HK1238166A1 - Apoaequorin-containing compositions and methods of using same - Google Patents

Description

Apoaequorin-containing compositions and methods of use thereof

The patent application of the invention is a divisional application of an invention patent application with the international application number of PCT/US2009/036767, the international application date of 2009, 3 month and 11 day, the application number of 200980108891.X entering the Chinese national stage and the name of apoaequorin-containing composition and a using method thereof.

Cross Reference to Related Applications

This application claims priority to U.S. provisional application 61/035,443 filed on 11/3/2008, which is incorporated by reference herein in its entirety for all purposes.

Statement regarding federally sponsored research or development

Not applicable.

Technical Field

The present invention relates generally to compositions useful for maintaining calcium homeostasis. In particular, the present invention relates to apoaequorin (apoaequorin) -containing compositions useful for preventing and/or alleviating diseases or symptoms associated with calcium imbalance.

Background

Calcium is the fifth most abundant element in the human body, mainly present in bones. More than 99% of the calcium in the body is stored in bone, the supply of which is constantly exchanged for 1% of the calcium remaining dissolved in body fluids and soft tissues, such as blood. This exchange is controlled in large part by the endocrine system which senses the concentration of ionized calcium in the plasma and directs calcium exchange to maintain this important balance. Only a small fraction of the 1% calcium in interstitial fluid and soft tissues is ionized and dissolved. Calcium retained in body fluids and tissues is bound to proteins, particularly calcium binding proteins (CaBP). CaBP is known to play a role in maintaining calcium homeostasis.

Since the body requires certain concentrations of calcium ions to perform the necessary physiological processes, maintaining calcium homeostasis is critical to body health. The medical community understands that appropriate ionized calcium concentrations in plasma and body fluids are critical to bodily functions including, but not limited to, neuronal excitability, muscle contraction, membrane permeability, cell division, hormone secretion, and bone mineralization. Disruption of calcium homeostasis, i.e., calcium imbalance, is associated with a number of diseases, syndromes and conditions, including but not limited to: cancer, heart disease and neurodegenerative diseases.

In the past, calcium channel antagonists that block calcium flow between the interior of cells and interstitial fluid have been prescribed drugs widely used to prevent calcium-related disorders, including hypertension, angina, asthma, migraine and neurodegeneration. For example, nimodipine has been found to improve clinical symptoms and cognitive function of dementia by alleviating calcium imbalance leading to neurodegeneration. However, many such calcium channel antagonists have adverse side effects including, but not limited to, discomfort, fluid retention, heartburn, heart rate instability (erratic heart rate), dizziness, regurgitation and rare fainting, fever, and excessive bleeding.

Despite these advances, there remains a need for new and alternative therapeutic agents that alleviate or prevent calcium disorders. In particular, there is a need for pharmaceutical or nutritional compositions with reduced side effects compared to existing agents, and if such pharmaceutical or nutritional compositions are found, they should meet the needs for long-term use in the medical and nutritional health community.

Summary of The Invention

The present invention provides compositions that are useful for alleviating and/or preventing symptoms or conditions associated with calcium imbalance. Such compositions comprise apoaequorin in combination with an acceptable carrier for administration to a subject via various routes.

Thus, the present invention relates to compositions comprising an effective amount of apoaequorin in combination with an acceptable carrier. In certain embodiments, the present invention relates to nutraceutical compositions comprising an effective amount of apoaequorin in combination with an acceptable carrier. In certain embodiments, the nutraceutical compositions comprise, in addition to apoaequorin, at least one other component recognized to provide a nutritional benefit, such as an immune potentiator, an anti-inflammatory agent, an antioxidant, an antiviral agent, or mixtures thereof. In certain embodiments, the apoaequorin composition is provided in a unit dosage form selected from the group consisting of a tablet, a capsule, a solution, a suspension, a syrup, a beverage, an oral or ophthalmic formulation, or an injection.

In another aspect, the invention relates to a method of treating a symptom or condition associated with calcium imbalance comprising administering to a subject in need of such treatment an effective amount of apoaequorin.

