Methods for treating mitochondrial disorders

Abstract

The present disclosure provides pharmaceutical and nutritional compositions and methods for treating mitochondrial disorders. The present invention relates to a pharmaceutical or nutritional composition comprising a stabilized sulforaphene (e.g., a sulforaphene-cyclodextrin complex) that improves the efficacy, biological activity, and stability of the isolated sulforaphene. The disclosure also includes the use of the stabilized sulforaphene as an effective therapeutic agent for the treatment of mitochondrial disorders, such as mitochondrial myopathy.

Description

[0001] This application is a divisional application of patent application No. 201980001164.7, filed on February 8, 2019, entitled "Method for Treating Mitochondrial Diseases".

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 628,353, filed February 9, 2018, the entire contents of which are incorporated herein by reference. Technical Field

[0004] This disclosure provides pharmaceutical compositions and nutritional compositions for treating mitochondrial diseases. More specifically, this disclosure relates to methods of treating a subject suffering from a mitochondrial disease by administering a pharmaceutical composition or nutritional composition comprising stable raphanin. Background Technology

[0005] Certain components found in the seeds of cruciferous plants (such as radish (Raphanus sativus)) are known to possess anticancer and antimicrobial properties. See PCT / US2006 / 010032. One such component, sulforaphane, exists in these plants in a bound form as sulforaphane glycoside (a glucosinolate). In nature, it has the following chemical structure. Sulforaphane (C6H) 11 NOS2 is formed from sulforaphane via an enzymatic reaction. However, while much research has focused on the isolation and uses of sulforaphane, other components (such as sulforaphane) have been neglected due to their lack of solubility, bioavailability, and stability after isolation.

[0006] Mitochondrial disorders are diseases or defects that occur when mitochondria are unable to produce adenosine triphosphate (ATP) through oxidative phosphorylation. Mitochondria are essential organelles present in all mammalian cells. In short, mitochondria transport electrons from NADH or FADH2 to O2 via electron carriers to produce ATP from adenosine diphosphate (ADP) during cellular respiration—a process known as the electron transport chain. Here, the energy released when electrons are transferred from the higher-energy NADH or FADH2 to the lower-energy O2 is needed to phosphorylate ADP and produce ATP. The energy used to produce ATP from ADP is controlled by the electron transport chain and is produced through oxidative phosphorylation. The electron transport chain is crucial for generating cellular energy and maintaining cell viability. Therefore, dysfunction in the mitochondrial electron transport chain leads to reduced cellular ATP production, increased anaerobic metabolism, and increased free radical production, resulting in oxidative stress and cell death.

[0007] Mitochondrial disorders can include one or more related symptoms, such as abnormal mitochondrial oxidative metabolism, abnormal aerobic metabolism, muscle weakness, fatigue, heart failure or dysfunction, activity limitation, and seizures.

[0008] Given the drawbacks associated with existing treatments and the increasing number of patients diagnosed with mitochondrial diseases, there is a need to develop new compositions and methods for the treatment of mitochondrial diseases. Summary of the Invention

[0009] The methods and compositions disclosed herein are based on the discovery that raphanin, while highly unstable when isolated from radish seeds, is an effective compound for treating certain mitochondrial disorders. More specifically, the inventors have found that the efficacy and bioavailability of raphanin are severely reduced within just a few days of isolation, a finding that eliminates any potential therapeutic use of raphanin. However, the inventors have also found that pharmaceutical and nutritional compositions consisting of stable raphanin prolong the biological activity of raphanin and significantly improve its ability to treat mitochondrial disorders by increasing cellular ATP production.

[0010] Therefore, one aspect of this disclosure provides a method for treating mitochondrial disorders, the method comprising administering to a subject a pharmaceutical composition or nutritional composition comprising stabilized raphanin. In one instance, this disclosure provides a method for increasing mitochondrial function by increasing cellular ATP production through administering to a subject a pharmaceutical composition or nutritional composition comprising stabilized raphanin.

[0011] The methods disclosed herein include using pharmaceutical or nutritional compositions comprising stabilized raphanin. In some embodiments, the stabilized raphanin may be chemically modified to improve the solubility of the raphanin compound. In other embodiments, raphanin may be stabilized by the presence of one or more solubilizers in the pharmaceutical or nutritional composition. In one embodiment, the stabilizer is a cyclodextrin, alcohol, glycol, ketone, oil, or a combination thereof.

[0012] In some embodiments, the pharmaceutical composition or nutritional composition includes raphanin and cyclodextrin. In some embodiments, the cyclodextrin is one or more of α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin.

[0013] In a preferred embodiment, the pharmaceutical or nutritional composition comprises raphanin and hydroxypropyl-β-cyclodextrin. Therefore, in a specific embodiment, the method includes administering to a subject a pharmaceutical composition comprising a complex of raphanin and hydroxypropyl-β-cyclodextrin (i.e., stabilized raphanin).

[0014] In one embodiment, the pharmaceutical composition or nutritional composition consists essentially of stabilized raphanin. In other embodiments, the composition consists essentially of raphanin complexed with cyclodextrin (e.g., hydroxypropyl-β-cyclodextrin).

[0015] In some cases, this disclosure relates to methods of administering a pharmaceutical composition or a nutritional composition to a subject. Administration may be, for example, oral, intravenous, intraperitoneal, or local administration. In a particular embodiment, the subject is given an oral pharmaceutical composition or nutritional composition containing a stable raphanin composition. In other embodiments, the composition is administered to the subject by injection (e.g., via intravenous or intraperitoneal injection). In some embodiments, the composition is administered orally in the form of pills, liquids, powders, or combinations thereof.

[0016] In some embodiments, the subject treated by the method of the present invention is a mammal. In some embodiments, the subject is a human, mouse, or rat. In one exemplary embodiment, the subject to be treated is a human subject suffering from mitochondrial disease. In one case, the subject to be treated may exhibit impaired mitochondrial function (e.g., ATP production). In a specific embodiment, the subject is a person diagnosed with mitochondrial disease, wherein the mitochondrial disease affects the subject's ability to produce ATP through mitochondrial oxidative phosphorylation. Mitochondrial disease may include one or more associated symptoms, such as abnormal mitochondrial oxidative metabolism, abnormal aerobic metabolism, muscle weakness, fatigue, heart failure or dysfunction, limited activity, and seizures.

[0017] Exemplary mitochondrial conditions that can be treated with this method include, but are not limited to, mitochondrial myopathy, essential tremor, Parkinson's disease, and metabolic disorders that cause abnormal metabolism of carbohydrates, lipids (fat), proteins, and / or nucleic acids in subjects, such as mitochondrial diabetes, chronic fatigue syndrome (CFS), skin aging, prostate diseases (such as benign prostatic hyperplasia (BPH)), hyperthyroidism, glucose intolerance, hypercholesterolemia, dyslipidemia, hyperinsulinemia, thyroid dysfunction, multiple sclerosis, polycystic ovary syndrome (PCOS), psoriasis, and coronary artery disease.

[0018] In one embodiment, the mitochondrial condition treated by administering dissolved raphanin is mitochondrial myopathy. In a specific embodiment, the method of this disclosure includes orally administering an effective amount of a pharmaceutical composition or nutritional composition comprising a complex of raphanin and hydroxypropyl-β-cyclodextrin to a subject diagnosed with mitochondrial myopathy.

