ʟ-ergothioneine cocrystal and derivatives

Abstract

The present disclosure is directed to ʟ‑Ergothioneine hemiascorbic acid, a cocrystal monohydrate form, and processes for the preparation of the cocrystal monohydrate form.

Description

Attorney Docket No. 110798.00057L-ERGOTHIONEINE COCRYSTAL AND DERIVATIVES CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 742,037 filed January 6, 2025, the entirety of which is herein incorporated for all purposes.FIELD

[0002] The present disclosure is directed to L-Ergothioneine hemiascorbic acid, a cocrystal monohydrate form, and processes for the preparation of the cocrystal monohydrate form.BACKGROUND

[0003] L-Ergothioneine is a naturally occurring thio-histidine betaine amino acid. The compound is only synthesized by certain microbes, including bacteria Actinomycetota and Cyanobacteria, and certain fungi. Human and animals must obtain L-Ergothioneine exclusively through diet, for example in kidney beans, black beans, oat bran, and mushrooms.L-Ergothioneine

[0004] Although humans do not produce L-Ergothioneine, it is reported to have antioxidant activity and cytoprotective properties. In fact, humans have evolved a selective transporter for L-Ergothioneine, solute carrier family 22, member 4 (SLC22A4). Once consumed, L-Ergothioneine accumulates in tissues high in SLC22A4, including erythrocytes, bone marrow, the liver, the kidney, and eyes.

[0005] There are multiple lines of evidence that L-Ergothioneine acts as a potent antioxidant and cellular protectant (Borodina et al. Nutr Res Rev 2020, 33: 190). For example, L-Ergothioneine has been shown to decrease oxidative stress levels in the liver and kidneys of rats (Deiana et al. Clin. Nutr. , 2004. 23 : 183). It has also been studied as an antioxidant in certain disease states, including acute respiratory distress (Repine et al. PrevMed 2012, 54: S79) and chronic obstructive pulmonary disease (COPD) (Rahman, Biochim Biophys Acta, 2012, 1822: 714). Its antioxidant properties also make it common ingredient in cosmetics for protection against oxidative stress.1110638946211 \AMERICASAttorney Docket No. 110798.00057

[0006] Typically, L-Ergothioneine is produced through biological extraction or chemical synthesis. A crystal form of L-Ergothioneine dihydrate was first reported in Sugihara et al. (Acta Cryst, 1976, B32: 181). In 2005, an anhydrate crystalline form of L-Ergothioneine was also reported (Hand et al. J. Nat. Prod. 2005, 68: 293).

[0007] Given the importance of L-Ergothioneine in the fields of food, medicine, and cosmetics, it would be beneficial to develop highly purified and stable forms of L-Ergothioneine that can be easily manufactured and formulated.SUMMARY

[0008] Provided herein is L-Ergothioneine hemiascorbic acid. The ascorbic acid of the L-Ergothioneine hemiascorbic acid can be L-ascorbic acid or D-ascorbic acid. In a preferred embodiment, the ascorbic acid is L-ascorbic acid.

[0009] In one embodiment, the L-Ergothioneine hemiascorbic acid is crystalline. In one embodiment, the L-Ergothioneine hemiascorbic acid is the L-Ergothioneine hemiascorbic acid (HACC) cocrystal monohy drate described herein. In one embodiment, the L-Ergothioneine hemiascorbic acid is L-Ergothioneine L-hemiascorbic acid (HACC) cocrystal monohydrate. Also provided herein is a method for the synthesis of L-Ergothioneine HACC cocrystal monohydrate.

[0010] In another aspect, provided herein are pharmaceutical compositions comprising L-Ergothioneine hemiascorbic acid. In one embodiment, the pharmaceutical composition comprises a crystalline form of L-Ergothioneine, including the L-Ergothioneine HACC cocrystal monohydrate described herein.

[0011] Also provided herein is a method for treating diseases or disorders caused by oxidative stress and / or inflammation comprising administering L-Ergothioneine hemiascorbic acid or a crystalline form of L-Ergothioneine, including the L-Ergothioneine HACC cocrystal monohydrate described herein.BRIEF DESCRIPTION OF FIGURES

[0012] FIG. 1 are XRPD patterns from select crystallization experiments that generated L-Ergothioneine HACC cocrystal monohydrate.

[0013] FIG. 2 is the XRPD pattern of L-Ergothioneine HACC cocrystal monohydrate.21106389462\1\AMERICASAttorney Docket No. 110798.00057

[0014] FIG. 3 is an atomic displacement ellipsoid diagram of L-Ergothioneine HACC cocrystal monohydrate.

[0015] FIG. 4 is a partial representation of the unit cell for L-Ergothioneine HACC cocrystal monohydrate that illustrates the 1:1 symmetry-imposed disorder of the ascorbic acid molecule with two water molecules.

[0016] FIG. 5 A is the packing diagram of L-Ergothioneine HACC cocrystal monohydrate viewed along the a crystallographic axis.

[0017] FIG. 5B is the packing diagram of L-Ergothioneine HACC cocrystal monohydrate viewed along the b cry stallographic axis.

[0018] FIG. 6 is the partial representation of the unit cell for L-Ergothioneine HACC cocrystal monohydrate that illustrates the alternating layers along the c crystallographic axis.

[0019] FIG. 7 is a comparison of the calculated and experimental L-Ergothioneine HACC cocrystal monohydrate XRPD patterns.

[0020] FIG. 8A is the DSC graph of L-Ergothioneine HACC cocrystal monohydrate.

[0021] FIG. 8B is the TGA graph of L-Ergothioneine HACC cocrystal monohydrate.

[0022] FIG. 9 is the1HNMR of L-Ergothioneine HACC cocrystal monohydrate.

[0023] FIG. 10 is a comparison of L-Ergothioneine HACC cocrystal monohydrate XRPD patterns showing a decrease in the intensity of the spurious peaks during analysis.DETAILED DESCRIPTION

[0024] Disclosed herein is L-Ergothioneine hemiascorbic acid. In one embodiment, the L-Ergothioneine hemiascorbic acid is crystalline. In one embodiment, the L-Ergothioneine hemiascorbic acid is the L-Ergothioneine hemiascorbic acid (HACC) cocrystal monohydrate described herein. In one embodiment, the L-Ergothioneine hemiascorbic acid isL-Ergothioneine L-hemiascorbic acid (HACC) cocrystal monohydrate. Also provided herein is a method for the synthesis of L-Ergothioneine HACC cocrystal monohydrate.

[0025] The present disclosure provides at least the following embodiments:a) L-Ergothioneine hemiascorbic acid;b) L-Ergothioneine hemiascorbic acid of (a) wherein the L-Ergothioneine hemiascorbic acid is crystalline;c) L-Ergothioneine hemiascorbic acid (HACC) cocrystal monohydrate;31106389462\1\AMERICASAttorney Docket No. 110798.00057d) the L-Ergothioneine hemiascorbic acid of (a), (b), or (c) wherein the ascorbic acid is L-ascorbic acid;e) A pharmaceutical composition comprising any one of (a)-(d);f) A method to treat a disease or disorder caused by oxidative stress and / or inflammation comprising administering to a patient in need thereof any one of (a)-(e);g) A method for the synthesis of L-Ergothioneine HACC cocrystal monohydrate comprising (a) mixing L-Ergothioneine and ascorbic acid in an organic solvent / water mixture and (b) allowing the organic solvent / water mixture to slowly evaporate to afford L-Ergothioneine HACC cocrystal monohydrate;h) The method of (g) wherein the ascorbic acid is L-ascorbic acid;i) The method of (g) or (h) wherein the organic solvent in step (a) is acetone and the ratio of acetone to water is about 3:1 (v / v);j) The method of any one of (g)-(i) further comprising (c) slurrying the L-Ergothioneine HACC cocrystal monohydrate from step (b) in an organic solvent / water mixture and additional ascorbic acid; (d) stirring the slurry; and (e) recovering the solids of the slurry to afford L-Ergothioneine HACC cocrystal monohydrate;k) The method of (j) wherein the additional ascorbic acid is L-ascorbic acid; and l) The method of (j) or (k) wherein the organic solvent in step (c) is ethanol and the ratio of ethanol to water is about 4: 1 (v / v).

