Apoequorin and Vitamin D-containing compositions and methods for using them.

Abstract

The present invention provides novel and alternative therapeutic agents for reducing or preventing calcium imbalance, particularly pharmaceutical compositions or nutritional supplement compositions with reduced side effects compared to conventional drugs, as well as methods for using them. [Solution] A composition is provided for treating symptoms or disorders associated with at least one of calcium imbalance and vitamin D deficiency, comprising (A) an effective amount of apoequorin; (B) an effective amount of vitamin D; and (C) an acceptable carrier. Also provided is the use of apoequorin and vitamin D for the manufacture of a nutritional supplement composition for treating symptoms or disorders associated with at least one of calcium imbalance and vitamin D deficiency in subjects administered the nutritional supplement composition.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 398,669, filed on September 23, 2016, which is hereby incorporated by reference in its entirety for all purposes.

[0002] Statement Regarding Federally Sponsored Research Not applicable.

Background Art

[0003] The present invention generally relates to compositions useful for maintaining calcium homeostasis. In particular, the present invention relates to apo - equorin and vitamin D - containing compositions useful for preventing and / or alleviating diseases or conditions associated with calcium imbalance and vitamin D deficiency.

[0004] Calcium is the fifth most abundant element in the human body and is mainly present in bones. More than 99% of the calcium in the body is stored in the skeleton, which constantly exchanges its supply with the remaining 1% that has dissolved into soft tissues such as body fluids and blood. The control of this exchange is largely influenced by the endocrine system that senses the concentration of ionized calcium in plasma and directs calcium exchange to maintain this important balance. Only a very small part of the 1% of calcium in interstitial fluid and soft tissues is ionized and soluble. The remaining calcium in body fluids and tissues is bound to proteins, especially calcium - binding proteins (CaBP). CaBP is known to function in maintaining calcium homeostasis.

[0005] Maintaining calcium homeostasis is crucial for bodily health because the body requires specific concentrations of calcium ions to perform necessary physiological processes. Appropriate ionic calcium concentrations in plasma and body fluids are understood in the medical community to be important for bodily functions such as nerve excitability, muscle contraction, membrane permeability, cell division, hormone secretion, and bone mineralization, but are not limited to these. Disruption of calcium homeostasis, i.e., calcium imbalance, is associated with many diseases, syndromes, and conditions, including but not limited to cancer, heart disease, and neurodegenerative diseases.

[0006] Calcium channel blockers, which block the flow of calcium between cells and interstitial fluid, have been widely prescribed as useful medications for preventing calcium-related disorders, including hypertension, angina, asthma, migraines, and neurodegeneration. For example, nimidopine has been found to improve clinical symptoms and cognitive function in dementia by reducing calcium imbalances that cause nerve deterioration. However, many of these calcium channel blockers, though not limited to these, have undesirable side effects such as fatigue, fluid retention, heartburn, unstable heart rate, dizziness, nausea, and, rarely, syncope, fever, and excessive bleeding.

[0007] Despite these advances, there is still a need for novel and alternative therapies to alleviate or prevent calcium imbalance. In particular, there is a desire for pharmaceutical or nutritional supplement compositions with reduced side effects compared to conventional drugs, and the discovery of such compositions would fulfill a long-awaited need in the medical and nutritional health communities. [Overview of the project]

[0008] This invention relates to the alleviation of symptoms or disorders associated with calcium imbalance and vitamin D deficiency. The present invention provides a composition advantageous for reduction and / or prevention. Such a composition comprises apoequorin and vitamin D, combined with a carrier acceptable for administration to the subject via various routes.

[0009] Accordingly, the present invention relates to compositions comprising an effective amount of apoequorin and vitamin D combined with a tolerable carrier. In certain embodiments, the present invention relates to a nutritional supplement composition comprising an effective amount of apoequorin and vitamin D combined with a tolerable carrier. In certain embodiments, the nutritional supplement composition comprises, in addition to apoequorin and vitamin D, at least one other component recognized to provide nutritional benefits, such as an immunostimulant, anti-inflammatory agent, antioxidant, antiviral agent, or a mixture thereof. The apoequorin and vitamin D composition in certain embodiments is provided in a single dose selected from tablets, capsules, liquids, suspensions, syrups, beverages, oral or ophthalmic formulations, or injections.