[0019] In other embodiments, the method of this disclosure includes orally administering an effective amount of a pharmaceutical composition to a subject diagnosed with essential tremor, the pharmaceutical composition comprising a complex of raphanin and hydroxypropyl-β-cyclodextrin.

[0020] In another embodiment, the method of this disclosure includes orally administering an effective amount of a pharmaceutical composition to a subject diagnosed with polycystic ovary syndrome (PCOS), the pharmaceutical composition comprising a complex of raphanin and hydroxypropyl-β-cyclodextrin.

[0021] In other embodiments, the method of this disclosure includes orally administering an effective amount of a pharmaceutical composition comprising a complex of raphanin and hydroxypropyl-β-cyclodextrin to a subject diagnosed with a skin disease (e.g., psoriasis). In one instance, the method of this disclosure is used to treat a subject suffering from psoriatic arthritis or aging skin by administering an effective amount of a pharmaceutical composition comprising a complex of raphanin and hydroxypropyl-β-cyclodextrin.

[0022] In other embodiments, the method of this disclosure includes orally administering an effective amount of a pharmaceutical composition to a subject diagnosed with chronic fatigue syndrome (CFS), the pharmaceutical composition comprising a complex of raphanin and hydroxypropyl-β-cyclodextrin. Attached Figure Description

[0023] Figure 1A-Figure 1B Stabilized raphanin induces mitochondrial ATP production in skin cells. SK-MEL-31 human skin cells were cultured and seeded in 96-well plates at a density of 15,000 cells per well. Cells were then treated with astrosaponin (STSP), a known inhibitor of mitochondrial function and ATP production. Cells were incubated with STSP followed by administration of a complex of 100 μM raphanin and hydroxypropyl-β-cyclodextrin (compound 1), a stabilized raphanin composition isolated from radish seed extract (compound 2), or a known ATP-inducing positive control (compound 3). Cells were then incubated for 4 h (data not shown), 48 h (A), or 72 h (B). Skin cells treated with the stabilized raphanin composition for more than 4 h showed similar improvements in mitochondrial function as cells treated with the positive control, and were significantly higher than cells treated with STSP alone.

[0024] Figures 2A-2BStabilized raphanin induces mitochondrial ATP production in muscle. A204 human muscle cells were cultured and seeded in 96-well plates at a density of 15,000 cells per well. Cells were then treated with astrosaponin (STSP), a known inhibitor of mitochondrial function and ATP production. After incubation with STSP, cells were administered 100 μM of a raphanin and hydroxypropyl-β-cyclodextrin complex (compound 1), 17.5 μg / μL of a stabilized raphanin composition isolated from radish seed extract (compound 2), or 17.5 μg / μL of a known ATP-inducing positive control (compound 3). Cells were then incubated for 4 h (data not shown), 48 h (A), or 72 h (B). Muscle cells treated with the stabilized raphanin composition showed similar improvements in mitochondrial function as cells treated with the positive control, and significantly higher than cells treated with STSP alone.

[0025] Figures 3A-3B Stabilized raphanin induces mitochondrial ATP production in pancreatic cells. PANC-1 human pancreatic cells were cultured and seeded at a density of 15,000 cells per well in 96-well plates. Cells were then treated with astrosaponin (STSP), a known inhibitor of mitochondrial function and ATP production. After incubation with STSP, cells were administered 100 μM of a raphanin and hydroxypropyl-β-cyclodextrin complex (compound 1), 17.5 μg / μL of a stabilized raphanin composition isolated from radish seed extract (compound 2), or 17.5 μg / μL of a known ATP-inducing positive control (compound 3). Cells were then incubated for 4 h (data not shown), 48 h (A), or 72 h (B). Pancreatic cells treated with the stabilized raphanin composition showed similar enhancements in mitochondrial function as those treated with the positive control, and significantly higher than cells treated with STSP alone. Detailed Implementation

[0026] Unbound by any particular theory, the present invention is based on the discovery that raphanin compounds become unstable and ineffective shortly after isolation from cruciferous plants (such as Raphanus sativus, where raphanin is naturally formed). Furthermore, the inventors have found that stabilized raphanin is an active compound in radish seed extract for treating mitochondrial disorders. For example, as shown herein, stabilized raphanin improves mitochondrial ATP production compared to radish seed extract, leading to increased and maintained cell viability. Therefore, the inventors have determined that pharmaceutical and nutritional compositions consisting of stabilized raphanin (e.g., raphanin complexed with hydroxypropyl-β-cyclodextrin) prolong the bioactivity of raphanin and significantly improve its ability to treat mitochondrial disorders such as mitochondrial myopathy (e.g., chronic fatigue syndrome (CFS)), essential tremor, mitochondrial diabetes, PCOS, and skin diseases (e.g., psoriasis and aging skin).

[0027] Therefore, this disclosure provides a method for treating mitochondrial diseases, the method comprising administering to a subject a pharmaceutical composition or nutritional composition consisting of stabilized raphanin.

[0028] The treatment methods disclosed herein include administering a pharmaceutical composition or nutritional composition comprising stabilized sulforaphene. As used herein, the term "sulforaphene" (or raphanin) refers to a compound with the molecular formula C6H9NOS2, the structure of which is as follows: Sulforaphen has several known aliases, such as sulforaphen, sulphoraphen, 4-isothiocyano-1-(methylsulfinyl)-1-butene, and 4-methylsulfinyl-3-butenyl isothiocyanate. The known molecular weight of sulforaphen is approximately 175.26 g / mol. The sulforaphen used in this article is intended to exclude sulforaphane, which has a different structure and activity. As mentioned above, sulforaphane has the molecular formula C6H. 11 NOS2, which, for example, lacks a double bond between the first and second carbons of the molecule, is also known as 1-isothiocyano-4-[(R)-methylsulfinyl]-butane.

[0029] Rhamnosine is a vinyl sulfoxide, typically having the structural formula RS(=O)-R', which acts as a Michael acceptor in organic chemistry. Vinyl sulfoxides are compounds that regulate anaerobic oxidative phosphorylation in mitochondria.

[0030] Rhus sulforaphane is a naturally occurring compound found, for example, in radish (Raphani semen) seeds, produced by the hydrolysis of sulforaphane (glucosinolate) by myrosinase. Therefore, in some embodiments, radis sulforaphane can be isolated from certain root vegetables (e.g., radish). The isolated naturally occurring radis sulforaphane can also be purified for use in this method.

[0031] When referring to compounds such as raphanin, the term "isolated" means that the compound has been removed from its natural environment or its formation environment and is substantially free of other molecular substances. "Substantially free" means that the isolated compound constitutes at least 60%, 70%, 80%, 90%, or 95% (by dry weight or volume) of the composition or formulation. For example, an isolated raphanin composition may be substantially free of other compounds (proteins, lipids, collagen) or the plant material from which it is obtained; that is, raphanin constitutes more than about 80%, more than about 90%, or more than about 95% of the volume of the formulation. The degree of purification may be based on the intended use.

