H2S oxidizing agent, a therapeutic carbon nanomaterial for synthesizing biological polysulfides

Abstract

A therapeutic method for catalytically forming persulfides and / or polysulfides from hydrogen sulfide using endogenously or exogenously released hydrogen sulfide in vitro and in vivo is disclosed. The method involves contacting cells in need, including cells under conditions of oxidative stress, traumatic brain injury (TBI), and hypoxia, or cells with excess hydrogen sulfide or defective protein persulfation, with oxidized carbon nanoparticle materials, which contain multiple carbonyl, hydroxyl, and carboxyl substituents. Oxidized carbon nanoparticle (OCN) materials can be prepared from any of a variety of sources, with activated carbon being preferred. Water-dispersible OCN materials are described that do not require the addition of hydrophilic polymers to provide dispersibility at concentrations of approximately 1-5 mg / mL for at least 7 days. These OCN materials also have a UV absorbance maximum of approximately 220 nm in water and pass through 0.22 μm pore PES membranes.

Description

[Technical Field]

[0001] Description of federally supported research or development This invention was made with government support under grant number R01 439371-00001, awarded by the National Institutes of Health, United States. The U.S. Government reserves certain rights in this invention.

[0002] This invention relates to the field of carbon nanomaterials and their therapeutic applications. [Background technology]

[0003] Explanation of related technologies Hydrogen sulfide (H2S) is a gaseous substance with several endogenous properties similar to nitric oxide and carbon monoxide. Hydrogen sulfide is an excellent reducing agent (it is easily oxidized), and in the past, attempts have been made to use it as a therapeutic agent. However, these attempts have been hampered by the inherent difficulties in handling H2S gas or its sulfide salts. In the former case, H2S is flammable, highly toxic, and potentially poisonous. The therapeutic usefulness of sulfide salts is limited because they decompose rapidly.

[0004] One attempt to address these shortcomings is the use of H2S donors that circulate and release H2S throughout the body. However, while donors can overcome the inherent problems of H2S as a gas or salt, they do not resolve the narrow therapeutic index, and to date, only one clinical trial using an H2S donor with a different mechanism of action, N-acetylcysteine, is known to have been completed.

[0005] H2S is metabolized into polysulfides within cells. Polysulfides are excellent reducing agents and have many potentially beneficial effects on oxidative stress. Polysulfides derived from the oxidation of hydrogen sulfide (Na x S y ) is an effective antioxidant in vitro.

[0006] Polysulfides can be excellent reducing agents due to their weak terminal SH bonds. Polysulfides target both one-electron and two-electron oxidizing agents, including superoxides and hydrogen peroxide (90, 92). In the case of two-electron oxidizing agents, polysulfides appear to react faster than thiols such as glutathione, and therefore may be important components of the cellular antioxidant network (90, 92).

[0007] The formation of polysulfides that act as reducing agents is not the only consequence of biologically important hydrogen sulfide metabolism. Protein persulfides (RSS) - (Here, R is a protein or polypeptide) has recently been identified as a product of H2S-based signaling, and therefore influences numerous protein functions that have significance for a wide range of biological processes and disease conditions.

[0008] In nature, polysulfides can be produced intracellularly by several enzymatic pathways, including the catalytic oxidation of hydrogen sulfide by superoxide dismutase (87), catalase (91), cystathionine B-synthase (CBS) (89), and cysteinyl-tRNA synthetase (CARS) (89, 88). Furthermore, polysulfides can be formed by the free radical oxidation of hydrogen sulfide, which forms HS·. HS· then undergoes the reaction (H2O + 2HS· -> HSS) - +H3O + ) or reacts with endogenous thiols (R-SH) to form R-SSH species (92).

[0009] Therefore, methods for rapidly generating polysulfides from H2S have the potential to overcome the limitations of administering H2S itself, making them a promising therapeutic option.

[0010] Another fate of polysulfides is to be oxidized by enzymatic oxidation via heme proteins, or by non-enzymatic oxidation via simple oxidation [Vitvitsky et al., J Biol Chem 2015;290(13):8310-8320], resulting in thiosulfate (S2O3) oxidation. 2-) is oxidized. Thiosulfate can act as an H2S donor and has protective properties in ischemia-reperfusion injury [Marutani et al., J Am Heart Assoc 2015;4(11); Epub 2015 / 11 / 08]. In other cases, S2O3 2- undergoes further enzymatic oxidation in mitochondria to form sulfite (SO3 2- ) or sulfate (SO4 2- ) [Mishanina et al., Nat Chem Biol 2015;11(7):457-464], and they are excreted outside the body.

[0011] Carbon monoxide can be used to catalyze the oxidation of hydrogen sulfide in those fuel products to form elemental sulfur polymers (polysulfides) as a means of removing hydrogen sulfide from natural gas and other petroleum raw materials. However, it has hitherto been unclear whether graphene oxide, another carbon nanomaterial, can enhance the oxidation of hydrogen sulfide catalytically or non-catalytically to form polysulfides in living cells.

[0012] Such an action may be beneficial under conditions of acute injury such as oxidative stress and chronic conditions such as diabetes. Such an action may provide a means of utilizing the beneficial effects of hydrogen sulfide as a substrate rather than as a main therapeutic agent.

[0013] A lack of H2S has been observed in several diseases. This lack and the consequent loss of sufficient polysulfides probably contribute to the pathology through both the loss of sufficient antioxidant power and the decrease in protein persulfidation, ultimately impairing the function of these proteins.

[0014] To provide a safe and effective means of generating reducing agents (antioxidants) in vivo for the elimination of free radicals and other potentially beneficial effects of polysulfides, including persulfidation necessary for proper protein function, alternative materials that can safely enhance the oxidation of hydrogen sulfide in vivo are needed in the medical field. Such materials may have various applications, particularly in the treatment of diabetes, inflammation-related diseases, such as Alzheimer's disease, arthritis, traumatic brain injury (TBI), and tissue damage resulting from ischemic or hemorrhagic events.

[0015] The present invention, as detailed herein, provides non-toxic materials, compositions, and therapeutic methods using these materials that offer a means to meet the need for safely and effectively removing harmful free radicals by catalytically and non-catalytically forming polysulfides from hydrogen sulfide in vitro and in vivo, and for restoring protein persulfidation by converting H2S to polysulfides. [Overview of the project]

[0016] The present invention envisions a therapeutic method for enhancing the production of persulfides and / or polysulfides from endogenously or exogenously released hydrogen sulfide in vitro and in vivo. The method comprises contacting an effective amount of oxidized carbon nanoparticle material with cells in need, the particles comprising multiple carbonyl, hydroxyl, and carboxyl substituents. Exemplary cells in need include cells under oxidative stress in traumatic brain injury (TBI), hypoxia, ischemia, reperfusion injury, and cells requiring sufficient protein persulfation to ensure proper protein function, as is required in numerous disease processes associated with a decrease in H2S and / or polysulfides and / or persulfides.

[0017] The intended carbon oxide nanoparticle materials can be prepared from any of a variety of sources, including graphene, graphene nanoribbons, graphene oxide, graphite, graphite oxide nanoribbons, carbon black, hydrophilic carbon clusters, coal oxide, and activated carbon (preferably, which forms activated carbon oxide / coal oxide particles (OAC)). These materials exhibit strong catalytic redox activity, and formulations of these materials have demonstrated efficacy in animal models of injury.

[0018] Means of enhancing the formation of polysulfides from endogenously or exogenously released H2S can increase effectiveness in conditions in which this active substance is pathologically involved. Compared to several preparations of oxidatively activated carbon nanomaterials, OAC made from activated carbon prepared according to Good Manufacturing Practices (GMP) contains little to no metal contamination that needs to be removed.

[0019] In one embodiment, an oxide carbon nanoparticle material may be used as part of a method for catalyzing the oxidation of hydrogen sulfide to persulfides and / or polysulfides in cell culture and / or living tissue environments. In one embodiment, the oxide carbon nanoparticle material is functionalized with one or more exogenously supplied specific substituents selected from the group consisting of hydrogen sulfide-releasing moieties, solubilizers, biological barrier transporter moieties, tissue targeting agents, assay identifiers, chelating agents, and pharmaceuticals. The exogenously supplied specific substituents may be non-covalently bonded or covalently linked to the oxide carbon nanoparticle material.

[0020] In another embodiment of this design, the carbon oxide nanoparticle material is poly(ethylene glycol) [PEG], poly(propylene glycol) [PPG], poly(ethyleneimine) [PEI], poly(vinyl alcohol) [PVA], PPG-PEG block copolymer, C 12 -C 18 It is functionalized with one or more solubilizers selected from the group consisting of -poly(ethylene oxide)-ether and poly(acrylic acid) [PAA].

[0021] In another aspect of this embodiment, the carbon nanometer particle material with oxidation is conjugated with a hydrogen sulfide releasing moiety, and the released hydrogen sulfide is oxidized in situ by the carbon nanometer particles to produce antioxidant products. The hydrogen sulfide releasing site is preferably covalently linked to the carbon core of the nanomaterial and can provide hydrogen sulfide by thiolysis or enzymatic degradation. Illustrative examples of such materials include N-benzoylthiol-benzamide, acylperthiol, arylthioamide, polysulfide, dithioperoxy anhydride, S-aroylthioxamine, geminal-dithiol, and trimethyloxonium prodrug.

[0022] In another aspect, the carbon nanometer material with oxidation is provided as part of a method for increasing the concentration of polysulfide and / or persulfide in vivo by catalytic oxidation of endogenous hydrogen sulfide. The resulting increase in the concentration of polysulfide and persulfide appears as a pool rich in therapeutic antioxidants and the restoration of protein persulfidation.

[0023] In another aspect, the rate of formation of polysulfide can be enhanced or inhibited by additional chemical reactions that may be desirable depending on the适应症. As an example, the rate of formation of polysulfide is enhanced by a covalent reaction with ethylenediamine.

[0024] Still further, in an aspect, the carbon nanometer particle material with oxidation is functionalized with a transporter moiety selected from one or more of the group consisting of adamantanyl, amantadiniyl, memantineyl, rimantadiniyl, dopamantineyl, tromantadiniyl, vildaglupetinyl, and carmantadiniyl groups.

[0025] In another embodiment, carbon oxide nanoparticles are functionalized with chelating agents (e.g., deferoxamine and deferasirox) intended to bind to toxic materials such as iron in conditions of excessive metal concentration, either systemically, such as in iron overload, or locally, such as in part of a pathological process like hemorrhage or dementia.

[0026] In a particular embodiment, the above method provides the use of persulfides and polysulfides in therapeutic applications such as the treatment of conditions associated with inflammation, ischemia, or neurodegeneration in a subject.