[0010] In another embodiment, the present invention relates to a method for treating symptoms or disorders associated with calcium imbalance and vitamin D deficiency, comprising administering an effective amount of apoequorin and vitamin D to a subject in need of such treatment.

[0011] The methods according to the present invention, though not limited to these, are useful for treating a wide variety of symptoms or disorders associated with calcium imbalance and vitamin D deficiency, such as sleep quality, energy quality, mood quality, pain, and memory quality. In certain embodiments, calcium imbalance and vitamin D deficiency are physiologically related to nerve excitability, muscle contraction, membrane permeability, cell division, hormone secretion, bone mineralization, or post-ischemic cell death. In such methods, apoequorin and vitamin D are preferably administered to the subject in the form of a nutritional supplement composition.

[0012] In yet another embodiment, the present invention includes the use of apoequorin and vitamin D for the manufacture of a nutritional supplement composition for treating symptoms or disorders associated with calcium imbalance and vitamin D deficiency in subjects administered the nutritional supplement composition. Examples of symptoms or disorders treated by such compositions include symptoms or disorders related to sleep, energy, mood, pain, or memory.

[0013] Accordingly, the present invention further relates to apoequorin and vitamin D for use in treating symptoms or disorders associated with calcium imbalance and vitamin D deficiency in a subject, including, for example, symptoms or disorders related to sleep, energy, mood, pain, or memory in the subject.

[0014] The present invention offers various advantages over conventional compositions and methods in that it brings about an overall improvement in the mental and physical health of the subject.

[0015] Other objects, features, and advantages of the present invention will become apparent after reviewing the specification and claims. [Brief explanation of the drawing]

[0016] [Figure 1] Figure 1 is a graph showing the percentage change from baseline in scores across the areas of overall sleep quality, energy, mood, pain, and general health, from day 0 to day 90. [Figure 2] Figure 2 shows a graph representing data from 56 participants who took apoequorin (10 mg) daily. Participants were evaluated from day 8 to day 30. Memory assessments showed statistically significant improvements in memory after 30 days (hp<0.05). 57% of participants showed improvement in overall memory, 51% in information retention, 84% in remembering directions, and 66% in word recall. N=56; 66% female, 34% male, mean age=56 years; range 20-78 years. [Figure 3] Figure 3 is a graph showing the percentage change from baseline in scores obtained from a standardized cognitive battery questionnaire, from day 0 to day 90. [Figure 4] Figure 4 shows neuroprotection induced by AQ (apoequorin) and vitamin D in rat hippocampal brain sections. [Modes for carrying out the invention]

[0017] I. General theory Before describing the materials and methods of the present invention, it should be understood that the present invention is not limited to specific methodologies and materials described, as these may change. Furthermore, the terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0018] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Similarly, the terms “a” (or “an”), “one or more,” and “at least one” are interchangeable herein. Also, the terms “comprising,” “including,” and “having” are interchangeable.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the invention, but preferred methods and materials are described herein. All publications and patents specifically described herein are incorporated by reference for all purposes.

[0020] II. The present invention Aequorin is a photoprotein originally isolated from bioluminescent jellyfish and other marine organisms. The aequorin complex contains 22,285 daltons of apoequorin protein, molecular oxygen, and the photophosphorus coelenterazine. 2 When ions bind to this complex, coelenterazine is oxidized to selenthermide, simultaneously releasing carbon dioxide and blue light. Aequorin is not excreted or secreted from cells, nor is it compartmentalized or sequestered within cells. Therefore, aequorin measurements occur over relatively long periods of time.2 It has been used to detect changes. In some experimental systems, the luminescence of aequorin was detectable from several hours to several days after cell loading. Furthermore, aequorin is known not to disrupt cell function or embryo development.

[0021] The aequorin complex has been widely used as an intracellular Ca 2 indicator due to its Ca 2 -dependent luminescence. Aequorea victoria aequorin from jellyfish has been used specifically for the following purposes: (1) Analyzing the secretory response of single adrenal chromaffin cells to nicotinic cholinergic agonists. (2) Clarifying the role of Ca 2 release in myocardial injury. (3) Demonstrating the massive release of Ca 2 during fertilization. (4) Studying the regulation of sarcoplasmic reticulum Ca 2 pump expression in developing chick myoblasts. (5) Calibrating a micropipette with an injection volume of only 3 picoliters.

