WO2024220998A1

WO2024220998A1 - Orally bio a vailable pharmaceutical crystals and salts

Info

Publication number: WO2024220998A1
Application number: PCT/US2024/025720
Authority: WO
Inventors: Daryl Davies; Paul Seidler; Hovhannes John Gukasyan; Isis JANILKARN-URENA
Original assignee: University of Southern California USC
Current assignee: University of Southern California USC
Priority date: 2023-04-20
Filing date: 2024-04-22
Publication date: 2024-10-24
Anticipated expiration: 2025-10-20
Also published as: WO2024220998A9

Abstract

A co-crystal or crystal salt formulation comprising dihydromyricetin (DHM) and a crystal coformer selected from triethanolamine, 2-amino-2-(hydroxymethyl)-1, 3-propanediol (Tris-Base), sodium hydroxide, calcium hydroxide, and an amino acid; and methods of using the same.

Description

ORALLY BIO A VAILABLE PHARMACEUTICAL CRYSTALS AND SALTS

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/460,793, filed April 20, 2023, which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant no. P41GM136508, 1R01AG070895-01A1, awarded by the (NIH) National Institutes of Health and grant no. R01AA022448, awarded by the (NIH/NIAAA) National Institute on Alcohol Abuse and Alcoholism. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Solubility is a crucial factor affecting the efficacy of pharmaceuticals and other bioactive compounds such as vitamins, natural products, and dietary supplements. The degree of absorption is directly proportional to the solubility of a compound, although the physical and chemical mechanisms underlying solubility are largely theoretical. Compounds that deviate from ideality pose a challenge to scientists seeking to troubleshoot and develop rational strategies to overcome poor chemical solubility. Approximately 40% of new pharmaceutical chemicals developed by the industry are described as practically insoluble in water and nearly 90% of new drugs are classified as Case II/IV drugs in the Biopharmaceutics Classification System (BCS) partly due to low solubility. Salt formulations are a common strategy for preparing stable, safe, and bioavailable dosage forms in the pharmaceutical industry. However, predicting optimal salt forms and how they will function remains challenging.

With millions aging into elevated risk for AD, an elderly population that is expected to double by 2050, and knowledge that AD pathophysiology accrues over the course of decades, there is pressing need for comprehensive public health action. The U.S. Department of Health and Human Services outlined a “National Plan to Address Alzheimer's Disease” aiming to “Prevent” and “Treat” AD by 2025 by dual focus on supporting healthier aging and developing pharmaceutical therapies to manage and cure AD. Epidemiological research suggests lifestyle and environmental factors convolve with genetics and aging to affect timing of molecular transformations that lead to AD. This, and realization that AD manifests after decades in the making inspires research to delay AD onset by managing lifestyle interventions in the decades that precede AD dementia. Current prevention strategies being investigated include dietary and exercise lifestyle adaptions, and attention to cardiovascular health and physical wellbeing. Lifestyle adaptions that support healthier aging paired with biologically active chemicals, such as pharmaceutics and dietary supplements could provide a tiered and currently missing framework to manage aging and AD by leveraging early chemical intervention.

Accordingly, there is a need for new compositions, formulations, and/or delivery systems thereof that have improved solubility in polar solvents, such as water, that may be used to treat diseases, such as Alzheimer’s Disease, in mammals. The present disclosure satisfies these needs.

SUMMARY OF THE INVENTION

In the present disclosure, the solubility, activity, and Microcrystal electron diffraction (MicroED) structure of salt formulations of the natural product dihydromyricetin (DHM), a brain-permeable flavonoid with high chemical similarity to a known in vitro tau inhibitor, EGCG, were investigated. DHM is a plant-derived polyphenol with antioxidant and antiinflammatory activity and potential benefits in ameliorating dyslipidemia and alcohol intoxication. The CNS effects of DHM are attributed to increased GABAergic transmission and synaptic functioning, reduced neuroinflammation, and restoration of redox imbalances in neurons through improved mitochondrial function. Despite its rich phenolic character, DHM behaves as a hydrophobic substance with an aqueous solubility of only ~0.4 mg/ml. While hydrophobic character of DHM allows for permeability to the CNS, poor water solubility impedes dosing and intestinal absorption. Water-soluble formulations of DHM could overcome dosing issues by enabling delivery of higher dosage concentrations to enable increased absorption.

Co-crystal forms of salt products typically allow for higher dissolved concentrations of chemicals in bulk solution than their respective free acid or base (non-ionized) forms. Suitable counterions are found on the FDA-approved list of Inactive Ingredients Database (www.fda.gov/drugs/drug-approvals-and-databases/inactive-ingredients-database-download). While natural flavonoids and DHM are generally regarded as safe (GRAS), feasibility of isolating them as salts or co-crystals with suitable counterions that improve poor physicochemical properties (e.g., low solubility, instability or hygroscopicity) has not been widely explored. Several examples of other pharmaceutical co-crystal formulations have been commercialized, e.g., Entresto® and Steglatro®, with the latter being a medicinal chemistry derived analog of phlorizin (i.e., another flavonoid). In this study, we aimed to improve DHM solubility by salt/co-crystal formulation and explored DHM’s in vitro activity to disrupt prionogenic seeding by AD tau tangles.

Tau tangles are thought to kill neurons in AD and other dozens of other neurodegenerative tauopathies, so suppressing tau spreading is seen as a top interest therapeutic mechanism of action. Despite having outstanding in vitro inhibitory activity towards seeding by AD tau, EGCG is limited by poor brain permeation. Based on the CryoEM structure of AD tau fibrils bound with EGCG and realized chemical of EGCG and DHM, we identified DHM as a possible alternative natural product and brain-permeable tau inhibitor, which spurred our investigations of its bioactivity towards AD tau.

Accordingly, the disclosure provides for crystal salts and co-crystals comprising dihydromyricetin (DHM) and a crystal salt or crystal coformer selected from the group consisting of triethanolamine, 2-amino-2-(hydroxymethyl)-l, 3-propanediol (Tris-Base), sodium hydroxide, calcium hydroxide, and an amino acid. In some embodiments, the crystal salt comprises DHM and triethanolamine, or DHM and 2-amino-2-(hydroxymethyl)-l, 3- propanediol (Tris-Base), or DHM and sodium hydroxide, or DHM and calcium hydroxide, or DHM and L-lysine or L-arginine. In other embodiments, the co-crystal comprises DHM and triethanolamine, or DHM and 2-amino-2-(hydroxymethyl)-l, 3-propanediol (Tris-Base), or DHM and sodium hydroxide, or DHM and calcium hydroxide, or DHM and L-lysine or L- arginine. In some embodiments, the salt crystal or co-crystal comprises a crystal lattice having a void volume of about 75% to about 90%, a unit cell volume of about 900 A³ to about 1200 A³, and a Matthews Coefficient (VM) of about 1.5 A³/Da to about 2.5 A³/Da.

In some embodiments, a salt crystal or co-crystal of the disclosure comprises a crystalline lattice of DHM-coformer having a void volume of about 80% to about 85%. In some embodiments, the unit cell volume is about 950 A³ to about 1000 A³ and the Matthews Coefficient (VM) of about 1.9 A³/Da to about 2.2 A³/Da. In other embodiments, the unit cell volume is about 971.5 A³ and the Matthews Coefficient (VM) is about 2.07 A³/Da.

In some embodiments, a crystal salt or co-crystal comprises a ratio of the DHM to the crystal coformer of about 1 :1 to about 1 :5, or about 1:1 to about 1:3, or about 1:1 to about 1 :1.25.

In some embodiments, the crystal salt or co-crystal of DHM is about 5 times to about 10 times more soluble in apolar solvent compared to the solubility of DHM in the polar solvent.

In some embodiments, a crystal salt or co-crystal may further comprise a pharmaceutically acceptable carrier or excipient. The disclosure also provides for methods of treating a disease comprising administering an effective amount of a crystal salt or co-crystal of dihydromyricetin (DHM) to a subject in need thereof, wherein the crystal salt or co-crystal of DHM comprises the DHM and a crystal coformer selected from the group consisting of triethanolamine, 2-amino-2-(hydroxymethyl)- 1, 3-propanediol, sodium hydroxide, calcium hydroxide, and an amino acid; wherein the cocrystal of DHM thereby treats the disease.

In some embodiments, the disease comprises one or more of alcohol use related disorders, age related diseases, or oxidative stress induced by one or more of disease, age, drug use, and environment, or poisoning, or liver damage, a disease caused by inflammation, or a neurological disease. In some embodiments, the neurological disease is Alzheimer’s Disease or otherwise a result of priogenic seeding or tau fibril aggregation.

These and other features and advantages of this invention will be more fully understood from the following detailed description of the invention taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention described herein.

Figure 1. (a) DHM binding sites on AD tau fibrils predicted by CB-Dock. AutoDock Vina docking scores are shown in the embedded table. Chemical structures comparing DHM and EGCG are shown below the CB-Dock model (b-c) Dose dependent seeding inhibition measured by transfecting AD crude brain homogenates in HEK293 tau biosensor cells that stably express P301S 4R1N tau fused to YFP. Seeding inhibition was determined by counting the number of fluorescent puncta as a function of inhibitor concentration. IC50 values were calculated by nonlinear curve fitting from dose-response plots. Experiments in b were performed by pre-incubating AD crude brain homogenates with DHM dissolved in DMSO. Experiments in c were performed identically except using the soluble fraction of DHM dissolved in water following centrifugation at 8,000 rpm. (d-f) Representative images from tau biosensor cells transfected with AD crude brain homogenate (d), or identically treated cells following pre- incubation of AD crude brain homogenate with DHM (10 mM final concentration on cells). Representative tau-4RlN cells containing aggregated tau puncta are marked with red arrows, and cells without are marked with white arrows.

Figure 2. Predicted and experimental pKa’s of DHM. (a) Ionizations occurring from neutral to anionic states produced using ADMET Predictor™. Predicted proportions of respective ionized states are indicated. Microstates for multiply ionized phenols are shown as a function of increasing pH. (b and c) UV-VIS spectroscopic pKa determination of DHM determined at pH values ranging from pH 3.1-13.3. (d) NaOH potentiometric titration curves with MeOH revealing an equivalence point at -pH 7.9.

Figure 3. (a) Schematic showing experimental design of experiments in Figure 3. Cocrystalline DHM formulations were dissolved in water to a target 10 mM concentration. Insoluble matter was removed by centrifugation as indicated. Supernatants containing water solubilized DHM were assayed for tau inhibitor activity, (b) Seeding inhibition measured by quantifying the number of fluorescent puncta as a function of indicated inhibitor. AD sample is AD brain homogenate without added inhibitor. Error bars represent standard deviations of triplicate measures, (c) Representative images from b of tau biosensor cells seeded by transfection by AD crude brain homogenates following pre-treatment with DHM or salt cocrystals. Inhibitors were added to a final concentration of 10 pM on cells. Example puncta are shown by red arrows, examples of non-seeded cells shown with white arrows, (d and e) IC50 dose dependent inhibition of puncta following pre-treatment with DHM-TEA and -Ca(b). (f) Seeding inhibition measured for DHM-TEA and -Ca(b), or TEA and -CaOI E. (g) Solubility determination of DHM, DHM-TEA and DHM-Ca(b) in water, expressed in mg/ml.

Figure 4. (A) DHM-mediated AD tau fibril disaggregation, measured by qEM. Fibrils were counted from N=90 randomly acquired EM images, which were split three ways and quantified in triplicate. Columns show numbers of fibrils counted as a function of inhibitor pre- incubation time, as indicated. Error bars represent standard deviations. (B-D) Example EM images used from qEM after 48 hr inhibitor incubation.

Figure 5. (a) X-Ray powder diffractogram comparing native dihydromyricetin (DHM) shown in red, overlayed against co-former dihydromyricetin with triethanolamine shown in orange, (b) Asymmetric unit of DHM complex with TEA determined by MicroED. (c) Crystal lattice of DHM-TEA co-crystal. Note solvent channels created by the intercalation of TEA in the lattice between DHM molecules, (d) X-ray structure of DHM dihydrate (Xu et al., Acta Crystallographica Section E 63, o4384-o4384). Differential scanning calorimetry thermographs of DHM-TEA and DHM dihydrate.

Figure 6. (a-c) DHM-TEA crystal structure. Non-bonded phenols are shown in a and b with green arrows, (d-f) X-ray structure of DHM dihydrate³¹. Contacts between phenols of the pyrogallol ring are shown in b and c for DHM-TEA and DHM dihydrate, respectively. Aromatic stacking interactions are shown in e and f for DHM-TEA and DHM dihydrate, respectively, (g-h) Pair correlation functions for DHM-TEA and DHM dihydrate, as labeled.

Figure 7. Proposed model for increased DHM delivery by salt formulation, (a) DHM aggregates resist dissolution in and partitioning to water, thereby impeding intestinal absorption, (b) DHM-TEA crystals partition and dissolved more readily dissolved in water, thereby increasing absorbed DHM, which is needed to reach circulation.

Figure 8. (A and B) CryoEM structure of AD brain-derived tau PHF bound by EGCG. (C) Manually generated model of DHM binding to EGCG binding site of AD tau made by superimposing DHM with bound EGCG. (D-F) Alternative predicted DHM binding sites generated by CB-Dock. (D) Site 1 overlaps with the binding volume predicted in the manually generated model in C, except bound DHM is predicted to orient with aromatic rings perpendicular to the fibril axis burying in a cavity between Lys340 and Glu338. (E) Site 2 of DHM binding predicted by CB-Dock occurs in a cavity lined by His362 and K369. (F) Site 3 predicted by CB-Dock occurs with DHM packing aromatic moieties with the aliphatic chain of K321, and H-bonding occurring between the phenolic moieties and the amide backbone at a sharp turn formed by G323.

Figure 9. IC50 curves generated as described in the main text, except using DHM- TEA and -Ca(b) formulations (left and right, respectively) dissolved in DMSO. Both formulations have IC50s similar to DHM when dissolved in DMSO (Fig. IB), thus suggesting DHM is the active component in tau inhibitor assays and that counterions themselves do not exhibit inhibitory activity towards tau seeding.

Figure 10. Seeding inhibition measured as described in the main text, without inhibitor pre- incubation with crude AD brain homogenates.

Figure 11. The crystal structure of DHM-TEA as solved with a resolution of 0.75 A. The blue mesh on atoms are 2F_O-F_C electrostatic potential maps at the level of 0.67 e- A^-3.