[0027] In some embodiments, compositions are provided that contain oxidized carbon nanomaterials in therapeutic preparations suitable for use in subjects. Here, the oxidized carbon nanoparticle material can be functionalized with pharmaceuticals that are cannabinoids. Illustrative cannabinoids include cannabigerol, cannabigerol monomethyl ether, cannabinerol acid A, cannabigerovaline, cannabigerol acid A, cannabigerol acid A monomethyl ether, cannabigerovaline acid A, cannabichromene, cannabichromene acid A, cannabivarichromene, cannabiclomevalin, cannabiclomevalin acid A, cannabidiol, cannabidiorcol, cannabidiolic acid, cannabidivalic acid, cannabinodivalin, cannabinodivalin, cannabicitran, 1,1-dimethylheptyl-11-hydroxytetrahydrocannabinol) (HU-210), and dexanabinol.

[0028] In certain embodiments, compositions of modified carbon nanomaterials, particularly carbon oxide nanomaterials (nanoparticles), include carboxy-functionalized carbon nanoparticles. These materials are provided as therapeutic preparations suitable for enhancing polysulfide production by cells. Examples of carbon nanomaterials in therapeutic preparations include graphene, graphene nanoribbons, graphene oxide, graphite, graphite oxide nanoribbons, carbon black, carbon oxide black, hydrophilic carbon clusters (HCCs), oxidized activated coal, oxidized activated carbon, or any combination thereof.

[0029] A preferred group of carbon oxide nanoparticles (CNPs) are prepared using activated carbon particles (PEG-OAC) oxidized with nitric acid or fuming nitric acid, which are then functionalized with polyethylene glycol. The activated carbon used is preferably made from pharmaceutical-grade and / or activated carbon prepared according to Good Manufacturing Practices (GMP). The oxidation process can also be carried out using a mixture of nitric acid or fuming nitric acid with sulfuric acid or fuming sulfuric acid as the oxidation medium.

[0030] Because the reactants can be explosive, the oxidation reactions discussed herein, which are potentially hazardous, have been carried out in a nitric acid still bath. The reaction process can also be carried out in a less hazardous flow device. By varying the concentration and temperature, desirable particle characteristics can be obtained.

[0031] Various quenching methods can be used. As with aqueous solutions of organic and inorganic bases, the addition of water is used, but glycine quenching is preferred because glycine acts as a buffer and rapidly removes nitric acid.

[0032] For example, some carbon nanoparticles include hydrophilic carbon clusters (HCCs) or polyethylene glycol-conjugated hydrophilic carbon clusters (PEG-HCCs). These compositions are described as having rapid antioxidant activity and high antioxidant activity, and are non-toxic in vitro and in vivo.

[0033] The previously reported, known carbon oxide nanoparticles form an aqueous dispersion. This aqueous dispersion exhibits particle separation from the dispersion within approximately one hour, remaining on a polyethersulfone (PES) filter membrane with a pore size of 0.22 μm in diameter, rather than passing through it, exhibiting a UV maximum of approximately 250 nm, and containing approximately 4–7 percent carbonyl groups as determined by X-ray photoelectron spectroscopy (XPS). PEG-OAC materials have been shown to possess antioxidant and in vivo protective properties similar to PEG-HCC.

[0034] A new type of carbon oxide nanoparticle material is also being considered. These particles are disc-shaped and form a non-separable aqueous dispersion with a concentration of approximately 1 to 5 mg / mL. This dispersion is stable against separation for at least 7 days at ambient room temperature. This carbon oxide nanoparticle aqueous dispersion exhibits a maximum UV absorbance at approximately 220 nm. These particles contain approximately 9 to 15 percent carbonyl groups as determined by X-ray photoelectron spectroscopy (XPS) and pass through a polyethersulfone (PES) filter membrane with a pore size of 0.22 μm.

[0035] Carbon oxide nanoparticle materials can be added directly to cell cultures or used as a powder in a spray. Preferably, the intended carbon oxide nanoparticle material is used dissolved or dispersed in a physiologically acceptable diluent or carrier, such as a solid or liquid pharmaceutical composition.

[0036] When formulated in this manner, the intended composition may be provided to a subject as a pill or tablet, as a liquid to be swallowed, or as something to be injected percutaneously, such as by intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal injection, or as a topical treatment to the skin and / or mucous membranes, or as an intranasal spray or mist, or as an inhalant. [Brief explanation of the drawing]

[0037] In the drawings that form part of this disclosure,

[0038] [Figure 1] Figure 1 is a graph showing the time course of cerebral blood flow recovery in a rat model of traumatic brain injury in the presence of PBS (solid line), PEG-OAC (dotted line), and PEG-HCC (dashed line). It can be seen that the results with PEG-OAC were equivalent to those with PEG-HCC. At time 0, traumatic brain injury occurs, followed by a decrease in blood pressure due to phlebotomy. This reduces cerebral perfusion due to the brain injury (y-axis). At PH (pre-hospitalization stage), saline solution is injected to transiently increase cerebral perfusion. At the "hospitalization stage," rats are resuscitated with stored blood along with injections of PBS (vehicle), PEG-HCC, or PEG-OAC. Perfusion in the PBS group increased transiently but then continued to decrease, while both nanoparticles (NPs) maintained increased perfusion throughout the experiment. The PEG-HCC group required a second injection, but the PEG-OAC group did not. [Figure 2] Figure 2 shows a plot of electron paramagnetic resonance (EPR) signals against the K2O concentration (star) of PEG-OAC present at 0.25 ng / L. The dashed fitted line passing through the measured values ​​is the Michaelis-Menten saturation curve, indicating the enzyme-like catalytic activity of the nanoparticles. An insert is included to show the confirmation of stable radicals. [Figure 3] Figure 3 shows a schematic alternative synthesis of PEG-OAC. Here, GMP AC is oxidized via fuming nitric acid (fuming sulfuric acid, which was used in PEG-HCC, is not used in this scheme) and then PEGylated. [Figure 4] Figure 4 is a micrograph showing as-synthesized OAC particles visualized by transmission electron microscopy. The scale bar represents 5 nm. [Figure 5] Figure 5 is a schematic representation of the synthesis scheme for oxidized activated carbon (OAC) using nitric acid. The left panel shows the synthesis used in previous studies to fabricate sensors (not in vivo or in cells). The right panel shows the synthesis scheme for preparing oxidized activated carbon for in vivo and intracellular use. [Figure 6]Figure 6 shows photographs of the filtrates of AC oxidized via 70% HNO3 (left) and AC oxidized via 90% HNO3 (fuming nitric acid; right). Note that OAC oxidized with 90% HNO3 yields a water-dispersible product that can pass through a 0.22 μm PES filter. The product obtained from the synthesis of OAC using 70% HNO3 is water-insoluble and remains on the filter film, resulting in a clear solution. [Figure 7] Figure 7 is a graph showing the thermogravimetric analysis (TGA) results for activated carbon (AC), OAC oxidized with 70% HNO3, and OAC oxidized with 90% HNO3, showing weight losses of 1.5%, 19.5%, and 45%, respectively. The OAC synthesized using 90% HNO3 showed the largest weight loss, indicating that the material was more thoroughly oxidized. Measurements were obtained under N2 conditions at a ramp rate of 10°C / min up to 600°C. [Figure 8] Figure 8 is a plot of current in milliamperes (mA) against volts, illustrating the electrochemical activity of OAC synthesized via oxidation with 90% HNO3 compared to OAC synthesized via oxidation with 70% HNO3. Both materials exhibit reduction potentials from an onset potential of >0V (approximately +0.2V). However, the reduction potential of the material oxidized with 90% HNO3 has various shoulder peaks, while the reduction potential of the material oxidized with 70% HNO3 is a single broad peak. [Figure 9A] Figure 9A is a graph showing the reduction of rezazurin using different thiol molecules as reducing agents, specifically PEG-HCC (pegylated hydrophilic carbon cluster) and dithiothreitol, demonstrating their ability to act as catalysts for the reduction of rezazurin (control, circles, -●-; 4 mg / L PEG-HCC, squares, -■-). [Figure 9B] Figure 9B is a graph showing the catalytic reduction of rezazurin to resorphine by PEG-OAC using 2 mM glutathione as a reducing agent. [Figure 10A]Figures 10A and 10B are graphs showing the preparation of polysulfides from the reactions of Na2S + PEG-OAC (Figure 10A), and PEG-HCC and EN-PEG-HCC (Figure 10B) in the presence of sulfur detection probe 4 (SSP4) [discussed in U.S. Patent No. 10,520,509, available from Dojindo Molecular Technologies, Rockville, MD] and Na2S, with or without OAC (no nanomaterial, bottom line). PEG-OAC has been shown to provide a highly effective catalyst for H2S. PEG-OAC was synthesized from a GMP pharmaceutical-grade source of activated carbon. This graph demonstrates that the rate of polysulfide [3',6'-di(O-thiosalicyl)-fluorescein; sulfan sulfur probe 4; SSP4], as measured by SSP4 fluorescence, was significantly improved in the presence of the PEG-OAC material. [Figure 10B] Figures 10A and 10B are graphs showing the preparation of polysulfides from the reactions of Na2S + PEG-OAC (Figure 10A), and PEG-HCC and EN-PEG-HCC (Figure 10B) in the presence of sulfur detection probe 4 (SSP4) [discussed in U.S. Patent No. 10,520,509, available from Dojindo Molecular Technologies, Rockville, MD] and Na2S. Figure 10B further shows how concentrations (0.1, 1, 4, 10 mg / L) of PEG-HCC and EN-PEG-HCC affect SSP4 fluorescence in cell-free solutions containing sodium sulfide. EN-PEG-HCC reacts more rapidly at equivalent concentrations. [Figure 11A]Figures 11A and 11B are graphs showing the SSP4 fluorescence of cultured b.End3 cells using EN-PEG-HCC (Figure 11A) and PEG-HCC (Figure 11B) at 10 mg / L NP, under normal oxygen and 90 minutes of hypoxia (t=0). Hypoxic samples were incubated for 90 minutes in deoxygenated buffer containing NP (bubbled with N2 for 30 minutes) before the initial readout (t=0), and then readout was performed for another 90 minutes in room air. Normal oxygen samples were mixed and readout was performed for 90 minutes. EN-PEG-HCC = ethylenediamine-blocked PEG-HCC. EN-PEG-HCC again showed higher SSP4 production compared to PEG-HCC. The overlapping of the SSP4 individual plots for normal and hypoxic atmospheres demonstrates that SSP4 fluorescence does not increase in the absence of oxidized nanomaterials. EN-PEG-HCC induces SSP4 fluorescence under both hypoxic and normoxic conditions, exhibiting different dynamics. [Figure 11B] Figures 11A and 11B are graphs showing the SSP4 fluorescence of cultured b.End3 cells using EN-PEG-HCC (Figure 11A) and PEG-HCC (Figure 11B) at 10 mg / L NP, under normal oxygen and 90 minutes of hypoxia (t=0). Hypoxic samples were incubated for 90 minutes in deoxygenated buffer containing NP (bubbled with N2 for 30 minutes) before the initial readout (t=0), and then readout was performed for another 90 minutes in room air. Normal oxygen samples were mixed and readout was performed for 90 minutes. EN-PEG-HCC = ethylenediamine-blocked PEG-HCC. EN-PEG-HCC again showed higher SSP4 production compared to PEG-HCC. The overlapping of the SSP4 individual plots for normal and hypoxic atmospheres demonstrates that SSP4 fluorescence does not increase in the absence of oxidized nanomaterials. EN-PEG-HCC induces SSP4 fluorescence under both hypoxic and normoxic conditions, exhibiting different dynamics. [Figure 12]Figure 12 is a graph showing the reduction of resolphins by dithiothreitol in the presence of PEG-HCC or PEG-OAC. PEG-HCC (circles), PEG-OAC (squares). Reduction using PEG-OAC is faster than reduction using PEG-HCC. [Figure 13] Figure 13 is a graph showing the polysulfide formation rates in cell-free PBS aqueous solutions containing 300 μM Na2S and a 10 μM fluorescent sulfur probe (SSP4) with and without the addition of 4 mg / L PEG-OAC (dashed line). Fluorescence was measured at 540 nm for 10 minutes. At that time, the polysulfide formation rate increased 30-fold due to the presence of PEG-OAC, as indicated by the fluorescence. [Figure 14] Figure 14 is a graph showing the dose-dependent (0, 1, 3, and 9 mg / L) production of thiosulfate in a cell-free PBS aqueous solution containing 300 μM Na2S, after adding PEG-OAC. Thiosulfate production was measured using the method described in Dong et al., ACS Sens. 2017;2(8):1152-1159. Thiosulfate is an oxidation product of polysulfides, suggesting the formation of polysulfides. [Figure 15] Figure 15 is a graph illustrating the particle distribution when OAC particles were prepared using fuming nitric acid (90% HNO3) at 100°C for 6 hours. The average diameter of the particles in the mixture is 2.8 nm. [Figure 16] Figure 16 shows a transmission electron microscope image of OAC nanoparticles on a lace-like carbon surface. These nanoparticles appear as dark circles with an average diameter of 2.8 nm. [Figure 17] Figure 17 is a graph illustrating that PEG-OAC increases the rate of polysulfide production in HEK293 cells without the need for further sulfide donors. Polysulfide production in HEK293 cells was measured by ssp4 (y-axis, arbitrary units) and catalyzed in a dose-dependent manner by PEG-OAC (0, 1, 3, 9 mg / L). [Figure 18]PEG-OAC reduces both the proliferative and toxic effects of 10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl)triphenylphosphonium bromide (AP39) [Szczesny et al., Nitric Oxide 2014;14:120-130], a mitochondrial H2S donor. As observed, PEG-OAC reduces 10 nM AP39-induced proliferation in bEnd.3 cells and reduces 1000 nM AP39-induced toxicity in those cells. [Figure 19] Figure 19 shows fluorescence micrographs of bEnd.3 cells treated with no additives [AP39(-) / PEG-OAC(-)], AP39 alone [AP39(+) / PEG-OAC(-)], PEG-OAC alone [AP39(-) / PEG-OAC(+)], or both [AP39(+) / PEG-OAC(+)]. Co-inclusion of 4 mg / L PEG-OAC with bEnd.3 cells increased the fluorescence of polysulfide-sensitive sulfur detection probe 4 (SSP4) compared to the control [AP39(-) / PEG-OAC(+)] in the absence of AP39. Adding AP39 increased the fluorescence signal, but this effect was amplified by the addition of PEG-OAC [AP39(+) / PEG-OAC(+)]. [Modes for carrying out the invention]

