WO2018183353A1 - Small molecule compounds to support healthy human aging - Google Patents

Abstract

Disclosed herein are methods of activating expression of the FOXO3 gene in a subject comprising administering to a subject small molecule compound (such as astaxanthin and/or one or more astaxanthin derivatives), or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject.

Description

TITLE: SMALL MOLECULE COMPOUNDS TO SUPPORT HEALTHY HUMAN

AGING BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to support healthy human aging. More specifically, the present invention relates to the use of small molecule compounds to activate genes associated with aging.

2. Description of the Relevant Art

Genetic factors may contribute as much as 50% to the variability in human lifespan, depending upon the population. In 1987, a research group in Okinawa published the first replicated study of specific genes that contribute to human longevity (Takataet al.,“Influence of major histocompatibility complex region genes on human longevity among Okinawan-Japanese centenarians and nonagenarians” The Lancet. 1987, 330(8563):824–826). Since that time, the most robust genetic findings in human aging and longevity involve alleles in the APOE and FOXO3 genes. Later, it was discovered that the APOE2 allele was over-represented and the APOE4 allele was under-represented in centenarians, and this led to a large number of studies that clearly outlined an important role for this gene in both human lifespan and healthspan. No other genetic findings were widely replicated in human aging until 2008. Our research group in Hawaii found that the evolutionarily conserved FOXO3 gene had alleles that doubled (heterozygotes) or tripled (homozygotes) the probability of living near one hundred years. FOXO3 is now the second most replicated gene in human aging and one of only two genes to reach genome-wide significance in case-control studies of longevity.

FOXO3, is 125 kb in size, located on chromosome 6q21, and codes for a forkhead Box O transcription factor. FOXO3 has four exons, where exons 2 and 3 code for the translated protein. Intron 2 alone accounts for over 95% of the FOXO3 gene. We have found that FOXO3 might be important in human aging and longevity based on cumulative model organism data. We reported strong associations with longevity with three single nucleotide polymorphisms (SNPs) of FOXO3 in linkage disequilibrium within intron 2. This association was found in male Americans of Japanese ancestry. The FOXO3 associations were confirmed in numerous other populations confirming an important role in human aging. However, the mechanism is not yet known.

An important role of FOXO3 in the inflammatory response was recently discovered. Carriers of the minor allele of a SNP in intron 2 of FOXO3, in LD with our longevity associated alleles, is associated with an ameliorated course of Crohn’s disease and rheumatoid arthritis, and an anti- inflammatory cytokine profile. Multiple studies have now shown that chronic inflammation, especially in the elderly, is an important mechanism for CHD, cancer, stroke, and dementia, among other major age-associated diseases. Our preliminary data support this finding and we hypothesize that FOXO3 impacts aging through a cytokine driven anti- inflammatory pathway.

Further studies were conducted that demonstrate that evolutionarily conserved longevity- associated genes in model organisms are also important for human longevity. These studies showed that FOXO3 as a longevity-associated gene in humans. In fact, eleven independent replications, including a case/control genome-wide significance study, have validated FOXO3 as one of only two confirmed longevity genes to date. FOXO3 is a crucial downstream target of the insulin/insulin-like growth factor signaling pathway. The importance of the insulin pathway in regulating lifespan has been demonstrated in studies that modified this signaling pathway via caloric restriction, administration of metformin (prescribed to enhance insulin sensitivity in diabetic patients), and late life doses of rapamycin. Whilst these studies have been replicated in model organisms, the relevance of the findings to human longevity and the specific mechanism by which they influence lifespan are not fully understood. Likewise, the mechanism of action of the FOXO3 protective variants that confer a greater chance a longer lifespan– particularly at the cell and molecular level– is presently unknown. Association of the FOXO3 component of the insulin signaling pathway with a molecular marker of lifespan– telomere dynamics– should greatly advance insight into the mechanisms responsible for longevity.

The contribution of adult stem cells to tissue homeostasis is at the core of one of the main theories of aging. The theory of stem cell aging arose partly from observations that populations of adult stem cells decline in number and function with organismal aging. Although a definitive connection between stem cell maintenance and lifespan has not been shown in humans, studies of mouse knockout strains have linked specific genes that maintain multiple stem cell populations with premature aging phenotypes. FOXO3 has been associated with the maintenance of hematopoietic, neural, and muscle stem cells in mouse knockout models. A demonstration that the human FOXO3 longevity-associated variant impacts the maintenance of these cell populations with age would add great clinical significance to the existing findings in model organisms, particularly when related to tissue-specific stem cell-related health outcomes.

FOXO3 is a member of the forkhead box O transcription factor family; these proteins share a common DNA-binding domain (forkhead box) and play critical regulatory roles for many genes. As such they can been found in almost all tissues of the body throughout development (GeneAtlas). FOXO3 has a wide variety of functions, including, but not limited to, energy homeostasis, oxidative stress management, proteostasis, lipid metabolism, apoptosis, cell cycle progression, and cellular potency. FOXO3 is a target of the insulin/insulin-like growth factor signaling (IIS) pathway. The signaling cascade modulates AKT, resulting in inhibition of FOXO3 activity by phosphorylation-dependent 14-3-3 exclusion from the nucleus. Conversely, FOXO3 activity is increased in response to oxidative stress via the MAP kinase pathway, specifically JNK, which reverses the 14-3-3 mediated nuclear exclusion and promotes nuclear translocation. FOXO has multiple sites susceptible to post-translational modification– e.g., phosphorylation, acetylation and ubiquitination– that allow it to serve as a signaling conduit for different cellular processes that can regulate the localization, sensitivity to other regulators, and turnover of FOXO3.

The relation between FOXO3 and human longevity was demonstrated by showing that variants in FOXO3 SNPs were strongly associated with longevity in the Kuakini HHP cohort of Japanese-American men. The mortality risk for homozygous carriers of the minor allele of SNP rs2802292 (the most robust SNP) was almost 3 times lower than their major allele carrying counterparts, and these longer-lived individuals displayed better health for a longer time (i.e., they had a greater healthspan). This finding has since been confirmed as significant in multiple and diverse human populations, making FOXO3 the second human gene with a confirmed relationship to human longevity. Deeper analysis shows the protective effect of the FOXO3 gene is partially via a significant reduction in coronary heart disease (CHD) risk, although the mechanism by which FOXO3 protects against CHD is unknown.

The majority (~66-75%) of the world’s population does not carry a copy of the protective FOXO3 variant, and therefore represents a large number of vulnerable persons who could particularly benefit from a consumable product that activates the FOXO3 gene. SUMMARY OF THE INVENTION

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject who would benefit from such treatment a therapeutically effective amount of one or more small molecule compounds, or pharmaceutically acceptable salts thereof, sufficient to at least partially increase the amount of FOXO3 expressed in the subject.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more small molecule compounds, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject. In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more carotenoid derivatives, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject.

In another embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more carotenoid derivatives, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject, wherein the carotenoid derivative has the structure:

where R¹ and R² are each independently:

where each R⁵ is independently hydrogen, -CH₃, -OH, or -OR⁶ wherein at least one R⁵ group in the carotenoid derivative is -OR⁶;

monounsaturated hydrocarbon]; -C(O)-[C₆-C₂₄ polyunsaturated hydrocarbon]; or an amino acid group; and where R⁷ is hydrogen, alkyl, or aryl. In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject astaxanthin and/or one or more astaxanthin derivatives, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FOXO3 expressed in the subject. In an embodiment, the method includes administering naturally occurring astaxanthin or synthetic astaxanthin to the subject. In an embodiment, the astaxanthin is dispersed in a carbohydrate-based matrix.

In an embodiment, the method further comprises administering one or more antioxidants to the subject substantially simultaneously with the administration of the astaxanthin and/or one or more astaxanthin derivatives.

In an embodiment, the method includes administering one or more astaxanthin derivatives having the structure:

-C(O)-amino acid, -C(O)-OR⁷; -P(O)(OR⁷)₂; -S(O)(OR⁷)₂; -C(O)-[C₆-C₂₄ saturated hydrocarbon]; -C(O)-[C₆-C₂₄ monounsaturated hydrocarbon]; -C(O)-[C₆-C₂₄ polyunsaturated hydrocarbon]; or an amino acid group; and where each R⁷ is independently hydrogen, alkyl, or aryl. In an embodiment, the one or more astaxanthin derivatives have the structure:

wherein each R⁶ is independently: -C(O)-alkyl-N(R⁷)₂; -C(O)-alkyl-N⁺(R⁷)₃; -C(O)-alkyl- CO₂R⁷; -C(O)-alkyl-CO - 2 ; -C(O)-amino acid; -C(O)-[C₆-C₂₄ saturated hydrocarbon]; - C(O)-[C₆-C₂₄ monounsaturated hydrocarbon]; or -C(O)-[C₆-C₂₄ polyunsaturated hydrocarbon]; and where each R⁷ is independently hydrogen, alkyl, or aryl. In an embodiment, the one or more astaxanthin derivatives have the structure:

wherein each R⁶ is independently -C(O)-alkyl-N(R⁷)₂ or -C(O)-alkyl-N⁺(R⁷)₃; where alkyl is a C₁-C₆ straight chain hydrocarbon, and where each R⁷ is independently hydrogen or C₁-C₃ alkyl. In an embodiment, the method further includes co-administration to the subject of one or more of the following compounds: ganoderic acids; caffeic acid; sulfated polysaccharides; curcuminoids; ginkgolides; astragenols; cycloastragenols, gypenosides, theaflavin, and theaflavin gallate. In an embodiment, the method further includes co-administration to the subject of one or more flavonoids.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more flavonoids, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FOXO3 expressed in the subject.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more ganoderic acids, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FOXO3 expressed in the subject.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject caffeic acid, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FOXO3 expressed in the subject.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more sulfated polysaccharides, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FOXO3 expressed in the subject.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more curcuminoids, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FOXO3 expressed in the subject.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more ginkgolides, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FOXO3 expressed in the subject.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more astragenols, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FOXO3 expressed in the subject.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more gypenosides, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FOXO3 expressed in the subject.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject theaflavin or theaflavin gallate, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FOXO3 expressed in the subject.