Figure 12. Pharmacokinetic Curve (Oral Dose): 5mg free DHM vs. 5mg DHM+TEA co-crystal (Isis 101).

Figure 13. Percent Fraction of Oral Dose Absorbed (%Fa).

Figure 14. Relative Oral Bioavailability: Free DHM vs DHM+TEA co-crystal. Figure 15. DHM TEA co-crystal: X-ray powder diffractogram demonstrates multiple, sharp diffraction peaks distinctive from native (free) DHM; free DHM crystal purchased in

2021 (bottom trace) overlayed with DHM + TEA co-crystal made in 2021 and analyzed in

2022 (top trace).

Figure 16. DHM + 1-lysine crystal: X-ray powder diffractogram demonstrates multiple, sharp diffraction peaks; DHM + 1-lysine crystal made in 2021 (bottom trace) overlayed with DHM + 1-lysine crystal made in 2024 (top trace).

Figure 17. DHM + Ca(OH)2: X-ray powder diffractogram demonstrates multiple, sharp diffraction peaks; DHM + Ca(OH)2 crystal made in 2021 (bottom trace) overlayed with DHM + Ca(OH)2 crystal made in 2021 and reanalyzed in late 2022 (top trace), and DHM + Ca(OH)2 crystal made in 2024 (middle trace).

Figure 18. Pharmacokinetic curves of 5mg P.O. dose in rats. (A) Shows a comparison of DHM + triethanolamine co-crystal formed using ethanol as a solvent, referred to as Isis 101 simulated (i.e. Calc) oral exposure from a 5mg total dose vs observed values, Liu et al., of native DHM in rats. Isis 101 predicted Cmax and AUC are approximately 119 and 65 times, resp., higher than those observed in rats. (B) Shows an overlay of the validated predictive GastroPlus® (i.e. Calc) vs Liu at al. (i.e., Obs) dosed orally at a 5mg total dose of native DHM PBPK model in rats with a -90% confidence interval.

Figure 19. Pharmacokinetic curves of 25mg P.O. dose in rats. (A) Shows a comparison of Isis 101 simulated (i.e. Calc) oral exposure from a 25mg total dose vs observed values, Tong et al., of native DHM in rats. Isis 101 predicted Cmax and AUC are approximately 20 and 27 times, resp., higher than those observed in rats. (B) Shows an overlay of the GastroPlus® (i.e. Calc) vs Tong at al. (i.e. Obs) dosed orally at 25mg total dose of native DHM PBPK model in rats, having poor confidence in accurately capturing the initial absorption intensity and extent of it at this higher dose (*at higher oral doses, in vivo, there may be enzyme/transporter saturation occurring or delayed absorption, that result in a discrepancy with PBPK model). Also, there is a lack of rat PO dosage from detail in (Tong et al. J Pharm Biomed Anal, 2015. 114: p. 455-61.) (e.g. DHM particle size and form data), and an IR Suspension for DHM was assumed.

Figure 20A-I. Parameter sensitity analysis 5, 25 mg doses, and Isis 101. Three stage global parameter sensitivity analysis (PSA) performed in GastroPlus® 9.8.3. (Simulations Plus, Inc., USA). (A-C) describe the AUC, Cmax, and %Fa profiles as a function of increasing DHM doses using the native DHM form and formulation from validated Liu et al. PBPK model which has 5mg PO IR Suspension as a PSA baseline. While there is a somewhat proportional relationship between doses and AUC, or Cmax, the predicted fraction of DHM absorbed is poor. At low doses of <lmg it is -35% Fa, and %Fa drops exponentially with increasing DHM dose (Panel C) to ~l-3%Fa, indicating inefficient absorption from oral route of administration, i.e. at meaningful >100mg DHM doses 97-99% of the total dose is not absorbed. Panels (D-F) describe the AUC, Cmax, and %Fa profiles as a function of increasing DHM doses using the native DHM form and formulation from Tong et al. PBPK model which has 25mg PO IR Suspension as a PSA baseline. While there is a somewhat-proportional relationship between doses and AUC, or Cmax, the predicted fraction of DHM absorbed is significantly and exponentially dropping, impacted with increasing DHM dose (Panel F). At low doses of <lmg it is estimated as complete, however %Fa drops exponentially with increase in DHM dose (Panel F), indicating inefficient absorption primarily related to solubility limitations from oral route of administration, i.e. at meaningful >200mg oral DHM doses, more than 90% of the total dose is not absorbed (i.e. lost). Panels (G-I) describe the AUC, Cmax, and %Fa profiles as a function of increasing Isis 101 doses using the validated Liu et al. rat PBPK model. Isis 101 yielding 5mg of DHM equivalents is used as the baseline. In contrast to native DHM, Isis 101 has a supra-proportional dose response relationship at low doses (e.g. 1- lOOmg) with AUC and Cmax, and maintains a higher fraction of the dose absorbed >25%Fa at lOOmg and >10%Fa at <500mg dose boundaries. Isis 101 parameter sensitivity analysis using 25mg Tong et al. model are not performed due to the lower reliability of that model (see Fig. 2). Panels G-I suggest that Isis 101 enables higher efficiency absorption of DHM especially at low doses, and it maintains a higher %Fa over native DHM up to 500mg.

Figure 21. Parameter sensitivity analysis (PSA) performed in GastroPlus® 9.8.3. (Simulations Plus, Inc., USA). The independent parameter is the dose, and the dependent parameter is the fraction of the initial PO dose absorbed (%Fa). Plots are constructed from twenty distinct %Fa calculations using different PK simulations at doses that bracket the shown baseline dose of DHM at either 5mg or 25mg, ten- fold below and ten-fold above with ten doses in each range. For example, 0.5mg to 5mg with ten doses in between, and 5mg-500mg with ten doses in between, is used for the 5mg Liu et al. rat data PBPK model. While the native DHM %Fa decreases from 0.5-500mg, in a sigmoidal manner, the Isis 101 %Fa is not sensitive to dose from 0.5- lOOmg (seen as a flat line). From 100mg-500mg there is a <15% change in %Fa with Isis 101. DETAILED DESCRIPTION

This disclosure pertains to optimized and improved physical and chemical characteristics of dihydromyricetin (DHM) which provide suitable biopharmaceutical performance in a pharmaceutically acceptable context (e.g. accurate and efficient dosing of DHM to treat a recognized medical condition, alcohol use related disorders, age related diseases, oxidative stress induced by disease/age/drug use/environment, poisoning, liver damage, etc.), including but not limited to physical and chemical stability and higher solubility in biorelevant aqueous systems to allow DHM to reach the bloodstream at therapeutically relevant concentrations. DHM, in its commercially available form, is isolated from natural sources and comes as a practically water insoluble solid of beige-brown color. However, no known salt forms have been characterized in terms of physical, chemical, and biopharmaceutical properties. Based on the known physicochemical properties and oral bioavailability of DHM, an evaluation of the biopharmaceutics of the molecule suggests it is best described as a Biopharmaceutics Classification System class 4 compound (Amidon, G.L. Pharm Res 1995) - a poor/low aqueous solubility and tissue-membrane permeability molecule.

Improvement of biopharmaceutical properties in this natural product can be fully applied to other known flavonoids with sufficiently (80-90%) similar structure and chemical properties: i.e. taxifolin, hovenitin I, dihydrorobinetin, 4H-l-Benzopyran-4-one, 2,3-dihydro- 3,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-, Aromadedrin, NSC 36398, 3'-O-Methyltaxifolin, Pinobanksin, Dihydrokaempferide, Flavanone, 3,3',4',5,7-pentahydroxy-6-methyl-, fustin, 4H- 1 -Benzopyran-4-one, 2,3-dihydro-3,5-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-7-methoxy-, (2R,3R)-, 4H-1 -Benzopyran-4-one, 2-(2,6-dihydroxyphenyl)-2,3-dihydro-3, 5, 7 -trihydroxy-, (2R,3R)-, 2',3,5,7-Tetrahydroxyflavanone, Aromadendrin 7-methyl ether, Alpinone; quercetin and >90% structural similarity to this 3,3’,4',5,7-Pentahydroxyflavone family; or (-)- epigallocatechin gallate (EGCG), a green tea polyphenol.

Improvement of biopharmaceutical properties in this context pertains to the discovery of acidic (based on Brpnsted-Lowry acid-base theory) nature of DHM. Specifically, with a log of acid dissociation constant pKa in the physiologically useful and relevant pH range of 7-8. This discovery enables the preparation of pharmaceutically and nutraceutically stable salt (solid or in situ) forms. Specifically, the invention can be widely applied in at least two applications: solid crystal form manufacturing and liquid in situ extemporaneous compounding approaches. Counterions (e.g., a Brpnsted-Lowry base) qualified in suitable pharmaceutical dosage forms may be used to crystallize racemic or enantiomerically pure solid crystal substance of DHM paired with a base where a full or partial transfer of proton has occurred. Furthermore, counterions can be used in situ, to form solutions, such as a powder sachet mixture, to prepare a liquid oral solution for extemporaneous use.

Based on preliminary physical analysis of dihydromyricetin (DHM from Master Herbs, Pomona CA), a naturally occurring and biologically active flavonoid, the commercially available product appears as crystalline material with >98% purity and needle shaped crystal morphology by light microscopy. The possibility of hydrates in this particular starting material needs further investigation using appropriate physical methods, such as thermogravimetric analysis (TGA). Furthermore, enantiomerically pure forms need methods of absolute configuration understanding, isolation with 99% e.e., and testing, since the molecular has two chiral centers and four possible enantiomers.

There are several disclosures of DHM (in its free acid form) formulations which claim improvements in solubility, including, but not limited to, any pharmaceutically suitable polymer used as a matrix material in hot-melt extrusion or amorphous spray dried dispersion, and cyclodextrins such as those exemplified on label claim of ‘morning recovery’ drink from More Labs (Los Angeles, CA; www.morelabs.com). These are fundamentally different pharmaceutical approaches to compound and delivery, which rely on employment of excipients (or inactive ingredients found in pharmaceutical dosage forms that are generally accepted as safe). In contrast, embodiments of the current invention describe salts and co-crystals of DHM having appropriate properties to enable oral dosage form compounding and dissolution in the gastrointestinal tract at site of absorption (putatively entire small intestine and proximal colon), without use of any excipient technology and only relying on the intrinsic physicochemical and biopharmaceutical properties of the novel salts and/or co-crystals (regardless of solid state or in situ aqueous solution state).

Definitions.

The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley’s Condensed Chemical Dictionary 14^th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and/or the recitation of claim elements or use of "negative" limitations.

The term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is disubstituted.

As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value without the modifier "about" also forms a further aspect.

Wherever the term “comprising” is used herein, options are contemplated wherein the terms “consisting of” or “consisting essentially of’ are used instead. As used herein, “comprising” is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the aspect element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the aspect. In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms. The disclosure illustratively described herein may be suitably practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent, or as otherwise defined by a particular claim. For integer ranges, the term "about" can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, composition, or embodiment. The term about can also modify the endpoints of a recited range as discussed above in this paragraph.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. It is therefore understood that each unit between two particular units are also disclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed, individually, and as part of a range. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

An "effective amount" refers to an amount effective to treat a disease, disorder, and/or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is well within the capacity of persons skilled in the art, especially in light of the detailed disclosure provided herein. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect. An appropriate "effective" amount in any individual case may be determined using techniques, such as a dose escalation study.

The terms "treating", "treat" and "treatment" include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and/or (iv) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms "treat", "treatment", and "treating" can extend to prophylaxis and can include prevent, prevention, preventing, lowering, stopping or reversing the progression or severity of the condition or symptoms being treated. As such, the term "treatment" can include medical, therapeutic, and/or prophylactic administration, as appropriate.

The terms "inhibit", "inhibiting", and "inhibition" refer to the slowing, halting, or reversing the growth or progression of a disease, infection, condition, or group of cells. The inhibition can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, compared to the growth or progression that occurs in the absence of the treatment or contacting.

The term "substantially" is typically well understood by those of skill in the art and can refer to an exact ratio or configuration, or a ratio or configuration that is in the proximity of an exact value such that the properties of any variation are inconsequentially different than those ratios and configurations having the exact value. The term "substantially" may include variation as defined for the terms "about" and "approximately", as defined herein above.

While in some embodiments, a therapeutic dosage of a composition as described herein may be described for a murine model, a person of ordinary skill in the art may obtain an equivalent dosage for other mammals, and in particular, for humans, using methods that are known in the art, for example, as described in Nair et al., J Basic Clin Pharma 2016;7:27-3, incorporated herein by reference in its entirety.

A “co-crystal” is a form of DHM comprising DHM and at least one other component (“coformer”), both in neutral form. Co-crystals are typically characterized by a crystalline structure, which is generally held together by freely reversible, non-covalent interactions (pi- stacking, guest-host complexation and van der Waals interactions.). Co-crystals are typically made up of DHM and at least one other component in a defined stoichiometric ratio. In some embodiments, co-crystals can encompass hydrates, solvates, and clathrates. Co-crystals can comprise DHM in combination with an organic and/or an inorganic component. Co-crystals can generally be distinguished from salts by the absence of a proton transfer between the components (i.e., the DHM and the one or more coformers) in a co-crystal. According to the U.S. Food and Drug Administration's Guidance for Industry (April 2013), a co-crystal is defined as a solid that is a crystalline material composed of two or more molecules in the same crystal lattice, where the components are in a neutral state and interact via nonionic interactions. See U.S. Department of Health and Human Services, Food and Drug Administration, Guidance for Industry: Regulatory Classification of Pharmaceutical CoCrystals (April 2013), which is incorporated herein by reference in its entirety.

A “DHM salt” is a form of DHM characterized by the interaction between DHM in ionic form and a coformer in ionic form (e.g., an acid or base) via the transfer of one or more protons from the coformer donor to the DHM acceptor. The stoichiometry of the salts, cocrystals, and salt crystals described herein can vary. For example, in certain embodiments, where two components (i.e., DHM and one coformer) are present, the DHM:coformer stoichiometry can range in certain embodiments from about 5: 1 to about 1:5 DHM:coformer. Where more than one coformer is used to form a DHM salt, co-crystal, or salt crystal, the ratios of the coformers with respect to both the DHM and to one another can also vary. In preferable embodiments, a given sample of the salts, co-crystals, and salt crystals provided according to the present disclosure exhibit substantially one single stoichiometry.

As used herein, the term “void volume” refers to the volume of unoccupied space in a crystal structure (i.e., not occupied by an atom of compound of the crystal structure.).