[0039] definition The following definitions will be used in the following explanation.

[0040] The aspects and embodiments of the present invention described herein are understood to include "consisting of" and / or "essentially consisting of" aspects and embodiments.

[0041] The articles "a" and "an" are used herein to indicate that the grammatical object of the article is one or more (i.e., at least one). For example, "an element" means one or more elements.

[0042] Any reference to a value or parameter "about" in this specification includes (and describes) the variability of that value or parameter itself. For example, any description referring to "about X" includes a description of "X".

[0043] As used herein, the term “anticancer agent” refers to a pharmacologically active substance capable of inhibiting cancer selected from the group consisting of melanoma, glioma and adenocarcinoma, bladder cancer, bone cancer, bone marrow cancer, brain cancer, spinal cord cancer, breast cancer, cervical cancer, gallbladder cancer, ganglion cancer, gastrointestinal cancer, stomach cancer, colon cancer, heart cancer, kidney cancer, liver cancer, lung cancer, muscle cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, prostate cancer, salivary gland cancer, skin cancer, spleen cancer, testicular cancer, thymic cancer, thyroid cancer, or uterine cancer. Detailed description of preferred embodiments

[0044] The present invention envisions a therapeutic method for catalytically forming persulfides and / or polysulfides from endogenously or exogenously released hydrogen sulfide in vitro and in vivo. The method comprises contacting an effective amount of oxidized carbon nanoparticle material with the cells to be used, wherein the particles contain multiple carbonyl, hydroxyl, and carboxyl substituents.

[0045] The exemplary cells in need are those present in mammalian subjects, transplant- or implant-ready organs, where the body is under oxidative stress resulting from hypoxia (but not limited to) that may occur as a result of traumatic brain injury (TBI), inflammation, sepsis, epileptic seizures, stroke or thrombosis, or shock. The exemplary cells are those in need within mammalian subjects where the body has insufficient H2S to produce adequate persulfur proteins, or insufficient innate ability to produce enough persulfur proteins for proper function in aging, cancer, inflammation, diabetes, stroke, trauma (but not limited to these), and to reduce potential toxicity resulting from excess H2S.

[0046] The intended carbon oxide nanoparticle material can be prepared from any of the following sources: graphene, graphene nanoribbons, graphene oxide, graphite, graphite oxide nanoribbons, carbon black, hydrophilic carbon clusters (HCC), oxidized coal, activated coal, and activated carbon (preferably which forms oxidized activated carbon particles (OAC)).

[0047] OACs exhibited strong catalytic redox activity, and formulations of these materials demonstrated efficacy in an in vivo rodent traumatic brain injury model shown in Figure 1. Compared to several preparations of oxidized activated carbon nanomaterials, OACs prepared from activated carbon prepared according to Good Manufacturing Practices (GMP) contained little to no metal contamination that needed to be removed.

[0048] Means of enhancing the formation of polysulfides from endogenously or exogenously released H2S can increase the effectiveness of this active substance in conditions in which it is pathologically involved.

[0049] In one embodiment, the carbon oxide nanoparticle material may be used as part of a method for catalyzing the oxidation of hydrogen sulfide to persulfides and / or polysulfides in cell culture and / or living tissue environments. In one embodiment, the carbon oxide nanoparticle material is functionalized with one or more exogenously supplied specific substituents selected from the group consisting of hydrogen sulfide-releasing moieties, solubilizers, biological barrier transporter moieties, tissue targeting agents, assay identifiers, chelating agents, and pharmaceuticals. The exogenously supplied specific substituents may be non-covalently bonded or covalently linked to the carbon oxide nanoparticle material.

[0050] In some embodiments, active agents may associate with carbon nanomaterials covalently or noncovalently. For example, carbon nanomaterials may be functionalized with one or more molecules, chelating agents, such as deferoxamine, polymers, or chemical moieties, and nanoparticles may contain functional groups or solubilizing groups, such as ketones, alcohols, epoxides, carboxylic acids, 1,2-diones, 1,4-diones, 1,2-quinones, 1,4-quinones, and combinations thereof.

[0051] More specifically, carbon oxide nanomaterials can be functionalized with any number of solubilizing polymers. Exemplary solubilizing polymers include one or more of poly(ethylene glycol) [PEG], poly(propylene glycol) [PPG], poly-(ethyleneimine) [PEI], poly(vinyl alcohol) [PVA], and poly(acrylic acid) [PAA], or any combination thereof. As is well known, these solubilizing polymers are available in many lengths (molecular weights), and several are used herein and illustrated in the figures. For example, solubilizing polymers or functionalizing groups can be covalently or noncovalently associated with carbon oxide nanomaterials so that the carbon oxide nanomaterials can be noncovalently encapsulated with multiple solubilizing groups such as PEG, PPG, or a block copolymer of PEG and PPG, e.g., Pluronic® polyol (PEG-PPG-PEG). In some embodiments, the solubilizing groups are covalently bonded to the particles.

[0052] Carbon oxide nanomaterials (nanoparticles) as part of a therapeutic composition may also include targeting agents that can be covalently or noncovalently attached to the nanoparticles. For example, such targeting agents may include antibodies, RNA, DNA, aptamers, small molecules, dendrimers, proteins or peptides, saccharides, polysaccharides, or any combination thereof.

[0053] Targeting agents exhibit recognition activity towards markers related to the needs that the contacted cells must satisfy, such as the mitigation of oxidative stress. For example, exemplary markers may be cell surface proteins that are upregulated in response to oxidative stress, or carbohydrate-recognizing elements. Specific targeting agents are selected based on their affinity and / or specificity to the target site of the cell requiring the action, or to a specific cell type or specific intracellular organelle within the target site.

[0054] The therapeutic compositions of the present invention may also be associated with chelating agents to enhance their ability to remove excess metals or other active substances as part of a pathological process in which a reduction in the level of the active substance is beneficial. For example, the chelating agents may be deferoxamine, diethylenetriaminepentaacetic acid (pentetic acid or DTPA), deferasirox, deferipron, aerobactin, triapine, salicylaldehyde isonicotinoyl hydrazone, (E)-N9-[1-(2-hydroxy-5-nitrophenyl)-ethylidene]isonicotinoyl hydrazone, dipyridylketone isonicotinoyl hydrazone, D-penicillamine, or structural derivatives thereof that enable conjugation to nanoparticles.

[0055] The therapeutic compositions of the present invention may also associate with transporter moieties to facilitate the transport of oxidized carbon nanomaterials across biological barriers, namely the blood-brain barrier (BBB) ​​and the blood-spinal cord barrier (BSCB). For example, these transporter moieties may include adamantine molecules or adamantine derivatives. Exemplary adamantine derivatives include amantadine, memantine, rimantadine, dopamantine, tromantadine, vildagliptin, carmantadine, and any combination thereof. These compounds are referred to as having substituents of adamantanyl, amantazinyl, memantinyl, rimantazinyl, dopamantinyl, tromantadinyl, vildagliptinyl, and carmantadinyl groups.