In an embodiment, a method of reducing the effects of aging in a subject comprises administering to a subject astaxanthin and/or one or more astaxanthin derivatives, or

pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject, wherein the astaxanthin and/or one or more astaxanthin derivatives are administered at least once a day to the subject regardless of the presence of one or more age related diseases in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

Advantages of the present invention will become apparent to those skilled in the art with the benefit of the following detailed description of embodiments and upon reference to the accompanying drawings in which:

FIG. 1 depicts HPLC traces from plasma samples taken after administration of astaxanthin or astaxanthin derivatives;

FIG. 2 shows the concentration of astaxanthin produced by metabolism of astaxanthin derivatives given orally to dogs; and

FIG.3 depicts the results of an efficacy test for FOXO3 expression by astaxanthin.

While the invention may be susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. The drawings may not be to scale. It should be understood, however, that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. DETAILED DESCRIPTION OF THE EMBODIMENTS

It is to be understood that the present invention is not limited to particular compounds, methods or biological systems, which may, of course, vary. It is also to be understood that, as used in this specification and the appended claims, the singular forms“a”,“an”, and“the” include singular and plural referents unless the content clearly dictates otherwise. It is to be yet further understood that any terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The terms used throughout this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the general embodiments of the invention, as well as how to make and use them. It will be readily appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed in greater detail herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term.

As used herein the term "carotenoid derivatives" may be generally defined as biologically active structural analogs and derivatives of carotenoids. Typical derivatives include molecules which demonstrate equivalent or improved biologically useful and relevant function, but which differ structurally from the parent (i.e., naturally occurring) compounds. Parent carotenoids are selected from the more than 600 naturally-occurring carotenoids described in the literature, and their stereo- and geometric isomers. Such analogs may include, but are not limited to, esters, ethers, carbonates, amides, carbamates, phosphate esters and ethers, sulfates, glycoside ethers, with or without spacers (linkers).

As used herein, the term“xanthophyll carotenoid” generally refers to a naturally occurring or synthetic 40-carbon polyene chain with a carotenoid structure that contains at least one oxygen-containing functional group. The chain may include terminal cyclic end groups. Exemplary, though non-limiting, xanthophyll carotenoids include astaxanthin, zeaxanthin, lutein, echinenone, lycophyll, canthaxanthin, and the like. Non-limiting examples of carotenoids that are not xanthophyll carotenoids include ^-carotene and lycopene.

As used herein the term“expression” is the process by which information from a gene is used in the synthesis of a gene product. For example, expression of the gene FOXO3 produces the protein FoxO3. As used herein the term“activation” is the process by which a gene is induced to begin production of the gene product. For example, activation of the gene FOXO3 will induce the production of the protein FoxO3 based on the FOXO3 gene.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein,“pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Pharmaceutically acceptable acid addition salts of the compounds of the invention include salts derived form inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorus, and the like, as well as the salts derived from organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, butyrate, caprylate, isobutyrate, oxalate, malonate, succinate, sulfosalicylate, salicylate, suberate, sebacate, fumarate, maleate, laurate, mandelate, benzoate, chlorobenzoate, hydroxybenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, embonate, stearate, hydroxynaphthoate, methanesulfonate, and the like. Also contemplated are the salts of amino acids such as arginate, gluconate, galacturonate, and the like; see, for example, Berge et al., "Pharmaceutical Salts," J. of Pharmaceutical Science, 1977; 66:1 19. The acid addition salts of the basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner. The free base form may be regenerated by contacting the salt form with a base, and isolating the free base in the conventional manner. The free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base for purposes of the present invention. Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metal hydroxides, or of organic amines. Examples of metals used as cations are sodium, potassium, calcium, aluminum, magnesium, titanium, ammonium and the like. Examples of suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, meglumine, guanidine, and procaine. The base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, the disclosure of which is hereby incorporated by reference. It is understood that the active compounds and their pharmaceutically acceptable salts mentioned can also be present, for example, in the form of their pharmaceutically acceptable solvates, in particular in the form of their hydrates.

The phrase“co-administration” as used herein refers to administering an effective active agent which is distinct from the primary active agent being administered. The co-administered compound may be given to the subject before, substantially simultaneously with, or after administration of the primary active agent.

The phrase“combination therapy” (or“co-therapy”), as used herein embraces the administration of small molecule compounds, and of one or additional agents suitable for the treatment of one or more conditions associated with the targeted medical condition, as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these therapeutic agents. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected). The term is intended to embrace administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a pharmaceutical preparation having a fixed ratio of each therapeutic agent or in multiple preparations for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection. The sequence in which the therapeutic agents are administered is not narrowly critical. "Combination therapy" also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients (such as, but not limited to, additional pharmacologic agents) and non-drug therapies (such as, but not limited to, surgery or radiation treatment).

As used herein the term“synergistic combination” may be generally defined as any composition two or more therapeutic compounds that exhibit an effect that is greater than the effect of the individual therapeutic components by themselves.

As used herein the terms“administration,”“administering,” or the like, when used in the context of providing a pharmaceutical or nutraceutical composition to a subject generally refers to providing to the subject one or more pharmaceutical, “over-the-counter” (OTC) or nutraceutical compositions in combination with an appropriate delivery vehicle by any means such that the administered compound achieves one or more of the intended biological effects for which the compound was administered. By way of non-limiting example, a composition may be administered parenteral, subcutaneous, intravenous, intracoronary, rectal, intramuscular, intra- peritoneal, transdermal, or buccal routes of delivery. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, weight, and/or disease state of the recipient, kind of concurrent treatment, if any, frequency of treatment, and/or the nature of the effect desired. The dosage of pharmacologically active compound that is administered will be dependent upon multiple factors, such as the age, health, weight, and/or disease state of the recipient, concurrent treatments, if any, the frequency of treatment, and/or the nature and magnitude of the biological effect that is desired.

As used herein, terms such as “pharmaceutical composition,” “pharmaceutical formulation,”“pharmaceutical preparation,” or the like, generally refer to formulations that are adapted to deliver a prescribed dosage of one or more pharmacologically active compounds to a cell, a group of cells, an organ or tissue, an animal or a human. Methods of incorporating pharmacologically active compounds into pharmaceutical preparations are widely known in the art. The determination of an appropriate prescribed dosage of a pharmacologically active compound to include in a pharmaceutical composition in order to achieve a desired biological outcome is within the skill level of an ordinary practitioner of the art. A pharmaceutical composition may be provided as sustained-release or timed-release formulations. Such formulations may release a bolus of a compound from the formulation at a desired time, or may ensure a relatively constant amount of the compound present in the dosage is released over a given period of time. Terms such as“sustained release,”“controlled release,” or“timed release” and the like are widely used in the pharmaceutical arts and are readily understood by a practitioner of ordinary skill in the art. Pharmaceutical preparations may be prepared as solids, semi-solids, gels, hydrogels, liquids, solutions, suspensions, emulsions, aerosols, powders, or combinations thereof. Included in a pharmaceutical preparation may be one or more carriers, preservatives, flavorings, excipients, coatings, stabilizers, binders, solvents and/or auxiliaries that are, typically, pharmacologically inert. It will be readily appreciated by an ordinary practitioner of the art that, included within the meaning of the term are pharmaceutically acceptable salts of compounds. It will further be appreciated by an ordinary practitioner of the art that the term also encompasses those pharmaceutical compositions that contain an admixture of two or more pharmacologically active compounds, such compounds being administered, for example, as a combination therapy.

As used herein the terms“subject” generally refers to a mammal, and in particular to a human.

The terms“in need of treatment,”“in need thereof,”“who would benefit from such treatment,” or the like when used in the context of a subject being administered a pharmacologically active composition, generally refers to a judgment made by an appropriate healthcare provider that an individual or animal requires or will benefit from a specified treatment or medical intervention. Such judgments may be made based on a variety of factors that are in the realm of expertise of healthcare providers, but include knowledge that the individual or animal is ill, will be ill, or is at risk of becoming ill, as the result of a condition that may be ameliorated or treated with the specified medical intervention.

The phrases“therapeutically effective amount” and“effective amount” are synonymous unless otherwise indicated, and mean an amount of a compound of the present invention that is sufficient to improve the condition, disease, or disorder being treated. Determination of a therapeutically effective amount, as well as other factors related to effective administration of a compound of the present invention to a patient in need of treatment, including dosage forms, routes of administration, and frequency of dosing, may depend upon the particulars of the condition that is encountered, including the patient and condition being treated, the severity of the condition in a particular patient, the particular compound being employed, the particular route of administration being employed, the frequency of dosing, and the particular formulation being employed. Determination of a therapeutically effective treatment regimen for a patient is within the level of ordinary skill in the medical or veterinarian arts. In clinical use, an effective amount may be the amount that is recommended by the U.S. Food and Drug Administration, or an equivalent foreign agency. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the mammalian host treated and the particular mode of administration.

By“prophylactically effective amount” is meant an amount of a pharmaceutical composition that will substantially prevent, delay or reduce the risk of occurrence of the biological or physiological event in a cell, a tissue, a system, animal or human that is being sought by a researcher, veterinarian, physician or other caregiver.

The term“pharmacologically inert,” as used herein, generally refers to a compound, additive, binder, vehicle, and the like, that is substantially free of any pharmacologic or“drug- like” activity.

A“pharmaceutically or nutraceutically acceptable formulation,” as used herein, generally refers to a non-toxic formulation containing a predetermined dosage of a pharmaceutical and/or nutraceutical composition, wherein the dosage of the pharmaceutical and/or nutraceutical composition is adequate to achieve a desired biological outcome. The meaning of the term may generally include an appropriate delivery vehicle that is suitable for properly delivering the pharmaceutical composition in order to achieve the desired biological outcome.

In an embodiment, the FOXO3 gene is activated using small molecule compounds. The activation of FOXO3 gene slows human aging, as well as slowing or reducing the onset of age- related diseases.

The pharmaceutical preparation of the small molecule compounds may be administered orally, in the form of a tablet, a capsule, an emulsion, a liquid, or the like. Alternatively, the pharmaceutical preparation may be administered via a parenteral route. A more detailed description of the types of pharmaceutical preparations that are suitable for some embodiments is described in detail below. Some embodiments may be particularly suited timed or sustained release pharmaceutical preparations, in which the preparation is adapted to deliver a known dosage of carotenoid derivatives at or over a predetermined time. In an embodiment, a pharmaceutical preparation may be a“soft drug”, in that the compound is active in the derivatized state, and may yield the effective small molecule compound after metabolic conversion in vivo. In an embodiment, a pharmaceutical preparation may be adapted to one drug, or a portion thereof, before delivering the second drug. For example, a pharmaceutical preparation may be adapted in such a way that at least a portion of the small molecule compound is released into the body of a subject before additional compositions or medicaments are released. One or more of the additional compositions or medicaments suitable for the treatment of the medical conditions presently contemplated may be formulated as a separate pharmaceutical composition to be administered in conjunction with the subject carotenoid derivatives as part of a therapeutic regimen, or may be formulated in a single preparation together with the one or more carotenoid derivatives. Such a composition may be administered orally, parenterally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. The term parenteral generally embraces non-oral routes of administration, including but not limited to, subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.

