Patent Application
20230248766
A portion of the disclosure of this patent contains material that is subject to copyright protection. The copyright owner has no objection to the reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent files or records, but otherwise reserves all copyright rights whatsoever.
The present invention relates to a composition for the modulation of oxidative stress or redox signaling. In particular, it relates to fullerene compositions and related enzyme mimics which modulate oxidative stress and redox balance.
Oxidative stress is a biological state that occurs when a cell’s antioxidant capacity is overwhelmed by reactive oxygen species (ROS) causing a redox imbalance. Reactive oxygen species are a type of free radical, which is formed with oxygen. Free radicals are chemical species that contain one or more unpaired orbital electrons and are therefore unstable and liable to react with other molecules to form more stable compounds with a lower energy state. In an attempt to achieve this stable state, ROS can react with proteins, lipids, and DNA within the cell. This can result in cell damage and even cell death by inactivation of cellular components such as enzymes, membranes, and DNA. As such, ROS and oxidative stress as a whole have been suggested to participate in the initiation and/or propagation of diseases such as cardiovascular and inflammatory diseases, cancer, and diabetes by causing or exacerbating cell death.
ROS can be produced on a regular basis during oxidative metabolism and in more potent levels during inflammatory processes. It can also be present in administered products. During oxidative metabolism, electrons are lost from the electron transport chain and combine with oxygen, resulting in the formation of superoxide radical anions (O2—′). At the time of inflammation, macrophages and neutrophils that contain the NADPH oxidase complex generate superoxide radicals and hydrogen peroxide to aid in the destruction of foreign agents. Environmental factors such as tobacco smoke, UV radiation and exposure to atmospheric oxygen, as well as overexertion during exercise and the consumption of alcohol and certain foods, can also result in the generation of too much ROS. Though many of these factors can be avoided or limited, as humans, our omnivorous diet exposes us to a variety of foods, some of which may contribute to increased oxidative stress in the gut. An uncontrolled increase of ROS in the gastrointestinal mucosa can lead to inflammatory or ischemic disorders. Oxidative stress has been postulated to play a role in inflammatory bowel disease (IBD) initiation and progression. The binding of an inflammatory stimulus to its cellular receptor triggers the activation of specific intracellular signaling pathways to upregulate the production of inflammatory mediators. Therefore, antioxidative stress mechanisms and antioxidants are key to limiting the proliferation of ROS and re-establishing a stable redox balance.
It would be useful to find compositions that could modulate/reduce oxidative stress or redox signaling in a cell to prevent damage or even death of cells (apoptosis) or an entire organism.
Biofullerene compositions are reported in detail in the Wilson 2000 Pat. No. 6,162,926, in the name of Murphy, Wilson, Lu, titled “Multisubstituted Fullerenes and Methods for Their Preparation and Characterization.” Biofullerene applications for neurodegenerative diseases have also been reported. Details of the use of fullerene additives in classical genomic experiments of transfection or transformation have been extensively described.
Both natural and artificial mixtures of C60 and C70 fullerenes exist. Fullerene concentration in carbon soot varies from parts per million in nature to much higher concentrations (1-14%) in soot from specially-designed manufacturing processes. This “as produced” fullerene soot is usually at least 70% C60 and about 25% C70. Other fullerenes such as C76, C78, and C84 are also present at less than 5% (see
The present invention relates to the discovery that a combination of C60 and C70 fullerenes in a formulation will reduce oxidative stress and modulate redox signaling. This prevents cell injury or cell death due to oxidative damage. Use of the compositions of the present invention allows for preventing cell damage and can therefore extend the life of the cell or organism once treated with the compositions of the present invention.
Accordingly, in one embodiment, there is a method of modulating oxidative stress or redox signaling in a living cell in need thereof comprising administering an effective fullerene composition which contains one of C60 or a C60/C70 mixture comprising at least about 70% C60 wherein the composition regulates at least one reactive oxygen species (ROS) and mimics superoxide dismutase (SOD).
In another embodiment, there is a process for the production of e,e,e-C60 fullerene tris-malonic acid (C3) comprising the steps of:
While this invention is susceptible to embodiment in many different forms, there is shown in the drawings, and will herein be described in detail, specific embodiments with the understanding that the present disclosure of such embodiments is to be considered as an example of the principles and not intended to limit the invention to the specific embodiments shown and described. In the description below, like reference numerals are used to describe the same, similar, or corresponding parts in the several views of the drawings. This detailed description defines the meaning of the terms used herein and specifically describes embodiments in order for those skilled in the art to practice the invention.
