WO2023028586A1

WO2023028586A1 - Use of taxifolin for treating systemic lupus erythematosus

Info

Publication number: WO2023028586A1
Application number: PCT/US2022/075518
Authority: WO
Inventors: Steven Chen; Linda Shuo MAY-ZHANG
Original assignee: Phyto Tech Corp.
Priority date: 2021-08-27
Filing date: 2022-08-26
Publication date: 2023-03-02

Abstract

Provided herein are methods of using taxifolin for treating or preventing systemic lupus erythematosus a thromboinflammatory disease, or cystic fibrosis in a subject.

Description

USE OF TAXIFOLIN FOR TREATING SYSTEMIC LUPUS ERYTHEMATOSUS

BACKGROUND

[0001] Taxifolin (5, 7, 3', 4'-flavan-on-ol) is a naturally occurring compound found in a variety of plant-based foods, including fruits, vegetables, and grains. Taxifolin belongs to the subclass flavanonols in the flavonoids. Taxifolin has two stereocenters on the C-ring, thus producing two pairs of enantiomers, including (+)-taxifolin and (-)-taxifolin.

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple organs with devastating pathological consequences. Current treatment regimens largely rely on immunosuppressants and corticosteroids to attenuate autoimmune activity. Such treatments have toxic side effects, lack efficacy, and/or makes the subject prone to infections.

[0003] SLE generates autoantibodies which induce an influx of neutrophils to different sites of the body, acting as a first line of defense. Neutrophil extracellular traps (NETs) generated by neutrophils through a process called NETosis play an important role in the pathogenesis of SLE. It has recently been reported that natural gingerols reduce release in models of SLE and APS in mice. Ali et al., JCI Insight. 2021;6(3):el38385. https://doi.org/10.1172/jci.insight.138385.

[0004] NETs may also play a role in thromboinflammatory diseases such as antiphospholipid syndrome or thrombosis, see, e.g., Ali et al., Nat. Commun. 70: 1916 (2019), and in cystic fibrosis, see, e.g., Cheng et al., Front. Immunol. 4 : 1 (2013).

[0005] There exists a need for other therapeutic agents to treat or prevent SLE, thromboinflammatory diseases, and/or cystic fibrosis.

SUMMARY OF THE INVENTION

[0006] In one embodiment, the present disclosure provides methods of treating or preventing systemic lupus erythematosus, a thromboinflammatory disease, or cystic fibrosis, or a symptom thereof, in a subject in need thereof, comprising administering a therapeutically effective amount of taxifolin to the subject. [0007] In some embodiments of any one of the methods or compositions provided herein, the composition comprises purified taxifolin, such as a natural form of purified taxifolin. In some embodiments of any one of the methods or compositions provided herein, the purified taxifolin has purity of about 98% or is at least 98%.

[0008] In some embodiments of any one of the methods or compositions provided herein, the composition is in a free-flowing powder form.

[0009] In some embodiments of any one of the methods provided herein, the methods comprise providing or administering an effective amount of a composition comprising taxifolin to a subject in need thereof, such as any one of the subjects provided herein.

[0010] Some aspects of the disclosure provide methods and compositions for treating systemic lupus erythematosus, a thromboinflammatory disease, or cystic fibrosis, or a symptom thereof, comprising providing or administering an effective amount of a composition comprising taxifolin to a subject in need thereof, optionally, wherein the taxifolin has a purity of at least or about 98%.

[0011] In some embodiments, the composition is any one of the orally consumable products provided herein. In some embodiments, the composition is a pharmaceutical composition.

[0012] In some embodiments, the subject is in need of a medicament for the treatment of systemic lupus erythematosus, a thromboinflammatory disease, or cystic fibrosis, or a symptom thereof.

[0013] Some aspects of the disclosure provide compositions, such as pharmaceutical formulations, comprising a therapeutically effective amount of taxifolin and, optionally, one or more pharmaceutically acceptable carriers.

[0014] In one embodiment, any one of the taxifolin compositions provided herein can be for any one of the purposes provided herein.

[0015] In one embodiment, any one of the taxifolin compositions provided herein can be used as part of a blend or other active and/or inactive agents.

[0016] Some aspects of the disclosure provide methods of producing taxifolin from naringenin via modified E. coli or other microbial strains. In one embodiment, the production of taxifolin is via bioconversion. In one embodiment, taxifolin can be produced by biosynthesis in microbial culture using a specific synthetic pathway.

[0017] In one embodiment, the cellular system for producing taxifolin is selected from the group consisting of bacteria, yeast, and a combination thereof, or any cellular system that would allow the genetic transformation with selected genes and thereafter the biosynthetic production of taxifolin, such as from naringenin. In one embodiment the microbial system comprises E. coli.

[0018] While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawing and will herein be described in detail. It should be understood, however, that the drawings and detailed description presented herein are not intended to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Fig. 1 shows the bioconversion pathway of naringenin to eriodictyol to taxifolin. F3'H and F3H represent Flavin reductase enzymes.

[0020] Fig. 2 is a bar graph showing the effect of resveratrol, mangiferin, DHQ (taxifolin), and quercetin on MPO activity in a bleomycin (BLM) pulmonary fibrosis animal model.

[0021] Fig. 3 is a set of four images showing the morphology of pancreatic tissue of mice infected with CVB4. Image "a" is a control showing an intact mouse. Image "b" is a CVB4 infected-mouse at day 5 p.i. Image "c" is a CVB4-infected mouse treated with DHQ (taxifolin) on day 5 p.i. Image "d" is a CVB4-infected mouse treated with ribavirin on day 5 p.i. Is = islets; Ac = acini; stars = areas of tissue destruction and cell infiltration. Haematoxilin-eosin, original magnification x300.

[0022] Fig. 4A is a bar graph showing the effect of taxifolin on NETosis in neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA) as measured by NET-associated myeloperoxidase (MPO) activity.

[0023] Fig. 4B is bar graph showing the effect of taxifolin on NETosis in neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA) as measured by fold-increase in extracellular DNA.

[0024] Fig. 5A is a bar graph showing the effect of taxifolin on NETosis in neutrophils stimulated by calcium ionophore (ionomycin) as measured by NET-associated myeloperoxidase (MPO) activity. [0025] Fig. 5B is a bar graph showing the effect of taxifolin on NETosis in neutrophils stimulated by calcium ionophore (ionomycin) as measured by fold-increase in extracellular DNA.

[0026] Fig. 6A is bar graph showing the effect of taxifolin on NETosis in neutrophils stimulated by human anti-phospholipid antibodies (APS IgG) as measured by NET- associated myeloperoxidase (MPO) activity.

[0027] Fig. 6B is a bar graph showing the effect of taxifolin on NETosis in neutrophils stimulated by human anti-phospholipid antibodies (APS IgG) as measured by foldincrease in extracellular DNA.

[0028] Fig. 7A is a bar graph showing the effect of taxifolin on NETosis in neutrophils stimulated by immune complexes formed from human lupus anti antibodies, anti- ribonucleoprotein (RNP IC), as measured by NET-associated myeloperoxidase (MPO) activity.

[0029] Fig. 7B is a bar graph showing the effect of taxifolin on NETosis in neutrophils stimulated by immune complexes formed from human lupus anti antibodies, anti- ribonucleoprotein (RNP IC), as measured by fold-increase in extracellular DNA.

[0030] Fig. 8A is a set of two scatter plots and a photograph showing spleen weight, spleen-body weight ratio, and spleen size in a lupus mouse model after 6 weeks of treatment with taxifolin or a control vehicle (Experimental Condition 1).

[0031] Fig. 8B is a set of two scatter plots and a photograph showing spleen weight, spleen-body weight ratio, and spleen size in a lupus mouse model wherein taxifolin or a control vehicle was administered at week 4 of lupus symptoms and for two weeks thereafter (Experimental Condition 2).

[0032] Fig. 9A is a set of three scatter plots showing white blood cell count, neutrophil count, and lymphocyte count in a lupus mouse model after 6 weeks of treatment with taxifolin or a control vehicle (Experimental Condition 1).

[0033] Fig. 9B is a set of three scatter plots showing white blood cell count, neutrophil count, and lymphocyte count in a lupus mouse model wherein taxifolin or a control vehicle was administered at week 4 of lupus symptoms and for two weeks thereafter (Experimental Condition 2).

[0034] Fig. 10A is a scatter plot showing MPO-DNA complex optical density in a lupus mouse model after 6 weeks of treatment with taxifolin or a control vehicle (Experimental Condition 1). [0035] Fig. 1 OB is a scatter plot showing MPO-DNA complex optical density in a lupus mouse model wherein taxifolin or a control vehicle was administered at week 4 of lupus symptoms and for two weeks thereafter (Experimental Condition 2).

[0036] Fig. 11 A is a set of three scatter plots showing anti-dsDNA IgG concentration, anti-P2GP I optical density, and total IgG concentration in a lupus mouse model after 6 weeks of treatment with taxifolin or a control vehicle (Experimental Condition 1).

[0037] Fig. 1 IB is a set of three scatter plots showing anti-dsDNA IgG concentration, anti-P2GP I optical density, and total IgG concentration in a lupus mouse model wherein taxifolin or a control vehicle was administered at week 4 of lupus symptoms and for two weeks thereafter (Experimental Condition 2).

[0038] Fig. 12 is a scatter plot and bar graph showing thrombus weight (mg) in C57BL/6 mice treated with control IgG, control IgG and taxifolin, anti-phospholipid antibodies (APS IgG), and APS IgG and taxifolin.

DETAILED DESCRIPTION

[0039] Aspects of the present disclosure relate to the use of taxifolin to treat or prevent systemic lupus erythematosus, a thromboinflammatory disease, or cystic fibrosis, or a symptom thereof, in a subject. Accordingly, methods and compositions, including pharmaceutical formulations, described herein are useful for such purpose.

[0040] The terms "improve," "improving," and "improvement" as used herein refers to a process or an action that results in enhanced results when compared with a corresponding counterpart that is not affected by said process or action. As used herein, the terms also refer to a process or an action that results in no change of results when compared with a corresponding counterpart that is not affected by said process or action.

[0041] As used herein, "purify" or "purified" refer to freeing something of extraneous contaminating or debasing matter. As used herein, a "substantially pure" preparation of something refers to a preparation having purity of the desired thing of at least 90% (i.e., 90% or greater than 90%). Any one of the taxifolin compositions provided herein may be substantially pure. Such pure or purified or the like taxifolin compositions may then be used in combination with other desired agents (active and/or inactive agents) in an ultimate composition that is provided or administered to a subject. Thus, the pure or purified or the like taxifolin compositions may be combined with other active and/or inactive agents, such as to form a blend, which blend is the composition that is provided or administered to a subject in any one of the methods or compositions provided herein.