[0022] Apoaequorin has a molecular weight of approximately 22 kDa. Apoaequorin can be used to regenerate aequorin by reducing the disulfide bonds in apoaequorin. Calcium-loaded apoaequorin retains the same compact scaffold and overall folding pattern as the unreacted luminescent protein containing the bound substrate.

[0023] Conventional purification of apoaequorin from jellyfish Aequorea victoria requires cumbersome extraction procedures and sometimes yields preparations that are substantially heterogeneous or toxic to the organism under study. Typically, about 125 mg of purified luminescent protein is obtained from 2 tons of jellyfish. In contrast, recombinant apoaequorin is preferably produced by purifying apoaequorin from genetically engineered Esclierichia coli and subsequently reconstituting the apoaequorin complex with pure coelenterazine in vitro. Apoaequorin useful in this invention is described and commercially available by purification schemes and / or synthesis known to those skilled in the art. S. Inouye, S. Zenno, Y. Sakaki, and F. Tsuji. High level expression and Purification of apoaequorin. (1991) Protein Expression and Purification 2, 122-126.

[0024] Vitamin D is a group of fat-soluble secosteroids that increase the intestinal absorption of calcium, iron, magnesium, phosphates, and zinc. Vitamin D is produced in the body in response to the skin being exposed to ultraviolet light from sunlight. It is also found in naturally occurring foods such as fish, fish liver oil, and egg yolks, as well as in fortified dairy and grain products. In nutritional supplements, the two most common forms of vitamin D compounds are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol).

[0025] Vitamin D is a fat-soluble vitamin that is biologically inactive and must undergo two hydroxylations in the body to become active. The first occurs in the liver, converting vitamin D to 25-hydroxyvitamin D[25(OH)D], also known as calcifediol. The second occurs mainly in the kidneys, forming the bioactive 1,25-dihydroxyvitamin D[1,25(OH)2D], also known as calcitriol. The active form of vitamin D, calcitriol, circulates in the blood as a hormone, regulating the concentrations of calcium and phosphate in the bloodstream and promoting healthy bone growth and remodeling.

[0026] Vitamin D promotes calcium absorption and enables normal bone mineralization, maintaining sufficient serum calcium and phosphate levels to prevent hypocalcemia tetany. It is also used in bone growth and bone remodeling by osteoblasts and osteoclasts. It has also been shown to play a role in cell proliferation, regulation of neuromuscular and immune function, and reduction of inflammation.

[0027] This invention relates to the administration of apoequorin and vitamin D-containing compositions to a subject for correcting or maintaining calcium balance and vitamin D levels in that subject. Vitamin D deficiency can contribute to calcium imbalance. Maintaining ionic calcium concentrations in plasma and body fluids is understood to be important for a wide variety of bodily functions, including but not limited to nerve excitability, muscle contraction, membrane permeability, cell division, hormone secretion, bone mineralization, or prevention of post-ischemia cell death. Disruption of calcium homeostasis, i.e., calcium imbalance, is understood to cause and / or correlate with many diseases, syndromes, and conditions. Such diseases, syndromes, and conditions include those related to sleep quality, energy quality, mood quality, as well as memory quality and pain perception. Studies of CaBP have led to their recognition as protective factors acting to maintain appropriate ionic calcium levels.

[0028] Maintaining vitamin D levels is understood to be important for calcium absorption, regulation of cell proliferation, neuromuscular and immune function, and reduction of inflammation. Vitamin D deficiency is most closely associated with rickets, a disease in which bone tissue does not calcify properly, leading to cartilage and skeletal deformities. The Institute of Medicine guidelines stipulate that the recommended dietary tolerance (RDA) for vitamin D to optimize bone health is 600 international units (IU) for adults aged 1–70 years and 800 IU for adults aged 70 years and older.

[0029] In certain embodiments, the present invention involves administering apoequorin in combination with vitamin D for the purpose of treating calcium imbalance, slowing the progression of calcium imbalance, preventing the onset of calcium imbalance, preventing and / or treating the recurrence of calcium imbalance, and treating vitamin D deficiency. In other embodiments, the present invention provides a method comprising administering apoequorin and vitamin D in combination with one or more additional agents having known therapeutic or nutritional value. Particularly preferred uses of apoequorin and vitamin D are to treat one or more symptoms and disorders related to sleep quality, energy, mood, memory, and pain perception.