The terms “about” and “essentially” mean ±10 percent.
The terms “a” or “an”, as used herein, are defined as one or as more than one. The term “plurality”, as used herein, is defined as two or as more than two. The term “another”, as used herein, is defined as at least a second or more. The terms “including” and/or “having”, as used herein, are defined as comprising (i.e., open language). The term “coupled”, as used herein, is defined as connected, although not necessarily directly, and not necessarily mechanically.
The term “comprising” is not intended to limit inventions to only claiming the present invention with such comprising language. Any invention using the term comprising could be separated into one or more claims using “consisting” or “consisting of” claim language and is so intended.
Reference throughout this document to “one embodiment”, “certain embodiments”, “an embodiment”, or similar terms means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments without limitation.
The term “or”, as used herein, is to be interpreted as an inclusive or meaning any one or any combination. Therefore, “X, Y, or Z” means any of the following: “X, Y, Z”; “X, Y” ; “Y, Z” ; “X, Z” etc. An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in some way inherently mutually exclusive.
The drawings featured in the figures are for the purpose of illustrating certain convenient embodiments of the present invention and are not to be considered as limitation thereto. The term “means” preceding a present participle of an operation indicates a desired function for which there is one or more embodiments, i.e., one or more methods, devices, or apparatuses for achieving the desired function and that one skilled in the art could select from these or their equivalent in view of the disclosure herein, and use of the term “means” is not intended to be limiting.
As used herein, the term “oxidative stress” refers to an imbalance between the production and introduction of reactive oxygen species (ROS) and scavenging systems of reactive oxygen species in the living body or cell (plant or animal) resulting in an excess of the reactive oxygen species. As this state worsens, nucleic acids, proteins, lipids, and the like — constituents of living cells — are oxidized resulting in damage to the cell or entire organism. Upon exposure to oxidative stress from internal or external sources, cells work toward a body’s defense by inducing the expression of antioxidant proteins or phase II detoxification enzymes such as glutathione peroxidase. Thus oxidative stress can damage cells and even cause cell death or cause the entire organism to die.
As used herein, the term “modulating” refers to modulation of “oxidative stress response” and refers to a physiological response in a biological sample (e.g., one or more cells in an organism) to an imbalance between the production of reactive oxygen species (ROS), chemically reactive molecules comprising oxygen ions including, but not limited to, superoxide anion (O2—′), hydroxyl anion (′—OH), peroxynitrite (′OONO), nitric oxide (NO-), and hydrogen peroxide (H2O2). The cellular regulation of ROS through enzymes, antioxidants, and other compounds facilitate ROS breakdown and keep the cell in a state of redox balance, or homeostasis.
As used herein, the term “redox signaling” refers to regulated alterations in the intracellular redox state (redox signaling) and can modulate events such as DNA synthesis, enzyme activation, selective gene expression, regulation of cell cycle, cell growth, and programmed cell death.
Oxidative stress and resulting signaling pathways have been known for quite a long time but their detailed operational biochemistry is quite complex. A commercial agricultural product known as Harpin [C. Oh, et. al. 2007. “Growth-enhancing effect of HrpN in Arabidopsis”. Plant Physiol. 145: 426 (2007)] turns on genes that control redox homeostasis in plants. Knowledge of similar functionality in animals has lagged. Recently some data has emerged on animal redox homeostasis (C. Lipina, et. al., “Modulation of cellular redox homeostasis by the endocannabinoid system,” Open Biology 150276 (2016). The endocannabinoid ROS signaling system uses cannabidiol (
As used herein, the term “living cell” refers to cells having an active metabolism, including cells capable of reproduction, wherein the cell is in a living organism. Death of a cell is often called apoptosis.