[0042] The term "free-flowing powder" or "free-flowing powder form" refers to powder compositions that have satisfying flowability, which is the ability of a powder to flow. For instance, a free-flowing powder may be pourable or may be easy to pour.

[0043] Generally, the term "about," as used herein when referring to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass in one example variations of ± 15% or ±10%, in another example ±5%, in another example ±1%, and in yet another example ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method and for the disclosed compositions.

[0044] As used herein, the term "stereoisomers" is a general term for all isomers of an individual molecule that differ only in the orientation of their atoms in space. It includes enantiomers and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereomers).

[0045] The term "chiral center" or "asymmetric carbon atom" refers to a carbon atom to which four different groups are attached.

[0046] The terms "enantiomer" and "enantiomeric" refer to a molecule that cannot be superimposed on its mirror image and hence is optically active wherein the enantiomer rotates the plane of polarized light in one direction and its mirror image compound rotates the plane of polarized light in the opposite direction.

[0047] The term "racemic" refers to a mixture of equal parts of enantiomers and which mixture is optically inactive.

[0048] The term "absolute configuration" refers to the spatial arrangement of the atoms of a chiral molecular entity (or group) and its stereochemical description, e.g., R or S.

[0049] The stereochemical terms and conventions used in the specification are meant to be consistent with those described in z/re & Appl. Chem. 65:2193 (1996), unless otherwise indicated.

[0050] The term "enantiomeric excess" or "ee" refers to a measure for how much of one enantiomer is present compared to the other. For a mixture of R and S enantiomers, the percent enantiomeric excess is defined as | R - S | * 100, where R and S are the respective mole or weight fractions of enantiomers in a mixture such that R + S = 1. With knowledge of the optical rotation of a chiral substance, the percent enantiomeric excess is defined as ([a]obs/[a]max)*100, where [a]obsis the optical rotation of the mixture of enantiomers and [a]max is the optical rotation of the pure enantiomer. Determination of enantiomeric excess is possible using a variety of analytical techniques, including NMR spectroscopy, chiral column chromatography, or optical polarimetry.

[0051] Aspects of the disclosure relate to taxifolin compositions and uses thereof.

Taxifolin may be isolated and purified from natural sources. Taxifolin may be also be produced synthetically. Methods of producing taxifolin are described in, for example, WO2013113329, and such methods of production are incorporate herein by reference in their entirety. Taxifolin is also referred to in the art as dihydroquercetin (DHQ).

[0052] The term "taxifolin" as used herein refers, collectively, to 2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one, all possible stereoisomers, e.g., enantiomers and diastereomers, and mixtures, e.g., racemic mixtures, thereof, and the pharmaceutically acceptable salts thereof. The structure of 2-(3,4-dihydroxyphenyl)- 3,5,7-trihydroxychroman-4-one is:

[0053] The present disclosure encompasses the use of stereoisomers of 2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one.

[0054] In some embodiments, taxifolin is (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7- trihydroxychroman-4-one (also known as (2A,3A)-3,3',4',5,7-pentahydroxyflavan-4-one; (2R,3R)-trans-dihydroquercetin, (2R,3R)-dihydroquercetin, (+)-dihydroquercetin;

(+)-taxifolin), which has the structure shown below:

[0055] In some embodiments, taxifolin is (2S,3R)-2-(3,4-dihydroxyphenyl)-3,5,7- trihydroxychroman-4-one (also known as (25,3A)-3,3',4',5,7-pentahydroxyflavan-4-one), which has the structure shown below:

[0056] In some embodiments, taxifolin is (2S,3S)-2-(3,4-dihydroxyphenyl)-3,5,7- trihydroxychroman-4-one (also known as (25,35)-3,3',4',5,7-pentahydroxyflavan-4-one), which has the structure shown below:

[0057] In some embodiments, taxifolin is (27?,35)-3,3',4',5,7-pentahydroxyflavan-4-one, which has the structure shown below:

[0058] In some embodiments, taxifolin is a single stereoisomer, e.g., (2R,3R)-2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one. In some embodiments, taxifolin is a mixture of one or more stereoisomers. In some embodiments, taxifolin is a racemic mixture.

[0059] In some embodiments, taxifolin is a single stereoisomer having an enantiomeric excess (ee) of about 50% or more. In another embodiment, the ee is about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more. In another embodiment, the ee is about 100%.

[0060] Other aspects of the present disclosure provide methods of producing taxifolin from naringenin via bioconversion. A bioconversion reaction is provided in the schematic in Fig. 1.

[0061] Taxifolin can be produced, for example, with coli. For example, flavin reductase can be overexpressed together with SAM5 allowing a modified microbial strain to catalyze the conversion of naringenin to eriodictyol and further to taxifolin with high efficiency. Microbial systems that can be utilized include E. coli BL21(DE3) strains and pRSF-BLK grown in LB medium with 30 pg/L kanamycin; the E. coli strains can be grown in LB medium containing 30 pg/L kanamycin and 100 pg/L spectinomycin, respectively. The cells can be grown to OD600=0.6 in a shaker at 37°C, and then changed to 30°C with addition of lactose to final concentration of 1.5% (w/v) to induce the expression of exogenous genes. After 3 hours of expression induction, naringenin (40% w/v) dissolved in DMSO can be added to the culture. The culture can be kept shaking under the same culture conditions.

[0062] DNA fragments of Sam5 and two uncharacterized Flavin reductases, PpFR and SeFR, which are codon optimized for E. coli expression, are provided with the sequences listed in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, respectively. Native HpaC DNA sequence is listed in SEQ ID NO: 7. Their corresponding protein sequences are listed in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, respectively. Sam5, PpFR and SeFR can be synthesized in Genscript Company and used as the templates for the following PCR amplification.

[0063] Sam5 is cloned into the Nde I/Xho I restriction site of pRSFDuet-1. PpFR, SeFR and HpaC are cloned into the Nde I/Xho I restriction sites of pCDFDuet-1. For enzyme overexpression and purification, HpaC, PpFR and SeFR are cloned into Nde I/Xho I restriction site of pET28a vector. HpaC is cloned from genomic DNA of E. coli strain MG1655 extracted using Bacterial DNA extraction kit. HpaC gene is amplified from the E. coli genomic DNA with PCR with introduction of Nde I site at the 5'-end and Xho I site at the end of 3'-end. The primers used are forward primer HpaC Ndel F (5'- GGGAATTCCATATGCAATTAGATGAACAACGCCTGCG) (SEQ ID NO: 9) and reverse primer HpaC XhoI R (5'- CTCGAGCGGTTAAATCGCAGCTTCCATTTCCAGC) (SEQ ID NO: 10). The PCR product digested with Nde I and Xho I is ligated with plasmid pCDFDuet-1 digested with the same enzymes and transformed into E. coli DH5a.

[0064] To make constructs with an operon of SAM5 and a specific Flavin reductase, the specific Flavin reductase gene is inserted downstream of SAM5 by Gibson assembly, yielding three constructs named SAM5-HpaC-pRSF, SAM5-PpFR-pRSF and SAM5- SeFR-pRSF. Sam5-pRSF is introduced into E. coli BL21 (DE3) cells with standard chemical transformation protocols, leading to the development of taxifolin producing E. coli strains. Sam5-pRSF is co-transformed into BL21 (DE3) with PCDFDuet-1, PpFR- pCDF, SeFR-pCDF and HpaC-pCDF respectively according to standard procedures, generating corresponding taxifolin producing E. coll strains. The three plasmids with the constructed operon, SAM5-HpaC-pRSF, SAM5-PpFR-pRSF, and SAM5-SeFR-pRSF, are transformed into BL21(DE3) respectively, yielding three E. coll strains.

[0065] The plasmids, HpaC-pET28a, PpFR-pET28a and SeFR-pET28a, are transformed into BL21(DE3) competent cells for heterologous protein expression with standard procedures, respectively. A single colony for each transformation is grown in 5 mL of LB medium with 50 mg/L of kanamycin at 37°C until OD600 reached about 1.0, and these seed cultures are transferred to 200 mL of LB medium with 50 mg/L of kanamycin. The cells are grown at 37°C at 250 rpm to OD600 of 0.6-0.8, and then IPTG is added to a final concentration of 0.5 mM and the growth temperature is changed to 16°C. The E. coli cells are harvested after 16 hours of IPTG induction for protein purification by centrifugation at 4000 g for 15 min at 4°C. The resultant pellet is re-suspended in 5 mL of 100 mM Tris-HCl, pH 7.4, 100 mM NaOH, 10% glycerol (v/v), and sonicated for 2 min on ice. The mixture is centrifuged at 4000 g for 20 min at 4°C. The recombination protein in the supernatant is purified with His60 Ni Superflow resin from Clontech Inc. per the manufacturer's protocol.

[0066] Flavin reductase activity can be determined by measuring the change of the absorbance at 340 nm at 30 °C, using SpectraMax i3. Fixed amounts of purified proteins (0.1 pg, respectively) are incubated with 400 pM NADH and 200 pM FAD in reaction buffer (Tris-HCl 20 mM pH 7.4, final volume 100 pL). Assay mixtures without FAD can be used as blanks.

[0067] In an embodiment, the expression vector includes those genetic elements for expression of the recombinant polypeptide in bacterial cells. The elements for transcription and translation in the bacterial cell can include a promoter, a coding region for the protein complex, and a transcriptional terminator.

[0068] A person of ordinary skill in the art will be aware of the molecular biology techniques available for the preparation of expression vectors. The polynucleotide used for incorporation into the expression vector can be prepared by routine techniques such as polymerase chain reaction (PCR). In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed (e.g., plasmid, cosmid, Lambda phages). A vector containing foreign DNA is considered recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

[0069] A number of molecular biology techniques have been developed to operably link DNA to vectors via complementary cohesive termini. In one embodiment, complementary homopolymer tracts can be added to the nucleic acid molecule to be inserted into the vector DNA. The vector and nucleic acid molecule are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

[0070] In an alternative embodiment, synthetic linkers containing one or more restriction sites provide are used to operably link the polynucleotide of the subject technology to the expression vector. In an embodiment, the polynucleotide is generated by restriction endonuclease digestion. In an embodiment, the nucleic acid molecule is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3 '-ends with their polymerizing activities, thereby generating blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the product of the reaction is a polynucleotide carrying polymeric linker sequences at its ends. These polynucleotides are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the polynucleotide.

[0071] Alternatively, a vector having ligation-independent cloning (LIC) sites can be employed. The required PCR amplified polynucleotide can then be cloned into the LIC vector without restriction digest or ligation (Aslanidis and de Jong, NUCL. ACID. RES. 18 6069-74, (1990), Haun, et al., BIOTECHNIQUES 13, 515-18 (1992), each of which are incorporated herein by reference).