[0056] The transporter moiety that can be used with oxidized carbon nanomaterials may include cannabinoid molecules or cannabinoid derivatives, such as, but are not limited to, cannabigerol, cannabigerol monomethyl ether, cannabinerol acid A, cannabigerovaline, cannabigerol acid A, cannabigerol acid A monomethyl ether, cannabigerovaline acid A, cannabichromene, cannabichromene acid A, cannabivarichromene, cannabiclomevalin, cannabiclomevalin acid A, cannabidiol, cannabidiolcol, cannabidiolic acid, cannabidivalic acid, cannabinodivalin, cannabinodivalin, cannabicitran, HU-210 (1,1-dimethylheptyl-11-hydroxytetrahydrocannabinol) or dexanabinol (HU-211).

[0057] Transporter portions can associate with carbon nanomaterials in various ways, such as by covalent bonding to the ends or faces of the carbon nanomaterials. Covalent bonding can occur directly between the portion and the particle, or via linker molecules, and can associate with the ends of solubilizing chains, or one or more transporter portions can be non-covalently bonded to the oxidized carbon nanomaterial.

[0058] In some embodiments, the therapeutic composition may include anti-inflammatory agents, anticancer agents, antidiabetic agents, and combinations thereof.

[0059] The carbon nanomaterials disclosed herein can be used as MRI tracers to enable real-time tracking, distribution, and delivery of therapeutic compositions administered to a subject. In this case, other elements such as gadolinium may be beneficial to enhance contrast, and these are often added via conjugated chelating agents.

[0060] Treatment method and pharmaceutical composition A method for forming persulfides and / or polysulfides from hydrogen sulfide is proposed. The method comprises contacting an effective amount of oxidized carbon nanoparticle material with the cells to be used, the particles comprising multiple carbonyl, hydroxyl, and carboxyl substituents.

[0061] As part of a method for treating a subject and its cells that require treatment, the proposed method reduces free radical species and therefore reduces free radical pathology typically associated with or within the subject's body at the site of injury. For example, the treatment method may be described as reducing the level of reactive oxygen species at the target site of the subject by approximately 5% to approximately 50%.

[0062] Another proposed method involves increasing the persulfurization of the protein, which is necessary for its proper function, thereby restoring its function.

[0063] Furthermore, carbon oxide nanomaterials offer minimal toxicity to the subjects being treated.

[0064] In some embodiments, oxidized carbon nanomaterials may be described as hydrophilic, hydrophobic, or amphiphilic (amphiphilic, allowing water solubility along with non-covalent bonding of hydrophobic molecules such as drugs or diagnostic labels), and may, for example, be analogous to micelles or liposomes, though not limited to these. Furthermore, carbon nanomaterials can capture additional active agents for delivery to sites, thus capturing the efficacy and antioxidant properties of active agents.

[0065] In other embodiments, the oxidized carbon nanomaterials may be modified to include a hydrogen sulfide donor moiety. H2S donor skeletons based on compounds with known biological activity are classified below according to their H2S release mechanism, as discussed in Powell et al., Biochem Pharmacol 149:110-123 (2018). These materials are discussed in Example 4 below this specification.

[0066] Thiol-triggered H2S donors react with thiols and possess a wide variety of reactive moieties, including N-benzoylthiol benzamide, acylperthiol, polysulfides, dithioperoxyanhydrides, and S-aloylthioxamine. Additionally, there are two enzyme-triggered structural motifs: geminal-dithiol and trimethyllock prodrug. All of these skeletons can be modified with unique R and R' groups, as is well known in the art, which facilitate direct conjugation to linker molecules or nanoparticles.

[0067] In some embodiments, the carbon oxide nanomaterial may contain an active agent, which is covalently or noncovalently associated with the carbon oxide nanomaterial. For example, the active agent may include anticancer drugs, chemotherapeutic agents, antioxidants, anti-inflammatory drugs, or any combination of these active agents may be small molecules, proteins, aptamers, DNA, antisense oligonucleotides, miRNAs, siRNAs, or combinations thereof.

[0068] For example, one or more active agents may covalently associate with oxidized carbon nanomaterials through cleavable moieties (i.e., ester / amide bonds), photocleavable moieties, or pH-sensitive cleavable moieties. Further modes of association with carbon nanomaterials are also conceivable.

[0069] In yet another embodiment, a method is provided for treating oxidative stress, in which the level of reactive oxygen species in a subject is reduced by administering a preparation of oxidized carbon nanoparticles to the subject. In a particular application, the preparation may be provided as a localized treatment preparation in and / or on a site on the subject (Figure 1). Methods are also provided for reducing the levels of reactive oxygen species (ROS), reactive sulfur species (RSS), or other radical stressors and those not yet characterized to homeostatic levels or pre-stress levels, the method comprising administering a preparation of oxidized carbon nanoparticles.

[0070] The above-described treatment preparations and methods may function to reduce ROS and RSS by any number of mechanisms, such as by weakening the generation of reactive species and / or mitigating the consumption of endogenous antioxidant enzymes induced by the reactive species. As part of a therapeutic treatment, the reduction in ROS resulting from the administration of the preparation is intended to be achieved, for example, by measuring changes in the intensity of one or more reactive oxygen species-specific dyes or markers.

[0071] In yet another embodiment, a method for restoring protein persulfidation is provided. The treatment preparations and methods of use thereof are thought to function by enhancing the production of persulfides and / or polysulfides from H2S by administering oxidized carbon nanoparticles to the subject. Insufficient persulfidation contributes to a range of diseases in which enhanced production of persulfides and / or polysulfides may be desirable. The degree of protein persulfidation can be measured by mass spectrometry or other means as an indicator of the treatment effect.

[0072] The above method of using the material, which metabolizes hydrogen sulfide within cells to produce therapeutic products, is combined with a hydrogen sulfide donor to insert beneficial polysulfides into the target site of the subject.

[0073] The intended carbon oxide nanoparticle material may be used at least in the manufacture of a drug (pharmaceutical composition) useful for forming persulfides and / or polysulfides from hydrogen sulfide. The intended carbon oxide nanoparticle material, the drug or pharmaceutical composition containing it, forms persulfides and / or polysulfides by contact with cells in vitro or in vivo, such as in a subject requiring it. When used in this manner, pharmaceutically acceptable diluents, collectively referred to as pharmaceutically acceptable salts, buffers, etc., are usually present for those that may be present in a composition not intended for pharmaceutically acceptable use.

[0074] The intended carbon oxide nanoparticle material often exists as a carboxylic acid and / or carboxylic acid anion. To balance the charge of the carboxylate group, cations such as monovalent lithium, sodium, potassium, and ammonium ions, or divalent ions such as magnesium or calcium, may be present.

[0075] Amine-containing compounds can also associate with the intended particles, be covalently or acovalently linked, or simply be bound by hydrophobic, hydrophilic, or other forces. If a solubilizer such as PEI is associated with these particles, the number of positive charges from the protonated amine groups of the polymer may outweigh the number of negative charges from the carboxylates of those particles at the pH values ​​of most intended body fluids or cell culture media, which are typically near neutral. Therefore, anionic groups may be present to balance the positive charges.

[0076] Examples of useful anions include, but are not limited to, sulfuric acid, hydrochloric acid, hydrobromic acid, acetic acid, adipic acid, alginic acid, citric acid, aspartic acid, benzoic acid, benzenesulfonic acid, bisulfuric acid, butyric acid, camphoric acid, camphor sulfonic acid, digluconic acid, cyclopentanepropionic acid, dodecyl sulfate, ethanesulfonic acid, glucoheptanoic acid, glycerophosphate, hemisulfuric acid, heptanoic acid, hexanoic acid, fumaric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, methanesulfonic acid, nicotinic acid, 2-naphthalenesulfonic acid, oxalic acid, palmoic acid, pectinic acid, persulfate, 3-phenylpropionic acid, picric acid, pivalic acid, propionic acid, succinic acid, tartaric acid, thiocyanic acid, tosylic acid, mesylic acid, and undecanoic acid.

[0077] A list of commonly used pharmaceutically acceptable acids and bases that form pharmaceutically acceptable salts with pharmaceutical compounds is published in Berge, J. Pharm. Sci. 1977 68(1):1-19.

[0078] The intended pharmaceutical composition comprises an intended carbon oxide nanoparticle material or a pharmaceutically acceptable salt thereof, dissolved or dispersed in a physiologically (pharmaceutically) acceptable carrier in an amount that forms persulfides and / or polysulfides. Such a composition can be brought into contact with mammalian cells by administration in vitro, such as in a cell culture, or in vivo, such as in a living host mammal requiring it.

[0079] The data in the figure shows that concentrations of approximately 0.1 to 10 mg / L and 2 mg / kg provide effective doses in this study. Those skilled in the art can easily use these amounts to determine effective doses for specific subjects or cells being treated.

[0080] The intended composition is typically administered in vivo to the subject requiring it multiple times within one month, for example, weekly, and may be administered over several months to several years. More commonly, the intended composition is administered multiple times throughout the course of treatment.

[0081] The intended pharmaceutical composition may, if desired, be administered orally (perorally) or parenterally (preferably) as a formulation comprising a conventional non-toxic, pharmaceutically acceptable carrier, adjuvant, and vehicle. As used herein, the term parenteral includes subcutaneous injection, intravenous injection (most preferred), intramuscular injection, intrasternal injection, or infusion method. The formulation of drugs is discussed, for example, in Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania; 1975 and Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980.

[0082] Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. The amount of the compound intended in a solid dosage form is the effective dose as discussed earlier. Furthermore, solid dosage forms can be administered multiple times a week.

[0083] In such solid dosage forms, the compounds of the present invention are typically combined with one or more adjuvants appropriate for the indicated route of administration. When administered orally, these compounds may be miscible with lactose, sucrose, starch powder, cellulose esters of alkanates, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfate, gelatin, gum arabic, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, and then tableted or encapsulated for convenient administration.

[0084] Such capsules or tablets may include controlled-release formulations that can be provided as a dispersion of the active compound in hydroxypropyl methylcellulose. In the case of capsules, tablets, and pills, their dosage forms may also include buffers such as sodium citrate, magnesium carbonate or calcium carbonate, or magnesium bicarbonate or calcium bicarbonate. Tablets and pills may further be prepared using enteric coatings.