Topical administration may involve the use of transdermal administration such as transdermal patches or iontophoresis devices. In some embodiments, a topical composition may include the small molecule compounds dissolved in an appropriate topical base for application to the skin of a subject. The topical base may include a thickening agent (e.g., polyethylene glycol), a skin emollient (e.g., an alpha hydroxyl acid), and an emulsifier (e.g., polysorbate 20).

In another embodiment, the small molecule compounds described herein may be used to induce FOXO3 expression in cells by ex vivo application of the small molecule compounds to the cells. In one embodiment, the small molecule compounds are applied, ex vivo, to cultured cells, more particularly to cultured stem cells. The collected cells may be suspended in a cell culture media and the small molecule compounds added to the media to induce FOXO3 expression in the cultured cells. The small molecule compounds may be dissolved in an appropriate solvent to form a solution that includes from 0.1% to 90% by weight of the small molecule compound dissolved in the solution. The absolute amount of the small molecule compounds applied to the cell culture media is between about 0.1 μg/mL of media to about 1 μg/mL of media. The small molecule compound solution may be added periodically to the cell culture media to induce FOXO3 expression. Addition of the small molecule composition to the cultured cells may induce a greater than 50% increase in the amount of FoxO3 expressed by the cells.

Therapeutic kits comprising the small molecule compounds, either alone or in combination with an additional composition suitable for the treatment of the medical condition are also contemplated herein. Such kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation of at least one small molecule compound. The kits also may contain other pharmaceutically acceptable formulations, such as those containing components to target the small molecule compounds to distinct regions of a patient where treatment is needed, or any one or more of a range of drugs which may work in concert with the small molecule compounds.

In some embodiments, small molecule compounds described herein may be administered in the form of nutraceuticals. “Nutraceuticals” as used herein, generally refers to dietary supplements, foods, or medical foods that: 1. possess health benefits generally defined as supporting or maintaining the general structures or function of the body or the overall health; and 2. are safe for human consumption in such quantity, and with such frequency, as required to realize such properties. Generally a nutraceutical is any substance that can be a food or a part of a food and provides health benefits. Such products may range from isolated nutrients, dietary supplements and specific diets to genetically engineered designer foods, herbal products, and processed foods such as cereals, soups and beverages. It is important to note that this definition applies to all categories of food and parts of food, ranging from dietary supplements such as folic acid, used for the prevention of spina bifida, to chicken soup, taken to lessen the discomfort of the common cold. This definition also includes a bio-engineered designer vegetable food, rich in antioxidant ingredients, and a stimulant functional food or pharmafood. Within the context of the description herein, where the composition, use and/or delivery of pharmaceuticals are described, nutraceuticals may also be composed, used, and/or delivered in a similar manner where appropriate.

In some embodiments, the small molecule compounds described herein may be administered to the subject in a nutraceutical formulation. The nutraceutical formulation may be chronically administered to the subject or occasionally administered to the subject. Examples of chronic administration include, but are not limited to, at least once a day, every other day, or every week. When administered as a nutraceutical formulation, the formulation may be administered regardless of the presence of one or more age related diseases in the subject. As used herein the phrase“age-related diseases” refers to disease which appear in subjects as subject grow older. Age related diseases include, but are not limited to, coronary heart disease, cancer, stroke, diabetes, and hypertension.

The small molecule compounds may be administered at a dosage level up to conventional dosage levels for such compounds, but will typically be less than about 1 μg/g of body weight per day. Suitable dosage levels may depend upon the overall systemic effect of the chosen small molecule compound, but typically suitable levels will be about 0.001 to 50 mg/kg body weight of the patient per day, from about 0.005 to 30 mg/kg per day, or from about 0.05 to 10 mg/kg per day. The small molecule compounds may be administered on a regimen of up to 6 times per day, between about 1 to 4 times per day, or once per day.

In the case where an oral composition is employed, a suitable dosage range is, e.g. from about 0.01 mg to about 100 mg of a small molecule compound per kg of body weight per day, preferably from about 0.1 mg to about 10 mg per kg of body weight per day.

It will be understood that the dosage of the small molecule compounds will vary with the nature and the severity of the condition to be treated, and with the particular small molecule compounds chosen. The dosage will also vary according to the age, weight, physical condition and response of the individual patient. The selection of the appropriate dosage for the individual patient is within the skills of a clinician.

In one embodiment, carotenoids and/or carotenoid analogs or derivatives, including pharmaceutically acceptable salts thereof, may be used to activate expression of FOXO3 in a subject who would benefit from such treatment. Carotenoids and/or carotenoid analogs may be administered in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject.

Carotenoid derivatives suitable for use in activating FOXO3 expression may include carotenoids having the general structure:

where R¹ and R² are each independently:

where each R⁵ is independently hydrogen, -CH₃, -OH, or -OR⁶ wherein at least one R⁵ group in the carotenoid derivative is -OR⁶; wherein each R⁶ is independently: alkyl; aryl; -alkyl- N(R⁷)₂; -aryl-N(R⁷)₂; -alkyl-N⁺(R⁷)₃; -aryl-N⁺(R⁷)₃; -alkyl-CO₂R⁷; -aryl-CO₂R⁷; -alkyl-CO₂-; - aryl-CO₂-; -C(O)-alkyl-N(R⁷)₂; -C(O)-aryl-N(R⁷)₂; -C(O)-alkyl-N⁺(R⁷)₃; -C(O)-aryl-N⁺(R⁷)₃; - C(O)-alkyl-CO₂R⁷; -C(O)-aryl-CO₂R⁷; -C(O)-alkyl-CO₂-; -C(O)-aryl-CO₂-; -C(NR⁷)-alkyl- N(R⁷)₂; -C(NR⁷)-aryl-N(R⁷)₂; -C(NR⁷)-alkyl-N⁺(R⁷)₃; -C(NR⁷)-aryl-N⁺(R⁷)₃; -C(NR⁷)-alkyl- CO₂R⁷; -C(NR⁷)-aryl-CO₂R⁷; -C(NR⁷)-alkyl-CO₂-; -C(NR⁷)-aryl-CO₂-; -C(NR⁷)-alkyl-N(R⁷)- alkyl-N(R⁷)₂; -C(O)-amino acid, -C(O)-OR⁷; -P(O)(OR⁷)₂; -S(O)(OR⁷)₂; -C(O)-[C₆-C₂₄ saturated hydrocarbon]; -C(O)-[C₆-C₂₄ monounsaturated hydrocarbon]; -C(O)-[C₆-C₂₄ polyunsaturated hydrocarbon]; or an amino acid group; where each R⁷ is independently hydrogen, alkyl, or aryl.

In some embodiments, carotenoid derivatives suitable for use with the present methods and uses may be derived from astaxanthin and have the structure

wherein each R⁶ is independently: alkyl; aryl; -alkyl-N(R⁷)₂; -aryl-N(R⁷)₂; -alkyl-N⁺(R⁷)₃; -aryl-N⁺(R⁷)₃; -alkyl-CO₂R⁷; -aryl-CO₂R⁷; -alkyl-CO₂-; -aryl-CO₂-; -C(O)-alkyl-N(R⁷)₂; - C(O)-aryl-N(R⁷)₂; -C(O)-alkyl-N⁺(R⁷)₃; -C(O)-aryl-N⁺(R⁷)₃; -C(O)-alkyl-CO₂R⁷; -C(O)- aryl-CO₂R⁷; -C(O)-alkyl-CO - 2; -C(O)-aryl-CO - 2 ; -C(NR⁷)-alkyl-N(R⁷)₂; -C(NR⁷)-aryl- N(R⁷)₂; -C(NR⁷)-alkyl-N⁺(R⁷)₃; -C(NR⁷)-aryl-N⁺(R⁷)₃; -C(NR⁷)-alkyl-CO₂R⁷; -C(NR⁷)- aryl-CO₂R⁷; -C(NR⁷)-alkyl-CO - 2; -C(NR⁷)-aryl-CO - 2; -C(NR⁷)-alkyl-N(R⁷)-alkyl-N(R⁷)₂; -C(O)-amino acid, -C(O)-OR⁷; -P(O)(OR⁷)₂; -S(O)(OR⁷)₂; -C(O)-[C₆-C₂₄ saturated hydrocarbon]; -C(O)-[C₆-C₂₄ monounsaturated hydrocarbon]; -C(O)-[C₆-C₂₄ polyunsaturated hydrocarbon]; or an amino acid group; and where each R⁷ is independently hydrogen, alkyl, or aryl. In some embodiments, carotenoid derivatives suitable for use with the present methods and uses may be derived from astaxanthin and have the structure

wherein each R⁶ is independently: -C(O)-alkyl-N(R⁷)₂; -C(O)-alkyl-N⁺(R⁷)₃; -C(O)-alkyl-CO₂R⁷; -C(O)-alkyl-CO - 2; -C(O)-amino acid; -C(O)-[C₆-C₂₄ saturated hydrocarbon]; -C(O)-[C₆-C₂₄ monounsaturated hydrocarbon]; or -C(O)-[C₆-C₂₄ polyunsaturated hydrocarbon]; where each R⁷ is independently hydrogen, alkyl, or aryl. In some embodiments, carotenoid derivatives suitable for use with the present methods and uses may be derived from lutein and have the structure

wherein each R⁶ is independently: -C(O)-alkyl-N(R⁷)₂; -C(O)-alkyl-N⁺(R⁷)₃; -C(O)-alkyl-CO₂R⁷; -C(O)-alkyl-CO - 2; -C(O)-amino acid; -C(O)-[C₆-C₂₄ saturated hydrocarbon]; -C(O)-[C₆-C₂₄ monounsaturated hydrocarbon]; or -C(O)-[C₆-C₂₄ polyunsaturated hydrocarbon]; where each R⁷ is independently hydrogen, alkyl, or aryl. In some embodiments, carotenoid derivatives suitable for use with the present methods and uses may be derived from zeaxanthin and have the structure