[0072] In an embodiment, in order to isolate and/or modify the polynucleotide of interest for insertion into the chosen plasmid, it is suitable to use PCR. Appropriate primers for use in PCR preparation of the sequence can be designed to isolate the required coding region of the nucleic acid molecule, add restriction endonuclease or LIC sites, place the coding region in the desired reading frame. [0073] In an embodiment, a polynucleotide for incorporation into an expression vector of the subject technology is prepared using PCR appropriate oligonucleotide primers. The coding region is amplified, whilst the primers themselves become incorporated into the amplified sequence product. In an embodiment, the amplification primers contain restriction endonuclease recognition sites, which allow the amplified sequence product to be cloned into an appropriate vector.

[0074] The expression vectors can be introduced into plant or microbial host cells by conventional transformation or transfection techniques. Transformation of appropriate cells with an expression vector of the subject technology is accomplished by methods known in the art and typically depends on both the type of vector and cell. Suitable techniques include calcium phosphate or calcium chloride co-precipitation, DEAE- dextran mediated transfection, lipofection, chemoporation or electroporation.

[0075] Successfully transformed cells, that is, those cells containing the expression vector, can be identified by techniques well known in the art. For example, cells transfected with an expression vector of the subject technology can be cultured to produce polypeptides described herein. Cells can be examined for the presence of the expression vector DNA by techniques well known in the art.

[0076] The host cells can contain a single copy of the expression vector described previously, or alternatively, multiple copies of the expression vector,

[0077] In some embodiments, the transformed cell is an animal cell, an insect cell, a plant cell, an algal cell, a fungal cell, or a yeast cell. In some embodiments, the cell is a plant cell selected from the group consisting of: canola plant cell, a rapeseed plant cell, a palm plant cell, a sunflower plant cell, a cotton plant cell, a corn plant cell, a peanut plant cell, a flax plant cell, a sesame plant cell, a soybean plant cell, and a petunia plant cell.

[0078] Microbial host cell expression systems and expression vectors containing regulatory sequences that direct high-level expression of foreign proteins are well-known to those skilled in the art. Any of these could be used to construct vectors for expression of a recombinant polypeptide in a microbial host cell. These vectors could then be introduced into appropriate microorganisms via transformation to allow for high level expression of a recombinant polypeptide.

[0079] Vectors or cassettes useful for the transformation of suitable microbial host cells are well known in the art. Typically the vector or cassette contains sequences directing transcription and translation of the relevant polynucleotide, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the polynucleotide which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. It is preferred for both control regions to be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a host.

[0080] Termination control regions may also be derived from various genes native to the microbial hosts. A termination site optionally may be included for the microbial hosts described herein.

[0081] Standard recombinant DNA and molecular cloning techniques are well known in the art and are described, for example, by Sambrook, J., Fritsch, E. F. and Maniatis, T. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter "Maniatis"); and by Silhavy, T. L, Bennan, M. L. and Enquist, L. W. EXPERIMENTS WITH GENE FUSIONS; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. et al., IN CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, published by Greene Publishing and Wiley-InterScience, 1987; (the entirety of each of which is hereby incorporated herein by reference).

[0082] In some embodiments of any one of the methods or compositions provided herein, the taxifolin in the composition as described herein is substantially purified. In some embodiments of any one of the methods or compositions provided herein, the taxifolin in the composition has a chemical purity (as determined by HPLC) greater than about 70%, greater than about 71%, greater than about 72%, greater than about 73%, greater than about 74%, greater than about 75%, greater than about 76%, greater than about 77%, greater than about 78%, greater than about 79%, greater than about 80%, greater than about 81%, greater than about 82%, greater than about 83%, greater than about 84%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99%, inclusive of all ranges and subranges therebetween. In some embodiments of any one of the methods or compositions provided herein, the taxifolin has a chemical purity of about 95% or more. In some embodiments of any one of the methods or compositions provided herein, the taxifolin in the composition as described herein has a chemical purity of 98% or more, e.g., 99% or more. In some embodiments of any one of the methods or compositions provided herein, this purity of the taxifolin refers to the taxifolin that is used in an ultimate product, which ultimate product can include other active and/or inactive agents.

[0083] In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be in a free-flowing powder form. Without wishing to be bound by theory, the flow of powder can be affected by factors such as shape of the particles, surface of the particles, and the presence of electrostatic charges. In some embodiments of any one of the methods or compositions provided herein, to improve the flowability, flow enhancers may be used in the composition to reduce interparticle friction. In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be in any form that is suitable for the methods or compositions as described herein.

[0084] In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be used in various forms for human consumption or animal consumption. In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be used in dietary supplements, including human or animal supplements. In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be used in food, including human food, pet food, animal feed (e.g., feed for equine, cattle). In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be used in beverages. In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be used in cosmetic products. In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be used in any dietary products suitable for the methods as described herein. In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be used in any pharmaceutical products, for humans or animals, suitable for the methods as described herein.

[0085] In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition is in an amount suitable for human subjects in need thereof. In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition is in an amount effective for any one of the methods as described herein. For example, the taxifolin composition is in an amount effective for treating, preventing, or reducing symptoms of systemic lupus erythematosus.

[0086] Any one of the compositions provided herein, as well as any one of the methods provided herein, can be used in treating, preventing, or reducing symptoms of systemic lupus erythematosus. Any one of the subjects provided herein may be one in need of any one of the possible endpoints provided herein.

[0087] In some embodiments, the symptoms of systemic lupus erythematosus are skin- related symptoms. Skin-related symptoms include, but are not limited to, skin inflammation, skin irritation, rashes, sores, scars, discoloration, hair loss, calcinosis, and mucosal ulcers. See, e.g., Villanueva et al., The Journal of Immunology 187(l):538-52 (2011); Guiducci et al., Journal of Experimental Medicine 207 73):2931-42 (2010).

[0088] The present disclosure also provides methods of treating a thromboinflammatory disease, disorder, or condition, or a symptom thereof, in a subject in need thereof, the method comprising administering an effective amount of taxifolin to the subject.

[0089] In some embodiments, the thromboinflammatory disease, disorder, or condition is anti-phospholipid syndrome or thrombosis.

[0090] The present disclosure also provides methods of treating cystic fibrosis, or a symptom thereof, in a subject in need thereof, the method comprising administering an effective amount of taxifolin to the subject.

[0091] As described herein, "effective amount" refers to an amount of the taxifolin composition that can treat, prevent, or alleviate the symptoms of any one of the conditions or states provided herein, e.g., systemic lupus erythematosus, including a disorder or disease or other condition provided herein, when administered to a subject in need thereof, and therefore, can achieve at least a desired outcome or effect as provided herein and/or understood by a skilled person in the art. Effective amounts also refer to an amount of the taxifolin composition that can be used for any one of the purposes provided in and achieve any one of the endpoints described herein.

[0092] Aspects of the present disclosure provide at least in part methods, and related compositions, of treating systemic lupus erythematosus. Any one of the compositions provided herein, as well as any one of the methods provided herein, can be used to treat systemic lupus erythematosus or a similar autoimmune disease. [0093] As described herein, "systemic lupus erythematosus" refers to an autoimmune disease in which a subject's immune system mistakenly mounts an immune response against functioning tissue.

[0094] The amount of a taxifolin composition required to achieve a particular outcome or effect, may vary based on several factors including, but not limited to: the route of administration, the body weight of the subject in need thereof, the level of taxifolin required to achieve a desired effect, the stability of the taxifolin composition, etc. One of skill in the art can readily determine an amount of the taxifolin composition to provide or administer to a subject for any one of the purposes provided herein, as relevant and needed. In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be in any amount suitable for human subjects in need thereof as described herein.

[0095] In some embodiments of any one of the methods provided herein, the disorder, disease or other condition is or is associated with inflammation.

[0096] In some embodiments of any one of the methods or compositions provided herein, the taxifolin is purified. In some embodiments of any one of the methods or compositions provided herein, the purified taxifolin has purity of about 95% or at least 98% or 99% or more. In some embodiments of any one of the methods or compositions provided herein, the purified taxifolin can have any purity as described herein.

[0097] In some embodiments of any one of the methods or compositions provided herein, the taxifolin can be in any amounts that are suitable for the methods as described herein.

[0098] In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be in a free-flowing powder form. In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be in any forms as described herein.

[0099] The taxifolin composition described herein can be used as or mixed with dietary supplements, medical compositions, cosmeceuticals, for nutrition, as well as in pharmaceutical products.

[0100] In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be formulated or prepared as a food supplement for oral consumption. In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be formulated or prepared for dietary products, such as food products, medical foods, etc. In some embodiments of any one of the methods or compositions provided herein, the taxifolin composition can be formulated or prepared for beverages.

[0101] Food compositions according to the invention include any preparations or compositions which are suitable for consumption and are used for nutrition or enjoyment purposes. They are generally products which are intended to be eaten by humans or animals and introduced into the body through the mouth, to remain there for a certain time and then either be eaten (e.g., ready-to-eat foodstuffs or feeds, see also herein below) or removed (e.g. chewing gums). Such products include any substances or products which in the processed, partially processed or unprocessed state are to be ingested by humans or animals. They also include substances which are added to orally consumable products during their manufacture, preparation or treatment and which are intended to be introduced into the human or animal oral cavity.

[0102] The food compositions according to the invention also include substances which in the unchanged, treated or prepared state are to be swallowed by a human or animal and then digested; in this respect, the orally consumable products according to the invention also include casings, coatings or other encapsulations which are to be swallowed at the same time or which may be expected to be swallowed. The expression "food composition" covers ready-to-eat foodstuffs, beverages and feeds, that is to say foodstuffs, beverages or feeds that are already complete in terms of the substances that are important for the taste. The expressions "ready-to-eat foodstuff and "ready-to-eat feed" also include drinks as well as solid or semi-solid ready-to-eat foodstuffs or feeds. Examples include frozen products, which must be thawed and heated to eating temperature before they are eaten. Products such as yoghurt or ice cream as well as chewing gums or hard caramels are also included among the ready-to-eat foodstuffs or feeds of the current invention.

