Pharmaceutical composition and method of treating disease using a molecular complex assembled on astaxanthin core

Abstract

A pharmaceutical composition including a therapeutically effective amount of esterified 3S, 3'S trans astaxanthin in combination with one or more constituent ingredients assembled on a molecular core of the astaxanthin to increase bioavailability of the composition, and methods of treating disease by administering a therapeutically effective amount of the pharmaceutical composition to a patient.

Description

PHARMACEUTICAL COMPOSITION AND METHOD OF TREATING DISEASE USING A MOLECULAR COMPLEX ASSEMBLED ON ASTAXANTHIN CORECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 594,214 filed October 30, 2023 and U.S. Provisional Application No. 63 / 549,091 filed February 2, 2024, the disclosures of which are hereby incorporated by reference herein in their entirety.BACKGROUND

[0002] The present general inventive concept relates to compositions of matter and methods of treating chronic disease. Along with the evolution of photosynthetic bacteria as early as 3.8 bilhon years ago, and archaea, the earliest eukaryotic cells evolved about 2.5 bilhon years ago, rising from the co-evolution of antioxidants, especially the xanthophyll carotenoids, of which astaxanthin is the highest form. These antioxidants address the molecular pathways of photosynthesis and mitochondrial function that require a variety of cofactors to produce energy and address harmful metabolic waste products. As cells became more complex and metabolically active, they produce higher levels of adenosine triphosphate (ATP) for energy from the consumption of oxygen, which produces higher amounts of metabolic waste products such as reactive oxygen species (ROS). ROS. including oxygen singlets and hydrogen peroxide derivatives, are free radicals that if uncontrolled, cause oxidative stress that causes damage to essential components of the cell, including proteins, lipids, carbohydrates and nucleic acids, leading to cell death. Accumulated unmitigated oxidative damage in the mitochondria over time is increasingly implicated in the development of chronic diseases such as cardiovascular disease, diabetes, neurodegenerative diseases and cancer.

[0003] One such example is type 2 diabetes mellitus (T2DM), where high blood sugar accumulates due to a lack of insulin-regulated processing, owing to the destruction of insulinproducing beta cells in the pancreas. This cell damage is exacerbated by overconsumption of high fructose and glucose that produces excessive ROS during mitochondrial respiration, and the lack of dietary antioxidants in modem processed foods to quench these free radicals. In addition to being the most potent antioxidant, ASX has many cell signaling properties that suppress destructive inflammation associated with oxidative stress, including inhibiting the NF-KB pathway thatproduces pro-inflammatory cytokines, and preventing apoptotic cell death via modulation of MAPKs and the PI3K / Akt pathway.

[0004] In order to address the problem of excess ROS with increasing metabolic needs, early life forms evolved antioxidants that could quench these free radicals and prevent cellular damage, such as vitamins A, C and E, and xanthophyll carotenoids such as beta-carotene, lycopene and astaxanthin. Carotenoids are responsible for the bright colors in fruits and vegetables, and in the case of astaxanthin, the pink color in salmon and other animals such as flamingos that consume astaxanthin-producing organisms such as brine shrimp. Astaxanthin (ASX) evolved to be the apex carotenoid, and as a free radical scavenger, ASX is more than 65 times stronger than vitamin C and 50 times more powerful than vitamin E in protecting cell membranes. ASX has been shown to be more effective than other carotenoids at singlet oxygen quenching by being up to 800 times stronger than coenzyme Q, 6000 times greater than vitamin C, 550 times more powerful than green tea catechins, and 11 times stronger than beta-carotene.BRIEF SUMMARYDisclosed herein is an astaxanthin nanoemulsion that is superior to other commercial forms of Astaxanthin (ASX) in cost of production, bioavailability and bioactivity. Due to the much higher bioavailability' and bioactivity' of the inventor’s ASX-NE, oral administration of the composition in patients can significantly build on prior clinical evidence of improvement in patients taking much less bioavailable and bioactive forms of ASX for treatment of chronic diseases such as cardiovascular disease, immune dysfunction, neurodegenerative diseases, inherited mitochondrial diseases, wound care and fertility7, among others. In T2DM, this can result in reduced blood sugar and A1C levels, reduced fatigue, decreased insulin resistance, and lower serum inflammatory markers compared to prior formulations of ASX.

[0005] These and other claims, embodiments, and descriptions of the present general inventive concept will become more clearly understood from the following detailed description and attached figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Example embodiments of the present general inventive concept can be more clearly understood by reading the present specification and claims in view of the following drawings in which:FIG. 1 A illustrates a 3S, 3’S stereoisomer of astaxanthin (ASX);FIG. IB illustrates a 3R, 3’S stereoisomer of ASX;FIG. 1C illustrates a 3R, 3’R stereoisomer of ASX;FIG. 2A illustrates an all trans (la, 2a, 3a) isomer of ASX;FIG. 2B illustrates a 13-cis (1c, 2c, 3c) isomer of ASX;FIG. 2C illustrates a 15-cis (Id, 2d, 3d) isomer of ASX;FIG. 2D illustrates a 9-cis (lb, 2b. 3b) isomer of ASX;FIG. 3 illustrates exemplary membrane-spanning properties of ASX;FIG. 4A illustrates Fuji 02 supplement astaxanthin;FIG. 4B illustrates extracted attritted supplement astaxanthin;FIG. 5 is a graph illustrating effectiveness of a bioeffective dose of astaxanthin;FIG. 6A illustrates Astaxanthin Oleoresin diluted in saltwater - 6 hours; andFIG. 6B illustrates ASX with 2.5% PVP - 6 hours.DETAILED DESCRIPTION

[0007] Reference will now be made to example embodiments of the present general inventive concept, examples of which are illustrated in the accompanying drawings and / or described herein. The example embodiments are intended to explain features of the present general inventive concept by way of example and not by way of limitation, noting the scope of the presentgeneral inventive concept can be understood with reference to the disclosed subject matter and equivalents thereof.

[0008] Astaxanthin (ASX) has a unique structure ideally suited for neutralizing reactive oxygen species (ROS). ASX contains a long nonpolar hydrophobic core, capped on either end with polar hydroxyl and keto groups that quench harmful singlet oxygen species, scavenge peroxyl and hydroxyl radicals and convert them into more stable compounds, preventing the formation of free radicals and inhibiting the auto-oxidation chain reaction. Due to its dual hydrophobic and hydrophilic properties it can pass easily from cell to cell and readily transit the blood brain barrier. Astaxanthin possesses two identical asymmetric atoms at C-3 and C-3' making possible three optical stereoisomers: 3S.3'S; 3R,3'S; and 3R,3'R.

[0009] FIGS. 1A to 1C illustrate the three stereoisomers of ASX, where FIG. 1A illustrates a 3S, 3’S stereoisomer of astaxanthin (ASX), FIG. IB illustrates a 3R, 3’S stereoisomer of ASX, and FIG. 1C illustrates a 3R, 3’R stereoisomer of ASX.

[0010] In addition, as illustrated in FIGS. 2A to 2D, ASX can exist in cis or trans form, where the all-trans form can readily isomerize into cis-trans mixtures and cis forms, especially the 9-cis and 13-cis isomers, due to increased temperature, exposure to light, or the presence of acids. Here, FIG. 2A illustrates an all trans (la, 2a, 3a) isomer of ASX, FIG. 2B illustrates a 13-cis (1c, 2c, 3c) isomer of ASX, FIG. 2C illustrates a 15-cis (Id, 2d, 3d) isomer of ASX, and FIG. 2D illustrates a 9-cis (lb. 2b, 3b) isomer of ASX.

