Functionalized carbon allotropes for treating homocysteine and cysteine related diseases and conditions

Abstract

Provided herein are compositions, systems, kits, and articles of manufacture for using various functionalized carbon allotropes for treating a cysteine or homocysteine related disease or condition. In particular embodiments, the compositions comprise: a functionalized carbon allotrope (FCA) that breaks down homocysteine and / or cysteine to produce H2S. In certain embodiments, the FCA comprises: i) graphene oxide, ii) functionalized graphene, iii) functionalized carbon nanotubes, iv) functionalized graphene nanoribbons, v) functionalized carbon onions, vi) functionalized carbon nanohorns, vii) carboxy fullerenes, viii) carbon dots, ix) fullertubes, and / or x) polyamino fullerenes.

Description

[0001] FUNCTIONALIZED CARBON ALLOTROPES FOR TREATING HOMOCYSTEINE AND CYSTEINE RELATED DISEASES AND CONDITIONS

[0002] The present application claims priority to U.S. Provisional application serial number 63 / 504,842, filed May 30, 2023, which is herein incorporated by reference in its entirety.

[0003] FIELD

[0004] Provided herein are compositions, systems, kits, and articles of manufacture for using various functionalized carbon allotropes for treating a cysteine or homocysteine related disease or condition. In particular embodiments, the compositions comprise: a functionalized carbon allotrope (FCA) that breaks down homocysteine and / or cysteine to produce H2S. In certain embodiments, the FCA comprises: i) graphene oxide, ii) functionalized graphene, iii) functionalized carbon nanotubes, iv) functionalized graphene nanoribbons, v) functionalized carbon onions, vi) functionalized carbon nanohorns, vii) carboxy fullerenes, viii) carbon dots, ix) fullertubes, and / or x) polyamino fullerenes. In some embodiments, the FCAs are functionalized with hydroxyl, carbonyl, carboxyl, hemi-ketal, epoxide, and / or amine functional groups.

[0005] BACKGROUND

[0006] Elevated levels of homocysteine in the blood, aka hyperhomocysteinemia (HHC), is a biomarker for cardiovascular diseases (e.g., hypertension, stroke and atherosclerosis) and is considered as a risk factor of stroke and heart failure as well as other pathologies, such as dementia, Alzheimer’s disease and chronic kidney disease (1-4). HHC promotes atherosclerotic lesion progression and endothelial dysfunction. High levels of homocysteine has been shown to inhibit NO production by reducing expression level of NO synthase (eNOS) and alter lipid metabolism by oxidative degradation of endothelium membrane lipids. Further, high levels of homocysteine in blood has been shown to increase proliferation of smooth muscle cells, alter platelet function, and promote monocyte activation (1).

[0007] The homocysteine levels are regulated by methionine metabolism and transsulfuration pathways (Fig 1). Homocysteine is synthesized from methionine by first conversion of methionine to S -adenosylmethionine (SAM) by SAM synthetase, conversion of SAM to S- adenosylhomocysteine (SAH) by methyltransferase (MT), and finally SAH to homocysteine by SAH hydrolase. Homocysteine, thus produced, can be catabolized by remethylation to for methionine, transsulfuration to form cysteine or broken down to produce hydrogen sulfide (H2S). Remethylation of homocysteine involves folate / vitamin Bl 2-dependent and vitamin B12-independent mechanisms. Transsulfuration of homocysteine is catalyzed by the vitamin B6-dependent enzymes: cystathionine P-synthase (CBS) and cystathionine y-lyase (CGL). Homocysteine is broken down by CGL to produce H2S, a-ketobutyrate and ammonia (5). HHC can be caused by deficiencies in enzyme, low level of enzyme co-factors, increase in methionine intake, and ingestion of certain drugs. However, deficiencies in enzyme, due to genetic, epigenetic or posttranslational modifications, associated with homocysteine metabolism is considered to be the most common cause of HHC (6). Deficiency in CBS causes homocystinuria (HCU), which is a rare inherited disorder with main clinical features of osteoporosis, dislocation of the optic lenses, learning difficulties and thromboembolism.

[0008] Currently, there is no treatment for HHC or HCU. Clinically, HHC is managed by vitamin supplementation (vitamin B6, B12 and B9) to reduce the risk of cardiovascular diseases. However, clinical studies have failed to show reduction in risk for cardiovascular diseases with vitamin supplementation (7, 8). Further, clinical treatment options for HCU are low-protein diet and betaine for severely affected patients, and vitamin supplementation for mildly affected adults. Artificial protein-based enzymes for Hey treatment are in clinical trials. However, protein-based artificial enzymes can be immunogenic, prone to intracellular modifications, and the treatment may not be viable after few months. Novel, safe and efficient strategies to replace enzyme deficiencies are needed.