As used herein, the term “Matthews Coefficient” refers to crystal volume per unit of protein molecular weight and may be calculated, for example, using the Wake Forest University Matthews coefficient calculator fund at www.csb.wfu.edu/tools/vmcalc/vm.html.

As used herein, the term “unit cell” refers to the smallest unit having full symmetry of the crystal structure.

Embodiments of the Invention.

The disclosure provides for compounds comprising co-crystals or crystal salts of dihydromyricetin (DHM) and one or more crystal conforming compounds and methods of using the same to treat diseases in mammals (e.g., humans) such, but not limited to, Alzheimer’s Disease or other diseases caused by tau protein aggregation. In preferred embodiment, the compound and/or compositions of the disclosure comprise a co-crystal of DMH and one or more crystal coformers.

In general, the crystal coformer is any co-crystal former that may be safely administered to humans. Such compositions may be identified on the GRAS list (also known as the “Generally Recognized As Safe” list) or the EAFUS list (also known as the “Everything Added to Food in the United States” list) maintained by the U.S. Food and Drug Administration or excipients approved for pharmaceutical use. More typically, however, the co-crystal former will be a pharmaceutically acceptable carbohydrate, amine, amide, sulfonamide, carboxylic acid, sulfonic acid, phenolic, polyphonic, aromatic heterocycle, xanthine (or a derivative thereof), or alcohol.

In some embodiments, the crystal coformer comprises one or more of 1,5-napthalene- disulfonic acid, l-hydroxy-2-naphthoic acid, 4- aminobenzoic acid, 4-aminopyridine, 4- chlorobenzene-sulfonic acid, 4-ethoxyphenyl urea, 7-oxo-DHEA, acesulfame, acetohydroxamic acid, adenine, adipic acid, alanine, allopurinaol, arginine, ascorbic acid, asparagine, aspartic acid, benzenesulfonic acid, benzoic acid, caffeine, camphoric acid, capric acid, chrysin, cinnamic acid, citric acid, clemizole, cyclamic acid, cysteine, dimethylglycine, D-ribose, fumaric acid, galactaric acid, genistein, gentisic acid, glucamine A-melhyl, gluconic acid, glucosamine, glucuronic acid, glutamic acid, glutamine, glutaric acid, glycine, glycolic acid, hippuric acid, histidine, hydroquinone, imidazole, ipriflavone, isoleucine, lactobionic acid, lauric acid, leucine, lysine, maleic acid, malic acid, malonic acid, mandelic acid, methionine, nicotinamide, nicotinic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phenylalanine, piperazine, procaine, proline, p-toluenesulfonic acid, pyridoxamine, pyridoxine (4-pyridoxic acid), pyroglutamic acid, quercetin, resveratrol, saccharin, salicylic acid, salicylic acid 4-amino, sebacic acid, serine, stearic acid, succinic acid, tartaric acid, threonine, TRIS, tryptophan, tyrosine, urea, valine, Vitamin K5, xylitol, L-ascorbic acid (Vitamin C), gallic acid, maleic acid, iso-nicotinamide, nicotinic acid, iso-nicotinic acid, theobromine, theophylline, caprolactam, lactose, glucose, and sucrose. Other exemplary crystal coformers are described, for example, in U.S. Patent Number 11,225,468 to Dull et al. and 10,842,797 to Zaworotko et al.

In another embodiment, the crystal or crystal salt coformer is selected from the group consisting of triethanolamine, 2-amino-2-(hydroxymethyl)-l, 3-propanediol (Tris-Base), sodium hydroxide, calcium hydroxide, and an amino acid. In one embodiment, the crystal coformer is triethanolamine or calcium hydroxide. In other embodiments, the crystal coformer is triethanolamine. In some embodiments, the coformer comprises an amino acid such as lysine or arginine. In some embodiments, the co-crystal or crystal salt comprises DHM-TEA, DHM- (L)-Lysine, DHM-Tris-Base, or DHM-Ca.

In some embodiments, the stoichiometry of the DHM and crystal coformer in the cocrystal or crystal salt may be a ratio of about 1 :5 to about 5: 1. In some embodiments, the stoichiometry of the DHM and crystal or crystal salt coformer may be a ratio of about 1 : 1, about 1:2, about 1 :3, about 1 :4, about 1:5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, or about 1 : 10. In some embodiments, the ratio of the DHM to the crystal coformer is about 1 : 1 to about 1 :5. In other embodiments, the stoichiometry of the DHM and crystal or crystal salt coformer may be a ratio of about 1:1.1, about 1:1.2, about 1 :1.3, about 1 : 1.4, about 1:1.5, about 1 : 1.6, about 1 :1.7, about 1: 1.8, about 1: 1.9, or about 1:2. For example, in one embodiment, the stoichiometry of DHM and triethanolamine in a co-crystal may be a ratio of about 1:1, about 1 :2, about 1 :3, about 1 :4, or about 1:5; or about 1 :1.1, about 1:1.2, about 1 :1.3, about 1:1.4, about 1: 1.5, about 1 : 1.6, about 1 : 1.7, about 1 : 1.8, about 1 :1.9, or about 1 :2. In another embodiment the stoichiometry of DHM and Ca²⁺ in a co-crystal may be a ratio of about 1: 1, about 1 :2, about 1 :3, about 1:4, or about 1:5; or about 1 :1.1 , about 1 : 1.2, about 1: 1.3, about 1 :1.4, about 1 :1.5, about 1 :1.6, about 1 :1.7, about 1:1.8, about 1 :1.9, or about 1:2. In some embodiments, the stoichiometry of the DHM to the crystal coformer is a ratio of about 1: 1 to about 1:3 or about 1:1 to about 1:1.25. In some embodiments, a co-crystal or crystal salt may include a crystal lattice comprising a void volume of about 75% to about 90%. For example, in some embodiments, the void volume may be about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%. In some embodiments, a co-crystal of DHM and a coformer comprise a void volume of about 80% to about 85%. In one embodiment, a co-crystal of DHM and a triethanolamine crystal coformer comprise a void volume of about 80% to about 85%.

In some embodiments, a co-crystal or crystal salt may include a crystal lattice o - o - comprising a unit cell volume of about 800 A to about 1200 A . For example, a co-crystal may include a unit cell volume of about 800 A³, about 825 A³, about 850 A³, about 875 A³, about 900 A³, about 925 A³, about 950 A³, about 975 A³, about 1000 A³, about 1025 A³, 1050 A³, about 1075 A³, about 1100 A³, about 1125 A³, about 1150 A³, about 1175 A³, or about 1200 A . In some embodiments, a co-crystal of DHM and a crystal coformer comprise a unit cell volume of about 950 A³ to about 1000 A³. In one embodiment, a co-crystal of DHM and a triethanolamine crystal coformer comprise a unit cell volume of about 950 A³ to about 1000 A³.

In some embodiments, a co-crystal or crystal salt may include a crystal lattice comprising a Matthews Coefficient (VM) of about 1.5 A /Da to about 2.5 A³/Da. For example, a co-crystal may include a Matthews Coefficient (VM) of about 1.5 A³/Da, about 1.6 A³/Da, about 1.7 A³/Da, about 1.8 A³/Da, about 1 .9 A³/Da, about 2 A³/Da, about 2.1 A³/Da, about 2.2 o o o

A /Da, about 2.3 A /Da, about 2.4 A /Da, or about 2.5 A /Da. In some embodiments, a co- crystal of DHM and a crystal coformer comprise a Matthews Coefficient of about 1.9 A /Da to about 2.2 A³/Da. In one embodiment, a co-crystal of DHM and a triethanolamine crystal coformer comprise a Matthews Coefficient of about 1.9 A³/Da to about 2.2 A³/Da.

In some embodiments, a co-crystal consists essentially of dihydromyricetin (DHM); and a crystal coformer selected from the group consisting of triethanolamine, 2-amino-2- (hydroxymethyl)-l, 3-propanediol (Tris-Base), sodium hydroxide, calcium hydroxide, and an amino acid. In some embodiments, a co-crystal consists essentially of dihydromyricetin (DHM) and triethanolamine.

In some embodiments, a co-crystal formulation may comprise dihydromyricetin (DHM); and a crystal coformer selected from the group consisting of triethanolamine, 2-amino- 2-(hydroxymethyl)-l, 3-propanediol (Tris-Base), sodium hydroxide, calcium hydroxide, and an amino acid, wherein the co-crystal comprises a crystal lattice having a void volume of about 75% to about 90%, a unit cell volume of about 900 A³ to about 1200 A³, and a Matthew Coefficient (VM) of about 1.5 A³/Da to about 2.5 A³/Da.

In some embodiments, a co-crystal of DHM and a crystal coformer comprise a crystal lattice having a void volume of about 80% to about 85%, a unit cell volume of about 965.5 A³ to about 980 A3, and a Matthews Coefficient (VM) is about 2.0 A³/Da to about 2.15 A³/Da.

In some embodiments, a co-crystal of DHM and triethanolamine comprise a crystal lattice having a void volume of about 83%, a unit cell volume of about 971.5 A³ and a Matthews Coefficient (VM) is about 2.07 A³/Da.

Salt and co-crystals of the disclosure increase the solubility, and therefore the bioavailability of DHM. In some embodiments, the crystal salt or co-crystal of DHM is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about nine times, about ten times, about 11 times, about 12, times, about 13 times, about 14 times, about 15 times or more than 15 times more soluble in a polar solvent compared to the solubility of DHM in the polar solvent. In some embodiments, the crystal salt or co-crystal of DHM is about 5 times to about 10 times more soluble in a polar solvent compared to the solubility of DHM in the polar solvent.

In some embodiments, the crystal salt or co-crystal of DHM is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about nine times, about ten times, about 11 times, about 12, times, about 13 times, about 14 times, about 15 times or more than 15 times more soluble as measured in blood plasma compared to the solubility of DHM in blood plasma. In some embodiments, the crystal salt or co-crystal of DHM is about 5 times to about 10 times more soluble as measured in blood plasma compared to the solubility of DHM in blood plasma.

The disclosure also provides for methods of treating a disease comprising administering an effective amount of a co-crystal or crystal salt of dihydromyricetin (DHM) to a subject in need thereof, wherein the co-crystal of DHM comprises the DHM and a crystal coformer selected from the group consisting of triethanolamine, 2-amino-2-(hydroxymethyl)-l, 3- propanediol, sodium hydroxide, calcium hydroxide, and an amino acid; wherein the co-crystal of DHM thereby treats the disease.