As used herein, the term “C60/C70 mixture” refers to mixtures of C60 and C70 fullerenes wherein there is at least about 70% C60 fullerene in the mixture and some portion of the rest is C70. It also, in one embodiment, contains other forms of fullerenes including C76, C78, and C84. The fullerenes are ball shaped fundamental forms of carbon that are formed whenever carbon is burned. Mixtures may be naturally occurring or mixed from individual component fullerenes. They can also be functionalized. For most biological applications (circulation in fluid or blood, entry into cells, and distribution through tissue) a water-soluble composition is usually desired. Modified versions of fullerenes which are both water soluble and non-water soluble are well known. Making the molecule polar makes it water soluble and making it non-polar makes it organic solvent or oil soluble. It is noted the compositions can be substituted or unsubstituted and be polar or non-polar, as desired. The key activity of this combination is an ROS scavenging antioxidant. This means its activity is like an enzyme in that it carries out a catalytic cycle by quenching the undesired oxidant and then is reduced by a cofactor, such as glutathione, back to its original state. This recycling effect allows the fullerenes of the present invention to be much more active per gram (up to 250 times) than other antioxidants such as Vitamin C.
As used herein, the term “reactive oxygen species” (ROS) refers to compositions which are oxidants in living cells and that includes, but are not limited to, hydrogen peroxide, hydroxyl radical, superoxide anion (O2—′), singlet oxygen, nitric oxide, peroxyl radical, and peroxynitrite radical anion.
As used herein, the term “superoxide dismutase” (SOD) refers to an enzyme that scavenges superoxide anions having the formula of O2—′. In normal cells, when expression of SOD is lowered or eliminated, cells are triggered to die. Under these conditions of low SOD, the fullerenes of the present invention mimic SOD such that they prevent cells from dying by replacing any missing naturally occurring SOD.
As used herein, the term “a glutathione peroxidase enzyme mimic” refers to compositions which have roughly the same activity profile as glutathione peroxidase. In one embodiment, the mimic is Ebselen (see
As used herein, the term “modulation of oxidative stress induced cell death” refers to both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the treatment to result in a desired biological effect in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side effects) resulting from administration of the treatment. On the other hand, the term “ineffective” indicates that a treatment does not provide a sufficient pharmacological effect to be therapeutically useful, even in the absence of deleterious effects, at least in the unstratified population. (However, a treatment may be deemed effective based on its effectiveness in the general population, even though it may be ineffective in a subgroup that can be identified by the expression profile or profiles.) “Less effective” means that the treatment results in a therapeutically significant lower level of pharmacological effectiveness and/or a therapeutically greater level of adverse physiological effects, e.g., greater liver toxicity.
The key observation of this disclosure is that fullerene compounds of the invention maintain redox balance or homeostasis by regulation of ROS in a manner similar to SOD. While fullerene compounds are super-antioxidants, the use of fullerenes for regulation of ROS is quite different from using massive doses of antioxidants such as vitamin C or E. Vitamin C and E can function as both antioxidants and prooxidants. The studies recently summarized [A. Gorlach, et al., “Reactive oxygen species, nutrition, hypoxia, and diseases: Problems solved?” Redox Biology, 6, 372-385 (2015)] highlight treatments that illustrate misunderstanding REDOX balance and the cellular signaling modulation methods reported herein.
Oxidation-induced cell damage is undesirable in both disease states and general health. In animals, examples of disease states and general health conditions include, but are not limited to, oxidative stress induced muscle fatigue, muscle strain, injury or damage to cells, macular degeneration, cataracts, sunburn, hair issues (graying, alopecia), inflammation, psoriasis, itch, fertility issues (sperm and egg life), wound healing, ischemia, sepsis, reperfusion injury, anxiety, aging, and the like. Human disease effects also include Alzheimer’s disease, ALS, Parkinson’s disease, and diabetes, In plants, conditions affected by oxidative stress include general growth rate and plant health, prevention of leaf or needle drop, damage from freezing, insect damage, physical injury (as occurs in turf grass), and post-harvest spoilage. Controlling oxidative stress will improve production, as well as mitigate post-harvest spoilage of crops as well as valuable products such as essential oils and plant components such as CBD from cannabis.
Due to their enzyme mimic, super-antioxidant, and free radical scavenging properties, several fullerene C60 formulations improve plant health and growth. One such formulation uses fullerene soot, the charcoal created by combustion of carbon to produce fullerenes. This fullerene soot has beneficial properties for both plants and animals
It is well known that charcoal itself is beneficial for human health. Currently charcoal is being studied for additional medical applications and is a widely available health supplement. Charcoal has also been used to improve plant health. We have discovered that charcoal fullerene soot used as a soil additive, absorbs toxins, improves carbon and mineral composition, and enhances soil nutrients, which all serve to improve fertility and productivity, leading to increased yields and improved plant health for most crops.