[0009] SUMMARY

[0010] Provided herein are compositions, systems, kits, and articles of manufacture for using various functionalized carbon allotropes for treating a cysteine or homocysteine related disease or condition. In particular embodiments, the compositions comprise: a functionalized carbon allotrope (FC A) that breaks down homocysteine and / or cysteine to produce H2S. In certain embodiments, the FC A comprises: i) graphene oxide, ii) functionalized graphene, iii) functionalized carbon nanotubes, iv) functionalized graphene nanoribbons, v) functionalized carbon onions, vi) functionalized carbon nanohorns, vii) carboxy fullerenes, viii) carbon dots, ix) fullertubes, and / or x) polyamino fullerenes. In some embodiments, the FCAs are functionalized with hydroxyl, carbonyl, carboxyl, hemi-ketal, epoxide, and / or amine functional groups. In some embodiments, provided herein are compositions comprising: a) a functionalized carbon allotrope (FCA) that breaks down homocysteine and / or cysteine to produce H2S, wherein the FCA optionally comprises: i) graphene oxide, ii) functionalized graphene, iii) functionalized carbon nanotubes, iv) functionalized graphene nanoribbons, v) functionalized carbon onions, vi) functionalized carbon nanohorns, vii) carboxy fullerenes, viii) carbon dots; ix) fullertubes, and / or x) polyamino fullerenes; and b) a physiologically tolerable aqueous solution; and c) optionally a carrier component for the FCA. Examples of fullertubes are described, for example, in Koenig et at., J. Am. Chem. Soc. 2020, 142, 36, 15614-15623, herein incorporated by reference for such fullertubes.

[0011] In particular embodiments, the functionalized graphene comprises graphene functionalized with at least one of the following functional groups: hydroxyl, carbonyl, carboxyl, hemi-ketal, epoxide, thiol, sulfide, persulfide, thioether, thiosulfate, thiazole, and thiazine, and amine. In further embodiments, the functionalized carbon nanotubes comprise carbon nanotubes functionalized with at least one of the following functional groups: hydroxyl, carbonyl, carboxyl, hemi-ketal, epoxide, thiol, sulfide, persulfide, thioether, thiosulfate, thiazole, and thiazine, and amine. In other embodiments, the functionalized carbon nanotubes are functionalized multi-walled carbon nanotubes (FMWCNs). In other embodiments, the functionalized carbon nanotubes are functionalized single-walled carbon nanotubes (FsWCNs). In further embodiments, the functionalized carbon nanotubes have an outer dimension of between about 5-100 nm (e.g., about 5 ... 15 ... 30 ... 45 ... 65 ... 85 ... or 100 nm). In other embodiments, the FCA has a widest dimension of between 1 nm and 1 um, and optionally wherein the widest dimension is about 5-25 nm.

[0012] In some embodiments, the physiologically tolerable aqueous solution comprises at least one of the following: a buffering agent, pyrogen-free water, isotonic saline, and / or Ringer’s solution. In additional embodiments, the carrier component comprises a material selected from: polymeric nanoparticles, mesoporous nanoparticles, dendrimers, liposomes, metallic nanoparticles, inorganic nanoparticles (e.g. composed of silica), and inorganic microparticles (e.g., composed of silica). In certain embodiments, the nanocarrier component targets the FCA to a desired location when administered to a mammal. In particular embodiments, the carboxy fullerenes have the following formula C60(C(COOH)2)3 in the C3 or D3 isoform.

[0013] In certain embodiments, the functionalized graphene nanoribbons, functionalized carbon onions, and / or functionalized carbon nanohorns are functionalized with at least one of the following functional groups: hydroxyl, carbonyl, carboxyl, hemi-ketal, epoxide, thiol, sulfide, persulfide, thioether, thiosulfate, thiazole, and thiazine, and amine.

[0014] In some embodiments, provided herein are systems, kits, and articles of manufacture comprising: a) a composition comprising: i) a functionalized carbon allotrope (FC A) that breaks down homocysteine and / or cysteine to produce H2S, wherein the FC A optionally comprises: A) graphene oxide, B) functionalized graphene, C) functionalized carbon nanotubes, D) functionalized graphene nanoribbons, E) functionalized carbon onions, F) functionalized carbon nanohoms, G) carboxy fullerenes, H) carbon dots; I) fullertubes, and / or J) polyamino fullerenes; and ii) a physiologically tolerable aqueous solution or lyophilized version thereof; and iii) optionally a carrier component for the FCA; and b) a container selected from the group consisting of: i) an IV fluid solution bag, ii) a syringe vial, iii) a syringe, iv) a sterile shipping container configured for shipping powder or liquid, v) an orally ingestible dosage form.

[0015] In certain embodiments, the composition is present inside the container. In other embodiments, the composition is in a liquid form. In other embodiments, the composition is in a powder form. In some embodiments, the container is the IV solution bag, and wherein the composition is present in the IV solution bag. In further embodiments, the container is the syringe vial, and wherein the composition is present in the syringe vial. In particular embodiments, the container is the sterile shipping container, wherein the composition is present in the sterile shipping container.

[0016] In additional embodiments, the orally ingestible dosage form is a capsule, or pill that comprises an enteric coating. In further embodiments, the FCA is incorporated in, or on, the carrier component.

