Pharmaceutical compositions and methods of treating diseases using astaxanthin core-assembled molecular complexes

Abstract

A pharmaceutical composition comprising an effective therapeutic amount of esterified 3S,3'S trans-astaxanthin and one or more components assembled on the core of an astaxanthin molecule to increase the bioavailability of the composition, and a method of treating a disease by administering an effective therapeutic amount of the pharmaceutical composition to a patient.

Description

Cross-references to related applications

[0001] This application claims priority to 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 incorporated herein by reference in their entirety. Background Technology

[0002] The present invention relates to material compositions and methods for treating chronic diseases. As early as 3.8 billion years ago, photosynthetic bacteria began to evolve. With the evolution of these, along with archaea, the earliest eukaryotic cells appeared approximately 2.5 billion years ago, evolving from antioxidants, particularly lutein-like carotenoids, with astaxanthin being the most abundant form. These antioxidants act on molecular pathways of photosynthesis and mitochondrial function, both of which require various cofactors to generate energy and process harmful metabolic waste. As cells become more complex and metabolically more active, they obtain energy by consuming oxygen to produce higher levels of adenosine triphosphate (ATP), which generates greater amounts of metabolic waste, such as reactive oxygen species (ROS). ROS, including singlet oxygen and hydrogen peroxide derivatives, are free radicals that, if left uncontrolled, cause oxidative stress, leading to damage to key cellular components, including proteins, lipids, carbohydrates, and nucleic acids, ultimately resulting in cell death. Unrelieved oxidative damage accumulated in mitochondria is increasingly believed to be associated with the development of chronic diseases such as cardiovascular disease, diabetes, neurodegenerative diseases, and cancer over time.

[0003] One such example is type 2 diabetes (T2DM), in which hyperglycemia accumulates due to the loss of insulin-regulating processes caused by the destruction of insulin-producing beta cells in the pancreas. This cellular damage is exacerbated by excessive intake of high fructose and glucose, which generate excessive amounts of reactive oxygen species (ROS) during mitochondrial respiration, and modern processed foods lack dietary antioxidants to scavenge these free radicals. In addition to being a potent antioxidant, astaxanthin possesses numerous cellular signaling properties that can inhibit destructive inflammation associated with oxidative stress, including inhibiting the NF-κB pathway that produces pro-inflammatory cytokines, and preventing apoptosis by regulating the MAPK and PI3K / Akt pathways.

[0004] To address the problem of excessive ROS (reactive oxygen species) resulting from increased metabolic demands, early life forms evolved antioxidants. These antioxidants scavenge these free radicals and prevent cell damage. Examples of these antioxidants include vitamins A, C, and E, as well as lutein-like carotenoids such as beta-carotene, lycopene, and astaxanthin. Carotenoids are responsible for the vibrant colors of fruits and vegetables. Astaxanthin, for instance, is responsible for the pink color of salmon and other animals, such as flamingos that consume astaxanthin-producing organisms like krill. Astaxanthin (ASX) has been developed into the most potent carotenoid, and as a free radical scavenger, ASX is more than 65 times stronger than vitamin C and more than 50 times stronger than vitamin E in protecting cell membranes. ASX has been shown to be more effective than other carotenoids in quenching singlet oxygen, up to 800 times stronger than coenzyme Q, 6000 times stronger than vitamin C, 550 times stronger than green tea catechins, and 11 times stronger than beta-carotene. Summary of the Invention

[0005] This paper discloses an astaxanthin nanoemulsion that is superior to other commercially available forms of astaxanthin (ASX) in terms of production cost, bioavailability, and bioactivity. Because the inventors' ASX-NE has significantly higher bioavailability and bioactivity, oral administration of this composition to patients can significantly further improve the treatment outcomes of patients with chronic diseases (cardiovascular disease, immune dysfunction, neurodegenerative diseases, hereditary mitochondrial diseases, wound care, and fertility, etc.) treated with ASX forms that have much lower bioavailability and bioactivity, building upon existing clinical evidence. In type 2 diabetes mellitus (T2DM), this results in reduced blood glucose and AIC levels, decreased fatigue, reduced insulin resistance, and decreased serum inflammatory markers compared to existing ASX formulations.

[0006] These and other claims, embodiments, and description of the present invention will become clearer and more readily understood through the following detailed description and accompanying drawings. Attached Figure Description

[0007] The exemplary embodiments of the general inventive concept of the present invention can be more clearly understood by referring to the following figures and by reading this specification and claims, wherein:

[0008] Figure 1A The 3S,3'S stereoisomer of astaxanthin (ASX) is shown;

[0009] Figure 1B The 3R,3'S stereoisomer of ASX is shown;

[0010] Figure 1C The 3R,3'R stereoisomer of ASX is shown;

[0011] Figure 2A The all-trans (la, 2a, 3a) isomers of ASX are shown;

[0012] Figure 2B The 13-cis (1c, 2c, 3c) isomers of ASX are shown;

[0013] Figure 2C The 15-cis (1d, 2d, 3d) isomers of ASX are shown;

[0014] Figure 2D The 9-cis (1b, 2b, 3b) isomers of ASX are shown;

[0015] Figure 3 Exemplary transmembrane properties of ASX are shown;

[0016] Figure 4A This shows Fuji 02 astaxanthin supplement;

[0017] Figure 4B The extracted, ground astaxanthin supplement is shown.

[0018] Figure 5 This is a graph showing the bioeffective dose of astaxanthin;

[0019] Figure 6A Astaxanthin oleoresin diluted in brine for 6 hours is shown; and

[0020] Figure 6B The ASX containing 2.5% PVP is shown – 6 hours. Detailed Implementation Plan

[0021] Exemplary embodiments of the general inventive concept of the present invention will now be described with reference to the accompanying drawings and / or description herein. These exemplary embodiments are intended to illustrate the features of the general inventive concept of the present invention by way of example rather than limitation; it should be noted that the scope of the general inventive concept of the present invention can be understood with reference to the disclosed subject matter and its equivalents.

[0022] Astaxanthin (ASX) possesses a unique structure particularly well-suited for neutralizing reactive oxygen species (ROS). ASX contains a long, nonpolar hydrophobic core capped at both ends by polar hydroxyl and ketone groups. These groups quench harmful singlet oxygen, scavenge superoxide radicals and hydroxyl radicals, and convert them into more stable compounds, thereby preventing free radical formation and inhibiting auto-oxidation chain reactions. Due to its dual hydrophobic and hydrophilic properties, it can be easily transported from cell to cell and readily crosses the blood-brain barrier. Astaxanthin has two identical asymmetric atoms at C-3 and C-3', thus potentially existing in three optical stereoisomers: 3S, 3'S, and 3R, 3'R.

[0023] Figures 1A to 1C Three stereoisomers of ASX are shown, among which, Figure 1A The 3S, 3'S stereoisomers of astaxanthin (ASX) are shown. Figure 1B The 3R, 3'S stereoisomer of ASX is shown, and Figure 1C The 3R, 3'R stereoisomers of ASX are shown.

[0024] In addition, such as Figures 2A to 2D As shown, ASX can exist in the cis configuration, wherein the all-trans form can readily isomerize to cis-trans mixtures and the cis form, especially the 9-cis and 13-cis isomers, due to increased temperature, exposure to light, or the presence of acid. Figure 2A The all-trans (1s, 2a, 3a) isomers of ASX are shown. Figure 2B The 13-cis (1c, 2c, 3c) isomers of ASX are shown. Figure 2C The 15-cis (ld, 2d, 3d) isomers of ASX are shown. Figure 2D The 9-cis (lb, 2b, 3b) isomers of ASX are shown.