In connection with the administration of a composition or drug, a drug which is “effective against” a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease signs or symptoms, extension of life, improvement in quality of life, or other effects generally recognized as positive by medical professionals familiar with treating the particular type of disease or condition.
As used herein, the term “fullerene” in its unsubstituted form refers to a compound composed entirely of carbon in the form of a hollow sphere or spheroid. Each carbon atom is connected by one double bond and two single bonds to other carbon atoms. Spherical fullerenes generally have a mixture of pentagonal faces and hexagonal faces, non-limiting examples which include C60, C70, C76, C78, C84 more-or-less spherical fullerenes. Fullerenes can be substituted or unsubstituted, especially to control solubility. They retain their antioxidant capacity when substituted and are included in the disclosure. Tubular fullerenes also exist and are called carbon nanotubes. They are not included in this disclosure.
As used herein, the term “C3” refers to e,e,e-C60 fullerene tris-malonic acid (see
As used herein, the term “antioxidant” refers to a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from an oxidizing agent to another substance. Such reactions can be promoted by and/or produce superoxide anions or peroxides. Oxidation reactions can produce free radicals, which start chain reactions that can damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions by being oxidized themselves. As a result, these “oxidized antioxidants” usually need reducing agents such as thiols, ascorbic acid, or polyphenols to reduce them back to their starting state. Antioxidants include, but are not limited to, α-tocopherol (vitamin E), ascorbic acid (vitamin C), porphyrin, α-lipoic acid, and n-acetylcysteine.
As used herein, the term “effective amount” refers to the amount of an agent to produce the intended pharmacological, therapeutic, or preventive result. The pharmacologically effective amount results in the amelioration of one or more signs or symptoms of a disease or condition, or causes a halt in the progression or the regression of the disease or condition. For example, a therapeutically effective amount refers to the amount of a therapeutic agent that increases cell life or reduces cell damage from oxidants. The effective amount can be given in single or multiple daily doses (or less frequently) sufficient to cause the intended effect.
As used herein, the term “polar” refers to compositions which are water soluble. Such compounds and methods to make them are well known. In one embodiment, cyclodextrin (See
As used herein, “non-polar” refers to fullerenes that are not soluble in water. For use in cells or any living organism (animal or plant), fullerenes can also be dissolved in an oil. Products on the market that contain only C60 are normally dissolved in olive oil. It has been discovered that the amount of polyunsaturated fatty acids is critical in optimizing solubilization. In one embodiment, an edible oil is selected for dissolution of the fullerenes of the present invention. That oil has, at a minimum, 50% polyunsaturated fatty acids. Oils such as corn, cottonseed, flaxseed, hempseed, and soybean all meet this criteria. In one embodiment, the oil is hemp oil. Compositions are formulated with appropriate excipient additives and the like, as well as the edible oils, for administration.
New process chemistry for C3 preparation has taken the original process and changed and optimized the chemistry, reaction conditions, materials, and solvents to a workable and scalable manufacturing process. The critical step (and worst step in the old process) was the preparation of a macrocyclic linker. Major fundamental improvements were (1) to change from malonyl dichloride to malonic acid, (2) to change from methylene chloride to ethyl acetate as solvent, and (3) the initial reaction conditions were made 5 times (5×) more concentrated (leading to potential savings in solvent and processing costs). The final compound of the present invention is the compound called C3 (See
A 5 L, 3-necked R.B. flask with a mechanical stirrer, N2 flow adapter, and a glass stopper, were charged with 1, 8-octanediol (14.6 g, 0.10 mol), malonic acid (10.4 g, 0.10 mol), and EtOAc (3 L). The mixture was stirred for 0.5 hrs at room temperature until all the solids were dissolved. To this solution, dicyclohexylcarbodiimide (DCC, 41.3 g, 0.20 mol) and dimethylaminopyridine (DMAP, 3.67 g, 0.03 mol) were added and the resulting white suspension was stirred for 24 hrs at room temperature. The reaction mixture was concentrated to about one-half volume at reduced pressure, then filtered. The filter cake was washed with ethyl acetate (2 × 300 ml). The filtrate was concentrated under reduced pressure to a small volume. The residue was slurried in 