[0017] In some embodiments, provided herein are articles of manufacture comprising an orally ingestible pill or capsule, wherein the orally ingestible pill or capsule comprises: a) a composition comprising: i) a functionalized carbon allotrope (FCA) that breaks down homocysteine and / or cysteine to produce H2S, wherein the FCA optionally comprises: A) graphene oxide, B) functionalized graphene, C) functionalized carbon nanotubes, D) functionalized graphene nanoribbons, E) functionalized carbon onions, F) functionalized carbon nanohoms, G) carboxy fullerenes, H) carbon dots, I) fullertubes, and / or J) polyamino fullerenes; and ii) a physiologically tolerable aqueous solution; and iii) optionally a carrier component for the FCA; and b) an enteric coating which surrounds the composition. In particular embodiments, the pill or capsule comprises a capsule, wherein the capsule comprises a softgel. In additional embodiments, the softgel comprises gelatin. In other embodiments, the composition further comprises a solvent.

[0018] In some embodiments, provided herein are methods of treating a cysteine or homocysteine related disease or condition comprising at least one of the following: a) administering any of the compositions above or otherwise herein to a subject with a cysteine or homocysteine related disease or condition; and / or b) treating the subject using the systems, kits, or articles of manufacture above or herein, or using the orally ingestible pills or capsules described above or otherwise herein; and / or c) providing to the subject the systems, kits, or articles of manufacture above or otherwise herein, or providing the orally ingestible pills or capsules described above or herein.

[0019] In certain embodiments, the subject has homocystinuria. In other embodiments, the subject has hyperhomocysteinemia. In further embodiments, the subject has cystinuria. In additional embodiments, the subject has cystine stones. In other embodiments, the subject is a human. In some embodiments, the treating, providing, or administering is repeated at least once.

[0020] DEFINITIONS

[0021] As used herein, the term “and / or” includes any and all combinations of listed items, including any of the listed items individually. For example, “A, B, and / or C” encompasses A, B, C, AB, AC, BC, and ABC, each of which is to be considered separately described by the statement “A, B, and / or C.”

[0022] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0023] As used herein, the term “amino” refers to a group -NRxRy, wherein Rxand Ryare selected from hydrogen and alkyl (e.g., C1-C4 alkyl). A group -NH(alkyl) may be referred to herein as “alkylamino” and a group -N(alkyl)2 may be referred to herein as “dialkylamino.”

[0024] As used herein, the term “hydroxy” or “hydroxyl” refers to an -OH group.

[0025] As used herein, the term “epoxide” refers to is a cyclic ether, where the ether forms a three-atom ring: two atoms of carbon and one atom of oxygen. The terms “administer,” “administering,” “administered,” or “administration” refer to any manner of providing a compound or a pharmaceutical composition (e.g., one described herein), to a subject. Routes of administration can be accomplished through any means known by those skilled in the art, including, but not limited to, oral, buccal, intravenous, subcutaneous, intramuscular, transdermal, by inhalation, and the like.

[0026] “Effective amount,” as used herein, refers to a dosage of composition effective for eliciting a desired effect. This term as used herein may also refer to an amount effective at bringing about a desired in vivo effect in a subject, such as a human.

[0027] As used herein, the term “subject” is intended to include human and non-human animals. Exemplary human subjects include a human patient having a homocysteine or cysteine related disorder or condition. The term “non-human animals” includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and / or agriculturally useful animals (such as sheep, dogs, cats, cows, pigs, etc.), and rodents (such as mice, rats, hamsters, guinea pigs, etc.).

[0028] As used herein, the term “treat” or “treating” a subject having a disorder refers to administering or providing a composition, system, or article of manufacture described herein to the subject, such that at least one symptom of the disorder is cured, healed, alleviated, relieved, altered, remedied, ameliorated, or improved. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, cure, improve or affect the disorder or the symptoms of the disorder. The treatment may inhibit deterioration or worsening of a symptom of a disorder.

[0029] FIGURES

[0030] Figure 1 : Homocysteine in methionine and transsulfuration pathway. The yellow highlighted pathway shows breakdown of cysteine and homocysteine by CGL enzyme to produce H2S.

[0031] Figure 2A-B. EMNs as catalyst for catabolism of cysteine. We have synthesized a catalyst that mimics CGL and CBS enzymes using fullerenes as scaffold (1.3 nm). The polyhydroxy fullerenes (PHF) contains hydroxyl and hemiketal groups on the surface of fullerenes. 2A) Confirmation of PHF’ s role as a catalyst. Cysteine (Cys) consumption with time from a reaction mixture consisting of Cys (100 mM) and PHF (1 mg / mL) in D2O was determined by 1H NMR. Catechol was used as internal standard. Every 24 hours, 100 mM Cys was added to reaction mixture without replenishing PHF. The absence of decline in Cys consumption rate indicates that PHF is a catalyst and not a reactant. 2B) H2S evolution from a reaction mixture consisting of Cys (10 mM) and PHF in PBS was determined by paper chromatography assay. Every 24 hours, 10 mM Cys was added to the mixture without replenishing CAE-A. Constant H2S production confirms that PHF is a catalyst. No further Cys additions were made after day 20. ND = Not detectable; MD = Maximum detectable with lead acetate paper. Note that less than 20% of H2S produced evolves as volatile gas detected by lead acetate paper, rest remains in solution at pH 7.4.