wherein each R⁶ is independently: -C(O)-alkyl-N(R⁷)₂; -C(O)-alkyl-N⁺(R⁷)₃; -C(O)-alkyl-CO₂R⁷; -C(O)-alkyl-CO - 2; -C(O)-amino acid; -C(O)-[C₆-C₂₄ saturated hydrocarbon]; -C(O)-[C₆-C₂₄ monounsaturated hydrocarbon]; or -C(O)-[C₆-C₂₄ polyunsaturated hydrocarbon]; where each R⁷ is independently hydrogen, alkyl, or aryl. In some embodiments, carotenoid derivatives suitable for use with the present methods and uses may be derived from lycophyll and have the structure

wherein each R⁶ is independently: -C(O)-alkyl-N(R⁷)₂; -C(O)-alkyl-N⁺(R⁷)₃; -C(O)-alkyl-CO₂R⁷; -C(O)-alkyl-CO - 2; -C(O)-amino acid; -C(O)-[C₆-C₂₄ saturated hydrocarbon]; -C(O)-[C₆-C₂₄ monounsaturated hydrocarbon]; or -C(O)-[C₆-C₂₄ polyunsaturated hydrocarbon]; where each R⁷ is independently hydrogen, alkyl, or aryl. In some embodiments, carotenoid derivatives suitable for use with the present methods and uses may be derived from astaxanthin and have the structure

wherein each R⁶ is independently -C(O)-alkyl-N(R⁷)₂ or -C(O)-alkyl-N⁺(R⁷)₃; where alkyl is a C₁-C₆ straight chain hydrocarbon, and where each R⁷ is independently hydrogen or C₁-C₃ alkyl. In some embodiments, carotenoid derivatives suitable for use with the present methods and uses may be derived from lutein and have the structure

wherein each R⁶ is independently -C(O)-alkyl-N(R⁷)₂ or -C(O)-alkyl-N⁺(R⁷)₃; where alkyl is a C₁-C₆ straight chain hydrocarbon, and where each R⁷ is independently hydrogen or C₁-C₃ alkyl. In some embodiments, carotenoid derivatives suitable for use with the present methods and uses may be derived from zeaxanthin and have the structure

wherein each R⁶ is independently -C(O)-alkyl-N(R⁷)₂ or -C(O)-alkyl-N⁺(R⁷)₃; where alkyl is a C₁-C₆ straight chain hydrocarbon, and where each R⁷ is independently hydrogen or C₁-C₃ alkyl. In some embodiments, carotenoid derivatives suitable for use with the present methods and uses may be derived from lycophyll and have the structure

wherein each R⁶ is independently -C(O)-alkyl-N(R⁷)₂ or -C(O)-alkyl-N⁺(R⁷)₃; where alkyl is a C₁-C₆ straight chain hydrocarbon, and where each R⁷ is independently hydrogen or C₁-C₃ alkyl. When R⁶ is -C(O)-amino acid, coupling of the amino acid or the peptide is accomplished through an ester linkage or a carbamate linkage. Specifically, an ester linked group -O-C(O)- amino acid has the general structures:

Depending on if the free form of the salt form is desired. A carbamate linked amino acid group–OR⁶ will have the general structure:

Depending on if the free form, acidic salt, or basic salt form is desired. For both ester linked and carbamate linked amino acids, the group R⁹ represents an amino acid side chain. Specifically, R⁸ can be:

-H (glycine); -CH₃ (alanine); -CH(CH₃)-CH₃ (valine); -CH₂-CH(CH₃)-CH₃ (leucine);

-CH(CH₃)-CH₂-CH₃ (isoleucine); -CH₂-Ph (phenylalanine); -CH₂-CH₂-S-CH₃ (methionine); -CH₂-OH (serine); -CH(CH₃)-OH (threonine); -CH₂-SH (cysteine); -CH₂-Ph-OH (tyrosine); -CH₂-C(O)-NH₂ (aspargine); -CH₂-CH₂-C(O)-NH₂ (glutamine); -CH₂-CO₂H (aspartic acid); -CH₂-CH₂-CO₂H (glutamic acid); -CH₂-CH₂-CH₂-CH₂-NH₂ (lysine); -CH₂-CH₂-CH₂-NH₂ (ornithine);

-CH₂-CH₂-CH₂-NH-C(NH)-NH₂ (arginine);

(tryptophan). Amino acid side chains can be in the neutral form (as depicted above) or in a salt form. When R⁸ represents the side chain from the amino acid proline, the following compounds result:

In one embodiment, R⁶ is -C(O)-amino acid, where the amino acid is lysine. In such an embodiment, the substituent–OR⁶ can be:

When R⁶ is -C(O)-alkyl-N(R⁷)₂ or -C(O)-alkyl-N⁺(R⁷)₃ in some embodiments, alkyl is a C₁-C₆ straight chain hydrocarbon and R⁷ is H or Me. Specific examples of -C(O)-alkyl-N(R⁷)₂ or -C(O)-alkyl-N⁺(R⁷)₃ include:

When R⁶ is -C(O)-[C₆-C₂₄ saturated hydrocarbon], the substituent, R⁶, is derived from coupling of a saturated fatty acid with the carotenoid parent structure. Examples of saturated fatty acids include, but are not limited to: hexanoic acid (caproic acid); octanoic acid (caprylic acid); decanoic acid (capric acid); dodecanoic acid (lauric acid); tridecanoic acid; tetradecanoic acid (myristic acid); pentadecanoic acid; hexadecanoic acid (palmitic acid); heptadecanoic acid (margaric acid); octadecanoic acid (stearic acid); eicosanoic acid (arachidic acid); docosanoic acid (behenic acid); tricosanoic acid; and tetracosanoic acid (lignoceric acid). When R⁶ is -C(O)-[C₆-C₂₄ monounsaturated hydrocarbon], the substituent, R⁶, is derived from coupling of a monounsaturated fatty acid with the carotenoid parent structure. Examples of monounsaturated fatty acids include, but are not limited to: 9-tetradecenoic acid (myristoleic acid); 9-hexadecenoic acid (palmitoleic acid); 11-octadecenoic acid (vaccenic acid); 9- octadenoic acid (oleic acid); 11-eicosenoic acid; 13-docosenoic acid (erucic acid); 15- tetracosanoic acid (nervonic acid); 9-trans-hexadecenoic acid (palmitelaidic acid); 9-trans- octadecenoic acid (elaidic acid); 8-eicosaenoic acid; and 5-eicosaenoic acid.

When R⁶ is -C(O)-[C₆-C₂₄ polyunsaturated hydrocarbon], the substituent, R⁶, is derived from coupling of a polyunsaturated fatty acid with the carotenoid parent structure. Examples of polyunsaturated fatty acids include, but are not limited to omega-3 polyunsaturated fatty acids, omega-6 polyunsaturated fatty acids; and conjugated polyunsaturated fatty acids. Examples of omega-3 polyunsaturated fatty acids include, but are not limited to: 9,12,15-octadecatrienoic acid (alpha-linolenic acid); 6,9,12,15-octadecatetraenoic acid (stearidonic acid); 11,14,17- eicosatrienoic acid (eicosatrienoic acid (ETA)); 8,11,14,17-eicsoatetraenoic acid (eicsoatetraenoic acid); 5,8,11,14,17-eicosapentaenoic acid (eicosapentaenoic acid (EPA)); 7,10,13,16,19-docosapentaenoic acid (docosapentaenoic acid (DPA)); 4,7,10,13,16,19- docosahexaenoic acid (docosahexaenoic acid (DHA)); 6,9,12,15,18,21-tetracosahexaenoic acid (nisinic acid); 9E,11Z,15E-octadeca-9,11,15-trienoic acid (rumelenic acid); 9E,11Z,13Z,15E- octadeca-9,11,13,15-trienoic acid (α-parinaric acid); and all trans-octadeca-9,11,13,15-trienoic acid (β-parinaric acid). Examples of omega-6 polyunsaturated fatty acids include, but are not limited to: 9,12-octadecadienoic acid (linoleic acid); 6,9,12-octadecatrienoic acid (gamma- linolenic acid); 11,14-eicosadienoic acid (eicosadienoic acid); 8,11,14-eicosatrienoic acid (homo- gamma-linolenic acid); 5,8,11,14-eicosatetraenoic acid (arachidonic acid); 13,16-docosadienoic acid (docosadienoic acid); 7,10,13,16-docosatetraenoic acid (adrenic acid); 4,7,10,13,16- docosapentaenoic acid (docosapentaenoic acid); 8E,10E, 12Z-octadecatrienoic acid (calendic acid); 10E,12Z-octadeca-9,11-dienoic acid; 8E,10E,12Z-octadecatrienoic acid (α-calendic acid); 8E,10E,12E-octadecatrienoic acid (β-calendic acid); 8E,10Z,12E-octadecatrienoic acid (jacaric acid); and 5Z,8Z,10E,12E,14Z-eicosanoic acid (bosseopentaenoic acid). Examples of conjugated polyunsaturated fatty acids include, but are not limited to: 9Z,11E-octadeca-9,11-dienoic acid (rumenic acid); 10E,12Z-octadeca-9,11-dienoic acid; 8E,10E,12Z-octadecatrienoic acid (α- calendic acid); 8E,10E,12E-octadecatrienoic acid (β-calendic acid); 8E,10Z,12E-octadecatrienoic acid (jacaric acid); 9E,11E,13Z-octadeca-9,11,13-trienoic acid (α-eleostearic acid); 9E,11E,13E- octadeca-9,11,13-trienoic acid (β-eleostearic acid); 9Z,11Z,13E-octadeca-9,11,13-trienoic acid (catalpic acid); 9E,11Z,13E-octadeca-9,11,13-trienoic acid (punicic acid); 9E,11Z,15E-octadeca- 9,11,15-trienoic acid (rumelenic acid); 9E,11Z,13Z,15E-octadeca-9,11,13,15-trienoic acid (α- parinaric acid); all trans-octadeca-9,11,13,15-trienoic acid (β-parinaric acid); and 5Z,8Z,10E,12E,14Z-eicosanoic acid (bosseopentaenoic acid).

In some embodiments, carotenoid analogs or derivatives may have increased water solubility and/or water dispersibility relative to some or all known naturally occurring carotenoids. Contradictory to previous research, improved results are obtained with derivatized carotenoids relative to the base carotenoid, wherein the base carotenoid is derivatized with substituents including hydrophilic substituents.