In some embodiments, the disease comprises one or more of alcohol use related disorders, age related diseases, or oxidative stress induced by one or more of disease, age, drug use, and environment, or poisoning, or liver damage, or an inflammatory disease, or a neurological disease. Exemplary neurological or neurocognitive disorders that may treated with a co-crystal or salt crystal as described herein include, but are not limited to, abarognosis (e.g., loss of the ability to detect the weight of an object held in the hand or to discern the difference in weight between two objects), acid lipase disease, acid maltase deficiency, acquired epileptiform aphasia, absence of the septum pellucidum, acute disseminated encephalomyelitis, adie's pupil, Adie’s syndrome, adrenoleukodystrophy, agenesis of the corpus callosum, agnosia, Aicardi syndrome, Aicardi-Goutieres syndrome disorder, AIDS - neurological complications, akathisia, alcohol related disorders, Alexander disease, Alien hand syndrome (anarchic hand), allochiria, Alpers' disease, altitude sickness, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis, anencephaly, aneurysm, Angelman syndrome, angiomatosis, anoxia, Antiphospholipid syndrome, aphasia, apraxia, arachnoid cysts, arachnoiditis, amold- chiari malformation, Asperger syndrome, arteriovenous malformation, ataxia, ataxias and cerebellar or spinocerebellar degeneration, ataxia telangiectasia, atrial fibrillation, stroke, attention deficit hyperactivity disorder, auditory processing disorder, autism, autonomic dysfunction, back pain, Barth syndrome, Batten disease, becker's myotonia, Behcet’s disease, bell's palsy, benign essential blepharospasm, benign focal amyotrophy, benign intracranial hypertension, Bernhardt-Roth syndrome, bilateral frontoparietal polymicrogyria, Binswanger's disease, blepharospasm, Bloch-Sulzberger syndrome, brachial plexus birth injuries, brachial plexus injury, Bradbury-Eggleston syndrome, brain or spinal tumor, brain abscess, brain aneurysm, brain damage, brain injury, brain tumor, Brown-Sequard syndrome, bulbospinal muscular atrophy, CADASIL (cerebral autosomal dominant arteriopathy subcortical infarcts and leukoencephalopathy), Canavan disease, Carpal tunnel syndrome, causalgia, cavernomas, cavernous angioma, cavernous malformation, Central cervical cord Syndrome, Central cord syndrome, Central pain syndrome, central pontine myelinolysis, centronuclear myopathy, cephalic disorder, ceramidase deficiency, cerebellar degeneration, cerebellar hypoplasia, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral beriberi, cerebral cavernous malformation, cerebral gigantism, cerebral hypoxia, cerebral palsy, cerebral vasculitis, Cerebro-Oculo-Facio-Skeletal syndrome (COFS), cervical spinal stenosis, Charcot- Marie-Tooth disease, chiari malformation, Cholesterol ester storage disease, chorea, choreoacanthocytosis, Chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic orthostatic intolerance, chronic pain, Cockayne syndrome type II, Coffin-Lowry syndrome, colpocephaly, coma, Complex regional pain syndrome, compression neuropathy, concussion, congenital facial diplegia, congenital myasthenia, congenital myopathy, congenital vascular cavernous malformations, corticobasal degeneration, cranial arteritis, craniosynostosis, cree encephalitis, Creutzfeldt- Jakob disease, cumulative trauma disorders, Cushing's syndrome, Cytomegalic inclusion body disease (CIBD), cytomegalovirus infection, Dancing eyes-dancing feet syndrome (opsoclonus myoclonus syndrome), Dandy- Walker syndrome (DWS), Dawson disease, decompression sickness, De morsier's syndrome, dejerine-klumpke palsy, Dejerine-Sottas disease, Delayed sleep phase syndrome, dementia, dementia - multi-infarct, dementia - semantic, dementia - subcortical, dementia with lewy bodies, dentate cerebellar ataxia, dentatorubral atrophy, depression, dermatomyositis, developmental dyspraxia, Devic's syndrome, diabetes, diabetic neuropathy, diffuse sclerosis, Dravet syndrome, dysautonomia, dyscalculia, dysgraphia, dyslexia, dysphagia, dyspraxia, dyssynergia cerebellaris myoclonica, dyssynergia cerebellaris progressiva, dystonia, dystonias, Early infantile epileptic, Empty sella syndrome, encephalitis, encephalitis lethargica, encephalocele, encephalopathy, encephalopathy (familial infantile), encephalotrigeminal angiomatosis, encopresis, epilepsy, epileptic hemiplegia, erb's palsy, erb- duchenne and dejerine- klumpke palsies, erythromelalgia, essential tremor, extrapontine myelinolysis, Fabry’s disease, Fahr's syndrome, fainting, familial dysautonomia, familial hemangioma, familial idiopathic basal ganglia calcification, familial periodic paralyses, familial spastic paralysis, Farber’s disease, febrile seizures, fibromuscular dysplasia, fibromyalgia, Fisher syndrome, floppy infant syndrome, foot drop, Foville's syndrome, friedreich's ataxia, frontotemporal dementia, Gaucher's disease, generalized gangliosidoses, Gerstmann's syndrome, Gerstmann-Straussler-Scheinker disease, giant axonal neuropathy, giant cell arteritis, Giant cell inclusion disease, globoid cell leukodystrophy, glossopharyngeal neuralgia, Glycogen storage disease, gray matter heterotopia, Guillain-Barre syndrome, Hallervorden-Spatz disease, head injury, headache, hemicrania continua, hemifacial spasm, hemiplegia alterans, hereditary neuropathies, hereditary spastic paraplegia, heredopathia atactica polyneuritiformis, herpes zoster, herpes zoster oticus, Hirayama syndrome, Holmes- Adie syndrome, holoprosencephaly, HTLV-1 associated myelopathy, HIV infection, Hughes syndrome, Huntington's disease, hydranencephaly, hydrocephalus, hydrocephalus - normal pressure, hydromyelia, hypercortisolism, hypersomnia, hypertension, hypertonia, hypotonia, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile hypotonia, infantile neuroaxonal dystrophy, Infantile phytanic acid storage disease, Infantile refsum disease, infantile spasms, inflammatory myopathy, inflammatory myopathies, iniencephaly, intestinal lipodystrophy, intracranial cyst, intracranial hypertension, Isaac's syndrome, Joubert syndrome, Karak syndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsbourne syndrome, Kleine-Levin syndrome, Klippel feil syndrome, Klippel- Trenaunay syndrome (KTS), Kluver-Bucy syndrome, Korsakoff s amnesic syndrome, Krabbe disease, Kugelberg-Welander disease, kuru, Lafora disease, lambert-eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral femoral cutaneous nerve entrapment, Lateral medullary (wallenberg) syndrome, learning disabilities, Leigh's disease, Lennox-Gastaut syndrome, Lesch- Nyhan syndrome, leukodystrophy, Levine-Critchley syndrome, lewy body dementia, Lipid storage diseases, lipoid proteinosis, lissencephaly, Locked-In syndrome, Lou Gehrig’s, lumbar disc disease, lumbar spinal stenosis, lupus - neurological sequelae, lyme disease - neurological sequelae, Machado- Joseph disease (spinocerebellar ataxia type 3), macrencephaly, macropsia, megalencephaly, Melkersson-Rosenthal syndrome, Menieres disease, meningitis, meningitis and encephalitis, Menkes disease, meralgia paresthetica, metachromatic leukodystrophy, metabolic disorders, microcephaly, micropsia, migraine, Miller fisher syndrome, mini-stroke (transient ischemic attack), misophonia, mitochondrial myopathy, Mobius syndrome, Moebius syndrome, monomelic amyotrophy, mood disorder, Motor neurone disease, motor skills disorder, Moyamoya disease, mucolipidoses, mucopolysaccharidoses, multi-infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, multiple system atrophy with orthostatic hypotension, muscular dystrophy, myalgic encephalomyelitis, myasthenia - congenital, myasthenia gravis, myelinoclastic diffuse sclerosis, myoclonic encephalopathy of infants, myoclonus, myopathy, myopathy - congenital, myopathy - thyrotoxic, myotonia, myotonia congenita, myotubular myopathy, narcolepsy, neuroacanthocytosis, neurodegeneration with brain iron accumulation, neurofibromatosis, Neuroleptic malignant syndrome, neurological complications of AIDS, neurological complications of lyme disease, neurological consequences of cytomegalovirus infection, neurological manifestations of AIDS, neurological manifestations of pompe disease, neurological sequelae of lupus, neuromyelitis optica, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorders, neuropathy - hereditary, neurosarcoidosis, neurosyphilis, neurotoxicity, neurotoxic insult, nevus cavemosus, Niemann-pick disease, Non 24-hour sleep-wake syndrome, nonverbal learning disorder, normal pressure hydrocephalus, O'Sullivan-McLeod syndrome, occipital neuralgia, occult spinal dysraphism sequence, Ohtahara syndrome, olivopontocerebellar atrophy, opsoclonus myoclonus, Opsoclonus myoclonus syndrome, optic neuritis, orthostatic hypotension, Overuse syndrome, chronic pain, palinopsia, panic disorder, pantothenate kinase- associated neurodegeneration, paramyotonia congenita, Paraneoplastic diseases, paresthesia, Parkinson’s disease, paroxysmal attacks, paroxysmal choreoathetosis, paroxysmal hemicrania, Parry-Romberg syndrome, Pelizaeus- Merzbacher disease, Pena shokeir II syndrome, perineural cysts, periodic paralyses, peripheral neuropathy, periventricular leukomalacia, persistent vegetative state, pervasive developmental disorders, photic sneeze reflex, Phytanic acid storage disease, Pick's disease, pinched nerve, Piriformis syndrome, pituitary tumors, PMG, polio, polymicrogyria, polymyositis, Pompe disease, porencephaly, Post-polio syndrome, postherpetic neuralgia (PHN), postinfectious encephalomyelitis, postural hypotension, Postural orthostatic tachycardia syndrome, Postural tachycardia syndrome, Prader-Willi syndrome, primary dentatum atrophy, primary lateral sclerosis, primary progressive aphasia, Prion diseases, progressive hemifacial atrophy, progressive locomotor ataxia, progressive multifocal leukoencephalopathy, progressive sclerosing pohodystrophy, progressive supranuclear palsy, prosopagnosia, Pseudo-Torch syndrome, Pseudotoxoplasmosis syndrome, pseudotumor cerebri, Rabies, Ramsay hunt syndrome type I, Ramsay hunt syndrome type II, Ramsay hunt syndrome type III, Rasmussen's encephalitis, Reflex neurovascular dystrophy, Reflex sympathetic dystrophy syndrome, Refsum disease, Refsum disease - infantile, repetitive motion disorders, repetitive stress injury, Restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, rheumatic encephalitis, rhythmic movement disorder, Riley-Day syndrome, Romberg syndrome, sacral nerve root cysts, saint vitus dance, Salivary gland disease, Sandhoff disease, Schilder's disease, schizencephaly, schizophrenia, Seitelberger disease, seizure disorder, semantic dementia, sensory integration dysfunction, septo-optic dysplasia, severe myoclonic epilepsy of infancy (SMEI), Shaken baby syndrome, shingles, Shy- Drager syndrome, Sjogren's syndrome, sleep apnea, sleeping sickness, snatiation, Sotos syndrome, spasticity, spina bifida, spinal cord infarction, spinal cord injury, spinal cord tumors, spinal muscular atrophy, spinocerebellar ataxia, spinocerebellar atrophy, spinocerebellar degeneration, Steele- Richardson-Olszewski syndrome, Stiff-Person syndrome, striatonigral degeneration, stroke, Sturge- Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, SUNCT headache, superficial siderosis, swallowing disorders, Sydenham's chorea, syncope, synesthesia, syphilitic spinal sclerosis, syringohydromyelia, syringomyelia, systemic lupus erythematosus, tabes dorsalis, tardive dyskinesia, tardive dysphrenia, tarlov cyst, Tarsal tunnel syndrome, Tay-Sachs disease, temporal arteritis, tetanus, Tethered spinal cord syndrome, Thomsen disease, thomsen’s myotonia, Thoracic outlet syndrome, thyrotoxic myopathy, tic douloureux, todd's paralysis, Tourette syndrome, toxic encephalopathy, transient ischemic attack, transmissible spongiform encephalopathies, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraparesis, Troyer syndrome, trypanosomiasis, tuberous sclerosis, ubisiosis, uremia, vascular erectile tumor, vasculitis syndromes of the central and peripheral nervous systems, viliuisk encephalomyelitis (VE), Von economo's disease, Von Hippel-Lindau disease (VHL), Von recklinghausen's disease, Wallenberg's syndrome, Werdnig-Hoffman disease, Wernicke-Korsakoff syndrome, West syndrome, Whiplash, Whipple's disease, Williams syndrome, Wilson's disease, Wolman's disease, X-linked spinal and bulbar muscular atrophy, or Zellweger syndrome. In some embodiments, the neurological disease is dementia or Alzheimer’s Disease. In other embodiments, the neurological disease is Alzheimer’s Disease. In some embodiments, the crystal salts and/or co-crystals described herein may be used to inhibit priogenic seeding or to inhibit tau protein aggregation or inhibit tau fibril aggregation.

Exemplary inflammatory disease that may be treated with the crystal salts or co-crystals as described herein include an inflammatory disease associated with an autoimmune disease, a central nervous system (CNS) inflammatory disease, a joint inflammation disease, an inflammatory digestive tract disease, inflammatory skin and other inflammatory diseases related to epithelial cells such as bronchitis or other respiratory diseases, inflammation associated with cancer, such as colon carcinoma, inflammation associated with irritation, and inflammation associated with injury. For example, inflammatory diseases include rheumatoid arthritis, osteoarthritis juvenile idiopathic arthritis, psoriasis, psoriatic arthritis, allergic airway disease (e.g. asthma, rhinitis), chronic obstructive pulmonary disease (COPD), inflammatory bowel diseases (e.g. Crohn's disease, ulcerative colitis), endotoxin-driven disease states (e.g. complications after bypass surgery or chronic endotoxin states contributing to e.g. chronic cardiac failure), and related diseases involving cartilage, such as that of the joints. Particularly the term refers to rheumatoid arthritis, osteoarthritis, allergic airway disease (e.g. asthma), chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases (e.g. Crohn's disease and ulcerative colitis). Autoimmune diseases refers to the group of diseases including obstructive airways disease, including conditions such as COPD, asthma (e.g. intrinsic asthma, extrinsic asthma, dust asthma, infantile asthma) particularly chronic or inveterate asthma (for example late asthma and airway hyperreponsiveness), bronchitis, including bronchial asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythrematosis, lupus nephritis, dermatomyositis, Sjogren’s syndrome, multiple sclerosis, psoriasis, dry eye disease, type I diabetes mellitus and complications associated therewith, atopic eczema (atopic dermatitis), thyroiditis (Hashimoto's and autoimmune thyroiditis), contact dermatitis and further eczematous dermatitis, inflammatory bowel disease (e.g. Crohn's disease and ulcerative colitis), atherosclerosis and amyotrophic lateral sclerosis. Particularly the term refers to COPD, asthma, systemic lupus erythematosis, type I diabetes mellitus and inflammatory bowel disease. Respiratory disease refers to diseases affecting the organs that are involved in breathing, such as the nose, throat, larynx, eustachian tubes, trachea, bronchi, lungs, related muscles (e.g., diaphragm and intercostals), and nerves. In particular, examples of respiratory diseases include asthma, adult respiratory distress syndrome and allergic (extrinsic) asthma, non-allergic (intrinsic) asthma, acute severe asthma, chronic asthma, clinical asthma, nocturnal asthma, allerGen-induced asthma, aspirin-sensitive asthma, exercise-induced asthma, isocapnic hyperventilation, child onset asthma, adult-onset asthma, cough-variant asthma, occupational asthma, steroid-resistant asthma, seasonal asthma, seasonal allergic rhinitis, perennial allergic rhinitis, chronic obstructive pulmonary disease, including chronic bronchitis or emphysema, pulmonary hypertension, interstitial lung fibrosis and/or airway inflammation, cystic fibrosis, and hypoxia. Allergy refers to the group of conditions characterized by a hypersensitivity disorder of the immune system including, allergic airway disease (e.g. asthma, rhinitis), sinusitis, eczema and hives, as well as food allergies or allergies to insect venom. As used herein the term ‘cardiovascular disease’ refers to diseases affecting the heart or blood vessels or both. In particular, cardiovascular disease includes arrhythmia (atrial or ventricular or both); atherosclerosis and its sequelae; angina; cardiac rhythm disturbances; myocardial ischemia; myocardial infarction; cardiac or vascular aneurysm; vasculitis, stroke; peripheral obstructive arteriopathy of a limb, an organ, or a tissue; reperfusion injury following ischemia of the brain, heart, kidney or other organ or tissue; endotoxic, surgical, or traumatic shock; hypertension, valvular heart disease, heart failure, abnormal blood pressure; vasoconstriction (including that associated with migraines); vascular abnormality, inflammation, insufficiency limited to a single organ or tissue.

In some embodiments, a crystal salt or co-crystal of DHM may be administered in an amount of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, or about 5 mg/kg to about 25 mg/kg. In some embodiments, a dose of a crystal salt or co-crystal may be about 0.5 mg, about 5 mg, about 10 mg, about 115 mg, or about 500 mg. In other embodiments, a dose of a co-crystal may be about 100 mg to about 1000 mg. Multiple doses may be administered within a 24-hour period. In some embodiments, the doses are administered orally or intravenously.

In one embodiment, a method of treating Alzheimer’s Disease comprises administering an effective amount of a crystal salt or co-crystal of dihydromyricetin (DHM) to a subject, wherein the co-crystal of DHM comprises the DHM and a crystal coformer selected from the group consisting of triethanolamine, 2-amino-2-(hydroxymethyl)- 1 , 3 -propanediol, sodium hydroxide, calcium hydroxide, and an amino acid; wherein the crystal salt of the co-crystal of DHM thereby treats the Alzheimer’s Disease. In another embodiment, a method of treating a Alzheimer’s Disease comprises administering an effective amount of a co-crystal of dihydromyricetin (DHM) to a subject, wherein the co-crystal of DHM comprises the DHM and triethanolamine.

In some embodiments, a method for treating a disease using a crystal salt or co-crystal as described herein may comprise administering to the subject a second therapeutic agent. For example, in some embodiments, the second therapeutic agent comprises epigallocatechin gallate.

Pharmaceutical Formulations.

