Injectable thermosensitive hydrogels for sustained release of iron nanochelators

Abstract

Disclosed herein are injectable hydrogel formulations prepared by incorporating cross-linked hyaluronic acid into Pluronic F127 for the sustained release of DFO nanochelators. Methods of manufacture and use are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 371,526, filed August 16, 2022, the entire contents of which are incorporated herein by reference. The present disclosure relates to an injectable hydrogel formulation prepared by incorporating cross-linked hyaluronic acid into Pluronic F127 for the sustained release of DFO nanochelator, as well as methods of making and using the same. [Background technology]

[0002] Iron is an essential metal, but high iron stores are toxic due to metal-induced oxidative stress, which promotes organ damage such as heart failure, liver cirrhosis, diabetes, and neurodegenerative diseases. Primary iron overload (hereditary hemochromatosis) is one of the most common genetic disorders, affecting approximately 1 million people worldwide, primarily in the Caucasian population. Secondary iron overload can occur in patients with hemoglobinopathies, such as thalassemia major, sickle cell anemia, aplastic anemia, myelofibrosis, myelodysplastic syndromes, and Diamond-Blackfan anemia, because such patients require chronic blood transfusions. Because no effective pathway for iron excretion has been identified, chelation therapy is widely used to improve the condition of patients with iron overload, especially those with transfusion-related iron accumulation.

[0003] Three small molecule-based iron chelators are currently in use: deferoxamine (DFO), deferiprone (DFP), and deferasirox (DFX). DFO, in particular, has demonstrated favorable therapeutic efficacy since its approval by the US FDA in 1968. However, its extremely short half-life (e.g., 5–15 minutes in rodents) necessitates repeated injections or continuous infusion, significantly reducing patient compliance and quality of life. The other two chelators, DFP and DFX, overcome this compliance issue by allowing oral administration. However, DFP and DFX cause significant side effects, such as gastrointestinal bleeding, agranulocytosis, neutropenia, thrombocytopenia, liver fibrosis, and renal failure. Other orally active iron chelators (e.g., desferrithiocin derivatives) are in clinical trials or preclinical stages, but they are known to be nephrotoxic. [8]

[0004] Currently, several sustained drug release systems (e.g., hydrogels, patches, implantable drug devices, and infusion pumps) have been developed, significantly improving patient compliance and adhesion issues. In particular, injectable thermosensitive hydrogels are frequently used for drug delivery because they do not require additional chemical reactions or external stress (e.g., light or pressure) for crosslinking. However, many polymeric hydrogels exhibit potential toxicity. Therefore, to accelerate the progress of clinical trials, it is necessary to develop hydrogel formulations using FDA-approved materials. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure relates to an injectable thermosensitive hydrogel for the long-term release of DFO-NPs for iron chelation therapy. [Means for solving the problem]

[0006] In some embodiments, the present disclosure provides a sustained release injectable composition comprising an iron chelator and at least one hydrogel.

[0007] In some embodiments, the present disclosure provides a sustained release injectable composition for iron chelation therapy in a human patient, comprising an iron chelator and a hydrogel, wherein the composition is releasable over a two-week period. In some embodiments, the present disclosure provides a sustained release injectable composition for iron chelation therapy in a human patient, comprising an iron chelator and a hydrogel, wherein the composition is released over a two-week period.

[0008] In some embodiments, the present disclosure provides a method of treating iron overload in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a sustained release injectable composition comprising an iron chelator and at least one hydrogel.

[0009] In some embodiments, the present disclosure provides a method for reducing the amount of free metal ions in a cell or tissue sample, comprising contacting the cell or tissue sample with a therapeutically effective amount of a sustained-release injectable composition comprising an iron chelator and at least one hydrogel.

[0010] In some embodiments, the present disclosure provides a method of treating a disease associated with an abnormal amount of free metal ions in a subject, the method comprising administering to a subject determined to have an abnormal level of free metal ions a therapeutically effective amount of a sustained-release injectable composition comprising an iron chelator and at least one hydrogel.

[0011] In some embodiments, the present disclosure provides a method for preparing a sustained release injectable composition useful for iron chelation therapy, comprising: a) cross-linking HA; b) forming the cross-linked HA into particles; c) adding particles of crosslinked HA to a solution of DFO-NPs; d) further adding F127 to the solution resulting from step c), A method is provided.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Methods and materials for use in this application are described herein; however, other suitable methods and materials known in the art can also be used. These materials, methods, and examples are merely illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. [Brief explanation of the drawings]

[0013] [Figure 1] Schematic diagram of the DFO-NP-loaded injectable hydrogel. DFO-NPs were loaded within cross-linked hyaluronic acid (xHA), and F127 was formulated as the outer hydrogel. The sustained release of DFO-NPs from the hydrogel significantly improved their pharmacokinetics and minimized their distribution to non-target tissues. [Figure 2] Figure 1H-NMR spectra of DFO, ZW-EPL+, ZW-EPL-, and DFO-NP. The DFO to ZW-EPL- ratio was calculated by peak integration of the ZW-EPL- proton (position α' at 4.12 ppm) and the DFO protons (positions e, l, and s at 3.6 ppm). The calculated DFO per chain was based on the assumption of 30 lysine units per ZW-EPL-. [Figure 3] Figure 1 shows the hydrodynamic diameter (HD) analysis of DFO-NPs. (a) Size-exclusion chromatography measured at 254 nm (black line) and 760 nm (red line). Arrowheads and numbers indicate the corresponding HDs of standard proteins: aprotinin: 1.96 nm, ribonuclease: 3.28 nm, ovalbumin: 6.10 nm, thyroglobulin: 17.00 nm. (b) Standard calibration curve of HD using standard proteins. [Figure 4]Photophysical properties of DFO-NP are shown. a) Optical properties including absorption and fluorescence spectra at a concentration of 100 μM. b) UV-vis absorption spectra of DFO-NP titrated with Fe(III). (The blue line indicates the DFO-NP solution before adding the iron solution, and the red line indicates the DFO-NP solution after titration with the iron solution. c) Titration curve (A-A) of absorbance change. [Figure 5] Figure 1 shows the results of in vitro cell uptake and viability tests for DFO-NP. (a) shows the results of cell uptake tests for ZW-EPL+ (5 μM) and DFO-NP (5 μM) in NIH3T3 and H23 cells. (b) shows the cell viability of NIH3T3 cells at various concentrations of DFO-NP. [Figure 6] Rheological properties of hydrogel formulations: a) cross-linked hyaluronic acid (HA, 7%), b) DFO-NP-loaded HA (DFO-NP / HA, 30 / 7%), c) Pluronic F127 (F127, 30%), d) DFO-NP-loaded F127 (DFO-NP / F127, 30 / 30%), e) HA and F127-incorporated hydrogel (HA / F127 7 / 30%), and f) DFO-NP-loaded HA / F127 (DFO-NP / HA / F127, 30 / 7 / 30%). [Figure 7] Figure 1 shows in vivo longitudinal monitoring of DFO-NP release from various hydrogel formulations. (a) Representative color and NIR fluorescence images (up to 14 days post-injection) of CD-1 mice subcutaneously injected with DFO-NP and DFO-NP-loaded hydrogels (e.g., DFO-NP / F127, DFO-NP / HA, and DFO-NP / HA / F127). White arrowheads indicate remaining hydrogel on day 14. Exposure time is 50 ms. (b) Longitudinal profiling of the signal-to-background ratio (SBR) of the hydrogel injection site (region of interest, ROI) relative to the background signal (Bg) (n = 3 per group, mean ± sem). (c) SBR of skin and hydrogel relative to muscle after skin peeling on day 14. A p-value < 0.05 was considered significant. *p < 0.05, ****p < 0.001. [Figure 8]Figure 1 shows the biodistribution of DFO-NPs 14 days after injection. (a) Color and NIR fluorescence images of the abdomen, chest, and excised organs of mice subcutaneously injected with DFO-NPs and DFO-NP-loaded hydrogel formulations. (b) Signal-to-background ratios (SBRs) of excised organs and hydrogels relative to muscle. He: heart, Lu: lung, Li: liver, Pa / Sp: pancreas / spleen, Ki: kidney, Du: duodenum, In: intestine, Mu: muscle. [Figure 9] Pharmacokinetic analysis of DFO-NP and DFO-NP / F127, DFO-NP / HA, and DFO-NP / HA / F127 hydrogels. a) Plasma concentration decay curves, b) Area under the curve (AUC). [Figure 10] H&E stained images (10x) of the heart, lung, liver, spleen, and kidney in the saline group and the DFO-NP / HA / F127 group are shown. [Figure 11] Representative fluorescence images of a blood sample in a capillary tube at each time point are shown. [Figure 12] Figure 1 shows the results of in vivo toxicity tests in mice injected with saline and DFO-NP / HA / F127 hydrogel. a) H&E stained images (20x) of the heart, lung, liver, spleen, and kidney in each group. b) Serum aspartate transferase (AST), alanine transferase (ALT), and AST / ALT ratio. c) Blood urea nitrogen (BUN) and creatinine (CREA). Mice were sacrificed and analyzed 14 days after injection. A p value of <0.05 was considered significant. *p<0.05. [Figure 13] The therapeutic effect of DFO-NP-loaded hydrogel formulations in an animal model is shown. Male CD-1 mice were fed a 1% carbonyl iron diet for 1 week and then treated with a single subcutaneous injection of saline, blank NP / HA / F127 (84 μmol / kg as NP), or DFO-NP / HA / F127 (84 μmol / kg as NP). After drug administration, mice were fed a regular institutional diet. Organs were harvested 3 weeks after drug administration (n=3, mean ± SEM). A p-value <0.05 was considered significant. *p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0014] composition In some embodiments, the present disclosure provides a sustained release injectable composition comprising an iron chelator and at least one hydrogel.