[0025] Despite the increasing volume of literature on the benefits of dietary ASX intake, the ability to extract concentrated amounts of ASX in a form with therapeutic bioavailability in humans has remained a challenge. The largest sources of astaxanthin used for extraction are *Phaffia rhodozyma* and *Haematococcus pluvialis* (HP), and also from the shells and biomass of marine arthropods such as shrimp and krill. HP-encysted erythrocytes are a rich source of ASX, containing 3-5% ASX by dry weight. Astaxanthin can also be obtained through chemical synthesis from petroleum byproducts. For example, DSM synthetic astaxanthin is described as follows: “CAROPHYLLR® Pink 10% CWS consists of free-flowing, purplish-brown to brownish-purple particles (beads). It contains astaxanthin encapsulated in a corn starch-coated matrix composed of lignin sulfonate and corn oil. Ethoxyquinoline is added as an antioxidant.” Synthetic ASX can also be further chemically modified to improve solubility, such as the clinically tested astaxanthin derivative CDX-085 (developed by Cardax Pharmaceuticals).

[0026] The chemical compositions of ASX derived from these sources are not identical; in fact, they produce different isomers, which can significantly affect their biological activity in animals and humans. ASX from *Phaeophyte rubrum* is 3R,3'R, while ASX from *H. pylori* algae is 3S,3'S. Synthetic forms of ASX, including the clinical Cardax candidate CDX-085, contain a racemic mixture of 3S,3'S; 3R,3'S; and 3R,3'R in a 1:2:1 ratio. DSM synthetic ASX has been found to be toxic at high doses.

[0027] Figure 3 Exemplary transmembrane properties of ASX are illustrated. ASX possesses a unique structure among antioxidant carotenoids and vitamins such as vitamin C, featuring a long hydrophobic chain that extends across the entire width of the cell membrane, with polar terminal groups located on both the inner and outer sides of the mitochondrial membrane to quench aqueous free radicals and participate in cell signaling. Figure 3 As shown, the exemplary bilayer membrane 35 may include an ASX core 12 and a polar head 18.

[0028] The polar ring structure is configured to anchor the nonpolar backbone to the center of the cytoplasmic membrane, thus allowing the molecule to be positioned very stably. However, not all isomers of ASX possess these transmembrane properties. The 3S,3'S form of ASX preferentially inserts into the cytoplasmic membrane and correctly positions the hydrophobic chain across the membrane due to the angles at the C-3 and C-3' atoms of this isomer. ASX with other isomer configurations cannot correctly insert into the cell membrane and thus remain extracellularly, exhibiting much lower biological activity and being expelled into the circulation. This explains why, when yeast-derived ASX (containing all 3R,3'R) is fed to fish, the pigment is easily washed away when the fish are cleaned to expose the white flesh, as the ASX is not properly taken up by the cell membrane. Synthetic ASX, including the modified water-soluble version of Cardax, contains only 25% of the correct 3S,3'S form. Furthermore, this may explain the drug's poor performance in recent human clinical trials.

[0029] Furthermore, the trans or cis configuration of ASX can also lead to differences in bioavailability and bioactivity from different sources. All-trans ASX has been found to be more readily absorbed in the intestine after oral administration compared to the more sterically hindered cis isomer, and is more easily transported across cell membranes and tissues due to its enhanced ability to penetrate lipid membranes. Esterification of ASX can also affect bioavailability, as esterified ASX has been found to have higher chemical stability and is more readily absorbed in the intestine than monoesterified or unesterified ASX. This explains the poor bioactivity of 3R,3'R ASX derived from yeast, which is the unesterified form and is primarily used in the feed industry as a red carotenoid pigment. The degree of esterification, geometric isomerism, and optical stereoisomerism of ASX are important considerations when selecting an appropriate ASX source. Therefore, ASX harvested from *H. pylori* algal cysts is a rich source of all-trans, fully esterified, and 3S,3'S ASX.

[0030] However, previous methods for extracting ASX from HP were costly and inefficient. The industry standard involved extracting ASX using high-temperature supercritical carbon dioxide (SCCO2) followed by particle reforming with the aid of a small amount of ethanol (ETOH). This process denatures the molecule and only reduces the particle size from 60 μM HP encapsulations to 4-6 μM particles, which is still too large for absorption by the human intestine and is mostly excreted. The applicant has developed a patented process for extracting ASX from HP using high-shear, low-temperature, and pressure milling to protect ASX from denaturation and degradation during heat or chemical treatment. This involves a container filled with small ceramic or steel balls and a rotor rotating at hundreds of revolutions per minute, followed by filling with ethanol and a cooling jacket. Dissolution is then performed using food-grade ethanol, reforming the ASX into a nanoemulsion with a particle size of less than 100 nm. Importantly, this process occurs without removing any components of the HP. Conversely, all components of the HP encapsulation, including carbohydrates, proteins, lipids, fatty acids, etc., are retained and reformulated into a unique molecular "complex" with significant bioavailability and bioactivity, referred to herein as astaxanthin nanoemulsion (ASX-NE).

[0031] One of the unique characteristics of astaxanthin molecules in their proper configuration is their ability to insert into the lipid bilayer without disrupting cell membrane structure, thereby protecting redox states while maintaining mitochondrial functional integrity. Using this method, ASX is esterified to stabilize lipids and encapsulated in micelles and liposomes, forming a proprietary configuration of essential carbohydrates and lipids to facilitate incorporation into the cell membrane—a beneficial additional property known as the "entourage effect." This is partly attributed to the inclusion of ester-rich sources, such as glycolipids and phospholipids, which support the structural integrity of the plasma membrane upon ASX insertion.

[0032] Among the most common fatty acids and their derivatives detected in *Helicobacter pylori* (HP) capsules, linolenic acid (14.83%) was the most prevalent, followed by pentadecanoic acid (14.25%), palmitoleic acid (7.77%), and linoleic acid (4.4%). Omega-3 polyunsaturated fatty acids such as linolenic acid and pentadecanoic acid are important precursors to beneficial eicosanoic acids (EAs), while linoleic acid, an Omega-6 polyunsaturated fatty acid, produces fewer EEAs. Eicosanoic acids are perhaps the oldest hormone system. They are ubiquitous in biology and likely participate in almost all metabolic processes. They are defined as signaling molecules generated from the oxidation of arachidonic acid and polyunsaturated fatty acids. Eicosanoic acid compounds participate in promoting or inhibiting inflammation, allergies, fever, and other immune responses; contribute to pain perception; regulate cell growth; control blood pressure; and regulate local blood flow to tissues. The main forms of EEA compounds are prostaglandins, thromboxanes, leukotrienes, lipoxygenin, eoxin, and eoxin. Like most mammals, humans cannot convert Omega-6 polyunsaturated fatty acids into Omega-3 polyunsaturated fatty acids. To support optimal eicosanoic acid (EA) metabolism, Omega-3 EEA must be incorporated into our diet at a higher proportion than Omega-6 EEA through the intake of fish and fish oil. Therefore, the higher Omega-3 EEA composition in ASX nanoemulsions is expected to result in more beneficial EEA synthesis and bioactivity.

[0033] Lipid oxidation can be harmful to cells, especially near the nucleus, and complex mechanisms exist to prevent unwanted oxidation caused by COX, lipoxygenases, and phospholipases. Enzymes involved in eicosanoic acid biosynthesis (e.g., glutathione S-transferases, epoxide hydrolases, and carrier proteins) belong to families whose functions are largely related to cellular detoxification. This suggests that eicosanoic acid signaling may have evolved from ROS detoxification, and that cells must derive some benefit from the production of lipid hydroperoxides near their nucleus. Indeed, PG and LT may signal or regulate DNA transcription at this site, while LTB4 is a ligand for PPARα. The ASX-NE molecular complex, containing fatty acids crucial for eicosanoic acid synthesis, embedded in the membrane and close to the nucleus, is also expected to confer regulatory benefits to cells.