Further details regarding the use and synthesis of carotenoid derivatives and analogs suitable for use in the presently described embodiments may be found in the following U.S. patent documents: U.S. Patent No. 7,145,025 entitled “STRUCTURAL CAROTENOID ANALOGS FOR THE INHIBITION AND AMELIORATION OF DISEASE” issued December 5, 2006; U.S. Patent Application Publication No. 2005/0113372 entitled“CAROTENOID ANALOGS OR DERIVATIVES FOR THE INHIBITION AND AMELIORATION OF DISEASE”; U.S. Patent Application Publication No. 2005/0075337, entitled “PHARMACEUTICAL COMPOSITIONS INCLUDING CAROTENOID ANALOGS OR DERIVATIVES FOR THE INHIBITION AND AMELIORATION OF DISEASE”; U.S. Patent Application Publication No. 2005/0261254 entitled “CAROTENOID ANALOGS OR DERIVATIVES FOR THE INHIBITION AND AMELIORATION OF INFLAMMATION”; U.S. Patent Application Publication No. 2006/0058269 entitled“CAROTENOID ANALOGS OR DERIVATIVES FOR THE INHIBITION AND AMELIORATION OF INFLAMMATION”; U.S. Patent Application Publication No. 2006/0178538 entitled“METHODS FOR THE SYNTHESIS OF CHIRAL DIHYDROXY INTERMEDIATES USEFUL FOR THE CHIRAL SYNTHESIS OF CAROTENOIDS”; U.S. Patent Application Publication No. 2006/0183947 entitled“METHODS FOR THE SYNTHESIS OF ASTAXANTHIN”; U.S. Patent Application Publication No. 2006/0155150 entitled“METHODS FOR THE SYNTHESIS OF LUTEIN”; U.S. Patent Application Publication No. 2006/0088905 entitled“METHODS FOR THE SYNTHESIS OF ZEAXANTHIN”; U.S. Patent Application Publication No. 2006/0167319 entitled “METHODS FOR THE SYNTHESIS OF UNSATURATED KETONE INTERMEDIATES USEFUL FOR THE SYNTHESIS OF CAROTENOIDS”; U.S. Patent Application Publication No. 2006/0183185 entitled“METHODS FOR THE SYNTHESIS OF ASTAXANTHIN”; U.S. Patent Application Publication No.2006/0111580 entitled“METHODS FOR THE SYNTHESIS OF CHIRAL DIHYDROXY KETONE INTERMEDIATES USEFUL FOR THE CHIRAL SYNTHESIS OF CAROTENOIDS”; U.S. Patent Application Publication No. 2006/0088904 entitled“METHODS FOR THE SYNTHESIS OF ASTAXANTHIN”; U.S. Patent Application Publication No. 2006/0270590 entitled“REDUCTION IN COMPLEMENT ACTIVATION AND INFLAMMATION DURING TISSUE INJURY BY CAROTENOIDS, CAROTENOID ANALOGS, OR DERIVATIVES THEREOF”; U.S. Patent Application Publication No. 2006/0270589entitled“CAROTENOIDS, CAROTENOID ANALOGS, OR CAROTENOID DERIVATIVES FOR THE STABILIZATION OR IMPROVEMENT OF VISUAL ACUITY”; U.S. Patent Application Publication No. 2007/0015735 entitled“WATER- DISPERSIBLE CAROTENOIDS, INCLUDING ANALOGS AND DERIVATIVES”; U.S. Patent Application Publication No. 2006/0276372 entitled“CAROTENOIDS, CAROTENOID ANALOGS, OR CAROTENOID DERIVATIVES FOR THE TREATMENT OF PROLIFERATIVE DISORDERS”; U.S. Application Serial No. 11/417,307 entitled“USE OF CAROTENOIDS AND/ORCAROTENOID DERIVATIVES/ANALOGS FOR REDUCTION/INHIBITION OF CERTAIN NEGATIVE EFFECTS OF COX INHIBITORS”; U.S. Serial No. 60/691,518 entitled“METHODS FOR SYNTHESIS OF CAROTENOIDS, INCLUDING ANALOGS, DERIVATIVES, AND SYNTHETIC AND BIOLOGICAL INTERMEDIATES”; U.S. Patent Application Publication No. 2006/0293545 entitled “METHODS FOR SYNTHESIS OF CAROTENOIDS, INCLUDING ANALOGS, DERIVATIVES, AND SYNTHETIC AND BIOLOGICAL INTERMEDIATES”; U.S. Application Serial No. 11/636,401 entitled“STRUCTURAL CAROTENOID ANALOGS OR DERIVATIVES FOR THE MODULATION OF SYSTEMIC AND/OR TARGET ORGAN REDOX STATUS”; and U.S. Serial No. 11/699,924 entitled“SYNTHESIS OF CAROTENOID ANALOGS OR DERIVATIVES WITH IMPROVED ANTIOXIDANT CHARACTERISTICS”, all of which are commonly owned with the present application and which are hereby expressly incorporated by reference in their entirety as though fully set forth herein.

Naturally occurring carotenoids such as xanthophyll carotenoids of the C40 series, which include commercially important compounds such as lutein, zeaxanthin, and astaxanthin, have poor aqueous solubility in the native state. Varying the chemical structure(s) of the esterified moieties may vastly increase the aqueous solubility and/or dispersibility of derivatized carotenoids.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more flavonoids, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FOXO3 expressed in the subject. Flavonoids have the general structure that includes a phenyl ring fused to a heterocyclic ring with a pendent phenyl ring extending from the fused ring system. Subgroups of flavonoids include, but are not limited to, anthoxanthins, flavanones, flavanonols, flavans, flavanols, theaflavins, anthocyanidins, isoflavonoids.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more anthoxanthins, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject. Anthoxanthins have a 2-phenylchromen-4-one core structure depicted below, where each R is independently–H, -OH, or–OAlkyl (e.g., -OMe).

Anthoxanthins where R₁ is a hydroxy group or alkylated hydroxy group are commonly known as flavonols, while anthoxanthins R₁ is hydrogen are commonly known as flavones. Exemplary flavones include, but are not limited to, Luteolin, Apigenin, and Tangeritin. Exemplary flavonols include, but are not limited to, Quercetin, Kaempferol, Myricetin, Fisetin, Galangin, Isorhamnetin, Pachypodol, and Rhamnazin. In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more flavanones, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FOXO3 expressed in the subject. Flavanones have a 2,3-dihydro-2-phenylchromen-4-one core structure depicted below, where each R is independently–H, -OH, or–OAlkyl (e.g., -OMe).

Exemplary flavanones include, but are not limited to, Hesperetin, Naringenin, Eriodictyol, and Homoeriodictyol. In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more flavanonols, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject. Flavanonols have a 2,3-dihydro-2-phenylchromen-4-one core structure depicted below, where each R is independently–H, -OH, or–OAlkyl (e.g., -OMe) and R₁ is -OH, or–OAlkyl (e.g., -OMe).

Exemplary flavanonols include, but are not limited to, Taxifolin (Dihydroquercetin) and Dihydrokaempferol.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more flavans, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject. Flavans have a 2,3-dihydro-2-phenylchromene core structure depicted below, where each R is independently–H, -OH,–OAlkyl (e.g., -OMe), or -C(O)-3,4,5-trihydroxyphenyl (gallic acid derivative).

Exemplary flavans include, but are not limited to, Catechin, Gallocatechin, Catechin 3-gallate, Gallocatechin 3-gallate, Epicatechin, Epigallocatechin, Epicatechin 3-gallate, Epigallocatechin 3- gallate, Theaflavin-3-gallate, Theaflavin-3'-gallate, and Theaflavin-3,3'-digallate.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more anthocyanidins, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject. Anthocyanidins have a 2-phenylchromene core structure depicted below, where each R is independently–H, -OH,–OAlkyl (e.g., -OMe), -monosaccharide, or -disaccharide.

Exemplary anthocyanidins include, but are not limited to, Aurantinidin, Capensinidin, Cyanidin, Delphinidin, Europinidin, Hirsutidin, Malvidin, Pelargonidin, Peonidin, Petunidin, Pulchellidin, and Rosinidin.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more isoflavonoids, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject. Isoflavonoids can be broken into two groups. Isoflavones have a 3-phenylchromen- 4-one core structure depicted below, where each R is independently–H, -OH, or–OAlkyl (e.g., - OMe). Isoflavans have a 3-phenylchroman core structure depicted below where each R is independently–H, -OH, or–OAlkyl (e.g., -OMe). Both isoflavones and isoflavans can be used to activate expression of FOXO3 in a subject.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more ganoderic acids, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject. The class of ganoderic acids includes compounds having triterpenoid structure depicted below, where =R₁ is =O or CH-OH; =R₂ is =O or CH-OH; =R₃ is =O or–CH-OH; and R₄ is H or OH.

Exemplary ganoderic acids include, but are not limited to:

Ganoderic acid A (R₁ is =O; R₂ is CH-OH; R₃ is CH-OH; R₄ is H);

Ganoderic acid B (R₁ is CH-OH; R₂ is =O; R₃ is CH-OH; R₄ is H);

Ganoderic acid C (R₁ is =O; R₂ is =O; R₃ is CH-OH; R₄ is H);

Ganoderic acid D (R₁ is CH-OH; R₂ is CH-OH; R₃ is CH-OH; R₄ is H);

Ganoderic acid E (R₁ is =O; R₂ is =O; R₃ is =O; R₄ is H);

Ganoderic acid F (R₁ is =O; R₂ is =O; R₃ is =O; R₄ is OH);

Ganoderic acid G (R₁ is CH-OH; R₂ is =O; R₃ is CH-OH; R₄ is OH);

Ganoderic acid H (R₁ is CH-OH; R₂ is =O; R₃ is =O; R₄ is OH);

Lucidenic acid A (R₁ is =O; R₂ is =O; R₃ is CH-OH; R₄ is H);

Lucidenic acid B (R₁ is =O; R₂ is =O; R₃ is CH-OH; R₄ is OH); and

Lucidenic acid C (R₁ is CH-OH; R₂ is =O; R₃ is CH-OH; R₄ is OH).