[0030] In this specification, the term “treating” includes preventive treatment and treatment of relapse of the disorder. Where used herein, the terms “reducing,” “alleviating,” “suppressing,” and “inhibiting” have their commonly understood meanings of reducing or decreasing. In this specification, the term “progression” means an increase in scope or severity, progression, growth, or worsening. In this specification, the term “recurrence” means a relapse of the disease after remission.

[0031] In this specification, the term “administering” refers to bringing a patient, tissue, organ, or cell into contact with apoequorin and vitamin D. In this specification, administration may be achieved in vitro, i.e., in a test tube, or in vivo, i.e., in the cells or tissues of an organism such as a human. In a preferred form, the present invention encompasses administering a composition useful in the present invention to a patient or subject. As used herein interchangeably, “patient” or “subject” refers to a mammal, preferably a human, that is either: (1) having a calcium imbalance-related disorder and / or vitamin D deficiency that is treatable or can be treated by administration of apoequorin and vitamin D; or (2) susceptible to a calcium imbalance-related disorder and / or vitamin D deficiency that is preventable by administration of apoequorin and vitamin D.

[0032] In this specification, the terms “effective dose” and “therapeutic effective dose” refer to the amount of an active agent sufficient to produce a desired therapeutic response without excessive adverse side effects such as toxicity, irritation, or allergic reactions. The specific “effective dose” will obviously vary depending on factors such as the particular condition being treated, the patient’s physical condition, the type of animal being treated, the duration of treatment, the nature of any concomitant therapy, and the structure of any particular formulations and compounds or derivatives used. In this case, a dose will be considered therapeutically effective if it produces one or more of the following: (1) prevention of calcium imbalance-related disorders and / or vitamin D deficiency; (2) recovery from or stabilization of calcium imbalance-related disorders and / or vitamin D deficiency. The optimal effective dose can be readily determined by those skilled in the art using routine experiments.

[0033] In a specific composition for oral administration to a subject, apoequorin is formulated with at least one acceptable carrier in a dose of approximately 10–50 mg / dose, preferably in capsule form, with a recommended dose of approximately 20 mg / day for the subject. In a specific preferred composition for oral administration to a subject, vitamin D (in the form of D3 cholecalciferol) is formulated in combination with apoequorin in a dose of approximately 25–75 mcg / dose, with a recommended dose of approximately 50 mcg / day for the subject.

[0034] The compositions of the present invention include liquid or freeze-dried or otherwise dried formulations, and include various buffering components (tris-HCl, acetate, phosphate, etc.), pH and ionic strength diluents, additives such as albumin and gelatin to prevent adsorption to surfaces, detergents (Tween 20, Tween 80, Pluronic F68, bile salts, etc.), solubilizers (glycerol, polyethyleneglycerol, etc.), antioxidants (ascorbic acid, sodium metabisulfite, etc.), preservatives (thimerosal, benzyl alcohol, parabens, etc.), bulk materials or osmotic pressure modifiers (lactose, mannitol, etc.), covalent bonding of polymers such as polyethylene glycol with proteins, complexes with metal ions, or the incorporation of materials onto the surface or interior of granular preparations of polymer compounds such as polylactic acid, polyglycolic acid, or hydrogels, or onto liposomes, microemulsions, micelles, layered or multilayer vesicles, erythrocyte ghosts, or spheroplasts. Such compositions may affect physical properties, solubility, stability, in vivo release rate, and in vivo clearance rate. Controlled-release compositions or sustained-release compositions include formulations in lipophilic depot agents (fatty acids, waxes, oils, etc.).

[0035] Methods of administering particulate compositions coated with polymers (such as poloxamer or poloxamine) are also included in the present invention. Other embodiments of the compositions include protective coatings for particulate forms, protease inhibitors, or permeabilis enhancers for various administration routes, including parenteral, pulmonary, nasal, and oral. In certain embodiments, the compositions are administered parenterally, paracancerally, transmucosally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, or intratumorally.