[0034] An exemplary embodiment of the general inventive concept of this invention can be achieved through an ASX-NE nanoemulsion molecular complex that is superior to other commercial forms of ASX in terms of production cost, bioavailability, and bioactivity. Due to the higher bioavailability and bioactivity of the ASX-NE described in this application, oral administration in patients is expected to significantly enhance the improvement effect based on existing clinical evidence concerning improvements in chronic diseases, including cardiovascular disease, immune dysfunction, neurodegenerative diseases, inherited mitochondrial diseases, wound care, and fertility, after patients take ASX forms with much lower bioavailability and bioactivity. In type 2 diabetes mellitus (T2DM), this will lead to a decrease in blood glucose and A1C levels. Compared with previous ASX formulations, fatigue is reduced, insulin resistance is reduced, and serum inflammatory markers are decreased.

[0035] The bioavailability of epigallocatechin gallate (EGCG), the richest and most active antioxidant in green tea, remains uncertain, and the nanoemulsified astaxanthin molecular complex of this invention can serve as a carrier. It can be used as an adjuvant and antioxidant. The synergistic effect of this molecular complex suggests that EGCG could be used to develop strategies for anti-aging or age-delaying agents, as well as therapeutics for neurodegenerative diseases such as Alzheimer's and Parkinson's.

[0036] Astaxanthin in Haematococcus pluvialis (HP) biomass is likely entirely in the trans configuration and entirely in esterified form in its original state. In the HP, the astaxanthin molecular complex likely contains lipids, as well as proteins and carbohydrates. A review of various methods indicates that, apart from this method, almost all methods are likely to convert the trans configuration to the cis configuration and deesterify it.

[0037] The cis form has a larger geometric configuration, which affects its bioavailability and target destination. After processing, the biomass is likely to remain in the esterified trans isomer; and the racemic ratio of stereoisomers in the synthetic product is likely to lead to cis deesterification and coupling, which polymerizes and has an affinity for the cell membrane surface, but does not insert and bridge like the esterified trans isomer, which makes it biologically active and facilitates cell entry and exit and physical reinforcement of the cell membrane. If the polymerized cis deesterified product accumulates on the cell membrane surface, it will hinder the passage of substances, and insufficient quantity will damage the cell membrane. Therefore, the cis form can ultimately exist in lipids and proteins and accumulate in and between tissues and organs (muscle, adipose tissue, liver, etc.). Furthermore, the esterified trans form can ultimately enter the cell membrane, effectively mitigating ROS near the source, near mitochondria, and even in the mitochondrial membrane.

[0038] As for EGCG, a catechin from green tea, it is a potent polyphenol, but it is also known to be one of the molecules with the lowest bioavailability because it is a highly reactive oxidant that reacts and denatures in the digestive tract and blood plasma. Although EGCG has been shown to effectively destroy tau fibers that may contribute to neuropathologies such as Alzheimer's disease, it cannot cross the blood-brain barrier (BBB).

[0039] The ASXNE oleoresin of the present invention can be considered an ideal carrier for EGCG and other relatively small but bioavailable molecules, enabling them to cross the BBB.

[0040] The FDA-set daily dose limit for EGCG is approximately 338 mg / day. If bioavailability is achieved as part of this molecular complex, this amount can be reduced by an order of magnitude or more, for example, to approximately 12 mg / day.

[0041] The molecular complex of this invention can carry EGCG across the blood-brain barrier and deliver unreacted EGCG to disrupt tau fibers that form obstructive amyloid structures and lead to Alzheimer's disease.

[0042] For example, administering a rapid dose of a mixture of processed astaxanthin and EGCG, typically a crystalline powder with a particle size of about 17 micrometers, to a 20-gram mouse can produce efficacy at a concentration of 50 ppm in 3 grams of feed.

[0043] Many “natural” molecules, such as catechins and EGCG, possess great potential, but require the bioavailability offered by the complexes of this invention. EGCG has been extensively reported to disrupt tau fibers, which bind to amyloid fat and contribute to Alzheimer's disease, but it is ineffective due to its exceptionally low bioavailability and inability to cross the BBB.

[0044] The esterified trans-astaxanthin molecular complex of the present invention, composed of micelles and liposomes rich in lipids, proteins and carbohydrates, has extremely high bioavailability, and EGCG can be nanoscaled in our nanoemulsion and protected by our molecular complex in the digestive tract and plasma.

[0045] For example, 1 kg of HP containing approximately 4% astaxanthin (i.e., approximately 40 g of astaxanthin) can be added to approximately 80 g of green tea extract containing 40 g of EGCG. The mixture is then ground for 30 minutes in 1.5 liters of food-grade ethanol (ETOH) using a 3 mm ball and 400 RPM in a one-gallon stirred mill. Then, after adding 200 g of an oil such as cod liver oil, the ETOH can be extracted at low temperature under high vacuum. The resulting oleoresin can then be prepared to provide astaxanthin and EGCG in feed at the desired ppm concentration. During the low-temperature, gentle extraction of ETOH under vacuum, the components are all submicron in size and will reassemble in a non-polymerized molecular structure within the micelle and liposome structures of lipids, proteins, carbohydrates, and EGCG small molecules.

[0046] Evidence could be found in the difference between plasma EGCG in this nanoemulsion (NE) and plasma EGCG in a common water-mixed green tea extract, as well as in evidence in clownfish and mice that the molecular complex of the present invention is configured to cross the blood-brain barrier.

[0047] Therefore, the low-temperature nanoemulsification process of the present invention can also be used with other natural extracts and materials derived from plants, algae, and animal tissues to form molecular complexes with high bioavailability. Using ethanol as a solvent to treat HP and green tea extracts, and then removing the ethanol by vacuum after adding some oil, is one of many exemplary methods of the overall inventive concept of the present invention.

[0048] Astaxanthin can be released from the capsules at the molecular level using both chemical and mechanical processes. This can be achieved using a closed mill with temperature-controlled solvents and mechanical energy. In some embodiments, the process does not exceed 50 degrees Celsius.

[0049] The extracted, ground astaxanthin molecules can be "coated" onto a biological carrier to maintain their molecular form, thereby enabling them to be delivered to the cell membrane via the gastrointestinal tract, digestive membrane, and blood system. This can be done during the grinding process described above or during a second grinding process.

[0050] HP biomass can be used, but given the small amount of bioeffective dose in this case, such as about 400 ppm, after milling, a large amount of oleoresin can be made available to coat almost any digestible biomass.

[0051] The resulting molecular compound complex can be a trans- or esterified version of astaxanthin, surrounded by micellar and liposome lipid structures that carry and retain various components from Haematococcus pluvialis biomass, as well as other additive components such as EGCG. The resulting molecule can provide numerous benefits, including but not limited to: acting as an adjuvant to facilitate and promote eicosanoic acid metabolism, which is of significant value for molecular metabolic health; it is also a potent antioxidant that protects multiple metabolic molecular processes, including mitochondrial, cytoplasmic membrane, and nearby eicosanoic acid functions; it can serve as an adjuvant antioxidant of significant value in vaccines and immunotherapy; and as an antioxidant of significant value to the thymus, it can stimulate the production of a large number of robust T cells. Furthermore, based on its structure and its ability to cross the blood-brain barrier, it can also be used as a carrier for potent biomolecules that would otherwise be unavailable, such as EGCG.