[0032] Figure 3A-B: EMNs as catalyst for catabolism of cysteine and homocysteine. 3 A) Catalysis kinetics for PHF compared to CGL enzyme with and without PLP as co-factor and cysteine as substrate. EMNs do not need a cofactor for catalysis. 3B) Catalysis kinetics for PHF as EMN and homocysteine as substrate. The markers are experimental data points obtained from slope of linear portion of H2S evolution vs time graph for each substrate concentration. The solid lines are kinetic model fit. n=6; Error bars +SD; ****p<0.0001

[0033] Figure 4: EMNs can enter cells and catalyze H2S production. Untreated, Na2S treated, and PHF treated 3T3 fibroblasts were incubated with P3 (H2S specific dye) probe for 30 minutes. The cells were then fixed and imaged with multi-photon microscopy at same settings. The right image in each panel shows the 3x zoom of inset in left image. P3 signal is enhanced from cells treated with H2S donor Na2S and PHF, which produces intracellular H2S with bioavailable cysteine.

[0034] Figure 5: Effect of EMN active site topology on amino acid catabolism and H2S evolution. Molecular structures of cysteine and homocysteine showing the spacing between thiol and amine groups are shown on the top. H2S evolution from different types of EMN: PHF (fullerene), hydroxylated multi-walled carbon nanotubes (MWNT-OH) and hydroxylated graphene (G-OH); and cysteine and homocysteine as substrates are shown in the bar graph. The under! vatized carbon nanomaterials are shown in the bottom to demonstrate the role of curvature.

[0035] DETAILED DESCRIPTION

[0036] Provided herein are compositions, systems, kits, and articles of manufacture for using various functionalized carbon allotropes for treating a cysteine or homocysteine related disease or condition. In particular embodiments, the compositions comprise: a functionalized carbon allotrope (FC A) that breaks down homocysteine and / or cysteine to produce H2S. In certain embodiments, the FC A comprises: i) graphene oxide, ii) functionalized graphene, iii) functionalized carbon nanotubes, iv) functionalized graphene nanoribbons, v) functionalized carbon onions, vi) functionalized carbon nanohorns, vii) carboxy fullerenes, viii) carbon dots, ix) fullertubes, and / or x) polyamino fullerenes. In some embodiments, the FCAs are functionalized with hydroxyl, carbonyl, carboxyl, hemi-ketal and / or amine functional groups.

[0037] Enzyme mimicking nanomaterials (EMNs) represent an emerging field with the potential to rescue aging-associated enzymatic deficiencies. Although nanomaterials lack the well-defined tertiary structures of enzymes, certain similarities in size, shape, and surface charge enable nanomaterials to mimic enzymes (9). Importantly, EMNs are not prone to genetic or post- translational modifications unlike natural or artificial enzymes. The first report of an EMN was for carboxyfullerene, which was demonstrated to mimic superoxide dismutase, an oxidoreductase (10-12). Since then several carbon based EMNs have been reported to mimic oxidoreductases (13-16). Metal and metal-oxide nanoparticles, which have been traditionally used as catalysts in industrial chemical reactions have been shown to catalyze biologically relevant reactions under physiological conditions (17-19). Hybrid metal- carbon based EMNs are also being developed (20-23). Thus far, nanomaterials have been reported to mimic enzymes mostly from oxidoreductases and a few from the hydrolases family (19, 24). Oxidoreductases (Enzyme Commission 1 ; ECI) and hydrolases (EC3) comprise only two out of seven families of enzymes. The active sites of EMNs thus far reported lack the complex 3D spatial arrangements of those in biological enzymes. Consequently, present EMNs tend to be highly non-specific, i.e., the same EMN (e.g., gold or graphene oxide) catalyzes many different reactions (9, 16, 25-27). While advantageous from an in vitro standpoint, such non- specificity is impermissible for in vivo applications. Work conducted during development of embodiments herein has developed novel EMNs that are the first to extend the purview of EMNs to the lyases (EC 4). In general, these EMNs mimic CGL enzyme that breaks down cysteine and homocysteine to produce H2S.

[0038] In regards various functionalized graphenes compounds, many of these can be purchased from commercial suppliers (e.g., Sigma 796034, 900726 and 900713). Another source for graphene and functionalized graphene is CheapTubes (see online catalog). In regard to various functionalized nanotubes, many of these can be purchased from commercial suppliers (e.g., Sigma 755125 and 652490). Another source for nanotubes and functionalized nanotubes is CheapTubes (see, online catalog) and U.S. Research Nanomaterials (see online catalog).

[0039] The disclosed functionalized carbon allotrope (FCAs) (e.g., i) graphene oxide, ii) functionalized graphene, iii) functionalized carbon nanotubes, iv) functionalized graphene nanoribbons, v) functionalized carbon onions, vi) functionalized carbon nanohoms, vii) carboxy fullerenes, and / or viii) polyamino fullerenes) and compositions herein, may be incorporated into pharmaceutical compositions suitable for administration to a subject (such as a patient, which may be a human or non-human). The pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactically effective amount” of the FCAs or compositions herein. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the FCAs herein are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease or condition, the prophylactically effective amount will be less than the therapeutically effective amount.