[0015] In some embodiments, the iron chelator is deferoxamine (DFO), which has the formula: [ka]

[0016] In some embodiments, the iron chelator is a pharmaceutical salt of DFO, e.g., the mesylate salt. In some embodiments, the DFO is a DFO nanochelator (DFO-NP).

[0017] In some embodiments, the DFO-NP is a compound of formula AB. A is a group comprising 1, 2, 3, or 4 anionic groups, each of which is a sulfonate salt. In some embodiments, A comprises 1 or 2 anionic groups, each of which is a sulfonate salt. In some embodiments, A is selected from the group consisting of formulas A-1, A-2, A-3, A-4, A-5, and A-6. [ka] During the ceremony, [ka] indicates the bond between A and B, X is a bond, CH2, NH, -NH-C 1-6 is selected from the group consisting of alkylene-, O, and S, and each R A are independently selected anionic groups, and each R C are independently selected cationic groups, and L 1 , L 2 , and L 3are independently selected C 1-6 It is an alkylene group.

[0018] In some embodiments, A is selected from: [ka]

[0019] In some embodiments, A is: [ka]

[0020] In some embodiments, B is selected from the group consisting of a biocompatible polypeptide and a biocompatible polyester, each of which is substituted with one or more C groups and one or more -DE groups. In some embodiments, B is selected from the group consisting of polylysine, polylactic acid, poly(lactic-co-glycolic acid), polyaspartic acid, polyglutamic acid, and polyglutamic acid-poly(ethylene glycol) copolymer, each of which is substituted with one or more C groups and one or more -DE groups. In some embodiments, B is polylysine substituted with one or more C groups and one or more -DE groups. In some embodiments, polylysine is ε-poly-L-lysine substituted with one or more C groups and one or more -DE groups. In some embodiments, B is: [ka] During the ceremony, [ka] represents a bond between A and B, and n is an integer of 5 to 30.

[0021] In some embodiments, C is an anionic group of the formula: [ka] During the ceremony, [ka] represents a bond between C and B, and p is an integer of 1 to 10.

[0022] In some embodiments, D is a linking group of the formula: [ka] During the ceremony, [ka] represents a bond between D and B or a bond between D and E, and q is an integer from 1 to 10. In some embodiments, q is an integer from 1 to 5.

[0023] In some embodiments, E is selected from the group consisting of an iron-chelating group, a lead-chelating group, a copper-chelating group, an arsenic-chelating group, a mercury-chelating group, and a manganese-chelating group. In some embodiments, E is an iron-chelating group. In some embodiments, E is selected from the group consisting of dimercaptosuccinic acid, dimercaprol, ethylenediaminetetraacetic acid, p-aminosalicylic acid, D-penicillamine, deferoxamine, deferiprone, and deferasirox. In some embodiments, E is deferoxamine (DFO). In some embodiments, B is polylysine, E is an iron chelating group, and the polylysine is substituted with one or more C groups and one or more -DE groups. In some embodiments, the polylysine is ε-poly-L-lysine (i.e., EPL) substituted with one or more C groups and one or more -DE groups. In some embodiments, B is ε-poly-L-lysine, E is deferoxamine (i.e., DFO), and the ε-poly-L-lysine is substituted with one or more C groups and one or more -DE groups.

[0024] In some embodiments, the molar ratio (i.e., stoichiometry) of the metal chelator E (e.g., an iron chelator) to the biocompatible polymer B in the compound of Formula I is from about 10:1 to about 1:1, e.g., from about 10:1 to about 2:1, from about 10:1 to about 4:1, from about 10:1 to about 6:1, from about 10:1 to about 8:1, from about 8:1 to about 1:1, from about 8:1 to about 2:1, from about 8:1 to about 4:1, from about 8:1 to about 6:1, from about 6:1 to about 1:1, from about 6:1 to about 2:1, from about 6:1 to about 4:1, from about 4:1 to about 1:1, from about 4:1 to about 2:1, or from about 2:1 to about 1:1.

[0025] In some embodiments, the molar ratio (i.e., stoichiometry) of the metal chelator E (e.g., iron chelator) to the biocompatible polymer B in the compound of Formula I is about 2:1, about 4:1, about 6:1, or about 8:1.

[0026] In some embodiments, the hydrogel comprises a hyaluronic acid matrix. The term "hyaluronic acid matrix" encompasses all variants and combinations of hyaluronic acid or hyaluronan of various chain lengths and charge states, as well as various chemical modifications. In some embodiments, the hyaluronic acid matrix is ​​chemically unmodified hyaluronic acid or hyaluronate. In some embodiments, the hyaluronic acid matrix is ​​sodium hyaluronate. In some embodiments, the hyaluronic acid matrix is ​​hyaluronic acid (HA). HA is a disaccharide polymer composed of D-glucuronic acid and DN-acetylglucosamine, linked by alternating β-1,4 and β-1,3 glycosidic bonds.

[0027] In some embodiments, the hyaluronic acid substrate has an average molecular weight in the range of 0.1-10 MDa. In some embodiments, the hyaluronic acid substrate has an average molecular weight in the range of 0.8-5 MDa. In some embodiments, the hyaluronic acid substrate has an average molecular weight more preferably in the range of 1.5-3 MDa. In some embodiments, the hyaluronic acid substrate has an average molecular weight in the range of 2-3 MDa. In some embodiments, the hyaluronic acid substrate is obtained from a non-animal source. In some embodiments, the hyaluronic acid substrate is obtained from bacteria.

[0028] In some embodiments, the HA is crosslinked. In some embodiments, the hyaluronic acid is crosslinked with one or more multifunctional crosslinkers selected from the group consisting of divinyl sulfone, multiepoxides, and diepoxides. In some embodiments, the multifunctional crosslinker is selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxyoctane. In some embodiments, the multifunctional crosslinker is 1,4-butanediol diglycidyl ether (BDDE). It is desirable that the one or more multifunctional crosslinkers provide ether crosslinks.

[0029] In some embodiments, the hyaluronic acid product is singly crosslinked. A singly crosslinked product has the advantage of being chemically well-defined. In some embodiments, the shaped hyaluronic acid product is multiply crosslinked. In certain embodiments, the shaped hyaluronic acid product is crosslinked by ether crosslinks. The ether crosslinked hyaluronic acid gel products of the present disclosure are stable and can be easily sterilized, for example, by autoclaving.