[0052] Biological processes involving amino acids, proteins, enzymes, and hormones may be susceptible to ROS and oxidative stress, and these related processes may be impaired by ROS and oxidative stress. Biological processes involving these components can benefit from the presence of astaxanthin.

[0053] When astaxanthin from the SCCO2 process is included in broodstock feed and compared with broodstock feed containing astaxanthin prepared by this process, significant differences in performance can be observed. This is an unexpected result that leads to significant differences in bioavailability and bioeffective dosage based on bioavailability.

[0054] Figure 4A and 4B The photographs show the effect of the bioeffective dose of astaxanthin in the broodstock diet on the blue tang and its effect on yolk size. Figure 4A Showing Fuji SCCO2 astaxanthin supplement. Figure 4B This shows a milled astaxanthin supplement extracted by SN.

[0055] See Figure 4A and 4B It can be seen that after three weeks of feeding the broodstock, the blue tangs ingested broodstock feed supplemented with Fuji astaxanthin biomass extracted and retained in the biomass by SCCO2 and SN extracted and ground astaxanthin. As a result, the individual astaxanthin molecules "coated" on the nanoscale substrate had a size of less than 1 micrometer, showing a significant difference in yolk quality.

[0056] It should be noted that in most oviparous animals, yolk formation is a rapid process lasting several hours: the ovary sends hormonal signals received by the thyroid gland, triggering the production of approximately three essential selenoproteins. The liver takes up these substances and uses them to generate vitellogenin (VTG). Subsequently, the VTG is secreted and taken up again, thus being converted into yolk. For most marine species, this involves hydrolysis to ensure that the eggs float to the surface, where plankton is abundant.

[0057] Taking the blue tang as an example, the parent fish spawn near sunset. The female performs a spiral dance two to three meters high, followed by the male to ensure that the sperm and eggs are fully mixed and fertilized. The eggs hatch the following afternoon. After hatching, the animal lacks normally functioning eyes, mouth, gastrointestinal tract, or nervous system. It must develop to the point where it can begin exogenous feeding within approximately three days. In the natural environment, astaxanthin is present in the food chain. However, this is not the case in the hatchery. The eggs on the right cannot survive for more than a few hours. The eggs on the right can be cultured until full development.

[0058] The difference lies in bioavailability. Molecules smaller than one micrometer, "coated" on biomass, can pass through the gastrointestinal system and digestive membranes and be transported via the bloodstream. However, once these molecules come into contact with cells, the astaxanthin portion of the molecule has a much stronger chemical affinity for the cell membrane, attaching to and effectively residing there. Once residing there, it significantly reduces oxidative stress and greatly enhances the function and responses of amino acids, proteins, hormones, and enzymes.

[0059] To demonstrate the bioeffective dosage of the astaxanthin composition of the present invention, this composition was used in clownfish broodstock feed and nest production. Results showed that when using the astaxanthin compound of the present invention, the number of nests in the aquaculture environment was approximately 130, while after two weeks without astaxanthin use, this number dropped to 80. Upon resuming astaxanthin use for another two weeks, the number recovered.

[0060] Figure 5 The bioeffective dose of astaxanthin in rotifers and their resistance to juglone are shown.

[0061] Because algae *Hydrocotyle spp.* form cysts from an evolutionary perspective to protect these cysts during periods of quiescence, and since they have no other defense mechanisms besides cystation, it is natural to understand that these cysts are robust, tough, and indigestible. To overcome this strategy, mechanical processes can be used to release molecules from the cysts. Specifically, hydrophobic molecules can be extracted from hydrophilic biomass through chemical processes, and molecules in very small molecular form (e.g., less than 1 micrometer) can be delivered through membranes to both aqueous streams and presented to the cytoplasmic membrane.

[0062] The methods and compositions of the present invention have demonstrated that astaxanthin ground in the manner described above, when provided by "extracted" biomass, has a bioavailability far exceeding that of fully extracted biomass containing all present astaxanthin, and also exceeding that of fully extracted biomass prepared by adding an oleoresin fraction extracted from the biomass using our own process.

[0063] In this process, the extracted biomass is typically light pink or almost white, and up to 99% of the astaxanthin has been extracted. It is precisely this biomass, containing only trace amounts of astaxanthin, that exhibits the highest bioavailability. In this case, the pairing of astaxanthin molecules with certain molecules in the biomass involves a minimum ratio between astaxanthin and biomass molecules.

[0064] For example, it is known that when using SCCO2 in a fish-containing diet, there is usually little or no effect, even with increased amounts. However, for this composition, it has been found that adding a smaller proportion of biomass and grinding it together to less than one micrometer, and "coating" the astaxanthin onto the biomass substrate, can surprisingly improve bioavailability and significantly reduce the required bioeffective dose.

[0065] Studies have shown that ASX exhibits significant ROS scavenging, NF-κB inhibition, enhanced Nrf2 / sirtuin / PPARγ activity, and properties that maintain thymic T cells and enhance immunity, all achieved through a form of ASX with far lower bioavailability. This article presents a significantly more bioavailable and bioactive ASX formulation that will demonstrate more significant effects in preventing and alleviating highly inflammatory diseases, suppressing inflammation-mediated immunosuppression and reactivity mediated by vaccine adjuvants and immunotherapy, and has a significant impact on inhibiting thymic involution and promoting lifelong adaptive immunity.

[0066] As shown and described herein, exemplary embodiments of the general inventive concept of the present invention can be achieved through a method of treating hyperinflammatory diseases using a composition of material assembled around an astaxanthin core in a nanoemulsion formulation, including but not limited to cytokine release syndrome and cytokine storm (CS), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and systemic inflammatory response syndrome (SIRS). These diseases are caused by, but are not limited to, viral and microbial infections, autoimmune diseases, and treatment with vaccine adjuvants and immunotherapies to enhance the immune response 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 virus vaccines, mRNA vaccines, vaccine adjuvants, nanoparticle vaccines and immunotherapies, bacterial therapies and bacteria-based vaccines, exosome vaccines and immunotherapies, cell therapy vaccines (including CAR-T) and immunotherapies, cytokine therapies and cytokine-drug conjugates, antibody therapies and antibody-drug conjugates, gene delivery vaccines and immunotherapies, and vaccines and therapeutics that induce pro-inflammatory cytokines.

[0067] Exemplary embodiments of the general inventive concept of this application can also be implemented by material compositions and methods configured to treat excessive NF-κB signaling with ASX-NE and inhibit the production of pro-inflammatory cytokines, alone or in combination with vaccines, vaccine adjuvants, and immunotherapies, thereby enhancing the immune response and limiting reactivity. 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 bacterial-based vaccines, exosome vaccines and immunotherapies, cell therapy vaccines and immunotherapies (including CAR-T), cytokine therapy and cytokine conjugate therapy, antibody therapy and antibody-drug conjugates, gene-delivered vaccines and immunotherapies, and vaccines and therapeutics that induce pro-inflammatory cytokines.

[0068] Exemplary embodiments of the general inventive concept of this application can also be achieved by a material composition and method, said method using ASX-NE to treat thymic atrophy and restore T cell function.

[0069] Many diseases are believed to be driven by reactive oxygen species (ROS), oxidative stress, and the downstream consequences of these conditions. These diseases include type 2 diabetes (T2DM), cancer, Alzheimer's disease, Parkinson's disease, various forms of dementia, heart disease, liver, kidney, and eye diseases, as well as many other diseases, including rare inherited mitochondrial diseases that lead to excessive ROS production. In many cases, treatments targeting these causes and progressions have been considered valuable and effective through the use of specific molecules. Most of these are hydrophobic antioxidants, such as astaxanthin, vitamins C and E, epigallocatechin gallate (EGCG), coenzyme Q10, etc. The limitations of these approaches have been identified and defined by several barriers and systems, including poor bioavailability and a lack of adequate delivery systems and methods, which are generally considered to hinder their progress and success.