[0040] For example, a therapeutically effective amount of the FCAs herein may be about 1 mg / kg to about 1000 mg / kg, about 5 mg / kg to about 950 mg / kg, about 10 mg / kg to about 900 mg / kg, about 15 mg / kg to about 850 mg / kg, about 20 mg / kg to about 800 mg / kg, about 25 mg / kg to about 750 mg / kg, about 30 mg / kg to about 700 mg / kg, about 35 mg / kg to about 650 mg / kg, about 40 mg / kg to about 600 mg / kg, about 45 mg / kg to about 550 mg / kg, about 50 mg / kg to about 500 mg / kg, about 55 mg / kg to about 450 mg / kg, about 60 mg / kg to about 400 mg / kg, about 65 mg / kg to about 350 mg / kg, about 70 mg / kg to about 300 mg / kg, about 75 mg / kg to about 250 mg / kg, about 80 mg / kg to about 200 mg / kg, about 85 mg / kg to about 150 mg / kg, about 90 mg / kg to about 100 mg / kg, and 1 microgram / kg to 1000 microgram / kg (e.g., 1 ... 10 ... 20 .... 30 .... 50 ... 100 ... 200 ... 300 ... 400 ... 500 ... 600 ... 700 ... 800 ... 900 ... 1000 micrograms / kg).

[0041] The pharmaceutical compositions may include pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0042] Thus, the FCAs and compositions herein may be formulated for administration by, for example, solid dosing, eye drop, in a topical oil-based formulation, injection, inhalation (either through the mouth or the nose), implants, or oral, buccal, parenteral, or rectal administration. Techniques and formulations may generally be found in “Remington’s Pharmaceutical Sciences,” (Meade Publishing Co., Easton, Pa.). Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage.

[0043] The route by which the disclosed FCAs and compositions are administered and the form of the composition will dictate the type of carrier to be used. The compositions may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., dermal, pulmonary, nasal, aural, ocular, liposome delivery systems, or iontophoresis).

[0044] Carriers for systemic administration typically include at least one of diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, and others. All carriers are optional in the compositions.

[0045] Suitable diluents include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol. The amount of diluent(s) in a systemic or topical composition is typically about 50 to about 90%.

[0046] Suitable lubricants include silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, com oil and oil of theobroma. The amount of lubricant(s) in a systemic or topical composition is typically about 5 to about 10%.

[0047] Suitable binders include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as com starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose. The amount of binder(s) in a systemic composition is typically about 5 to about 50%.

[0048] Suitable disintegrants include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmellose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins. The amount of disintegrant(s) in a systemic or topical composition is typically about 0.1 to about 10%.

[0049] Suitable colorants include a colorant such as an FD&C dye. When used, the amount of colorant in a systemic or topical composition is typically about 0.005 to about 0.1%. Suitable flavors include menthol, peppermint, and fruit flavors. The amount of flavor(s), when used, in a systemic or topical composition is typically about 0.1 to about 1.0%. Suitable sweeteners include aspartame and saccharin. The amount of sweetener(s) in a systemic or topical composition is typically about 0.001 to about 1%. Suitable antioxidants include butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E. The amount of antioxidant(s) in a systemic or topical composition is typically about 0.1 to about 5%. Suitable preservatives include benzalkonium chloride, methyl paraben and sodium benzoate. The amount of preservative(s) in a systemic or topical composition is typically about 0.01 to about 5%. Suitable glidants include silicon dioxide. The amount of glidant(s) in a systemic or topical composition is typically about 1 to about 5%.

[0050] Suitable solvents include water, isotonic saline, ethyl oleate, glycerin, hydroxylated castor oils, alcohols such as ethanol, and phosphate buffer solutions. The amount of solvent(s) in a systemic or topical composition is typically from about 0 to about 100%. Suitable suspending agents include AVICEL RC-591 (from FMC Corporation of Philadelphia, PA) and sodium alginate. The amount of suspending agent(s) in a systemic or topical composition is typically about 1 to about 8%.

[0051] Suitable surfactants include lecithin, Polysorbate 80, and sodium lauryl sulfate, and the TWEENS from Atlas Powder Company of Wilmington, Delaware. Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient Handbook, 1992, pp.587-592; Remington’s Pharmaceutical Sciences, 15th Ed. 1975, pp. 335-337; and McCutcheon’s Volume 1, Emulsifiers & Detergents, 1994, North American Edition, pp. 236-239. The amount of surfactant(s) in the systemic or topical composition is typically about 0.1% to about 5%.

[0052] Although the amounts of components in the systemic compositions may vary depending on the type of systemic composition prepared, in general, systemic compositions include 0.01% to 50% of the FCAs herein, and 50% to 99.99% of one or more carriers. Compositions for parenteral administration typically include 0.1% to 10% of actives and 90% to 99.9% of a carrier including a diluent and a solvent.

[0053] Compositions for oral administration can have various dosage forms. For example, solid forms include tablets, capsules, granules, and bulk powders. These oral dosage forms include a safe and effective amount, usually at least about 5%, and more particularly from about 25% to about 50% of actives. The oral dosage compositions include about 50% to about 95% of carriers, and more particularly, from about 50% to about 75%.