[0085] The intended pharmaceutical composition is preferably suited for parenteral administration. Therefore, when administered, the pharmaceutical composition is preferably in liquid form, most preferably an aqueous liquid, although other liquids are also intended as discussed below, and currently the most preferred composition is an injectable preparation.

[0086] Therefore, injectable preparations, such as sterile injectable aqueous or oily solutions or suspensions, can be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents. The sterile injectable preparations may also be, for example, a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol.

[0087] Acceptable vehicles and solvents that can be used include water, Ringer's solution, isotonic sodium chloride solution, and phosphate-buffered saline. Sterilized solutions can be prepared by dissolving the active component in the desired solvent system and then sterilizing the resulting solution by passing it through a membrane filter, or by dissolving the sterilized compound in a pre-sterilized solvent under sterile conditions.

[0088] Other liquid pharmaceutical compositions include, for example, solutions suitable for parenteral administration. Examples of liquid compositions suitable for parenteral administration include sterile aqueous solutions of the active component of carbon oxide nanoparticles, or sterile solutions of the active component in a solvent containing water, ethanol, or propylene glycol. In some embodiments, the intended carbon oxide nanoparticle material is provided as a dry powder that is dissolved (dispersed) in a suitable liquid medium, such as sodium chloride for injection, before use.

[0089] In another embodiment, the composition being considered is designed for external use. Therefore, the viscosity of the composition is preferably higher than that of drinking water. The viscosity is preferably between that of heavy cream or motor oil and that of mayonnaise at room temperature. Otherwise, these viscosities are equivalent to those of hand cream or hand lotion, compared to ointment. Thus, one preferred composition can be easily poured at room temperature. Other preferred compositions, such as gels, tend to be very difficult to pour, even at room temperature, requiring scooping from a container, transferring to another container, and maintaining a pointed shape for several seconds.

[0090] The intended topically administered pharmaceutical composition does not need to be applied, for example, by hand or with a spatula, but can be applied via a transdermal patch that adheres to the skin or mucous membrane to be treated. In such a patch, the pharmaceutical composition is typically held within a composition-permeable reservoir arranged on a flexible plane, the reservoir being surrounded on its flexible surface by an adhesive. The reservoir and the adhesive flexible surface do not need to be a single entity and may be separate entities.

[0091] The desired viscosity range is usually achieved with the help of one or more polymer thickeners. Exemplary polymer thickeners include the starch derivatives Zeina B862 hydroxypropyl starch phosphate and Zeina B860 hydroxypropyl starch. Polysaccharide gums are another useful thickener that may be present in the intended composition.

[0092] A suitable representative gum belongs to the galactomannan gum category. Galactomannan gum is a carbohydrate polymer containing D-galactose and D-mannose units, or other derivatives of such polymers. Galactomannan is relatively numerous, and its composition varies depending on its origin. Galactomannan gum is characterized by a chain-like structure in which β-D-mannopyranosyl units are linked (1→6). One-membered rings of α-D-mannopyranosyl units are present as lateral branches linked (1→6) to the main chain. Galactomannan gum includes guar gum, which is the finely ground endosperm of the seeds of either of two leguminous plants (Cyamposis tetragonalobus and psoraloids), and locust bean gum, which is found in the endosperm of the seeds of carob (Ceratonia siliqua).

[0093] Other suitable representative gums include agar gum, carrageenan gum, gatti gum, karaya gum, rhamsan gum, and xanthan gum. The compositions of the present invention may include mixtures of various gums, or mixtures of gum and acidic polymers.

[0094] Gum, particularly galactomannan gum, is a well-known material. See, for example, *Industrial Gums: Polysaccharides & Their Derivatives*, Whistler RL and BeMiller JN (eds.), 3rd Ed. Academic Press (1992) and Davidson RL, *Handbook of Water-Soluble Gums & Resins*, McGraw-Hill, Inc., New York (1980). Most gums are commercially available in various forms, generally as powders, and are ready for use in food and topical compositions. For example, powdered locust bean gum is available from Tic Gums Inc. (Belcam, Md.).

[0095] When used, polysaccharide gum is typically present in an amount of about 0.5 percent to about 5 percent based on the total weight of the composition, with a preferred amount being about 0.5 percent to about 2 percent.

[0096] A substitute or additive for polysaccharide gum is polyacrylic acid polymer. A common type of polyacrylic acid polymer is commonly known as "carbomer," which is a polyacrylic acid polymer loosely crosslinked with polyalkenyl polyethers. These materials are commercially available from BFGoodrich Company (Akron, Ohio) under the name "CARBOPOL®". Particularly preferred types of carbomer are those named "CARBOPOL® 940" and "CARBOPOL® 934".

[0097] Other polyacrylic acid polymers suitable for use in the implementation of the present invention are commercially available under the names "Pemulen®" (BFGoodrich Company) and "POLYCARBOPHIL®" (AHRobbins, Richmond, Va.). Pemulen® polymers are copolymers of C10-C30 alkyl acrylates crosslinked with sucrose allyl ether or pentaerythritol allyl ether and one or more monomers from acrylic acid, methacrylic acid, or simple esters thereof. POLYCARBOPHIL® is polyacrylic acid crosslinked with divinyl glycol.

[0098] The level of polymer thickeners and their incorporation into the above formulations are based on established pharmaceutical principles. Other formulation components such as penetrating agents, colorants, antioxidants, emollients, lubricants, and antimicrobial agents may also be added.

[0099] It is understood that in therapeutic formulations, active oxidized carbon nanoparticle materials can be dispersed, dissolved, emulsified, suspended, freeze-dried, encapsulated, micronized, and nanoparticleized. Furthermore, the formulation may contain solvents, such as alcohols, polyols, esters, water, thickeners and viscosity modifiers, such as galactomannan gum, carbohydrate polymers, such as starch, cellulose derivatives, alginic acid and alginic acid derivatives, alkyl acrylate polymers and alkyl acrylate copolymers, polyvinyl alcohol and polyvinyl alcohol derivatives, film-forming agents, such as cellulose ethers and carbomers, chelating agents, emulsifiers, such as long-chain fatty acids, ordinary alcohols or fatty alcohols and / or esters, amino acids, and buffering agents.

[0100] In further embodiments, the intended carbon oxide nanoparticle material composition is formulated as a nasal spray or inhalant for administering the active carbon oxide nanoparticle material to the lungs or to the brain via a cribriform. Compositions for such applications are generally less viscous than compositions for topical applications and have a water-like viscosity at the body temperature of a mammalian subject.

[0101] In nasal spray administration, the carbon oxide nanoparticle material is delivered by inhalation. For example, carbon oxide nanoparticle material or carbon oxide nanoparticle material derivatized with PEG and / or a chelating agent may be prepared for dry dispersion, for example, by freeze-drying or spray-drying of a solution containing a conjugate, using methods such as those described in U.S. Patents 6,509,006, 6,592,904, 7,097,827 and 6,358,530. Exemplary dry powder excipients include low molecular weight carbohydrates or polypeptides that are mixed with the carbon oxide nanoparticle material to aid dispersion.

[0102] Typically, the composition is prepared as an injectable liquid solution or suspension. A solid form suitable for dissolving or suspending in a liquid vehicle before injection may also be prepared. Injectable preparations are preferred, but topical, nasal spray, or powder compositions can also be dispersed, emulsified, or encapsulated in liposomes or microparticles such as polylactides, polyglycolides, or copolymers [Langer, (1990) Science 249(4976):1527-1533].

[0103] In addition to the oxidized carbon nanoparticle material, the intended liposomes may contain one or more of the following: sterols, phosphatidylethanolamine having a fatty acid moiety with 12 to 18 carbon atoms, and phosphatidylcholine having a fatty acid moiety with 12 to 18 carbon atoms. More specifically, the components of the liposome may include phosphatidylserine, phosphatidyl-inositol, sphingomyelin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, dipalmitoyl-phosphatidylglycerol, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glyceryl ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymer, fatty acids, fatty acid amides, diacylglycerol, diacylglycerol succinate, DMPC (1,2-dimiristoyl-sn-glycero-3-phosphocholine) or DMPG (1,2-dimiristoyl-sn-glycero-3-phospho-rac-[1-glycerol]).

[0104] In alternative embodiments, useful types of pharmaceutically acceptable excipients as carriers for dry powder dispersions include stabilizers such as human serum albumin (HSA), which is also a useful dispersant; extenders such as carbohydrates, amino acids, and polypeptides; pH adjusters or buffers; and salts such as sodium chloride. These carriers may be in crystalline or amorphous form, or a mixture of the two. Devices that may be used to deliver powder or aerosol formulations include, for example, those described in U.S. Patents No. 5,605,674 and No. 7,097,827, which may be nebulizers.

[0105] Carbon oxide nanoparticle materials and their formulations used in carrying out the uses and methods provided herein can also be freeze-dried. Therefore, stable freeze-dried pharmaceutical compositions such as those provided herein can be prepared by freeze-drying a solution containing carbon oxide nanoparticle material alone, or a solution mixed with an extender, such as mannitol, trehalose, raffinose, and sucrose, or a mixture thereof. Many other conventional freeze-drying agents exist. Among sugars, lactose is the most common. Citric acid, sodium carbonate, EDTA, benzyl alcohol, glycine, and sodium chloride are also used [see, for example, Baheti et al., (2010) J Excip Food Chem 1(1):41-54].

[0106] Furthermore, sterilized fixatives are used as solvents or suspensions in conventional methods. For this purpose, any non-irritating fixative, including synthetic monoglycerides or synthetic diglycerides, can be used. In addition, fatty acids such as oleic acid are used in the preparation of injectable compositions. Dimethylacetamide, surfactants including ionic and nonionic detergents, and polyethylene glycol can be used. Mixtures of solvents and wetting agents, such as those discussed above, are also useful.

[0107] Mammals (subjects) that require treatment and are administered a pharmaceutical composition containing the compound of interest may include primates such as humans, apes such as chimpanzees or gorillas, monkeys such as crab-eating macaques or macaques, laboratory animals such as rats, mice or rabbits, companion animals such as dogs, cats or horses, or food animals such as dairy cows or male calves, sheep, lambs, pigs, goats or llamas.

[0108] When an in vitro assay is intended, the sample to be assayed, such as cells and tissues, can be used. As is well known, these in vitro compositions typically contain water, sodium chloride or potassium chloride, and one or more buffer salts such as acetates and phosphates, Hepes, and a metal ion chelating agent such as EDTA, which is buffered to a desired pH value such as pH 4.0 to 8.5, preferably about pH 7.2 to 7.4, depending on the assay being performed.

[0109] Preferably, the pharmaceutical composition exists in unit dosage form. In such form, the composition is divided into unit doses containing an appropriate amount of the active compound. The unit dosage form may be a packaged preparation, the package containing, for example, separate quantities of the preparation in vials or ampoules.