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject caffeic acid, esters of caffeic acid, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject sulfated polysaccharides, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject. An exemplary sulfated polysaccharide is fucoidan.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject curcuminoids, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject. Curcuminoids have the general structure below, where each R is independently:–H, -OH, or– OAkyl. Exemplary curcuminoids include, but are not limited to, Curcumin, Demethoxycurcumin, and Bisdemethoxycurcumin.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject who would benefit from such treatment a therapeutically effective amount of one or more ginkgolides, or pharmaceutically acceptable salts thereof, sufficient to at least partially increase the amount of FoxO3 expressed in the subject. Ginkgolides have the general structure below, where each R₁, R₂, and R₃ is independently:–H or -OH. Exemplary ginkgolides include, but are not limited to: Ginkgolide A (R₁is -OH; R₂ is -H; R₃ is -H); Ginkgolide B (R₁is -OH; R₂ is -OH; R₃ is -H);

Ginkgolide C (R₁is -OH; R₂ is -OH; R₃ is -OH);

Ginkgolide J (R₁is -OH; R₂ is -H; R₃ is -OH); and

Ginkgolide M (R₁is -H; R₂ is -OH; R₃ is -OH)

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more astragenols or cycloastragenols, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject. Astragenols and cycloastragenols have the general structures below, where each R₁, R₂, and R₃ is independently: CH-OH or =O.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject one or more gypenosides, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject. Gypenosides have the structure depicted below, where each R is independently H, Alkyl (e.g., Me), monosaccharide, or disaccharide.

In an embodiment, a method of activating expression of FOXO3 in a subject comprises administering to a subject theaflavin or theaflavin gallate, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject. Theaflavins have the structure depicted below, where each R is independently H, Alkyl, or C(O)-3,4,5-trihydroxyphenyl (gallic acid derivative).

Although the above structure, and subsequent structures, depict alkenes in the E configuration this should not be seen as limiting. Compounds discussed herein may include embodiments where alkenes are in the Z configuration or include alkenes in a combination of Z and E configurations within the same molecule. The compounds depicted herein may naturally convert between the Z and E configuration and/or exist in equilibrium between the two configurations.

Compounds described herein embrace isomers mixtures, racemic, optically active, and optically inactive stereoisomers and compounds, even when a single stereoisomer is depicted. In some embodiments, a single stereoisomer of a small molecule compound may be administered to a human subject. Administering a single stereoisomer of a particular compound (e.g., as part of a pharmaceutical composition) to a human subject may be advantageous (e.g., increasing the potency of the pharmaceutical composition). Administering a single stereoisomer may be advantageous due to the fact that only one isomer of potentially many may be biologically active enough to have the desired effect. Examples

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Synthesis of Carotenoid Derivatives

Example 1: Synthesis of the disuccinic acid ester of astaxanthin

To a solution of astaxanthin (6.0 g, 10.05 mmol) in DCM (“dichloromethane”) (50 mL) at room temperature was added DIPEA (“N,N-diisopropylethylamine”) (35.012 mL, 201 mmol), succinic anhydride (10.057 g, 100.5 mmol), and DMAP (“4-(dimethylamino)pyridine”) (0.6145 g, 5.03 mmol). The reaction mixture was stirred at room temperature for 48 hours, at which time the reaction was diluted with DCM, quenched with brine/ 1M HCl (60 mL/ 10 mL), and then extracted with DCM. The combined organic layers were dried over Na₂SO₄ and concentrated to yield astaxanthin disuccinate (100%) HPLC retention time: 10.031 min., 82.57% (AUC); LRMS (ESI) m/z (relative intensity): 798 (M⁺+2H) (52), 797 (M⁺+H) (100); HPLC retention time: 10.595 min., 4.14% (AUC); LRMS (ESI) m/z (relative intensity): 797 (M⁺+H) (40), 697 (100); HPLC retention time: 10.966 min., 5.68% (AUC); LRMS (ESI) m/z (relative intensity): 797 (M⁺+H) (100), 679 (31); HPLC retention time: 11.163 min., 7.61% (AUC); LRMS (ESI) m/z (relative intensity): 797 (M⁺+H) (38), 679 (100), and no detectable astaxanthin. Example 2: Synthesis of the disodium salt of the disuccinic acid ester of astaxanthin

Disuccinic acid ester of astaxanthin (2 g, 2.509 mmol) and 200 mL ethanol were stirred at room temperature under nitrogen in a 500 mL round-bottom flask. Sodium ethoxide (340 mg, 5.019 mmol, Acros #A012556101) was added as a solid in a single portion and the solution was allowed to stir overnight. The following day, the precipitate was filtered off and washed with ethanol followed by methylene chloride to afford a purple solid, the disodium salt of the disuccinic acid ester of astaxanthin, [1.41 g, 67%] and was placed on a high vacuum line to dry. 1H-NMR (Methanol-d₄) ^ 6.77-6.28 (14 H, m), 5.53 (2 H, dd, J=12.6, 6.8), 2.68-2.47 (8 H, m), 2.08-1.88 (22 H, m), 1.37 (6 H, s), 1.24 (6H, s); ¹³C NMR (CDCl₃) δ 196.66, 180.80, 175.01, 163.69, 144.12, 141.38, 138.27, 136.85, 136.12, 135.43, 132.35, 129.45, 126.22, 124.71, 72.68, 44.09, 38.63, 34.02, 32.34, 31.19, 26.86, 14.06, 13.19, 12.91; Mass spectroscopy +ESI, 819.43 monosodium salt, 797.62 disuccinic acid ester of astaxanthin; HPLC 7.41 min (99.84%). Example 3: Synthesis of the BocLys(Boc)OH ester of astaxanthin.

To a mixture of astaxanthin (11.5 g, 19.3 mmol) and BocLys(Boc)OH (20.0 g, 57.7 mmol) in methylene chloride (500 mL) were added 4-dimethylaminopyridine (DMAP) (10.6 g, 86.6 mmol) and 1,3-diisopropylcarbodiimide (“DIC”) (13.4 g, 86.7 mmol). The round-bottomed flask was covered with aluminum foil and the mixture was stirred at ambient temperature under nitrogen overnight. After 16 hours, the reaction was incomplete by HPLC and TLC. [HPLC: Column: Waters Symmetry C18 3.5 micron 4.6 mm ^ 150 mm; Temperature: 25^oC; Mobile phase: (A = 0.025 % TFA in H₂O; B = 0.025% TFA in MeCN), 95% A/5% B (start); linear gradient to 100% B over 12 min, hold for 4 min; linear gradient to 95% B/5% A over 2 min; linear gradient to 95% A/5% B over 4 min; Flow rate: 2.5 mL/min; Detector wavelength: 474 nm.] An additional 1.5 equivalents of DMAP and DIC were added to the reaction and after 2 hours, the reaction was complete by HPLC. The mixture was then concentrated to 100 mL and a white solid (1,3-diisopropylurea) was filtered off. The filtrate was flash chromatographed through silica gel (10% to 50% Heptane/EtOAc) to give the desired BocLys(Boc)OH ester of astaxanthin as a dark red solid (28.2 g, >100% yield). ¹H NMR (DMSO-d₆) δ 7.24 (2 H, t, J=6.3 Hz), 6.78 (2 H, d, 5.0 Hz), 6.57-6.27 (14 H, m), 5.50-5.41 (2 H, m), 3.99-3.97 (2 H, d, 6.0 Hz), 2.90 (4 H, m), 2.03 (4 H, m), 2.00 (6 H, s), 1.97 (6 H, s), 1.82 (6 H, s), 1.70-1.55 (4 H, m), 1.39- 1.33 (36 H, m), 1.24-1.13 (8 H, m), 1.01-0.99 (6 H, m), 0.86-0.83 (6 H, m). HPLC: 21.3 min (24.6% AUC)); 22.0 min (48.1% (AUC)); 22.8 min (20.6% (AUC)). TLC (1:1 Heptane/EtOAc: R_f 0.41; R_f 0.5; R_f 0.56). LC/MS analysis was performed on a Agilent 1100 LC/MSD VL ESI system by flow injection in positive mode; Mobile Phase: A=0.025% TFA in H₂O; B=0.025%TFA in MeCN, 10%A/90%B(start); Starting pressure: 10 bar; PDA Detector 470 nm. +ESI, m/z=1276.1(M+Na⁺).

A mixture of DiBocLys(Boc) ester of astaxanthin (20.0 g, 16.0 mmol) and HCl in 1,4- dioxane (4.00 M, 400 mL, 1.60 mol, 100 eq) was stirred at ambient temperature under a nitrogen atmosphere. The round-bottomed flask was covered with aluminum foil and the reaction was stirred for 1 hour, at which time the reaction was complete by HPLC. The title compound precipitated and was collected by filtration, washed with ether (3 ^ 100 mL) and dried (14.7 g, 92%, 91.6% purity by HPLC). A portion (13.5 g) of the crude solid was dissolved in 500 mL of a 1:2 methanol/methylene chloride mixture and stirred under nitrogen. Diethyl ether (168 mL) was then added dropwise and the precipitated solid was collected by filtration to afford BocLys(Boc)OH ester of astaxanthin hydrochloride salt as a dark red solid (8.60 g, 63.7% yield). ¹H NMR (DMSO-d₆) δ 8.65 (6 H, s), 8.02 (6 H, s), 6.78-6.30 (14 H, m), 5.59-5.51 (2 H, m), 4.08 (2 H, m), 2.77 (4 H, m), 2.09-2.07 (4 H, m), 2.01 (6 H, s), 1.97 (6 H, s), 1.90-1.86 (4 H, m), 1.84 (6 H, s), 1.61-1.58 (8 H, m), 1.37 (6 H, s), 1.22 (6 H, s). HPLC: 7.8 min (97.0% (AUC)). LC/MS analysis was performed on an Agilent 1100 LC/MSD VL ESI system with Zorbax Eclipse XDB-C18 Rapid Resolution 4.6 x 75mm, 3.5 microns, USUT002736; Temperature: 25 ^oC; Mobile Phase: (%A=0.025% TFA in H₂O; %B=0.025% TFA in MeCN), 70%A/30%B (start); linear gradient to 50%B over 5 min, linear gradient to 100%B over 7 min; Flow rate: 1.0 mL/min; Starting pressure: 108 bar; PDA Detector 470 nm. Mass spectrometry +ESI, m/z=853.9(M+H⁺), m/z=875.8(M+Na⁺); LC 4.5 min.

A similar process can be used to manufacture other amino acid astaxanthin derivatives. Using a similar process, Astaxanthin disarcosinate and astaxanthin di(methylglycine) were also synthesized. Example 4: Synthesis of lutein disuccinate.