[0030] In some embodiments, the hydrogel further comprises a poloxamer, which is a non-ionic triblock copolymer consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide) (PPO)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide) (PEO)). In some embodiments, the poloxamer is selected from the group consisting of pluronic L31, L35, F38, L42, L43, L44, L61, L62, L63, L64, P65, F68, L72, P75, F77, L81, P84, P85, F87, F88, L92, F98, L101, P103, P104, P105, F108, L121, L122, L123, F127, 10R5, 10R8, 12R3, 17R1, 17R2, 17R4, 17R8, 22R4, 25R1, 25R2, 25R4, 25R5, 25R8, 31R1, 31R2, 31R, and mixtures thereof. In some embodiments, the poloxamer is poloxamer 407 (Pluronic F127). Pluronic® F127 poloxamer is a synthetic triblock copolymer (PEO 99 -PPO 67 -PEO 99 )

[0031] Pluronic F127 and HA were selected as materials for the injectable hydrogel formulation because they are FDA-approved and widely used in various fields, including drug delivery systems. Pluronic F127 exhibits a unique thermosensitive sol-gel transition, driven by a micellar mechanism, when concentrated in water. HA has been clinically used as a dermal filler and drug delivery vehicle due to its excellent biocompatibility, minimal toxicity, and low inflammatory potential. Therefore, HA was introduced into the hydrogel to control the initial burst release of F127.

[0032] In some embodiments, the composition comprises about 15% to about 45% by weight of the iron chelator. In some embodiments, the composition comprises about 25% to about 35% by weight of the iron chelator. In some embodiments, the composition comprises about 28% to about 32% by weight of the iron chelator. In some embodiments, the composition comprises about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or 45% by weight of the iron chelator. In some embodiments, the composition comprises about 30% by weight of the iron chelator. In some embodiments, the composition comprises about 30% by weight of DFO-NP.

[0033] In some embodiments, the composition comprises about 1% to about 20% by weight of the hyaluronic acid substrate. In some embodiments, the composition comprises about 5% to about 15% by weight of the hyaluronic acid substrate. In some embodiments, the composition comprises about 6% to about 10% by weight of the hyaluronic acid substrate. The composition comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% by weight of the hyaluronic acid substrate. In some embodiments, the composition comprises about 7% by weight of the hyaluronic acid substrate. In some embodiments, the composition comprises about 7% by weight of HA.

[0034] In some embodiments, the composition comprises about 15% to about 45% by weight of poloxamer, in some embodiments, about 25% to about 35% by weight of poloxamer, in some embodiments, about 28% to about 32% by weight of poloxamer. In some embodiments, the composition comprises about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or 45% by weight of poloxamer. In some embodiments, the composition comprises about 30% by weight of poloxamer. In some embodiments, the composition comprises about 30% by weight of F127.

[0035] In some embodiments, the composition comprises about 1% to about 70% water by weight. In some embodiments, the composition comprises about 5% to about 15% water by weight. In some embodiments, the composition comprises about 10% to about 25% water by weight. In some embodiments, the composition comprises about 20% to about 35% water by weight. In some embodiments, the composition comprises about 30% to about 45% water by weight. In some embodiments, the composition comprises about 40% to about 55% water by weight. In some embodiments, the composition comprises about 50% to about 65% water by weight. In some embodiments, the composition comprises about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 16%, about 18%, about 20%, about 20%, about 20%, about 20%, about 20%, about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, about 42%, about 44%, about 46%, about 48%, about 50%, about 52%, about 54%, about 56%, about 58%, about 60%, about 62%, about 64%, about 66%, about 66%, or about 70% water by weight. In some embodiments, the composition comprises about 33% water by weight.

[0036] In some embodiments, the composition comprises 30% by weight poloxamer, 7% by weight HA, 30% by weight DFO-NP, and 33% by weight water.

[0037] Treatment method In some embodiments, the present disclosure provides a sustained release injectable composition for iron chelation therapy in a human patient, comprising an iron chelator and a hydrogel, wherein the composition is releasable over a two-week period. In some embodiments, the present disclosure provides a sustained release injectable composition for iron chelation therapy in a human patient, comprising an iron chelator and a hydrogel, wherein the composition is released over a two-week period.

[0038] Some embodiments provide methods of treating iron overload in a patient in need thereof. The method comprises administering to the patient an effective amount of a composition described herein. Other embodiments provide methods of treating acute kidney injury in a patient in need thereof. See, e.g., Leaf and Swinkels, Catalytic Iron and Acute Kidney Injury, Am J Physoi Renal Physiol 2016 Nov 1;311(5):F871-F876. The method comprises administering to a patient with acute kidney injury an effective amount of a composition described herein.

[0039] The present application further provides a method for chelating metal ions in a sample (e.g., a cell or tissue sample) or in a subject, comprising contacting the sample or administering to the subject a compound provided herein (e.g., a compound of Formula I) or a pharmaceutically acceptable salt thereof.

[0040] As used herein, the term "metal ion" refers to free metal ions in a sample (e.g., a cell or tissue sample) or in a subject, or metal ions bound to a low affinity ligand (e.g., citrate), or a combination thereof.

[0041] As used herein, the term "subject" or "patient" refers to any animal (such as a mammal). Exemplary subjects include, without limitation, mice, rats, rabbits, dogs, cats, pigs, cows, sheep, horses, primates, and humans. In some embodiments, the subject is a human. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a composition described herein.

[0042] In some embodiments, the method is a method of chelating a metal ion in a cell or tissue sample, comprising contacting the cell or tissue sample with a composition described herein, in some embodiments, the contacting forms a metal compound chelate.

[0043] The present disclosure further provides methods for reducing the amount of free metal ions in a cell or tissue sample, comprising contacting the cell or tissue sample with a composition described herein, in some embodiments, the contacting forms a metal compound chelate, thereby reducing the amount of free metal ions in the cell or tissue sample.

[0044] The present disclosure further provides methods for chelating metal ions in a subject, comprising administering to the subject a therapeutically effective amount of a composition described herein. In some embodiments, the metal ions are free metal ions or metal ions bound to a low affinity ligand (e.g., citrate).

[0045] The present disclosure further provides a method for reducing the amount of free metal ions in a subject, the method comprising administering to the subject a composition described herein.

[0046] The present disclosure further provides methods for reducing the amount of metal ions in the bloodstream of a subject in need of a reduction in the amount of metal ions (e.g., free metal ions or metal ions bound to a low-affinity ligand (e.g., citrate)). In some embodiments, the method is a method for reducing the amount of iron ions (e.g., free iron ions or iron ions bound to a low-affinity ligand (e.g., citrate)) in a subject in need of a reduction in the amount of iron ions in the bloodstream. In some embodiments, the subject has been determined to have a high level of iron ions (e.g., free iron ions or iron ions bound to a low-affinity ligand (e.g., citrate)) in their bloodstream compared to a subject with a normal level of iron ions in their bloodstream.

[0047] The present disclosure further provides a method of reducing iron in a subject in need thereof, comprising administering to the subject a composition described herein. In some embodiments, the method is a method of reducing iron overload in a subject in need thereof.

[0048] The present disclosure further provides a method for reducing the amount of metal ions bound to low affinity ligands in a subject, the method comprising administering to the subject a composition described herein.

[0049] The present disclosure further provides methods for treating a disease associated with an abnormal amount of metal ions in a subject. In some embodiments, the disease is associated with an abnormal amount of free metal ions, an abnormal amount of metal ions bound to a low-affinity ligand (e.g., citrate), or a combination thereof. In some embodiments, the method comprises administering to the subject a compound provided herein, or a pharmaceutically acceptable salt thereof.

[0050] In some embodiments, the disease is associated with an abnormally high amount of metal ions (e.g., free metal ions, metal ions bound to low-affinity ligands, or a combination thereof) in a subject compared to subjects with normal levels of metal ions. In some embodiments, the disease is associated with an abnormally high amount of free metal ions in a subject compared to subjects with normal levels of free metal ions. In some embodiments, the disease is associated with an abnormally high amount of metal ions bound to low-affinity ligands in a subject compared to subjects with normal levels of metal ions bound to low-affinity ligands in a subject. In some embodiments, the disease is associated with an abnormally high amount of free metal ions in combination with metal ions bound to low-affinity ligands in a subject compared to subjects with normal levels of free metal ions and metal ions bound to low-affinity ligands.

[0051] In some embodiments, an abnormal amount of a metal ion in a subject refers to an increase in the concentration of the metal ion in the subject of about 5% to about 100% compared to the concentration of the metal ion in a normal subject, for example, an increase in the concentration of the metal ion in the subject of about 5% to about 100%, about 5% to about 75%, about 5% to about 50%, about 5% to about 25%, about 5% to about 10%, about 10% to about 100%, about 10% to about 75%, about 10% to about 50%, about 10% to about 25%, about 25% to about 100%, about 25% to about 75%, about 25% to about 50%, about 50% to about 100%, about 50% to about 75%, or about 75% to about 100% compared to the concentration of the metal ion in a normal subject.