[0070] Hereditary mitochondrial mutations affecting various proteins and complexes I through IV in the mitochondrial electron transport chain (ETC) all tend to lead to excessive mitochondrial ROS production, resulting in degenerative diseases. Certain cell types containing large amounts of mitochondria, such as nerve cells, retinal ganglion cells and cochlear ganglion cells, pancreatic β cells, muscle cells, and cardiomyocytes, are particularly susceptible to damage caused by excessive mitochondrial oxidative stress.

[0071] For example, Friedreich ataxia (FA) is a progressive neurodegenerative disease caused by a genetic mutation in the mitochondrial electron transport chain (ETC) protein frataxin (FXN). This protein is essential for maintaining the iron-sulfur clusters of enzymes involved in oxidative phosphorylation, the tricarboxylic acid cycle, and other cellular events important for mitochondrial iron metabolism and iron homeostasis. Cells deficient in FXN exhibit oxidative stress, mitochondrial iron accumulation, reduced ATP production, and cellular dysfunction. FA patients present with neurological and skeletal ataxia, diabetes, hearing and vision loss, and often fatal cardiomyopathy.

[0072] Another example is Leber hereditary optic neuropathy (LHON) and Leigh syndrome, where hereditary mutations affect the function of complex I, leading to excessive production of reactive oxygen species (ROS). In LHON, this primarily affects retinal ganglion cells and causes progressive blindness; while in Leigh syndrome, it leads to neuropathy and fatal lung failure. Hereditary mutations in complex I can also cause maternally inherited diabetes and deafness (MIDD), which can sometimes progress to more severe mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like events (MELAS).

[0073] Antioxidants have been clinically tested in these rare inherited mitochondrial diseases, but with limited efficacy. Ubiquinone analogs, such as coenzyme Q10, which act as carriers of electrons from complex I to complex II in the electron transport chain (ETC), are considered promising for mitigating ROS damage in these patients. However, due to the poor intestinal absorption of this highly lipophilic molecule, evidence of clinical benefit for coenzyme Q10 in LHON patients is insufficient. Idebenone, a short-chain, water-soluble ubiquinone derivative more readily absorbed orally, provides protection by bypassing complex I, maintaining ATP production, and preventing mitochondrial oxidative damage. Idebenone is approved for LHON in the EU but not yet in the US. The vitamin E derivative EPI-743 (PTC-743) also failed to meet the Phase III primary endpoint in the treatment of LHON. Omaveloxolone (an Nrf2 activator) has recently been approved in the US for the treatment of FA in patients aged 16 years and older. In particular, for FA, the lack of antioxidant reserves associated with abnormal activation of the transcription factor NRF2 may play a more important role than direct ROS production.

[0074] Astaxanthin, with a publicly disclosed ORAC value of 2.9 million, is 800 times stronger than coenzyme Q10 in singlet oxygen quenching and 50 times stronger than vitamin E in protecting cell membranes. Astaxanthin is also an Nrf2 activator and possesses NF-κB inhibitory properties. Through our patented milling process on *Hypericum hirsutum* algae, the astaxanthin in this application is made more readily bioavailable. As a result, the *Hypericum hirsutum* components are recombined into a molecular complex with astaxanthin at its core, surrounded by lipid-rich micelles and liposomes, and these structures also include proteins, carbohydrates, salts, ions, and other beneficial components. It has been found that the combination of bioactive molecules offers significant benefits for the treatment of rare mitochondrial genetic diseases.

[0075] To preclinically validate the activity of the molecular complex of this application in these disease areas, cell culture assays using patient cells can be employed. The astaxanthin complex is ground, extracted with ethanol, and then formulated in rapeseed oil, cod liver oil, or other suitable oil to achieve a final astaxanthin concentration of 2.1% or 21,000 ppm. For these assays, the oleoresin must be diluted from 21,000 ppm in the cell culture medium to a concentration between 5 ppm and 200 ppm.

[0076] As described herein, the addition of surfactants to the molecular complex can prevent aggregation and flocculation.

[0077] Surfactants consist of both hydrophilic and hydrophobic groups. Emulsifiers are a type of surfactant, but fall within a specific range of surfactant functions. Surfactants have a broader scope and include emulsifiers. Surfactants can coat surfaces, while emulsifiers cannot. Optimal oil / water emulsification can involve both surfactants and emulsifiers.

[0078] The hydrophilic-lipophilic balance (HLB) of a surfactant is known as an indicator of its degree of hydrophilicity or lipophilicity, and it is determined by calculating the percentage of the molecular weight of the hydrophilic and lipophilic portions of the surfactant molecule.

[0079] The surfactant has an HLB value between 0 and 40, while the emulsifier typically has a value between approximately 2 and 15, where 2-8 indicates a water-in-oil emulsifier. Furthermore, 9-15 can indicate an oil-in-water emulsifier.

[0080] If we assume that most of the HP eventually becomes oil, that is, when 250 grams of HP is ground, the product includes approximately 220 grams of oleoresin, with the remainder being the removed water. As a next step, approximately 220 grams of cod liver oil are added, yielding approximately 500 grams of oil with an astaxanthin content of approximately 21,000 ppm.

[0081] The process involves diluting the mixture with cod liver oil or rapeseed oil to an initial concentration of approximately 5000 ppm. This means that approximately 1500 grams of oil should be added to dilute the concentration from approximately 20,000 ppm to 5000 ppm, resulting in approximately 2000 grams of oil. Subsequently, the compound can be diluted to a final concentration of approximately 50 ppm in cell culture medium, resulting in a total liquid volume of approximately 200,000 grams. Here, approximately 1% of the oil can be added, which, assuming dilution with water to approximately 50 ppm, would be approximately 20 grams.

[0082] In some embodiments, the surfactant may be polyvinylpyrrolidone (PVP). A high molecular weight nonpolar emulsifier and protective colloid, it forms a thin molecular layer on the surface of individual colloidal particles to impart a positive charge and prevent aggregation. It improves stability, increases viscosity, and reduces sedimentation by lowering the zeta potential. While it does not charge the emulsion droplets, it can form a thick protective layer around the droplets and minimize the possibility of aggregation.

[0083] Given that the PVP polymer chain length is 40, the grinding process of this invention reduces it to monomers. This means that its effectiveness will be up to 40 times greater.

[0084] Furthermore, while rapeseed oil is an abundant and inexpensive excipient, recent literature has described previously unrecognized biological functions of oil lipid compositions, and not all lipids have been found to be beneficial.

[0085] Lipids are heterogeneous, water-insoluble metabolites, primarily composed of cholesterol and fatty acids such as glycerophospholipids, sphingolipids, and sterol lipids. They are important energy sources and fundamental components of the plasma membrane, and participate in many cellular processes, such as metabolism, immune function, and stress responses. Different classes of lipids can act as metabolic intermediates, membrane components, and signaling molecules in immune cells, and can produce significantly different functional outcomes.

[0086] 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) that is associated with promoting chronic inflammation and impairing T cell metabolic function and limiting cell membrane fluidity (which may impair immune cell antigen presentation).

[0087] Omega-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are essential fatty acids that enhance multiple aspects of cellular metabolism and immune function. Metabolites produced after processing these fatty acids, such as decanoins, maresin, and protectins, play additional beneficial roles in regulating immune homeostasis.