[0032] Methods for isolating and purifying raphanin from radish seeds are known to those skilled in the art, and any such method may be used herein. For example, a known defatting method, as described in West, L. et al., J Agric. Food Chem. (2004) 52, pp. 916-926 (the entire contents of which are incorporated herein by reference), may be used to defatt the radish seeds before the formation of an aqueous extract. In this document, the plant or a portion thereof may be ground, pulverized, or mixed before or simultaneously with the addition of the aqueous extract. The extraction of raphanin may be carried out with water or water containing an organic solvent, such as ethanol. Specifically, in some cases, an aqueous extract of cruciferous plants is formed by contacting cruciferous plant seeds with water at a temperature of 60°C to 110°C for at least 5 minutes. The aqueous extract may then be contacted with an adsorbent that preferentially adsorbs raphanin rather than other compounds in the extract, such as activated carbon, silica, chemically modified silica, bleached clay, and mixtures thereof. Once the desired compound is adsorbed, it can be separated from high molecular weight proteins or compounds.

[0033] In other embodiments, raphanin can be produced synthetically, for example, by click chemistry, combinatorial chemistry, cycloaddition reaction, or solid-phase synthesis. However, those skilled in the art will readily recognize other known methods for forming synthetic raphanin.

[0034] As described above, the pharmaceutical or nutritional compositions disclosed herein comprise "stabilized raphanin". As used herein, "stabilized raphanin" or "stable raphanin" are used interchangeably and refer to a raphanin compound or raphanin containing a compound (e.g., a complex) that becomes more biologically active over time than naturally isolated raphanin. Biological activity can be determined by those skilled in the art using known methods, such as cell culture assays, in vivo studies, receptor binding assays, spectrophotometric assays, etc. In some cases, raphanin is stabilized by altering its chemical structure using known methods, such as modifying side groups, changing the charge of the molecule, or adding a methyl group. In other cases, raphanin is stabilized by using a stabilizer.

[0035] As used herein, the term "agent / pharmaceutical" refers to any kind of composition or combination of compositions. In one embodiment of this disclosure, the agent / pharmaceutical is a small molecule. In another embodiment of this disclosure, the agent / pharmaceutical is a biomolecule, including but not limited to proteins, peptides, antibodies, or nucleic acids. In some embodiments, the stabilizer is a cyclodextrin, alcohol, glycol, ketone, oil, or a combination thereof. In a specific embodiment, stabilized raphanin comprises a complex of raphanin and a stabilizer. In an exemplary embodiment, the stabilizer is hydroxypropyl-β-cyclodextrin.

[0036] In some embodiments, the pharmaceutical or nutritional composition comprises stabilized raphanin, said stabilized raphanin being a raphanin / cyclodextrin complex. The cyclodextrin may be one or more of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, or their hydroxyalkyl derivatives.

[0037] In a specific embodiment, the pharmaceutical composition or nutritional composition comprises stabilized raphanin, said stabilized raphanin being selected from one or more of the following raphanin / cyclodextrin complexes: raphanin and β-cyclodextrin or their hydroxyalkyl derivatives.

[0038] In one embodiment, the pharmaceutical composition or nutritional composition may consist essentially of stabilized raphanin, meaning that the only essential element in the composition is stabilized raphanin (i.e., the active agent). In other embodiments, the pharmaceutical composition consists essentially of stabilized raphanin, wherein the stabilized raphanin is raphanin complexed with cyclodextrin or its hydroxyalkyl derivative, meaning that the only essential elements in the composition are raphanin (i.e., the active agent) and cyclodextrin.

[0039] In other embodiments, the composition consists essentially of stabilized raphanin, wherein the stabilized raphanin is raphanin complexed with β-cyclodextrin or its hydroxyalkyl derivative, meaning that the only essential elements in the composition are raphanin (i.e., the active agent) and β-cyclodextrin. While these pharmaceutical compositions may contain other elements, such as additives or solvents, these other elements can be readily substituted for other similar or analogous elements.

[0040] In one exemplary embodiment, the pharmaceutical composition or nutritional composition comprises a stabilized raphanin / cyclodextrin complex consisting of hydroxypropyl-β-cyclodextrin and raphanin.

[0041] In some embodiments, the method of the present invention uses a pharmaceutical composition or nutritional composition comprising stabilized raphanin, wherein the stabilized raphanin has a purity greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99%. In some cases, the molar ratio of the stabilized raphanin to other elements of the pharmaceutical composition is in the range of 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, or 0.9:1 or higher.

[0042] A pharmaceutical or nutritional composition comprising stabilized rapofrucin and cyclodextrin can be formed by mixing rapofrucin or its natural precursor with cyclodextrin in a suitable solvent to form a precipitate. Any suitable solvent known in the art can be used. In some cases, the solvent is an aqueous solvent containing water and optionally one or more water-miscible solvents, such as ethanol. In other cases, the solvent is water. The dissolution of cyclodextrin in the solvent can be accomplished by any dissolution method known in the art. For example, cyclodextrin can be completely or partially dissolved in the solvent by placing it in the solvent and heating the mixture. In another example, sonication can be used to completely or partially dissolve cyclodextrin in the solvent. Once rapofrucin and cyclodextrin are combined in solution, they can be mixed and cooled to form a precipitate (stabilized rapofrucin). The precipitate can then be filtered to obtain a stable rapofrucin-cyclodextrin complex.

[0043] In some embodiments, the method of the present invention utilizes a pharmaceutical composition comprising raphanin in a cyclodextrin complex containing greater than 75%, 80%, 85%, 90%, 95%, or 99% pure raphanin. In some cases, the molar ratio of raphanin to cyclodextrin in the resulting complex is within the range of 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, or 0.9 or higher. In specific cases, the raphanin to cyclodextrin ratio is 0.8:1 to 1:1, 0.9:1 to 1:1, 0.95:1 to 1:1, or 0.98:1 to 1:1.

[0044] In some embodiments, the pharmaceutical composition is formulated as a unit dose composition, such as a tablet, powder, or liquid, which may contain about 0.1 wt% to 100 wt%, 0.1 wt% to 90 wt%, 0.1 wt% to 80 wt%, 0.1 wt% to 70 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 50 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, or 0.1 wt% to 10 wt% of the active compound, i.e., stabilized raphanin. In one embodiment, the pharmaceutical composition contains 5.0 wt% to 30 wt%, 10 wt% to 25 wt%, or 10 wt% to 20 wt% of stabilized raphanin. In a specific embodiment, the pharmaceutical composition contains 10 wt% to 22 wt% of stabilized raphanin.

[0045] In some non-limiting embodiments, the pharmaceutical or nutritional compositions of this disclosure comprise stabilized raphanin and at least one additive. In specific embodiments, the pharmaceutical composition comprises a stabilized raphanin / cyclodextrin complex and at least one additive. The additive may include a carrier, stabilizer, antioxidant, colorant, diluent, and excipient that will not alter the properties of raphanin to the point of loss of therapeutic effect.