[0054] Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically include an active component, and a carrier comprising ingredients selected from diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, glidants, and combinations thereof. Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose and cellulose. Specific binders include starch, gelatin, and sucrose. Specific disintegrants include alginic acid and croscarmellose. Specific lubricants include magnesium stearate, stearic acid, and talc. Specific colorants are the FD&C dyes, which can be added for appearance. Chewable tablets preferably contain sweeteners such as aspartame and saccharin, or flavors such as menthol, peppermint, fruit flavors, or a combination thereof.

[0055] Capsules (including implants, time release and sustained release formulations) typically include an FCA herein, and a carrier including one or more diluents disclosed above in a capsule comprising gelatin. Granules typically comprise a disclosed FCA, and preferably glidants such as silicon dioxide to improve flow characteristics. Implants can be of the biodegradable or the non-biodegradable type. The selection of ingredients in the carrier for oral compositions depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this disclosure.

[0056] Solid compositions may be coated by conventional methods, typically with pH or time-dependent coatings, such that a disclosed FCA is released in the gastrointestinal tract in the vicinity of the desired application, or at various points and times to extend the desired action. The coatings typically include one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, EUDRAGIT coatings (available from Evonik Industries of Essen, Germany), waxes and shellac.

[0057] Compositions for oral administration can have liquid forms. For example, suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non-effervescent granules, suspensions reconstituted from non-effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like. Liquid orally administered compositions typically include FCA compound and a carrier, namely, a carrier selected from diluents, colorants, flavors, sweeteners, preservatives, solvents, suspending agents, and surfactants. Peroral liquid compositions preferably include one or more ingredients selected from colorants, flavors, and sweeteners.

[0058] Other compositions useful for attaining systemic delivery of the subject FCA include sublingual, buccal and nasal dosage forms. Such compositions typically include one or more of soluble filler substances such as diluents including sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose. Such compositions may further include lubricants, colorants, flavors, sweeteners, antioxidants, and glidants.

[0059] The amount of the carrier employed in conjunction with a disclosed FCA is sufficient to provide a practical quantity of composition for administration per unit dose of the FCA. Techniques and compositions for making dosage forms useful in the methods of this disclosure are described in the following references: Modem Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

[0060] A carrier may include a single ingredient or a combination of two or more ingredients. In the topical compositions, the carrier includes a topical carrier. Suitable topical carriers include one or more ingredients selected from phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil, combinations thereof, and the like. More particularly, carriers for skin applications include propylene glycol, dimethyl isosorbide, and water, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols.

[0061] Suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinations thereof. Specific solvents include ethyl alcohol and homotopic alcohols. The amount of solvent(s) in a topical composition is typically about 0% to about 95%.

[0062] For use in the methods described herein, kits and articles of manufacture are also provided, which, for example, include an FCA or pharmaceutical composition described herein. In some embodiments, such kits comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers are formed from a variety of materials such as glass or plastic.

[0063] The articles of manufacture provided herein may contain, for example, packaging materials. Packaging materials for use in packaging pharmaceutical products include those found in, e.g., U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. For example, in some embodiments the container(s) includes the compositions herein. The container(s) optionally have a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprise an FCA herein with an identifying description or label or instructions relating to its use in the methods described herein.

[0064] For example, a kit typically includes one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and / or devices) desirable from a commercial and user standpoint for use of an FCA described herein. Non-limiting examples of such materials include, but not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or tube labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included. A label is optionally on or associated with the container. For example, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In addition, a label is used to indicate that the contents are to be used for a specific therapeutic application. In addition, the label indicates directions for use of the contents, such as in the methods described herein. In certain embodiments, the pharmaceutical composition is presented in a pack or dispenser device which contains one or more unit dosage forms containing an FCA provided herein. The pack, for example, contains metal or plastic foil, such as a blister pack. Or, the pack or dispenser device is accompanied by instructions for administration. Or, the pack or dispenser is accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. In some embodiments, compositions containing an FCA provided herein formulated in a compatible pharmaceutical carrier are prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0065] EXAMPLE

[0066] EXAMPLE 1

[0067] Carbon Nanomaterials for Substrate Specific EMNs

[0068] Fullerenes, carbon nanotubes and graphenes are the nanoscale allotropes of carbon that have multitude of sp2 carbons, which can be sites for functionalization with desired chemical groups. Studies conducted with polyhydroxy fullerenes (PHF) suggest that suitably spaced functional groups and double bonds constitute active sites for catabolism of thiol containing amino acids. The role of PHF as a catalyst was determined by multi-day experiments where cysteine was added every day and PHF was not replenished. The consumption of cysteine was measured with NMR (Fig 2A) and in a separate experiment, H2S production was measured using paper chromatography assay (Fig 2B).

[0069] Figure 3 shows the enzyme kinetics for PHF as EMN and cysteine and homocysteine as substrates. It is important to note that EMNs do not need pyridoxal-5 ’ -phosphate (PLP) as a co-factor. PHF catalytically breaks down both cysteine and homocysteine to produce H2S. However, the rate suggests that PHF has higher affinity towards catabolism of cysteine than homocysteine.