[0052] In some embodiments, an abnormal amount of a metal ion in a subject refers to an increase in the concentration of the metal ion in the subject that is about 2-fold to about 10-fold compared to the concentration of the metal ion in a normal subject, for example, an increase in the concentration of the metal ion in the subject that is about 2-fold to about 10-fold, about 2-fold to about 8-fold, about 2-fold to about 5-fold, about 2-fold to about 3-fold, about 3-fold to about 10-fold, about 3-fold to about 8-fold, about 3-fold to about 5-fold, about 5-fold to about 10-fold, about 5-fold to about 8-fold, or about 8-fold to about 10-fold compared to the concentration of the metal ion in a normal subject.

[0053] Methods for determining the concentration of metal ions in a subject are routine in the art and include, for example, measuring metal ions in cell samples (e.g., NIR microscopy) or tissue samples (e.g., biopsy samples by NIR spectroscopy) and / or measuring metal ions in a subject using imaging techniques (e.g., magnetic resonance imaging and / or optical fluorescence imaging).

[0054] The present application further provides a method for treating a disease associated with an abnormal amount of free metal ions in a subject. In some embodiments, the method comprises administering to the subject a compound provided herein or a pharmaceutically acceptable salt thereof.

[0055] In some embodiments, the disease is associated with abnormal amounts of iron ions, abnormal amounts of lead ions, abnormal amounts of copper ions, abnormal amounts of arsenic ions, abnormal amounts of manganese ions, abnormal amounts of cadmium ions, abnormal amounts of nickel ions, abnormal amounts of chromium ions, abnormal amounts of gold ions, or abnormal amounts of antimony ions, or any combination thereof, in some embodiments, the disease is associated with abnormal amounts of iron ions, abnormal amounts of lead ions, or abnormal amounts of copper ions, or any combination thereof, in the subject.

[0056] In some embodiments, the disease is associated with an abnormal amount of iron ions, an abnormal amount of lead ions, or an abnormal amount of copper ions in the subject. In some embodiments, the disease is associated with an abnormal amount of iron ions in the subject.

[0057] In some embodiments, the disease is selected from the group consisting of transfusion hemosiderosis (e.g., resulting from a blood transfusion in a subject with one or more diseases selected from the group consisting of thalassemia, myelodysplastic syndrome, sickle cell anemia, and Blackfan Diamond anemia), hemochromatosis (e.g., inherited or acquired), Wilson's disease, copper poisoning, and heavy metal poisoning (e.g., lead poisoning, mercury poisoning, cadmium poisoning, arsenic poisoning, manganese poisoning, etc.).

[0058] As used herein, the term "treating" or "treatment" refers to one or more of the following: (1) inhibiting a disease, e.g., inhibiting a disease, condition, or disorder (i.e., preventing further development of the condition and / or symptom) in an individual experiencing or exhibiting a symptom or pathology of the disease, condition, or disorder, and (2) ameliorating a disease, e.g., ameliorating a disease, condition, or disorder (i.e., causing the condition and / or symptom to improve) in an individual experiencing or exhibiting a symptom or pathology of the disease, condition, or disorder, e.g., reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease.

[0059] The term "effective amount," as used herein, refers to the amount of agent(s) necessary to alleviate at least one or more symptoms of a disease or disorder, and refers to a sufficient amount of a pharmacological composition to provide a desired effect (e.g., alleviating iron overload). An effective amount, as used herein, would also include an amount sufficient to delay the onset of, alter the course of, or reverse a disease symptom (e.g., without limitation, slow the progression of a disease symptom). Thus, an exact "effective amount" cannot be specified.

[0060] The compositions described herein are isolated agents, i.e., the agents are substantially pure and substantially free of other substances with which they may be found in nature or in vivo systems to a degree practical and appropriate for their intended use. In particular, the agents are sufficiently pure and sufficiently free of other components to be useful, for example, in the manufacture of pharmaceutical formulations. Because isolated compositions can be combined with a pharmaceutically acceptable carrier in a pharmaceutical formulation, the composition (i.e., the active agent) may constitute only a small weight percent of the formulation.

[0061] In some embodiments, the compositions described herein are administered to a subject, preferably by injection or infusion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In preferred embodiments, compositions for use in the methods described herein are administered by intravenous infusion or injection. The terms "parenteral administration" and "administered parenterally," as used herein, refer to methods of administration (usually by injection) other than enteral and topical administration. The terms "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered," as used herein, refer to administration of a drug other than direct administration into a target site, tissue, or organ, such that the drug enters the subject's circulatory system and is therefore subject to systemic metabolism and other similar processes.

[0062] For clinical use of the methods described herein, administration of the agent or composition can include formulation into a pharmaceutical composition or formulation for parenteral administration (e.g., intravenous or other modes of administration). In some embodiments, the agent can be administered with any pharmaceutically acceptable carrier compound, material, or composition that provides effective treatment in the subject. Thus, pharmaceutical formulations for use in the methods described herein can include an agent described herein in combination with one or more pharmaceutically acceptable ingredients.

[0063] The phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle (e.g., liquid or diluent, excipient, solvent) that is responsible for maintaining the stability, solubility, or activity of the composition. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can function as pharmaceutically acceptable carriers include the following: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) excipients, such as cocoa butter and suppository wax; (8) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (9) glycols, such as propylene glycol; (10) polio (11) esters, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG), (12) esters, such as ethyl oleate and ethyl laurate, (13) buffers, such as magnesium hydroxide and aluminum hydroxide, (14) alginic acid, (15) unpasteurized water, (16) isotonic saline, (17) Ringer's solution, (19) pH buffer solutions, (20) polyesters, polycarbonates, and / or polyanhydrides, (21) bulking agents, such as polypeptides and amino acids, (22) serum components, such as serum albumin, HDL, and LDL, (23) C2-C12 alcohols, such as ethanol, and (24) other non-toxic compatible substances used in pharmaceutical formulations. Release agents, coating agents, preservatives, and antioxidants may also be present in the formulation.Terms such as "excipient," "carrier," and "pharmaceutically acceptable carrier" are used interchangeably herein. The agents described herein can be specially formulated for administering the compound to a subject in liquid form, including, for example, as a sterile solution or suspension, or as a sustained-release formulation adapted for parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection. Parenteral dosage forms of the agent can also be administered to a subject by various routes (e.g., without limitation, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial). Because administration of parenteral dosage forms typically circumvents a patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized before administration to a patient. Examples of parenteral dosage forms include, without limitation, solutions ready for injection, dry products ready for dissolution or suspension in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, controlled-release parenteral dosage forms, and emulsions. Suitable vehicles that can be used to provide parenteral dosage forms of the present disclosure are well known to those skilled in the art. Examples include, without limitation, the following: Sterile water, Water for Injection USP, saline, glucose solution, aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection, water-miscible vehicles (such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol), and non-aqueous vehicles (such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate).

[0064] Process for preparing the composition In yet another aspect, the present disclosure includes a method for making a sustained release injectable composition useful for iron chelation therapy.

[0065] "Sustained release" or "sustained release" means that iron chelation therapy or an iron chelator is released from a composition at a controlled rate, thereby maintaining therapeutically useful (but below toxic) blood levels of the iron chelator for an extended period of time. Alternatively, "sustained release" or "sustained release" means that a desired pharmacological effect is maintained for an extended period of time. Experiments using the compositions described herein surprisingly revealed that the duration of delivery of the iron chelator was longer than expected. However, experiments described herein surprisingly revealed that release of the iron chelator from experimental injectable compositions occurred over a period of 1 to 30 days, at least two weeks, and in some experiments, longer than two weeks. In some cases, the iron chelating effect was at least about two weeks, allowing for the possibility of fewer dosing cycles not previously thought possible.