[0026] Definitions

[0027] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures know n in the art that are described or referenced herein are generally w ell understood and commonly employed using conventional methodologies by those skilled in the art.

[0028] As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise.

[0029] As used herein, the term “about” refers to the stated value plus or minus 10%, plus or minus 5%, or plus or minus 1%. For example, a value of “about 10” can encompass a41106389462\1\AMERICASAttorney Docket No. 110798.00057range of 9 to 11. For logarithmic scales, the term “about” refers to the stated value plus or minus 0.3 log units, or plus or minus 0.2 log units, or plus or minus 0.1 log units. For example, a value of “about pH 4.6” can encompass a pH range of 4.5-4.7.

[0030] The term “substantially free of or “substantially in the absence of’ with respect to a composition refers to a composition that includes at least about 85 or 90% by weight, in certain embodiments at least about 95%, 98 %, 99% or 100% by weight, of a designated enantiomer or stereoisomer of a compound. For example, “substantially free of’ or “substantially in the absence of’ with respect to a composition can refer to a composition that includes about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% by weight of a designated enantiomer or stereoisomer of a compound. In certain embodiments, in the methods and compounds provided herein, the compounds are substantially free of other enantiomers or stereoisomers.

[0031] Similarly, the term “isolated” with respect to a composition refers to a composition that includes at least 85%, 90%, 95%, 98%, or 99% to 100% by weight, of a designated compound, enantiomer, or stereoisomer, the remainder comprising other chemical species, enantiomers, or stereoisomers. For example, “isolated” with respect to a composition can refer to a composition that includes about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% by weight of a designated compound, enantiomer, or stereoisomer, the remainder comprising other chemical species, enantiomers, or stereoisomers.

[0032] As used herein, the terms “subject” and “patient” are used interchangeably herein. The terms “subject” and “subjects” refer to an animal, such as a mammal including a nonprimate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey such as a cynomolgous monkey, a chimpanzee and a human), and for example, a human. In another embodiment, the subject is a farm animal e.g., a horse, a cow, a pig. etc.) or a pet (e.g, a dog or a cat). In certain embodiments, the subject is a human.

[0033] “Therapeutically effective amount” refers to an amount of a form or composition that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. A “therapeutically effective amount” can vary depending on, inter alia, the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated.51106389462\1\AMERICASAttorney Docket No. 110798.00057

[0034] “Treating” or “treatment” of any disease or disorder refers, in certain embodiments, to ameliorating a disease or disorder that exists in a subject. In another embodiment, “treating” or “treatment” includes ameliorating at least one physical parameter, which may be indiscernible by the subject. In yet another embodiment, “treating” or “treatment” includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. In yet another embodiment, “treating” or “treatment” includes delaying the onset of the disease or disorder.

[0035] As used herein, the terms “prophylactic agent” and “prophylactic agents” as used refer to any agent(s) which can be used in the prevention of a disorder or one or more symptoms thereof. In certain embodiments, the term “prophylactic agent” includes a form provided herein. In certain other embodiments, the term “prophylactic agent” does not refer a form provided herein. For example, a prophylactic agent is an agent which is known to be useful for, or has been or is currently being used to prevent or impede the onset, development, progression and / or severity of a disorder.

[0036] As used herein, the phrase “prophylactically effective amount” refers to the amount of a therapy (e.g., prophylactic agent) which is sufficient to result in the prevention or reduction of the development, recurrence or onset of one or more symptoms associated with a disorder (or to enhance or improve the prophylactic effect(s) of another therapy (e.g., another prophylactic agent).

[0037] L-Ergothioneine hemiascorbic acid

[0038] Provided herein is L-Ergothioneine hemiascorbic acid. In one embodiment, the ascorbic acid of L-Ergothioneine hemiascorbic acid is L-ascorbic acid. In one embodiment, theL-Ergothioneine hemiascorbic acid is crystalline. In one embodiment, the crystalline form is L-Ergothioneine hemiascorbic acid (HACC) cocrystal monohydrate. In one embodiment, the crystalline form is L-Ergothioneine L-hemi ascorbic acid (HACC) cocrystal monohydrate.

[0039] In one embodiment, L-Ergothioneine HACC cocrystal monohydrate is characterized by an X-ray pow der diffraction (XRPD) pattern substantially similar to that set forth in FIG. 2. In one embodiment, L-Ergothioneine HACC cocrystal monohydrate is characterized by an X-ray powder diffraction (XRPD) pattern comprising peaks substantially61106389462\1\AMERICASAttorney Docket No. 110798.00057similar to those set forth in Table 4. In certain embodiments, L-Ergothioneine HACC cocrystal monohydrate is characterized by an XRPD pattern comprising:a) 20 values of about 4.83, 7.39, 14.45, 15.50, 19.14, 19.38, 19.97, 20.68, 21.66. 23.05, 24.24, 26.25. 27.31, and 29.28 + 0.2° 20;b) 20 values including at least or selected from about 4.83, 7.39, 14.45, 15.50, 19.14, 19.38, 19.97, 20.68, 21.66, 23.05, 24.24, 26.25, 27.31, and 29.28 ± 0.2° 20;c) at least two, three, four, or five 20 values selected from about 4.83, 7.39, 14.45, 15.50, 19.14, 19.38, 19.97, 20.68. 21.66, 23.05. 24.24, 26.25, 27.31, and 29.28 + 0.2° 20; d) at least six, seven, or eight 20 values selected from about 4.83, 7.39, 14.45, 15.50, 19.14, 19.38, 19.97, 20.68, 21.66, 23.05, 24.24, 26.25, 27.31, and 29.28 + 0.2° 20;e) 20 values of about 4.83, 19.14, 19.38, 20.68, 23.05, and 24.24 + 0.2° 20;f) 20 values including at least or selected from about 4.83, 19.14. 19.38, 20.68. 23.05, and 24.24 ± 0.2° 20;g) 20 values of about 19.14, 19.38, 20.68, and 24.24 + 0.2° 20;h) 20 values including at least or selected from about 19.14, 19.38, 20.68, and 24.24 + 0.2° 20;i) 20 values of about 19.14. 20.68, and 24.24 ± 0.2° 20;j) 20 values including at least or selected from about 19.14, 20.68, and 24.24 + 0.2° 20; k) 20 values of about 4.83, 7.39, 14.45, 15.50, 16.41, 19.97, 20.68, and 21.66 ± 0.2° 20;orl) 20 values including at least or selected from about 4.83, 7.39, 14.45, 15.50, 16.41, 19.97, 20.68. and 21.66 ± 0.2° 20.

[0040] In one embodiment, L-Ergothioneine HACC cocrystal monohydrate is characterized by a differential scanning calorimetry (DSC) thermograph having two endotherms wherein the first has an endotherm maximum between about 85 °C and 95 °C and wherein the second has an endotherm maximum between about 215 °C and 225 °C. In one embodiment, L-Ergothioneine HACC cocrystal monohydrate is characterized by a differential scanning calorimetry (DSC) thermograph having two endotherms wherein the first has an endotherm maximum at about 88 °C and wherein the second has an endotherm maximum at about 217 °C. In one embodiment, L-Ergothioneine HACC cocrystal monohydrate is characterized by a DSC thermograph substantially similar to that set forth in FIG. 8A.71106389462\1\AMERICASAttorney Docket No. 110798.00057

[0041] In one embodiment, L-Ergothioneine HACC cocrystal monohydrate is characterized by a weight loss in the range of about 1% to 5% when heated to about 150 °C, an additional weight loss in the range of about 1% to 5% when heated from about 150 °C to 175 °C, and an additional weight loss in the range of about 1% to 5% when heated from about 175 °C to 200 °C in a thermogravimetric analysis (TGA). In one embodiment, L-Ergothioneine HACC cocrystal monohydrate is characterized by a weight loss of about 4.7% when heated to about 150 °C, an additional weight loss in the range of about 2% when heated from about 150 °C to 175 °C, and an additional weight loss in the range of about 4.8% when heated from about 175 °C to 200 °C in a thermogravimetric analysis (TGA). In one embodiment, L-Ergothioneine HACC cocrystal monohydrate is characterized by a TGA substantially similar to that set forth in FIG. 8B.