[0011] While the vast body of literature on the benefits of dietary’ consumption of ASX continues to grow, the ability to extract concentrated amounts ASX in a form that is therapeutically bioavailable to humans has been a challenge. The largest sources of astaxanthin for extraction come from the yeast Phaffia rhodozyma. the green algae Haematococcus pluvialis (HP), and by extraction from the shells and biomass of marine arthropods such as shrimp and krill. HP-encysted red cells are a rich source of ASX, containing 3-5% of their dry weight as ASX. Astaxanthin can also be chemically synthesized from petroleum byproducts, such as DSM synthetic astaxanthin described as, ‘ CAROPHYLL® Pink 10% CWS consists of violet-brown to brown-violet, free flowing particles (beadlets). They contain astaxanthin in a cornstarch-coated matrix of lignosulfonate and com oil. Ethoxyquin is added as an antioxidant.” Synthetic ASX can be furtherchemically modified to enhance solubility, such as in the clinically tested astaxanthin derivative CDX-085. developed by Cardax Pharmaceuticals.

[0012] The chemical composition of ASX derived from these sources is not identical, and in fact, produces different isomeric forms that can greatly impact bioactivity in animals and humans. ASX from Phaffia yeast is 3R.3'R, while ASX from HP algae is 3S,3'S. The synthetic forms of ASX, including the clinical Cardax candidate CDX-085, contain a racemic mixture of a 1 :2: 1 ratio of 3S,3'S; 3R,3'S; and 3R,3'R. DSM synthetic ASX was found to be toxic in high doses.

[0013] FIG. 3 illustrates exemplary membrane-spanning properties of ASX. ASX has a unique structure among the antioxidant carotenoids and vitamins such as vitamin C. with a long hydrophobic chain that inserts across the entire width of the cell membrane, with polar end groups that situate inside and outside the mitochondrial membrane to quench aqueous free radicals and participate in cell signaling. As illustrated in FIG. 3, the exemplary bi-layer membrane 35 can include the ASX cores 12 and polar heads 18.

[0014] The polar ring structures are configured to anchor the non-polar backbone in the center of the cell plasma membrane so that the molecule is very stably positioned. However, not all isomers of ASX contain these membrane-spanning properties. Recent studies have described the 3S, 3'S form of ASX to preferentially insert into the cell plasma and properly anchor the hydrophobic chain across the membrane, due to the angles on the C-3 and C-3' atoms of this isomer. ASX with other isomer configurations is unable to properly insert into the cell membrane, and thus remains outside of the cell with much lower bioactivity and is excreted into circulation. This may explain why the red pigment from yeast-derived ASX, containing all 3R, 3’R, when fed to fish is easily washed away when the fish are cleaned to reveal white flesh, as the ASX is not properly taken up by the cell membranes. Synthetic ASX, including Card ax's modified water- soluble versions, contains only 25% of the proper 3S,3'S form, and this may explain the drug's poor performance in recent human clinical trials.

[0015] Additionally, whether the ASX is found in the trans vs. cis configuration may also contribute to differences in bi oavai 1 ability and bioactivity of ASX from different sources. All-trans ASX was found to be more easily absorbed in the gut following oral administration, and more easily able be transported across cell membranes and tissues due its enhanced ability to permeate the lipid membrane, as compared to the sterically bulkier cis isomer. Esterification of ASX can alsoimpact bioavailability, as esterified ASX was found to be more chemically stable and more readily absorbed in the gut than mono- or non-esterified ASX. This explains the poor bioactivity of yeast- derived 3R, 3’R ASX, which is non-esterified and is used mainly as a red carotenoid pigment in the feed industry. The degree of esterification, geometrical isomer and optical stereoisomer forms of ASX are important considerations in choosing an appropriate source of ASX. As such, ASX harvested from HP algae cysts is a rich source of all-trans, all-esterified, 3S,3'S ASX.

[0016] However, prior extraction methods from HP have been costly and inefficient. The industry' standard is using high temperature super-critical carbon dioxide (SCCO2) to extract ASX and reformulate the particles aided by a small amount of ethanol (ETOH). This process denatures the molecule while only reducing the particle size from the 60 pM HP cyst to a 4-6 pM particle, which is too large to be absorbed by the human gut and is mostly excreted. The present applicants developed a patented process to extract ASX from HP using high-shear, low-temperature and pressure milling to protect ASX from denaturing and degradation through thermal or chemical processing. This involves a vessel filled with small ceramic or steel balls and rotating bars spinning at several hundred RPM, then filled with ethanol and a cooled jacket. Subsequent solubilization using food-grade ethanol reformulates it into a nanoemulsion of particles less than 100 nm. Importantly, this occurs without removing any of the constituents in the HP. Instead, all of the constituents of the HP cysts, including carbohydrates, proteins, lipids, fatty acids, etc., are retained and reformulated into a unique molecular “complex.” with novel bioavailable and bioactive properties, referred to herein as astaxanthin nanoemulsion (ASX-NE)

[0017] One of the unique features of astaxanthin molecules in the proper configuration is their ability to insert into the lipid bilayer without degrading the cell membrane structure, thereby shielding the redox state while maintaining the functional integrity of the mitochondria. With this method, ASX is esterified for stability and encased in micelles and liposomes in a proprietary configuration of necessary' carbohydrates and lipids to facilitate incorporation into cell membranes, a beneficial additive property known as the ‘"entourage effect." This is partly due to the inclusion of a rich source of esters such as glycolipids and phospholipids which support the structural integrity of the plasma membrane as the ASX is inserted.

[0018] Some of the most common fatty acids and their derivatives detected in HP cysts were linolenic acid (14.83%), followed by pentadecanoic acid (14.25%), palmitoleic (7.77%) andlinoleic acid (4.4%). Omega-3 PUFAs such as linolenic acid and pentadecanoic acid are important precursors of beneficial eicosanoids, while linoleic acid is an Omega-6 PUFA that produces less beneficial eicosanoids. Eicosanoids are perhaps the most ancient hormone system, ubiquitous in biology and perhaps involved in virtually all metabolic processes. They are defined as signaling molecules made by the oxidation of arachidonic acid and PUFAs. Eicosanoids are involved in promoting or inhibiting inflammation, allergy, fever and other immune responses; contributing to the perception of pain; regulating cell growth; controlling blood pressure; and modulating the regional flow of blood to tissues. The main forms of eicosanoids are prostaglandins, thromboxanes, leukotrienes, lipoxins, resolvins, and eoxins. Humans, as most mammals, are unable to convert Omega-6 PUFAs into Omega-3 PUFAs, and Omega-3 PUFAs must be incorporated into our diet at a higher rate than Omega-6 PUFAs by consuming fish and fish oils, in order to support optimal eicosanoid metabolism. As such, the higher composition of Omega-3 PUFAs in the ASX nanoemulsion would be expected to yield more beneficial eicosanoid synthesis and bioactivity.

[0019] The oxidation of lipids can be hazardous to cells, particularly when close to the nucleus, and there are elaborate mechanisms to prevent unwanted oxidation by COX, the lipoxygenases, and the phospholipases. The enzymes that are biosynthetic for eicosanoids (e.g., glutathione-S-transferases, epoxide hydrolases, and carrier proteins) belong to families whose functions are involved largely with cellular detoxification. This suggests that eicosanoid signaling might have evolved from the detoxification of ROS and the cell must realize some benefit from generating lipid hydroperoxides in close proximity to its nucleus. Indeed, PGs and LTs may signal or regulate DNA-transcription there, and LTB4 is ligand for PPARa. The inclusion of fatty acids essential to eicosanoid synthesis in the ASX-NE molecular complex, embedded in the membrane and close to the nucleus also would be expected to confer a regulatory benefit to the cell.