[0066] In some embodiments, the sustained release of iron chelators from the compositions disclosed herein is for a period of 1 day to about 30 days. In some embodiments, the sustained release of iron chelators from the compositions disclosed herein is for a period of about 10 days to about 21 days. In some embodiments, the sustained release of iron chelators from the compositions disclosed herein is for a period of about 14 days to about 21 days. In some embodiments, the sustained release of iron chelators from the compositions disclosed herein is for a period of about 10 days to about 15 days. In some embodiments, the sustained release of iron chelators from the compositions disclosed herein is for a period of at least 5 days. In some embodiments, the sustained release of iron chelators from the compositions disclosed herein is for a period of at least 10 days. In some embodiments, the sustained release of iron chelators from the compositions disclosed herein is for a period of at least 12 days. In some embodiments, the sustained release of iron chelators from the compositions disclosed herein is for a period of at least 14 days (2 weeks). In some embodiments, the sustained release of iron chelators from the compositions disclosed herein is for a period of at least 16 days. In some embodiments, the sustained release of iron chelator from the compositions disclosed herein is at least 18 days. In some embodiments, the sustained release of iron chelator from the compositions disclosed herein is at least 20 days. In some embodiments, the sustained release of iron chelator from the compositions disclosed herein is at least 21 days (3 weeks). In some embodiments, the sustained release of iron chelator from the compositions disclosed herein is at least 22 days. In some embodiments, the sustained release of iron chelator from the compositions disclosed herein is at least 24 days. In some embodiments, the sustained release of iron chelator from the compositions disclosed herein is at least 26 days. In some embodiments, the sustained release of iron chelator from the compositions disclosed herein is at least 28 days. In some embodiments, the sustained release of iron chelator from the compositions disclosed herein is at least 30 days.

[0067] In some embodiments, the present disclosure provides a method for preparing a sustained release injectable composition useful for metal chelation therapy, comprising: a) cross-linking the hyaluronic acid matrix; b) forming the cross-linked hyaluronic acid matrix into particles; c) adding particles of crosslinked hyaluronic acid matrix to a solution of a metal chelator; d) further adding a poloxamer to the solution resulting from step c).

[0068] In some embodiments, the present disclosure provides a method for preparing a metal chelated hydrogel composition, comprising: a) cross-linking the hyaluronic acid matrix; b) forming the cross-linked hyaluronic acid matrix into particles; c) freeze-drying the particles; d) adding particles of crosslinked hyaluronic acid matrix to a solution of a metal chelator; e) adding further poloxamer to the solution resulting from step d); f) isolating the hydrogel comprising the metal chelator loaded within crosslinked hyaluronic acid matrix particles combined with poloxamer.

[0069] In some embodiments, the hyaluronic acid substrate is HA. In some embodiments, the metal chelator is DFO or DFO-NP. In some embodiments, the poloxamer is F127.

[0070] In some embodiments, an injectable hydrogel was prepared by incorporating cross-linked hyaluronic acid (xHA) into Pluronic F127 (F127) (both FDA-approved) to load a high dose of DFO-NPs and reduce the initial burst release. The hydrogel exhibited thermosensitive rheological properties, and its release kinetics was evaluated over time using a near-infrared (NIR) fluorescence imaging system. This sustained-release hydrogel formulation achieves long-term release of the iron nanochelator with short retention in non-target tissues, thereby improving therapeutic efficacy while minimizing toxicity. This exceeds the capabilities of current small molecule chelators. Considering the need for lifelong administration of chelators for patients with iron overload, hydrogel-based nanochelators offer significant advantages over current chelation therapies. This injectable hydrogel formulation exhibited a thermosensitive sol-gel transition at body temperature and released the renally clearable iron nanochelator over a two-week period, resulting in a half-life 47-fold longer than that of the nanochelator alone. [Example]

[0071] Materials: Epsilon-poly-L-lysine (EPL, MW approximately 3,900) was purchased from BOC Sciences (Shirley, NY). Ninhydrin reagent, hyaluronic acid (100 kDa), and succinic anhydride (SA) were purchased from Acros Organics (Morris Plains, NJ). Ethyl acetate (EA), deuterium oxide (DO), anhydrous dimethyl sulfoxide (DMSO), Pluronic F127, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), ferric chloride, sodium acetate, and sodium hydroxide were purchased from Sigma-Aldrich (Saint Louis, MO). DFO and assay reagent kits (for aspartate aminotransferase, alanine aminotransferase, and serum creatinine) were purchased from Cayman Chemical (Ann Arbor, MI).

[0072] Example 1: Synthesis and characterization of DFO-NPs DFO-NPs were prepared as described in WO 2018 / 147894, which is incorporated herein by reference in its entirety.

[0073] Before designing the injectable hydrogel formulation, DFO-NPs were prepared. To monitor the in vivo behavior of DFO-NPs, a zwitterionic NIR fluorophore (ZW800-1C) was attached to ε-poly-L-lysine (ZW-EPL). + ) conjugated to ZW-EPL. + All primary amines in ZW-EPL were succinylated. - ), converting the side chain to a negatively charged carboxylate group, thus forming ZW-EPL - DFO conjugation via an amide bond was then possible (DFO-NP). See Scheme 1 below. [ka] Scheme 1 ZW800-1C Conjugate EPL (ZW-EPL + ) synthesis: Preparation of ZW800-1C conjugate EPL. 1 g of epsilon poly-L-lysine (EPL) was dissolved in 100 mL of phosphate-buffered saline. The pH of the EPL solution was adjusted to approximately 8.0 with 6 M NaOH solution. ZW800-1C-succinic acid amide ester (ZW800-1C-NHS) was added at 25 mg mL. -1 ZW800-1C-NHS was dissolved in DMSO at a concentration of 0.5 mL. 5 mL of the ZW800-1C-NHS solution was added dropwise to the EPL solution under vigorous stirring at room temperature. After 3 hours, the reaction mixture was added to 1 L of acetone / ethyl acetate (EA) (4 / 1) to precipitate ZW800-EPL. The mixture was centrifuged at 3000 rpm at 4°C for 15 minutes. The supernatant was discarded, and the precipitate was redissolved in DW (>50 mL) and reprecipitated in 1 L of acetone / EA. The precipitation was repeated two more times to complete the purification. The final precipitate was dried overnight in vacuum.

[0074] Succinylated ZW800-EPL (ZW-EPL - ) synthesis: ZW800-EPL + ZW-EPL (1 g, 0.2 mmol) and EPL (19 g, 4.75 mmol) were dissolved together in 2 L of PBS. 45 g of succinic acid (450 mmol) in 180 mL of DMSO (250 mg / mL) was added to the ZW-EPL solution. The pH of the reaction mixture was adjusted to approximately 7.0 with 6 M NaOH solution, if necessary. The reaction mixture was stirred at room temperature for 30 minutes. The succinylation rate was then confirmed by the ninhydrin test. For purification, the reaction mixture was precipitated using the same procedure as described in the previous section.

[0075] Synthesis of renally clearable nanochelator (DFO-NP): 50 g of deferoxamine (76 mmol) was dissolved in 500 mL of DW. The DFO solution was neutralized by adding 6 M NaOH solution. 20 g of ZW-EPL - (2.5 mmol) was dissolved in 1.25 L of DW. 42 g of DMTMM and the prepared DFO solution were added to the ZW-EPL - The reaction mixture was stirred at 60°C for 2 hours and then dialyzed against DW using a cellulose dialysis membrane with a MWCO of 6 to 8 kDa. After dialysis, the solution was freeze-dried.

[0076] 1 H-NMR analysis: To determine the DFO conjugation ratio on the nanochelators, 10 mg of DFO-NPs was dissolved in 600 μL of DO. 1 H-NMR spectroscopy was performed on a Varian 500 MHz spectrometer. The number of DFOs on succinylated EPL was calculated by comparing the peak integrals of the protons of succinylated EPL at 4.1 ppm with the e, l, and s protons of DFO at 3.6 ppm (Figure 2).

[0077] Each product was analyzed and quantified by nuclear magnetic resonance (NMR) spectroscopy to have a DFO number of approximately 1.0 on the DFO-NPs (see Figure 2). The peaks at 4.12 ppm (position α′) and 3.6 ppm (positions e, l, s) correspond to the EPL and DFO protons, respectively.

[0078] Optical properties of DFO-NP: DFO-NP was dissolved in DW at a concentration of 100 μM. Absorbance and fluorescence emission spectra were observed from 500 nm to 1000 nm using a UV / Vis / NIR spectrometer (USB2000, Ocean Insight, Dunedin, FL). For fluorescence emission spectra, a 760 nm laser (Nawoo, Gwangju, Korea) was used for excitation.