[0110] Chemotherapy agents The intended compositions may include chemotherapeutic agents together with carbon oxide nanoparticles. These chemotherapeutic agents may include alkaloids, covalent DNA binders, non-covalent DNA binders, antimetabolites, enzymes, hormones, platinum compounds, anticancer drugs, monoclonal antibodies conjugated to toxins and / or radionuclides, biological response modifiers, hematopoietic growth factors, angiogenesis inhibitors, DNA crosslinkers, topoisomerase I inhibitors, or microtubule inhibitors. Further examples of chemotherapeutic agents include 2-chlorodeoxyadenosine, 2'-deoxycoformycin, 5-fluorouracil, 5-fluorodeoxyuridine, 4-dimethoxydaunomycin, 11-deoxy-daunorubicin, 13-deoxy-daunorubicin, adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14-naphthalene acetate, 6-mercaptopurine, 6-thioguanine, acyclovir, amsacrine, anastrozole, asparaginase, azathioprine, busulfan, camptothecin, canbusil, carboplatin, and carminomycin. The possible candidates are inomycin, carmustine, chlorambucil, chlorodeoxyadenosine, cisplatin, cyclophosphamide, cytarabine, dactinomycin, dacarbazine, daunorubicin, doxorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, flutamide, hydroxymethylmelamine, idarubicin, ifosfamide, irinotecan, lomustine, lovastatin, mechloretamine, melphalan, methotrexate, mitoxantrone, mitomycin, oxaliplatin, semustine, thiotepa, tamoxifen, taxol, taxotere, teniposide, thioguanine, topotecan, vincristine, vinblastine, and vindesine.

[0111] Examples The following embodiments are intended to illustrate various specific aspects of the intended invention and are not intended to limit the intended invention.

[0112] Example 1 - Materials and Method Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich and used without further purification unless otherwise stated. Single-walled carbon nanotubes (SWCNTs; Batch HPR187.4) were obtained from HiPco Laboratory at Rice University. GMP AC was obtained from EnviroSupply & Service. Fuming (90%) nitric acid was obtained from Alfa Aesar.

[0113] Other suitable PEGylated or functionalized carbon nanomaterials can be used, including, but are not limited to, PEGylated graphite oxide nanoribbons (PEG-GONR), PEGylated carbon oxide black (PEG-OCB), and PEGylated carbon black (PEG-CB).

[0114] The above nanomaterials can be produced from various commercially available carbonaceous starting materials using established synthesis methods. The synthesis and characterization of PEG-HCC can be found in Berlin et al. (2010) ACS Nano. 4(8):4621-4636, which is incorporated herein by reference. The synthesis and characterization of PEG-OAC are listed in Example 3.

[0115] Example 2: Nanozyme material Nanozymes, which are catalytic nanoparticles, can carry out various chemical reactions in a non-stoichiometric manner. In this example, the nanozyme is derived from an oxide carbon nanomaterial equivalent to PEG-HCC (size of 40 nm or less, highly conjugated planar graphene domains and an optimally balanced oxygen-containing portion). Existing nanozymes derived from oxide carbon nanomaterials have been shown to effectively disproportionate superoxides (SO) and catalyze reactions of mitochondrial substrates such as nicotinamide adenine dinucleotide:cytochrome c oxidoreductase.

[0116] sp decorated with oxygen-containing parts 2Hybrid carbon-rich carbon nanoparticles (CNPs) function as nanozyme catalysts for superoxide disproportionation and electron transfer in mitochondria. PEG-HCC catalytically disproportionates superoxides via a two-step reaction with a lower total activation energy than the spontaneous disproportionation of SO alone. This process occurs through oxidation of SO to O2 and simultaneous reduction by PEG-HCC. In the second step, SO is reduced to H2O2 by PEG-HCC. This process occurs because PEG-HCC readily accepts and donates electrons (6, 16).

[0117] Because single-walled carbon nanotubes (SWCNTs) are expensive and considered toxic, reducing reliance on them as a starting material will be a driving force for alternative carbon sources. Even after severe oxidation, which resulted in fairly small, non-tubular HCCs, no toxicity was observed. While effective CNPs were developed from bituminous coal from a previous cycle, variability in the raw material may complicate clinical translation.

[0118] In this invention, CNPs with properties similar to HCCs and CNPs with different properties are synthesized starting from materials prepared using Good Manufacturing Practice (GMP) activated carbon, by adjusting the oxidation of the particles to achieve an optimal balance of oxygen-containing moieties for improved antioxidant activity and mitochondrial activity. The nanozyme candidates address concerns related to SWCNTs.

[0119] However, GMP-certified activated carbon (AC) remains a safe and inexpensive candidate for forming oxidized activated carbon (OAC). Low-ash content activated carbon derived from NSF / ANSI-certified processed bituminous coal can serve as an alternative source to activated coal, which is the activating material.

[0120] OACs such as HCC or PEG-HCC analogs include (1) a highly conjugated carbon core, (2) a balance of oxygen-containing functional groups, particularly 1,2-quinones at the ends for preferred electrochemical activity and a balance of carboxylic acids for easy additional attachment such as PEGylation, (3) a biocompatible size range from approximately less than 100 nm, such as the particles in Example 3, to an average diameter of 2.8 nm, as shown in Figures 15 and 16, and (4) the ability to catalytically disproportionate superoxides (Figure 2). Further preferred properties of OACs that are not present in HCCs are discussed hereafter and, as shown in Figure 6, that the OAC is stably water-dispersible.

[0121] To date, the inventors have developed promising PEG-HCC substitutes from quantum dots (GQDs) and oxygen-containing aerosols (OACs) derived from bituminous coal-derived graphene. The inventors have published several papers on coal-derived GQDs (27, 28) and the production of PEG-GQDs as antioxidants (17) and neuroprotective agents (7) by oxidation and PEGylation. Since quinones have been identified as the central redox activity of HCCs (8), a balance must be maintained between quinone groups and other oxygen-containing functional groups. The oxidation of ACs can be optimized for quinones and carboxylic acids. Ultimately, the most desirable outcome is the preparation of particles that mimic the electrochemical properties of PEG-HCCs.

[0122] The PEGylation reaction is typically carried out as described in U.S. Patent No. 9,572,834 and in detail in Berlin et al., ASC Nano 2010;4:4621-4636. Roughly, 5000 MW of methoxy-(polyethylene glycol)amine is bonded to the carboxyl groups of OAC particles to form amides using N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) in dimethylformamide (DMF). Similar materials are prepared by other coupling chemistry and by reacting the ketone functional groups of the particles with the same amine in the presence of a reducing agent such as sodium cyanoborohydride.

[0123] PEG-OAC is synthesized by reacting AC with 90% HNO3 at approximately 100-140°C for 1-24 hours, while varying the oxidation conditions and time (Figure 3). These particles were used as prepared. The reaction is intended to occur in less than one hour in a flow reactor. Furthermore, the activated carbon particles are intended to be more uniformly sized, averaging approximately 20 nm, by vigorously shaking them before the reaction to obtain OAC particles of a more uniform size.

[0124] Each batch is subjected to quality control and characterized to measure electrochemical activity by cyclic voltammetry (CV), oxygen-containing functional groups by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy (XPS and FTIR), PEGylation efficiency by thermogravimetric analysis (TGA), size by high-resolution transmission electron microscopy (HRTEM), particle height profile by atomic force microscopy (AFM), hydrodynamic volume by dynamic light scattering (DLS) or nanoparticle tracking analysis (NTA), and acceptable trace metal levels by inductively coupled plasma mass spectrometry (ICP-MS).

[0125] To mitigate concerns about metal contamination (such as that previously observed in graphene oxide (GO) (31, 32)), all materials are subjected to the filtration method previously described (17). Trace metal analysis by ICP-MS is performed as reported (32). Endotoxins are minimized by following published guidelines (33). Both biological validity and reproducibility are ensured by adhering to MIRIBEL standards (34).

[0126] When OACs have a highly conjugated carbon core, oxygen-containing functional groups that result in a broad reduction potential with a high onset potential, and a suitable size (diameter) in the range of 100 nm or less, such as approximately 1 to 40 nm or approximately 3 to 30 nm, the particles passing through a 0.22 μm filter exhibit both antioxidant, persulfide and / or polysulfide enhancement, and electron transport shuttle properties in solution, intracellularly, and in vivo.

[0127] Carbon oxide nanoparticles (CNPs) can be heterogeneous. To minimize variability, each synthesis is performed in triplets to establish the expected mean and standard deviation values ​​of the target factors listed above.

[0128] OACs are typically disc-shaped, with a diameter of approximately 1 to 30 nm, which can vary depending on the reaction conditions. Like discs, the particles can be round. However, not all particles are round; rather, they are flattened oval in shape. In this specification, the longest dimension of the latter particles is considered to be their diameter.

[0129] The broader, disc-like shape of OACs may hinder cellular uptake compared to thinner, ribbon-like HCCs (2), suggesting that shape effects may alter the nanomaterial's ability to penetrate the cell membrane, requiring further investigation. However, since the PEG portion forms a micelle-like structure around the hydrophobic conjugated carbon core of the nanomaterial, potential shape effects may become less significant after PEGylation. Diameter can also affect cellular uptake and, for example, intracellular distribution into mitochondria, and the ideal size and shape depend on the targeted pathology. Graphene quantum dots (GQDs) may also be used. Graphene quantum dots have been prepared from a variety of raw materials and have been effective in the TBI models investigated.

[0130] The analytical techniques used are as reported in the references (16, 35) explicitly incorporated herein by reference for this purpose. CVs are collected as reported in (16). High-resolution spectra for elemental analysis of carbon-containing functional groups are obtained using XPS and X-ray emission from the 1s electron orbital of carbon. FTIR is obtained for analysis of oxygen-containing functional groups. AFM is used to measure particle height profiles, and HRTEM is used for lateral shape measurements as described (Figure 4) (27).

[0131] CNP was evaluated for toxicity to each cell line tested, with cytotoxicity based on the lowest concentration that did not result in more than 10% cell loss (10-fold higher). Only replicates that did not show toxicity in vitro proceeded to in vivo testing.

[0132] Example 3 - Uniqueness of the biological process of oxidation of activated carbon with nitric acid This embodiment demonstrates a novel synthetic method for producing carbon oxide nanomaterials that provide biologically active carbon oxide nanoparticle products without substituents such as solubilizing polymers. Synthesis using other methods has yielded non-biologically active preparations of carbon oxide materials, making these materials useless in compositions for reducing reactive oxygen species in vivo or for treating diseases / pathologies associated with these conditions.