[0020] Example embodiments of the present general inventive concept can be achieved by an ASX-NE nanoemulsion molecular complex that is superior to other commercial fonns of ASX in terms of cost of production, bioavailability and bioactivity. Due to the much higher bioavailability7and bioactivity of the present ASX-NE, it is expected that oral administration in patients will significantly build on prior clinical evidence of improvement in patients taking much less bioavailable and bioactive forms of ASX in chronic diseases such as cardiovascular disease, immune dysfunction, neurodegenerative diseases, inherited mitochondrial diseases, wound care and fertility, among others. In T2DM. this will result in reduced blood sugar and A1C levels, reducedfatigue, decreased insulin resistance and lower serum inflammatory' markers compared to prior formulations of ASX.

[0021] The bioavailability' of the most abundant and most active green tea antioxidant, epigallocatechin gallate (EGCG) remains uncertain, and considering the present molecular complex of nano emulsified astaxanthin can be a carrier, it can be used as an example of the adjuvant, antioxidant, entourage effect of the present molecular complex. It is suggested that EGCG may be exploited to craft strategies for the development of an antiaging or age-delaying agent, and a therapeutic for neurodegenerative diseases such as Alzheimer’s and Parkinson’s.

[0022] The Elaematococcus pluvialis (HP) biomass astaxanthin is likely all trans and all esterified when raw. In the HP, the astaxanthin molecular complex is likely lipids but also proteins and carbohydrates. A review of the various processes suggests it is likely that virtually all processes other than the present methods convert trans to cis and de-esterifies.

[0023] The cis has a larger geometry, and this affects its bioavailability as well as its targeted destination. It is likely that after the processing stage the biomass is still esterified and trans, and it is likely that the racemic ratio of the stereoisomers in the synthetic results in cis de- esterified and coupled and would polymerize and have an affinity for the surface of the cell plasma membrane but not the insertion and bridging the esterified trans does which makes it bioactive and conductive to passage in and out of the cell and physically reinforcing the cell plasma membrane, which the polymerized cis de-esterified if accumulating on the surface of the cell plasma membrane would block passage and in sufficient quantity destroy the cell plasma membrane. Accordingly, the cis can end up in lipids and proteins accumulating in and among tissues and organs, muscle, fatty tissue, liver etc., and the esterified trans can end up in the cell plasma membrane where it can be maximally effective to mitigate ROS near the source, near the mitochondria or even in the membranes of the mitochondria.

[0024] As to the green tea catechin EGCG, a potent polyphenol, yet know n to be one of the most biologically unavailable molecules since it is a powerfully reactive oxidant that reacts and denatures both in the digestive tract and the blood plasma. While EGCG has been shown to effectively break up tau fibers that may contribute to neuropathologies such as Alzheimer’s, it cannot cross the blood brain barrier (BBB).

[0025] The present oleoresin of ASX NE can be considered an ideal carrier for EGCG and other relatively small but bio-unavailable molecules to enable the ability to cross the BBB.

[0026] The FDA limit in dose for EGCG per day is about 338 mg per day. If bioavailable as part of the present molecular complex, this could be reduced by an order or more, such as about 12 mg a day.

[0027] The present molecular complex can carry the EGCG through the blood brain barrier and present unreacted EGCG to destroy the tau fibers creating the blocking amyloid structures causing Alzheimer's.

[0028] For example, a quick dose to a mouse of a mix of our processed astaxanthin milled with EGCG, typically a crystalline powder of about 17 microns at 50 ppm in 3 grams of feed to a 20 gram mouse can demonstrate efficacy.

[0029] There are many “natural” molecules like catechins and EGCG which have great potential power but need bioavailability that the present complex can provide. It has been widely reported that EGCG destroys the tau fibers that bind the amyloid fats causing Alzheimer’s disease, but it is ineffective because of exceptionally low bioavailability and inability to cross the BBB.

[0030] The present molecular complex of esterified trans astaxanthin composed of micelle and liposome structures rich in lipids, proteins and carbohydrates among others is exceptionally bioavailable and EGCG can be nano sized in our nano emulsion and protected in the digestive tract and the blood plasma by our molecular complex.

[0031] It is possible to take, for example, a kilo of HP having about 4% astaxanthin, i.e., about 40 grams of astaxanthin, and add about 80 grams of green tea extract with 40 grams of EGCG and mill in a liter and a half of food grade ethanol (ETOH) in a gallon size attrition mill with 3 mm balls and 400 RPM for 30 minutes. The ETOH can then be extracted at low temperature under a hard vacuum after adding 200 grams of an oil such as cod liver oil. The resulting oleoresin could then be prepared to dose the astaxanthin and EGCG at the desired ppm in the feed. In the low temperature gentle extraction of the ETOH under vacuum, the constituents, all sub-micron size, will reassemble in a non-polymerized molecular structure of lipids with proteins, carbohydrates and the small molecule of EGCG in micelle and liposome structures.

[0032] Proofs of this can be the difference of blood plasma EGCG in the present nano emulsion (NE) and in normal water mixed green tea extract, and proofs in clown fish and mice that the present molecular complex is configured to cross the blood brain barrier.

[0033] Accordingly, one can use the present low temperature nano emulsification process with other natural extracts and materials from plants, algae, and animal tissue to create and form molecular complexes with high bioavailability. The processing with HP and green tea extract using ETOH as a solvent and vacuum removal of the ETOH after adding some oil is one of many exemplary' approaches of the present general inventive concept.

[0034] One can use both chemical and mechanical processes to liberate the astaxanthin at the molecular level from the cyst. This can be done by a closed mill using a temperature-controlled solvent and mechanical energy. In some embodiments the process does not exceed 50 degrees C.

[0035] One can preserve the molecular form of the extracted attritted molecule of astaxanthin so to deliver it to the cell plasma membrane through the GI tract, digestive membrane and blood system by “Plating” it on a bio carrier, and this can be done during the above milling process or in a second milling process.

[0036] One can use the HP biomass, but given the small amount of a bioeffective dose in this case, e.g., about 400 ppm, after milling, a large amount of oleoresin can be made available to be plated on most any digestible biomass.

[0037] The resulting molecular compound complex can be a trans version of the astaxanthin, esterified, surrounded by micelle and liposome lipid structures carrying a wide variety7of constituents retained from the Haematococcus pluvialis biomass as well as other constituents that can be added, such as EGCG. The resulting molecule can provide many benefits, including, but not limited to, enabling and promoting eicosanoid metabolism as an adjuvant, of great value to molecular metabolic health. It is also a powerful antioxidant, protecting a wide variety' of metabolic molecular processes, including that of the mitochondria, eicosanoid function on and near the cell plasma membrane. It can be an adjuvant antioxidant of great value to vaccines and immunotherapies, and as an antioxidant of great value to the thymus making abundant robust T cells. It also serves as a carrier of very unavailable biomolecular powerful molecules such as EGCG based on its structure and power to transit the blood brain barrier.

[0038] Biological processes involving amino acids, proteins, enzymes and hormones can be vulnerable to oxidative stress and processes involving these can be impaired by ROS and oxidative stress. Biological processes involving these items can benefit from the presence of astaxanthin.