[0036] Furthermore, in this specification, “pharmaceutically acceptable carriers” are well known to those skilled in the art and are not limited to these, but include 0.0141 M, preferably 0.05 M, phosphate buffer or 0.9% saline solution. Further examples of such pharmaceutically acceptable carriers include aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Examples of aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions (including saline solution and buffering media).

[0037] Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's solution, and non-volatile oils. Intravenous vehicles include liquids and nutritional supplements, and electrolyte supplements such as those based on ringer's dextrose. Furthermore, they may contain preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases.

[0038] The controlled-release or sustained-release compositions administered according to the present invention include formulations in lipophilic depot agents (fatty acids, waxes, oils, etc.). Furthermore, the present invention includes particulate compositions coated with polymers (poloxamer or poloxamine, etc.), and compounds that are bound to antibodies against tissue-specific receptors, ligands, or antigens, or bound to ligands of tissue-specific receptors.

[0039] Other embodiments of compositions administered according to the present invention include particle forms, protective coatings, protease inhibitors, or permeabilis enhancers for various routes of administration, including parenteral, pulmonary, nasal, intraocular, and oral.

[0040] Chemicals modified by covalent bonding of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, or polyproline are known to exhibit a substantial extension of the blood half-life after intravenous injection compared to the corresponding unmodified compounds. Such modifications can also increase the solubility of the chemical in aqueous solutions, prevent aggregation, enhance the physical and chemical stability of the compound, and significantly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity can be achieved by administering such polymer-substance abducts at lower frequencies or doses than with unmodified substances.

[0041] In yet another method of the present invention, the composition can be delivered by a controlled-release system. For example, the drug can be administered by intravenous infusion, implantable osmotic pump, transdermal patch, liposome, or other mode of administration. In one embodiment, a pump can be used. In another embodiment, a polymer material can be used. In yet another embodiment, the controlled-release system can be placed in close proximity to the therapeutic target, i.e., the brain, in which case only a fraction of the systemic dose is required.

[0042] The composition may contain apoequorin and vitamin D, or further contain a pharmaceutically acceptable carrier, and may be in solid or liquid form such as tablets, powders, capsules, pellets, solutions, suspensions, elixirs, syrups, beverages, emulsions, gels, creams, ophthalmic preparations, or suppositories, including rectal and urethral suppositories. Pharmacologically acceptable carriers also include gums, starches, sugars, cellulosic materials, and mixtures thereof. The composition containing apoequorin and vitamin D can be administered to a patient, for example, by subcutaneous implantation of pellets. In a further embodiment, the pellets provide sustained release of apoequorin and / or vitamin D over a period of time. The composition can also be administered by intravenous, intra-arterial, or intramuscular liquid injection, oral administration of liquid or solid, or topical application. Administration can also be achieved by the use of rectal or urethral suppositories.

[0043] The compositions that can be administered according to the present invention can be prepared by known methods of dissolution, mixing, granulation, or tablet formation. For oral administration, physiologically acceptable derivatives such as apoequorin or its salts, esters, N-oxides, and / or physiologically acceptable derivatives such as vitamin D or its salts, esters, N-oxides are mixed for these purposes with conventional additives such as vehicles, stabilizers, or inert diluents, and converted by conventional methods into administration-suitable forms such as tablets, coated tablets, hard or soft gelatin capsules, or aqueous, alcoholic, or oily solutions.

[0044] Examples of preferred inert vehicles include conventional tablet bases such as lactose, sucrose, or corn starch, combined with binders such as acacia, corn starch, or gelatin, disintegrants such as corn starch, potato starch, or alginic acid, or lubricants such as stearic acid or magnesium stearate.

[0045] Suitable oily vehicles or solvents include vegetable oils such as sunflower oil or fish liver oil, or animal oils. The composition can be prepared in either dry or wet granule form. For parenteral administration (subcutaneous, intravenous, intra-arterial, or intramuscular injection), the chemical substance or its physiologically acceptable derivatives, such as salts, esters, or N-oxides, can be converted to solutions, suspensions, or expulsions using, if desired, substances commonly and appropriately used for this purpose, such as solubilizers or other aids.

[0046] Examples include sterile liquids such as water and oil, with or without surfactants and other pharmaceutically acceptable excipients. Examples of oils include petroleum, animal, plant, or synthetic oils, specifically, peanut oil, soybean oil, or mineral oil. Generally, water, saline solution, aqueous dextrose and related sugar solutions, and glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions.