[0046] Exemplary carriers include, but are not limited to, physiological saline, Ringer's solution, phosphate solution or buffer, buffered saline, and other carriers known in the art. The carrier may be solid or liquid, or both, and may be formulated with the pharmaceutical composition into a unit-dose composition, such as tablets, powders, or liquids, which may contain about 0.1 wt% to 100 wt%, 0.1 wt% to 90 wt%, 0.1 wt% to 80 wt%, 0.1 wt% to 70 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 50 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, or 0.1 wt% to 10 wt% of the active compound, namely stabilized raphanin. In one embodiment, the pharmaceutical composition contains 5.0 wt% to 30 wt%, 10 wt% to 25 wt%, or 10 wt% to 20 wt% of stabilized raphanin. In a specific embodiment, the pharmaceutical composition contains 10 wt% to 22 wt% of stabilized raphanin.

[0047] The pharmaceutical or nutritional composition can be formulated based on the desired route of administration of stabilized raphanin. The desired route of administration can be one or more of oral, enteral, parenteral, injection, oral, and topical administration. For example, in some embodiments, the composition is suitable for oral administration. In some embodiments, the composition includes additives and / or carriers suitable for promoting delivery of the composition to the skin, intestines, or bloodstream of a subject.

[0048] In particular, the pharmaceutical or nutritional compositions disclosed herein, or formulations containing them, can be administered orally, for example as tablets, coated tablets, troche, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use can be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more additives selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives to provide a pharmaceutically acceptable and palatable formulation.

[0049] Tablets may be uncoated, or they may be coated using known techniques to delay disintegration and adsorption in the gastrointestinal tract, thereby providing sustained action over a longer period. For example, delay-release materials such as glyceryl monostearate or glyceryl distearate may be used.

[0050] The pharmaceutical or nutritional compositions disclosed herein may also contain non-toxic excipients. Such excipients may be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrants such as corn starch or alginic acid; binders such as starch, gelatin, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc.

[0051] Pharmaceutical or nutritional compositions can also be formulated into hard gelatin capsules, wherein the active ingredient (stabilized raphanin, such as raphanin / cyclodextrin complex) is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate, or kaolin); or formulated into soft gelatin capsules, wherein the active ingredient is present in or mixed with an aqueous or oily medium, such as peanut oil, liquid paraffin, milk, olive oil, or any of various herbal extracts.

[0052] Aqueous suspensions can be prepared containing an active ingredient (stabilized raphanin) incorporated with excipients suitable for preparing aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone tragacanth gum, and gum arabic; dispersants or wetting agents can be naturally occurring phospholipids (e.g., lecithin), or condensation products of alkyl esters and fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide and long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), or condensation products of ethylene oxide and esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitan monooleate), or condensation products of ethylene oxide and esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene dehydrated sorbitan monooleate).

[0053] Aqueous suspensions may also contain one or more preservatives (such as ethylparaben or n-propylparaben), one or more coloring additives, one or more flavoring additives, or one or more sweeteners (such as sucrose, glycerin, sorbitol or saccharin).

[0054] Oily suspensions can be formulated by suspending the active ingredient (stabilized raphanin, such as raphanin / cyclodextrin complex) in omega-3 fatty acids, vegetable oils (such as peanut oil, olive oil, sesame oil, or coconut oil), or mineral oils (such as liquid paraffin). Oily suspensions may contain thickening additives such as beeswax, hard paraffin, or cetyl alcohol.

[0055] Sweeteners and flavorings, such as those mentioned above, can be added to provide a palatable oral formulation.

[0056] Dispersible powders and granules suitable for preparing pharmaceutical compositions by adding water provide the active ingredient (stabilized raphanin) when mixed with a dispersant or wetting agent, a suspending agent, and one or more preservatives. Examples of suitable dispersants or wetting agents and suspending agents are those already mentioned above. Additional excipients, such as sweeteners, flavoring agents, and coloring agents, may also be present.

[0057] Syrups and elixirs containing stabilized raphanin (e.g., raphanin / cyclodextrin complexes) can be formulated using sweeteners such as glycerol, sorbitol, or sucrose. Such formulations may also contain modifiers, preservatives, and / or flavoring agents, as well as coloring agents. Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and / or elixirs containing inert diluents commonly used in the art (e.g., water). Such compositions may also contain adjuvants, such as wetting agents, emulsifiers, and / or suspending agents, as well as sweeteners, flavoring agents, and / or aromatizers.

[0058] Pharmaceutical or nutritional compositions suitable for oral administration can exist in discrete units, each unit containing a predetermined amount of stabilized raphanin for effective treatment: as powder or granules; as a solution or suspension of an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. As noted, such compositions can be prepared by any suitable pharmaceutical method, which may include the step of binding the active ingredient (stabilized raphanin, such as a raphanin / cyclodextrin complex) to a carrier (which may constitute one or more additives).

[0059] For example, tablets can be prepared by compressing or molding stabilized raphanin powder or granules, optionally with one or more excipients. Compressed tablets can be prepared by compressing a compound (e.g., powder or granules) in a free-flowing form in a suitable machine, the compound optionally being mixed with a binder, lubricant, inert diluent, and / or surfactant / dispersant. Molded tablets can be prepared by molding a powdered compound wetted with an inert liquid diluent in a suitable machine.

[0060] The pharmaceutical or nutritional compositions disclosed herein may be coated or uncoated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a delayed effect over a longer period. For example, delaying materials such as glyceryl monostearate or glyceryl distearate may be used.

[0061] The compositions used in the methods of the present invention can be injectable. For example, the pharmaceutical composition can be a sterile injectable solution or suspension in a non-toxic, parenteral diluent or solvent, such as a solution in 1,3-butanediol. Water, Ringer's solution, and isotonic sodium chloride solution can be used in acceptable carriers and solvents. Additionally, sterile fixative oils are commonly used as solvents or suspension media. For this purpose, any mild fixative oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, n-3 polyunsaturated fatty acids have been found to be useful in the preparation of injectable formulations.

[0062] Pharmaceutical and nutritional compositions may also comprise sterile aqueous formulations prepared by mixing stable raphanin with water and sterilizing the resulting solution to be isotonic with blood. Injectable pharmaceutical compositions according to the invention typically contain 5% w / w to 40% w / w of stable raphanin.

[0063] The injectable composition may also contain saline, glucose, or water as a suitable carrier. A suitable dose of stabilized raphanin (e.g., raphanin / cyclodextrin complex) is the dose that achieves a relatively similar serum level as described above.

[0064] The pharmaceutical compositions disclosed herein can also be topical formulations for use on the skin, and can be in the form of ointments, creams, lotions, pastes, gels, sprays, powders, gels, collyrium eye washes, solutions, suspensions, aerosols, or oils. Carriers can be used, including petrolatum (e.g., petrolatum). Lanolin, polyethylene glycol, alcohols, and combinations of two or more of these. The active ingredient (stabilized raphanin, for example, raphanin / cyclodextrin complex) is typically present at a concentration of about 0.1 wt% to 100 wt%, 0.1 wt% to 90 wt%, 0.1 wt% to 80 wt%, 0.1 wt% to 70 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 50 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, or 0.1 wt% to 10 wt% of the active compound (i.e., stabilized raphanin). In one embodiment, the pharmaceutical composition contains 5.0 wt% to 30 wt%, 10 wt% to 25 wt%, or 10 wt% to 20 wt% of stabilized raphanin. In a specific embodiment, the pharmaceutical composition contains 10 wt% to 22 wt% of stabilized raphanin.