[0039] When astaxanthin from the SCCO2 process is included in broodstock feeds and compared to broodstock feeds including astaxanthin prepared by the present process, dramatic differences in performance can be shown, which is an unexpected result of dramatically different bioavail ability and bioeffective doses based on bioavailability.

[0040] FIGS. 4A and 4B are photos depicting astaxanthin bioeffective dose in broodstock diets for Blue tangs and effect on yolk size. FIG. 4A illustrates Fuji SCCO2 supplement astaxanthin. FIG. 4B illustrates SN extracted attritted supplement astaxanthin.

[0041] Referring to FIGS. 4A and 4B, it can be seen that after three weeks on broodstock feed blue tangs fed a broodstock feed supplemented with biomass of Fuji Astaxanthin extracted by SCCO2 and left in the biomass, and SN’s extracted and attritted astaxanthin, there by single molecules of astaxanthin “plated” upon the nano sized substrate in sizes below 1 micron, show a dramatic difference in yolk mass.

[0042] It is to be noted that the construction of the yolk in most egg layers is a rapid process of hours: the ovaries send a hormonal message picked up by the thyroid that triggers the making of approximately three essential seleno-proteins. The liver picks these up and uses them to make vitellogenin (VTG). The VTG is then secreted and picked up again to be converted into the egg yolk. In the case of most marine species this involves hydrolysis to assure the egg will float up to the columns at the surface rich in plankton.

[0043] In the case of the blue tang, the broodstock spawn at near sundown, the females doing a spiraling dance of two to three meters height, followed by the males who assure that the milt and the eggs are well mixed and fertilized. The eggs hatch the next afternoon. Upon hatching the animal has no working eye, mouth, GI Tract, or nervous system. It must develop to be able to begin exogenous feeding within about three days. In nature there is astaxanthin in the food chain. In hatcheries not so much. The eggs on the right cannot survive for more than a few hours. The eggs on the right are capable of being raised through grow out.

[0044] The difference is bioavailability. The molecules “Plated” on the biomass in sizes below one micron can pass through the GI system and the digestive membranes, through the blood, but once the molecules come into contact with a cell, the chemical attraction of the astaxanthin part of this molecule pair is much stronger for the cell plasma membrane and it launches there and takes up effective residence. Once there it dramatically reduces oxidative stress and the functions and reactions involving amino acids, proteins, hormones and enzymes are enhanced enormously.

[0045] To demonstrate the bioeffective dose of the present astaxanthin composition, the composition was used in broodstock diets of clown fish and nest production where it was found that using the present astaxanthin compound resulted in about 130 nests in residence, whereas in two weeks without astaxanthin it dropped to 80. After two weeks back on astaxanthin it recovered.

[0046] FIG. 5 illustrates a bioeffective dose of astaxanthin in rotifers and resistance to Juglone.

[0047] Since the algae HP encysts from an evolutionary developmental point of view to safeguard the cyst during stasis, where it has no defenses other than the encystment, it is natural to understand that the cyst is strong, robust, not digestible, and that in order to overcome this strategy, it is possible to break the molecules out of the cysts using a mechanical process, where one can extract the hydrophobic molecule from the hydrophilic biomass using a chemical process and deliver very small molecular forms of the molecule, e.g., less than one micron through two aqueous streams, through a membrane and present it to the cell plasma membrane.

[0048] It has been shown by the present process and composition that the bio availability of astaxanthin milled as described above, when presented from the “extracted” biomass was much higher than the fully extracted biomass with all the astaxanthin present, or by addition of portions of the oleoresin extracted from the biomass by our own process.

[0049] In such a case the extracted biomass is typically light pink, almost while, and as much as 99% of the astaxanthin has been extracted. It is this biomass containing very small amounts of astaxanthin that shows the highest bioavailability. The pairing of the astaxanthin molecule with some molecule(s) of biomass in this case involves a minimal ratio of astaxanthin to biomass molecules.

[0050] For example, it is known that using SCCO2 in diets with fish, adding more usually has little or no effect. However, with the present composition, it has been found that adding less in proportion to the biomass and all milled to less than one micron together and having the astaxanthin ‘■plated” on the biomass substrates can increase the bio availability at surprising ratios and reduces the required bioeffective dose dramatically.

[0051] Studies have shown that ASX demonstrates significant ROS -scavenging, NF-KB inhibiting, Nrf2 / sirtuin / PPAR-y-boosting activity', as well as thymic T cell maintenance and immune enhancing properties, all with far less bioavailable versions of ASX. Provided herein is a significantly more bioavailable and bioactive formulation of ASX that will demonstrate a more pronounced effect on preventing and mitigating hyperinflammatory disorders, inhibiting vaccine adjuvant and immunotherapy inflammation-mediated immunosuppression and reactogenicity, and have a significant impact on inhibiting thymic involution and promoting life-long adaptive immunity.

[0052] As illustrated and described herein, example embodiments of the present general inventive concept can be achieved by a composition of matter and method of treating hyperinflammation disorders using ASX-NE assembled on an astaxanthin core in a nanoemulsion formulation for disorders including but not limited to cytokine release syndrome and cytokine storm (CS), Acute Lung Injury (ALI), Acute Respiratory Disease Syndrome, (ARDS), Systemic Inflammatory Response Syndrome (SIRS), resulting from but not limited to viral and microbial infections, autoimmune disorders, and treatment with vaccine adjuvants and immunotherapies in order to boost immune responses to these therapies. These therapies include but are not limited to STING agonists and STING pathway activators, RIG-I / MDA5 agonists. TLR agonists, live- attenuated viral vaccines, mRNA vaccines, vaccine adjuvants, nanoparticle vaccines and immunotherapies, bacterial therapies and bacterially-based vaccines, exosomal vaccines and immunotherapies, cell therapy vaccines and immunotherapies including CAR-T, cytokine therapies and cytokine-conjugated therapies, antibody therapies and antibody-drug conjugates, gene- delivered vaccines and immunotherapies, vaccines and therapeutics that induce pro-inflammatory cytokines.

[0053] Example embodiments of the present general inventive concept can also be achieved by a composition of matter and method configured to treat excessive NF-KB signaling and inhibitproduction of pro-inflammatory cytokines using ASX-NE, alone or in combination with vaccines, vaccine adjuvants and immunotherapies in order to boost immune responses and limit reactogenicity. These therapies include but are not limited to STING agonists and STING pathway activators, RIG-I / MDA5 agonists, TLR agonists, live-attenuated viral vaccines, mRNA vaccines, vaccine adjuvants, nanoparticle vaccines and immunotherapies, bacterial therapies and bacterially- based vaccines, exosomal vaccines and immunotherapies, cell therapy vaccines and immunotherapies including CAR-T, cytokine therapies and cytokine-conjugated therapies, antibody therapies and antibody-drug conjugates, gene-delivered vaccines and immunotherapies, vaccines and therapeutics that induce pro-in fl ammatory cytokines.

[0054] Example embodiments of the present general inventive concept can also be achieved by a composition of matter and method to treat thymic involution and restore T cell function using ASX-NE.