1:1 toluene: hexanes (ca. 65 ml) and the resulting solids (mostly pure dimer) were filtered off and the filtrate was concentrated. The residue was purified by flash column chromatography (150 g, 5.5×15 cm), using a gradient elution starting with hexane (0.5 L), then 10% EtOAc/ hexane (1 L), then 20% EtOAc/ hexane (2.0 L). The product eluted with 20% EtOAc/ hexane. Yield 1.44 gm (6.7%). Reaction of C60 with macrocyclic trimer (cyclo-[3]-octylmalonate): Synthesis of the e,e,e- (C3-precursor) and trans-4, trans-4, trans-4- (C3V-precursor) trisadducts in a dry 3-necked flask equipped with gas inlet, 250 ml dropping funnel and magnetic stirrer, 255 mg (0.354 mmol / 1.0 eq.) C60 were dissolved under argon in 400 ml of dry toluene. Subsequently, 205 mg (0.319 mmol / 0.9 eq.) of the macrocycle and 243 mg (0.956 mmol / 2.7 eq.) of iodine were added to the solution. Then, a solution of 404 mg (397 µl / 2.65 mmol / 7.5 eq.) DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) in 160 ml of dry toluene was added dropwise over a period of 3 hours. The color of the solution turned to a deep orange. After additional stirring at room temperature for about 10 mins, the crude mixture was subjected to flash chromatography on silica gel (6 × 25 cm). Traces of C60 and other impurities were eluted with toluene, then the eluant was changed to toluene/ ethylacetate 98:2 and the trisadducts C3-precursor and C3V-precursor were eluted together as a bright orange band. C3-precursor and C3V-precursor have been separated by preparative HPLC on Nucleosil (toluene/ ethylacetate 98:2). The product fractions were evaporated, precipitated from CH2Cl2/ pentane, washed three times with pentane, and dried at 60° C. in high vacuum.
A formulation of a water soluble fullerene compound such as polyhydroxy-C60 or FC4S (
Formulation of C60 in Hemp oil (~ 1 g/liter) can be used as a skin cream for treatment of skin sores, abrasions, cuts, and even blemishes. One example uses the C60 skin treatment for preventing skin damage (peeling) after sunburn. Before and after results are shown in
Formulation of water soluble C60 compound polyhydroxy-C60 or FC4S (
Numerous health benefits result from all of these regimens and the formulations could also be employed for more serious conditions such as Parkinson’s, ALS, or Alzheimer’s.
Formulation of C60/C70 natural mixed fullerene or other fullerenes can be used in combination with cannabidiol (
Formulation of water soluble C60 compound polyhydroxy-C60 (
For plants, fullerene soot is a soil additive utilizing the combination of effects of carbon soot in soil composition and fullerene super-antioxidant activity that results in faster growing and stronger plants. A new discovery has been that there are positive plant enhancement effects of fullerenes present in the fullerene soot - the charcoal created by combustion of carbon in the manufacturing process for fullerenes. While charcoal itself is widely used in agriculture and is known to have a beneficial effect on soil fertility and productivity, we now reveal that “charcoal fullerene soot” combines the considerable benefits of charcoal as a soil nutrient with the super-antioxidant plant health effect of fullerene to result in an increase in yields for most crops. One typical formulation is fullerene soot charcoal containing 1-14% fullerenes with the remainder typical amorphous carbon. A soil enhancement treatment involves amendment of the top few inches of soil to ~5% charcoal. This amended layer absorbs toxins, improves available carbon nutrient content and both the soot and the fullerenes produce their redox balance effect on the growing plants.
Now referring to the drawings,
Those skilled in the art to which the present invention pertains may make modifications resulting in other embodiments employing principles of the present invention without departing from its spirit or characteristics, particularly upon considering the foregoing teachings. Accordingly, the described embodiments are to be considered in all respects only as illustrative, and not restrictive, and the scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description or drawings. Consequently, while the present invention has been described with reference to particular embodiments, other modifications of structure, sequence, materials, and the like, apparent to those skilled in the art, still fall within the scope of the invention as claimed by the applicant.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCTUS2060388 | Nov 2020 | WO | international |
This application is a continuation-in-part of U.S. Non-Provisional Application No. 16/685,729 filed on Nov. 15, 2019; this application also claims priority of a 371 international PCT application No. PCT/US20/60388, filed on Nov. 13, 2020; and which is incorporated herein in its entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16685729 | Nov 2019 | US |
| Child | 18066596 | US |