[0103] Non-limiting examples of food and beverage products include carbonated soft drinks, ready to drink beverages, energy drinks, isotonic drinks, low-calorie drinks, zerocalorie drinks, sports drinks, teas, fruit and vegetable juices, juice drinks, dairy drinks, yoghurt drinks, alcohol beverages, powdered beverages, bakery products, cookies, biscuits, baking mixes, cereals, confectioneries, candies, toffees, chewing gum, dairy products, flavored milk, yoghurts, flavored yoghurts, cultured milk, soy sauce and other soy base products, salad dressings, mayonnaise, vinegar, frozen-desserts, meat products, fish-meat products, bottled and canned foods, tabletop sweeteners, fruits and vegetables. [0104] The compositions can be used "as-is" or in combination with sweeteners, flavors and food ingredients. Dry compositions, such as powders, granules or tablets are stable indefinitely when stored under dry conditions at room temperature. Compositions in the form of aqueous solutions are stable indefinitely when frozen. If a preservative such as benzoic acid or its salts, sulphur dioxide or sodium meta-bi sulphite is added to such a composition, it may be stored almost indefinitely at room temperature.

[0105] Non-limiting examples of flavors include lemon, orange, fruity, banana, grape, pear, pineapple, bitter almond, cola, cinnamon, sugar, cotton candy, vanilla flavors.

[0106] Non-limiting examples of other food ingredients include flavors, acidulants, organic and amino acids, coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents.

[0107] Aspects of the present disclosure provide, in some embodiments, a composition comprising a therapeutically effective amount of taxifolin. The composition can be a pharmaceutical composition. In some embodiments of any one of the methods or compositions provided herein, the pharmaceutical composition can include one or more pharmaceutically acceptable carriers.

[0108] In another embodiment, the taxifolin is prepared in the form of a topical formulation, such as a cream, a lotion, a gel, an ointment, a paste, an aerosol foam or spray, a powder, or a transdermal patch. Topical formulations are known to those of skill in the art and may comprise one or more excipients, including, but not limited to, emollients, hydrophilic and/or lipophilic solubilizing agents, emulsifiers, viscosity modifying agents, matrix builders, film formers, and skin penetrants.

[0109] In some embodiments of any one of the methods or compositions provided herein, the pharmaceutical composition comprising a therapeutically effective amount of taxifolin can comprise other pharmaceutical ingredients, such as preservatives, or chemical stabilizers.

[0110] Formulation of pharmaceutically-acceptable excipients and carrier solutions is known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein. The amount of active compound in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[OHl] The forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. In many cases, the form is sterile and fluid to the extent that easy syringeability exists. It may be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0112] Routes of administration are known to those of skill in the art and may be combined, if desired. In another embodiment, the taxifolin is administered orally. In another embodiment, the taxifolin is administered topically.

[0113] Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

[0114] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

[0115] Purified taxifolin can have purity of about 98%, 99% or more, or any other purity level that is suitable for the methods and the formulating of the compositions as disclosed herein.

[0116] Suitable carriers may be readily selected by one of skill in the art in view of the type of ultimate product and agents. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.

[0117] Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin. In some embodiments of any one of the methods or compositions provided herein, the composition can comprise flavoring agents or sweeteners.

[0118] Methods described herein comprise providing or administering the composition in sufficient amounts for any one of the endpoints provided herein without undue adverse effects.

[0119] The taxifolin composition can be formulated or prepared in a neutral or salt form. [0120] The disclosure also provides the following particular embodiments.

[0121] Embodiment 1. A method of treating or preventing systemic lupus erythematosus, or a symptom thereof, in a subject in need thereof, the method comprising administering an effective amount of taxifolin to the subject.

[0122] Embodiment 2. The method of Embodiment 1, wherein the taxifolin has a chemical purity of at least 95%. [0123] Embodiment 3. The method of Embodiment 1 or 2, wherein the taxifolin has a chemical purity of at least about 98%.

[0124] Embodiment 4. The method of any one of Embodiments 1-3, wherein the taxifolin is administered in an amount that provides about 10 mg/day to about 1,500 mg/day to the subject.

[0125] Embodiment 5. The method of any one of Embodiments 1-4, wherein the taxifolin is administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

[0126] Embodiment 6. The method of any one of Embodiments 1-5, wherein the taxifolin is administered orally.

[0127] Embodiment 7. The method of Embodiment 6, wherein the taxifolin is administered as an orally consumable product.

[0128] Embodiment 8. The method of Embodiment 7, wherein the orally consumable product comprises a casing or a coating.

[0129] Embodiment 9. The method of Embodiment 6, wherein the taxifolin is administered as a dietary capsule or tablet, a sachet, a functional beverage, gummies, chewables, or a nutritional bar.

[0130] Embodiment 10. The method of Embodiment 6, wherein the taxifolin is administered in a carrier.

[0131] Embodiment 11. The method of Embodiment 10, wherein the carrier comprises liposomes, micelles, lipid emulsions, milks, lipids, phospholipids, microparticles, or nanoparticles, or a combination thereof.

[0132] Embodiment 12. The method of any one of Embodiments 6-11, wherein the taxifolin is administered one, two, or three times per day.

[0133] Embodiment 13. The method of Embodiment 12, wherein the taxifolin is administered one time per day.

[0134] Embodiment 14. The method of Embodiment 12, wherein the taxifolin is administered three times per day.

[0135] Embodiment 15. The method of any one of Embodiments 1-14, wherein the taxifolin is (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one.

[0136] Embodiment 16. The method of Embodiment 15, wherein the (2R,3R)-2-

(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one has an enantiomeric excess of about 95% or more. [0137] Embodiment 17. The method of any one of Embodiments 1-16 for treating systemic lupus erythematosus.

[0138] Embodiment 18. The method of any one of Embodiments 1-16 for preventing systemic lupus erythematosus.

[0139] Embodiment 19. The method of any one of Embodiments 1-16 for treating a symptom of systemic lupus erythematosus.

[0140] Embodiment 20. The method of any one of Embodiments 1-16 for preventing a symptom of systemic lupus erythematosus.

[0141] Embodiment 21. Taxifolin for use in treating or preventing systemic lupus erythematosus, or a symptom thereof, in a subject.

[0142] Embodiment 22. The taxifolin of Embodiment 21, wherein the taxifolin has a chemical purity of at least 95%.

[0143] Embodiment 23. The taxifolin of Embodiment 21 or 22, wherein the taxifolin has a chemical purity of at least about 98%.

[0144] Embodiment 24. The taxifolin of any one of Embodiments 21-23, wherein the taxifolin is to be administered in an amount that provides about 10 mg/day to about 1,500 mg/day to the subject.

[0145] Embodiment 25. The taxifolin of any one of Embodiments 21-24, wherein the taxifolin is to be administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

[0146] Embodiment 26. The taxifolin of any one of Embodiments 21-25, wherein the taxifolin is to be administered orally.

[0147] Embodiment 27. The taxifolin of Embodiment 26, wherein the taxifolin is to be administered as an orally consumable product.

[0148] Embodiment 28. The taxifolin of Embodiment 27, wherein the orally consumable product comprises a casing or a coating.

[0149] Embodiment 29. The taxifolin of Embodiment 26, wherein the taxifolin is to be administered as a dietary capsule or tablet, a sachet, a functional beverage, gummies, chewables, or a nutritional bar.

[0150] Embodiment 30. The taxifolin of Embodiment 26, wherein the taxifolin is to be administered in a carrier. [0151] Embodiment 31. The taxifolin of Embodiment 30, wherein the carrier comprises liposomes, micelles, lipid emulsions, milks, lipids, phospholipids, microparticles, or nanoparticles, or a combination thereof.

[0152] Embodiment 32. The taxifolin of any one of Embodiments 26-31, wherein the taxifolin is to be administered one, two, or three times per day.

[0153] Embodiment 33. The taxifolin of Embodiment 32, wherein the taxifolin is to be administered one time per day.

[0154] Embodiment 34. The taxifolin of Embodiment 32, wherein the taxifolin is to be administered three times per day.

[0155] Embodiment 35. The taxifolin of any one of Embodiments 21-24, wherein the taxifolin is (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one.

[0156] Embodiment 36. The taxifolin of Embodiment 35, wherein the (2R,3R)-2-

(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one has an enantiomeric excess of about 95% or more.

[0157] Embodiment 37. The taxifolin of any one of Embodiments 21-36 for treating systemic lupus erythematosus.

[0158] Embodiment 38. The taxifolin of any one of Embodiments 21-36 for preventing systemic lupus erythematosus.

[0159] Embodiment 39. The taxifolin of any one of Embodiments 21-36 for treating a symptom of systemic lupus erythematosus.

[0160] Embodiment 40. The taxifolin of any one of Embodiments 21-36 for preventing a symptom of systemic lupus erythematosus.

[0161] Embodiment 41. Use of taxifolin in the manufacture of a medicament for treating or preventing systemic lupus erythematosus, or a symptom thereof, in a subject.

[0162] Embodiment 42. The use of Embodiment 41, wherein the taxifolin has a chemical purity of at least 95%.

[0163] Embodiment 43. The use of Embodiment 41 or 42, wherein the taxifolin has a chemical purity of at least about 98%.

[0164] Embodiment 44. The use of any one of Embodiments 41-43, wherein the taxifolin is to be administered in an amount that provides about 10 mg/day to about 1,500 mg/day to the subject. [0165] Embodiment 45. The use of any one of Embodiments 41-44, wherein the taxifolin is to be administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

[0166] Embodiment 46. The use of any one of Embodiments 41-45, wherein the taxifolin is to be administered orally.

[0167] Embodiment 47. The use of Embodiment 46, wherein the taxifolin is to be administered as an orally consumable product.

[0168] Embodiment 48. The use of Embodiment 47, wherein the orally consumable product comprises a casing or a coating.

[0169] Embodiment 49. The use of Embodiment 46, wherein the taxifolin is to be administered as a dietary capsule or tablet, a sachet, a functional beverage, gummies, chewables, or a nutritional bar.

[0170] Embodiment 50. The use of Embodiment 46, wherein the taxifolin is to be administered in a carrier.

[0171] Embodiment 51. The use of Embodiment 50, wherein the carrier comprises liposomes, micelles, lipid emulsions, milks, lipids, phospholipids, microparticles, or nanoparticles, or a combination thereof.

[0172] Embodiment 52. The use of any one of Embodiments 46-51, wherein the taxifolin is to be administered one, two, or three times per day.

[0173] Embodiment 53. The use of Embodiment 52, wherein the taxifolin is to be administered one time per day.

[0174] Embodiment 54. The use of Embodiment 52, wherein the taxifolin is to be administered three times per day.

[0175] Embodiment 55. The use of any one of Embodiments 41-154, wherein the taxifolin is (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one.

[0176] Embodiment 56. The use of Embodiment 55, wherein the (2R,3R)-2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one has an enantiomeric excess of about 95% or more.

[0177] Embodiment 57. The use of any one of Embodiments 41-56 for treating systemic lupus erythematosus.