The methods of the invention may be used in methods of treating various symptoms or conditions associated with calcium imbalance, including but not limited to, quality of sleep, quality of energy (energy quality), quality of mood, pain, quality of memory. In certain embodiments, the calcium imbalance is associated with cell death following ischemia, neuronal excitability, muscle contraction, membrane permeability, cell division, hormone secretion, bone mineralization. In such methods, the apoaequorin is preferably administered to the subject in the form of a nutraceutical composition.

In yet another embodiment, the present invention includes the use of apoaequorin for the preparation of a nutraceutical composition for treating a symptom or condition associated with calcium imbalance in a subject administered the nutraceutical composition. Exemplary symptoms or conditions that can be treated by such compositions include those associated with sleep, energy, mood, pain, or memory.

Thus, the present invention also includes the use of apoaequorin to treat symptoms or conditions associated with calcium imbalance in a subject, including those symptoms or conditions associated with, for example, sleep, energy, mood, pain, or memory in a subject.

Various advantages of the present invention over existing compositions and methods are that it improves the mental and physical health of the subject as a whole.

Other objects, features and advantages of the present invention will become apparent after review of the specification and claims.

Brief Description of Drawings

Figure 1 shows the percent change from baseline in the following regional scores from day 0 to day 90: sleep, energy, mood, pain, and overall quality of overall health.

FIG. 2 shows data for the daily administration of apoaequorin (10mg) by 56 participants. Participants were evaluated from day 8 to day 30. Memory studies showed a statistically significant improvement in memory after 30 days (hp <. 05). Improvements in overall memory of 57% participants, retention information of 51%, memory driven direction of 84% and word memory of 66%. N56; 66% female, 34% male, average age 56 years; the range is 20-78 years old.

Figure 3 shows the percent change in standardized cognitive testing questionnaire (standardized cognitive testing questionnaire) scores from day 0 to day 90 compared to baseline.

Detailed Description

1. Overview

Before the present materials and methods are described, it is to be understood that this invention is not limited to the particular methodology and materials described, as such methodologies and materials may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. The terms "a" (or an), "one or more" and "at least one" are also used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" are used interchangeably.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications and patents specifically mentioned herein are incorporated herein by reference for all purposes, including describing and disclosing the chemicals, instruments, statistical analyses and methods reported in the publications that might be used in connection with the invention. All references cited in this specification should be considered as indicating the state of the art. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

II. the invention

Aequorin is the first photoprotein isolated from photojellyfish and other marine organisms. The aequorin complex comprises 22,285-dalton apoaequorin, dioxygen, and the luminophore coelenterazine (coelenterazine). When three Ca²⁺Upon ionic binding to the complex, coelenterazine oxidizes to coelenteramide (coelenteramide), with carbon dioxide release and blue light. Aequorin is not exported or secreted by cells, nor compartmentalized or sequestered within cells. Therefore, measurement of aequorin has been used to detect Ca occurring over a longer period²⁺And (6) changing. In several experimental systems, the luminescence of aequorin can be detected from hours to days after cell loading. Aequorin is also known to not disrupt cell function or embryonic development.

Since its luminescence depends on Ca²⁺The aequorin complex has been widely used as intracellular Ca²⁺An indicator. Aequorin of victoria multiphoton luminescent jellyfish (Aequorea victoria) is used exclusively for: (1) analyzing the secretory response of the monadrechne cell to the nicotinic cholinergic agonist; (2) elucidation of Ca²⁺Release effects in myocardial destruction; (3) demonstration of Ca during fertilization²⁺Release of a large amount; (4) study of sarcoplasmic reticulum Ca in developing chick myoblasts²⁺Regulation of pump expression; and (5) correcting for micropipettes with injection volumes of at least 3 picoliters.

The apoaequorin has a molecular weight of about 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 unreacted photoprotein with bound substrate.