[0065] The pharmaceutical and nutritional compositions of the present invention may also contain a safe and effective amount of isotonic agents, including salts (e.g., sodium chloride) and / or non-electrolyte isotonic agents (e.g., sorbitol and mannitol).

[0066] The pharmaceutical and nutritional compositions of the present invention can also be enhanced by incorporation of surfactants or co-solvents. Such co-solvents include polysorbate 20, polysorbate 60, and polysorbate 80, and polyoxyethylene / polyoxypropylene surfactants (e.g., available from...). The co-solvents may be obtained from Pluronic F-68, Pluronic F-84, and Pluronic P-103, or other reagents known to those skilled in the art. The amount of these co-solvents may be from about 0.01 wt% to about 2 wt%.

[0067] Effective formulations and methods of administration are well known in the art and described in standard textbooks. See, for example, Gennaro, AR, Remington: The Science and Practice of Pharmacy, 20th ed. (Lippincott, Williams, and Wilkins), 2000; Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1975; Liberman et al., eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Kibbe et al., eds., Handbook of Pharmaceutical Excipients (3rd ed.), American Pharmaceutical Association, Washington, 1999.

[0068] As described above, in some embodiments, stabilized raphanin is included in the nutritional composition. As used herein, "nutritional composition" refers to a multi-pharmaceutical composition in which one agent is stabilized raphanin and other agents can target one or more different biochemical pathways to provide multiple therapeutic effects to the subject.

[0069] In some embodiments, the nutritional composition comprises stable raphanin and at least one, at least two, at least three, at least four, at least five, or at least six agents from the group consisting of members that have therapeutic effects on one or more mammalian diseases (e.g., mitochondrial diseases). Group 1: Minerals, vitamins, and dietary supplements; Group 2: Herbal products, such as garlic (allicin), ginger, echinacea, ginseng, licorice, onion, senna, turmeric (curcumin), or portions thereof; Group 3: Dietary enzymes, such as bromelain and papain; Group 4: Fiber; Group 5: Hydrolyzed protein; Group 6: Phytonutrients, such as resveratrol; Group 7: Carotenoids, such as lycopene; Group 8: Prebiotics and probiotics.

[0070] In another embodiment, the nutritional composition comprises stable raphanin and at least one active ingredient from the group described above. In a specific embodiment, the nutritional composition comprises stable raphanin and at least one active ingredient from the group described above, and is provided as a food additive (e.g., powder). In some embodiments, the nutritional composition comprises a raphanin / cyclodextrin complex and at least one active ingredient from at least one group described above. In one embodiment, the nutritional composition comprises stable raphanin and at least one active ingredient from each of the groups described above.

[0071] The method of the present invention includes administering to a subject a nutritional composition comprising stabilized raphanin, said stabilized raphanin including, but not limited to, raphanin / cyclodextrin complexes. For example, administering to a subject a nutritional composition consisting of a raphanin / hydroxypropyl-β-cyclodextrin complex.

[0072] According to the method of the present invention, any method for preparing nutritional preparations can be used. In some cases, the method for preparing the nutritional composition of this disclosure is the same as or substantially the same as the method for preparing pharmaceutical compositions. In one embodiment, the nutritional composition of this disclosure is prepared by the same method used to prepare the pharmaceutical compositions described above. In other embodiments, the nutritional composition is formulated and administered according to the methods described above. In yet another embodiment, the nutritional composition of this disclosure is prepared using methods known to those skilled in the art.

[0073] The term “subject” as used herein may be used interchangeably with “patient” and may refer to a human or any other mammal, including but not limited to primates, rats, mice, rabbits, pigs, cattle, sheep, goats, cats, or dogs. As used herein, “subject” refers to any subject with a condition (direct, related, or other) that is indicated as a mitochondrial disorder in the judgment of a physician (e.g., a clinician or veterinarian).

[0074] In some embodiments, the subject treated by the method of the present invention is a mammal. In some embodiments, the subject is a human, mouse, or rat. In one exemplary embodiment, the subject to be treated is a human subject suffering from symptoms indicative of mitochondrial disease. In a specific embodiment, the subject is a person diagnosed with mitochondrial disease, wherein mitochondrial disease affects the subject's ability to metabolize carbohydrates, lipids (fats), proteins, and / or nucleic acids, as well as to produce ATP.

[0075] In some cases, subjects suffered from one or more of the following conditions associated with mitochondrial disorders, such as abnormal mitochondrial oxidative metabolism, reduced ATP production, and / or decreased cell viability.

[0076] Administration can be, for example, oral, intravenous, intraperitoneal, or local administration. In a specific embodiment, the subject is given a stable raphanin composition orally. In other embodiments, the composition is administered to the subject by injection (e.g., via intravenous or intraperitoneal injection). In yet another embodiment, the pharmaceutical composition is administered locally.

[0077] In this method, a subject requiring treatment and / or prevention of one or more mitochondrial diseases or their symptoms described herein may be treated by administering a pharmaceutical or nutritional composition consisting of stable raphanin, such that the amount of the active ingredient (stable raphanin, such as raphanin / cyclodextrin complex) is sufficient to constitute a dose or amount (i.e., “effective amount”) for the treatment or prevention of mitochondrial diseases or their symptoms.

[0078] As used herein, "effective amount" refers to the dose or amount of a pharmaceutical or nutritional composition containing stable raphanin administered to a subject to reduce or eradicate mitochondrial disease or its symptoms. An effective amount of a composition can be readily determined by those skilled in the art using known techniques and observations obtained under similar conditions.

[0079] In one exemplary embodiment, an effective amount of a pharmaceutical or nutritional composition containing stable raphanin administered to a subject results in an increase in ATP production in the subject's cells.

[0080] The effective amount of a pharmaceutical or nutritional composition consisting of stabilized raphanin (e.g., raphanin / cyclodextrin complex) can depend on many factors, such as the specific stabilizer or method used, the subject, the route of administration, and the disease being treated. For example, the dosage may be adjusted based on the subject's weight, age and health, and tolerance to the administered composition.

[0081] Certain non-limiting examples of suitable dosage ranges for the injectable pharmaceutical composition include, but are not limited to: for individuals weighing less than 40 kg, a dose of 0.06 mg / kg / day, which may be increased or decreased by 0.02 mg / kg up to a maximum daily dose of 0.13 mg / kg; for individuals weighing more than 40 kg, a dose of 2.5 mg / day, which may be increased or decreased by 1.25 mg up to 2.5 mg / day up to a maximum daily dose of 10 mg / day; for women weighing more than 40 kg, a dose of 5 mg / day is suitable, which may be increased or decreased by 1.25 mg up to 2.5 mg / day up to a maximum daily dose of 10 mg / day. In yet another example, the pharmaceutical composition of this disclosure can be delivered to a subject at doses of 30 mcg / mL, 60 mcg / mL, 90 mcg / mL, or 120 mcg / mL.