[0055] A number of diseases are thought to have etiologies and progress fueled by reactive oxidative species (ROS), oxidative stress, and downstream consequences of these conditions. These diseases include Type 2 Diabetes Mellitus (T2DM), cancer, Alzheimer’s Disease, Parkinson Disease, many forms of dementia, heart disease, diseases of the liver, kidney and eyes, and many others, including rare genetic mitochondrial diseases resulting in excessive ROS production. In many cases approaches to addressing these etiologies and progressions are believed to be valuable and effective through the use of specific molecules. Most of these are hydrophobic antioxidants such as astaxanthin, vitamins C and E, Epigall ocatechin gallate (EGCG), co-en / mc Q10 among others. The limits of these approaches are identified and defined by several barriers and systems, including poor bioavailability and a lack of adequate delivery systems and methods that are widely seen as hindering their progress and success.

[0056] Inherited mitochondrial mutations that affect various proteins and complexes I through IV of the mitochondrial electron transport chain (ETC) converge on excessive mitochondrial ROS production that leads to degenerative disease. Certain cell types containing larger amounts of mitochondria, such as neural, retinal and cochlear ganglion cells, pancreatic [1- cells, myocytes and cardiomyocytes, are particularly vulnerable to damage from excessive mitochondrial oxidative stress.

[0057] For example, Friedreich’s Ataxia (FA) is a progressive neurodegenerative disease caused by inherited mutations in the mitochondrial electron transport chain (ETC) protein frataxin (FXN), which is required for maintenance of iron-sulfur clusters for enzymes involved in oxidative phosphorylation, the Krebs Cycle, and other cellular events important for mitochondrial iron metabolism and iron homeostasis. FXN-deficient cells exhibit oxidative stress, mitochondrial iron accumulation, decreased ATP production, and cellular dysfunction. FA patients exhibit neural and skeletal ataxias, diabetes, hearing and vision loss, and cardiomyopathies which are typically fatal.

[0058] Another example is Leber’s Hereditary Optic Neuropathy (LHON) and Leigh Syndrome, where inherited mutations affect the function of complex I. resulting in excessive ROS production that for LHON affects mainly the retinal ganglion cells and causes progressive blindness, and neuropathies and lethal lung failure for Leigh Syndrome. Inherited mutations in complex I also cause maternally inherited diabetes and deafness (MIDD), which can sometimes progress to more severe mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS).

[0059] Antioxidants have been clinically tested in these rare genetic mitochondrial diseases with limited success. Ubiquinone analogues such as co-enzyme Q10 that function as carriers of electrons from complex I to complex II of the ETC were seen as promising for mitigating ROS damage in these patients. However, evidence for the clinical benefits of co-enzyme Q10 in LHON patients was found to be lacking due to the poor intestinal absorption of this highly lipophilic molecule. Idebenone, a short-chained water-soluble ubiquinone derivative that is more easily absorbed through the oral route, provides protection by bypassing complex I, maintaining ATP production, and protecting against mitochondrial oxidative damage. Idebenone is approved for LHON in the EU but not in the US. The vitamin E derivative EPI-743 (PTC-743) also missed its Ph3 primary' endpoint for LHON. Omav el oxoIone, aNrf2 activator, was recently approved for FA in the US in patients 16+. For FA in particular, the lack of antioxidant reserves associated with abnormalities of activation of the transcription factor NRF2 may play an even more important role than direct ROS production.

[0060] Astaxanthin, with an ORAC value published at 2.9 million, is 800x stronger than coenzyme Q10 at single oxygen quenching and 50x more powerful than vitamin E in protecting cell membranes. Astaxanthin is also a Nrf2 activator, with NF-kB inhibiting properties as well. Thepresent astaxanthin becomes even more bioavailable through our patented milling process of the HP algae. The result is a reassembly of the HP constituents into a molecular complex of astaxanthin at the core, surrounded by lipid-rich micelle and liposome structures that also include proteins, carbohydrates, salts, ions and other beneficial constituents. It has been discovered that the combination of the bioactive molecules brings tremendous benefit for treating rare mitochondrial genetic diseases.

[0061] In order to preclinically validate the activity of the present molecular complex in these disease areas, cell culture assays using diseased human cells can be tested. The astaxanthin complex is milled, ethanol extracted, then formulated in rapeseed oil, cod liver oil or other suitable oil, for a final astaxanthin concentration of 2. 1%. or 21,000 ppm. For these assays, the oleoresin must be diluted into cell culture media from 21,000 ppm to between 5 and 200 ppm.

[0062] As described herein, the addition of a surfactant to the molecular complex can prevent aggregation and flocculation.

[0063] Surfactants are composed of both hydrophilic and hydrophobic bases. An emulsifier is a surfactant but fits within a specific range of surfactant functions. Surfactants have a large range and contain emulsifiers. While a surfactant can coat the surfaces an emulsifier does not. Optimal oil / water emulsification can include both a surfactant and an emulsifier.

[0064] The hydrophilic-lipophilic balance (HLB) of a surfactant is known as a measure of its degree of hydrophilicity or lipophilicity, determined by calculating percentages of molecular weights for the hydrophilic and lipophilic portions of the surfactant molecule.

[0065] The HLB value of the surfactant is between 0-40, while an emulsifier is typically between about 2-15, with 2-8 being an oil-coated water emulsifier, and 9-15 can represent a water- coated oil emulsifier.

[0066] If one assumes that most of the HP ends up as oil, meaning when 250 grams of HP is milled, the product comprises about 220 grams of oleoresin, the balance being removed moisture. As a next step, about 220 grams of cod liver oil is added, resulting in about 500 grams oil, which is about 21,000 ppm astaxanthin.

[0067] The process includes diluting the mixture with cod liver oil or rapeseed oil to an initial concentration of about 5000 ppm, which means about 1500 grams of oil should be added to dilute from about 20,000 ppm to 5000 ppm, resulting in about 2000 grams of oil. Subsequently, the compound can be diluted to a final concentration of about 50 ppm in cell culture media, resulting in about 200,000 grams of total liquid. Here, about 1% of the oil can be added which would be about 20 grams presuming diluting to about 50 ppm with water.

[0068] In some embodiments, the surfactant can be polyvinylpyrrolidone (PVP), a high molecular weight nonpolar emulsifier and protective colloid that forms a thin molecular layer over the surface of the individual colloidal particles to impart a positive charge and prevent aggregation. It enhances stability by reducing zeta potential, increases viscosity and reduces sedimentation. While it does not put a charge to emulsion droplets, it can form a thick protective layer around the droplets and minimizes the possibility of coalescence.

[0069] Given that the PVP polymer was 40 chains long, the present milling process reduced this to a monomer, meaning it would be as much as 40 times more powerful.

[0070] In addition, while rapeseed oil is an abundant and inexpensive excipient, recent literature has described previously unappreciated biological functions for the lipid compositions of oils, and not all lipids were found to be beneficial.

[0071] Lipids are heterogeneous water insoluble metabolites mainly comprised of cholesterol and fatty’ acids, such as glycerophospholipids, sphingolipids, and sterol lipids. They are important energy sources and essential plasma membrane components and are also involved in many cellular processes such as metabolism, immune function and stress responses. Different classes of lipids can act as metabolic intermediates, constituents of membranes, and signaling molecules in immune cells, and can produce dramatically different functional outcomes.

[0072] For example, linoleic acid is an omega-6 polyunsaturated fatty acid (PUFA) that is a positive regulator of T-cell activation, proliferation, and metabolic function. In contrast, palmitic acid is a saturated fatty acid (SFA) implicated in promoting chronic inflammation, impairing T-cell metabolic function, and restricting cell membrane fluidity that can impair immune cell antigen presentation.

[0073] Omega-3 PUFAs such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EP A) are essential fatty- acids that can enhance many aspects of cell metabolism and immune function. Metabolites that result from the processing of these fatty acids, such as resolvins, maresins and protectins, perform additional beneficial functions in regulating immune homeostasis.