The compounds described herein can be used to prepare therapeutic pharmaceutical compositions, for example, by combining the compounds with a pharmaceutically acceptable diluent, excipient, or carrier. The compounds may be added to a carrier in the form of a salt or solvate. For example, in cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a-ketoglutarate, and [3- glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable ionic compound. Alkali metal (for example, sodium, potassium, or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods.

The compounds of the formulas described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms. The forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes.

The compounds described herein may be systemically administered in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier. For oral administration, compounds can be enclosed in hard- or soft-shell gelatin capsules, compressed into tablets, or incorporated directly into the food of a patient's diet. Compounds may also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations typically contain at least 0.1% of active compound. The percentage of the compositions and preparations can vary and may conveniently be from about 0.5% to about 60%, about 1% to about 25%, or about 2% to about 10%, of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions can be such that an effective dosage level can be obtained.

The tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; and a lubricant such as magnesium stearate. A sweetening agent such as sucrose, fructose, lactose, or aspartame; or a flavoring agent such as peppermint, oil of Wintergreen, or cherry flavoring, may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

The active compound may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil. Under ordinary conditions of storage and use, preparations may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions, dispersions, or sterile powders comprising the active ingredient adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and/or 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, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, optionally followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation can include vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the solution.

For topical administration, compounds may be applied in pure form, e.g., when they are liquids. However, it will generally be desirable to administer the active agent to the skin as a composition or formulation, for example, in combination with a dermatologically acceptable carrier, which may be a solid, a liquid, a gel, or the like.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Useful liquid carriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, or water- alcohol/gly col blends, in which a compound can be dissolved or dispersed at effective levels, optionally with the aid of non- toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of dermatological compositions for delivering active agents to the skin are known to the art; for example, see U.S. Patent Nos. 4,992,478 (Geria), 4,820,508 (Wortzman), 4,608,392 (Jacquet et al.), and 4,559,157 (Smith et al.). Such dermatological compositions can be used in combinations with the compounds described herein where an ingredient of such compositions can optionally be replaced by a compound described herein, or a compound described herein can be added to the composition.

Useful dosages of the compounds described herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent No. 4,938,949 (Borch et al.). The amount of a compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will be ultimately at the discretion of an attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently formulated in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. In one embodiment, the invention provides a composition comprising a compound of the invention formulated in such a unit dosage form.

The compound can be conveniently administered in a unit dosage form, for example, containing 5 to 1000 mg/m², conveniently 10 to 750 mg/m², most conveniently, 50 to 500 mg/m² of active ingredient per unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

Results and Discussion.

The polyphenol DHM has a similar chemical structure to EGCG, a known polyphenolic ligand and inhibitor of tau (Fig. 1A). Despite rich phenolic character, DHM is notoriously hydrophobic with a reported water solubility of -0.2-0.4 mg/ml and enhanced ability to partition to the CNS compared to other polyphenols. Therefore, we evaluated the in silico predicted binding of DHM to AD tau PHFs using CB-Dock, a suite that incorporates cavity detection with AutoDock Vina. Using an AD tau PHF co-cryoEM structure with EGCG as a starting model, we evaluated the most likely binding cavities for DHM. The top-scoring predicted DHM binding cavity matched the primary EGCG binding volume formerly determined by CryoEM (Fig. 1 A), although the DHM binding pose output by CB-Dock differs from models we generated manually by superimposing DHM with EGCG from the liganded CryoEM structure (Fig. 8). Both modeling approaches predict interactions with Lys340, although models generated by CB-Dock orient the 71 orbitals of aromatic moieties of DHM perpendicular to the fibril axis whereas manually generated models orient stacks of aromatic moieties parallel to strand forming P sheets of the fibril. Additional possible DHM binding sites detected by CB-Dock include cavities labeled Sites 2 and 3 (Fig. 1 A). However, both Sites 2 and 3 scored lower by AutoDock Vina. Site 2 is lined by histidine and lysine residues, which could support hydrogen bonding interactions with DHM, similar to the EGCG binding site of Tau PHFs. Site 3 is created by a sharp turn at a histidine and glycine interface and scores significantly lower favorably compared to Sites 1 and 2.

We tested the in vitro activity of DHM to inhibit prionogenic seeding by AD tau (Fig. Ib-f) since modeling suggests DHM can bind to AD tau fibrils in a manner similar to EGCG. Transfecting AD crude brain homogenates in tau biosensor cells, which express an aggregation-prone fragment of tau fused with YFP, produces a punctated cellular phenotype, shown in Fig. Id that results from the aggregation of intracellular tau that is seeded by tau from AD brain homogenate. Pre-treating AD crude brain homogenates with DHM dissolved in DMSO inhibited seeding in a dose-dependent manner with an IC50 of 1.9 pM (Fig. lb). In comparison, DHM that was dissolved in water elicited less potent inhibition with an apparent IC50 of 10.5 pM (Fig. 1c). These results were consistent with limiting solubilities observed for DHM stock solutions prepared in water. We observed that 10 mM master stock solutions of DHM prepared in water were unstable, resulting in DHM precipitation and eventual equilibration to a stock solution with actual measured concentrations of 0.65 mg/ml (2 mM). In contrast, stock solutions prepared in DMSO remained soluble. Our observation that only about one-fifth of DHM remained dissolved in aqueous solutions after clearing insoluble matter by centrifugation may account for the lowered measured IC50 of DHM solutions prepared in water. These data illustrate two important points: (1) DHM effectively inhibits seeding by AD tau, particularly when solubilized in nonpolar solvents, and (2) the inhibitory power of DHM is limited by poor aqueous solubility. Compounds with limiting aqueous solubilities exhibit low bioavailability and compromised bioactivity. Therefore, we investigated the physicochemical factors underlying the poor aqueous solubility of DHM with the goal of developing formulations to overcome the solubility limitation. An analysis of pKa shown in Fig. 2a reveals plausible ionization states of DHM. A first acid dissociation event is predicted by ADMET Predictor (Simulations Plus, Inc., Lancaster, CA) to occur between pH 7.05 7.83, with DHM transitioning from neutral to a negatively charged ion. Four primary sites of possible ionization shown in Fig. 2a are predicted in varying ratios.

The predicted near neutral pKa for DHM was verified experimentally by two methods: potentiometric titration and UV-Vis spectroscopy (Pandey et al., Spectrochim Acta A Mol Biomol Spectrosc 115, 887-890). Experimental pKa values presented in Fig. 2b-c were obtained by plotting the absorbance ratio of DHM at 240 and 260 nm across a range of pH values (3.1-13.3) in various buffer solutions (as listed in Table 1). The determined pKa, pH 7.9, was taken as the Y intercept of the line of best fit of the log ratio (Fig. 2c). To confirm the accuracy of the pKa value obtained via this method, DHM was subjected to potentiometric titration with sodium hydroxide in methanol, and the resulting pKa value was found to be in agreement with the pH 7.9 value (as depicted in Fig. 2d).

We investigated the solubility-enhancing properties of the following five complexes with DHM: triethanolamine (TEA), sodium hydroxide (NaOH), TRIS Base (2-Amino-2- (hydroxymethyl)-l,3-propanediol), L-lysine, and calcium hydroxide (CaOH2), each combined with DHM in one of four solvents (ethanol, methanol, isopropanol, or acetone) and rendered by slow evaporation. New salt or co-crystal forms of DHM were made by setting up a reaction in a solvent system which allows DHM and counterion to fully dissolve and facilitate proton transfer from DHM (proton donor) to a base (proton acceptor). We set up a counterion screen and produced several slurries, using various pharmaceutically relevant counterions in different solvent systems (Table 2). Five of the combinations yielded powders, which were taken as potential salt formulations: DHM-TEA prepared from ethanol (also referred to as Isis 101 or DHM- la), DHM-Tris prepared from isopropanol (also referred to as DHM-3c), DHM-Lysine prepared from water (also referred to as DHM-X), and two DHM-Ca formulations: DHM-Ca(b) and DHM-Ca(c), prepared from methanol and 2-propanol, respectively (also referred to as DHM-b and DHM-c, respectively).

The five salt forms were tested for tau inhibitor activity by dissolving in water to a target concentration of 10 mM. As shown in the schematic in Fig. 3a outlining our workflow, insoluble DHM was removed from concentrated stocks by centrifugation to yield supernatants that were tested for tau inhibitor activity. We reasoned that aqueously soluble fractions of DHM salts would exhibit tau inhibitor activities with potencies that approached DHM dissolved in DMSO. DHM-TEA, -Lysine, -Tris, -Ca all exhibited inhibitor activity compared with DHM (Fig 3b-e). DHM-TEA, -DHM-Ca were of greatest improved tau inhibitor potencies. The IC50s of DHM-TEA and DHM-Ca(b) were 2.6 and 0.87 uM, respectively, (Fig. 3d and e), similar to the IC50 we measured for DHM dissolved in DMSO.

As a control to ensure counterions themselves do not contribute to the measured tau inhibitory activities, we measured tau seeding after pre-incubating AD brain homogenates with DHM salts and counterions. As shown in Fig 3f, neither TEA nor CaOHi counterion had inhibitory activity towards tau seeding by crude AD brain homogenates, confirming that the DHM inhibitor activity is attributable to improved solubility of DHM and not the result of synergistic inhibition by accompanying counterions. Also supporting this are data in Fig. 9 showing DHM salts DHM-TEA and -DHM-Ca(b) possess IC50s of ~1 pM similar to DHM, indicating that the counterions themselves do not enhance tau inhibitor activity beyond what is possible for DHM itself dissolved at the target concentration. Thus, counterions improve inhibitory activity of DHM by effecting solubility. Experimental solubilities of DHM-TEA and -DHM-Ca shown in Fig 3g reveals 4-6 fold improved water solubility, consistent with the fold improvements in IC50 that were seen for these salts.

Since DHM shares a similar structure to EGCG, we tested the hypothesis that DHM inhibits seeding by a similar mechanism by AD tau disaggregating fibrils. AD tau fibril disaggregation by DHM was measured by quantitative electron microscopy (qEM). As shown in micrographs in Fig. 4, incubation with EGCG reduces the average number of AD tau fibrils observed by negative-stain EM imaging by 70-80%. DHM-TEA and -Ca(b) both reduced fibril density by 50%, from 300 to 160 fibrils after 48 hrs incubation with DHM-TEA and from 227 total fibrils to 111 fibrils after 48 hrs incubation with DHM-Ca(b). These data suggest fibril disaggregation is one plausible mechanism of inhibition, although they leave open the possibility that DHM binding to AD tau is inhibitory towards tau seeding since fibril disaggregation occurs to a lesser extent compared to EGCG. Further supporting the possibility that ligand binding exerts inhibitory effects absent of fibril disaggregation are data showing that inhibitor pre-incubation is not needed to realize seeding inhibition (Fig. 10). These data showing that seeding inhibition by DHM occurs despite some fraction of remaining fibrils in solution thus suggest it is possible that ligand binding itself suppresses seeding, at least to a partial degree. The mechanism of enhanced solubility of DHM salt was investigated by structural chemistry. DHM-TEA and -Ca exhibited X-ray powder diffraction indicating crystallinity (Fig. 5a, 13, and 14), although only DHM-TEA produced suitable diffraction by MicroED to yield an atomic structure. TEA is seen in the 0.74 A resolution MicroED structure in a 1:1 molar ratio positioned 2.6 A from the 02 of the phenol of the resorcinol that is predicted to first become ionized (Fig. 5b and Fig. 2a). Although crystals of DHM-Ca(b) also diffracted, the diffraction power was insufficient to enable atomic structure determination. It is possible the tridentate nature of triethanolamine reinforced the crystal lattice enabled stronger diffraction and structure elucidation compared with DHM-Ca salts, which were crystalline but exhibited weaker diffraction.

Comparing the DHM-TEA vs. dihydrate (Xu et al., Acta Crystallographica Section E 63, o4384-o4384,) lattices in (Fig. 5c and d) reveals large solvent channels and high solvent content in the TEA lattice. Analysis of porosity using CrystalMaker reveals the DHM dihydrate unit cell is 20.6% filled space with 79.4% void. Co-crystallizing with TEA alters DHM packing leading to a fractured network of intermolecular phenol-phenol H-bonds that otherwise distinguishes the dihydrate lattice. As a result, the DHM-TEA lattice is relatively porous with void volume increased to 83.3% of the unit cell volume. The unit cell volume of DHM dihydrate, 2899. 1 A- , has a low solvent content described by a Matthew Coefficient (VM) of 1.02 A /Da, which is typical of small molecule crystals. The VM of DHM with a unit cell volume of 971.5 A³ is 2.07 A³/Da assuming a mass of 469 da, which is atypically large for a small molecule crystals and more closely mirrors the VM of protein crystals which are entrenched with large solvent channels enabling high solvent content (Matthews et al., J Mol Biol 33, 491-497). Large solvent channels permeating the lattice of DHM-TEA lattice are seen in Fig. 5c and 6a, and likely enable water molecules to infuse and dihydrate molecules of DHM more readily than for the DHM dihydrate. Correspondingly, DHM-TEA exhibits a cooperative transition at lower temperature suggesting DHM-TEA is less thermodynamically stable than DHM dihydrate (Fig. 5e).

Shown in Fig. 5 a, two trolamines pack between DHM molecules disrupting H-bonding between the three phenols of the pyrogallol ring, which are otherwise all H-bonded in the dihydrate crystal. Trolamine occupies this space without making any H-bond interactions with the pyrogallol, and hence leaves the three phenols of the pyrogallol ring unpaired with gaps of 3.3-3.8 A between the trolamines and phenols. Moreover, the angles at which the trolamines orient relative to the pyrogallol ring are -145-155°, thus leaving space and geometry for water molecules to permeate the lattice of the co-crystal to dihydrate and H-bond with the non-paired phenols of the pyrogallol ring.