[0047] The preparation of compositions containing active ingredients is well understood in the art. Such compositions are administered by injection as an aerosol delivered to the nasopharynx, or as a liquid solution or suspension. The composition may be prepared as an agent; however, a solid form suitable for dissolving or suspending in liquid before injection may also be prepared. The composition may also be emulsified. The active therapeutic component is often mixed with excipients that are pharmaceutically acceptable and compatible with the active component. Suitable excipients include, for example, water, saline solution, dextrose, glycerol, ethanol, or any combination thereof. Furthermore, the composition may contain small amounts of auxiliary substances such as wetting agents or emulsifiers, or pH buffers that enhance the efficacy of the active component.

[0048] The active ingredient can be incorporated into the composition in the form of a neutralized, pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, tartaric acid, or mandelic acid. Salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, as well as organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0049] For example, for topical administration to the body surface using creams, gels, drops, etc., apoequorin or its physiologically acceptable derivatives and / or vitamin D or its physiologically acceptable derivatives are prepared and applied as solutions, suspensions, or emulsions in physiologically acceptable diluents, with or without a pharmaceutical carrier.

[0050] In another method of the present invention, the active ingredient can be delivered as vesicles, particularly liposomes (see Langer, Science 249:1527-1533 (1990); Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, NY, pp.353-365 (1989)).

[0051] Salts of apoequorin and / or vitamin D are preferably pharmaceutically acceptable salts. However, other salts can also be used in the preparation of compositions according to the present invention or their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts include, for example, acid addition salts that can be formed by mixing a solution of apoequorin and / or vitamin D with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid.

[0052] Furthermore, the apoequorin and vitamin D-containing compositions described herein may be provided in the form of dietary supplement compositions in which apoequorin and vitamin D prevent, mitigate, or stabilize the development of various harmful calcium imbalance-related disorders and vitamin D deficiencies. For the purposes of this specification, the terms “dietary supplement” or “dietary supplement composition” refer to a food or part of a food that provides medical health benefits, including the prevention and / or treatment of disease. The dietary supplement compositions according to the present invention may contain only apoequorin and vitamin D as active ingredients, or they may further contain nutritional supplements that supplement the diet by increasing the total intake of vitamins, coenzymes, minerals, herbs, amino acids, etc.

[0053] Accordingly, the present invention provides a method for providing a nutritional supplement to a patient, comprising the step of administering to the patient a nutritional supplement composition containing apoequorin and vitamin D. Such a composition generally comprises a “nutritionally acceptable carrier,” which is any carrier suitable for oral delivery, including the aforementioned pharmaceutically acceptable carriers suitable for the oral route, as referred to herein. In certain embodiments, the nutritional supplement composition according to the present invention comprises a nutritional supplement comprising a functionally defined immunostimulant, anti-inflammatory agent, antioxidant, antiviral agent, or a mixture thereof.

[0054] Immunostimulants and / or antivirals are useful for promoting wound healing and improving immune function. These include extracts from cornflower or Echinacea herbs, Sambuca herbs, and goldenseal extract. Astragalus herbs, both in their natural and processed forms, are also effective immunostimulants. Astragalus stimulates the development of stem cells in bone marrow and lymphoid tissue-active immune cells. Zinc, and bioactive salts of zinc such as zinc gluconate and zinc acetate, also act as immunostimulants in the treatment of the common cold.

[0055] Antioxidants include allicin, a naturally occurring sulfur-containing amino acid that works to increase the levels of antioxidant enzymes in the blood. Herbs and herbal extracts containing allicin, such as garlic, are also effective antioxidants. Catechins, and extracts of herbs such as green tea that contain catechins, are also effective antioxidants. Extracts of the Astragalus genus also exhibit antioxidant activity. Bioflavonoids such as quercetin, hesperidin, rutin, and mixtures thereof are also effective antioxidants. The main beneficial role of bioflavonoids is to protect vitamin C from oxidation in the body. This makes more vitamin C, or ascorbic acid, available for use in the body.