[0079] Size-exclusion chromatography analysis: To measure the purity and hydrodynamic diameter (HD) of DFO-NPs, size-exclusion chromatography (SEC) was performed using a Waters HPLC system consisting of a Waters e2695 separation module and a Waters 2998 PDA detector. The column used was an Xbridge BEH 125Å 3.5 μm (7.8 x 150 mm, Waters) SEC column. The mobile phase was 10 mM PBS at 0.75 mL min. -1 The reaction mixture was isocratic for 15 minutes at a flow rate of 1000 kJ / min. Each component in the reaction mixture could be identified by its retention time and absorbance wavelength.

[0080] A standard calibration curve for HD was calculated by injecting 10 μl of protein standards containing aprotinin (6.5 kDa, 1.96 nm), ribonuclease (13.7 kDa, 3.28 nm), ovalbumin (44 kDa, 6.10 nm), and thyroglobulin (669 kDa, 9.6 nm) into the HPLC using the same mobile phase and flow rate as above. av was obtained from the following equation (1).

[0081] K av (V e -V0) / (V c-V0) (1) In the formula, V0, V c , and V e are the column void volume, geometric column volume, and eluent volume, respectively. The HD of DFO-NP was calculated by the following equation (2):

[0082]

number

[14]

[0083] The HD of DFO-NP was calculated to be 5.2 nm based on a calibration curve of a set of known standard proteins, suggesting that DFO-NP is renal clearable (see Figure 3). Furthermore, the purity of DFO-NP was confirmed to be 96%, suggesting the absence of impurities such as unconjugated DFO and ZW800-1C. The optical properties and iron-binding effect of DFO-NP were evaluated by spectrophotometry.

[0084] The absorption and fluorescence emission maxima for DFO-NP were 760 and 780 nm, respectively, as can be seen in Figure 4a. This allows DFO-NP to be detected in the NIR channel with minimal tissue scattering and autofluorescence. The iron-binding effect of DFO-NP was confirmed in an in vitro iron-binding assay using ferric chloride (FeCl3). 3+ The chelation of DFO-NPs with iron could be easily observed by measuring UV-Vis absorbance, since the complex with the ion absorbs at a wavelength of 430 nm.

[0085] The sequential addition of 5 μL of FeCl3 solution (4 mM) to the DFO-NP solution (1000 μL, 100 μM) resulted in an increase in the absorbance at 430 nm (see Figure 4b). After adding 25 μL of FeCl3 solution, which is equimolar to the DFO-NP solution, the increase in absorbance at 430 nm was shown to decrease (Figure S4c, Supporting Information). These results indicate that DFO can induce the absorption of Fe 3+ Due to the formation of monodentate complexes with ions, the stoichiometry of DFO-NP is shown to be approximately 1.0, which is consistent with the 1H-NMR results for proton quantification.

[0086] Example 2: In vitro iron chelating effect of DFO-NP To confirm the iron chelating effect of DFO-NP, a solution of DFO-NP was prepared at a concentration of 100 μM. 5 μL of iron chloride solution (4 mM) was added to the DFO-NP solution to measure the iron chelating effect. 3+ The absorbance change at 430 nm, which is the absorbance value of the complex between α- and DFO, was continuously measured. The functional group stoichiometry was calculated based on the titration curve generated by using the absorbance change at 430 nm.

[0087] Cell culture: NIH3T3 and H23 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Mediatech, Herndon, VA) containing 10% fetal bovine serum and 1% penicillin-streptomycin. NIH3T3 and H23 cells were cultured in 75 cm 2 The cells were incubated in tissue culture flasks (Corning, NY) at 37°C under 5% CO2.

[0088] In vitro cellular uptake and cytotoxicity of DFO-NPs: NIH3T3 (1 × 10 4 ) cells and H23 (1 × 10 4) cells were seeded in 96-well plates (Corning, NY) and cultured for 24 hours. Cells were washed twice with 200 μL of PBS. 100 μL of fresh complete medium containing ZW800-EPL+ (5 μM) or DFO-NP (5 μM) was added to each well. Cells were incubated for 30 minutes and 2 hours. Untreated cells served as controls. At the designated time points, cells were washed with PBS and subsequently fixed with 4% paraformaldehyde. Fixed cells were observed in the bright and near-infrared fluorescence channels using Cytation 5 (BioTek, Winooski, VT) and a NanoenTek JuLi Stage (Seoul, South Korea).

[0089] For cytotoxicity tests, NIH3T3 cells were treated with DFO-NPs ranging from 1 to 100 μM. After 24 hours, the NIH3T3 cells were washed twice with PBS. 100 μL of fresh complete medium was added to each well. Then, 10 μL of CCK8 solution was added to each well. After 4 hours, the absorbance was measured at 450 nm using a microplate reader (Cytation 5). All experiments were performed in triplicate.

[0090] Next, an in vitro cellular uptake experiment was performed to confirm the non-adhesiveness of DFO-NPs within cells (see Figure 5a). NIH3T3 cells and NCL-H23 (H23) cells were selected as typical normal and cancer cells, respectively, and positively charged ZW-EPL + was used as a positive control. + The treated cells showed a strong NIR fluorescence signal after 2 hours, whereas the DFO-NP-treated cells showed no signal, suggesting that the negatively charged surface of DFO-NP minimizes nonspecific cellular uptake. Furthermore, the cytotoxicity of DFO-NP was tested by incubating NIH3T3 cells with various concentrations of DFO-NP (1, 5, 10, 50, and 100 μM) for 24 hours, demonstrating no cytotoxicity associated with DFO-NP. See Figure 5b.

[0091] Example 3: Preparation and characterization of injectable hydrogels To begin the hydrogel formulation process, HA was cross-linked with 1,4-butanediol diglycidyl ether (BDDE), see Scheme 2 below. [ka] Scheme 2 Preparation of crosslinked HA: 100 mg of HA was dissolved in 600 μL of 0.3 M NaOH solution. 20 μL of 1,4-butanediol diglycidyl ether (BDDE) was added to the HA solution. The reaction mixture was vortexed for 1 minute and incubated at 40°C for 2 hours. The mixture was then neutralized to pH 7.0 with 0.1 M HCl. The neutralized reaction mixture was dialyzed against deionized water (DW) using a 6-8 kDa molecular weight cutoff (MWCO) cellulose dialysis membrane to remove residual BDDE. After dialysis, the HA hydrogel was dissolved to injectable particle sizes using a syringe with an 18-23 G needle. The injectable crosslinked HA hydrogel particles (xHA) were then freeze-dried.

[0092] Because coarse xHA is barely injectable, it was crushed by applying pressure through a 23-25 ​​gauge needle, resulting in crushed xHA microparticles (HAP), which could be injected using a syringe with a 23G needle. The HAP was freeze-dried and then immersed in a DFO-NP solution to load DFO-NP into the HAP. The dried DFO-NP-loaded HAP was added to a Pluronic F127 solution to obtain the final hydrogel formulation. The final weight percentages of DFO-NP, HAP, and Pluronic F127 were 30, 7, and 30 (denoted as DFO-NP / HA / F127:30 / 7 / 30%), and the final concentration of DFO-NP in the formulation was 40 mM. To compare the effects of hybrid HA and F127 hydrogels, DFO-NP / HA:30 / 7% and DFO-NP / F127:30 / 30% were prepared as controls. All of the prepared formulations were injectable using a syringe equipped with a 23G needle.

[0093] To evaluate the thermosensitivity of the prepared hydrogels, their rheological properties (e.g., loss modulus, storage modulus, and viscosity) were measured over a temperature gradient from 4 to 40 °C (Figure 6). Because a high drug content in a thermosensitive hydrogel generally suppresses its thermogelation properties, the corresponding hydrogels without DFO-NP (HA: 7%, F127: 30%, and HA / F127: 7 / 30%) were also tested to confirm the effect of DFO-NP on gelation. HA and DFO-NP / HA did not show significant changes in their rheological properties as the temperature increased (Figure 6a, b), with the storage and loss moduli remaining below 1,000 Pa. F127 showed a continuous increase in storage modulus and viscosity over the temperature range from 14 to 32 °C, indicating a thermosensitive sol-gel transition at 15 °C (Figure 2c, d). In contrast, no temperature-responsive behavior was observed in DFO-NP / F127. This indicates that a high content of DFO-NP prevented the hydrophobic aggregation of Pluronic micelles. Interestingly, both HA / F127 and DFO-NP / HA / F127 exhibited thermogelation properties (Figure 6e, f). For HA / F127, all rheological parameters significantly increased above 25 °C. This is attributed to the reinforcement by the HAP network. Despite the high DFO-NP content, the elastic modulus and viscosity values ​​for DFO-NP / HA / F127 were higher than those for HA / F127 at low temperatures (below 25 °C). Furthermore, DFO-NP / HA / F127 exhibited a thermosensitive rheological change above 30 °C, with the storage modulus increasing to 8,500 Pa. These results suggest that DFO-NP / HA / F127 is not only injectable but also capable of forming firm hydrogels at body temperature due to its high elastic modulus.