[0178] Embodiment 58. The use of any one of Embodiments 41-56 for preventing systemic lupus erythematosus. [0179] Embodiment 59. The use of any one of Embodiments 41-56 for treating a symptom of systemic lupus erythematosus.

[0180] Embodiment 60. The use of any one of Embodiments 41-56 for preventing a symptom of systemic lupus erythematosus.

[0181] Embodiment 61. The method of any one of Embodiments 1-5, wherein the taxifolin is administered topically.

[0182] Embodiment 62. The method of Embodiment 61, wherein the taxifolin is administered as a topical cream, a lotion, a gel, an ointment, a paste, an aerosol foam or spray, a powder, or a transdermal patch.

[0183] Embodiment 63. The taxifolin of any one of Embodiments 21-25, wherein the taxifolin is administered topically.

[0184] Embodiment 64. The taxifolin of Embodiment 63, wherein the taxifolin is administered as a topical cream, a lotion, a gel, an ointment, a paste, an aerosol foam or spray, a powder, or a transdermal patch.

[0185] Embodiment 65. The use of any one of Embodiments 41-45, wherein the taxifolin is administered topically.

[0186] Embodiment 66. The use of Embodiment 65, wherein the taxifolin is administered as a topical cream, a lotion, a gel, an ointment, a paste, an aerosol foam or spray, a powder, or a transdermal patch.

[0187] Embodiment 67. The method of any one of Embodiments 1-20, 61, or 62, wherein the symptom is skin inflammation.

[0188] Embodiment 68. The taxifolin of any one of Embodiments 21-40, 63, or 64, wherein the symptom is skin inflammation.

[0189] Embodiment 69. The use of any one of Embodiments 41-60, 65, or 66, wherein the symptom is skin inflammation.

[0190] Embodiment 70. A method of treating a thromboinflammatory disease, disorder, or condition, or a symptom thereof, in a subject in need thereof, the method comprising administering an effective amount of taxifolin to the subject.

[0191] Embodiment 71. The method of Embodiment 70, wherein the taxifolin has a chemical purity of at least about 95%.

[0192] Embodiment 72. The method of Embodiment 70 or 71, wherein the taxifolin has a chemical purity of at least about 98%. [0193] Embodiment 73. The method of any one of Embodiments 70-72, wherein the taxifolin is administered in an amount that provides about 10 mg/day to about 1,500 mg/day to the subject.

[0194] Embodiment 74. The method of any one of Embodiments 70-73, wherein the taxifolin is administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

[0195] Embodiment 75. The method of any one of Embodiments 70-74, wherein the taxifolin is administered orally.

[0196] Embodiment 76. The method of Embodiment 75, wherein the taxifolin is administered as an orally consumable product.

[0197] Embodiment 77. The method of Embodiment 76, wherein the orally consumable product comprises a casing or a coating.

[0198] Embodiment 78. The method of Embodiment 75, wherein the taxifolin is administered as a dietary capsule or tablet, a sachet, a functional beverage, gummies, chewables, or a nutritional bar.

[0199] Embodiment 79. The method of Embodiment 75, wherein the taxifolin is administered in a carrier.

[0200] Embodiment 80. The method of Embodiment 79, wherein the carrier comprises liposomes, micelles, lipid emulsions, milks, lipids, phospholipids, microparticles, or nanoparticles, or a combination thereof.

[0201] Embodiment 81. The method of any one of Embodiments 75-80, wherein the taxifolin is administered one, two, or three times per day.

[0202] Embodiment 82. The method of Embodiment 81, wherein the taxifolin is administered one time per day.

[0203] Embodiment 83. The method of Embodiment 81, wherein the taxifolin is administered three times per day.

[0204] Embodiment 84. The method of any one of Embodiments 70-83, wherein the taxifolin is (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one.

[0205] Embodiment 85. The method of Embodiment 84, wherein the (2R,3R)-2-

(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one has an enantiomeric excess of about 95% or more. [0206] Embodiment 86. The method of any one of Embodiments 70-83, wherein the thromboinflammatory disease, disorder, or condition is anti-phospholipid syndrome or thrombosis.

[0207] Embodiment 87. A method of treating cystic fibrosis, or a symptom thereof, in a subject in need thereof, the method comprising administering an effective amount of taxifolin to the subject.

[0208] Embodiment 88. The method of Embodiment 87, wherein the taxifolin has a chemical purity of at least about 95%.

[0209] Embodiment 89. The method of Embodiment 87 or 88, wherein the taxifolin has a chemical purity of at least about 98%.

[0210] Embodiment 90. The method of any one of Embodiments 87-89, wherein the taxifolin is administered in an amount that provides about 10 mg/day to about 1,500 mg/day to the subject.

[0211] Embodiment 91. The method of any one of Embodiments 87-90, wherein the taxifolin is administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

[0212] Embodiment 92. The method of any one of Embodiments 87-91, wherein the taxifolin is administered orally.

[0213] Embodiment 93. The method of Embodiment 92, wherein the taxifolin is administered as an orally consumable product.

[0214] Embodiment 94. The method of Embodiment 93, wherein the orally consumable product comprises a casing or a coating.

[0215] Embodiment 95. The method of Embodiment 92, wherein the taxifolin is administered as a dietary capsule or tablet, a sachet, a functional beverage, gummies, chewables, or a nutritional bar.

[0216] Embodiment 96. The method of Embodiment 92, wherein the taxifolin is administered in a carrier.

[0217] Embodiment 97. The method of Embodiment 96, wherein the carrier comprises liposomes, micelles, lipid emulsions, milks, lipids, phospholipids, microparticles, or nanoparticles, or a combination thereof.

[0218] Embodiment 98. The method of any one of Embodiments 92-97, wherein the taxifolin is administered one, two, or three times per day. [0219] Embodiment 99. The method of Embodiment 98, wherein the taxifolin is administered one time per day.

[0220] Embodiment 100. The method of Embodiment 98, wherein the taxifolin is administered three times per day.

[0221] Embodiment 101. The method of any one of Embodiments 87-100, wherein the taxifolin is (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one.

[0222] Embodiment 102. The method of Embodiment 101, wherein the (2R,3R)-2-

[0223] Embodiment 103. Taxifolin for use in treating a thromboinflammatory disease, disorder, or condition, or a symptom thereof, in a subject.

[0224] Embodiment 104. The taxifolin of Embodiment 103, wherein the taxifolin has a chemical purity of at least about 95%.

[0225] Embodiment 105. The taxifolin of Embodiment 103 or 104, wherein the taxifolin has a chemical purity of at least about 98%.

[0226] Embodiment 106. The taxifolin of any one of Embodiments 103-105, wherein the taxifolin is administered in an amount that provides about 10 mg/day to about 1,500 mg/day to the subject.

[0227] Embodiment 107. The taxifolin of any one of Embodiments 103-106, wherein the taxifolin is administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

[0228] Embodiment 108. The taxifolin of any one of Embodiments 103-107, wherein the taxifolin is administered orally.

[0229] Embodiment 109. The taxifolin of Embodiment 108, wherein the taxifolin is administered as an orally consumable product.

[0230] Embodiment 110. The taxifolin of Embodiment 109, wherein the orally consumable product comprises a casing or a coating.

[0231] Embodiment 111. The taxifolin of Embodiment 108, wherein the taxifolin is administered as a dietary capsule or tablet, a sachet, a functional beverage, gummies, chewables, or a nutritional bar.

[0232] Embodiment 112. The taxifolin of Embodiment 108, wherein the taxifolin is administered in a carrier. [0233] Embodiment 113. The taxifolin of Embodiment 112, wherein the carrier comprises liposomes, micelles, lipid emulsions, milks, lipids, phospholipids, microparticles, or nanoparticles, or a combination thereof.

[0234] Embodiment 114. The taxifolin of any one of Embodiments 108-113, wherein the taxifolin is administered one, two, or three times per day.

[0235] Embodiment 115. The taxifolin of Embodiment 114, wherein the taxifolin is administered one time per day.

[0236] Embodiment 116. The taxifolin of Embodiment 114, wherein the taxifolin is administered three times per day.

[0237] Embodiment 117. The taxifolin of any one of Embodiments 103-116, wherein the taxifolin is (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one.

[0238] Embodiment 118. The taxifolin of Embodiment 117, wherein the (2R,3R)-2-

[0239] Embodiment 119. The taxifolin of any one of Embodiments 102-118, wherein the thromboinflammatory disease, disorder, or condition is anti -phospholipid syndrome or thrombosis.

[0240] Embodiment 120. Taxifolin for use in treating cystic fibrosis, or a symptom thereof, in a subject.

[0241] Embodiment 121. The taxifolin of Embodiment 120, wherein the taxifolin has a chemical purity of at least about 95%.

[0242] Embodiment 122. The taxifolin of Embodiment 120 or 121, wherein the taxifolin has a chemical purity of at least about 98%.

[0243] Embodiment 123. The taxifolin of any one of Embodiments 120-122, wherein the taxifolin is administered in an amount that provides about 10 mg/day to about 1,500 mg/day to the subject.

[0244] Embodiment 124. The taxifolin of any one of Embodiments 120-123, wherein the taxifolin is administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

[0245] Embodiment 125. The taxifolin of any one of Embodiments 120-124, wherein the taxifolin is administered orally.

[0246] Embodiment 126. The taxifolin of Embodiment 125, wherein the taxifolin is administered as an orally consumable product. [0247] Embodiment 127. The taxifolin of Embodiment 126, wherein the orally consumable product comprises a casing or a coating.

[0248] Embodiment 128. The taxifolin of Embodiment 120, wherein the taxifolin is administered as a dietary capsule or tablet, a sachet, a functional beverage, gummies, chewables, or a nutritional bar.

[0249] Embodiment 129. The taxifolin of Embodiment 128, wherein the taxifolin is administered in a carrier.

[0250] Embodiment 130. The taxifolin of Embodiment 129, wherein the carrier comprises liposomes, micelles, lipid emulsions, milks, lipids, phospholipids, microparticles, or nanoparticles, or a combination thereof.

[0251] Embodiment 131. The taxifolin of any one of Embodiments 126-130, wherein the taxifolin is administered one, two, or three times per day.

[0252] Embodiment 132. The taxifolin of Embodiment 131, wherein the taxifolin is administered one time per day.

[0253] Embodiment 133. The taxifolin of Embodiment 131, wherein the taxifolin is administered three times per day.

[0254] Embodiment 134. The taxifolin of any one of Embodiments 120-133, wherein the taxifolin is (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one.

[0255] Embodiment 135. The taxifolin of Embodiment 134, wherein the (2R,3R)-2-

[0256] Embodiment 136. Use of taxifolin for use in manufacture of a medicament for treating a thromboinflammatory disease, disorder, or condition, or a symptom thereof, in a subject.