The number of in crystallo phenol H-bonds in DHM-TEA is 5 compared to 9 in DHM dihydrate (Fig. 6d). DHM dihydrate crystals make 5 intermolecular H-bonds between the phenolic moieties of DHM, and an additional 4 water mediated H-bonds bridge neighboring DHM molecules. A single peak at 2.7 A dominates the 0-0 pair correlation function, g(r), of the DHM hydrate lattice (Fig. 6h), which reflects the extensive intermolecular network of H- bonded phenols that anneals the 0-0 pair correlation function. Similarly, a peak at 2.85 A is in the C-0 pair correlation function in Fig. 6j, which arises from intermolecular H-bonding between phenols. Phenols of the DHM-TEA lattice are less extensively H-bonded, with no peak in the 0-0 pair correlation function (Fig. 6g) and minor peaks in the C-0 function 2.75 and 2.85 A (Fig. 6j). Corresponding, 3 H-bonds are seen between phenols with neighboring DHM molecules and 2 H-bonds with trolamines located on diametric surfaces of DHM (Fig. 6a). The lower overall contact frequency of DHM-TEA in the crystal lattice is consistent with its lower transition temperature measured by DSC for DHM-TEA (Fig. 5e), and explains increased solubility of DHM-TEA. Hydration of DHM-TEA is thermodynamically favored compared with the dihydrate since added enthalpic gains are possible by H-bonds formed between water molecules and non-bonded phenols upon solvation (Fig 6a, green arrows). Solvating DHM dihydrate requires the exchange of intermolecular lattice H-bonds with water molecules, which is kinetically and thermodynamically disfavored since there is no net enthalpy gain to offset the entropic penalty of solvation.

Hydrophobic contacts further contribute to the thermodynamics of DHM solubilization. The C-0 pair correlation function shows a peak at 3.7 A corresponding to the n stacking distances of aromatic rings of DHM in the dihydrate lattice (Fig. 6f and j). The ring-to-ring distance increases to 7.5 A in the DHM-TEA lattice (Fig. 6e) indicating loss of n stacking. Loss of n stacking is expected to lower the entropic barrier to solvation since aromatic rings of DHM could be partially solvated in the crystal, and moreover the configuration in DHM-TEA eliminates the energetic barrier associated with breaking n stacking in order to solubilize DHM.

Despite tremendous overall H-bonding potential, DHM exhibits remarkable hydrophobic behavior at a macroscopic level. The hydrophobic tendency of DHM is explained by its dihydrate crystal structure, which shows phenols of DHM totally satisfying the H- bonding potentials of neighboring DHM molecules through an extensive network H-bond pairing. In addition, the aromatic rings of DHM form favorable pi-stacking and Van der Waals interactions in the DHM hydrate, resulting in a stable form that is thermodynamically costly to solubilize in water. Our data establishes that trolamine physically disrupts packing of DHM co-crystals and the H-bonding network. These data provide a physical and thermodynamic basis explaining the transformed macroscopic properties of DHM salts, which exhibit up to five times increased water solubility, as counterions replace and only satisfy partially the hydrogen bonding potential of DHM, thereby leading to a more favorable thermodynamic state for solubilization. The cryptic hydrogen bonding potential that allows DHM to mask phenols by aggregation may also explain its tendency to better permeate the CNS compared to other polyphenols.

DHM has been investigated for effects in mitigating alcohol use-related disorders, age- related diseases, oxidative stress, poisoning, and liver damage, but its commercial form is a practically water-insoluble solid of beige -brown color. Thus, enhancing the physical properties of DHM to enable improved delivery and biopharmaceutical performance as a pharmaceutically acceptable formulation is of great interest. By behaving as a Brpnsted-Lowry acid-base with a log of acid dissociation constant pKa in the useful and relevant pH range of 7-8, we discovered that DHM salt formulations are possible. Ionized species of DHM enable the preparation of a wide range of possible DHM salt forms with bases that satisfy full or partial proton transfer to enable solid and liquid in situ extemporaneous pharmaceutical dosages. As illustrated in Fig. 7, DHM co-crystals described here have appropriate composition to enable improved biopharmaceutical performance in a pharmaceutically acceptable context (e.g. more accurate and efficient dosing of DHM by increasing solubility to allow more DHM to reach the bloodstream). Regarding the CNS permeability solubility-enhanced DHM, we anticipate circulating DHM could re-establish H-bonding reminiscent the intermolecular phenol-phenol interactions that are seen in the dihydrate crystal form and these assemblies will be important for shielding the hydrophilic character of DHM to enable CNS absorption.

As a possible tool in the management of Alzheimer’s Disease (AD), DHM holds potential in two possible realms. (1) DHM is a lead for medicinal chemistry structure-activity studies to generate pharmaceutical-grade anti-tau medications with improved potency and CNS permeability. (2) DHM natural products as dietary supplements offer a more immediate possible approach to combine with other lifestyle adaptations to aid healthier aging. Natural products are affordable and readily accessible chemicals, and in the case of DHM, could be rapidly developed and deployed commercially to support studies investigating healthier aging alongside lifestyle modifications (i.e. attention to diet, exercise, and cardiovascular health). The MicroED structure shows that co-crystalline formulations of DHM remain unaltered in chemical structure despite improvements in aqueous solubility and apparent potency. Thus, the formulations we describe do not alter the natural product status of DHM. DHM is sold as a dietary supplement in the United States and is generally regarded as safe (GRAS) by the FDA. A human clinical trial of 60 adults with non-alcoholic fatty liver disease assigned DHM 150 mg twice daily vs. placebo for three months identified no significant safety concerns (Chen et al. Pharmacol Res 99, 74-81). Although, as with most flavonoids, the major obstacle to DHM’s clinical application is its suboptimal pharmacokinetics (PK). The new formulations of DHM described herein, which have enhanced aqueous solubility, can provide forms of DHM with improved bioavailability.

In summary, by strategically coupling structural and formulations chemistry, DHM was realized as a possible natural product inhibitor lead compound for targeting prionogenic seeding by AD tau. These studies open the door for near-term investigations of the effects of DHM salts as dietary supplements for testing in humans to support healthier ageing, and in the broader context, suggests co-crystalline salt formulations may be extended to formulate other pseudo-hydrophobic medicinal chemicals to enhance solubility and extend dosing ranges.

The following Examples are intended to illustrate the above invention and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the invention could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the invention.

EXAMPLES

Example 1. Materials and Methods.

A commercially available batch of dihydromyricetin [(DHM) IUPAC Name (2R,3R)- 3,5,7-trihydroxy-2-(3,4,5-trihydroxyphenyl)-2,3-dihydrochromen-4-one, of > 98% purity] was purchased from Master Herbs Inc (Pomona, CA). Counterions, triethanolamine (99%) (2, 2', 2''- nitrilotriethanol or tris(2-hydroxyethyl)amine; (HOCHiCHijsN) was purchased from Lab Alley (Spice wood, TX); sodium hydroxide, calcium hydroxide, and L-lysine were all purchased from Sigma-Aldrich (Burlington, MA); and TRIS-base was purchased from Gold Biotechnology (St. Louis, MO). Solvents, DMSO, pure ethyl alcohol (200 proof, molecular grade), methanol (>99.5% purity, molecular grade), 2-propanol (>99.5% purity, molecular grade), acetone (>99.5% purity, molecular grade), and anti-solvent, chloroform (anhydrous, >99%, containing 0.5-1.0% ethanol as stabilizer) were all purchased from Sigma- Aldrich (Burlington, MA). The instruments used were a Mettler-Toledo ME54TE/00 analytical balance and TE412 Top-loading balance (Switzerland), VWR sypHony B10P pH meter, Fischer Scientific Stirring Hotplate 11-500-49SH (Walthamm MA); magnetic stir bars, flat bottom 96- well plates, 20mL glass scintillation vials, Vacubrand ME 2 NT vacuum pump, and the Biotek Agilent Synergy HTX Multi-mode Reader (Santa Clara, CA); qualitative Whatman Filter Papers (47mm diameter) was purchased from Sigma-Aldrich (Burlington, MA). Polarized light microscopy was performed using Olympus BX51 Microscope, and the analyzer (U-ANT; Analyzer for transmitted light U-Pl 15) and polarizing (U-POT; Polarizer for transmitted light, 45mm U- P110) filters were purchased from Olympus (Waltham, MA). Melting point determinations were performed using PerkinElmer DSC 8500 with HyperDSC and cooling accessory (IntraCooler 2), (PerkinElmer, Inc., Valencia, CA). X-ray powder diffraction studies were performed using MiniFlex™ 600, (Rigaku, Inc., The Woodlands, TX).

Counterion Screen. DHM co-crystals with various counterions indicated in the main text were screened by preparing in a variety solvents (methanol, ethanol, 2-propanol, or acetone) to facilitate crystallization by slow evaporation. Crystallization was carried out at ambient temperature (22-25°C) in a 250mL Erlenmey er flasks. A 1: 1, 1: 1.25, or 1:2 molar ratio of DHM to counterion were tested to optimize formation and crystallization. The counterion was first dissolved in the respective solvent and DHM added and to it with stirring for at least 45 min until homogenous slurries were observed. Slurries were then shielded from light slowly evaporated for two weeks. Resulting powders were washed with 50mL of chloroform over a vacuum pump membrane filtration. Filtrate residue was dried, recovered, and stored at ambient temperature until further use.

Polarized Light Microscopy. An Olympus (Olympus, Waltham, MA) microscope equipped with a polarizer and analyzer was used to initially evaluate isolated solids from slurries. lOOOx magnification was used with an analyzer (U-ANT) slider insert and a polarizer (U-POT). Samples were qualitatively assessed for particle size and shape, as well as birefringence for initial evidence of existence of orientation-dependent differences in refractive index. pKa Determination. pKa values were calculated using ADMET Predictor™ version 10.3.0.7 64-bit edition module of Simulations Plus software (Lancaster, CA), and ChemDraw® Professional version 20.1.1.125 (PerkinElmer Informatics, Inc.). Potentiometric titration curves of DHM were obtained by adding O.lmL of a 0.5N NaOH standard solution to three different 2mg/mL DHM analyte solutions. All solutions were made in 50mL beakers using deionized water and prepared at 22-25 °C ambient room temperature. The 2mg/mL DHM standard solution was titrated with 0.5N NaOH. The NaOH standard solution was prepared by dissolving 3g of sodium hydroxide in 150mL of water for a final concentration of 0.5N NaOH. A lOOmg/mL stock solution of DHM was prepared by weighing 0.5g of DHM using an analytical balance and dissolving it in 5mL of methanol in a 15mL conical tube. Three analyte solutions were prepared using methanol and water mixtures to yield 25mL of solutions with 25, 50, and 75% v/v methanol content. Five hundred pL of DHM stock solution was then added to the three analyte solutions to yield final DHM concentration of 2mg/mL, the initial pH value was measured and recorded. pKa was orthogonally determined through UV-VIS spectroscopy optical absorption (Pandey et al., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 115, 887-890). Briefly, a 5mM analyte stock solution of DHM was prepared by measuring 16 mg of DHM dissolved in lOmL of DMSO and was further diluted to 2mM and used as the working stock solution. Buffer solutions of various pH values were prepared as described in Table 1. Using a 96-well plate, 0.2mL of each pH buffer solution was added to duplicate wells, then 0.5pL/well of the DHM stock solution was added for a final DHM concentration of 5pM in each sample well. A spectral scan was conducted ( = 230-700nm), and absorbance was measured in lOnm increments. The pKa was calculated using pKa=pH+log [(DHM⁰*- DHM^pHabs)/(DHM^pHabs-DHM"abs), where DHM°_abs is the absorbance of the unionized molecule at lowest pH, DHM^pH _abs is the absorbance of the molecule in respective buffers tested, and DHM abs is the absorbance of the ionized molecule at highest pH.

Differential Scanning Calorimetry. Samples (3-5mg, small enough to avoid saturating the apparatus) were analyzed using a PerkinElmer DSC 8500, (PerkinElmer, Inc., Waltham, MA), that is equipped with a cooling system that uses a continuous dry nitrogen purge at 25mL/min. The instrument was calibrated for temperature and enthalpy change with indium, tin, and lead samples provided by the manufacturer. The samples and reference (i.e., blank) were placed in the furnace. Data was collected from 25-300 °C with a heating rate of 10 °C/min. Onset and peak of observed thermal events were recorded and qualitatively analyzed for relative comparison.

X-Ray Powder Diffractometry. Solid material isolated from DHM slurries were broken down into a fine powder using a glass coverslip or a spatula. Approximately 5-10 mg of the powder was placed onto a zero background, 5mm diameter x 0.2mm deep well, silicone sample holder (Rigaku, Inc., The Woodlands, TX). Analysis was performed on a Rigaku Miniflex 600 Benchtop XRD System (Rigaku, Inc., The Woodlands, TX). Diffractograms were collected operating at a scanning rate of 2.00° min ¹. The diffraction spectra were recorded at the diffraction angle, 20 from 0° to 50° at room temperature.

In silico modeling. Although DHM has a similar chemical structure to EGCG (Fig. la), the binding site of DHM remains unknown. Therefore, molecular docking of DHM on paired helical filament (PHF) tau (PDB: 5O3L) was performed to uncover potential sites of binding using Cavity-detection guided Blind Docking (CB-Dock), a protein-ligand docking method that uses AutoDock Vina (Fig. lb) (Cao et al., Bioinformatics, Jun 15 ;30(12): 1674-80 2014). The top five potential binding cavities were identified.

Preparation of crude Alzheimer's brain-derived tau seeds. Human Alzheimer’s brain autopsy samples were obtained from the UCLA Pathology Department according to HHS regulation from patients who consented to autopsy. Approximately 0.2 g of hippocampal tissue was excised, and a Kinematica PT 10-35 POLYTRON was used to homogenize the tissue with sucrose buffer supplemented with 1 mM Ethylene glycol tetraacetic acid (EGTA) and 5 mM Ethylenediaminetetraacetic acid (EDTA) at level 4-5 in 15 ml falcon tubes.

K18CY cell culture. HEK293T cell lines that stably express tau-K18CY labeled with green fluorescent protein (GFP) were used. The cells were cultured in a T25 flask in Dulbecco’ s Modified Eagle Medium (DMEM) (Life Technologies, cat. 11965092) supplemented with 10% (vol/vol) Fetal Bovine Serum (FBS) (Life Technologies, cat. A3160401), 1% penicillin/streptomycin (Life Technologies, cat. 15140122), and 1% Glutamax (Life Technologies, cat. 35050061) at 37°C and 5% CO2 in a humidified incubator. To test the inhibitors on the biosensor cells, 100 pl of cells were plated 1 :10 in 96 well plates and stored in the 37°C, 5% CO2 incubator for 16 to 24 hours prior to transfection.