[0056] Bioflavonoids such as quercetin are also effective anti-inflammatory agents and can be used in the compositions of the present invention. Anti-inflammatory herbal supplements and anti-inflammatory compounds derived from plants or herbs can also be used as anti-inflammatory agents in the compositions of the present invention. These include bromelain, a proteolytic enzyme found in pineapple; nettle tea and extracts; turmeric, turmeric extracts, or curcumin, a yellow pigment isolated from turmeric.

[0057] Another supplement that can be used in this invention is ginger, derived from herbs of the genus Zingiber. It has been found to have cardiotonic effects due to compounds such as gingerol and related compounds such as shogaol, and to be effective in treating dizziness and vestibular disorders. Ginger is also effective in treating nausea and other stomach ailments.

[0058] Supplements that aid in the reconstruction of soft tissue structures, particularly cartilage, are useful in compositions for treating pain associated with arthritis and other joint disorders. Glucosamine, glucosamine sulfate, and chondroitin can be obtained from various sources, such as elk velvet antlers. Marine lipid complexes, omega-3 fatty acid complexes, and fish oil are also known to be useful in treating pain associated with arthritis.

[0059] Supplements useful for treating migraines include feverfew and ginkgo biloba. The main active ingredient in feverfew is sesquiterpene lactone parthenolide, which inhibits the secretion of prostaglandins that cause pain by constricting blood vessels. Feverfew also exhibits anti-inflammatory effects. Fish oil may also be useful in treating migraines due to its platelet-stabilizing and anti-sedative effects. The herb ginkgo also promotes the treatment of migraines by stabilizing arteries and improving blood circulation.

[0060] While some of the supplements listed above have been described in terms of their pharmacological effects, other supplements can also be used in this invention, and their effects are well documented in the scientific literature.

[0061] This invention takes into consideration the following non-limiting examples which describe and disclose chemical substances, instruments, statistical analyses, and methodologies reported in publications that may be used in connection with this invention. It should be understood more fully. All references cited herein should be construed as representing the state of the art for those skilled in the art. Nothing herein should be construed as an acknowledgment that the present invention has no prior rights to such disclosures for the sake of prior art. [Examples]

[0062] [Example 1] Administration of apoequolin over 90 days improves the quality of life of the subjects in the study. This open-label analysis of 32 patients over 90 days shows improvements in overall sleep quality, energy, mood, pain, and overall health. Performance changes were measured via a standardized battery, which included qualitative cognitive tests, sleep index, headache index, and quality of life questionnaire assessments. The study demonstrated improved performance. No participants discontinued the study due to adverse events.

[0063] The results shown in Figure 1 represent the percentage change from baseline in scores in the areas mentioned; memory scores were excluded for the sake of other graphs. The analysis here is marked on the graph as 1, 2, 3, 4, and 5 for days 0–90. The graphs show improvements in overall sleep quality, energy, mood, pain, and overall health. Baseline was known from the pre-test phase.

[0064] [Example 2] Administration of apoequolin over 30 days improves the quality of life of the subjects in the study. This study was an open-label trial involving 56 participants over 30 days. Changes in performance were measured using a memory screening tool. As shown in Figure 2, the study showed improvement in memory performance as early as day 8, but demonstrated statistically significant improvement by day 30. No participants discontinued the study due to adverse events.

[0065] [Example 3] Administration of apoequolin over 90 days improves cognition in test subjects. This analysis of an open-label trial involving 32 patients demonstrates an increase in cognitive ability. Performance changes were measured via a standardized cognitive battery. The study showed cognitive improvement as early as day 8, but statistically better improvement was observed at day 30, as well as at days 60–90. No participants discontinued the study due to adverse events. The results shown in Figure 3 demonstrate a significant percentage increase in scores from baseline in cognitive ability. Note: More than 51% of participants showed an increase in cognitive ability.

[0066] [Example 4] Administration of a composition containing apoequorin and vitamin D. The apoequorin and vitamin D-containing composition is administered to patients in capsule form containing a mixture of apoequorin and vitamin D3. The composition contains 50 mcg of vitamin D3 (in the form of D3 cholecalciferol) and 20 mg of apoequorin. The dietary supplement composition is carried in a vegetable capsule (vegetable cellulose, water) that is acceptable as a dietary supplement. The composition further contains microcrystalline cellulose, sugar, and small amounts of acacia (gum arabic), casein peptone, corn starch, lactose, magnesium stearate (vegetable source), medium-chain triglycerides (vegetable oil), salt, soy peptone, DL-α-tocopherol, tricalcium phosphate, and water. One capsule per day is the recommended dose. Such a dose contains approximately 250% of the recommended daily dose of vitamin D. Vitamin D suitable for use in this invention is available from BASF and is marketed as "Dry Vitamin D3 100 GFP / HP". Furthermore, single doses may be formulated to contain other amounts of apoequorin, including single doses containing 10 mg or 40 mg of apoequorin.