[0070] The potential for in vivo application of EMNs for breakdown of cysteine and homocysteine was tested in 3T3 fibroblasts. P3 multi-photon fluorescent probe was utilized to detect intracellular H2S levels28. Fluorescence intensity of the fibroblasts exposed to PHF increased in a concentration dependent manner between 0 and 0.1 mg / mL of PHF. This represents an increase in H2S production of up to 2.4 times that of background level (control) due to PHF catalysis and suggests that H2S production rate is controllable by the PHF concentration. Neither cells nor PHF exhibited auto-fluorescence under multi-photon microscopy.

[0071] Nanomaterials with Higher Specificity for Homocysteine

[0072] Although PHF can break down homocysteine in vivo, its affinity towards homocysteine is lower than cysteine. To allow successful formation of a catalyst-substrate complex, the thiol and amine groups of the substrate molecule should align with active sites on the fullerene surface. Changing the spacing and curvature of active site or geometry of the substrate molecule would thus potentially affect the ability of substrate to interact with EMNs, either slowing or accelerating the reaction. To test this hypothesis, we compared substrates cysteine, which has the thiol-amine spacing of 2.5-3.5 A with homocysteine with a spacing of 4-5 A (Fig 5). Further, we tested EMNs with different curvatures: PHF, which has a OD fullerene cage with an estimated curvature K~ 1.4 nm-1; hydroxylated multi-walled carbon nanotubes (MWNT-OH), which is ID high aspect ratio tubes with an estimated curvature K~0.1 nm1; and hydroxylated graphene (G-OH), which is a 2D sheet like nanomaterial with an estimated curvature K~0 nm1. As shown in Figure 5, H2S was produced from homocysteine at a rate substantially less than achieved with cysteine with PHF as EMN. In the case of MWNT-OH as EMN, H2S production was similar with both substrates. However, in the case of G-OH, the trend was reversed with H2S production substantially higher with homocysteine than cysteine. While the present disclosure is not limited to any particular mechanism, and an understanding of the mechanism is not necessary to practice the invention, these results suggest that low curvature is preferred for homocysteine catabolism. Decrease in curvature in the range of nanotubes or graphene sheets can also be obtained with larger fullerenes, such as C70, Cs4, C120, C200, C500 or carbon onions.

[0073] REFERENCES

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[0083] 10. Dugan et al., Carboxyfullerenes as neuroprotective agents. Proceedings of the National Academy of Sciences. 1997;94:9434-9439.

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[0091] 17. Benedetti et al;, Electrocatalytic Nanoparticles That Mimic the Three-Dimensional Geometric Architecture of Enzymes: Nanozymes. J Am Chem Soc. 2018.

[0092] 18. Nasir et al., An overview on enzyme-mimicking nanomaterials for use in electrochemical and optical assays. Microchim Acta. 2017;184:323-342.

[0093] 19. Korschelt K, Tahir MN and Tremel W. A Step into the Future: Applications of Nanoparticle Enzyme Mimics. Chem-Eur J. 2018;24:9703-9713.

[0094] 20. Zhang et al., Bioorthogonal Nanozymes: Progress towards Therapeutic Applications. Trends in Chemistry. 2019;1:90-98. 21. Tonga et al., Supramolecular regulation of bioorthogonal catalysis in cells using nanoparticle-embedded transition metal catalysts. Nature Chemistry. 2015;7:597-603.

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Claims

CLAIMSWe claim:

1. A composition comprising: a) a functionalized carbon allotrope (FCA) that breaks down homocysteine and / or cysteine to produce H2S, wherein said FCA optionally comprises: i) graphene oxide, ii) functionalized graphene, iii) functionalized carbon nanotubes, iv) functionalized graphene nanoribbons, v) functionalized carbon onions, vi) functionalized carbon nanohoms, vii) carboxy fullerenes, viii) carbon dots, ix) fullertubes, and / or x) polyamino fullerenes; and b) a physiologically tolerable aqueous solution; and c) optionally a carrier component for said FCA.

2. The composition of claim 1 , wherein said functionalized graphene comprises graphene functionalized with at least one of the following functional groups: hydroxyl, carbonyl, carboxyl, hemi-ketal, epoxide, thiol, sulfide, persulfide, thioether, thiosulfate, thiazole, and thiazine, and amine.

3. The composition of claim 1, wherein said functionalized carbon nanotubes comprise carbon nanotubes functionalized with at least one of the following functional groups: hydroxyl, carbonyl, carboxyl, epoxide, hemi-ketal, thiol, sulfide, persulfide, thioether, thiosulfate, thiazole, and thiazine, and amine.

4. The composition of claims 1 or 3, wherein said functionalized carbon nanotubes are functionalized multi-walled carbon nanotubes (FMWCNs).

5. The composition of claims 1 or 3, wherein said functionalized carbon nanotubes are functionalized single-walled carbon nanotubes (FsWCNs).

6. The composition of any of claims 1 and 3-5, wherein said functionalized carbon nanotubes have an outer dimension of between about 5-100 nm.

7. The composition of any of claims 1-6, wherein said FCA has a widest dimension of between 1 nm and 1 um, and optionally wherein said widest dimension is about 5-25 nm.

8. The composition of claim 1, wherein said physiologically tolerable aqueous solution comprises at least one of the following: a buffering agent, pyrogen-free water, isotonic saline, and / or Ringer’s solution.