[0088] Rapeseed oil contains harmful palmitic acid and lacks beneficial omega-3 fatty acids such as DHA and EPA. In some embodiments, rapeseed oil can be replaced with cod liver oil, which is typically lacking in harmful saturated fatty acids (SFAs) such as palmitic acid and stearic acid, but instead contains high levels of beneficial omega-3 DHA and EPA. The optimal lipid composition of cod liver oil is expected to enhance the bioactivity of the astaxanthin molecular complex of this invention while achieving solubility of our nanoparticles after grinding and ethanol extraction.

[0089] A single oral administration of a large molecular complex may exceed the emulsifying capacity of the gastrointestinal tract (GI) and the limits that bile acids can provide as they travel from the liver to the small intestine via the gallbladder.

[0090] The digestive system is a complex set of interacting systems and subsystems, some of which impose strict constraints on various processes. Consequently, the system has been compelled to continuously evolve to address digestive challenges, such as improving the bioavailability of a constantly changing diet. The use of bile salts in our digestive processes is one example of these supporting system evolutions.

[0091] Bile salts are potent surfactants and emulsifiers that form highly bioavailable molecular complexes for transport across the gastrointestinal epithelium. These complexes consist of highly lipophilic and non-bioavailable molecules, including carotenoids (such as astaxanthin), vitamin E, carbohydrates, EGCG, etc. Even bile salts themselves have powerful metabolic functions.

[0092] Specific metabolic functions of gut bacteria in the production of bile salts have been identified. For example, certain conjugated bile salts produced by the liver have been found to be modified by gut bacteria, thus providing specific benefits in the treatment of dementia and ROS-based defects in mitochondria, and possessing the advantage of crossing the blood-brain barrier, similar to astaxanthin.

[0093] The rate of evolution among different organisms can be limited by differences of up to several orders of magnitude. Viruses and bacteria, however, are able to evolve so rapidly that they can adapt to unique biological communities within the lifespan of their host species. For example, while human biology is challenged on evolutionary timescales to adapt to rapid changes in diet, making it difficult for molecular and endocrine processes to adjust, the bacterial-rich microbiome in the human gut may be providing adjustments to address these issues on even faster timescales. In this context, the bacterial gut microbiome offers a greater opportunity to elucidate how people process their diet.

[0094] Conjugated bile salts, such as taurocholic acid, tauroursodeoxycholic acid (TUDCA), and glycocholic acid, are considered the primary bile salts that facilitate the passage of lipids and fats across the gastrointestinal tract membrane. They are potent hydrophilic surfactants and emulsifiers, and can even selectively include bile salts with specific metabolic and therapeutic effects. For example, TUDCA is a potent remedy for mitochondrial dysfunction and, similar to astaxanthin, a potent neurotransmitter capable of crossing the BBB. While taurocholic acid is produced in the liver, TUDCA is produced by bacteria in the gut through the addition of NO, thereby increasing the molecular weight from 515 grams per mole to 499 grams per mole. These bile salts are FDA-approved, primarily obtained as byproducts of animal slaughterhouses, and are commercially available as crystalline powders formed from very large polymers.

[0095] Unfortunately, much of the data and scientific conclusions related to human digestion are flawed due to the methodological limitations imposed by animal alternative models. Therefore, understanding the biological differences between humans and animal alternative models is crucial when drawing conclusions about human biology.

[0096] For example, mice and rats are often used as alternative models for oral medications that require digestion. A 20-gram mouse might ingest 15% of its body weight in food during a nighttime feeding, meaning a daily dose of the medication could be added to a single feeding. If the digestion of the medication requires bile acids, it is worth noting that mice and rats lack gallbladders, thus failing to provide a reservoir for on-demand bile acid release, and their bile acid profiles differ significantly from those of humans. Therefore, in these animals, the uptake of fats, and hydrophobic or amphiphilic micronutrients that depend on fat to form micelles and liposomes, thereby facilitating their passage through the bloodstream, may be limited.

[0097] To improve poor intestinal absorption in mouse biodistribution studies and poor solubility in cell culture media, PVP and bile acids conjugated with taurine can be added during the HP milling process. The bile salt is 95% water, and the salt itself is a crystalline powder; therefore, the preparation of nanoemulsions would be advantageous for this process, in which HP encapsulations are milled from approximately 60 micrometers (μm) to less than 100 nanometers (nm). This process can also mill PVP with a molecular weight (MW) of 40,000 into nano-sized molecules.

[0098] The addition of these surfactants and bile acid emulsifiers is expected to improve water solubility for cell culture and enable greater astaxanthin delivery via the intestines and into circulation. Furthermore, formulation of the oleoresin with cod liver oil, which is more immunomodulatory and metabolically advantageous, is anticipated to enhance the efficacy of our astaxanthin molecular complex, together with bioactive taurine-based bile salts, in treating chronic diseases caused by mitochondrial dysfunction and conditions resulting from excessive inflammation.

[0099] The blood-brain barrier (BBB) ​​is a very strong barrier. It not only protects the brain from a range of risks but also prevents some potent molecules from reaching therapeutic targets. For example, epigallocatechin gallate (EGCG) is a known tau fiber disruptor that binds to amyloid structures that contribute to Alzheimer's disease but cannot cross the blood-brain barrier.

[0100] By incorporating EGCG into an astaxanthin molecular complex containing PVP, conjugated bile salts, and cod liver oil, EGCG can cross the BBB, and its activity can be further enhanced by the overall bioactivity of astaxanthin and the other components in the molecular complex.

[0101] Numerous studies document the metabolic benefits of astaxanthin in achieving bioavailability for a wide range of mitochondrial and metabolic diseases. These benefits may involve the potent targeting and integration of the astaxanthin in the form of this invention into the cytoplasmic and mitochondrial membranes, exerting strong antioxidant and gene regulatory functions. As described herein, the astaxanthin molecule may be esterified, all-trans (all-E configuration) 3S,3'S astaxanthin, intended for the treatment of rare inherited mitochondrial diseases, cancer, inflammation, neurological disorders, heart disease, type 2 diabetes mellitus (T2DM), and skin, liver, kidney, and eye diseases.

[0102] Some embodiments of the general inventive concept of this invention involve adding safe, naturally occurring bile salts to algae. Adding a small amount of complementary emulsifier (e.g., PVP), followed by milling in ethanol, allows for the addition of other bioactive components, such as EGCG. Subsequently, the ethanol is removed via cryogenic vacuum processing while adding cod liver oil rich in omega-3 polyunsaturated fatty acids (PUFAs). All of this enhances the bioavailability of astaxanthin and EGCG, and results in potent bioactivity in the astaxanthin itself. The bile salts can be processed into crystalline polymers with high molecular weights, much like PVP. Cryogenic milling of these macromolecules in ethanol to prevent denaturation is expected to produce more effective bile salts, thereby significantly improving the bioavailability of our molecular complex. The applied mixture of surfactants and emulsifiers significantly enhances intestinal uptake and targeted delivery of astaxanthin. In our formulation, the selection of taurine-based TUDCA as the bile salt and the substitution of high-omega-3 polyunsaturated fatty acid cod liver oil with palmitic acid-containing rapeseed oil further enhances the immune and metabolic properties.

[0103] An exemplary embodiment of the general inventive concept of this invention provides a highly bioavailable molecular complex composed of astaxanthin and other Haematococcus pluvialis (HP) algal derivatives, with PVP and taurine-based conjugated bile acids added to a cod liver oil excipient. Further examples may include EGCG.