[0042] “Substantially’' when describing XRPD patterns is meant that the reported peaks can vary by + 0.2°. In certain embodiments, the reported peaks vary by + 0.1°.

[0043] “Substantially” when describing differential scanning calorimetry (DSC) thermographs and a thermogravimetric analysis (TGA) is meant that the reported temperatures can vary by + 0.5 ° C.

[0044] L-Ergothioneine HACC cocrystal monohydrate can be synthesized as described in Example 1 and Example 4. In certain embodiments, L-Ergothioneine HACC cocrystal monohydrate is synthesized by a method comprising: (a) mixing L-Ergothioneine and ascorbic acid in an organic solvent / water mixture and (b) allowing the organic solvent / water mixture to slowly evaporate to afford L-Ergothioneine HACC cocrystal monohydrate.

[0045] In a preferred embodiment, the ascorbic acid of step (a) is L-ascorbic acid. In an alternative embodiment, the ascorbic acid of step (a) is n-ascorbic acid. In an alternative embodiment, the ascorbic acid of step (a) is a mixture of L-ascorbic acid and D-ascorbic acid.

[0046] In certain embodiments, the method further comprises: (c) slurrying theL-Ergothioneine HACC cocrystal monohydrate from step (b) in an organic solvent / water mixture and additional ascorbic acid; (d) stirring the slurry: and (e) recovering the solids of the slurry.

[0047] In a preferred embodiment, the ascorbic acid of step (c) is L-ascorbic acid. In an alternative embodiment, the ascorbic acid of step (c) is D-ascorbic acid. In an alternative embodiment, the ascorbic acid of step (c) is a mixture of L-ascorbic acid and D-ascorbic acid.81106389462\1\AMERICASAttorney Docket No. 110798.00057

[0048] In certain embodiments, the organic solvent is independently selected at each occurrence from the group consisting of methanol, ethanol, acetone, and acetonitrile.

[0049] In certain embodiments, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 3: 1 to 1: 1. In certain embodiments, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 3: 1. In certain embodiments, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 2.5:1. In certain embodiments, the molar ratio ofL-Ergothioneine to ascorbic acid in step (a) is about 2:1. In certain embodiments, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 1.5:1.

[0050] In certain embodiments, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 1: 1 to 1:2. In one embodiment, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 1 : 1 to 1.6. In one embodiment, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 1:1 to 1.5. In one embodiment, the molar ratio ofL-Ergothioneine to ascorbic acid in step (a) is about 1:1 to 1.3. In one embodiment, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 1: 1. In one embodiment, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 1 :2. In one embodiment, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 1:3. In one embodiment, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 1 :4. In one embodiment, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 1:5. In one embodiment, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 1 :6.

[0051] In one embodiment, L-Ergothioneine and ascorbic acid are mixed in an organic solvent / water mixture at room temperature. In one embodiment, L-Ergothioneine and ascorbic acid are mixed in an organic solvent / water mixture and the organic / solvent water mixture is heated to about 35 °C, about 40 °C, about 45 °C, about 50 °C, or about 55 °C.

[0052] In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 3: 1 to 10: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 3: 1 to 6: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 6: 1 to 9: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 9: 1 to 10: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture is about 3:1 to 3:2 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 3: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 3:2 (v / v). In one91106389462\1\AMERICASAttorney Docket No. 110798.00057embodiment, the ratio of the organic solvent to water mixture in step (a) is about 4:1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture is about 6: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 8: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 9: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 10:1 (v / v).

[0053] In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 70-85% organic solvent / 30-15% water (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 70-75% organic solvent / 30-25% water (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 70-80% organic solvent / 30-20% water (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 80-85% organic solvent / 20-15% water (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 70% organic solvent / 30% water (v / v). In one embodiment, the ratio of the organic solvent to water mixture is about 72% organic solvent / 28% water (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 80% organic solvent / 20% water (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (a) is about 81% organic solvent / 19% water (v / v).

[0054] In one embodiment, the organic solvent in step (a) is acetone and the ratio of acetone to water is about 3:2 to 3:1 (v / v). In one embodiment, the organic solvent in step (a) is acetone and the ratio of acetone to water is about 3:2 (v / v). In one embodiment, the organic solvent in step (a) is acetone and the ratio of acetone to water is about 3:1 (v / v).

[0055] In one embodiment, the organic solvent in step (a) is acetonitrile and the ratio of acetonitrile to water is about 75-85% / 25-15% water (v / v). In one embodiment, the organic solvent in step (a) is acetonitrile and the ratio of acetonitrile to water is about 75-80% / 25-20% water (v / v). In one embodiment, the organic solvent in step (a) is acetonitrile and the ratio of acetonitrile to water is about 81 % / l 9% water (v / v).

[0056] In one embodiment, the organic solvent in step (a) is methanol and the ratio of methanol to water is about 10:1 to 8:1 (v / v). In one embodiment, the organic solvent in step (a) is methanol and the ratio of methanol to water is about 9: 1 (v / v).

[0057] In one embodiment, the organic solvent in step (a) is ethanol and the ratio of ethanol to water is about 70-80% / 30-20% water (v / v). In one embodiment, the organic101106389462\1\AMERICASAttorney Docket No. 110798.00057solvent in step (a) is ethanol and the ratio of ethanol to water is about 70-75% / 30-25% water (v / v). In one embodiment, the organic solvent in step (a) is ethanol and the ratio of ethanol to water is about 75% / 25% water (v / v).

[0058] In one embodiment, the concentration of L-Ergothioneine in the organic solvent / water mixture of step (a) is between about 0.01 M and 0.07 M. In one embodiment, the concentration of L-Ergothioneine in the organic solvent / water mixture of step (a) is between about 0.01 M and 0.05 M. In one embodiment, the concentration of L-Ergothioneine in the organic solvent / water mixture of step (a) is between about 0.01 M and 0.04 M. In one embodiment, the concentration of L-Ergothioneine in the organic solvent / water mixture of step (a) is between about 0.01 M and 0.03 M. In one embodiment, the concentration of L-Ergothioneine in the organic solvent / water mixture of step (a) is between about 0.01 M and 0.02 M. In one embodiment, the concentration of L-Ergothioneine in the organic solvent / water mixture of step (a) is about 0.065 M. In one embodiment, the concentration of L-Ergothioneine in the organic solvent / water mixture of step (a) is about 0.045 M. In one embodiment, the concentration of L-Ergothioneine in the organic solvent / water mixture of step (a) is about 0.035 M. In one embodiment, the concentration of L-Ergothioneine in the organic solvent / water mixture of step (a) is about 0.01 M.

[0059] In one embodiment, the solvent / water mixture is evaporated in step (b) at room temperature for at least one day. at least two days, at least three days, at least four days, at least five days, or more.

[0060] In one embodiment, the solvent / water mixture is evaporated in step (b) at about 32 °C for at least one day, at least two days, at least three days, at least four days, at least five days, or more.

[0061] In one embodiment, the solvent / water mixture is evaporated in step (b) at room temperature for at least one day, at least two days, at least three days, at least four days, at least five days, or more and at about 32 °C for at least one day.

[0062] In one embodiment, the ratio of the organic solvent to water mixture in step (c) is about 3: 1 to 10: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (c) is about 3:1 to 6: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (c) is about 6: 1 to 9: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (c) is about 9: 1 to 10: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (c) is about 3: 1 to 3:2 (v / v). In one embodiment,11110638946211 \AMERICASAttorney Docket No. 110798.00057the ratio of the organic solvent to water mixture in step (c) is about 3:1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (c) is about 3:2 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (c) is about 4: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (c) is about 6: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (c) is about 8: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (c) is about 9: 1 (v / v). In one embodiment, the ratio of the organic solvent to water mixture in step (c) is about 10: 1 (v / v).

[0063] In one embodiment, the organic solvent in step (c) is ethanol and the ratio of ethanol to water is about 5: 1 to 3: 1 (v / v). In one embodiment, the organic solvent in step (c) is ethanol and the ratio of ethanol to water is about 4: 1 (v / v).