Screening of DHM alongside EGCG. EGCG and DHM were dissolved in dimethyl sulfoxide (DMSO) to 10 mM at room temperature. Previously homogenized human-derived AD crude brain extracts were diluted 1 to 20 with Opti-MEM (Thermo Fisher Scientific, cat. 31985062) and sonicated in a Qsonica multiplate horn water bath for 3 minutes at 40% power. The diluted brain extracts were then incubated with the inhibitors for 16 to 24 hours at 4°C to yield a final EGCG or DHM concentration of 10 mM on the tau K18CY biosensor cells. Inhibitor-treated seeds were sonicated again in a Cup Horn water bath for 3 minutes at 40% power and then mixed with a 1 to 20 solution of Lipofectamine 2000 (Thermo Fisher Scientific, cat. 11668019) and Opti-MEM. The Lipofectamine creates a liposome around the fibrils to allow delivery into the cells. After 20 minutes, 10 pl of inhibitor- treated fibrils were added to the previously plated 100 pl of cells in triplicate, avoiding use of the perimeter wells. Screening of DHM crystalline salts. DHM and five salt formulations were screened alongside EGCG in triplicate using the same tau prionogenic seeding assay workflow, except inhibitors were dissolved in sterile, deionized water rather than DMSO, at 37°C or on the benchtop at room temperature. Dissolved inhibitors were then centrifuged for 10 minutes at 8,000 or 15,000 rpm to pellet the insoluble fraction of DHM and the salts. The supernatants were removed and used to test the soluble fraction of the small molecules’ inhibitory effect.

Plate reading. The number of seeded aggregates was determined by using the BioTek Cytation 5 Imaging Multimode Reader in the GFP channel to image the entire 96 well plate. A 3x2 montage was used to capture as much area of each well as possible. Exposure and contrast were adjusted to allow puncta to distinguish the seeded aggregates from the cells. Seeded aggregates appear as bright green puncta (Fig. 1c)

Quantification of tau aggregation. Seeded aggregates in a given image were quantified using a Java-based image processing program ImageJ 2.3.0 (Schneider el al., Nat Methods 9, 671-675 (2012)) script, which subtracts the background fluorescence from unseeded cells, and then uses a built-in particle analyzer to quantify the number of puncta (seeded aggregates) as peaks with fluorescence that contrast the background. The puncta count was then normalized across all images according to cell confluence by using a separate ImageJ 2.3.0 script for confluence. Normalizing to confluence is necessary in the event that an inhibitor is toxic to cells, so that toxic inhibitors are not considered to be effective against tau seeding. The average number of seeded aggregates of each well and standard deviations from triplicate measurements, normalized to confluence, was plotted to compare inhibitory activity.

Determination of potencies. The same tau prionogenic seeding assay workflow with Alzheimer’s crude brain extracts was conducted to determine the ICso’s - half maximal inhibition - of EGCG, DHM, and DHM analog. Ten different final inhibitor concentrations were tested - 0, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, 20.0, and 40.0 pM - in triplicate. Inhibitors were dissolved in both DMSO and DI water. After aggregation in each well was quantified using ImageJ, the GraphPad Prism 9 software was used to generate dose-response curves for IC50 determination.

Determination of solubility. Solubility determinations of DHM and salt co-crystal formulations were conducted by spectrophotometric analysis. Inhibitors were first dissolved to 10 mM in DMSO, and the absorbance spectra were collected to determine the wavelength at which absorbance peaked. The 10 mM stocks were then diluted 1:3, 1:6, 1 :12, 1:30, 1:60, 1: 120, and 1:300 and absorbance of each was measured using the NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer at peak wavelength alongside a DI water negative control to produce a calibration curve. The working linear range was determined by eliminating absorbance measurements above the limit of linearity. The 10 mM stocks of DHM and salt cocrystal forms previously used for the tau prionogenic seeding assays were diluted as necessary to fall within the linear range and their measured absorbances were used to calculate the true concentrations of stocks solutions.

Transmission electron microscopy (TEM). Transmission electron microscopy was used to determine whether DHM crystalline salt formulations exert inhibitor effects on seeding through tau disaggregation like EGCG. Purified Alzheimer’s brain-derived tau fibrils were incubated with inhibitor and added to negative stain grids to be imaged with the JEOL-2100 TEM.

Preparation of purified Alzheimer’s brain-derived tau fibrils. The same homogenization process of human Alzheimer’s brain autopsy samples with sucrose buffer supplemented with 1 mM EGTA and 5 mM EDTA to prepare the crude brain extracts was conducted. Protein content was then precipitated by heating in the Biorad thermal cycler to 95 °C for 20 minutes. Precipitated protein homogenates were then combined and centrifuged at 20,100 x g for 30 minutes at 4°C in an Eppendorf centrifuge, and the resulting supernatants were transferred to airfuge tubes and ultracentrifuged at 95 K for one hour. Ultrapellets containing the purified fibrils were resuspended in IX phosphate buffered saline (PBS), pH 7.4.

Negative stain grid preparation. Purified Alzheimer’s brain-derived tau fibrils were diluted 1 : 10 in PBS and incubated with EGCG or DHM ligands for 48 hours at 4°C. Negatively stained EM grids were prepared by depositing 6pl of fibril samples on formvar/carbon-coated copper grids (400 mesh) for 3 minutes with inhibitor pre-incubation times of either 0 hours (negative control) or 48 hours (positive control). The sample was rapidly and carefully removed by fast blot using filter paper without drying the grid and stained with 4% uranyl acetate for 2 minutes, then wicked dry by filter paper.

Quantitative EM (qEM) imaging. For quantitative EM image (qEM), negatively stained EM grids of each sample were screened on the JEOL 2100 TEM at a magnification of xl2,000, collecting 99 images in consistent increments. Visible fibrils were counted manually and analyzed in triplicate groups of 33 micrographs for each experimental condition. MicroED sample preparation, data collection and processing.

The DHM-TEA co-crystal was prepared for MicroED as described in ones, Jones et al., ACS Cent Sci 4, 11, 1587-1592 (2018). Around 1 mg ground powder was transferred into a lOmL scintillation vial and mixed with a carbon-coated copper grids (400-mesh, 3.05 mm O.D., Ted Pella Inc.) which was pretreated with glow-discharge plasma at 15 mA for 60 s on the negative mode using PELCO easiGlow (Ted Pella Inc.). After a gentle shaking of the vial, the grid was taken out and clipped at room temperature (see Fig. 11).

The clipped grid was loaded in an aligned Thermo Fisher Talos Arctica Cryo-TEM (200 kV, -0.0251 A) at 100 K, equipped with a CetaD CMOS camera (4096 x 4096 pixels). Screening of size-suitable microcrystals was done in imaging mode (SA 3400x). The MicroED data was collected in the diffraction mode with 829 mm diffraction length, 70pm C2 aperture and a 100pm selected area aperture in the parallel beam condition (45.2% C2 intensity) which resulted a beam size at approximately 2.5 pm. Typical data collection was performed using a constant rotation rate of -1 deg/s over an angular wedge of 130° from -65° to +65°, with Is exposure time per frame. Crystals as selected for MicroED data collection were isolated and calibrated to eucentric height to maintain the crystal inside the beam during the rotation.

The MicroED data was saved in MRC format and converted to SMV format using the mrc2smv software (https://cryoem.ucla.edu/microed). The converted frames were indexed and integrated by XDS (Kabsch et al., Acta Crystallogr D Biol Crystallogr 66, 125-132). Then two datasets were scaled and merged using XSCALE (Kabsch, et al., Acta Crystallogr D Biol Crystallogr 66, 133-144, (2010)), and the intensities were converted to SHELX hkl format using XDSCONV. The merged dataset can be ab initio solved by SHELXT (Sheldrick., et al. Acta Crystallogr A Found Adv 71, 3-8, (2015)) and refined by SHELXL (Sheldrick et al., Acta Crystallogr C Struct Chem 71, 3-8, (2015)) to yield the final MicroED structure.

Table 1. List of pH buffers.

Table 2. List of counterions and solvents used for counterion screen study.

Table 3. MicroED data processing statistics of DHM-TEA.

Stoichiometric formula C21 H27 N OH

Mr 469.44

Temperature (K) 100 Crystal system, space group Triclinic, P-1 Unit cell lengths a, b, c (A) a 8.0(10) b 11.1(10) c 12.2(10) angles a, b, g ( ° ) a 98.1(10) b 101.4(10) g 106.4(10) Cell volume (A³) 996.241 Overserved reflections (#) 10956 Unique reflections (#) 4415 Robs (%) 14.5 Rmeas (%) 18.5

CCI/2 98.9

Resolution (A) 0.74 Completeness (%) 86.7 Ri (%) 19.5

WR₂ (%) 46.6

GooF 1.562

Table 4. Chemical structure of dihydromyricetin.

Example 2. Formation of dihydromyricetin salts with improved solubility in polar solvents.

Understanding of the macrostates and microstates suggests there is a possibility of forming a salt with DHM in the pH range relevant from a gastrointestinal physiology perspective. pKa’s are calculated, however the important ones appear to be 7.05, 7.83, 9.24 using ADMET Predictor module of Simulations Plus software (Lancaster, CA), or 7.94, 8.31 from ChemDraw (PerkinElmer Informatics, Inc.). These need to be experimentally verified using a potentiometric or colorimetric titration technique.

DHM forms a stable crystal structure as witnessed by light microscopy images (needle like crystal units) and differential scanning calorimetry (DSC) trace. Several exotherms exist, while there is a need to further confirm, based on initial observations DHM may decompose without observable melting (solid->liquid phase transition not recorded).

Salt forms of DHM can be made by setting up a reaction in a solvent system which allows DHM and counterion to fully dissolve and facilitate proton transfer from DHM (the acid) to a pharmaceutically useful base. Initial example was done in ethanol (EtOH) using trolamine (triethanolamine) as a proton acceptor and DHM as a donor. A 1 :1, 1 : 1.25, and 1:2 molar ratio of DHM to trolamine were tested to optimize formation and crystallization of a putative salt. Crystals formed readily from ethanol slurry, however very rapidly with minimal cooling and EtOH evaporation. This generated a sufficiently crystalline (according to DSC trace for a putative DHM triethanolamine salt form) with an apparent melting onset at 155°C, while this needs to be confirmed by orthogonal techniques this is strong evidence that a new, ionic chemical form of DHM is formed as a triethanolamine- salt with significantly different DSC profile, e.g. melting point shift. A vapor diffusion driven crystal growth study, used water and propylene glycol mixtures to drive vapor phase water transfer from droplet to saturated osmolyte solution, generated better quality and morphology crystals for electron/x-ray diffraction analysis, which showed diffraction occurred. Higher ratios of triethanolamine are needed to be washed with chloroform or EtOH to remove excess triethanolamine.

Scheme 1 : At physiological pH, i.e. small intestinal ideally, avoid stomach/acid by enteric coat, the dissociation above will occur forming free acid form of DHM in unstirred layer (proton exchange with water, FhO⁺ and OH ).

Proposed scheme of DHM triethanolamine ionic compound production - location of H+ transfer is predicted based on macroconstants calculated in ADME Predictor module. MicroED may be able to confirm actual site of proton transfer.

Sample of this putative salt form was freely soluble in deionized water as opposed to DHM which is essentially insoluble. A quantitative estimation of aqueous solubility at various pH values needs to be confirmed which would enable compounding a wide range of doses and dosage forms for oral administration.

Additional putative salts, solid state or in situ liquid (aqueous) state: Investigate salt form and solid-state polymorph landscape of DHM:basic ionic compounds; understand phase boundaries and most stable from (in order to assess compounding/formulatability), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC); understand transitions between anhydrous and states of hydration as a function of water activity or %RH and temperature; resolve absolute structure/configuration (unit-cell) for possible polymorphs and hydrates; prediction theoretical xrpd pattern or generate experimentally; generate enantiomeric excess (e.e. >98%) as a measurement of purity, of four possible enantiomers of DHM:basic. Basic defined as a salt formed from BASE = NaOH, KOH, Ca(OHh, or more complex and chiral counterions to help define optical rotation in DHM like basic amino acids:

Other pharmaceutically acceptable basic counterions with defined optical rotation:

Hydrabamine

Other non-chiral basic counterions which are pharmaceutically acceptable or precedented:

Propose a salt screen using pharmaceutically acceptable counterions (listed basic counterions as feasible based on precedence in dosage forms and a difference in at least pKa of 2 units from DHM macroconstant prediction plot); option to perform a polymorph-&-stable form screen on potential salt hit (most stable salt, based on melting pt. and hygroscopicity) from salt counterion step, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). These could generate more than one salt product for solid dosage form manufacture; however, all should generate in situ aqueous salts that are kinetically stable for extemporaneous preparation and oral dosing. Option to resolve single crystal structure (unitcell) for possible polymorphs and hydrates with prediction of theoretical or experimental xrpd pattern. Example 3. Orally available flavonoid dihydromyricetin crystals

Fig. 12. shows a physiologically based pharmacokinetic (PBPK) model of DHM orally dosed to rats (Liu et al. Pharm Biol 55, 657-662, (2017)). The pharmacokinetic parameters of clearance (CL) and volume of distribution (Vd) in rats were derived from intravenous (IV) solution bolus injection dose study using 2mg/kg in rats by employing mechanistic deconvolution in PKPlus® module of GastroPlus® 9.8.3 (Simulations Plus, Inc, USA). IV PK curve from Liu et al., is not shown, however PKPlus® extrapolation of noncompartmental model fitting is in good agreement with published numbers for CL and Vd. Compound properties, physicochemical descriptors, formulation configuration, biorelevant solubilities, in vitro permeability, and metabolic/transporter data, were imported from GastroPlus® Checklist, where values can be found in previously published experimental data reports. The PBPK model uses an oral immediate release (IR) suspension for native DHM doses due to the low aqueous solubility and referenced methodology in Liu et al., and an oral IR solution for Isis 101 (DHM:TEA cocrystal) due to the significantly higher solubility of the cocrystal. Graph shows a comparison of Isis 101 simulated (i.e. Calc) oral exposure from a 5mg total dose vs observed values, Liu et al., of native DHM in rats. Isis 101 predicted Cmax and AUC are approximately 119 and 65 times, resp., higher than those observed in rats. Table 5 display Obs and Calc pharmacokinetic result summaries for the Liu et al. publication vs GastroPlus® model predictions for native DHM vs Isis 101, resp. Results: Cmax Is the maximum concentration of DHM in the blood. For the free DHM: 20.73 ng/ml; and DHM+TEA co-crystal: 2,476.3 ng/ml (-1 19 times higher). T_max is the time it takes to reach Cmax. For free DHM: 2 hours; for DHM+TEA co-crystal: 1.56 hours. AUC (area under the curve) refers to the bioavailability of a compound/drug for a given dose. For free DHM: 166.5 ng/ml; for DHM+TEA co-crystal: 10,860 ng/ml-hour (-65 times higher).