The purification of aequorin from aequorea victoria by conventional methods requires cumbersome extraction procedures and sometimes results in products that are substantially heterogeneous or toxic to the organism under study. Two tons of jellyfish typically yield about 125mg of purified photoprotein. In contrast, apoaequorin is preferably purified from genetically engineered E.coli (Escherichia coli) and then recombinant aequorin complexes are reconstituted in vitro with pure coelenterazine to produce recombinant aequorin. Aequorin useful in the present invention has been described and can be obtained commercially by purification schemes and/or synthetic methods 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.

The present invention relates to the administration of apoaequorin-containing compositions to a subject to correct or maintain the calcium balance in the subject. It is believed that maintaining ionic calcium concentrations in plasma and body fluids is critical to various bodily functions including, but not limited to, neuronal excitability, muscle contraction, membrane permeability, cell division, hormone secretion, bone mineralization, or prevention of cell death following ischemia. Disruption of calcium homeostasis, i.e., calcium imbalance, is believed to result in and/or be associated with a variety of diseases, syndromes and conditions. Such diseases, syndromes and conditions include those associated with quality of sleep, quality of energy, quality of memory and perception of pain. CaBP studies recognize that they can act as protective factors that play a role in maintaining proper ionic calcium levels.

In certain embodiments, the methods of the invention comprise apoaequorin as the sole active ingredient for treating, delaying the progression of, preventing the onset of, and preventing and/or treating the recurrence of a calcium imbalance. In other embodiments, the invention provides methods comprising administering apoaequorin in combination with one or more additional agents of known therapeutic or nutritional value. A particularly preferred use of apoaequorin is in the treatment of one or more symptoms and conditions associated with sleep, energy, mood, memory quality and perception of pain.

The term "treatment" as used herein includes prophylactic as well as palliative treatment of a disease. The terms "reduce", "alleviate", "suppress" and "inhibit" as used herein have the commonly understood meaning of alleviating or reducing. The term "develop" as used herein means that the score or severity increases, progresses, increases or becomes worse. The term "relapse" as used herein means a return of disease after regression.

The term "administering" as used herein refers to exposing a patient, tissue, organ or cell to apoaequorin. Administration as used herein may be performed in vitro, i.e., in vitro, or in vivo, i.e., in cells or tissues of a living organism, such as a human. In a preferred embodiment, the present invention comprises administering to a patient or subject a composition useful in the present invention. As equivalently used herein, a "patient" or "subject" refers to a mammal, preferably a human, having: (1) having a calcium imbalance-related disorder that can be alleviated or treated by administration of apoaequorin; or (2) susceptibility to a calcium imbalance-related disorder that can be prevented by administration of apoaequorin.

The terms "effective amount" and "therapeutically effective amount" as used herein refer to an amount of active agent sufficient to produce a desired therapeutic response without adverse side effects, such as toxicity, irritation, or allergic response. The specific "effective amount" will obviously vary with such factors as the particular condition being treated, the type of animal being treated, the duration of the treatment, the nature of the current treatment (if any), and the specific formulation employed and the structure of the compound or derivative thereof. In this example, an amount is considered therapeutically effective if it results in one or more of the following: (1) preventing disorders associated with calcium imbalance; and (2) reversing or stabilizing a calcium imbalance-related disorder. The optimal effective amount can be readily determined by one of ordinary skill in the art using routine experimentation.

In certain preferred compositions for oral administration to a subject, the apoaequorin is formulated with at least one acceptable carrier at a dosage of about 10mg per dose, preferably in the form of a capsule, and the recommended dosage for the subject is about 10mg per day (i.e., one capsule per day).

Compositions of the invention include liquid or lyophilized or other dry formulations, diluents containing various buffer levels (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent adsorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, cholate), solubilizing agents (e.g., glycerol, polyethylene glycol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking agents or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to proteins, formation of complexes with metal ions, or incorporation of materials into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid or hydrogels, or incorporated into liposomes, microemulsions, micelles, lamellar or multilamellar vesicles, erythrocytic ghosts or spheroids. Such compositions can affect physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include lipophilic depots (e.g., fatty acids, waxes, oils).