[0133] In this embodiment, the carbon oxide material used was GMP activated carbon (AC). However, similar results can be expected using any other carbon material.

[0134] Oxidized activated carbon (OAC) was synthesized by oxidizing AC via HNO3. Typically, existing literature on non-biological hydrocarbon studies using graphene oxide-like materials involves oxidation using non-fuming nitric acid (70% HNO3). However, in this study, set in a biological environment, a considerably higher concentration, in this case fuming (90%) nitric acid, is required. This study demonstrates a significant difference between biological and non-biological applications.

[0135] To determine whether the strength of the oxidizing agent affects the characteristics of the product, AC was oxidized with 70% HNO3 or 90% HNO3 (Figure 5). The resulting OAC products were analyzed by TGA, XPS, and CV to clarify the differences between materials.

[0136] Characterization of synthesized OACs by varying the strength of the oxidizing agent. The most significant difference between OAC synthesized using 70% HNO3 and OAC synthesized using 90% HNO3 is the water dispersibility of the resulting oxidized activated carbon nanoparticle material (Figure 6). The AC product oxidized with 90% HNO3 is completely water dispersible and can pass through a 0.22 μm PES filter, whereas the AC product oxidized with 70% HNO3 is water-insoluble and cannot pass through the filter (Figure 6). This result is likely due to the harsher oxidation conditions that result in higher oxygen content and smaller particles.

[0137] TGA analysis showed that OAC oxidized with 90% HNO3 was more effectively oxidized than activated carbon matrix and 70% HNO3 OAC, due to a greater weight loss around 190°C to 210°C (the temperature range in which oxygen functional groups decompose) (Figure 7). XPS analysis showed no significant difference between the oxygen-containing functional groups of the two OAC materials, but the product oxidized with 90% HNO3 had almost twice the carbonyl content of the product oxidized with 70% HNO3 (Table 1 below).

[0138] Of the carbonyl moieties, the 1,2-quinone group is most interesting because the aromatic quinone portion exhibits a reduction potential at a higher onset potential than the hydroxyl or carboxyl functional groups. Therefore, the presence of a quinone group is important for the electrochemical activity of OAC materials. Although 90%HNO3OAC shows a higher carbonyl atom percentage, the onset potential to reduction potential of both OAC products is comparable (approximately +0.2V) (Figure 8).

[0139] [Table 1]

[0140] Furthermore, the presence of a 1,2-quinone group provides a means for preparing ethylenediamine derivatives of oxidized carbon nanoparticle materials. Exemplary ethylenediamine-blocked PEG-HCC [EN-PEG-HCC] or non-PEG-modified OAC can be prepared as schematically shown below.

[0141] [ka]

[0142] In an illustrative synthesis, an aqueous solution of PEG-HCC (5.0 mL, 1.2 mg / mL, 6 mg) was added to ethylenediamine (5.0 mL, 4.5 g, 75 mmol). Water was removed by rotovap, and PEG-HCC precipitated in the ethylenediamine. PEG-HCC was dispersed by adding MeOH (6 mL) along with molecular sieves.

[0143] The above reaction mixture was stirred at room temperature for 5 days, then diluted with DI H2O and filtered through a 0.22 μm polyethersulfone (PES) membrane. The above material was purified by cross-flow filtration using a 50 kDa mPES dialysis column (intermembrane pressure of approximately 1 atm) (Spectrum® Labs Krosflo®, Research IIi TFF System) to obtain 15 mL of EN-PEG-HCC with a carbon core concentration of 400 mg / L.

[0144] Example 4 - Oxidation of sulfides This embodiment illustrates the use of carbon nanomaterials in intracellular synthesis of polysulfides in a manner that goes beyond the endogenous ability of cells to treat various injury conditions, and may demonstrate therapeutic applications for such treatment. These injury conditions include, but are not limited to, inflammation, sepsis, stroke, and other diseases in which free radical generation is involved as a multiple cause.

[0145] Polysulfides are partially endogenously produced as antioxidants by superoxide dismutase (84) and are formed under hypoxic conditions (83). These active sulfur species (RSS) can quench free radicals both in vitro and in materials such as rubber (81, 84). Catalytic formation of polysulfides has been demonstrated using superoxide dismutase and catalase (87, 91), but to the best of our knowledge, has not been demonstrated using exogenously supplied catalysts.

[0146] These carbon-based nanozymes (PEG-HCC, PEG-OAC) have been found to readily oxidize thiols (Figures 9A, 9B), suggesting potential activity towards persulfidation and H2S. The aforementioned nanozymes dramatically increase the rate of polysulfide formation in a cell-free environment using sodium sulfide as the H2S donor (Figures 10A and B). Similarly, several preparations have been shown to significantly improve the intracellular polysulfide formation rate (Figures 11A and B). The multiplicative increase in polysulfide generation depends on the particle preparation method. In the example shown in Figure 11A, PEG-HCC treated with ethylenediamine [EN-PEG-HCC] proved to be the most effective compared to PEG-HCC in Figure 11B.

[0147]

number

[0148] The possible mechanism of action is shown in equations 1-3 above. Electrons are HS - Since it can move from anion to OAC particle (E0 = +0.46V), HSS - / HS - The reduction potential of the OAC· / OAC- half-cell is preferred. The exact mechanism of this reaction is currently unknown. The resulting product, a polysulfide (Figure 10A) as shown in the study of SSP4, is HSS - It is presumed that this is the case.

[0149] As described in Kleinjan et al., Water Res 2005;39(17):4093-4100, under normal oxygen conditions, polysulfides are attacked by oxygen and converted to S2O3. 2- Can it be formed?

[0150]

number

[0151] Alternatively, as discussed in Vivitsky et al., J Biol Chem. 2015;290(13):8310-8320, it can be oxidized by heme proteins such as hemoglobin (Hb).

[0152]

number

[0153] Under normal oxygen conditions, both PEG-HCC and EN-PEG-HCC were shown to catalyze polysulfide formation (Figures 11A and 11B). However, under hypoxic conditions, both EN-PEG-HCC and PEG-HCC increased polysulfide yield considerably more rapidly, suggesting their usefulness in acute conditions involving oxygen deficiency (Figures 11A and 11B).

[0154] Furthermore, these nanoparticles were also found to function as antioxidants. Specifically, by directly attaching H2S donor molecules to the particles, the antioxidant and persulfurization properties of those particles as H2S sources can be further enhanced.

[0155] This example illustrates a range of known biologically active compounds that can be used as the skeletal structure of a hydrogen sulfide donor in the preparation of modified compounds having a unique R group, and that can subsequently be used to facilitate the conjugation of the modified biologically active compound into a linker molecule or nanoparticles. Exemplary skeletal structures are shown below.

[0156] Current methods for sulfide oxidation using carbon-based materials have been demonstrated in the study described in Powell et al., Biochem.Pharmacol (2018), 110-123. In detail, H2S donors are classified according to their H2S release mechanism. Thiol-triggered H2S donors react with thiols and have a wide variety of reactive moieties, including N-benzoylthiol benzamide, acylperthiol, polysulfides, dithioperoxyanhydrides, and S-aloylthioxamine. There are also two enzyme-triggered structural motifs: geminal-dithiol and trimethyllocprodrug.

[0157] [ka]

[0158] All of these skeletons can be modified with unique R groups that facilitate conjugation with linker molecules or nanoparticles. Importantly, despite high PEGylation, small molecules such as dithiothreitol (Figures 9A and 9B) and glutathione (Figure 9B) can reach the surface of these particles, which is a necessary property for the use of thiolysic H2S donors. Another aspect of the present invention may be as follows: [1] Carbon oxide nanoparticles that do not contain exogenously supplied functionalizing solubilizers and form a non-settling aqueous dispersion at a concentration of about 1 to about 5 mg / mL, wherein the dispersion is stable against sedimentation at ambient room temperature for at least 7 days, exhibits a maximum absorbance at about 220 nm, the carbon oxide nanoparticles contain about 9 to about 15 percent carbonyl groups as determined by X-ray photoelectron spectroscopy (XPS), and pass through a polyethersulfone (PES) filter membrane with a pore size of 0.22 μm. [2] The carbon oxide nanoparticles described in [1] above, which are generally disc-shaped particles. [3] The carbon oxide nanoparticles described in [2], wherein the particles have a diameter of about 5 to about 30 nm and a thickness of about 0.3 to about 2 nm. [4] The carbon oxide nanoparticles described in [1], which, when measured by cyclic voltammetry (CV), exhibit a reduction potential including an onset potential greater than approximately 0.05 V and a maximum reduction potential of approximately -2 V. [5] A method for forming persulfides and / or polysulfides from hydrogen sulfide, comprising contacting cells containing hydrogen sulfide with an effective amount of carbon oxide nanoparticles described in [1] above. [6] The method according to [5], wherein the carbon oxide nanoparticles are prepared from one or more nanoparticle materials selected from the group consisting of graphene, graphene nanoribbons, graphene oxide, graphite, graphite oxide nanoribbons, carbon black, hydrophilic carbon clusters, coal, activated coal, and activated carbon. [7] The method according to [5], wherein the persulfide and / or polysulfide is prepared from hydrogen sulfide released endogenously or exogenously. [8] The method according to [5], wherein the cells are contacted in vitro or in vivo. [9] The method according to [5], wherein the oxidized carbon nanoparticles are functionalized with one or more exogenously supplied specific substituents selected from the group consisting of hydrogen sulfide-releasing moieties, solubilizers, biological barrier transporter moieties, tissue targeting agents, assay identifiers, chelating agents, and pharmaceuticals.

[10] The carbon oxide nanoparticles are poly(ethylene glycol) [PEG], poly(propylene glycol) [PPG], poly(ethyleneimine) [PEI], poly(vinyl alcohol) [PVA], PPG-PEG block copolymer, C 12 -C 18 -The method according to [9], which is functionalized with one or more solubilizers selected from the group consisting of poly(ethylene oxide) ether and poly(acrylic acid) [PAA].

[11] The method according to [9], wherein the oxidized carbon nanoparticles are functionalized with an exogenously provided hydrogen sulfide-releasing moiety that provides hydrogen sulfide by thiolysis or enzymatic decomposition.

[12] The method according to

[11] , wherein the hydrogen sulfide-releasing portion is selected from one or more of the group consisting of N-benzoylthiol benzamide, acylperthiol, arylthioamide, dithioperoxyanhydride, S-aloylthioxamine, geminal-dithiol, and trimethyllocprodrug.

[13] The method according to [9], wherein the oxidized carbon nanoparticles are functionalized with a transporter moiety selected from one or more of the group consisting of adamantanil, amantadinil, memantinil, rimantazinil, dopamantinil, tromantadinil, vildagliptinil, and carmantadinil groups.