[0064] In certain embodiments, the molar ratio of L-Ergothioneine in step (a) to ascorbic acid in step (c) is about 8:1 to 3: 1. In certain embodiments, the molar ratio of L-Ergothioneine in step (a) to ascorbic acid in step (c) is about 7: 1 to 5: 1. In certain embodiments, the molar ratio of L-Ergothioneine in step (a) to ascorbic acid in step (c) is about 6: 1 to 5: 1. In certain embodiments, the molar ratio of L-Ergothioneine in step (a) to ascorbic acid in step (c) is about 5.5:1. In certain embodiments, the molar ratio of L-Ergothioneine in step (a) to ascorbic acid in step (c) is about 5:1.

[0065] In one embodiment, the concentration of additional ascorbic acid in step (c) in the organic solvent / water mixture in step (c) is between about 0.02 M and 0.06 M. In one embodiment, the concentration of additional ascorbic acid in step (c) in the organic solvent / water mixture in step (c) is between about 0.03 M and 0.06 M. In one embodiment, the concentration of additional ascorbic acid in step (c) in the organic solvent / water mixture in step (c) is between about 0.04 M and 0.06 M. In one embodiment, the concentration of additional ascorbic acid in step (c) in the organic solvent / water mixture in step (c) is about 0.025 M. In one embodiment, the concentration of additional ascorbic acid in step (c) in the organic solvent / water mixture in step (c) is about 0.045 M. In one embodiment, the concentration of additional ascorbic acid in step (c) in the organic solvent / w ater mixture in step (c) is about 0.05 M. In one embodiment, the concentration of additional ascorbic acid in step (c) in the organic solvent / water mixture in step (c) is about 0.055 M.121106389462\1\AMERICASAttorney Docket No. 110798.00057

[0066] In one embodiment, the slurry is stirred for at least 30 minutes, at least 45 minutes, at least 1 hour, at least 90 minutes, or at least 2 hours at room temperature in step (d).

[0067] In one embodiment, the solid are recovered in step (e) by filtration. In one embodiment, the solid are recovered in step (e) by vacuum filtration.

[0068] In one embodiment, L-Ergothioneine HACC cocrystal monohydrate is synthesized by a method comprising:a) mixing L-Ergothioneine and ascorbic acid in an acetone / water mixture (3:1 (v / v)) at room temperature;b) allowing the organic solvent / water mixture to slowly evaporate to affordL-Ergothioneine HACC cocrystal monohydrate;c) slurrying the L-Ergothioneine HACC cocrystal monohydrate from step (b) in an ethanol / water mixture (4: 1 (v / v)) and additional ascorbic acid;d) stirring the slurry at room temperature; ande) recovering the solids of the slurry to afford L-Ergothioneine HACC cocrystal monohydrate.

[0069] In one embodiment, the ascorbic acid in step (a) and step (c) is L-ascorbic acid. In one embodiment, the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 2:1. In one embodiment, the concentration of L-Ergothioneine in step (a) is about 0.1 M. In one embodiment, the molar ratio of L-Ergothioneine in step (a) to additional ascorbic acid in step (c) is about 5.5:1. In one embodiment, the concentration of ascorbic acid in step (c) in the ethanol / water mixture is about 0.055 M. In one embodiment, the slurry is step (d) is stirred for at least one hour at room temperature. In one embodiment, the solid in step (e) is recovered via vacuum filtration.

[0070] In an alternative embodiment, L-Ergothioneine HACC cocrystal monohydrate is synthesized by a method comprising:a) mixing L-Ergothioneine and ascorbic acid in an acetone / water mixture (3:2 (v / v)) and heating the mixture to about 45 °C wherein the molar ratio of L-Ergothioneine to ascorbic acid is about 1:1.1; andb) allowing the organic solvent / water mixture to slowly evaporate to affordL-Ergothioneine HACC cocrystal monohydrate.131106389462\1\AMERICASAttorney Docket No. 110798.00057

[0071] In one embodiment, the ascorbic acid in step (a) L-ascorbic acid.

[0072] In an alternative embodiment, L-Ergothioneine HACC cocrystal monohydrate is synthesized by a method comprising:a) mixing L-Ergothioneine and ascorbic acid in an acetonitrile / water mixture (81% acetonitrile / 19% water (v / v)) at room temperature wherein the molar ratio of L- Ergothioneine to ascorbic acid is about 1:1.1; andb) allowing the organic solvent / water mixture to slowly evaporate to affordL-Ergothioneine HACC cocrystal monohydrate.

[0073] In one embodiment, the ascorbic acid in step (a) is L-ascorbic acid.

[0074] In an alternative embodiment, L-Ergothioneine HACC cocry stal monohydrate is synthesized by a method comprising:a) mixing L-Ergothioneine and ascorbic acid in a ethanol / water mixture (72% acetonitrile / 28% water (v / v)) at room temperature wherein the molar ratio of L- Ergothioneine to ascorbic acid is about 1:1.1; andb) allowing the organic solvent / water mixture to slowly evaporate to affordL-Ergothioneine HACC cocrystal monohydrate.

[0075] In one embodiment, the ascorbic acid in step (a) is L-ascorbic acid.

[0076] In an alternative, L-Ergothioneine HACC cocrystal monohydrate is synthesized by a method comprising:a) mixing L-Ergothioneine and ascorbic acid in a methanol / water mixture (9: 1 (v / v)) at room temperature wherein the molar ratio of L-Ergothioneine to ascorbic acid is about 1:1.1; andb) allowing the organic solvent / water mixture to slowly evaporate to affordL-Ergothioneine HACC cocrystal monohydrate.

[0077] In one embodiment, the ascorbic acid in step (a) is L-ascorbic acid.

[0078] The L-Ergothioneine for the synthesis, isolation, or purification of L-Ergothioneine HACC cocrystal monohydrate can be from any liquid or solid mixture, including, but not limited to, crude material from a synthetic reaction mixture or a fermentation mixture, for example, from a biosynthesis. In one embodiment, the L-Ergothioneine for the synthesis of L-Ergothioneine HACC cocrystal monohydrate can be crude material biologically extracted.141106389462\1\AMERICASAttorney Docket No. 110798.00057

[0079] The forms of L-Ergothioneine described herein can be in a form that is at least 90% free of the opposite D-Ergothioneine enantiomer excluding the weight of the coformer. In one embodiment, the forms of L-Ergothioneine is at least 95%, 98%, 99%, or even 100% free of the opposite D-Ergothioneine enantiomer excluding the weight of the coformer. For example, in certain embodiments, the L-Ergothioneine hemiascorbic acid is at least 90%, 95%, 98%, 99%, or even 100% free of the opposite D-Ergothioneine excluding the weight of the hemiascorbic acid.

[0080] Unless described otherwise the forms described herein are substantiallyL-Ergothioneine. In an alternative embodiment, the form is a racemic mixture ofL-Ergothioneine and D-Ergothioneine. In a further alternative embodiment, the form is in a form that is substantially the D-Ergothioneine. In one embodiment, the D-Ergothioneine is at least 90%, 95%, 98%, 99%, or even 100% free of the opposite L-Ergothioneine excluding the weight of the coformer.

[0081] Further, the ascorbic acid of L-Ergothioneine hemiascorbic acid or a cocrystal form thereof can be L-ascorbic acid, D-ascorbic acid, or a mixture of L-ascorbic acid and D-ascorbic acid. In one embodiment, the ascorbic acid is substantially L-ascorbic acid. In one embodiment, L-Ergothioneine L-hemiascorbic acid or a cocrystal form thereof comprises at least 90%, 95%, 98%, 99%, or even 100% the L-ascorbic acid.

[0082] Pharmaceutical Compositions

[0083] The forms of L-Ergothioneine hemiascorbic acid provided herein, including L-Ergothioneine HACC cocrystal monohydrate, can be formulated as pharmaceutical compositions using methods available in the art and those disclosed herein. Any of the compositions disclosed herein can be provided in the appropriate pharmaceutical composition and be administered by a suitable route of administration. The compositions provided herein can also be formulated as nutraceutical or nutritional formulations with additives such as nutraceutically or nutritionally acceptable excipients, nutraceutically or nutritionally acceptable carriers, and nutraceutically or nutritionally acceptable vehicles.