[0257] Embodiment 137. The use of Embodiment 136, wherein the taxifolin has a chemical purity of at least about 95%.

[0258] Embodiment 138. The use of Embodiment 136 or 137, wherein the taxifolin has a chemical purity of at least about 98%.

[0259] Embodiment 139. The use of any one of Embodiments 136-138, wherein the taxifolin is administered in an amount that provides about 10 mg/day to about 1,500 mg/day to the subject. [0260] Embodiment 140. The use of any one of Embodiments 136-139, wherein the taxifolin is administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

[0261] Embodiment 141. The use of any one of Embodiments 136-140, wherein the taxifolin is administered orally.

[0262] Embodiment 142. The use of Embodiment 141, wherein the taxifolin is administered as an orally consumable product.

[0263] Embodiment 143. The use of Embodiment 142, wherein the orally consumable product comprises a casing or a coating.

[0264] Embodiment 144. The use of Embodiment 141, wherein the taxifolin is administered as a dietary capsule or tablet, a sachet, a functional beverage, gummies, chewables, or a nutritional bar.

[0265] Embodiment 145. The use of Embodiment 141, wherein the taxifolin is administered in a carrier.

[0266] Embodiment 146. The use of Embodiment 145, wherein the carrier comprises liposomes, micelles, lipid emulsions, milks, lipids, phospholipids, microparticles, or nanoparticles, or a combination thereof.

[0267] Embodiment 147. The use of any one of Embodiments 141-146, wherein the taxifolin is administered one, two, or three times per day.

[0268] Embodiment 148. The use of Embodiment 147, wherein the taxifolin is administered one time per day.

[0269] Embodiment 149. The use of Embodiment 147, wherein the taxifolin is administered three times per day.

[0270] Embodiment 150. The use of any one of Embodiments 136-149, wherein the taxifolin is (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one.

[0271] Embodiment 151. The use of Embodiment 150, wherein the (2R,3R)-2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one has an enantiomeric excess of about 95% or more.

[0272] Embodiment 152. The use of any one of Embodiments 134-151, wherein the thromboinflammatory disease, disorder, or condition is anti-phospholipid syndrome or thrombosis.

[0273] Embodiment 153. Use of taxifolin in the manufacture of a medicament for treating cystic fibrosis, or a symptom thereof, in a subject. [0274] Embodiment 154. The use of Embodiment 153, wherein the taxifolin has a chemical purity of at least about 95%.

[0275] Embodiment 155. The use of Embodiment 153 or 154, wherein the taxifolin has a chemical purity of at least about 98%.

[0276] Embodiment 156. The use of any one of Embodiments 153-155, wherein the taxifolin is administered in an amount that provides about 10 mg/day to about 1,500 mg/day to the subject.

[0277] Embodiment 157. The use of any one of Embodiments 153-156, wherein the taxifolin is administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

[0278] Embodiment 158. The use of any one of Embodiments 153-157, wherein the taxifolin is administered orally.

[0279] Embodiment 159. The use of Embodiment 158, wherein the taxifolin is administered as an orally consumable product.

[0280] Embodiment 160. The use of Embodiment 159, wherein the orally consumable product comprises a casing or a coating.

[0281] Embodiment 161. The use of Embodiment 158, wherein the taxifolin is administered as a dietary capsule or tablet, a sachet, a functional beverage, gummies, chewables, or a nutritional bar.

[0282] Embodiment 162. The use of Embodiment 158, wherein the taxifolin is administered in a carrier.

[0283] Embodiment 163. The use of Embodiment 162, wherein the carrier comprises liposomes, micelles, lipid emulsions, milks, lipids, phospholipids, microparticles, or nanoparticles, or a combination thereof.

[0284] Embodiment 164. The use of any one of Embodiments 158-163, wherein the taxifolin is administered one, two, or three times per day.

[0285] Embodiment 165. The use of Embodiment 164, wherein the taxifolin is administered one time per day.

[0286] Embodiment 166. The use of Embodiment 164, wherein the taxifolin is administered three times per day.

[0287] Embodiment 167. The use of any one of Embodiments 153-166, wherein the taxifolin is (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one. [0288] Embodiment 168. The taxifolin of Embodiment 167, wherein the (2R,3R)-2- (3,4-dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one has an enantiomeric excess of about 95% or more.

EXAMPLES

Example 1 Taxifolin Reduces Neutrophil Infiltration in Lungs

[0289] Intratracheal instillation of the anti-tumor agent bleomycin (BLM) is the most commonly used animal model for pulmonary fibrosis. The BLM-induced lung inflammation is orchestrated in part by neutrophils and macrophages. After BLM injection, these inflammatory cells migrate into the inflammatory foci where they release ROS, proteases and inflammatory cytokines that contribute to the fibrotic processes. Impellizzeri et al. (Protective effect of polyphenols in an inflammatory process associated with experimental pulmonary fibrosis in mice. British Journal of Nutrition 774:853-865 (2015), incorporated herein by reference) demonstrated that resveratrol at 50 mg/kg, mangiferin and taxifolin (DHQ) at 10 mg/kg significantly reduced the degree of polymorphonuclear leucocytes infiltration in the inflamed lung, as determined in part by an MPO assay, shown in Fig. 2. Treatment with quercetin did not significantly affect neutrophil infiltration in inflamed lungs.

Example 2 Taxifolin Reduces Neutrophil and Macrophage Infiltration and Tissue Damage in a Mouse Model of Viral Pancreatitis

[0290] Galochkina et al. (Virus-inhibiting activity of dihydroquercetin, a flavonoid from Larix sibirica, against coxsackievirus B4 in a model of viral pancreatitis. Archives of Virology 767:929-938 (2016), incorporated herein by reference) studied the effect of dihydroquercetin (DHQ, taxifolin), a flavonoid from larch wood, on the course of pancreatitis of white mice caused by coxsackievirus B4 (CVB4). DHQ (taxifolin) was applied intraperitoneally at doses of 75 or 150 mg/kg/day once a day for 5 days postinfection (p.i.) starting on day 1 p.i., and its effect was compared to that of the reference compound ribavirin. The application of DHQ (taxifolin) resulted in a dosedependent decrease in the virus titer in pancreatic tissue, reaching, at the highest dose, 2.4 logs on day 5 p.i. Also, the application of DHQ (taxifolin) led to restoration of antioxidant activity of pancreatic tissue that was impaired in the course of pancreatitis. Morphologically, pancreatic tissue of DHQ (taxifolin)-treated animals demonstrated less infiltration with inflammatory cells and no signs of tissue destruction compared to placebo-treated mice, as shown in Fig. 3. The effect of DHQ (taxifolin) was comparable to or exceeded that of ribavirin.

Example 3 Taxifolin Decreased the Functional Activity of PMN from Patients with Non-Insulin Dependent Diabetes

[0291] Fedosova et al. (Mechanisms Underlying Diquertin-Mediated Regulation of Neutrophil Function in Patients with Non-Insulin-Dependent Diabetes Mellitus. Bulletin of Experimental Biology and Medicine 137: 143-146 (2004), incorporated herein by reference) studied the effects of dihydroquercetin (taxifolin) on functional activity of polymorphonuclear neutrophils from patients with non-insulin-dependent diabetes mellitus. Flavonoids (quercetin and its derivative dihydroquercetin) dose-dependently suppressed generation of anion radicals and hypochlorous acid and production of malonic dialdehyde (MDA) during oxidation of neutrophil membranes, as shown in Tables 1 and 2. Dihydroquercetin decreased activities of protein kinase C and myeloperoxidase in activated polymorphonuclear neutrophils and could bind transition metals (Fe²⁺). These properties determine the ability of dihydroquercetin to decrease in vitro functional activity of polymorphonuclear neutrophils from patients with non-insulin-dependent diabetes mellitus.

TABLE 1. Effects of Quercetin (QV) and Dihydroquercetin (DQV) on Generation of Superoxide Radicals in PMA-Stimulated Human PMN (nmol 0’710⁶ PMN, M±m)

Note. Here and in Table 2: /?<0.01 : *compared to healthy donors; **compared to the control.

TABLE 2. Formation of Hypochi orous Acid in PMA-Stimulated Human PMN and Accumulation of MDA in the System of Fe²⁺ and Ascorbate under the Influence of QV and DQV (M±m)

Example 4

Inhibition of NETosis by Taxifolin

[0292] Neutrophils and neutrophil extracellular traps (NETs) play a critical role in lupus pathogenesis (Knight J.S., et al. Current Opinion in Rheumatology. 24, 441-450 (2012); Grayson, P.C., et al. J Leukoc Biol. 99, 253-264 (2016)). Rising levels of blood neutrophils predict glomerulonephritis (Banchereau, R., et al. Cell 165, 551-565 (2016)), while NETs themselves promote type I interferons (Lood, C., et al. Nat Med. 22, 146-153 (2016); Garcia-Romo, G.S., et al. Sci TranslMed. 3, 73ra20 (2011)), autoantibody formation (Lande, R., et al. Sci Transl Med. 3, 73ral9 (2011); Knight, J.S., et al. The Journal of Clinical Investigation. 123, 2981-2993 (2013)), endothelial damage (Carmona- Rivera, C., et al. Ann Rheum Dis. 74, 1417-1424 (2015)), and thrombosis (Meng, H., et al. Arthritis Rheumatol . 69, 655-667 (2017); Yalavarthi, S., et al. Arthritis Rheumatol . 67, 2990-3003 (2015)). At the same time, lupus autoantibodies — anti-ribonucleoprotein (Garcia-Romo, G.S., et al. Sci TranslMed. 3, 73ra20 (2011)), anti-LL-37 (Van Avondt, K., et al. PLoS One. 8, e78459 (2013)) and antiphospholipid (Yalavarthi, S., et al. Arthritis Rheumatol . 67, 2990-3003 (2015)) — promote NET release, thereby setting up a vicious cycle. Pathways implicated in the mechanism underlying autoantibody-mediated NET release include: reactive oxygen species (ROS) formation, TLR7, and Fc gamma receptor Ila (for anti-ribonucleoprotein immune complexes) (Garcia-Romo, G.S., et al. Sci Transl Med. 3, 73ra20 (2011)) and NF-KB, TLR4, and the beta-2 integrin Mac-1 (for antiphospholipid antibodies) (Yalavarthi, S., et al. Arthritis Rheumatol . 67, 2990-3003 (2015)).

[0293] The ability of taxifolin to inhibit NET release in neutrophils that are activated with various stimuli, including lupus autoantibodies isolated from lupus patients, was tested. Taxifolin was tested within the concentrations of 100 nM to 100 pM to determine therapeutic effect. The primary readouts from these experiments are NET release measured by myeloperoxidase enzymatic activity as well as fold-increase in extracellular DNA.