[0104] By using safe, natural surfactants and emulsifiers in the astaxanthin-based molecular complexes of this invention, a large number of lipid-rich molecular complexes generated from the processing of HP-derived astaxanthin are emulsified into a highly bioavailable form, enhancing the active molecular metabolic activity of these bioactive molecules and the bioactivity of astaxanthin. This combination provides an extremely powerful delivery system throughout the body, including the ability to cross the BBB, while delivering the most potent natural active agents and antioxidants to the areas of the body most in need of treatment.

[0105] An exemplary embodiment of the general inventive concept of this invention can be achieved through a method of treating a patient's disease, the method comprising (orally or otherwise) administering a pharmaceutical composition comprising a therapeutically effective amount of esterified 3S,3'S trans-astaxanthin combined with one or more constituent components assembled on the core of an astaxanthin molecule. The esterified trans-astaxanthin may be derived from Haematococcus pluvialis (HP) algae, and the molecular core may include a 3S,3'S stereoisomer to promote the assembly of the constituent components into the molecular core, and due to membrane integration and esterification, increase the bioavailability and bioactivity of the composition relative to astaxanthin compounds having 3R,3'R stereoisomers.

[0106] In some embodiments, one or more of the components are epigallocatechin-3-gallate (EGCG). The pharmaceutical composition may include the surfactant polyvinylpyrrolidone (PVP) and / or the emulsifier tauroursodeoxycholic acid (TUDCA) to improve the bioavailability of the composition and facilitate its transport across the patient's blood-brain barrier (BBB).

[0107] An exemplary embodiment of the general inventive concept of the present invention can also be achieved by a pharmaceutical composition for treating a patient's disease, the pharmaceutical composition comprising an effective therapeutic amount of esterified trans-3S,3'S astaxanthin, and combined with one or more components assembled on the molecular core of the astaxanthin, wherein the molecular core is configured to promote the assembly of the one or more components onto the molecular core to improve the bioavailability of the composition and / or the one or more components, thereby alleviating the symptoms of the disease.

[0108] The one or more components may be epigallocatechin gallate (EGCG), and the molecular core may have a 3S, 3'S stereoisomer to facilitate the crossing of the EGCG across the patient's blood-brain barrier (BBB).

[0109] The esterified trans-3S,3'S astaxanthin may be derived from Haematococcus pluvialis (HP) algae, and its molecular core may include a 3S,3'S stereoisomer to promote the assembly of one or more components onto the molecular core, and due to membrane incorporation and esterification, the bioactivity of the composition is improved relative to astaxanthin compounds having 3R,3'R stereoisomers.

[0110] The composition may include the surfactant polyvinylpyrrolidone (PVP) and / or the emulsifier tauroursodeoxycholic acid (TUDCA) to improve the bioavailability of the composition compared to astaxanthin compounds obtained from HP algae using supercritical CO2 extraction, thereby promoting the cross-section of the composition across the patient's blood-brain barrier (BBB).

[0111] The following examples are intended to describe and illustrate various aspects of the general inventive concept of the present invention.

[0112] Example 1: Characterization and comparison of astaxanthin derived from *H. pylori* obtained using different extraction methods.

[0113] Astaxanthin harvested from pristine, unruptured HP capsule biomass and extracted using industry-standard high-temperature supercritical carbon dioxide (ASX-CO2) or Adjuvia's patented process, namely high-shear, low-temperature, and pressure grinding (ASX-NE), was characterized and compared astaxanthin by molecular complex analysis. The composition of the molecular complexes surrounding the ASX formulations was characterized using chromatographic techniques, with rapeseed oil excipients used as a diluent reference.

[0114] Table 1 summarizes the comparative analysis results of gas chromatography-mass spectrometry (GC / MS) from astaxanthin samples. The samples were diluted in appropriate solvents before GC / MS analysis. The relative percentage of the reported peak area for each sample is given. Analytes for each sample were calculated independently of the other sample.

[0115] Table 1 1 ND=not detected. The attribution of the material structures was based on a search and analysis of the parent ion internal database. Within the scope of this study, no analytical grade reference standards were used to confirm the presence of the listed compounds.

[0116] As shown in Table 1, both ASX-CO2 and ASX-NE share identified compounds including substituted benzenes and aromatics, fatty acids, and dl-α-tocopherol. However, ASX-NE exhibits higher relative concentrations of several beneficial fatty acids and contains numerous compounds including microterpenes, long-chain alkanes and alkenes, fatty acid methyl esters, and phthalates, which were not observed in ASX-CO2. Importantly, ASX-NE includes the δ- and γ-isomers of tocopherol (vitamin E), which is crucial for protecting the ASX molecule from oxidation.

[0117] Table 2 summarizes the results of ion chromatography (IC) analysis of astaxanthin samples. The samples were extracted with water prior to IC analysis. Rapeseed oil was used as a method blank. These data indicate the presence of important inorganic salts in ASX-NE that are absent in ASX-CO2.

[0118] Table 2

[0119] The data in Table 2 show that, compared with the astaxanthin complex obtained by supercritical CO2 separation, the astaxanthin molecular complex ASX-NE obtained by patented grinding method has a unique and superior composition.

[0120] Example 2: Evaluation and comparison of the quenching effect of astaxanthin preparations on mitochondrial ROS.

[0121] ASX has previously been demonstrated to be a potent quencher of reactive oxygen species (ROS) produced by mitochondrial respiration. To assess whether the ASX-NE molecular complex is superior to supercritical CO2 (ASX-CO2), ASX derived from *Phaefflera hepatis* (ASX-PF), and synthetic ASX (ASX-SYN) in ROS quenching, the ability of oxidative stress and ASX to quench ROS was determined using human HepG2 hepatocyte assays. For this purpose, HepG2 cells were seeded in 384-well plates and allowed to recover for 24 hours, followed by a 2-hour pretreatment with one of the following: 5 mM N-acetyl-L-cysteine ​​(NAC, soluble in H2O) as a positive control, or 50 μM of an ASX compound (soluble in rapeseed oil). Subsequently, the cells were treated for 4 hours with 30 μM of the ROS inducer menadione (soluble in 0.1% DMSO) or a solvent control. Subsequently, cells were stained live with the cell-permeable fluorescent dye CellROX to measure oxidative stress. Cells were then fixed and stained with Hoechst. Plates were imaged only within 24 hours of staining.

[0122] As shown in Table 3, menadione treatment induced ROS, which could be detected by an increase in CellROX signal intensity, and this ROS was subsequently quenched to below baseline levels by the addition of NAC. Even in the absence of menadione, cell culture conditions generated oxidative stress, and pretreatment with NAC reduced oxidative stress to below baseline levels compared to the untreated and solvent controls. After pretreatment with ASX compounds, the ASX-NE molecular complex was the only ASX that significantly reduced oxidative stress under cell culture conditions to levels below those observed in the positive control NAC (p<0.0001). After menadione treatment, only ASX-NE reversed oxidative stress, and its effect was more significant than that of NAC (p<0.0001).

[0123] Table 3

[0124] The data in Table 3 show that the ASX-NE extraction method is more potent than the ASX-CO2 extraction method. It showed no activity in this assay. ASX-PF containing 100% of the R,R isomers exhibited ROS quenching ability under cell culture conditions, but its activity was extremely low in the presence of menadione. These data suggest that ASX-NE containing 100% of the S,S isomers has superior quenching ability compared to the R,R isomers that cannot be incorporated into the membrane; the superior components in its novel molecular complex may also contribute to this compared to supercritical CO2 extraction materials. ASX-SYN containing 25% of the S,S isomers showed partial efficacy and significant activity at the highest dose in the presence of menadione, although it was not higher than NAC or 100% S,S ASX-NE. These data suggest that ASX-NE has superior bioactivity compared to other forms of astaxanthin and NAC. It is used as a standard of care for mitochondrial inherited degenerative diseases such as Leigh syndrome.