[0094] Example 4: In vivo release and pharmacokinetics of DFO-NP-loaded injectable hydrogels Preparation of DFO-NP-loaded injectable hydrogel: DFO-NP (prepared as described in WO 2018 / 147894, the disclosure of which is incorporated herein by reference) was added to 1 g mL -1DFO-NP was dissolved in DW at a concentration of 0.75 mg. DFO-NP-loaded xHA was prepared by adding 70 mg of xHA to 300 μL of DFO-NP solution. The DFO-NP-loaded xHA was lyophilized. The lyophilized DFO-NP-loaded xHA was added to 1 mL of DW (DFO-NP / HA) or 1 mL of 30 wt% Pluronic F127 solution (DFO-NP / HA / F127) on ice before use. 300 mg of DFO-NP was added to 1 mL of 30 wt% F127 (DFO-NP / F127).

[0095] Rheological characterization of hydrogels: The rheological properties of the hydrogel formulations were analyzed using a Haake Viscotester IQ Rheometer (Thermo Scientific, Germany) equipped with a Peltier temperature-controlled base plate and a 25.0 mm stainless steel parallel plate measurement system. All measurements were performed at 4-40°C with a 0.5 mm gap. The temperature was 0.03°C s -1 The γ strain and frequency were set to 0.01 and 1 Hz, respectively.

[0096] In vivo pharmacokinetics of DFO-NPs released from hydrogel formulations: Animals were housed in an AAALAC-accredited facility and studied under the supervision of the MGH IACUC according to approved institutional protocols (2016N000136). Prior to injection of DFO-NPs and DFO-NP-loaded hydrogel formulations, 6-week-old CD-1 mice (male, 25-30 g) (Charles River Laboratories, Wilmington, MA) were anesthetized with isoflurane and oxygen, and a small cut was made at the tip of the tail. A capillary tube (Fisher) was inserted at 0 min. Blood samples were collected at a laboratory (Scientific, Pittsburgh, PA). For subcutaneous injection, DFO-NP was dissolved in DW at a concentration of 30% by weight, and DFO-NP / F127, DFO-NP / HA, and DFO-NP / HA / F127 were prepared as described above. Mice were divided into four groups (n = 3). 100 μL of each formulation was injected subcutaneously into the back of the mice. After injection, in vivo fluorescence images were captured at designated time points using a NIR imaging system (K-FLARE) with an 800 nm channel, and blood samples were simultaneously collected using capillary tubes. The fluorescence intensity of serum samples in the capillary tubes was measured using Cytation 5. Mice were sacrificed 14 days after injection to observe the biodistribution of DFO-NP and organs (liver, lung, spleen, kidney, intestine, and bladder).

[0097] Iron chelating effect of DFO-NP / HA / F127. CD-1 mice (male; 30-35 g; from Charles River Laboratories) were fed a high-iron diet (10,000 ppm Fe per kg) for 1 week. Subsequently, all mice were fed institutional chow (300 ppm Fe per kg) and administered saline, blank NP / HA / F127 (84 μmol / kg as NP), or DFO-NP / HA / F127 (84 μmol / kg as NP) via SC injection. Three weeks after administration, mice were euthanized, and the heart, liver, and spleen were subsequently harvested and analyzed for iron content using a non-heme iron colorimetric assay using bathophenanthroline disulfonic acid.

[0098] Histological analysis of organs: The excised organs (heart, lungs, liver, spleen, and kidneys) were stored at -80°C. The frozen organs were fixed in 10% neutral buffered formalin, dehydrated in ethanol, embedded in paraffin, and cut into slices (5 μm). These sections were then stained with hematoxylin and eosin (H&E) and pathologically observed under a light microscope system (Cytation 5, BioTek Instruments).

[0099] To evaluate the sustained release properties of the above hydrogel formulations, the DFO-NP-loaded injectable formulation and DFO-NP solution were subcutaneously injected into the backs of mice (Figure 1).

[0100] The injected dose of DFO-NPs was 125 μmol kg in all mice -1The fluorescence signal of DFO-NP in mice was observed 14 days after injection using a real-time NIR fluorescence imaging system (K-FLARE) (Figure 7). The fluorescence signal in all experimental groups rapidly decreased after an initial burst release on day 1 after injection. In mice injected with DFO-NP solution, the fluorescence signal became negligible after 5 days, and even after skin peeling at the injection site, almost no fluorescence signal was observed after 14 days. The fluorescence intensity and signal-to-background ratio (SBR) of DFO-NP / F127 slowly decreased from 10 to 5 over days 1 to 14, and no hydrogel remained at the injection site. In DFO-NP / HA, the observed fluorescence signal and SBR were lower than those of DFO-NP / F127 after 1 day and gradually decreased over time. However, hydrogel was found at the injection site after 14 days (white arrow in Figure 3a). The fluorescence signal in the DFO-NP / HA / F127 group decreased more slowly than that in the DFO-NP and DFO-NP / HA groups, and its SBR pattern was similar to that of the DFO-NP / F127 group (Figure 7b). The SBR of DFO-NP / HA / F127 remained at approximately 7.0 from days 9 to 14, significantly higher than that of the other groups. Furthermore, the hydrogel still remained in the DFO-NP / HA / F127 group, and relatively high fluorescence intensity was observed around the hydrogel at the injection site after 14 days (Figures 7a and 7b). For a more detailed comparison, the SBR of the skin and hydrogel at the injection site after 14 days was calculated (Figure 7c).

[0101] Although the hydrogel formulation did not completely suppress the initial burst release of DFO-NPs, it is noteworthy that rapid removal of non-transferrin-bound iron (the main form of iron circulating in the blood) is important for preventing recurrence in patients undergoing treatment for iron overload. Therefore, the initial release of some of the nanochelator from the hydrogel may actually have a positive effect on the treatment of iron overload. Interestingly, F127 micelles tended to be absorbed into the skin near the injection site, which may explain the high fluorescence signals on the skin in the DFO-NP / F127 and DFO-NP / HA / F127 treatment groups (Figure 7a). In addition to the higher skin SBR, the SBR of the remaining hydrogel in the DFO-NP / HA / F127 group was higher than in the others, suggesting that DFO-NPs may be released as the hydrogel degrades. Furthermore, biodistribution of DFO-NPs was observed in all experimental groups on day 14 after injection (Figure 8). No fluorescence signals were observed in organs other than the kidney and bladder in all groups. This clearly demonstrates that our nanochelator (DFO-NP) is exclusively excreted in urine and is free of potential off-target toxicity.

[0102] Pharmacokinetic studies To further investigate the sustained release of DFO-NP from the injectable hydrogel formulation, a PK study was performed. Blood samples were collected from mice at predetermined time points over a 14-day period, and the concentration of DFO-NP in the blood was determined by NIR fluorescence signal intensity at each time point. This was used to generate plasma concentration decay curves for the injected formulation (Figures 9a and 10).