The invention also includes methods of administering a particulate composition coated with a polymer, such as a poloxamer or poloxamine. Other embodiments of such compositions comprise particulate forms, protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral. In certain embodiments, parenterally, paracarcinemia, transmucosally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, or intratumorally.

In addition, as used herein, a "pharmaceutically acceptable carrier" is well known to those skilled in the art and includes, but is not limited to: 0.01-0.1M, preferably 0.05M phosphate buffer or 0.9% saline. Further, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.

Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer and fixed oils. Intravenous carriers include liquid and nutritional supplements, electrolyte supplements such as those based on ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, contrast agents (gelling agents), inert gases and the like.

Controlled or sustained release compositions that can be administered according to the invention include lipophilic depots (e.g., fatty acids, waxes, oils). The invention also contemplates compounds in which the particulate composition coated with a polymer (e.g., a poloxamer or a poloxamine) is coupled to an antibody directed against a tissue-specific receptor, ligand or antigen or to a ligand for a tissue-specific receptor.

Other embodiments of compositions administered according to the present invention comprise particulate forms, protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, ocular and oral.

It is known that the half-life in blood after intravenous injection is significantly increased by covalently linking to a water-soluble polymer, such as polyethylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone or polyproline as a modified chemical entity, compared to the corresponding unmodified compound. Such modifications may also increase the solubility of chemical entities in aqueous solutions, eliminate agglomeration, enhance the physical and chemical stability of the compounds, and greatly reduce the immunogenicity and reactivity of the compounds. Thus, such polymer-entity adducts can be administered less frequently or in lower doses compared to unmodified entities or to obtain the desired in vivo biological activity.

In another method of the invention, the composition can be delivered in a controlled release system. For example, the agent may be administered by intravenous infusion, implantable osmotic pump, transdermal patch, liposome, or other mode of administration. In one embodiment, a pump may be utilized. In another embodiment, polymeric materials may be utilized. In yet another embodiment, the controlled release system can be placed in the vicinity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose.

The composition may comprise apoaequorin alone or in combination with 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 formulations or suppositories, including rectal and urethral suppositories. Pharmaceutically acceptable carriers also include gums, starches, sugars, cellulosic materials, and mixtures thereof. The apoaequorin-containing composition can be administered to a patient, for example, by subcutaneous implantation into a pellet. In another embodiment, the pellet provides controlled release of apoaequorin over a period of time. The compositions may also be administered by intravenous, intraarterial, intramuscular injection of liquids, oral administration of liquids or solids, or by topical application. Administration can also be achieved using rectal or urethral suppositories.

The compositions administrable by the present invention can be prepared by known dissolving, mixing, granulating or tablet-forming processes. For oral administration, apoaequorin or its physiologically tolerated derivatives, such as salts, esters, N-oxides, etc., can be mixed with the customary additives for this purpose, such as carriers, stabilizers or inert diluents, and converted by customary methods into the appropriate form for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions.

Examples of suitable inert carriers are conventional tablet bases, such as lactose, sucrose or corn starch, mixed with binding agents, such as acacia, corn starch, gelatin, disintegrating agents, such as corn starch, potato starch, alginic acid, or with wetting agents, such as stearic acid or magnesium stearate.

Examples of suitable oily carriers or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil. The composition may be formulated as dry granules or wet granules. For parenteral administration (subcutaneous, intravenous, intraarterial or intramuscular injection), the chemical entity or a physiologically tolerable derivative thereof, for example a salt, ester, N-oxide or the like, is converted into a solution, suspension or emulsion, if desired in admixture with customary and suitable substances for this purpose, for example solubilizers or other auxiliary substances.

Examples are sterile liquids, such as water and oils, with or without the addition of surfactants and other pharmaceutically acceptable adjuvants. Exemplary oils are those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil or mineral oil. Water, saline, aqueous dextrose and related sugar solutions, and glycerol, such as propylene glycol or polyethylene glycol, are generally preferred liquid carriers, particularly for injectable solutions.