[0074] The composition of rapeseed oil contains harmful palmitic acid, while lacking beneficial omega-3 fatty' acids such as DHA and EP A. In some embodiments, rapeseed oil can be substituted with cod liver oil, which typically lacks harmful SFAs such as palmitic and stearic acid, and instead contains high levels of beneficial omega-3 DHA and EP A. It is expected that the optimal lipid components of cod liver oil enhance the bioactivity of the present astaxanthin molecular complex while enabling the solubility of our nanoparticles following milling and ethanol extraction.

[0075] Orally administering large amounts of molecular complexes at one time may exceed the emulsifying capacity of the gastro-intestinal (GI) tract and what bile acids arriving from the liver via the gall bladder can supply to the small intestine.

[0076] The digestive system is a set of complex interacting systems and subsystems, some of which place severe limits on processes. As such, systems have been forced to evolve to solve digestive problems such as enhancing the bioavailability of an ever-changing diet. The use of bile salts in our digestion is one example of the evolution of these support systems.

[0077] Bile salts are powerful surfactants and emulsifiers that create highly bioavailable molecular complexes of highly lipophilic and non-bioavailable molecules for transport across the GI epithelium, including carotenoids such as astaxanthin, vitamin E, carbohydrates, EGCG, among others, and even contribute powerful metabolic functions themselves.

[0078] Specific metabolic functions of bile salts evolved by bacteria in the gut have been identified. For example, certain conjugated bile salts made by the liver have been found to have been modified by bacteria in the gut, with specific benefits for treating dementia and ROS-based defects in the mitochondria, and like astaxanthin, have the advantage of transiting the blood brain barrier.

[0079] The evolution of different organisms can be rate limited by differences as large as several orders of magnitude. Viruses and bacteria can evolve so quickly that they can adjust tounique biomes over the lifetime of a host species. For example, while human biology has been challenged to adjust to what in evolutionary timelines is an instant change in diet, such that the molecular and endocrinal process are at a loss to adjust, the bacteria-rich microbiome in the human gut may be offering adjustments that are addressing these matters in a more rapid timeline. In this case, the bacterial gut microbiome offers a greater opportunity to evolve how people process their diet.

[0080] Conjugated bile salts such as taurocholic acid, tauroursodeoxycholic acid (TUDCA) and glycolic acid can be considered the main bile salts moving lipids and fats through GI membranes. They are hydrophilic and powerful surfactants and emulsifiers that can even be selected to include those with specific metabolic and therapeutic effects. For example, TUDCA is a powerful mitigator of mitochondrial dysfunction as well as being a powerful neural agent passing through the BBB like astaxanthin. While the taurocholic acid is made in the liver, TUDCA is made by bacteria in the gut by adding an NO, and the resulting molecular weight goes from 515 to 499 grams per mole. These bile salts are FDA approved, coming mostly as a byproduct from animal slaughterhouses, and can be commercially obtained as crystalline powders of very large polymers.

[0081] Unfortunately, much of the data and science relating to studying human digestion has been flawed by the methodology of using animal surrogates. Therefore, it is important to understand biological differences between humans and animal surrogates when making conclusions about human biology.

[0082] For example, mice and rats are often used as surrogates for orally administered treatments that require digestion. A 20-gram mouse might consume 15% of its body mass in an evening feeding, meaning a day’s dose of a therapeutic might be added to a single feeding. If this therapeutic requires bile acids for digestion, it is noteworthy to consider that mice and rats do not have gall bladders to provide a reservoir of bile acids to release on demand and have bile acid profiles quite distinct from humans. As such, uptake of fats and hydrophobic or amphiphilic micronutrients that rely on fat to form micelles and liposomes needed to transverse the bloodstream may be limited in these animals.

[0083] In order to address the poor gut absorption in the mouse biodistribution studies, as well as the poor solubility in cell culture media, it is possible to add PVP and conjugated taurine- based bile acids to the HP milling process. Bile salts are 95% water and the salts themselves are acrystalline powder, so making a nanoemulsion would favor a process in this regard, whereby the cysts of HP are milled from about 60 microns (pM) to less than 100 nanometers (nm). This process can additionally mill the 40,000 molecular weight (MW) PVP to nm size molecules.

[0084] It is expected that addition of these surfactants and bile acid emulsifiers will improve water solubility for cell culture and deliver much more astaxanthin through the intestine and into circulation in vivo. Additionally, formulating the oleoresin with the more immunomodulatory and metabolically favorable cod liver oil, which we predict along with the bioactive taurine-based bile salts will enhance the efficacy of our astaxanthin molecular complex in treating chronic diseases arising from mitochondria dysfunction, as well as treating disorders resulting from excessive inflammation.

[0085] The Blood Brain Barrier (BBB) can be a very' powerful barrier, protecting the brain from a wide range of risks, but also preventing some powerful molecules from reaching targets for therapy. For example, epigallocatechin gallate (EGCG) is a known disrupter of the tau fibers that bind amyloid structures that cause Alzheimer’s, yet cannot cross the BBB.

[0086] By incorporating EGCG into the astaxanthin molecular complex containing PVP, conjugated bile salts, and cod liver oil, it has enabled EGCG to transit the BBB. and its activity can be further enhanced by the overall bioactivity of astaxanthin and the other individual components in the molecular complex.

[0087] There is abundant literature describing the metabolic benefits that astaxanthin can bring to a wide range of mitochondrial and metabolic diseases if made bioavailable. These benefits can relate to the powerful targeting of the present form of astaxanthin to incorporate into the cell plasma and mitochondrial membranes and exercise powerful antioxidant and gene regulatory functions. As described herein, the astaxanthin molecule can be an esterified, all-trans (all-E) 3S, 3’S astaxanthin, intended to treat rare genetic mitochondrial diseases, cancer, inflammation, neurological disorders, heart disease, T2DM, skin, liver, kidney and eye disorders, and many others.

[0088] Some embodiments of the present general inventive concept include adding safe, naturally occurring bile salts to algae, milling them in ethanol with a small amount of complementary emulsifiers such as PVP, thus enabling the addition of other bioactive componentssuch as EGCG, and subsequently removing the ethanol by low temperature vacuum processing while adding omega-3 polyunsaturated fatty acid (PUFA)-rich cod liver oil, all of which will enhance astaxanthin and EGCG bioavailability and will be powerfully bioactive by themselves. The bile salts can be processed as a crystalline polymer, with high molecular weight, much like PVP. It is expected that low temperature milling of these large molecules in ethanol so that they are not denatured will result in more efficacious bile salts that will significantly enhance the bioavailability of our molecular complex. A mix of applied surfactants and emulsifiers provides significant enhancement in gut uptake and targeted delivery of astaxanthin. The selection of taurine-based TUDCA as the bile salt and the substitution of palmitic acid-containing rapeseed oil for high omega-3 PUFA cod liver oil in our formulation can add additional immune and metabolic enhancing properties.

[0089] Example embodiments of the present general inventive concept provide a highly bioavailable molecular complex composed of astaxanthin, along with the other Haematococcus pluvialis (HP) algae-derived components, with the addition of PVP and taurine-based conjugated bile acids in a cod liver oil excipient. Additional examples can also include EGCG.