[0294] Figs. 4A and 4B show that taxifolin inhibits NETosis in neutrophils activated by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C and consequently NAPDH oxidase (Parker, H., et al. J Leukoc Biol. 92, 841-849 (2012)), starting at 1 pM with quite dramatic inhibition at 100 pM.

[0295] In an assay of neutrophil activation independent of NADPH oxidase, Figs. 5 A and 5B show that taxifolin inhibits NETosis in neutrophils activated by a calcium ionophore A23187, which triggers NETosis via calcium influx, PALM translocation, and mitochondrial ROS (Parker, H., et al. J Leukoc Biol. 92, 841-849 (2012), Pinsky, D.J., et al. Proc Natl Acad Sci U.S.A. 91, 12086-12090 (1994)), starting at 1 pM.

[0296] In a third assay, neutrophils were activated by human antiphospholipid antibodies. IgG purified and pooled from patients with primary antiphospholipid syndrome (Meng, H., et al. Arthritis Rheumatol . 69, 655-667 (2017); Yalavarthi, S., et al. Arthritis Rheumatol. 67, 2990-3003 (2015)). Human antiphospholipid antibodies have been shown to trigger Fc receptor-independent NET release from human neutrophils (Meng, H., et al. Arthritis Rheumatol . 69, 655-667 (2017); Yalavarthi, S., et al. Arthritis Rheumatol . 67, 2990-3003 (2015)), and that the mechanism is dependent upon TLR4, NF-KB, and ROS (Yalavarthi, S., et al. Arthritis Rheumatol. 67, 2990-3003 (2015)). Figs. 6A and 6B show that taxifolin starting at 1 pM inhibits neutrophils stimulated by human antiphospholipid IgG. [0297] In a fourth assay, neutrophils were activated by human lupus autoantibodies. IgG purified from lupus patients with positive testing for anti-ribonucleoprotein (RNP) antibodies, but negative testing for antiphospholipid antibodies. Anti-RNP antibodies from these patients were combined with commercial RNP to form immune complexes. RNP/anti-RNP-mediated NET release is dependent upon TLR7 and ROS (Garcia-Romo, G.S., et al. Sci Transl Med. 3, 73ra20 (2011)). Figs. 7A and 7B show that taxifolin inhibits NETosis starting at 1 pM. At 100 pM, NETosis is almost completely inhibited compared to no stimuli.

Example 5 Suppression of lupus-associated autoantibody formulation and NET release in a mouse model of lupus

[0298] The ability of taxifolin to suppress lupus-associated autoantibody formulation and NET release in a mouse model of lupus was tested. The model used is the TLR7-agonist (R848)-induced mouse model using female BALB/c mice. Yokogawa, et al., Arthritis & Rheumatology 66(3):694-706 (2014). Two experimental setups were tested:

[0299] Experimental Condition 1) Mice were treated with the TLR7 agonist for 6 weeks to induce lupus, while taxifolin at low (20 mg/kg/day) and high (50 mg/kg/day) doses compared to vehicle were given orally at the beginning of lupus induction. This experimental design tested the hypothesis that taxifolin can inhibit lupus development and progression of disease; and

[0300] Experimental Condition 2) Mice were treated with the TLR7 agonist for 6 weeks to induce lupus, while taxifolin at low (20 mg/kg/day) and high (50 mg/kg/day) doses compared to vehicle were treated at week 4 of lupus symptoms. Taxifolin (or vehicle) oral treatment continued for 2 weeks. This tested the hypothesis that taxifolin can treat or reverse progression of disease.

[0301] The doses of taxifolin selected for the mice were estimated to be comparable to human doses of 100 mg/day (low) or 250 mg/day (high). These doses meet GRAS criteria for taxifolin's use as a dietary supplement.

[0302] Oral taxifolin treatment dose-dependently reduced splenomegaly (lupus-induced spleen enlargement) in both experimental conditions, as shown in Fig. 8A (Experimental Condition 1) and Fig. 8B (Experimental Condition 2). [0303] Oral taxifolin treatment tended to rescue lupus-induced hematology derangement in both experimental conditions, as shown in Fig. 9A (Experimental Condition 1) and Fig. 9B (Experimental Condition 2).

[0304] Oral taxifolin treatment attenuated NET release in both experimental conditions, which confirms in vitro data, as shown in Fig. 10A Experimental Condition 1) and Fig. 10B (Experimental Condition 2).

[0305] Oral taxifolin treatment suppressed autoantibodies formation in both experimental conditions, which confirms in vitro data, as shown in Fig. 11 A (Experimental Condition 1) and Fig. 1 IB (Experimental Condition 2).

Example 6

Effect of Taxifolin on Thrombus Weight in Anti-Phospholipid (aPL)-Accelerated Inferior Vena Cava Thrombosis Model

[0306] In a model of anti-phospholipid (aPL)-accelerated inferior vena cava thrombosis model using C57BL/6 mice (Ali 2020), taxifolin (20 mg/kg/day) almost completely negated the acceleration of large-vein thrombosis by human aPL, as shown in Fig. 12; mean thrombus weight was 3 ± 0.2 mg in control mice, 6.6 ± 1 mg in aPL-treated mice, and 3 ± 0.3 mg in aPL-treated mice that additionally received taxifolin (p<0.001).

EQUIVALENTS AND SCOPE

[0307] In the claims articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0308] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms "comprising" and "containing" are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0309] This application refers to various issued patents, published patent applications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0310] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims. [0311] SEQ ID NO: 1 : Nucleic Acid Sequence of SAM5

ATGACGATTACCTCTCCGGCCCCGGCTGGTCGCCTGAACAATGTGCGTCCGAT

GACGGGTGAAGAATACCTGGAATCCCTGCGTGACGGTCGTGAAGTGTATATTTACGG

CGAACGCGTCGATGACGTGACCACGCATCTGGCGTTCCGCAACAGCGTTCGTTCTAT

CGCCCGCCTGTATGATGTCCTGCACGATCCGGCCTCCGAAGGTGTTCTGCGCGTCCC

GACCGATACCGGTAATGGTGGTTTTACCCATCCGTTTTTCAAAACGGCGCGTAGCTC

TGAAGACCTGGTGGCGGCCCGTGAAGCCATTGTCGGTTGGCAACGCCTGGTGTATGG

CTGGATGGGTCGTACCCCGGATTACAAGGCAGCGTTTTTCGGTACGCTGGACGCTAA

CGCGGAATTTTATGGCCCGTTCGAAGCCAATGCACGTCGCTGGTATCGTGATGCACA

GGAACGCGTTCTGTACTTCAACCATGCTATCGTGCATCCGCCGGTCGATCGTGACCG

TCCGGCTGATCGTACCGCCGACATTTGCGTCCATGTGGAAGAAGAAACGGATTCAGG

CCTGATCGTGTCGGGTGCCAAAGTGGTTGCAACCGGTTCTGCTATGACGAACGCGAA

TCTGATTGCCCACTATGGTCTGCCGGTTCGCGATAAAAAGTTTGGCCTGGTGTTCACC

GTTCCGATGAACAGTCCGGGTCTGAAACTGATCTGTCGTACCTCCTATGAACTGATG

GTGGCCACGCAGGGCTCACCGTTTGATTACCCGCTGAGTTCCCGCCTGGATGAAAAT

GACAGCATTATGATCTTTGATCGTGTTCTGGTCCCGTGGGAAAACGTTTTCATGTACG

ACGCAGGCGCGGCCAATAGCTTTGCTACCGGCTCTGGTTTCCTGGAACGCTTTACCT

TTCATGGCTGCACGCGTCTGGCAGTGAAACTGGATTTTATTGCAGGCTGTGTTATGA

AGGCTGTGGAAGTTACCGGCACCACGCACTTCCGCGGTGTTCAGGCGCAAGTCGGC

GAAGTGCTGAACTGGCGTGATGTCTTTTGGGGTCTGTCGGACGCTATGGCGAAAAGT

CCGAACAGCTGGGTGGGCGGTAGCGTTCAGCCGAACCTGAATTATGGCCTGGCCTAC

CGCACCTTTATGGGCGTGGGTTATCCGCGTATTAAAGAAATTATCCAGCAAACGCTG

GGCTCTGGTCTGATCTACCTGAACTCATCGGCAGCTGATTGGAAGAATCCGGACGTT

CGCCCGTATCTGGATCGTTACCTGCGCGGCAGTCGTGGTATTCAGGCAATCGATCGT

GTCAAACTGCTGAAGCTGCTGTGGGACGCGGTGGGCACCGAATTTGCCGGTCGTCAT

GAACTGTATGAACGCAACTACGGCGGTGATCACGAAGGCATTCGTGTGCAGACCCT

GCAAGCCTATCAGGCAAATGGTCAAGCGGCGGCACTGAAAGGCTTTGCGGAACAGT

GCATGAGCGAATACGACCTGGATGGCTGGACCCGCCCGGACCTGATTAACCCGGGC ACCTGA

[0312] SEQ ID NO: 2 Amino Acid Sequence of SAM5

MTITSPAPAGRLNNVRPMTGEEYLESLRDGREVYIYGERVDDVTTHLAFRNSVRS

IARLYDVLHDPASEGVLRVPTDTGNGGFTHPFFKTARSSEDLVAAREAIVGWQRLVYG WMGRTPDYKAAFFGTLDANAEFYGPFEANARRWYRDAQERVLYFNHAIVHPPVDRDR

PADRTADICVHVEEETDSGLIVSGAKVVATGSAMTNANLIAHYGLPVRDKKFGLVFTVP

MNSPGLKLICRTSYELMVATQGSPFDYPLSSRLDENDSIMIFDRVLVPWENVFMYDAGA ANSFATGSGFLERFTFHGCTRLAVKLDFIAGCVMKAVEVTGTTHFRGVQAQVGEVLNW RDVFWGLSDAMAKSPNSWVGGSVQPNLNYGLAYRTFMGVGYPRIKEIIQQTLGSGLIYL NSSAADWKNPDVRPYLDRYLRGSRGIQAIDRVKLLKLLWDAVGTEFAGRHELYERNYG GDHEGIRVQTLQAYQANGQAAALKGFAEQCMSEYDLDGWTRPDLINPGT.