[0084] The methods provided herein encompass administering pharmaceutical or nutraceutical compositions containing at least one form of L-Ergothioneine hemiascorbic acid described herein, including L-Ergothioneine HACC cocrystal monohydrate, either alone or in the form of a combination with one or more compatible and pharmaceutically acceptable carriers, such as diluents or adjuvants.151106389462\1\AMERICASAttorney Docket No. 110798.00057

[0085] The term “nutraceutical” has been used to refer to any substance that is a food or a part of a food and provides medical or health benefits, including the prevention and treatment of disease. Hence, compositions falling under the label “nutraceutical” may range from isolated nutrients, dietary supplements and specific diets to genetically engineered designer foods, herbal products, and processed foods such as cereals, soups and beverages. In a more technical sense, the term has been used to refer to a product isolated or purified from foods, and generally sold in medicinal forms not usually associated with food and demonstrated to have a physiological benefit or provide protection against chronic disease.

[0086] In pharmaceutical compositions, use may be made, as solid compositions for oral administration, of tablets, pills, hard gelatin capsules, powders or granules. In these compositions, the active product is mixed with one or more inert diluents or adjuvants, such as sucrose, lactose or starch.

[0087] In clinical practice the compositions provided herein may be administered by any conventional route, in particular orally, parenterally, rectally or by inhalation (e.g. in the form of aerosols). In certain embodiments, the compositions provided herein are administered orally.

[0088] These compositions can comprise substances other than diluents, for example a lubricant, such as magnesium stearate, or a coating intended for controlled release.

[0089] In certain embodiments, a composition provided herein is a pharmaceutical composition or a single unit dosage form. Pharmaceutical compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., a compound provided herein, or other prophylactic or therapeutic agent), and a typically one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment and in this context, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” includes a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil. mineral oil, sesame oil and the like. Water can be used as a carrier when the pharmaceutical composition is administered intravenously.161106389462\1\AMERICASAttorney Docket No. 110798.00057Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in ‘'Remington’s Pharmaceutical Sciences” by E.W. Martin.

[0090] Typical pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are well-known to those skilled in the art of pharmacy, and non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a subject and the specific active ingredients in the dosage form. The composition or single unit dosage form, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0091] Lactose free compositions provided herein can comprise excipients that are well known in the art and are listed, for example, in the U.S. Pharmacopeia (USP) SP (XXI) / NF (XVI). In general, lactose free compositions comprise an active ingredient, a binder / filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts.Exemplary lactose free dosage forms comprise an active ingredient, microcr stalline cellulose, pre gelatinized starch, and magnesium stearate.

[0092] Further encompassed herein are anhydrous pharmaceutical compositions and dosage forms comprising active ingredients, since water can facilitate the degradation of some compounds. For example, the addition of water (e.g, 5%) is widely accepted in the pharmaceutical arts as a means of simulating long term storage in order to determine characteristics such as shelflife or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker. NY, NY. 1995, pp. 379 80. In effect, water and heat accelerate the decomposition of some compounds. Thus, the effect of water on a formulation can be of great significance since moisture and / or humidity are commonly encountered during manufacture, handling, packaging, storage, shipment, and use of formulations.

[0093] Anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose171106389462\1\AMERICASAttorney Docket No. 110798.00057and at least one active ingredient that comprises a primary or secondary amine can be anhydrous if substantial contact with moisture and / or humidity during manufacturing, packaging, and / or storage is expected.

[0094] An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions can be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0095] Generally, the ingredients of compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity' of active agent.

[0096] Typical dosage forms comprise a form provided herein and he w ithin the range of from about 0.1 mg to about 1000 mg per day, given as a single once-a-day dose in the morning or as divided doses throughout the day taken with food. Particular dosage forms can have about 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 100, 200, 250, 500 or 1000 mg of the active compound(s).

[0097] Further, the forms of L-Ergothioneine hemiascorbic acid as described herein can be administered in a liquid dosage form suitable for oral administration, including, but not limited to a solution or suspension. The forms of L-Ergothioneine as described herein can be administered intravenously.

[0098] Topical and mucosal delivery

[0099] Topical and mucosal dosage forms include, but are not limited to, solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art. See, e.g., Remington’s Pharmaceutical Sciences, 16th, 18th and 20theds., Mack Publishing, Easton PA (1980, 1990 & 2000); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985). In certain embodiments, the dosage form is selected from creams, lotions, gels, ointments, serum, face and body powder, foundation, color makeup, eyeliner, mascara, antiperspirant, deodorant, and micro-sponge.181106389462\1\AMERICASAttorney Docket No. 110798.00057

[0100] The products may be designed to reduce inflammation of the skin, even skin tone, hydrate or moisturize the skin, reduce fine lines and wrinkles, improve skin and such.

[0101] Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal. topical, and mucosal dosage forms encompassed herein are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied. With that fact in mind, ty pical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane 1,3 diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form lotions, tinctures, creams, emulsions, gels or ointments, which are nontoxic and pharmaceutically acceptable. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., Remington’s Pharmaceutical Sciences. 16th, 18th and 20theds., Mack Publishing. Easton PA (1980, 1990 & 2000).

[0102] Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with active ingredients provided. For example, penetration enhancers can be used to assist in delivering the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).

[0103] The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, may also be adjusted to improve delivery of one or more active ingredients. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity' of one or more active ingredients so as to improve delivery'. In this regard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery enhancing or penetration enhancing agent. Different hydrates or solvates of the active ingredients can be used to further adjust the properties of the resulting composition.191106389462\1\AMERICASAttorney Docket No. 110798.00057

[0104] Methods of Treatment

[0105] In one embodiment, a form of L-Ergothioneine hemiascorbic acid provided herein, including L-Ergothioneine HACC cocrystal monohydrate, is used to treat a disease or disorder caused by oxidative stress and / or inflammation. Non-limiting examples of disorders, diseases or other conditions associated with oxidative stress include cancer, neurodegenerative disease (Parkinson’s disease, Alzheimer’s disease, multiple sclerosis and amyolotrophic lateral sclerosis), eye disorders cardiovascular disease, atherosclerosis, sickle cell disease, thrombotic thrombocytopenic purpura, sepsis, cystic fibrosis, chronic fatigue syndrome, kidney disease, diabetes, acute respiratory distress syndrome, gout, arthritis, and other inflammatory diseases. Oxidative stress is involved in several age-related conditions (cardiovascular diseases, chronic obstructive pulmonary’ disease, chronic kidney disease, neurodegenerative diseases, and cancer), including sarcopenia and frailty. Oxidative stress is also involved in viral infections (HCV infection, HIV infection).

[0106] Non-limiting examples of inflammatory diseases or disorders include Alzheimer's, arthritis, asthma, atherosclerosis, Crohn's disease, colitis, cystic fibrosis, dermatitis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus erythematous, muscular dystrophy, nephritis, Parkinson's, rheumatoid arthritis, shingles and ulcerative colitis.Inflammatory diseases also include, for example, stroke, cardiovascular disease, chronic obstructive pulmonary disease (COPD). bronchiectasis, chronic cholecystitis, tuberculosis. Hashimoto’s thyroiditis, kidney fibrosis, sepsis, sarcoidosis, silicosis and other pneumoconioses.EXAMPLES

[0107] Methods

[0108] DSC Sample Preparation and Analysis

[0109] DSC was performed using a TA Instruments model Q10 differential scanning calorimeter. The instrument was calibrated using indium. The sample was placed into a standard aluminium DSC pan, covered with a lid, and the weight was accurately recorded. An aluminium pan configured as the sample pan was placed on the reference side of the cell. The pan lid was crimped prior to sample analysis. Samples were analysed in a single run from 25 to 300 °C at a heating rate of 10 or 20 °C / min under nitrogen gas.

[0110] Dynamic Vapor Sorption (D VS)201106389462\1\AMERICASAttorney Docket No. 110798.00057

[0111] Moisture sorption / desorption data were collected on a VTI SGA-100 Vapor Sorption Analyzer. Samples were not dried prior to analysis. Sorption and desorption data were collected over a range from 5% to 95% RH at 10% RH increments under a dry air purge. The equilibrium criterion used for analysis was less than 0.0100% weight change in 5 minutes w ith a maximum equilibration time of 3 hours with a 2-minute data logging interval. Data were not corrected for the initial moisture content of the samples.