[0125] Example 3: Adding PVP to prevent the aggregation of astaxanthin molecular complexes in water-based excipients.

[0126] Figure 6A The results show the effect of astaxanthin oleoresin diluted in brine for 6 hours. Figure 6A As shown, preliminary tests of diluting the oleoresin in a saline solution revealed significant aggregation and flocculation after 6 hours, which would likely hinder the bioavailability of the compound upon entry into cells. We propose that adding a surfactant to the molecular complex would prevent aggregation and flocculation.

[0127] Due to the high molecular weight (MW) of PVP (40,000), we chose to encapsulate it with HP and grind it together, thereby reducing the 17 μM PVP particles to a scale of less than 100 nm. The molecular complex was dissolved in rapeseed oil after HP grinding and ethanol extraction.

[0128] Figure 6B The results for ASX containing 2.5% PVP over 6 hours are shown. Figure 6B As shown, the addition of PVP prevented particles from agglomerating for at least 6 hours, demonstrating the importance of adding this surfactant to the astaxanthin molecular complex.

[0129] Example 4: The addition of PVP and TUDCA enhanced the bioavailability of the astaxanthin molecular complex in vivo.

[0130] The ASX-NE astaxanthin molecular complex extracted using a proprietary milling method was compared with the ASX-CO2 compound to enhance in vivo uptake and bioavailability. Furthermore, the ability of the surfactant PVP and emulsifier TUDCA to improve bioavailability was tested. For this purpose, a study was conducted in mice to compare the plasma bioavailability of astaxanthin nanoemulsions formulated with cod liver oil, wherein the astaxanthin in the nanoemulsions was isolated individually using the proprietary milling method (ASX-NE), or isolated with the addition of PVP or PVP+TUDCA, or isolated using supercritical CO2 (ASX-CO2), administered via a single oral gavage at a dose of 4 mg / kg. Plasma was collected 2 hours after administration, and the presence of ASX was assessed by high-performance liquid chromatography.

[0131] As shown in Table 4, ASX-NE exhibited significantly higher plasma bioavailability than ASX-CO2 (p=0.04). Adding PVP to ASX-NE further improved plasma bioavailability, and PVP, in combination with TUDCA, significantly enhanced bioavailability compared to ASX-NE alone (p=0.05). These data indicate that the ASX-NE astaxanthin molecular complex possesses superior bioavailability in vivo, which can be further enhanced by the addition of surfactants and emulsifiers.

[0132] Table 4.

[0133] Example 5: Adding EGCG ground together with astaxanthin from HP enables EGCG to cross the blood-brain barrier in vivo.

[0134] EGCG, a green tea catechin, shows promising potential for applications in Alzheimer's disease and other neurodegenerative diseases. However, this molecule cannot cross the blood-brain barrier (BBB). We hypothesized that using our proprietary grinding method to co-grind EGCG with ASX would allow EGCG to be incorporated into the astaxanthin molecular complex, enabling it to cross the BBB together with ASX for delivery. To test this, a study was conducted in mice to compare the tissue bioavailability of astaxanthin and EGCG formulations prepared in cod liver oil using PVP and TUDCA, with both ASX and EGCG administered orally at a single dose of 4 mg / kg via gavage. Tissues were collected at 2, 4, 8, 12, 24, and 48 hours after oral administration, and ASX and EGCG were determined by HPLC. A peak concentration of ASX (15.7 ± 9.2 ng / g) was detected in the brain at 8 hours after administration. The temporal variation of EGCG in the brain also followed the timeline of ASX detection, with a peak EGCG level (0.6 ± 0.1 ng / g) detected in the brain 8 hours after administration. These data demonstrate for the first time that EGCG can be detected in the brain; when this molecule forms a complex with ASX nanoemulsion, it has the ability to cross the brain-body barrier (BBB), thus holding promise for the treatment of neurodegenerative diseases associated with the presence of harmful β-amyloid plaques and tau fiber accumulation.

[0135] As shown and described herein, it should be understood that the description of some embodiments and methods of the general inventive concept of the present invention does not limit the various alternative, modification, and equivalent embodiments and methods that will occur to those skilled in the art as shown in this disclosure. Furthermore, certain details have been set forth in the description and illustrations provided herein to facilitate understanding of various embodiments of the general inventive concept of the present invention. However, some embodiments of the general inventive concept of the present invention may be practiced without these specific details.

[0136] The accompanying drawings illustrate exemplary embodiments of the general inventive concept of the present invention. The designs shown are illustrative and not limiting. Those skilled in the art will understand that various modifications and / or configurations of the general inventive concept can be employed based on reasonable engineering judgment, and that all such modifications and / or configurations fall within the scope of the general inventive concept of the present invention.

[0137] In certain sections, this written description and accompanying drawings illustrate examples of certain embodiments of the disclosed technology. However, the scope of patentability may also include other examples that would occur to a person skilled in the art. Such other examples are intended to fall within the scope of the patent claims. For example, various alternative examples may be defined as substantially equivalent in structural and / or functional elements to the example embodiments described and / or illustrated herein, or may include structural and / or functional elements that are not substantially different from the example embodiments described and / or illustrated herein.

Claims

1. A method of treating a patient’s disease, comprising oral administration of a pharmaceutical composition, said pharmaceutical composition comprising an effective therapeutic amount of esterified 3S, 3'S trans-astaxanthin and one or more components assembled on the core of said astaxanthin molecule.

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

3. The method according to claim 1, wherein, The molecular core includes a 3S, 3'S stereoisomer to facilitate the assembly of the constituent components onto the molecular core, and improves the bioavailability and bioactivity of the composition relative to astaxanthin compounds having 3R, 3'R stereoisomers due to membrane incorporation and esterification.

4. The method according to claim 1, wherein, One or more of the components are epigallocatechin-3-gallate (EGCG).

5. The method according to claim 1, wherein, The pharmaceutical composition includes the surfactant polyvinylpyrrolidone (PVP) and / or the emulsifier tauroursodeoxycholic acid (TUDCA) to improve the bioavailability of the composition and facilitate its transport across the patient's blood-brain barrier (BBB).

6. A pharmaceutical composition for treating a patient's disease, comprising: A therapeutically effective amount of esterified 3S,3'S trans-astaxanthin and one or more components assembled on the molecular core of astaxanthin, wherein the molecular core is configured to promote the assembly of the one or more components to the core of the molecule to improve the bioavailability of the composition and / or the one or more components, thereby alleviating the symptoms of the disease.

7. The composition according to claim 6, wherein, The one or more components are epigallocatechin-3-gallate (EGCG), and the molecular core includes a 3S,3'S stereoisomer to facilitate the crossing of the EGCG across the patient's blood-brain barrier (BBB).

8. The composition according to claim 6, wherein, The esterified trans-3S,3'S-astaxanthin is derived from Haematococcus pluvialis (HP) algae.

9. The composition according to claim 6, wherein, The molecular core includes a 3S, 3'S stereoisomer to facilitate the assembly of the constituent components into the molecular core, and enhances the bioactivity of the composition relative to astaxanthin compounds having 3R, 3'R stereoisomers due to membrane incorporation and esterification.

10. The composition of claim 6, further comprising the surfactant polyvinylpyrrolidone (PVP) and / or the emulsifier tauroursodeoxycholic acid (TUDCA) to improve the bioavailability of the composition compared to astaxanthin compounds extracted from HP algae using supercritical CO2, thereby promoting the transport of the composition across the patient's blood-brain barrier (BBB).

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