[0090] By using safe, natural surfactants and emulsifiers in the present astaxanthin-based molecular complex to emulsify large quantities of lipid-rich molecular complexes resulting from processing of HP-derived astaxanthin into a highly bioavailable form, it is possible to enhance the positive molecular metabolic effects of these bioactive molecules along with the bioactivity of astaxanthin. This combination provides a very7powerful delivery system throughout the body, including the ability to transit the BBB. while bringing nature's most powerful agents and antioxidants to areas of the body most in need of treatment.

[0091] Example embodiments of the present general inventive concept can be achieved by a method of treating disease in a patient administering a pharmaceutical composition (orally or otherwise) including a therapeutically effective amount of esterified 3S, 3’S trans astaxanthin in combination with one or more constituent ingredients assembled on a molecular core of the astaxanthin. The esterified trans astaxanthin can be derived from Haematococcus pluvialis (HP) algae, and the molecular core can include a 3S, 3’S stereoisomer to facilitate assembly of the constituent ingredient to the molecular core and to increase bioavailability and bioactivity of thecomposition relative to astaxanthin compounds having a 3R, 3’R stereoisomer due to membrane incorporation and esterification.

[0092] In some embodiments, the one or more constituent ingredients is epigallocatechin-3- gallate (EGCG). The pharmaceutical composition can include surfactant polyvinylpyrrolidone (PVP) and / or emulsifier tauroursodeoxy cholic acid (TUDCA) to increase bioavailability’ of the composition and to facilitate transit of the composition across the patient’s blood brain barrier (BBB).

[0093] Example embodiments of the present general inventive concept can also be achieved by a pharmaceutical composition for the treatment of disease in patients, including a therapeutically effective amount of esterified trans 3S, 3’S astaxanthin combined with one or more constituent ingredients assembled on a molecular core of the astaxanthin, wherein the molecular core is configured to facilitate assembly of the one or more constituent ingredients to the molecular core to increase bioavailability of the composition and / or the one or more constituent ingredients to reduce symptoms of the disease.

[0094] The one or more constituent ingredients can be epigallocatechin-3-gallate (EGCG), and the molecular core can possess a 3S, 3’S stereoisomer to facilitate transit of the EGCG through a blood brain barrier (BBB) of the patient.

[0095] The esterified trans 3S, 3’S astaxanthin can be derived from Haematococcus pluvialis (HP) algae, and the molecular core can include a 3S, 3’S stereoisomer to facilitate assembly of the one or more constituent ingredients to the molecular core and to increase bioactivity of the composition relative to astaxanthin compounds having a 3R, 3’R stereoisomer due to membrane incorporation and esterification.

[0096] The composition can include surfactant polyvinylpyrrolidone (PVP) and / or emulsifier tauroursodeoxy cholic acid (TUDCA) to increase bioavailability’ of the composition compared to an astaxanthin compound derived from HP algae using super critical CO2 extraction to facilitate transit of the composition across the patient’s blood brain barrier (BBB).

[0097] The following examples are included to describe and demonstrate aspects of the present general inventive concept.EXAMPLE 1: Characterization and Comparison of HP-Derived Astaxanthin Using Different Extraction Methods,

[0098] A molecular complex characterization and comparison study was performed of astaxanthin harvested from raw, uncracked HP cyst biomass and extracted using either the industry standard of high temperature super-critical carbon dioxide (ASX-CO2), or Adjuvia’s patented process using high-shear, low-temperature and pressure milling (ASX-NE). The composition of the molecular complexes surrounding the ASX formulations was characterized using chromatography techniques, with the rapeseed oil excipient as the diluent reference control.

[0099] Table 1 summarizes the results from the gas chromatography with mass spectrometry (GC / MS) comparative analysis of the astaxanthin samples. The samples were diluted in an appropriate solvent and analyzed by GC / MS. The relative area percentages of the reported peaks in each sample are presented, with each sample’s analytes calculated independently of the other sample.TABLE 1

[0100] As shown in Table 1, both ASX-CO2 and ASX-NE shared identified compounds including substituted benzenes and aromatics, fatty acids, and dl-a-Tocopherol. However, ASX-NE demonstrates higher relative concentrations of several beneficial fatty acids, and contains manycompounds including small terpenes, long alkanes and alkenes, fatty acid methyl esters, phthalates, that were not observed in the ASX-CO2. Importantly, the ASX-NE included the 5- and y- isomers of tocopherol (vitamin E), important for protecting the ASX molecule from oxidation.

[0101] Table 2 summarizes the results from ion chromatography (IC) analysis of the astaxanthin samples. The samples were extracted with water prior to analysis by IC. Rapeseed oil was used as a method blank for analysis. These data demonstrate the presence of important inorganic salts present in the ASX-NE that are not present in ASX-CO2.TABLE 2

[0102] The data of Table 2 demonstrate the unique and superior composition of the astaxanthin molecular complex isolated using the patented milling method, ASX-NE, as compared to the astaxanthin complex isolated using super critical CO2.EXAMPLE 2: Assessment and Comparison of Mitochondrial ROS Quenching by Astaxanthin Formulations.

[0103] ASX has been previously demonstrated to be a potent quencher of reactive oxygen species (ROS) generated by mitochondrial respiration. In order to assess the ability of the ASX-NE molecular complex to be superior in ROS-quenching as compared to the super critical CO2 (ASX- CO2), phaffia yeast-derived ASX (ASX-PF) and synthetic ASX (ASX-SYN), oxidative stress and the ability of ASX to quench ROS was measured using a human liver HepG2 hepatocyte cell-based assay. For this, HepG2 cells were seeded and allowed to recover for 24 hours in 384-well plates, then were subjected to a 2-hour pre-treatment with either 5 mM N-acetyl-L-cysleine (NAC) as a positive control (in H2O), or the ASX compounds (in rapeseed oil) in at a dose of 50 pM. This was followed by a 4-hour treatment with 30 pM of the ROS-inducing agent menadione (in 0. 1% DMSO), or vehicle control. Cells were then live-stained with the cell-permeable fluorogenic dye CellROX to measure oxidative stress, then fixed and stained with Hoechst. Plates were imaged within 24 hours of staining.

[0104] As shown in Table 3, treatment with menadione induced ROS as detected by increased CellROX signal intensity, which could then be quenched by the addition of NAC to below baseline. Even in the absence of menadione, cell culture conditions can generate oxidative stress, and pre-treatment with NAC reduced oxidative stress to below baseline from the untreated and vehicle controls. Following pre-treatment with the ASX compounds, the ASX-NE molecular complex was the only ASX that significantly reduced oxidative stress from cell culture conditions to below that observed with the positive control NAC (p <0.0001). Following treatment with menadione, only ASX-NE reversed oxidative stress, and more significantly than NAC (p <0.0001).TABLE 3

[0105] The data of Table 3 demonstrate the enhanced potency of the ASX-NE extraction method compared to the ASX-CO2 extraction method, which had no activity in this assay. The ASX-PF with 100% of the R,R isoform demonstrated ROS quenching ability in cell culture conditions, but it had minimal activity in the presence of menadione. These data demonstrate the superior quenching ability of the ASX-NE with 100% of the S,S isomer compared to the R,R isomer which cannot incorporate into the membrane, along with the potential contribution of its novel molecular complex with superior ingredients as compared to the super critical CO2 extracted material. The ASX-SYN containing 25% of the S,S isomer demonstrated partial efficacy, and at the highest dose demonstrated significant activity in the presence of menadione, although not more than NAC or 100% S,S ASX-NE. These data demonstrate the superior bioactivity' of ASX-NE compared to other forms of astaxanthin and to NAC. used as the standard of care treatment in mitochondrial inherited degenerative diseases such as Leigh Syndrome.EXAMPLE 3: Addition of PVP prevented aggregation of the astaxanthin molecular complex in water-based excipients

[0106] FIG. 6 A illustrates Astaxanthin Oleoresin diluted in saltwater - 6 hours. As shown in Figure 6A, initial tests of diluting the oleoresin into a saltwater solution resulted in significant aggregation and flocculation after 6 hrs, which would likely impede the bioavailability of the compound in getting into cells. We proposed that the addition of a surfactant to this molecular complex would prevent aggregation and flocculation.