[0067] [Example 5] Evaluation of neuroprotection by AQ (apoequorin) and vitamin D in rat hippocampal brain slices. Preliminary results from a subset of experimental data show significant cell death after 5 minutes of oxygen-glucose deprivation (OGD, Figure 4; top left image) and minimal cell death in slices that did not receive 5 minutes of OGD (Figure 4; top right image). Direct injection of AQ into the hippocampus reduces dead and dying cells (i.e., cells stained blue). Rats fed a diet supplemented with vitamin D (0.0125 mg / kg) for approximately 10 days also appear to have fewer dead and dying cells. The combination of AQ and vitamin D can further reduce the amount of cell death (Figure 4; bottom left image). Control slices (those not subjected to OGD) appear to be less altered from the function of the treated state.

[0068] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes taking them into account should be suggested to those skilled in the art and should be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. A composition for treating symptoms or disorders associated with at least one of calcium imbalance and vitamin D deficiency, comprising (A) an effective amount of apoequorin; (B) an effective amount of vitamin D; and (C) an acceptable carrier.

2. The composition according to claim 1, wherein the composition is in the form of a single dose containing the effective amount of apoequorin and vitamin D.

3. The composition according to claim 2, wherein (a) the effective amount of apoequolin in a single dose is about 10 mg to about 50 mg, and (b) the effective amount of vitamin D in a single dose is about 25 mcg to about 75 mcg.

4. The composition according to claim 3, wherein (a) the effective amount of apoequolin in a single dose is about 20 mg, and (b) the effective amount of vitamin D in a single dose is about 50 mcg.

5. The composition according to any one of claims 1 to 4, wherein vitamin D is in the form of D3 cholecalciferol.

6. The composition according to any one of claims 1 to 5, wherein the single dose is in the form of a capsule.

7. The composition according to any one of claims 1 to 6, wherein the composition is a nutritional supplement composition.

8. A method for treating symptoms or disorders associated with at least one of calcium imbalance and vitamin D deficiency, comprising administering an effective amount of the composition according to any one of claims 1 to 7 to a subject in need of such treatment.

9. The method according to claim 8, wherein the symptoms or disorder are related to sleep, and administration of the composition to the subject improves the quality of sleep in the subject.

10. The method according to claim 8, wherein the symptoms or disorder are related to vitality, and administration of the composition to the subject improves the quality of vitality in the subject.

11. The method according to claim 8, wherein the symptoms or disorder are related to mood, and administration of the composition to the subject improves the mood quality in the subject.

12. The method according to claim 8, wherein the symptoms or disorder are related to pain, and administration of the composition to the subject reduces pain in the subject.

13. The method according to claim 8, wherein the symptoms or impairment are related to memory, and administration of the composition to the subject improves the quality of memory in the subject.

14. The method according to claim 8, wherein the symptoms or disorders are related to nerve excitability, muscle contraction, membrane permeability, cell division, hormone secretion, bone mineralization, or post-ischemic cell death.

15. Use of apoequorin and vitamin D for the manufacture of a nutritional supplement composition for treating symptoms or disorders associated with at least one of calcium imbalance and vitamin D deficiency in subjects administered the nutritional supplement composition.

16. The use of apoequorin and vitamin D for the manufacture of a nutritional supplement composition for treating symptoms or disorders related to sleep, energy, mood, pain, or memory in a subject administered the aforementioned nutritional supplement composition.

17. Apoequorin and vitamin D for use in the treatment of symptoms or disorders associated with at least one of calcium imbalance and vitamin D deficiency in a subject.

18. Apoequorin and vitamin D for use in treating symptoms or disorders related to sleep, energy, mood, pain, or memory in subjects administered the aforementioned nutritional supplement composition.

Images