[0112] Optical Microscopy

[0113] Samples were observed under a dissecting stereomicroscope with crossed polarizers.

[0114] Proton NMR Spectroscopy (!H NMR)

[0115] The solution NMR spectra were acquired with a Bruker 500 MHz spectrometer. The samples were prepared by dissolving approximately 8 mg of sample in either D2O or DMSO- e.

[0116] TGA Sample Preparation and Analysis

[0117] TG analyses were performed using a TA Instruments model 55. Temperature calibration was performed using Alumel™ Each sample was placed in a platinum pan then inserted into the TG furnace. The furnace was heated under nitrogen. Samples were analysed in a single run from ambient to 300 °C at a heating rate of 10 °C / min.

[0118] X-ray Powder Diffraction (XRPD)

[0119] XRPD patterns were collected with a PANalytical Empyrean diffractometer in Bragg-Brentano geometry using a Cu radiation source generated at 45 kV / 40 mA. A silicon standard was analysed to check the instrument alignment. Prior to the analy sis, a specimen of the sample was packed into a silicon zero background diffraction holder with a 10x0.2 mm well and analysed in reflection geometry. The X-ray source was configured with Soller slits of 0.04 radians, a fixed anti-scatter slit of ! °, a mask of 4 mm, and a fixed divergence slit of 1 / 16°. The diffracted beam passed through a 7.5 mm anti-scatter slit and large Soller slit of 0.02 radians to the detector. Diffraction patterns were collected with Data Collector software v. 6.1 b using a PlXcel3D-Medipix3 1x1 detector located 240 mm from the specimen. The data was acquired using up to 6 repetitions from 2-40° 2G with sample spinning at a revolution time of 2 seconds.21110638946211 \AMERICASAttorney Docket No. 110798.00057

[0120] XRPD Peak Picking

[0121] The location of the peaks along the x-axis (° 29) were determined using measurement data viewer software (PowDLL Converter v2.97.0.0 and Data Viewer vl.9A). Rounding algorithms were used to round each peak to the nearest 0.01 ° 29. Peak position variabilities are given to within ±0.2° 29 based upon recommendations outlined in the USP discussion of variability in X-ray powder diffraction (United States Pharmacopeia, USP 43-NF 38 through S2, <941> Characterization of Crystalline and Partially Crystalline Solids by X-Ray Powder Diffraction (XRPD), 1 GUID-14EBB55E-0D24-45A1-A84F-FE4DCAAEE3E8_l_en-US, official prior to 2013). The accuracy and precision associated with any particular measurement reported herein has not been determined. The wavelength used to calculate d-spacings was 1.5405929 . the Cu-Kai wavelength. Variability associated with d-spacing estimates was calculated from the USP recommendation, at each d-spacing.

[0122] Per USP guidelines, variable hydrates and solvates may display peak variances greater than 0.2° 29 and therefore peak variances of 0.2° 29 are not applicable to these materials.

[0123] Particle statistics and / or preferred orientation were assessed and the XRPD pattern appears representative of the powder average intensi ty for the sample. The room temperature refinement parameters were used to generate the Miller Indices (hkl) and respective d-spacings for all the allowed peaks (Mercury 2021.2.0 (Build 327561). The allowed peaks were then used to guide the selection of prominent peaks, a subset of the entire observed peak list with preferably non-overlapping, low-angle peaks, with strong intensity.

[0124] Example 1. Solubility and Evaporative Studies

[0125] L-Ergothioneine was sparingly soluble in most solvents and solubility values for L-Ergothioneine in water, water / methanol mixtures, and fluorinated solvents at ambient temperature are provided in Table 1. L-Ergothioneine was shown to oxidize / decompose in isopropanol solvent systems at elevated temperatures and therefore its use was avoided.

[0126] Table 1. Approximate Solubility Estimates of L-Ergothioneine<<<>>221106389462\1\AMERICASAttorney Docket No. 110798.00057<<<<<<

[0127] Cocrystals are distinguished from salts because unlike salts, the components that co-exist in the cocrystal lattice with a defined stoichiometry interact nonionically.

[0128] A monohydrate of a hemiascorbic acid cocrystal (L-Ergothioneine hemiascorbic acid (HACC) cocrystal monohydrate) was identified through evaporative experiments in aqueous acetone or alcohol mixtures (Table 2A-Table 2B). Select XRPD patterns of the monohydrate are shown in FIG. 1.

[0129] Table 2A. Experimental Conditions and Results using L-Ascorbic Acid in Acetone / Water23110638946211 \AMERICASAttorney Docket No. 110798.00057

[0130] Table 2B. Experimental Conditions and Results using L-Ascorbic Acid in EtOH / Water

[0131] Attempts to provide L-Ergothioneine HACC cocrystal monohydrate using other experimental methods, including seeding, were not successful; instead, mixtures of various anhydrate forms and the dihydrate of L-Ergothioneine were observed.

[0132] Example 2. Characterization of L-Ergothioneine HACC cocrystal monohydrate

[0133] Characterization of L-Ergothioneine HACC cocrystal monohydrate, including single crystal structure elucidation, was performed on HAAC sample 1 from Table 2A.Briefly, a solution of 29.7 mg of L-Ergothioneine lot and 25.6 mg of L-ascorbic acid in 2 mL of 40:60 v / v water / acetone was provided with heating at about 45 °C. The clear solution was stored in a 20-rnL scintillation vial covered with parafilm with several pinholes and allowed to slowly evaporate to dryness at room temperature to afford L-Ergothioneine HACC cocrystal monohydrate. A cry stal of sufficient size and quality' for single cry stal structure determination was culled.

[0134] A redacted diffractogram of the L-Ergothioneine HACC cocrystal monohydrate is shown in FIG. 2. The observed peaks for XRPD pattern are provided in Table 3.

[0135] Table 3. Peaks of the XRPD pattern of L-Ergothioneine HACC cocrystal monohydrate°20 d space (A) Intensity (%) 4.83 ± 0.20* 18.281 ± 0.758 327.39 ± 0.20* 11.953 ± 0.323 169.57 ± 0.20 9.234 ± 0.193 512.76 ± 0.20 6.932 ± 0.108 1213.40 ± 0.20 6.602 ± 0.098 614.45 ± 0.20* 6.125 ± 0.084 1914.93 ± 0.20 5.929 ± 0.079 1015.50 ± 0.20* 5.712 ± 0.073 1415.79 ± 0.20 5.608 ± 0.071 716.41 ± 0.20* 5.397 ± 0.065 10241106389462\1\AMERICASAttorney Docket No. 110798.00057°20 d space (A) Intensity (%) 17.61 ± 0.20 5.032 ± 0.057 519.14 ± 0.20 4.633 ± 0.048 10019.38 ± 0.20 4.576 ± 0.047 3319.97 ± 0.20* 4.443 ± 0.044 1220.68 ± 0.20* 4.292 ± 0.041 5421.05 ± 0.20 4.217 ± 0.040 821.66 ± 0.20* 4.100 ± 0.037 1422.46 ± 0.20 3.955 ± 0.035 822.88 ± 0.20 3.884 ± 0.033 1123.05 ± 0.20 3.855 ± 0.033 2324.24 ± 0.20 3.669 ± 0.030 6725.31 ± 0.20 3.516 ± 0.027 1125.77 ± 0.20 3.454 ± 0.026 626.25 ± 0.20 3.392 ± 0.025 1527.03 ± 0.20 3.296 ± 0.024 827.31 ± 0.20 3.263 ± 0.023 1427.44 ± 0.20 3.248 ± 0.023 1228.00 ± 0.20 3.184 ± 0.022 529.11 ± 0.20 3.065 ± 0.021 1129.28 ± 0.20 3.048 ± 0.020 1829.76 ± 0.20 3.000 ± 0.020 5 *Prominent peaks

[0136] The crystal system is orthorhombic and the space group is P2i2t2t (19). At 150(2) K, the cell parameters and calculated volume are: a = 6.043(5) A, b = 6.945(5) A, c = 36.46(3) A, a = 90°, [3 = 90°, y = 90°, V = 1530(2) A3. The formula weight is 670.75 g mol-1 with Z = 2. resulting in a calculated density of 1.456 g em3(Table 4). An atomic displacement ellipsoid drawing of the cocrystal is shown in FIG. 3.