[0082] In other instances, the dosage of the compositions and methods of the invention provided herein can be determined and adjusted based on the efficacy demonstrated in providing therapeutic effects. Furthermore, those skilled in the art will know how to measure and quantify the presence or absence of mitochondrial disorders or their symptoms after treatment according to the methods of the invention. In some cases, the effective therapeutic dose will improve or reduce one or more of the following abnormalities in a subject: mitochondrial oxidative metabolism, amino acid metabolism, organic acid metabolism, ATP production in cells (e.g., skin cells or muscle cells), cell viability, fatty acid metabolism, carbohydrate metabolism, urea formation, and peroxisome metabolism.

[0083] For example, in cases of mitochondrial myopathy (such as chronic fatigue syndrome or essential tremor), an effective amount of stable raphanin will reduce or eliminate irregularities in cellular metabolism, improve mitochondrial function (i.e., ATP production), increase the secretion of one or more amino acids, or any combination of the aforementioned symptoms.

[0084] In cases of skin conditions such as psoriasis, aging skin, or psoriatic arthritis, effective treatment can improve skin damage or inflammation.

[0085] In the case of PCOS, effective treatment with a stable raphanin / cyclodextrin complex will reduce or eliminate one or more of the following symptoms in the subject: irregular cycles, androgen abnormalities, polycystic ovary syndrome, or related symptoms as described below.

[0086] The effective dose can also be extrapolated from dose-response curves derived from in vitro or animal model systems (e.g., in mouse models). Those skilled in the art will understand that the dose can also be determined with the guidance of Goodman & Gilman's, The Pharmacological Basis of Therapeutics, 9th edition (1996), Appendix II, pp. 1707-1711.

[0087] The pharmaceutical or nutritional compositions disclosed herein can be administered once or multiple times daily as needed. The frequency of administration can vary from a single daily dose to multiple daily doses. In one embodiment, the composition can be administered weekly, every two weeks, every three weeks, monthly, every two months, or every six months. In some embodiments, for example, the composition can be administered when a subject with mitochondrial disease experiences inflammation, skin lesions, muscle tremors, fatigue, or other symptoms that may require treatment.

[0088] This method includes the treatment of mitochondrial diseases.

[0089] The term "treatment" refers to the prevention or improvement of mitochondrial disease or one or more of its symptoms in a patient or subject. It does not mean that "treatment" of mitochondrial disease requires a complete cure or eradication. Treatment only needs to reduce or suppress the disease to have a "therapeutic effect." Similarly, if, in the practitioner's judgment, one or more characteristic markers of disease progression are reduced or suppressed, the progression of the disease is considered to be "reduced" or "suppressed." The term "therapeutic effect" refers to the inhibition, activation, or substitution of factors that cause or contribute to mitochondrial disease or its symptoms in a subject, such as mitochondrial myopathy, skin disease, muscle disease, or metabolic disease (e.g., type 2 diabetes).

[0090] The terms “mitochondrial disorder” or “mitochondrial disease” are used interchangeably herein to refer to a condition or defect in which the subject’s cells are unable to properly metabolize carbohydrates, lipids (fats), proteins, or nucleic acids through oxidative phosphorylation. Therefore, in the context of the methods of this invention, all conditions relating to an abnormality in the subject’s ability to produce ATP are included in the term “mitochondrial disorder.” Examples of mitochondrial disorders include, but are not limited to, mitochondrial myopathy, metabolic disorders, skin diseases, prostate disorders, or central nervous system disorders. For the purposes of this disclosure, it is not intended that metabolic disorders include cancer.

[0091] Exemplary mitochondrial conditions treatable by the methods of this invention include, but are not limited to, mitochondrial myopathy. As used herein, the term "mitochondrial myopathy" should be understood as a dysfunction in the production of ATP through mitochondrial oxidative phosphorylation in the muscle cells and / or central nervous system (CNS) cells of a subject. Symptoms of mitochondrial myopathy include muscle weakness or exercise intolerance, heart failure or rhythm disturbances, dementia, movement disorders, stroke-like episodes, deafness, blindness, ptosis, limited eye movement, vomiting, and seizures. Several exemplary mitochondrial myopathy conditions treatable by administration of an effective amount of raphanin / hydroxypropyl-β-cyclodextrin complex include, but are not limited to, chronic fatigue syndrome (CFS), prostate disease (i.e., benign prostatic hyperplasia), essential tremor, Parkinson's disease, and multiple sclerosis.

[0092] In an exemplary embodiment, a subject given a composition comprising stabilized raphanin (e.g., raphanin / hydroxypropyl-β-cyclodextrin complex) according to this disclosure suffers from mitochondrial myopathy, such as chronic fatigue syndrome (CFS). As used herein, “chronic fatigue syndrome (CFS)” means a mitochondrial disorder characterized by prolonged physical and mental fatigue. CFS is associated with one or more of the following symptoms: increased pain and sensitivity, metabolic abnormalities (e.g., energy metabolism, amino acid metabolism, nucleotide metabolism, nitrogen metabolism, hormone metabolism, and oxidative stress metabolism), decreased mitochondrial function, and reduced cellular amino acid and nitrogen secretion. Those skilled in the art can readily diagnose CFS in subjects using known methods by detecting changes in mitochondrial cellular metabolism (e.g., ATP production or cell viability) using metabolomics.

[0093] In an exemplary embodiment, a subject given a composition comprising stabilized raphanin (e.g., raphanin / hydroxypropyl-β-cyclodextrin complex) according to this disclosure suffers from mitochondrial myopathy, such as prostate disease. “Benign prostatic hyperplasia” or “prostate disease” is used interchangeably herein to refer to a mitochondrial condition presenting as an enlarged prostate, such as the mitochondrial condition present in “benign prostatic hyperplasia (BPH)”. Benign prostatic hyperplasia is associated with one or more of the following symptoms: reduced or obstructed flow of urine from the bladder, increased frequency of urination, inability to empty the bladder, urinary tract infection, or hematuria. Those skilled in the art can readily diagnose BPH in subjects using known methods by detecting changes in mitochondrial cellular metabolism (e.g., ATP production or cell viability) using metabolomics.

[0094] Mitochondria play a crucial role in providing the energy necessary for the normal functioning of dopaminergic neurons. Therefore, in some embodiments of this disclosure, subjects given a composition consisting of stabilized raphanin (e.g., raphanin / hydroxypropyl-β-cyclodextrin complex) suffer from mitochondrial myopathy of the central nervous system. Exemplary mitochondrial myopathy of the central nervous system includes, but is not limited to, essential tremor, multiple sclerosis, dementia, and Parkinson's disease.

[0095] In an exemplary embodiment, a subject given a composition comprising stabilized raphanin (e.g., raphanin / hydroxypropyl-β-cyclodextrin complex) according to this disclosure suffers from mitochondrial myopathy, such as essential tremor. The term "essential tremor" as used herein should be understood as a neurological disorder causing involuntary and rhythmic shaking of the limbs. Essential tremor manifests when the hands, head, or voice shake involuntarily during use, or when the subject exhibits an abnormal gait.