[14] The method according to [9], wherein the oxidized carbon nanoparticles are functionalized with a pharmaceutical selected from one or more of the group consisting of cannabigerol, cannabigerol monomethyl ether, cannabinerol acid A, cannabigerovaline, cannabigerol acid A, cannabigerovaline, cannabichromene, cannabichromene acid A, cannabivarichromene, cannabiclomevalin, cannabiclomevalin, cannabidiol, cannabidiolcol, cannabidiolic acid, cannabidivalic acid, cannabinodivalin, cannabinodivalin, cannabicitran, HU-210, and dexanabinol.

[15] The method according to [5], wherein the contact is repeated multiple times.

[16] A pharmaceutical composition comprising the carbon oxide nanoparticles described in [1], which are present in an amount effective for generating persulfides and / or polysulfides, dissolved or dispersed in a pharmaceutically acceptable diluent.

[17] The pharmaceutical composition according to

[16] , wherein the pharmaceutically acceptable diluent is an aqueous composition suitable for parenteral administration.

[18] The pharmaceutical composition according to

[17] , wherein the pharmaceutically acceptable diluent has an osmotic pressure that is isotonic with the blood of the intended recipient.

[19] The pharmaceutical composition according to

[18] , wherein the osmotic pressure is approximately 275 to approximately 295 mOsm / kg.

[20] A method for preparing an aqueous composition of carbon oxide nanoparticles as described in [1], wherein the method is: a) A step of forming an acidic reaction composition by mixing carbon nanoparticles with one or more of the following in an oxidatively effective amount: concentrated nitric acid alone, fuming nitric acid alone, concentrated sulfuric acid or concentrated nitric acid dissolved in fuming sulfuric acid, or fuming nitric acid dissolved in concentrated sulfuric acid or fuming sulfuric acid. b) A step of stirring the acidic reaction composition at atmospheric pressure at a temperature of about 22°C and reflux temperature for a sufficient time to form carbon oxide nanoparticles containing about 9 to about 15 percent carbonyl groups by X-ray photoelectron spectroscopy (XPS), c) A step of cooling the refluxed acidic reaction composition and quenching it in an aqueous medium containing a base if necessary to form an aqueous reaction composition. d) Dialyzing the aqueous reaction composition with water using a membrane having a molecular weight cutoff of about 1000 Da for a sufficient time to remove water-soluble materials with a molecular weight of less than about 1000 Da and to form an aqueous composition of carbon oxide nanoparticles, and e) The aqueous composition of carbon oxide nanoparticles is filtered through a polyethersulfone (PES) filter membrane with a pore size of 0.22 μm to obtain a filtrate of the aqueous composition of SOD-like carbon oxide nanoparticles. Methods that include...

[21] The preparation method according to

[20] , comprising the step of separating the water and the carbon oxide nanoparticles and recovering the carbon oxide nanoparticles.

[0159] Bibliography The following publications are expressly incorporated herein by reference in their entirety. Any reference or specification of any document in this application does not constitute an endorsement that such document is available as prior art of the present invention.

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Claims

1. Oxidized activated carbon nanoparticles that form a non-settling aqueous dispersion at a concentration of 1 to 5 mg / mL, wherein the dispersion is stable against sedimentation at ambient room temperature for at least 7 days and exhibits a maximum absorbance at 220 nm, the oxidized activated carbon nanoparticles are disc-shaped particles having a diameter of 5 to 30 nm and a thickness of 0.3 to 2 nm, containing 9 to 15 percent carbonyl groups as determined by X-ray photoelectron spectroscopy (XPS), and passing through a polyethersulfone (PES) filter membrane with a pore size of 0.22 μm.

2. The oxidized activated carbon nanoparticles according to claim 1, wherein the reduction potential measured by cyclic voltammetry (CV) shows an initial potential greater than 0.05 V and a maximum reduction potential of -2 V.

3. A method for forming persulfides and / or polysulfides from hydrogen sulfide, comprising contacting the required cells in vitro with an effective amount of oxidative activated carbon nanoparticles according to claim 1 or 2.

4. (i) The oxidized activated carbon nanoparticles are prepared from one or more nanoparticle materials selected from the group consisting of graphene, graphene nanoribbons, graphene oxide, graphite, graphite oxide nanoribbons, carbon black, coal, activated coal, and activated carbon; (ii) The persulfide and / or polysulfide is prepared from hydrogen sulfide released endogenously or exogenously; and / or (iii) The method according to claim 3, wherein the contact is repeated multiple times.

5. The method according to claim 3 or 4, wherein the oxidized activated carbon nanoparticles are functionalized with one or more exogenously supplied specific substituents selected from the group consisting of hydrogen sulfide-releasing moieties, solubilizers, biological barrier transporter moieties, tissue targeting agents, assay identifiers, chelating agents, and pharmaceuticals.

6. The aforementioned activated carbon nanoparticles (i) Poly(ethylene glycol) [PEG], poly(propylene glycol) [PPG], poly(ethyleneimine) [PEI], poly(vinyl alcohol) [PVA], PPG-PEG block copolymer, C 12 -C 18 - Functionalized with one or more solubilizers selected from the group consisting of poly(ethylene oxide) ethers and poly(acrylic acid) [PAA]; (ii) Functionalized with an exogenously provided hydrogen sulfide-releasing moiety that provides hydrogen sulfide by thiolysis or enzymatic decomposition, wherein the hydrogen sulfide-releasing moiety is selected from one or more of the group consisting of N-benzoylthiol benzamide, acylperthiol, arylthioamide, dithioperoxyanhydride, S-aloylthioxamine, geminal-dithiol, and trimethyllocprodrug: (iii) Functionalized with a transporter moiety selected from one or more of the group consisting of adamantanil, amantadinil, memantinil, rimantazinil, dopamantinil, tromantadinil, vildagliptinil, and carmantadinil groups; and / or The method according to claim 5, wherein the pharmaceutical is functionalized with one or more pharmaceuticals selected from the group consisting of (iv) cannabigerol, cannabigerol monomethyl ether, cannabinerol acid A, cannabigerovaline, cannabigerol acid A, cannabigerovaline, cannabichromene, cannabichromene acid A, cannabivalichromene, cannabiclomevalin, cannabiclomevalin, cannabidiol, cannabidiolcol, cannabidiolic acid, cannabidivalic acid, cannabinodivalin, cannabinodivalin, cannabicitran, HU-210, and dexanabinol.

7. Oxidized activated carbon nanoparticles according to claim 1 or 2 for use in pharmaceuticals, wherein the use is in a method for forming persulfides and / or polysulfides from hydrogen sulfide, the method comprising contacting the required cells in vitro with an effective amount of oxidized activated carbon nanoparticles.

8. (ii) The persulfide and / or polysulfide is prepared from hydrogen sulfide released endogenously or exogenously; and / or (iii) The contact is repeated multiple times, , oxidized activated carbon nanoparticles for use according to claim 7.

9. The oxidized activated carbon nanoparticles for use according to claim 7 or 8, wherein the oxidized activated carbon nanoparticles are functionalized with one or more exogenously supplied specific substituents selected from the group consisting of hydrogen sulfide-releasing moieties, solubilizers, biological barrier transporter moieties, tissue targeting agents, assay identifiers, chelating agents, and pharmaceuticals.

10. The aforementioned activated carbon nanoparticles (i) Poly(ethylene glycol) [PEG], poly(propylene glycol) [PPG], poly(ethyleneimine) [PEI], poly(vinyl alcohol) [PVA], PPG-PEG block copolymer, C 12 -C 18 - Functionalized with one or more solubilizers selected from the group consisting of poly(ethylene oxide) ethers and poly(acrylic acid) [PAA]; (ii) Functionalized with an exogenously provided hydrogen sulfide-releasing moiety that provides hydrogen sulfide by thiolysis or enzymatic decomposition, wherein the hydrogen sulfide-releasing moiety is selected from one or more of the group consisting of N-benzoylthiol benzamide, acylperthiol, arylthioamide, dithioperoxyanhydride, S-aloylthioxamine, geminal-dithiol, and trimethyllocprodrug: (iii) Functionalized with a transporter moiety selected from one or more of the group consisting of adamantanil, amantadinil, memantinil, rimantazinil, dopamantinil, tromantadinil, vildagliptinil, and carmantadinil groups; and / or (iv) Oxidized activated carbon nanoparticles for use according to claim 9, functionalized with a pharmaceutical selected from one or more of the group consisting of cannabigerol, cannabigerol monomethyl ether, cannabinerol acid A, cannabigerovaline, cannabigerol acid A, cannabigerovaline, cannabichromene, cannabichromene acid A, cannabibarichromene, cannabiclomevalin, cannabiclomevalin, cannabidiol, cannabidiolcol, cannabidiolic acid, cannabidivalic acid, cannabinodivalin, cannabinodivalin, cannabicitran, HU-210, and dexanabinol.

11. A pharmaceutical composition comprising oxidized activated carbon nanoparticles according to claim 1 or 2, which are present in an amount effective for generating persulfides and / or polysulfides when dissolved or dispersed in a pharmaceutically acceptable diluent.

12. The pharmaceutically acceptable diluent is an aqueous composition suitable for parenteral administration. The pharmaceutically acceptable diluent has an osmotic pressure that is isotonic with the blood of the intended recipient, and further The pharmaceutical composition according to claim 11, wherein the osmotic pressure is 275 to 295 mOsm / kg.

13. A method for preparing an aqueous composition of oxidized activated carbon nanoparticles according to claim 1 or 2, wherein the method is: a) A step of forming an acidic reaction composition by mixing activated carbon with an oxidizing amount of fuming nitric acid alone. b) A step of refluxing the acidic reaction composition at room temperature and atmospheric pressure for a sufficient amount of time to form oxidative activated carbon nanoparticles containing 9 to 15 percent carbonyl groups by X-ray photoelectron spectroscopy (XPS), c) A step of cooling the refluxed acidic reaction composition and quenching it in an aqueous medium containing a base if necessary to form an aqueous reaction composition. d) A step of dialyzing the aqueous reaction composition with water using a membrane having a molecular weight cutoff of 1000 Da for a sufficient time to remove water-soluble materials with a molecular weight of less than 1000 Da and to form an aqueous composition of oxidized activated carbon nanoparticles, and e) The aqueous composition of oxidized activated carbon nanoparticles is filtered through a polyethersulfone (PES) filter membrane with a pore size of 0.22 μm to obtain a filtrate of the aqueous composition of SOD-like oxidized activated carbon nanoparticles. Methods that include...

14. The preparation method according to claim 13, comprising the step of separating the water and the activated carbon nanoparticles and recovering the activated carbon nanoparticles.

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