[0137] Table 4. Crystal Data and Structure Refinement for L-Ergothioneine HACC cocrystal monohydrate251 106389462\1\AMERICASAttorney Docket No. 110798.00057< << <>

[0138] Each ascorbic acid molecule is disordered with two water molecules in a symmetry imposed 1:1 ratio (FIG. 4). The ascorbic acid molecule is compatible with water molecules created by a screw-translation along the A-axis (FIG. 5B), and with water molecules created by translation along the n-axis (FIG. 5A), indicating that the acid and the water molecules alternate along these two directions in an A-B-A-B-A-B-etc pattern. Along the c-directions, the ascorbic acid / water layers are separated by the zwitterions of the amino acid (FIG. 6). The alternating A-B ordering info of the ascorbic acid / water is lost between the channels separated by the amino acid. The disorder provides an average structure stoichiometry of 2:1:2 amino acid zwitterion / ascorbic acid / water, thus affordingL-Ergothioneine HACC cocrystal monohydrate.

[0139] Using GSAS-II (B.H. Toby & R.B. Von Dreele, J. Appl. Cry st. 46, 544-549 (2013)), the known crystallographic cell parameters were refined for the experimental powder pattern obtained at room temperature as: a = 6.003 A, b = 7.057 A, c = 36.848 A, a = 90°, [3 = 90°, y = 90°, V = 1561 A3. By means of the refinement, the calculated XRPD pattern from261106389462\1\AMERICASAttorney Docket No. 110798.00057the single crystal structure was adjusted to room temperature. The calculated and experimental powder patterns are compared in FIG. 7.

[0140] Some shift in peak position between the low temperature calculated and room temperature experimental powder diffraction patterns are expected because each data set were collected at different temperatures. Low temperatures are used in single crystal analysis to improve the quality of the structure, but can contract the crystal resulting in a change in the unit cell parameters, which is reflected in the calculated powder diffraction pattern. These shifts are particularly evident at high diffraction angles. Once adjusted for temperature, the peak positions agree well. Note that the shark-fin-shaped peak near 7.4° is a systematic absence violation of h,k,l = 0,0,3 from the ascorbic acid / water disorder that does not follow the crystallographic ‘system' of stacking, discussed above. Systematic absences are not normally observed and would not be evident in the calculated pattern.

[0141] The thermograms of L-Ergothioneine HACC cocr stal monohydrate are provided in FIG. 8A and FIG. 8B. The TGA showed 4.7% weight loss up to 150 °C and an additional 6.9% weight loss up to 200 °C. A broad DSC endotherm, concurrent with the volatilization in the TGA. exhibited a peak maximum of 88 °C. The weight loss up to 150 °C is consistent with the volatilization of ~0.9 mol / mol water. L- Ascorbic acid is known to decompose near 190 °C. Therefore, without being bound to any one theory', the final endotherm with an onset of 207 °C is likely from the decomposition of both L-ascorbic acid and L-Ergothioneine.

[0142] The solutionNMR spectrum was consistent with the chemical structure of L-Ergothioneine and contained peaks attributed to L-ascorbic acid (FIG. 9). The 1 : 1 molar mixture was evaporated to complete dryness; therefore, the peak integrations provide the 1:1 zwitterion / acid ratio of the experimental mixture rather than the hemicocrystal stoichiometry'. A summary' of the characterization is provided in Table 5.

[0143] Table 5. Characterization Summary for L-Ergothioneine HACC cocrystal monohydrate271106389462\1\AMERICASAttorney Docket No. 110798.00057

[0144] Example 3. Physical Stability of L-Ergothioneine HACC cocrystal monohydrate

[0145] Occasionally, the cocrystal was generated with spurious peaks in its XRPD pattern near 6.2°. The spurious peaks were noted to decrease in intensity while exposed to ambient laboratory conditions during XRPD analysis (FIG. 10). L-Ergothioneine HACC cocrystal monohydrate containing the spurious peaks was purposefully selected for thermal characterization, physical stability, and hygroscopicity' assessment to help identify environmental conditions in which L-Ergothioneine HACC cocrystal monohydrate remains stable but also allows the removal of the spurious peaks on exposure. Approximately 1 gram of material was prepared using rotary vacuum evaporation from 75:25 v / v acetone / water at room temperature. A summary of the characterization of this form of L-Ergothioneine HACC cocrystal monohydrate is provided in Table 6.

[0146] Table 6. Characterization Results for L-Ergothioneine HACC cocrystal monohydrate + Peaks near 6.2°

[0147] The thermograms of L-Ergothioneine HACC cocrystal monohydrate containing the spurious peaks are nearly identical to those of the L-Ergothioneine HACC cocrystal 281106389462\1\AMERICASAttorney Docket No. 110798.00057monohydrate characterized in Example 2. However, the TGA showed slightly more weight loss up to 150 °C. Assuming the complete 5.9% weight loss is from the volatilization of water, the loss is consistent with the volatilization of ~ 1.1 mol / mol water.

[0148] DVS with post-DVS XRPD analysis was conducted to determine relative hygroscopicity. The analysis indicated that the monohydrate was relatively stable between 15% and 85% RH within the timescale of the experiment; however, significant moisture loss or gain is observed at the extreme conditions of 5% and 95% RH. respectively. The sample of L-Ergothioneine HACC cocrystal monohydrate containing the spurious peaks lost 3.7 wt% upon equilibration at 5% RH, corresponding to slightly more than 0.5 mol / mol water loss. The material regained all the lost moisture upon reaching 45% RH on the sorption cycle. Equilibrium weight was not achieved at the prior RH step, suggesting that the material may have regained the lost moisture at lower RH conditions if sustained at that condition for longer. The weight was nearly stable up though 85% RH. An additional 2.3 wt% gain was achieved at 95% RH (corresponding to an additional gain of about 0.5 mol / mol water), suggesting the material exhibits significant hygroscopicity at the condition (equilibrium weight was not achieved, indicating that additional weight gain may be possible if sustained at 95% RH for longer). Upon desorption, the original weight was achieved again at the 85% RH step and nearly sustained down through 15% RH (the stable weight plateau region shows about 1.6 wt% loss). Again, a loss of 0.5 mol / mol water (2.6 wt%) was shown upon reaching 5% RH. By XRPD, the material recovered from the DVS experiment was still predominantly L-Ergothioneine HACC cocrystal monohydrate + peaks with a minor amount of another form of L-Ergothioneine not characterized herein .

[0149] The physical stability of this form of L-Ergothioneine HACC cocrystal monohydrate (L-Ergothioneine HACC cocrystal monohydrate + Peaks near 6.2° post-DVS) was investigated further.

[0150] By XRPD, L-Ergothioneine HACC cocrystal monohydrate was shown to remain unchanged, but the intensities of the spurious peaks were nearly eliminated upon exposure to 75% RH for 18 days.

[0151] The physical stability assessment of L-Ergothioneine HACC cocrystal monohydrate indicated that the form was relatively stable between 15% and 85% RH within the timescale studied; however, significant moisture loss or gain is observed at the extreme conditions of 5% and 95% RH, respectively. It was also determined that sustained exposure291106389462\1\AMERICASAttorney Docket No. 110798.00057to 75% RH removes the spurious peaks near 6.2° that are sometimes evident in the XRPD patterns of L-Ergothioneine HACC cocrystal monohydrate.

[0152] Example 4. Production of L-Ergothioneine HACC cocrystal monohydrate on a 2.6-Gram Scale

[0153] L-Ergothioneine HACC cocrystal monohydrate was produced at 2.6-gram scale by a two-step procedure using rotary vacuum evaporation from 3: 1 v / v acetone / water followed by a slurry wash in 4: 1 v / v ethanol / water (at a water activity of 0.70).