[0096] In one embodiment, a subject given a composition comprising stabilized raphanin (e.g., raphanin / hydroxypropyl-β-cyclodextrin complex) according to this disclosure suffers from mitochondrial myopathy, such as multiple sclerosis (MS). As used herein, the term "multiple sclerosis" should be understood as a neurodegenerative disease caused by abnormal mitochondrial oxidative phosphorylation, leading to reduced ATP production, axonal damage, neuronal loss (reduced cell viability), and central nervous system atrophy.

[0097] Mitochondrial function also regulates hormone production in the endocrine system. The thyroid gland, located in the front of the neck, secretes two thyroid hormones: T4 (thyroxine) and T3 (triiodothyronine). Thyroid hormones are known to regulate cellular basal metabolic state and oxidative metabolism. For example, thyroid hormones are associated with oxidative stress and antioxidant status because they can alter oxidative respiration in mitochondria. Furthermore, the thyroid gland can produce hormones that signal oxidative stress, leading to reduced mitochondrial ATP production and consequently cell death. This oxidative stress is present in conditions such as polycystic ovary syndrome (PCOS), mitochondrial diabetes, hypothyroidism, and hyperthyroidism.

[0098] Therefore, in an exemplary embodiment, subjects given a composition consisting of stabilized raphanin (e.g., raphanin / hydroxypropyl-β-cyclodextrin complex) according to this disclosure have PCOS. As used herein, "polycystic ovary syndrome" refers to mitochondrial disorders present in female subjects with one or more of the following symptoms: irregular cycles, androgen abnormalities, and polycystic ovaries. PCOS also presents with other common symptoms (symptoms / conditions) such as insulin resistance, obesity, impaired glucose tolerance, dyslipidemia, and / or hypertension.

[0099] Mitochondria play a vital role in providing the energy necessary for the normal functioning of skin cells. Therefore, in some embodiments of this disclosure, subjects given a composition consisting of stabilized raphanin (e.g., raphanin / hydroxypropyl-β-cyclodextrin complex) have cutaneous mitochondrial myopathy. Exemplary cutaneous mitochondrial myopathy includes, but is not limited to, psoriasis, psoriatic arthritis, and aging skin.

[0100] Therefore, in one embodiment, a subject given a composition of stabilized raphanin (e.g., raphanin / hydroxypropyl-β-cyclodextrin complex) according to this disclosure suffers from mitochondrial myopathy, such as psoriasis. As used herein, the term "psoriasis" refers to Th-1 mediated and Th-17 mediated diseases associated with mitochondrial disorders. Psoriasis is characterized by inflammation, irritation, scaling of the skin, and one or more of the following symptoms: xerotic lesions, joint stiffness or swelling, obesity, hypertension, dyslipidemia, and insulin resistance.

[0101] In another embodiment, a subject given a composition of stabilized raphanin (e.g., raphanin / hydroxypropyl-β-cyclodextrin complex) according to this disclosure suffers from mitochondrial myopathy, such as aging skin. As used herein, the term "aging skin" should be understood as a progressive structural and functional degeneration of the epidermis. Skin aging is primarily caused by the accumulation of reactive oxygen species (ROS), which are byproducts of mitochondrial cell metabolism. ROS, in turn, damage key cellular components such as membranes, enzymes, and deoxyribonucleic acid (DNA), leading to cell death. Furthermore, in aging skin, the rate of cell proliferation in the epidermis decreases, resulting in a stable deterioration of skin structure and function. Aging skin can be a precursor to other skin conditions such as eczema, dermatitis, keratosis, and various forms of tumors, such as basal cell carcinoma, squamous cell carcinoma, and malignant melanoma.

[0102] Other embodiments within the scope of the claims herein will be apparent to those skilled in the art from the practice or description of the methods disclosed herein. The following description and experimental results are intended to be considered exemplary only, and the scope and spirit of the invention are indicated by the appended claims.

[0103] Example

[0104] Example 1. Materials and Methods.

[0105] Cell cultures. For all cell-based assays, the following cell lines and culture methods were used. From SK-MEL-31 human skin cells and A204 human muscle cells were obtained, cultured, and maintained in MoCoy-5A medium containing 10% FBS and 1% penicillin and streptomycin. PANC-1 human pancreatic cancer cells were obtained, cultured, and maintained in DMEM medium containing 10% FBS and 1% penicillin and streptomycin.

[0106] Before plating into 96-well plates, cells were treated with trypsin and a cell counter was used. Automated Cell Counter (Invitrogen) was used for counting. Then, for each cell type used in the test, the cells were resuspended to the appropriate cell density before seeding. Specifically, the cell suspensions were formulated to contain 15,000 cells per 100 μL of cell culture medium for SK-MEL-31 and A204 cells, and 12,000 cells per 100 μL of cell culture medium for PANC-1 cells. 100 μL of the cell suspension was then seeded into each well of a white-walled, tissue culture-treated 96-well plate (Corning) and incubated at 37°C for 24 hours.

[0107] CellTiter-Glo (CTG) ATP assay. Twenty-four hours after cell seeding, cells were administered 1 / 3 diluted 100 μL of 1 μM astrococcus (Sigma-Aldrich) and one of the following combinations using a Tecan D300e digital dispenser (Tecan): 100 μM of a synthetic raphanin / hydroxypropyl-β-cyclodextrin complex (compound 1); 17.5 μg / mL of raphanin isolated from radish seed extract (compound 2); and 17.5 μg / mL of a positive control (compound 3). Nine sites were used for each cell line tested.

[0108] After treating cells with the test compound for 4 hours (data not shown), 48 hours, or 72 hours, the cells were incubated at room temperature for approximately 30 minutes. Then, 100 μL of CellTiter-Glo reagent (Promega) was added to each well of the plate, and each plate was mixed on an orbital shaker for 2 minutes to induce cell lysis. After shaking, each plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal. The signal was then read according to the manufacturer's protocol on the EnVision Multilabel Reader (Perkin Elmer). The effect of each composition on mitochondrial function was determined based on the quantification of ATP present in each well, proportional to the amount of cells present. Specifically, the effect of each compound was calculated using the following formula: Effect % = (X-Control Medium Only) / (Control - Control DMSO Effect) × 100%.

[0109] Example 2: Stable raphanin increases the production of mitochondrial ATP in skin cells.

[0110] To determine whether stabilized raphanin (i.e., the synthetic raphanin / hydroxypropyl-β-cyclodextrin complex) could treat mitochondrial disorders of the skin (such as psoriasis or skin aging), experiments were performed on SK-MEL-31 human skin cells as described above. Luminescent cell viability assay. This assay determines the number of surviving skin cells present in each well by measuring the amount of ATP present in each well. In short, the detection is based on measuring the amount of ATP present in surviving cells using a luciferase reaction. If mitochondrial function (i.e., ATP production from oxidative phosphorylation) is improved after administration of stabilized raphanin compared to cells treated with astrocytocin alone, then an exemplary stabilized raphanin composition can be used to treat mitochondrial disorders of the skin.

[0111] Asteroidin (STSP) is a cell-permeable alkaloid isolated from Streptomyces staurosporeus. Asteroidin is a potent, non-selective inhibitor of protein kinases (including protein kinase C) that induces apoptosis. Therefore, cells treated with asteroidin alone were used as a negative control.