[0107] Due to the high 40,000 molecular weight (MW) of PVP, we chose to mill it with the HP cysts, which reduced the 17 pM PVP particles to a <100nm scale. This molecular complex was dissolved in rapeseed oil following HP milling and ethanol extraction.

[0108] FIG. 6B illustrates ASX with 2.5% PVP - 6 hours. As shown in Figure 6B, the addition of PVP prevented the particles from aggregating for at least 6 hrs, demonstrating the importance of adding this surfactant to the astaxanthin molecular complex.EXAMPLE 4: Addition of PVP and TUDCA enhanced bioavailability of the astaxanthin molecular complex in vivo

[0109] The ASX-NE astaxanthin molecular complex extracted using the proprietary milling method was compared to the ASX-CO2 compound for enhanced in vivo uptake and bioavailability.Additionally, the ability of the surfactant PVP and emulsifier TUDCA to enhance bioavailability was tested. For this, a study was performed in mice to compare the plasma bioavailability of astaxanthin nanoemulsions formulated in cod liver oil either isolated using the patented milling method (ASX-NE), alone or with the addition of PVP, or PVP + TUDCA, or ASX isolated using super critical CO2 (ASX-CO2), administered as a single oral gavage of 4 mg / kg. Plasma was collected at 2 hrs post-dosing and assessed for the presence of ASX by HPLC.

[0110] As shown in Table 4, ASX-NE demonstrated significantly enhanced plasma bioavailability' than the ASX-CO2 (p = 0.04). The addition of PVP to the ASX-NE enhanced plasma bioavailability, and PVP with TUDCA significantly enhanced the bioavailability compared to ASX-NE alone (p = 0.05). These data demonstrate the superiority of the ASX-NE astaxanthin molecular complex for in vivo bioavailability', further enhanced with the addition of surfactants and emulsifiers.TABLE 4.EXAMPLE 5: Addition of EGCG milled with HP-derived Astaxanthin Enables EGCG to Cross the Blood-Brain Barrier In Vivo

[0111] EGCG is a green tea catechin with promising properties against Alzheimer’s and other neurodegenerative diseases, however the molecule suffers from an inability to cross the blood brain barrier (BBB). We hypothesized that milling of EGCG along with ASX using our proprietary milling method would allow the EGCG to be incorporated into the astaxanthin molecular complex, enabling it to be transmitted across the blood brain barrier along with ASX. To test this, a study was performed in mice to compare the tissue bioavailability of astaxanthin and EGCG formulated with PVP and TUDCA in cod liver oil, with both the ASX and EGCG at 4 mg / kg as a single oralgavage. Tissues were collected at 2, 4, 8, 12, 24, and 48 hrs post oral dosing and ASX and EGCG measured by HPLC. Peak concentrations of ASX in the brain were detected at 8 hrs post dosing (15.7 ± 9.2 ng / g). The time course of EGCG in the brain also followed the time course of ASX detection, with peak EGCG also detected in the brain at 8 hrs post-dosing (0.6 ± 0.1 ng / g). These data are the first demonstration of the ability of EGCG to be detected in the brain, where the molecule when complexed with the ASX nanoemulsion has the ability to be shuttled across the BBB, with the potential to treat neurodegenerative diseases associated with the presence of harmful beta-amyloid plaques and the accumulation of tau fibers.

[0112] As illustrated and described herein, it will be understood that the descriptions of some embodiments and methods of the present general inventive concept do not limit various alternative, modified, and equivalent embodiments and methods which will occur to those skilled in the art in view of the present disclosure. Furthermore, in the description and illustrations provided herein, certain details have been set forth to provide an understanding of various embodiments of the present general inventive concept. However, some embodiments of the present general inventive concept may be practiced without such specific details.

[0113] The attached figures illustrate example embodiments of the present general inventive concept. The illustrated designs are included by way of example and not by way of limitation. Those skilled in the art will appreciate that various modifications and / or configurations of the present general inventive concept can be included based on sound engineering judgement and such modifications and / or configurations are encompassed within the scope of the present general inventive concept.

[0114] In some parts, this written description and the accompanying figures illustrate examples of certain implementations of the disclosed technology. However, the patentable scope may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the patent claims. For example, various alternative examples may define structural and / or functional elements essentially equivalent to the example embodiments described and / or illustrated herein, or may include structural and / or functional elements with insubstantial differences from the example embodiments described and / or illustrated herein.

Claims

CLAIMSWhat is claimed is:

1. A method of treating disease in a patient comprising orally administering a pharmaceutical composition comprising a therapeutically effective amount of esterified 3S. 3’S trans astaxanthin in combination with one or more constituent ingredients assembled on a molecular core of the astaxanthin.

2. The method of claim 1, wherein the esterified trans astaxanthin is derived from Haematococcus pluvialis (HP) algae.

3. The method of claim 1, wherein the molecular core comprises a 3S, 3’S stereoisomer to facilitate assembly of the constituent ingredient to the molecular core and to increase bioavailability and bioactivity of the composition relative to astaxanthin compounds having a 3R, 3’R stereoisomer due to membrane incorporation and esterification.

4. The method of claim 1, wherein the one or more constituent ingredients is epigallocatechin-3-gallate (EGCG).

5. The method of claim 1, wherein the pharmaceutical composition includes surfactant polyvinylpyrrolidone (PVP) and / or emulsifier tauroursodeoxy cholic acid (TUDCA) to increase bioavailability of the composition and to facilitate transit of the composition across the patient’s blood brain barrier (BBB).

6. A pharmaceutical composition for the treatment of disease in a patient, comprising: a therapeutically effective amount of esterified 3S, 3’S trans astaxanthin combined with one or more constituent ingredients assembled on a molecular core of the astaxanthin, wherein the molecular core is configured to facilitate assembly of the one or more constituent ingredients tothe molecular core to increase bioavailability of the composition and / or the one or more constituent ingredients to reduce symptoms of the disease.

7. The composition of claim 6, wherein the one or more constituent ingredients is epigallocatechin-3-gallate (EGCG) and the molecular core comprises a 3S, 3’S stereoisomer to facilitate transit of the EGCG through a blood brain barrier (BBB) of the patient.

8. The composition of claim 6, wherein the esterified trans 3S, 3’S astaxanthin is derived from Haematococcus pluvialis (HP) algae.

9. The composition of claim 6. wherein the molecular core comprises a 3 S. 3’S stereoisomer to facilitate assembly of the constituent ingredient to the molecular core and to increase bioactivity of the composition relative to astaxanthin compounds having a 3R, 3’R stereoisomer due to membrane incorporation and esterification.

10. The composition of claim 6. further comprising surfactant polyvinylpyrrolidone (PVP) and / or emulsifier tauroursodeoxy cholic acid (TUDCA) to increase bioavailability of the composition compared to an astaxanthin compound derived from HP algae using super critical CO2 extraction to facilitate transit of the composition across the patient’s blood brain barrier (BBB).

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