9. The composition of any of claims 1-8, wherein said carrier component comprises a material selected from: polymeric nanoparticles, mesoporous nanoparticles, dendrimers, liposomes, metallic nanoparticles, inorganic nanoparticles, and inorganic microparticles.

10. The composition of any of claims 1-9, wherein said nanocarrier component targets said FCA to a desired location when administered to a mammal.

11. The composition of any of claims 1-9, wherein said carboxy fullerenes have the following formula C6o(C(COOH)2)s in the C3 or D3 isoform.

12. The composition of claim 1, wherein said functionalized graphene nanoribbons, functionalized carbon onions, and / or functionalized carbon nanohoms are functionalized with at least one of the following functional groups: hydroxyl, carbonyl, carboxyl, hemi-ketal, epoxide, thiol, sulfide, persulfide, thioether, thiosulfate, thiazole, and thiazine, and amine.

13. A system, kit, or article of manufacture comprising: a) a composition comprising: i) a functionalized carbon allotrope (FCA) that breaks down homocysteine and / or cysteine to produce H2S, wherein said FCA optionally comprises: A) graphene oxide, B) functionalized graphene, C) functionalized carbon nanotubes, D) functionalized graphene nanoribbons, E) functionalized carbon onions, F) functionalized carbon nanohoms, G) carboxy fullerenes, H) carbon dots, I) fullertubes, and / or J) polyamino fullerenes; and ii) a physiologically tolerable aqueous solution; and iii) optionally a carrier component for said first FCA; and b) a container selected from the group consisting of: i) an IV fluid solution bag, ii) a syringe vial, iii) a syringe,iv) a sterile shipping container configured for shipping powder or liquid, v) an orally ingestible dosage form.

14. The system, kit, or article of manufacture of claim 13, wherein said composition is present inside said container.

15. The system, kit, or article of manufacture of any of claims 13-14, wherein said composition is a liquid.

16. The system, kit, or article of manufacture of any of claims 13-15, wherein said composition is a powder.

17. The system, kit, or article of manufacture of any of claims 13-15, wherein said container is said IV solution bag, and wherein said composition is present in said IV solution bag.

18. The system, kit, or article of manufacture of any of claims 13-15, wherein said container is said syringe vial, and wherein said composition is present in said syringe vial.

19. The system, kit, or article of manufacture of any of claims 13-18, wherein said container is said sterile shipping container, wherein said composition is present in said sterile shipping container.

20. The system, kit, or article of manufacture of claim 13, wherein said orally ingestible dosage form is a capsule, or pill that comprises an enteric coating.

21. The system, kit, or article of manufacture of any of claims 13-20, wherein said FCA is incorporated in, or on, said carrier component.

22. An article of manufacture comprising an orally ingestible pill or capsule, wherein said orally ingestible pill or capsule comprises: a) a composition comprising: i) a functionalized carbon allotrope (FCA) that breaks down homocysteine and / or cysteine to produce H2S, wherein said FCA optionally comprises: A) graphene oxide, B) functionalized graphene, C) functionalized carbon nanotubes, D) functionalized graphene nanoribbons, E) functionalized carbon onions, F) functionalized carbon nanohoms, G) carboxy fullerenes, H) carbon dots, T) fullertubes, and / or J) poly amino fullerenes; and ii ) a physiologically tolerable aqueous solution; and iii) optionally a carrier component for said first FCA; and b) an enteric coating which surrounds said composition.

23. The article of manufacture of claim 22, wherein said pill or capsule comprises a capsule, wherein said capsule comprises a softgel.

24. The article of manufacture of claim 23, wherein said softgel comprises gelatin.

25. The article of manufacture of claim 22, wherein said composition further comprises a solvent.

26. A method of treating a cysteine or homocysteine related disease or condition comprising at least one of the following: a) administering said composition of any of claims 1-12 to a subject with a cysteine or homocysteine related disease or condition; and / or b) treating said subject using said system, kit, or article of manufacture of any of claims 13-21, or said orally ingestible pill or capsule of any of claims 22-25; and / or c) providing to said subject said system, kit, or article of manufacture of any of claims 13-21, or said orally ingestible pill or capsule of any of claims 22-25.

27. The method of claim 26, wherein said subject has homocystinuria.

28. The method of claim 26, wherein said subject has hyperhomocysteinemia.

29. The method of claim 26, wherein said subject has cystinuria.

30. The method of claim 26, wherein said subject has cystine stones.

31. The method of claim 26, wherein said subject is a human.

32. The method of claim 26, wherein said subject is administered said composition of any of claims 1 -12.

33. The method of claim 26, wherein said subject is treated using said system, kit, or article of manufacture of any of claims 13-21.

34. The method of claim 26, wherein said subject is treated with said orally ingestible pill or capsule of any of claims 22-25.

35. The method of claim 26, wherein said subject is provided said system, kit, or article of manufacture of any of claims 13-21.

36. The method of claim 26, wherein said subject is provided said orally ingestible pill or capsule of any of claims 22-25.

37. The method of claim 26, wherein said treating, providing, or administering is repeated at least once.

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