[0112] After STSP treatment, SK-MEL-31 human skin cells were administered 100 μM of a synthetic raphanin / hydroxypropyl-β-cyclodextrin complex (compound 1), 17.5 μg / mL of raphanin isolated from radish seed extract (compound 2), or 17.5 μg / mL of a positive control (compound 3), and incubated for 4 hours (data not shown), 48 hours, or 72 hours, as... Figure 1A-Figure 1B As shown.

[0113] Figure 1A and Figure 1B The data shown indicate that the two compounds containing stabilized raphanin (i.e., compounds 1 and 2) increased ATP production in skin cells after treatment with STSP, comparable to the results observed after treatment with the positive control compound (compound 3). Therefore, the stabilized raphanin compositions of this disclosure have been shown to enhance mitochondrial function by increasing in vitro oxidative phosphorylation and ATP production. In summary, these data suggest that the stabilized raphanin compositions of this disclosure may be used to treat mitochondrial disorders of the skin, such as psoriasis and skin aging.

[0114] Example 3: Stable raphanin increases the production of mitochondrial ATP in muscle cells.

[0115] To determine whether stabilized raphanin (i.e., the synthetic raphanin / hydroxypropyl-β-cyclodextrin complex) could treat mitochondrial myopathy in muscle tissue, such as chronic fatigue syndrome (CFS), essential tremor, or Parkinson's disease, as described above, experiments were performed on A204 human muscle cells. Luminescent cell viability assay. The experiment was performed as described in Example 2 above. Therefore, if mitochondrial function (i.e., ATP production from oxidative phosphorylation) is improved after administration of stable raphanin compared to cells treated with astrocytocin alone, then the exemplary stabilized raphanin composition may be used to treat muscle mitochondrial myopathy.

[0116] After STSP treatment, A204 human muscle cells were given 100 μM of a synthetic raphanin / hydroxypropyl-β-cyclodextrin complex (compound 1), 17.5 μg / mL of raphanin isolated from radish seed extract (compound 2), or 17.5 μg / mL of a positive control (compound 3), and incubated for 4 hours (data not shown), 48 hours, or 72 hours, as... Figures 2A-2B As shown.

[0117] Figures 2A-2B The data shown indicate that, after treatment with STSP, the two compounds containing stabilized raphanin (i.e., compounds 1 and 2) increased ATP production in muscle cells, comparable to the results observed after treatment with the positive control compound (compound 3). Therefore, the stabilized raphanin compositions of this disclosure have been shown to enhance mitochondrial function by increasing in vitro oxidative phosphorylation and ATP production. In summary, these data suggest that the stabilized raphanin compositions of this disclosure may be used to treat mitochondrial myopathy, such as CFS and essential tremor.

[0118] Example 4: Stable raphanin increases mitochondrial ATP production in other (pancreatic) cells.

[0119] To determine whether stabilized raphanin (i.e., the synthetic raphanin / hydroxypropyl-β-cyclodextrin complex) could treat mitochondrial diseases in other cell types (e.g., the pancreas, which regulates endocrine function), experiments were performed on PANC-1 human pancreatic cells as described above. Luminescent cell viability assay. Experiments were performed as described in Examples 2 and 3 above. Therefore, if mitochondrial function (i.e., ATP production from oxidative phosphorylation) is improved after administration of stable raphanin compared to cells treated with astrocytocin alone, the exemplary stabilized raphanin composition may be used to treat mitochondrial disorders originating from other organs or based on hormones, such as PCOS, prostate myopathy, and thyroid disorders.

[0120] Here, as in Examples 2 and 3, after STSP treatment, PANC-1 cells were given 100 μM of the synthetic raphanin / hydroxypropyl-β-cyclodextrin complex (compound 1), 17.5 μg / mL of raphanin isolated from radish seed extract (compound 2), or 17.5 μg / mL of the positive control (compound 3), and incubated for 4 hours (data not shown), 48 hours, or 72 hours.

[0121] Figures 3A-3B The data shown indicate that treatment with STSP resulted in increased ATP production in pancreatic cells from the two compounds containing stabilized raphanin (i.e., compounds 1 and 2), comparable to the results observed after treatment with the positive control compound (compound 3). Therefore, the stabilized raphanin composition of this disclosure has been shown to enhance mitochondrial function by increasing in vitro oxidative phosphorylation and ATP production. In summary, these data suggest that the stabilized raphanin composition of this disclosure may be used to treat mitochondrial disorders in other tissues, such as the pancreas.

Claims

1. Use of a stable raphanin composition in the preparation of a medicament for the prevention or improvement of mitochondrial diseases, wherein: An effective amount of the stable raphanin composition was administered to a subject suffering from mitochondrial disease, the stable raphanin composition comprising isolated raphanin and a stabilizer, and wherein the administration effectively prevented or improved the mitochondrial disease. The stable raphanin composition consists of a raphanin / cyclodextrin complex. The cyclodextrin is β-cyclodextrin or its hydroxyalkyl derivative.

2. The use according to claim 1, wherein, The mitochondrial disease mentioned is mitochondrial myopathy.

3. The use according to claim 2, wherein, The mitochondrial myopathy is selected from the group consisting of: chronic fatigue syndrome, essential tremor, psoriasis, skin aging, polycystic ovary syndrome, mitochondrial diabetes, thyroid disease, benign prostatic hyperplasia, and multiple sclerosis.

4. The use according to claim 3, wherein, The mitochondrial myopathy is chronic fatigue syndrome, essential tremor, polycystic ovary syndrome, or psoriasis.

5. The use according to claim 4, wherein, The mitochondrial myopathy mentioned is a chronic fatigue syndrome.

6. The use according to claim 1, wherein, The raphanin / cyclodextrin complex is a complex of raphanin and hydroxypropyl-β-cyclodextrin.

7. The use according to claim 1, wherein, The stable raphanin composition is given as a pharmaceutical preparation or nutritional composition.

8. The use according to claim 7, wherein, The pharmaceutical composition or nutritional composition may be administered orally, by injection, or topically.

9. The use according to claim 8, wherein, The pharmaceutical composition or nutritional composition may be administered orally.

10. The use according to claim 7, wherein, The pharmaceutical composition or nutritional composition is a powder, pill, or liquid preparation.

11. The use according to claim 1, wherein, The subjects were human.

12. The use according to any one of claims 1-11, wherein, The stable raphanin composition was administered to the subject at least once daily.

13. The use according to claim 12, wherein, The stable raphanin composition was administered to the subjects twice daily for at least two weeks.

14. Use of stable raphanin compositions in the preparation of reagents for increasing ATP production in cells, wherein: The stabilized raphanin composition is used for administration to cells, wherein the stabilized raphanin composition comprises isolated raphanin and a stabilizer, and wherein, compared to cells not administered the stabilized raphanin composition, the administration increases ATP production through the cells. The stable raphanin composition consists of a raphanin / cyclodextrin complex, and The cyclodextrin is β-cyclodextrin or its hydroxyalkyl derivative.

15. The use according to claim 14, wherein, The raphanin / cyclodextrin complex is a complex of raphanin and hydroxypropyl-β-cyclodextrin.

16. The use according to claim 14 or 15, wherein, The stable raphanin composition is given as a pharmaceutical preparation or nutritional composition.

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