[0154] The detailed procedure is as follows. Three separate batches of 723 mg of L-Ergothioneine and 278 mg of L-ascorbic acid were dissolved in 285 mL of 3:1 acetone / water at room temperature. The clear solutions were rotary vacuum evaporated at room temperature to dryness affording HAAC Monohydrate + peaks near 6.2°. The solids from the three rotovaps were combined and then slurried in 10 mL of 4:1 ethanol / water solution (0.7 water activity) containing 100 mg of dissolved L-ascorbic acid. The slurry was stirred for 1 hour on a stir plate with a stir bar spinning at 300 RPM at room temperature. The solids were recovered by water aspirated vacuum filtration over 55 mm Whatman 1 filter paper until visibly dry. The solids were stored under 75% RH until XRPD analysis was performed 4 days later.

[0155] The wash step was successful at removing the spurious peaks near 6.2° and any excess amount of free L-ascorbic acid. However, without being bound to any one theory, the solids were likely triturated in the wash solvent for too long (1 hour) and a small amount of 1-Ergothioneine Form B Dihydrate was evident.

[0156] The embodiments and examples descnbed above are intended to be merely illustrative and non-limiting. Those skilled in the art will recognize or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials and procedures. All such equivalents are considered to be within the scope and are encompassed by the appended claims.301106389462\1\AMERICAS

Claims

Attorney Docket No. 110798.00057WHAT IS CLAIMED IS:

1. L-Ergothioneine hemiascorbic acid.

2. The L-Ergothioneine hemiascorbic acid of claim 1, wherein the ascorbic acid is L- ascorbic acid.

3. The L-Ergothioneine hemiascorbic acid of claim 1 or 2, wherein the L-Ergothioneine hemiascorbic acid is crystalline.

4. The L-Ergothioneine hemiascorbic acid of claim 3, wherein the crystalline L- Ergothioneine hemiascorbic acid is L-Ergothioneine hemiascorbic acid cocrystal monohydrate.

5. The L-Ergothioneine hemiascorbic acid of claim 4, wherein the crystalline L- Ergothioneine hemiascorbic acid is L-Ergothioneine L-hemiascorbic acid cocrystal monohydrate.

6. The L-Ergothioneine hemiascorbic acid cocrystal monohydrate of claim 4, characterized by an X-ray powder diffraction (XRPD) comprising peaks at 19.14, 20.68, and 24.24 + 0.2° 20 using Cu Ka radiation.

7. The cocrystal of claim 6, characterized by an XRPD comprising peaks at 19.14, 19.38, 20.

68. and 24.24 ± 0.2° 20.

8. The cocrystal of claim 7, characterized by an XRPD comprising peaks at 4.83, 19.14, 19.38, 20.68, 23.05, and 24.24 + 0.2° 20.

9. The cocrystal of claim 8, characterized by an XRPD comprising peaks at 4.83, 7.39, 14.45, 15.50, 19.14, 19.

38. 19.97, 20.68.21.66, 23.05, 24.24, 26.25, 27.31, and 29.28 ± 0.2° 20.

10. The L-Ergothioneine hemiascorbic acid cocrystal monohydrate of claim 4, characterized by an XRPD comprising at least six, seven, or eight peaks selected from at 4.83, 7.39, 14.45, 15.50, 19.14, 19.

38. 19.97, 20.

68. 21.66, 23.05, 24.24, 26.25, 27.31, and 29.28 ± 0.2° 20.

11. The L-Ergothioneine hemiascorbic acid cocrystal monohydrate of claim 4, characterized by an XRPD comprising at least two, three, four, or five peaks selected from at 4.83, 7.39, 14.45, 15.50, 19.14, 19.38, 19.97, 20.68, 21.66, 23.05, 24.24, 26.25, 27.31, and 29.28 + 0.2° 20.

12. The L-Ergothioneine hemiascorbic acid cocrystal monohydrate of claim 4, characterized by an XRPD comprising peaks at 4.83, 7.39, 14.45, 15.50, 16.41, 19.97, 20.68, and 21.66 + 0.2° 20.311106389462\1\AMERICASAttorney Docket No. 110798.0005713. The L-Ergothioneine hemiascorbic acid cocrystal monohydrate of claim 4, characterized by an XRPD pattern substantially similar to that of FIG. 2.

14. The co-crystal of any one of claims 4 and 6-13, characterized by a differential scanning calorimetry (DSC) thermograph having two endotherms wherein the first has an endotherm maximum between about 85 °C and 95 °C and wherein the second has an endotherm maximum between about 215 °C and 225 °C.

15. The co-crystal of claim 14. characterized by a differential scanning calorimetry (DSC) thermograph having two endotherms wherein the first has an endotherm maximum at about 88 °C and wherein the second has an endotherm maximum at about 217 °C.

16. The co-crystal of any one of claims 4 and 6-13, characterized by a DSC thermograph substantially similar to that of FIG. 8A.

17. The co-crystal of any one of claims 4 and 6-16, characterized in a thermogravimetric analysis (TGA) by a weight loss in the range of about 1% to 5% when heated to about 150 °C, an additional weight loss in the range of about 1% to 5% when heated from about 150 °C to 175 °C, and an additional weight loss in the range of about 1% to 5% when heated from about 175 °C to 200 °C.

18. The co-crystal of claim 17, characterized in a thermogravimetric analysis (TGA) by a weight loss of about 4.7% when heated to about 150 °C, an additional weight loss in the range of about 2% when heated from about 150 °C to 175 °C, and an additional weight loss in the range of about 4.8% when heated from about 175 °C to 200 °C.

19. The co-crystal of any one of claims 4 and 6-18, characterized by TGA substantially similar to that of FIG. 8B.

20. A pharmaceutical composition comprising the L-Ergothioneine hemiascorbic acid of any one of claims 1-3 or the L-Ergothioneine hemiascorbic acid cocrystal monohydrate of any one of claims 4-19 in a pharmaceutically acceptable carrier.

21. A method for the treatment of a disease or disorder caused by oxidative stress and / or inflammation comprising administering the L-Ergothioneine hemiascorbic acid of any one of claims 1-3 or the L-Ergothioneine hemiascorbic acid cocrystal monohydrate of any one of claims 4-19, or the pharmaceutical composition of claim 18 to a patient in need thereof.

22. A method for the synthesis of L-Ergothioneine HACC cocrystal monohydrate comprising (a) mixing L-Ergothioneine and ascorbic acid in an organic solvent / water mixture and (b) allowing the organic solvent / water mixture to slowly evaporate to afford L-Ergothioneine HACC cocrystal monohydrate.321106389462\1\AMERICASAttorney Docket No. 110798.0005723. The method of claim 22, wherein the ascorbic acid is L-ascorbic acid.

24. The method of claim 22 or 23, wherein the organic solvent in step (a) is acetone and the ratio of acetone to water is about 3:1 (v / v).

25. The method of any one of claims 22-24, wherein the molar ratio of L-Ergothioneine to ascorbic acid in step (a) is about 2:1.

26. The method of any one of claims 22-25, wherein the concentration of L-Ergothioneine in step (a) is about 0.1 M.

27. The method of any one of claims 22-26, further comprising (c) slurrying theL-Ergothioneine HACC cocrystal monohydrate from step (b) in an organic solvent / water mixture and additional ascorbic acid; (d) stirring the slurry; and (e) recovering the solids of the slurry to afford L-Ergothioneine HACC cocrystal monohydrate.

28. The method of claim 27, wherein the additional ascorbic acid is L-ascorbic acid.

29. The method of claim 27 or 28, wherein the organic solvent in step (c) is ethanol and the ratio of ethanol to water is about 4: 1 (v / v).

30. The method of any one of claims 27-29, the molar ratio of L-Ergothioneine in step (a) to additional ascorbic acid in step (c) is about 5.5:1.

31. The method of any one of claims 27-30, the concentration of ascorbic acid in step (c) in the ethanol / water mixture is about 0.055 M.

32. The method of any one of claims 27-31, the slurry is step (d) is stirred for at least one hour at room temperature.

33. The method of any one of claims 27-32, the solid in step (e) is recovered via vacuum filtration.331106389462\1\AMERICAS

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