Fig. 13 shows Parameter sensitivity analysis (PSA) performed in GastroPlus® 9.8.3. (Simulations Plus, Inc., USA). The independent parameter is the dose, and the dependent parameter is the fraction of the initial PO dose absorbed (%Fa). Plots are constructed from twenty distinct %Fa calculations using different PK simulations at doses that bracket the shown baseline dose of DHM at either 5mg or 25mg, ten- fold below and ten-fold above with ten doses in each range. For example, 0.5mg to 5mg with ten doses in between, and 5mg-500mg with ten doses in between, is used for the 5mg Liu et al. rat data PBPK model. While the native DHM %Fa decreases from 0.5-500mg, in a sigmoidal manner, the Isis 101 %Fa is not sensitive to dose from 0.5~100mg (seen as a flat line). From 100mg-500mg there is a <15% change in %Fa with Isis 101. Results summary: Absorption of 0.5-100mg oral dose range of DHM+TEA co- crystal is continuous compared to free DHM. ~100-500mg oral dose range of DHM+TEA cocrystal demonstrates <15% change in absorption compared to free DHM. A lot of free DHM is being wasted at higher doses (above 2mg) whereas DHM+TEA co-crystal maintains near constant absorption (up to -lOOrng).

Table 5. Pharmacokinetic summaries for the Liu et al. publication vs GastroPlus® model predictions for native DHM vs Isis 101 (DHM + TEA co-crystal).

Fig. 14 shows the relative oral bioavailability of free DHM versus DHM + triethanolamine co-crystals. Relative (oral) Bioavailability: Is used to compare the amount of compound/drug from a novel formulation (DHM+TEA) that is absorbed into systemic circulation, relative to another reference formulation (free DHM). These histograms show the oral bioavailability of the DHM+TEA co-crystals, relative to free DHM at a dose range of 0.5- 500mg (see Table 6).

Table 6. Relative oral bioavailability of free DHM versus DHM + triethanolamine co-crystals.

Fig. 15-17 show X-ray power diffractogram of DHM + triethanolamine co-crystals, DHM + lysine co-crystals, and DHM + Ca(OH) co-crystals, respectively. Example 4. Mechanistic modeling and GastroPlus® simulation software

GastroPlus® software is a validated method used to create physiologically based pharmacokinetic (PBPK) models to assist in formulation development and regulatory evaluations by the FDA. We imported compound properties, physicochemical descriptors, formulation configuration, biorelevant solubilities, in vitro permeability, and metabolic/transporter data for free DHM and DHM+TEA co-crystals into GastroPlus®. Using data collected from published reports, we produced a pharmacokinetic curve that compares the bioavailability of free DHM and DHM+TEA co-crystals and another curve that compares the % fraction absorbed following oral administration of free DHM vs. DHM+TEA co-crystals.

Figure 18 shows physiologically based pharmacokinetic (PBPK) model of DHM orally dosed to rats (Tong et al. J Pharm Biomed Anal, 2015. 114: p. 455-61.). The pharmacokinetic parameters of clearance (CL) and volume of distribution (Vd) in rats were derived from intravenous (IV) solution bolus injection dose study using 2mg/kg in rats by employing mechanistic deconvolution in PKPlus® module of GlastroPlus® 9.8.3 (Simulations Plus, Inc, USA). IV PK curve from Liu et al., is not shown, however PKPlus® extrapolation of noncompartmental model fitting is in good agreement with published numbers for CL and Vd. Compound properties, physicochemical descriptors, formulation configuration, biorelevant solubilities, in vitro permeability, and metabolic/transporter data, were imported from GastroPlus Checklist where values can be found in previously published experimental data reports. The PBPK model uses an oral immediate release (IR) suspension for native DHM doses due to the low aqueous solubility and referenced methodology in Liu et al., and an oral IR solution for Isis 101 (DHM:TEA cocrystal) due to the significantly higher solubility of the cocrystal. (A) Shows a comparison of Isis 101 simulated (i.e. Calc) oral exposure from a 25mg total dose vs observed values, Tong et al., of native DHM in rats. Isis 101 predicted Cmax and AUC are approximately 20 and 27 times, resp., higher than those observed in rats. (B) Shows an overlay of the GastroPlus® (i.e. Calc) vs Tong at al. (i.e. Obs) dosed orally at 25mg total dose of native DHM PBPK model in rats, having poor confidence in accurately capturing the initial absorption intensity and extent of it at this higher dose (*at higher oral doses, in vivo, there may be enzyme/transporter saturation occurring or delayed absorption, that result in a discrepancy with PBPK model). Also, there is a lack of rat PO dosage from detail in Tong et al. (e.g. DHM particle size and form data), and an IR Suspension for DHM was assumed. Result summaries for the Tong et al. publication vs GastroPlus® model predictions for native DHM vs Isis 101, resp: Observed (20mg/kg):C_max (ng/mL): 20.73; T_max (h): 2; AUCo-<» (ng/mL -h): 166.5. Calculated/Optimized 20mg/kg): Cmax (ng/mL): 25.11; T_max (h): 2.56; AUCo ® (ng/mL •h): 130.2. Calculated Isis 101 : Cmax (ng/mL): 2476.3; T_max (h): 1.56; AUCo-» (ng/mL -h): 1.086 x 10⁴.

Fig. 19 shows physiologically based pharmacokinetic (PBPK) model of DHM orally dosed to rats (Tong et al.). The pharmacokinetic parameters of clearance (CL) and volume of distribution (Vd) in rats were derived from intravenous (IV) solution bolus injection dose study using 2mg/kg in rats by employing mechanistic deconvolution in PKPlus® module of GlastroPlus® 9.8.3 (Simulations Plus, Inc, USA). IV PK curve from Liu et al., is not shown, however PKPlus® extrapolation of noncompartmental model fitting is in good agreement with published numbers for CL and Vd. Compound properties, physicochemical descriptors, formulation configuration, biorelevant solubilities, in vitro permeability, and metabolic/transporter data, were imported from GastroPlus Checklist, where values can be found in previously published experimental data reports. The PBPK model uses an oral immediate release (IR) suspension for native DHM doses due to the low aqueous solubility and referenced methodology in Liu et al., and an oral IR solution for Isis 101 (DHM:TEA cocrystal) due to the significantly higher solubility of the cocrystal. Observed (lOOmg/kg): Cmax (ng/mL): 90.5; Tmax (h): 0.5; AUCo-® (ng/mL h): 530.2. Calculated/Optimized (lOOmg/kg): Cmax (ng/mL): 85.757; Tmax (h): 1.76; AUCo-«. (ng/mL -h): 397.26. Calculated Isis 101 : Cmax (ng/mL): 2476.3; Tmax (h): 1.56; AUCo-<» (ng/mL -h): 1.086 x 10⁴.

Fig. 20A-F shows three stage global parameter sensitivity analysis (PSA) performed in GastroPlus® 9.8.3. (Simulations Plus, Inc., USA). The independent parameter is the dose, and the dependent parameters evaluated are the AUC, Cmax, and fraction of the initial PO dose absorbed (%Fa). Plots are constructed from ten distinct AUC, Cmax, and %Fa calculations using different PK simulations at doses that bracket the shown baseline dose of DHM at either 5mg or 25mg, ten-fold below and ten-fold above with five doses in each range. For example, 0.5mg to 5mg with four doses in between, and 5mg-500mg with four doses in between, is used for the 5mg Liu et al. rat data PBPK model.

Fig. 21 shows parameter sensitivity analysis (PSA) performed in GastroPlus® 9.8.3. (Simulations Plus, Inc., USA). The independent parameter is the dose, and the dependent parameter is the fraction of the initial PO dose absorbed (%Fa). Plots are constructed from twenty distinct %Fa calculations using different PK simulations at doses that bracket the shown baseline dose of DHM at either 5mg or 25mg, ten- fold below and ten-fold above with ten doses in each range. For example, 0.5mg to 5mg with ten doses in between, and 5mg-500mg with ten doses in between, is used for the 5mg Liu et al. rat data PBPK model. While the native DHM %Fa decreases from 0.5-500mg, in a sigmoidal manner, the Isis 101 %Fa is not sensitive to dose from 0.5~100mg (seen as a flat line). From 100mg-500mg there is a <15% change in %Fa with Isis 101.

Example 5. Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of a compound of the DHM cocrystal described herein, a composition or a compound of DHM co-crystal specifically disclosed herein (hereinafter referred to as “Composition X’ or 'Compound X'):

(i) Tablet 1 mg/tablet

'Compound X' 100.0

Lactose 77.5

Povidone 15.0

Croscarmellose sodium 12.0

Microcrystalline cellulose 92.5

Magnesium stearate 3.0

300.0

(ii) Tablet 2 mg/tablet

'Compound X' 20.0

Microcrystalline cellulose 410.0

Starch 50.0

Sodium starch glycolate 15.0

Magnesium stearate 5.0

500.0

(iii) Capsule mg/capsule

'Compound X' 10.0

Colloidal silicon dioxide 1.5

Lactose 465.5

Pregelatinized starch 120.0

Magnesium stearate 3.0

600.0

(iv) Iniection 1 (1 mg/mL) mg/mL

'Compound X' (free acid form) 1.0

Dibasic sodium phosphate 12.0

Monobasic sodium phosphate 0.7

Sodium chloride 4.5

1.0 N Sodium hydroxide solution q.s.

(pH adjustment to 7.0-7.5)

Water for injection q.s. ad 1 mL (v) Injection 2 (10 mg/mL) mg/mL

'Compound X' (free acid form) 10.0

Monobasic sodium phosphate 0.3

Dibasic sodium phosphate 1.1

Polyethylene glycol 400 200.0

0.1 N Sodium hydroxide solution q.s.

(pH adjustment to 7.0-7.5)

Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can

'Compound X' 20

Oleic acid 10

Trichloro monofluoromethane 5,000

Dichlorodifluoromethane 10,000

Dichlorotetrafluoroethane 5,000

(vii) Topical Gel 1 wt.%

'Composition X' 5%

Carbomer 934 1.25%

Triethanolamine q.s.

(pH adjustment to 5-7)

Methyl paraben 0.2%

Purified water q.s. to 100g

(viii) Topical Gel 2 wt.%

'Composition X' 5%

Methylcellulose 2%

Methyl paraben 0.2%

Propyl paraben 0.02%

Purified water q.s. to 100g

(ix) Topical Ointment wt.%

'Composition X' 5%

Propylene glycol 1 %

Anhydrous ointment base 40%

Polysorbate 80 2%

Methyl paraben 0.2%

Purified water q.s. to 100g

(x) Topical Cream 1 wt.%

'Composition X' 5%

White bees wax 10%

Liquid paraffin 30%

Benzyl alcohol 5%

Purified water q.s. to 100g (xi) Topical Cream 2 wt.%

'Composition X' 5%

Stearic acid 10%

Glyceryl monostearate 3%

Polyoxyethylene stearyl ether 3%

Sorbitol 5%

Isopropyl palmitate 2 %

Methyl Paraben 0.2%

Purified water q.s. to 100g

These formulations may be prepared by conventional procedures well known in the pharmaceutical art. It will be appreciated that the above pharmaceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Compound X’. Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest.

While specific embodiments have been described above with reference to the disclosed embodiments and examples, such embodiments are only illustrative and do not limit the scope of the invention. Changes and modifications can be made in accordance with ordinary skill in the art without departing from the invention in its broader aspects as defined in the following claims.

All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. No limitations inconsistent with this disclosure are to be understood therefrom. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

What is claimed is:

1. A co-crystal comprising dihydromyricetin (DHM); and a crystal coformer selected from the group consisting of triethanolamine, 2-amino-2- (hydroxymethyl)-l,3-propanediol (Tris-Base), sodium hydroxide, calcium hydroxide, and an amino acid, wherein the co-crystal comprises a crystal lattice having a void volume of about 75% to about 90%, a unit cell volume of about 900 A³ to about 1200 A³, and a Matthews Coefficient (VM) of about 1.5 A³/Da to about 2.5 A³/Da.

2. The co-crystal of claim 1, wherein the amino acid is lysine or arginine.

3. The co-crystal of claim 1, wherein the crystal coformer is triethanolamine or Ca²⁺.

4. The co-crystal of claim 3, wherein the crystal coformer is triethanolamine.

5. The co-crystal of claim 4, wherein the co-crystal comprises a crystalline lattice of DHM-triethanolamine having a void volume of about 80% to about 85%.

6. The co-crystal of claim 4, wherein the unit cell volume is about 950 A to about 1000 A³ and the Matthews Coefficient (VM) of about 1.9 A³/Da to about 2.2 A³/Da.

7. The co-crystal of claim 6, wherein the unit cell volume is about 971.5 A³ and the Matthews Coefficient (VM) is about 2.07 A³/Da.

8. The co-crystal of claim 1 , wherein the co-crystal of DHM is about 5 times to about 10 times more soluble in a polar solvent compared to the solubility of DHM in the polar solvent.

9. The co-crystal of claim 1, wherein the ratio of the DHM to the crystal coformer is about 1: 1 to about 1 :5.

10. The co-crystal of claim 9, wherein the ratio of the DHM to the crystal coformer is about 1: 1 to about 1 :3.

11. The co-crystal of claim 10, wherein the ratio of the DHM to the crystal coformer is about 1: 1 to about 1 :1.25.

12. A composition comprising the co-crystal of claim 1 and a pharmaceutically acceptable carrier or excipient.

13. A method of treating a disease comprising administering an effective amount of a co-crystal of dihydromyricetin (DHM) to a subject in need thereof, wherein the cocrystal of DHM comprises the DHM and a crystal coformer selected from the group consisting of triethanolamine, 2-amino-2-(hydroxymethyl)-l, 3-propanediol, sodium hydroxide, calcium hydroxide, and an amino acid; wherein the co-crystal of DHM treats the disease.

14. The method of claim 13, wherein the disease comprises one or more of alcohol use related disorders, age related diseases, or oxidative stress induced by one or more of disease, age, drug use, and environment, or poisoning, or liver damage, or an inflammatory disease, or a neurological disease.

15. The method of claim 14, wherein the neurological disease is Alzheimer’s Disease.

16. The method of claim 13, wherein the crystal coformer is triethanolamine.

17. The method of claim 13, further comprising a second therapeutic agent, wherein the second therapeutic agent comprises epigallocatechin gallate.

18. The method of claim 13, wherein the bioavailability of the co-crystal of DHM is about 5 times to about 10 times greater in a polar solvent compared to the bioavailability of DHM in the polar solvent.