies for Experimental Biology 28: 1317-1330 Doi 10.1096 / fj.13-235440 Guddadarangavvanahally K. Jayaprakasha, Lingamullu Jagan Mohan Rao, and & Sakariah*, K. K. Improved HPLC Method for the Determination of Curcumin, Demethoxycurcumin, and Bisdemethoxycurcumin. (2002). doi:10.1021 / JF025506A Guo L, Salt TE, Luong V, Wood N, Cheung W, Maas A, Ferrari G, Russo-Marie F, Sillito AM, Cheetham ME et al (2007) Targeting amyloid-beta in glaucoma treatment. Proc Natl Acad Sci U S A 104: 13444-13449 Doi 10.1073 / pnas 0703707104 Guo L, Normando EM, Nizari S, Lara D, Cordeiro MF (2010) Tracking longitudinal retinal changes in experimental ocular hypertension using the cSLO and spectral domain-OCT. Investigative ophthalmology & visual science 51: 6504-6513 Doi 10.1167 / iovs.10-5551 Jahoor A, Patel R, Bryan A, Do C, Frier J, Watters C, Wahli W, Li G, Williams SC, Rumbaugh KP (2008) Peroxisome Proliferator-Activated Receptors Mediate Host Cell Proinflammatory Response to Pseudomonda aeruginosa Autoinducer. Journal of Bacteriology 13: 4408-4415 Karlsson T, Turkina M, Yakymenkko, Magnusson KE, Vikstrom E (2012) The Pseudomonas aeruginosa N-Acylhomoserine Lactone Quorum Sensing Molecules Target IQGAP1 and Modulate Epithelial Cell Migration PLoS Pathogens 8, e1002953 Levkovitch-Verbin, H. et al. A model to study differences between primary and secondary degeneration of retinal ganglion cells in rats by partial optic nerve transection. 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Claims

1. 1. A pharmaceutical composition comprising a peroxisome proliferator-activated receptor (PPAR) modulator and a polymeric nanocarrier component, the polymeric nanocarrier component is capable of solubilizing the PPAR modulator in an aqueous medium; A composition, characterized in that in the polymeric nanocarrier component, micelles form a non-ionic surfactant.

2. The composition according to claim 1, characterized in that the micelle-forming surfactant is selected from one or more of D-α-tocopherol polyethene glycol 1000 succinate, pegylated phospholipid derivatives (e.g., DSPE-PEG, DSPS-PEG, PLGA-PEG, etc.), poloxamers (e.g., Lutrol F68, Lutrol F127, etc.), lactic acid-glycolic acid copolymers (PLGA), chitosan derivatives (chitosan-PEG, etc.), or biodegradable polymer-PEG.

3. The composition of claim 1 or 2, wherein the polymeric nanocarrier component further comprises one or more additional substances selected from ethylphosphatidylcholine and a cationic lipid (e.g., N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP)), 18:1 DGS-NTA(Ni)[1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl], or Solutol HS.

4. The composition according to any one of claims 1 to 3, characterized in that the composition is in the form of a micellar composition.

5. The composition of claim 4 , wherein the micelles have a diameter of about 30 nm or less.

6. The composition is in the form of a three-component system comprising an aqueous continuous phase; The composition of claim 1 , wherein the PPAR modulator and the polymeric nanocarrier components are predominantly present in a dispersed phase distributed therein.

7. 7. The composition of claims 1 to 6, wherein said composition is sterile.

8. The composition according to any one of claims 1 to 7, wherein the PPAR modulator is a PPARγ agonist or a compound having PPARγ agonist activity.

9. The composition according to any one of claims 1 to 8, wherein the PPAR modulator is a thiazolidinedione, curcumin, or resveratrol.

10. 10. The composition of claim 9, wherein the PPAR modulator is selected from one or more of the following: pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, darglitazone, englitazone, netoglitazone, rivoglitazone, the phytocannabinoid Δ9-THCA, and troglitazone.

11. 11. The composition according to any one of claims 1 to 10, further comprising one or more pharmacologically acceptable carriers or excipients.

12. 12. The composition according to any one of claims 1 to 11, characterized in that it is suitable for non-injectable delivery, such as topical, oral or parenteral delivery.

13. 13. The composition according to any one of claims 1 to 12, characterized in that it is suitable for intranasal delivery.

14. 14. A composition according to any one of claims 1 to 13 for use in therapy.

15. 15. A composition according to any one of claims 1 to 14 for use in the treatment or prevention of a CNS disorder.

16. 16. The composition for use according to claim 15, wherein the CNS disease is a neurodegenerative disease, a retinal disease, or a brain disorder.

17. 17. The composition for use according to claim 16, wherein the neurodegenerative condition is Parkinson's disease, Alzheimer's disease, or Huntington's disease.

18. 18. A composition for use according to any one of claims 15 to 17, characterized in that the composition is administered topically, for example intraocularly or intranasally.

19. 14. A method for treating a CNS disorder, comprising administering to a patient a composition according to any one of claims 1 to 13.

20. 20. The method of claim 19, wherein the CNS disease is a neurodegenerative disease, a retinal disease, or a brain disorder.

21. 21. The method of claim 20, wherein the neurodegenerative condition is Parkinson's disease, Alzheimer's disease, or Huntington's disease.

22. 22. The method according to any one of claims 19 to 21, characterized in that the composition is administered topically, for example intraocularly, intranasally or transdermally.

23. A method for preparing a PPAR agonist composition according to any one of claims 1 to 13, characterized in that said method comprises the following steps: (1) dissolving one or more polymeric nanocarrier components in a first solvent mixture; (2) dissolving a PPAR modulator in a second solvent mixture; (3) combining the dissolved polymeric nanocarrier components and the dissolved PPAR modulator and drying the combination to form a membrane. (4) Rehydrating the membrane with a buffer to form a micellar solution. (5) disrupting the micellar solution with ultrasound to form a suspension. (6) Stabilizing the suspension (7) filtering the suspension to remove unencapsulated PPAR modulator.

24. 24. The method of claim 23, wherein the first solvent mixture is ethanol.

25. 25. The method of claim 23 or 24, wherein the first solvent mixture and the second solvent mixture are the same.

26. 14. A composition according to any one of claims 1 to 13, as described in the examples.

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