[0103] The plasma concentrations of DFO-NP in the DFO-NP / F127 and DFO-NP groups were barely detectable after 3 days. This result indicates that Pluronic F127 alone is not suitable for sustained release of DFO-NP, and that DFO-NP absorbed into the skin together with Pluronic F127 micelles was hardly released. The concentration in the DFO-NP / HA group was well maintained up to 3 days. However, the fluorescent signal from DFO-NP was barely observed on the 5th day after injection (0.05 nmol ml-1 In the DFO-NP / HA / F127 group, the concentration of DFO-NP showed a similar pattern to that of the DFO-NP / F127 group, mainly due to the presence of Pluronic F127 micelles in both groups. In contrast to the previously described formulations, the signal intensity gradually decreased until day 14, but at 1 hour post-injection, it reached the level of intravenously injected DFO-NP alone (2 μmol kg ). -1 ) equivalent to 1 nmol ml -1 The concentration decay suggested that DFO-NP / HA / F127 had sustained-release properties. For further comparison, the PK parameters of the formulations were calculated from the decay curves. As shown in Figure 9b, the area under the curve (AUC) of DFO-NP / HA / F127 was the highest among the different formulations, and was 4-fold higher than that of DFO-NP alone (***p<0.001 compared to the DFO-NP group, and **p<0.005 compared to the DFO-NP / F127 group). All calculated PK parameters (half-life, bioavailability, C max , T max , and K slow ) demonstrated that the PK of DFO-NP was significantly improved when formulated with HA / F127 hydrogel (Table 1). Notably, the half-life of DFO-NP / HA / F127 was 47-fold, 35-fold, and 4-fold longer, and the bioavailability was 4-fold, 2.5-fold, and 1.3-fold higher, compared with DFO-NP, DFO-NP / HA, and DFO-NP / F127, respectively. These results confirmed that DFO-NP / HA / F127 successfully delayed the release of DFO-NP. [Table 1]

[0104] Example 5: In vivo toxicity testing of DFO-NP-loaded injectable hydrogel formulations Toxicity testing: To evaluate the toxicity of DFO-NPs and DFO-NP-loaded hydrogel formulations, blood samples were collected by cardiac puncture 14 days after injection. These blood samples were stored at room temperature without anticoagulant for 30 minutes and then centrifuged at 3,000 rpm for 15 minutes. Serum was stored at -80°C until further assay. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured for hepatotoxicity, and blood urea nitrogen (BUN) and creatinine (CREA) for nephrotoxicity. All assays were performed using commercially available assay kits, and absorbance was immediately measured using a plate reader.

[0105] Statistical analysis: Fluorescence intensity and background intensity in regions of interest for each organ were quantified using customized imaging software and ImageJ v1.52i (National Institutes of Health, Bethesda, MD). Signal-to-background ratio (SBR) was calculated as SBR = fluorescence / background, where background is the fluorescence intensity of the muscle or system background. Data are presented as the mean ± sem of a minimum of three replicates. Statistical analysis was performed using Student's t-test to assess the significance of experimental data. Differences between groups were determined using one-way ANOVA analysis with Bonferroni's multiple comparison correction to evaluate statistical differences between more than two groups. A p value of less than 0.05 was considered significant. Data were indicated by *p<0.05, **p<0.01, and ***p<0.001.

[0106] The chronic toxicity of DFO-NP was evaluated when DFO-NP was formulated with DFO-NP / HA / F127 at a ratio of 30 / 7 / 30% and injected subcutaneously. For histological analysis, major organ samples (heart, lung, liver, spleen, and kidney) collected 14 days after injection were stained with hematoxylin and eosin (H&E) (Figures 9a and 10). No pathological differences were observed between the saline and DFO-NP / HA / F127-injected groups, even in the kidney, the organ most exposed to DFO-NP.

[0107] To further quantitatively evaluate hepatotoxicity and nephrotoxicity, biochemical analyses of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CREA) were performed. As shown in Figure 12b, there was no difference in AST levels between the saline and DFO-NP / HA / F127 groups. The ALT level in the DFO-NP / HA / F127 group was slightly higher than that in the saline control group (*p<0.05), but was within the normal range for mice (25-60 U / L).

[24] ), suggesting that DFO-NP / HA / F127 does not induce hepatotoxicity. Furthermore, there was no significant difference in CREA and BUN levels between the saline and DFO-NP / HA / F127 groups (Fig. 12c). This indicates that high doses of the DFO-NP / HA / F127 formulation do not induce any chronic toxicity.

[0108] Example 6: Therapeutic Efficacy in Vivo of DFO-NP-Loaded Injectable Hydrogel Formulations To confirm the in vivo therapeutic effect of the DFO-NP formulation, DFO-NP / HA / F127 hydrogel was subcutaneously injected into mice with diet-induced iron overload (DIO). For comparison, an empty hydrogel (i.e., a nanochelator without DFO (blank NP) containing the same hydrogel formulation) was subcutaneously injected. Saline was used as a control. Organs (spleen, liver, and heart) were harvested 3 weeks after administration of each sample, and the iron content in the organs was quantitatively measured using ICP-MS (Figure 13). There was a significant difference in splenic iron levels between the DFO-NP / HA / F127 and saline groups (Δ156.9 μg iron / g tissue; p<0.05 compared with the saline control group), and a trend toward lower iron content in the liver (Δ33.0 μg iron per g tissue) was confirmed. The greater iron excretion effect in the spleen compared with the liver is likely due to greater iron overload in the spleen than in the liver.

[0109] Other embodiments While the present application has been disclosed in particular embodiments, those skilled in the art will recognize that certain substitutions, modifications, and / or omissions may be made to the embodiments without departing from the spirit of the invention. Accordingly, the foregoing description is intended to be merely illustrative and not limiting of the scope of the invention. All references, scientific papers, patent publications, and any other documents cited herein are incorporated herein by reference in their entirety.

Claims

1. A sustained release injectable composition comprising an iron chelator and at least one hydrogel.

2. The composition of claim 1, wherein the composition is capable of releasing the iron chelator for about 1 day to about 30 days.

3. 3. The composition of claim 2, wherein the composition releases the iron chelator over a period of at least two weeks.

4. The composition of any one of claims 1 to 3, wherein the hydrogel comprises hyaluronic acid (HA).

5. The composition of claim 3 , wherein the HA is crosslinked.

6. 6. The composition of claim 4 or 5, wherein the HA is crosslinked with 1,4 butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), ethylene glycol diglycidyl ether (EGDE), 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), or diepoxyoctane.

7. The composition of any one of claims 1 to 6, wherein the hydrogel further comprises F127.

8. 8. The composition of claim 7, wherein the composition comprises 30% by weight F127, 7% by weight HA, and 30% by weight iron chelator.

9. The composition of any one of claims 1 to 8, wherein the iron chelator is deferoxamine (DFO).

10. The composition of claim 9, wherein the DFO is a DFO nanochelator (DFO-NP).

11. 11. A method of treating iron overload in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of the composition of any one of claims 1 to 10.

12. A method for chelating metal ions in a cell or tissue sample, comprising contacting the cell or tissue sample with a composition according to any one of claims 1 to 10.

13. 11. A method for reducing the amount of free metal ions in a cell or tissue sample, comprising contacting the cell or tissue sample with a composition according to any one of claims 1 to 10.

14. 11. A method of treating a disease associated with an abnormal amount of free metal ions in a subject, comprising administering to a subject determined to have an abnormal level of free metal ions a composition of any one of claims 1 to 10.

15. 15. The method of claim 14, wherein the disease is associated with an abnormal amount of iron ions, an abnormal amount of lead ions, or an abnormal amount of copper ions in the subject, or any combination thereof.

16. 15. The method of claim 14, wherein the disease is associated with an abnormal amount of iron ions in the subject.

17. 15. The method of claim 14, wherein the disease is selected from transfusion hemosiderosis, hemochromatosis, Wilson's disease, copper poisoning, and heavy metal poisoning.

18. 1. A method for preparing a sustained release injectable composition useful for iron chelation therapy, comprising: a) cross-linking HA; b) forming the crosslinked HA into particles; c) adding particles of cross-linked HA to a solution of DFO-NPs; d) further adding F127 to the solution resulting from step c).

19. 1. A sustained release injectable composition for iron chelation therapy in a human patient, comprising an iron chelator and a hydrogel, said composition being releasable over a period of two weeks.

20. The composition of claim 13, wherein the hydrogel consists of HA and F127.

21. The composition of claim 14, wherein the HA is crosslinked.

22. 16. The composition of claim 15, wherein the HA is cross-linked with BDDE.

23. The composition of claims 14-16, wherein the weight percentages of the ingredients are 30% by weight F127, 7% by weight HA, and 30% by weight DFO-NP.

24. The composition of claims 14 to 17, wherein the iron chelator is DFO.

25. The composition of claim 18, wherein the DFO is DFO-NP.

26. 20. A method of treating iron overload in a patient, comprising administering to said patient an effective amount of the composition of any one of claims 13 to 19.

27. All compositions, articles of manufacture, methods and uses disclosed and / or described herein.

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