[0313] SEQ ID NO:3 Nucleic Acid Sequence of SeFR

ATGATGACCGTTTATGATAGCGCACTGACAATGGAAGAAACCACCCTGCGTG

ATGCAATGAGCCGTTTTGCAACCGGTGTTAGCGTTGTTACCGTTGGTGGTGAACATA CACATGGTATGACCGCAAATGCCTTTACCTGTGTTAGCCTGGATCCGCCTCTGGTTCT

GTGTTGTGTTGCACGTAAAGCAACCATGCATGCAGCAATTGAAGGTGCACGTCGTTT

TGCAGTTAGCGTTATGGGTGGTGATCAAGAACGTACCGCACGTTATTTTGCAGATAA

ACGTCGTCCGCGTGGTCGTGCACAGTTTGATGTTGTTGATTGGCAGCCTGGTCCGCA

TACAGGTGCACCGCTGCTGAGCGGTGCGCTGGCATGGCTGGAATGTGAAGTTGCAC

AGTGGCATGAAGGTGGCGATCATACCATTTTTCTGGGTCGTGTTCTGGGTTGTCGTCG TGGTCCGGATAGTCCGGCACTGCTGTTTTATGGTAGCGATTTTCATCAGATCCGCTAA

[0314] SEQ ID NO:4 Amino Acid Sequence of SeFR

MMTVYDSALTMEETTLRDAMSRFATGVSVVTVGGEHTHGMTANAFTCVSLDPP LVLCCVARKATMHAAIEGARRFAVSVMGGDQERTARYFADKRRPRGRAQFDVVDWQP GPHTGAPLLSGALAWLECEVAQWHEGGDHTIFLGRVLGCRRGPDSPALLFYGSDFHQIR.

[0315] SEQ ID NO:5 Nucleic Acid Sequence of 5 PfFR

ATGAATGCAGCAACCGAAACCAAAGTTCATGATCTGCTGGATGCCGAAGGTC

GTGATGTTCGTGATGCACGTGAACTGCGTAATGTTCTGGGTCAGTTTGCAACCGGTG

TTACCGTTATTACCACCCGTACCGCAGATGGTCGTAATGTTGGTGTGACCGCAAATA

GCTTTAGCAGCCTGAGCCTGAGTCCGGCACTGGTTCTGTGGTCACTGGCACGTACCG

CACCGAGCCTGAAAGTTTTTTGTAGCGCAAGCCATTTTGCCATTAATGTGCTGGGTG

CACATCAGCTGCATCTGAGCGAACAGTTTGCACGTGCCGCAGCAGATAAATTTGCCG

GTGTTGCACATAGTTATGGTAAAGCGGGTGCACCGGTTCTGGATGATGTTGTTGCAG

TTCTGGTTTGCCGTAATGTTACCCAGTATGAAGGTGGTGATCATCTGATTTTTATCGG CGAAATTGAGCAGTATCGTTATAGCGGTGCAGAACCGCTGGTTTTTCATGCAGGTCA GTATCGTGGTCTGGGTAGCAATCGTGCAGAAAGCGTTCTGAAACATGAATAA

[0316] SEQ ID NO:6 Amino Acid Sequence of PfFR

MNAATETKVHDLLDAEGRDVRDARELRNVLGQFATGVTVITTRTADGRNVGVT ANSFSSLSLSPALVLWSLARTAPSLKVFCSASHFAINVLGAHQLHLSEQFARAAADKFAG VAHSYGKAGAPVLDDVVAVLVCRNVTQYEGGDHLIFIGEIEQYRYSGAEPLVFHAGQY RGLGSNRAESVLKHE.

[0317] SEQ ID NO:7 Nucleic Acid Sequence of HpaC

ATGCAATTAGATGAACAACGCCTGCGCTTTCGTGACGCAATGGCCAGCCTGT CGGCAGCGGTAAATATTATCACCACCGAGGGCGACGCCGGACAATGCGGGATTACG GCAACGGCCGTCTGCTCGGTCACGGATACACCACCATCGCTGATGGTGTGCATTAAC GCCAACAGTGCGATGAACCCGGTTTTTCAGGGCAACGGTAAGTTGTGCGTCAACGTC CTCAACCATGAGCAGGAACTGATGGCACGCCACTTCGCGGGCATGACAGGCATGGC GATGGAAGAGCGTTTTAGCCTCTCATGCTGGCAAAAAGGTCCGCTGGCGCAGCCGGT GCTAAAAGGTTCGCTGGCCAGTCTTGAAGGTGAGATCCGCGATGTGCAGGCAATTG GCACACATCTGGTGTATCTGGTGGAGATTAAAAACATCATCCTCAGTGCAGAAGGTC ACGGACTTATCTACTTTAAACGCCGTTTCCATCCGGTGATGCTGGAAATGGAAGCTG CGATTTAA

[0318] SEQ ID NO: 8 Amino Acid Sequence of HpaC

MQLDEQRLRFRDAMASLSAAVNIITTEGDAGQCGITATAVCSVTDTPPSLMVCIN ANSAMNPVFQGNGKLCVNVLNHEQELMARHFAGMTGMAMEERFSLSCWQKGPLAQP VLKGSLASLEGEIRDVQAIGTHLVYLVEIKNIILSAEGHGLIYFKRRFHPVMLEMEAAI. [0319] SEQ ID NO:9 Nucleic Acid Sequence of Forward Primer HpaC Ndel F GGGAATTCCATATGCAATTAGATGAACAACGCCTGCG

[0320] SEQ ID NO: 10 Nucleic Acid Sequence of Reverse Primer HpaC XhoI R

CTCGAGCGGTTAAATCGCAGCTTCCATTTCCAGC

Claims

- 43 -

WHAT IS CLAIMED IS: A method of treating or preventing systemic lupus erythematosus, or a symptom thereof, in a subject in need thereof, the method comprising administering an effective amount of taxifolin to the subject. The method of claim 1, wherein the taxifolin has a chemical purity of at least about 95%. The method of claim 1 or 2, wherein the taxifolin has a chemical purity of at least about 98%. The method of any one of claims 1-3, wherein the taxifolin is administered in an amount that provides about 10 mg/day to about 1,500 mg/day to the subject. The method of any one of claims 1-4, wherein the taxifolin is administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients. The method of any one of claims 1-5, wherein the taxifolin is administered orally. The method of claim 6, wherein the taxifolin is administered as an orally consumable product. The method of claim 7, wherein the orally consumable product comprises a casing or a coating. The method of claim 6, wherein the taxifolin is administered as a dietary capsule or tablet, a sachet, a functional beverage, gummies, chewables, or a nutritional bar. The method of claim 6, wherein the taxifolin is administered in a carrier. - 44 - The method of claim 10, wherein the carrier comprises liposomes, micelles, lipid emulsions, milks, lipids, phospholipids, microparticles, or nanoparticles, or a combination thereof. The method of any one of claims 1-5, wherein the taxifolin is administered topically. The method of claim 12, wherein the taxifolin is administered as a topical cream, a lotion, a gel, an ointment, a paste, an aerosol foam or spray, a powder, or a transdermal patch. The method of any one of claims 6-13, wherein the taxifolin is administered one, two, or three times per day. The method of claim 14, wherein the taxifolin is administered one time per day. The method of claim 14, wherein the taxifolin is administered three times per day. The method of any one of claims 1-16, wherein the taxifolin is (2R,3R)-2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one. The method of claim 17, wherein the (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7- trihydroxychroman-4-one has an enantiomeric excess of about 95% or more. The method of any one of claims 1-18 for treating systemic lupus erythematosus. The method of any one of claims 1-18 for preventing systemic lupus erythematosus. The method of any one of claims 1-18 for treating a symptom of systemic lupus erythematosus. The method of any one of claims 1-18 for preventing a symptom of systemic lupus erythematosus. The method of claim 21 or 22, wherein the symptom is skin inflammation. - 45 - A method of treating a thromboinflammatory disease, disorder, or condition, or a symptom thereof, in a subject in need thereof, the method comprising administering an effective amount of taxifolin to the subject. The method of claim 24, wherein the taxifolin has a chemical purity of at least about 95%. The method of claim 24 or 25, wherein the taxifolin has a chemical purity of at least about 98%. The method of any one of claims 24-26, wherein the taxifolin is administered in an amount that provides about 10 mg/day to about 1,500 mg/day to the subject. The method of any one of claims 24-27, wherein the taxifolin is administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients. The method of any one of claims 24-28, wherein the taxifolin is administered orally. The method of claim 29, wherein the taxifolin is administered as an orally consumable product. The method of claim 30, wherein the orally consumable product comprises a casing or a coating. The method of claim 29, wherein the taxifolin is administered as a dietary capsule or tablet, a sachet, a functional beverage, gummies, chewables, or a nutritional bar. The method of claim 29, wherein the taxifolin is administered in a carrier. The method of claim 33, wherein the carrier comprises liposomes, micelles, lipid emulsions, milks, lipids, phospholipids, microparticles, or nanoparticles, or a combination thereof. The method of any one of claims 29-34, wherein the taxifolin is administered one, two, or three times per day. The method of claim 37, wherein the taxifolin is administered one time per day. The method of claim 37, wherein the taxifolin is administered three times per day. The method of any one of claims 24-39, wherein the taxifolin is (2R,3R)-2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one. The method of claim 40, wherein the (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7- trihydroxychroman-4-one has an enantiomeric excess of about 95% or more. The method of any one of claims 24-41, wherein the thromboinflammatory disease, disorder, or condition is anti-phospholipid syndrome or thrombosis. A method of treating cystic fibrosis, or a symptom thereof, in a subject in need thereof, the method comprising administering an effective amount of taxifolin to the subject. The method of claim 43, wherein the taxifolin has a chemical purity of at least about 95%. The method of claim 43 or 44, wherein the taxifolin has a chemical purity of at least about 98%. The method of any one of claims 43-45, wherein the taxifolin is administered in an amount that provides about 10 mg/day to about 1,500 mg/day to the subject. The method of any one of claims 43-46, wherein the taxifolin is administered as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients. The method of any one of claims 43-47, wherein the taxifolin is administered orally. The method of claim 48, wherein the taxifolin is administered as an orally consumable product. The method of claim 49, wherein the orally consumable product comprises a casing or a coating. The method of claim 48, wherein the taxifolin is administered as a dietary capsule or tablet, a sachet, a functional beverage, gummies, chewables, or a nutritional bar. The method of claim 48, wherein the taxifolin is administered in a carrier. The method of claim 52, wherein the carrier comprises liposomes, micelles, lipid emulsions, milks, lipids, phospholipids, microparticles, or nanoparticles, or a combination thereof. The method of any one of claims 48-53, wherein the taxifolin is administered one, two, or three times per day. The method of claim 54, wherein the taxifolin is administered one time per day. The method of claim 54, wherein the taxifolin is administered three times per day. The method of any one of claims 43-56, wherein the taxifolin is (2R,3R)-2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxychroman-4-one. The method of claim 57, wherein the (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7- trihydroxychroman-4-one has an enantiomeric excess of about 95% or more.