The preparation of compositions containing active ingredients is well known in the art. Such compositions may be prepared as aerosols for delivery to the nasopharynx or as injectables, such as liquid solutions or suspensions; however, solid forms suitable for dissolution or suspension in a liquid prior to injection may also be prepared. The composition may be emulsified. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, or the like, or any combination thereof. In addition, the compositions may contain minor amounts of auxiliary substances which enhance the effectiveness of the active ingredient, for example wetting or emulsifying agents, pH buffering agents.

The active ingredient may be formulated into compositions, for example in the form of neutralized pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids, such as hydrochloric or phosphoric acids, or organic acids, such as acetic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups may also be obtained from inorganic bases such as sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine.

For topical administration to a body surface, using, for example, creams, gels, drops, and the like, the apoaequorin or its physiologically tolerable derivatives may be prepared as a solution, suspension, or emulsion and applied with or without a pharmaceutical carrier, using a physiologically acceptable diluent.

In another method of the invention, the active ingredient may be delivered using vectors, particularly Liposomes (see Langer, Science 249: 1527-.

The salt of apoaequorin is preferably a pharmaceutically acceptable salt. However, other salts may be utilized to prepare the compositions of the present invention or their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts include acid addition salts which may be formed, for example, by mixing a solution of apoaequorin 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.

In addition, the apoaequorin-containing compositions described herein may be in the form of nutraceutical compositions in which apoaequorin prevents the onset of or alleviates or stabilizes various deleterious calcium imbalance-related disorders. For the purposes of this specification, the term "nutraceutical" or "nutraceutical composition" refers to a food product, or a portion of a food product, that provides a medical health benefit, including the prevention and/or treatment of disease. The nutraceutical composition of the present invention may contain apoaequorin alone as an active ingredient, or may further comprise a mixture with dietary supplements including vitamins, coenzymes, minerals, herbs, amino acids, etc., which supplement the diet by increasing the total intake of the substance.

Accordingly, the present invention provides a method of providing a nutritional benefit to a patient comprising the step of administering to the patient a nutraceutical composition comprising apoaequorin. Such compositions typically comprise a "nutritionally acceptable carrier," which refers herein to any vehicle suitable for oral delivery, including pharmaceutically acceptable carriers suitable for the oral route described above. In certain embodiments, the nutraceutical compositions of the present invention comprise dietary supplements including, depending on the functional definition, immune boosters, anti-inflammatory agents, antioxidants, antiviral agents, or mixtures thereof.

Immunopotentiators and/or antiviral agents may be used to accelerate wound healing and improve immune function; they include extracts of Chrysanthemum, or herbs of the genus Echinacea (Echinacea), extracts of herbs of the genus Foeniculum vulgare (Sambuca) and extracts of Ranunculus japonicus. Natural or processed forms of the herb of the genus Astragalus (Astragalus) are also effective immunopotentiators. Astragalus stimulates the production of stem cells and lymphoid tissue active immune cells in the bone marrow. Zinc and its biologically active salts, such as zinc gluconate and zinc acetate, also act as immunopotentiators in the treatment of the common cold.

Antioxidants include the natural sulfur-containing amino acid allicin, which acts to increase the level of antioxidant enzymes in the blood. Herbs or plant extracts containing allicin, such as garlic, are also effective antioxidants. Catechins and extracts of catechin-containing herbs, such as green tea, are also effective antioxidants. Extracts of astragalus also show antioxidant activity. Bioflavonoids such as quercetin, hesperidin, purple quercitrin and mixtures thereof are also effective antioxidants. The main beneficial effect of bioflavonoids may be to protect vitamin C in the body from oxidation. This allows more vitamin C or ascorbic acid to be available to the body.

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

Another supplement useful in the present invention is ginger, derived from herbs of the genus Zingiber. It has now been found that it possesses cardiotonic activity due to related compounds such as gingerol and shogaol, and provides benefits in the treatment of vertigo and vestibular disorders. Ginger is also effective in treating nausea and other stomach disorders.