To a solution of lutein (0.010 g, 0.018 mmol) in DCM (2 mL) at room temperature was added DIPEA (0.063 mL, 0.360 mmol), succinic anhydride (0.036 g, 0.360 mmol), and DMAP (0.021 g, 0.176 mmol). The reaction mixture was stirred at room temperature for 48 hours, at which time the reaction was diluted with DCM, quenched with brine/ 1M HCl (6mL/ 1 mL), and then extracted with DCM. The combined organic layers were dried over Na₂SO₄ and concentrated to yield lutein disuccinate (93.09%) HPLC retention time: 11.765 min., 93.09% (AUC); LRMS (ESI) m/z (relative intensity): 769 (M⁺) (24), 651 (100), and no detectable lutein. Example 5: Synthesis of succinic acid esters of zeaxanthin.

To a solution of zeaxanthin (0.010 g, 0.018 mmol) in DCM (2 mL) at room temperature was added DIPEA (0.063 mL, 0.360 mmol), succinic anhydride (0.036 g, 0.360 mmol), and DMAP (0.021 g, 0.176 mmol). The reaction mixture was stirred at room temperature for 48 hours, at which time the reaction was diluted with DCM, quenched with brine/ 1M HCl (6 mL/1 mL), and then extracted with DCM. The combined organic layers were dried over Na₂SO₄ and concentrated to yield zeaxanthin monosuccinate (2.86%) HPLC retention time: 12.207 min., 2.86% (AUC); LRMS (ESI) m/z (relative intensity): 669 (M⁺+H) (53), 668 (M⁺) (100), zeaxanthin disuccinate (97.14%) HPLC retention time: 11.788 min., 67.42% (AUC); LRMS (ESI) m/z (relative intensity): 792 (M⁺+Na) (42), 769 (M⁺) (73), 651 (100); HPLC retention time: 13.587 min., 11.19% (AUC); LRMS (ESI) m/z (relative intensity): 792 (M⁺+Na) (36), 769 (M⁺) (38), 663 (100); HPLC retention time: 13.894 min., 18.53% (AUC); LRMS (ESI) m/z (relative intensity): 769 (M⁺) (62), 663 (77), 651 (100), and no detectable zeaxanthin. Example 6: Synthesis of dimethylaminobutyric acid monoester of astaxanthin.

To a suspension of 4-(dimethylamino)-butyric acid hydrochloride (0.2816 g, 1.68 mmol) in DCM/ DMF (3 mL /3 mL) at room temperature was added DIPEA (0.878 mL, 5.04 mmol), HOBT (“1-hydroxybenzotriazole”)-H₂O (0.3094 g, 2.02 mmol), DMAP (0.4105 g, 3.36 mmol), and astaxanthin (0.100 g, 0.168 mmol). The reaction mixture was stirred at room temperature for 36 hours, at which time the reaction was diluted with DCM, quenched with brine/ 1M HCl (20 mL/ 3 mL), and then extracted with DCM. The combined organic layers were concentrated to yield 4-(dimethylamino)butyric acid monoester (24.50%) HPLC retention time: 9.476 min., 20.32% (AUC); LRMS (ESI) m/z (relative intensity): 732 (M⁺+Na) (13), 729 (100); HPLC retention time: 9.725 min., 4.18% (AUC); LRMS (ESI) m/z (relative intensity): 732 (M⁺+Na) (50), 729 (100), and astaxanthin (61.21%). Example 7: Synthesis of lycophyll disuccinate.

To a stirring solution of lycophyll (28.0 mg, 0.0492 mmol) in 10 mL of dichloromethane were added succinic anhydride (12.3 mg, 0.123 mmol) and dimethylaminopyridine (15.0 mg, 0.123 mmol). The reaction vessel was wrapped in aluminum foil and stirred at ambient temperature overnight. After 16 hours, the reaction was complete by TLC. The mixture was then concentrated to give a crude red solid. Mass spectroscopic analysis of the crude solid detected the mass ion of the desired product (-APCI, m/z 767((M-H)-)). LC/MS analysis was performed on an Agilent 1100 LC/MSD VL ESI system with Zorbax Eclipse XDB-C18 Rapid Resolution 4.6x75mm, 3.5µm, USUT002736; Temperature: 25^oC; Mobile Phase:(%A=0.025%TFA in H₂O;%B=0.025%TFA in MeCN), 70%A/30%B(start); hold at 30%B for 1 min, linear gradient to 98%B over 10 min, hold at 98%B for 9 min; Flow rate: 1.0mL/min; Starting pressure: 112 bar; PDA Detector 470 nm, 373nm, 214nm. LRMS: - mode, APCI. Studies of the Bioavailability of Carotenoid Derivatives

Pharmacokinetic data was obtained showing that the carotenoids derivatives described above, when administered to animals, result in the accumulation of the parent carotenoid in the plasma. For example, it was determined that when astaxanthin derivatives are introduced into animals, the astaxanthin derivates are metabolized to produce free astaxanthin in the blood stream of the animal.

In one experiment, astaxanthin; all-trans 3S,3’S-astaxanthin diester disuccinate disodium salt (hereinafter“ADS”); and all-trans 3S,3’S-astaxanthin diester dilysinate tetrahydrochloride salt (hereinafter“ADL”) were administered orally to separate rats as a lipid suspension. A single dose that included 500 mg/kg of the carotenoid/carotenoid derivative was administered to each rat. Plasma from each of the rats was collected 4 hours after ingestion and 8 hours after ingestion and all samples were analyzed by high performance liquid chromatography (“HPLC”). HPLC analysis was used to detect the presence of free underivatized carotenoid (e.g., in this example, astaxanthin) in the plasma. HPLC chromatograms were collected for each plasma sample taken. The HPLC chromatograms are presented in FIG.1. Four hours after ingestion of astaxanthin, there was no significant amount of free astaxanthin in the rat plasma collected. Eight hours after ingestion of astaxanthin, there was no significant amount of free astaxanthin in the rat plasma collected. This indicates that very little (if any) astaxanthin is absorbed by the rats through oral dosage. Four hours after ingestion of ADS and ADL, a significant amount of free astaxanthin is seen in the rat plasma collected. Eight hour after ingestion of ADS and ADL also shows a significant amount of free astaxanthin in the rat plasma collected. This indicates that carotenoid derivatives are absorbed and metabolized by the rats to produce underivatized carotenoid.

In a second experiment, all-trans 3S,3’S-astaxanthin diester diglycinate dihydrochloride salt (hereinafter“ADG”); all-trans 3S,3’S-astaxanthin diester disarcosinate dihydrochloride salt (hereinafter“ADSa”); and ADL were administered orally to separate rats as an aqueous suspension containing 0.5% carboxymethylcellulose. A single dose was administered to the rats that included either 5 mg/kg of the carotenoid when administered intravenously, or 40 mg/kg of the carotenoid derivative when administered orally.

For each carotenoid derivative given to the rats in the second experiment, the amount of all astaxanthin isomers, the amount of trans astaxanthin isomers, and the amount of cis astaxanthin isomers in the plasma was determined at predefined time intervals for 3 days (72 hours). C_max (the peak plasma concentration of astaxanthin, trans astaxanthin and cis astaxanthin); T_max (the time it took for the plasma concentration to reach C_max); AUC (the area under the concentration curve); and T_1/2 (elimination half life) was determined for each sample and is presented in TABLE 1.

TABLE 1

As shown in TABLE 1, oral dosages of ADG, ADSa, and ADL were absorbed by the rats and metabolized to produce various C_max concentrations of astaxanthin. Intravenous dosages of ADG, ADSa, and ADL were also absorbed by the rats and metabolized to produce various C_max concentrations of astaxanthin. These experiments indicate that carotenoid derivatives ADG, ADSa, and ADL are absorbed and metabolized by the rats to produce underivatized carotenoid (in this example, astaxanthin).

In a fourth experiment, ADL was administered to non-naïve beagle dogs orally (as an aqueous suspension containing 0.5% carboxymethylcellulose) and intravenously as an aqueous solution. Oral doses that included 10 mg/kg of the carotenoid derivative, 100 mg/kg of the carotenoid derivative, and 500 mg/kg of the carotenoid derivative were administered to each dog. The intravenous dosage was 5 mg/kg. For each carotenoid derivative given to the dogs in the fourth experiment, the amount of all astaxanthin isomers, the amount of trans astaxanthin isomers, and the amount of cis astaxanthin isomers in the plasma was determined at predefined time intervals for 3 days (72 hours). C_max; T_max; AUC; and T_1/2 was determined for each sample and is presented in TABLE 2. TABLE 2

As shown in TABLE 2, intravenous and oral dosages of ADL were absorbed by the dogs and metabolized to produce various C_max concentrations of astaxanthin. This indicates that carotenoid derivatives ADL are absorbed and metabolized by the dogs to produce underivatized carotenoid (in this example, astaxanthin).

In a fifth experiment, ADL was administered to naïve beagle dogs orally (as an aqueous suspension containing 0.5% carboxymethylcellulose) twice a day for six days. Each oral dose included 75 mg/kg of the carotenoid derivative. For each carotenoid derivative given to the dogs in the fifth experiment, the amount of all astaxanthin isomers (TABLE 3), the amount of trans astaxanthin isomers (TABLE 4), and the amount of cis astaxanthin isomers (TABLE 5) in the plasma was determined at predefined time intervals for 6 days. C_max; T_max; AUC; and T_1/2 was determined for each sample.

TABLE 3

TABLE 4

TABLE 5

As shown in TABLES 3-5, oral dosages of ADL were absorbed by the dogs and metabolized to produce various C_max concentrations of astaxanthin over the six-day period. This indicates that carotenoid derivatives ADL are absorbed and metabolized by the dogs to produce underivatized carotenoid (in this example, astaxanthin).

In a sixth experiment, all-trans 3S,3’S-astaxanthin diester di-beta-alanine dihydrochloride salt (hereinafter“ADA”); ADG; ADL; ADSa; and ADS were administered orally to separate non-naïve dogs as a aqueous suspension containing 0.5% carboxymethylcellulose. A single dose that included 20 mg/kg of the carotenoid derivative was administered to each dog. For each carotenoid derivative given to the dogs in the sixth experiment, the amount of all astaxanthin isomers was determined at predefined time intervals for 3 days (72 hours). C_max was determined for each sample and a graph representing the obtained data is presented in FIG.2.

As shown in FIG. 2, oral dosages of ADA, ADG, ADL, ADSa, and ADS were absorbed by the dogs and metabolized to produce various C_max concentrations of astaxanthin. This indicates that carotenoid derivatives ADA, ADG, ADL, ADSa, and ADS are absorbed and metabolized by the dogs to produce underivatized carotenoid (in this example, astaxanthin).

In a seventh experiment, ADG or ADSa was administered to non-naïve beagle dogs orally (as an aqueous suspension containing 0.5% carboxymethylcellulose). Oral doses included either 50 mg/kg of the carotenoid derivative or 100 mg/kg of the carotenoid derivative. For each carotenoid derivative given to the dogs in the seventh experiment, the amount of all astaxanthin isomers, the amount of trans astaxanthin isomers, and the amount of cis astaxanthin isomers in the plasma was determined at predefined time intervals for 3 days (72 hours). C_max; T_max; AUC; and T_1/2 was determined for each sample and is presented in TABLE 6.

As shown in TABLE 6, oral dosages of ADG or ADSa were absorbed by the dogs and metabolized to produce various C_max concentrations of astaxanthin. This indicates that carotenoid derivatives ADL are absorbed and metabolized by the dogs to produce underivatized carotenoid (in this example, astaxanthin).

In an eighth experiment, ADSa was administered to non-naïve beagle monkeys orally (as an aqueous suspension containing 0.5% carboxymethylcellulose) and intravenously as an aqueous solution. Oral doses that included 10 mg/kg of the carotenoid derivative, 300 mg/kg of the carotenoid derivative, and 500 mg/kg of the carotenoid derivative were administered to each monkey. The intravenous dosage was 5 mg/kg. For each carotenoid derivative given to the monkeys in the eighth experiment, the amount of all astaxanthin isomers (TABLE 7), the amount of trans astaxanthin isomers (TABLE 8), and the amount of cis astaxanthin isomers (TABLE 9) in the plasma was determined at predefined time intervals for 3 days (72 hours). C_max; T_max; AUC; and T_1/2 was determined for each sample.

TABLE 8

As shown in TABLES 7-9, intravenous and oral dosages of ADSa were absorbed by the monkeys and metabolized to produce various C_max concentrations of astaxanthin. This indicates that carotenoid derivatives ADL are absorbed and metabolized by the monkeys to produce underivatized carotenoid (in this example, astaxanthin).

We have shown through these, and other unreported experiments, that carotenoid ester derivatives are readily metabolized by a variety of animals. Metabolism of carotenoid derivatives produces biologically significant amounts of the parent carotenoid in the blood stream of the animal. Efficacy of Astaxanthin to Affect FOXO3 Expression

Astaxanthin was shown to activate the FOXO3 gene in mammals (mice). In this study, C57BI6 mice were fed either regular chow (control) or chow containing an Astaxanthin disarcosinate ester at a low dose (0.08% w/w) or a high dose (0.4% w/w) for 2 weeks. Each group contained 6 mice. Following 2 weeks of dosing, all mice were sacrificed and organs, namely skeletal muscle, bone marrow, brain, and heart, were harvested, and snap frozen in liquid nitrogen. Tissues were homogenized in Trizol reagent, followed by extraction of RNA. FOXO3 expression level was assessed for all samples using real time RT-PCR. In brief, cDNA was generated from equal amounts of RNA from each sample by reverse transcription. The cDNA was then used as a template in real time PCR reactions (Cycle 1: (1X) Step 1: 95C for 3:00; Cycle 2: (40X) Step 1: 95C for 00:15, 52C for 00:30, Step 3: 72C for 00:30) using primers specific for the FOXO3 gene. Expression of the HPRT gene was also measured for all samples, as an internal reference. The highest level of FOXO3 activation was observed in the heart tissue (P=0.024), with positive trends also observed in the bone marrow and skeletal muscle. The results of the tests are presented in FIG.3. Astaxanthin Composition for Oral Delivery

Astaxanthin beadlets were formed by dispersing astaxanthin in a corn starch-coated matrix of modified food starch and glucose syrup with DL-alpha-tocopherol and sodium ascorbate added as antioxidants. Astaxanthin beadlets provide a source of astaxanthin to the blood stream of the subject when ingested.

In this patent, certain U.S. patents, U.S. patent applications, and other materials (e.g., articles) have been incorporated by reference. The text of such U.S. patents, U.S. patent applications, and other materials is, however, only incorporated by reference to the extent that no conflict exists between such text and the other statements and drawings set forth herein. In the event of such conflict, then any such conflicting text in such incorporated by reference U.S. patents, U.S. patent applications, and other materials is specifically not incorporated by reference in this patent.

Further modifications and alternative embodiments of various aspects of the invention may be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as the presently preferred embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description to the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims. In addition, it is to be understood that features described herein independently may, in certain embodiments, be combined.

Claims

WHAT IS CLAIMED IS: 1. A method of activating expression of FOXO3 in a subject comprising administering to a subject astaxanthin and/or one or more astaxanthin derivatives, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject.

2. A method of treating a subject at risk for coronary heart disease comprising administering to a subject who would benefit from such treatment a therapeutically effective amount of astaxanthin and/or one or more astaxanthin derivatives, or pharmaceutically acceptable salts thereof, sufficient to at least partially increase the amount of FoxO3 expressed in the subject.

3. A method of reducing the effects of aging in a subject comprising administering to a subject astaxanthin and/or one or more astaxanthin derivatives, or pharmaceutically acceptable salts thereof, in an amount sufficient to at least partially increase the amount of FoxO3 expressed in the subject, wherein the astaxanthin and/or one or more astaxanthin derivatives are chronically administered to the subject regardless of the presence of one or more age related diseases in the subject.

4. The method of any one of claims 1 to 3, wherein the astaxanthin and/or one or more astaxanthin derivatives are administered in an amount of 0.001 to 50 mg/kg body weight of the subject per day.

5. The method of any one of claims 1 to 4, wherein the astaxanthin and/or one or more astaxanthin derivatives are administered in a consumable form.

6. The method of any one of claims 1 to 4, wherein astaxanthin and/or one or more astaxanthin derivatives are administered as a topical composition that is applied to the skin of the subject.

7. A method of activating expression of FOXO3 in cultured cells comprising adding an effective amount of astaxanthin and/or one or more astaxanthin derivatives, or pharmaceutically acceptable salts thereof, to the media of the cultured cells, wherein the amount of astaxanthin and/or one or more astaxanthin derivatives is sufficient to at least partially increase the amount of Fox03 expressed in the cultured cells.

8. The method of claim 7, wherein the astaxanthin and/or one or more astaxanthin derivatives are added to the media of the cultured cells periodically until the FOX03 gene has been activated in the cultured cells.

9. The method of claim 7 or 8, wherein the cultured cells are cultured stem cells.

10. The method of any one of claims 1 to 9, wherein naturally occurring astaxanthin or synthetic astaxanthin is administered to the subject or the cultured cells.

11. The method of claim 10, wherein the astaxanthin is dispersed in a carbohydrate based matrix.

12. The method of any one of claims 1 to 11, further comprising administering one or more antioxidants to the subject or the cultured cells substantially simultaneously with the administration of the astaxanthin and/or one or more astaxanthin derivatives.

13. The method of any one of claims 1 to 12, wherein the one or more astaxanthin derivatives have the structure:

wherein each R⁶ is independently: alkyl; aryl; -alkyl-N(R⁷)₂; -aryl-N(R⁷)₂; -alkyl-N⁺(R⁷)₃; -aryl-N⁺(R⁷)₃; -alkyl-CO₂R⁷; -aryl-CO₂R⁷; -alkyl-CO₂-; -aryl-CO₂-; -C(O)-alkyl-N(R⁷)₂; - C(O)-aryl-N(R⁷)₂; -C(O)-alkyl-N⁺(R⁷)₃; -C(O)-aryl-N⁺(R⁷)₃; -C(O)-alkyl-CO₂R⁷; -C(O)- aryl-C0₂R⁷; -C(O)-alkyl-C0₂-; -C(O)-aryl-C0₂-; -C(NR⁷)-alkyl-N(R⁷)₂; -C(NR⁷)-aryl- N(R⁷)₂; -C(NR⁷)-alkyl-N⁺(R⁷)₃; -C(NR⁷)-aryl-N⁺(R⁷)₃; -C(NR⁷)-alkyl-C0₂R⁷; -C(NR⁷)- aryl-C0₂R⁷; -C(NR⁷)-alkyl-C0₂-; -C(NR⁷)-aryl-C0₂-; -C(NR⁷)-alkyl-N(R⁷)-alkyl-N(R⁷)₂; -C(O)-amino acid, -C(O)-OR⁷; -P(O)(OR⁷)₂; -S(O)(OR⁷)₂; -C(O)-[C₆-C₂₄ saturated hydrocarbon]; -C(O)-[C₆-C₂₄ monounsaturated hydrocarbon]; -C(O)-[C₆-C₂₄

polyunsaturated hydrocarbon]; or an amino acid group; and where each R⁷ is independently hydrogen, alkyl, or aryl.

14. The method of any one of claims 1 to 12, wherein the one or more astaxanthin derivatives have the structure:

wherein each R⁶ is independently: -C(O)-alkyl-N(R⁷)₂; -C(O)-alkyl-N⁺(R⁷)₃; -C(O)-alkyl- CO₂R⁷; -C(O)-alkyl-CO₂-; -C(O)-amino acid; -C(O)-[C₆-C₂₄ saturated hydrocarbon]; - C(O)-[C₆-C₂₄ monounsaturated hydrocarbon]; or -C(O)-[C₆-C₂₄ polyunsaturated hydrocarbon]; and where each R⁷ is independently hydrogen, alkyl, or aryl.

15. The method of any one of claims 1 to 12, wherein the one or more astaxanthin derivatives have the structure:

wherein each R⁶ is independently -C(O)-alkyl-N(R⁷)₂ or -C(O)-alkyl-N⁺(R⁷)₃; where alkyl is a C₁-C₆ straight chain hydrocarbon, and where each R⁷ is independently hydrogen or C₁-C₃ alkyl.

16. The method of any one of claims 1 to 15, further comprising co-administration to the subject or cultured cells one or more of the following compounds: ganoderic acids; caffeic acid; sulfated polysaccharides; curcuminoids; ginkgolides; astragenols; cycloastragenols, gypenosides, theaflavin, and theaflavin gallate.

17. The method of any one of claims 1 to 16, further comprising co-administration to the subject of one or more flavonoids.

18. The method of any one of claims 1 to 15, wherein astaxanthin and/or one or more astaxanthin derivatives are administered in an amount sufficient to induce at least a 50% increase in FoxO3 expression.