Supplements that aid in the reconstruction of soft tissue structures, particularly cartilage, are useful in compositions for the treatment of arthritis and other joint disease pain. Glucosamine, glucosamine sulfate, chondroitin can be derived from various sources, such as moose deer Antler (elkvellet Antler). Marine lipid complexes, omega 3 fatty acid complexes and fish oils are also known to be useful in the treatment of pain associated with arthritis.

Supplements useful for treating migraine include feverfeverfei (feverfew) and ginkgo biloba (Gingko biloba). The major active ingredient in feverfew is the sesquiterpene lactone parthenolide, which inhibits the secretion of prostaglandins that cause pain through vasospastic activity in blood vessels. Feverfew also exhibits anti-inflammatory properties. Fish oil can be used for treating migraine due to its platelet stabilizing and anti-vasospasm effects. The herb gingko biloba also helps to treat migraine headache by stabilizing the arteries and improving blood circulation.

Although the pharmacological effects of some of the above supplements have been described, other supplements may be used in the present invention, the effects of which are well documented in the scientific literature.

The invention may be more fully understood in view of the following non-limiting examples.

Examples

Example 1 administration of apoaequorin over a 90 day period improves the quality of life of test subjects

This analysis (open label study) of 32 patients showed an improvement in the overall quality of sleep, energy, mood, pain, general health over a 90 day period. Behavioral changes were detected by standardizing the questionnaire set. Including assessment of qualitative cognitive tests, sleep index, headache index, and quality of life questionnaire. The study showed improved behavior. None of the participants stopped the study due to adverse events.

The results shown in fig. 1 show the change in percentage of the area score relative to baseline; we excluded the memory score of another graph. This analysis is labeled on the graph as 1, 2, 3, 4 and 5 with day 0 to day 90. The figure shows an improvement in overall quality of sleep, energy, mood, pain and overall health. The baseline is known from the pre-study phase.

Example 2 administration of apoaequorin over a period of 30 days improved the quality of life of the test subjects

The study was an open label study in which 56 patients participated during a 30 day period. Behavioral changes were detected by a memory screening tool (memosyreening tool). As shown in figure 2, the study showed an improvement in memory behavior as early as day 8, but a statistically higher improvement at day 30. None of the participants stopped the study due to adverse events.

Example 3 administration of apoaequorin over a 90 day period improves cognition in test subjects

This analysis (an open-label study of 32 patients) showed an improvement in cognitive performance. Behavioral changes were detected by standard cognitive suite testing (static cognitive testing). The study showed cognitive improvement as early as day 8, but statistically higher improvements at day 30 and 60-90. None of the participants stopped the study due to adverse events. The results shown in figure 3 demonstrate a significant percentage increase in cognitive score relative to baseline. Note that: greater than 51% of the participants had improved cognitive performance.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims (5)

1. Use of apoaequorin in the manufacture of a composition for administration to a subject for treating a symptom or condition associated with calcium imbalance in the subject, wherein treating the symptom or condition associated with calcium imbalance comprises improving sleep quality in the subject, improving energy quality in the subject, improving mood quality in the subject, relieving pain in the subject, or improving memory quality in the subject, wherein the composition does not contain coelenterazine.

2. The use of claim 1, wherein the symptom or disorder associated with calcium imbalance is associated with neuronal excitability, muscle contraction, membrane permeability, cell division, hormone secretion, bone mineralization, or cell death following ischemia.

3. Use of apoaequorin in the preparation of a nutraceutical composition for treating a symptom or condition associated with calcium imbalance in a subject administered the nutraceutical composition, wherein the treating the symptom or condition associated with calcium imbalance comprises improving sleep quality in the subject, improving energy quality in the subject, improving mood quality in the subject, relieving pain in the subject, or improving memory quality in the subject, wherein the composition does not contain coelenterazine.

4. The use of claim 3, wherein the composition is administered to the subject in the form of a nutraceutical composition.

5. The use of claim 3, wherein said composition is administered to said subject in the form of an oral delivery capsule.