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WO2025056246A1 - Polymorphs and compositions comprising same - Google Patents

Polymorphs and compositions comprising same Download PDF

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Publication number
WO2025056246A1
WO2025056246A1 PCT/EP2024/072112 EP2024072112W WO2025056246A1 WO 2025056246 A1 WO2025056246 A1 WO 2025056246A1 EP 2024072112 W EP2024072112 W EP 2024072112W WO 2025056246 A1 WO2025056246 A1 WO 2025056246A1
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WIPO (PCT)
Prior art keywords
bhb
salt form
crystalline
peaks
xrpd pattern
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PCT/EP2024/072112
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French (fr)
Inventor
Labdhi PARIKH
Wang XIAOYANG
Philippe Delerive
Li MEIQI
Fan JUN
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Societe Des Produits Nestle S.A.
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Publication of WO2025056246A1 publication Critical patent/WO2025056246A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C59/01—Saturated compounds having only one carboxyl group and containing hydroxy or O-metal groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C211/00—Compounds containing amino groups bound to a carbon skeleton
    • C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
    • C07C211/02—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C211/03—Monoamines
    • C07C211/07—Monoamines containing one, two or three alkyl groups, each having the same number of carbon atoms in excess of three
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/26—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one amino group bound to the carbon skeleton, e.g. lysine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/14—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • the invention relates to polymorphs of D-beta-hydroxybutyrate (D-BHB).
  • D-beta-hydrxybutyrate which may also be referred to as R-beta- hydrxy butyrate or (R)-3 -hydroxy butrate and is the conjugate base of D-beta-hy dr oxybutyric acid (shown below).
  • D-BHB is of interest as a ketone body that has been associated with a variety of health benefits.
  • due to high doses of D-BHB that may be associated with achieving a therapeutic benefit there is a concern about high mineral intake from safety/compliance perspective.
  • crystalline forms of D-BHB or a salt thereof including free base crystalline forms, crystalline salts and crystalline solvates.
  • crystalline sodium salt forms A, B, and C crystalline magnesium salt forms A and C; crystalline L-lysine form A, crystalline erbumine salt forms A and B; and crystalline L-arginine salt forms A and B.
  • compositions comprising any one or more of the crystalline salt forms disclosed herein and a pharmaceutically acceptable carrier.
  • Figure 1 depicts an X-ray powder diffraction (“XRPD”) pattern of free base crystalline forms of D-BHB obtained from condition 1 (A) (XRPD Method 2) and condition 2 (B) (XRPD Method 2).
  • Figure 2 depicts a differential scanning calorimetry (“DSC”) thermograph of free base crystalline forms of D-BHB obtained from condition 1 (A) and condition 2 (B).
  • FIG. 3 depicts a thermogravimetric analysis (“TGA”) trace of free base crystalline forms of D-BHB obtained from condition 1 (A) and condition 2 (B).
  • TGA thermogravimetric analysis
  • Figure 4 depicts 'H-NMR spectrum of free base crystalline forms of D-BHB obtained from condition 1 (A) and condition 2 (B).
  • Figure 5 XRPD (Method 2) of (A) sodium salt screening; (B) magnesium salt screening; (C) L-arginine salt screening; (D) L-lysine salt screening; (E) erbumine salt screening; (F) betaine salt screening; and (G) re-slurry screening of L-arginine salt screening.
  • FIG. 16 D-BHB sodium salt Form C XRPD (Method 2) (A) FR03684-SUl-NaOH-1.5- THF and (B) FR03684-SU9-NaOH-1.5-THF. [023] Figure 17 D-BHB sodium salt Form C (FR03684-SU9-NaOH-1.5-THF) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ' H-NMR spectrum.
  • Figure 18 D-BHB L-arginine salt Form B (FR03684-SU2-L-arginine-acetone-water-95-5- re ) (A) overlay of XRPD (Method 2) of scale up Trial land (B) 'H-NMR spectrum.
  • Figure 21 D-BHB L-lysine salt Form A FR03684-SU3-L-lysine-EtOH-re (A) overlay XRPD (Method 2) from scale up and (B) 'H-NMR spectrum.
  • FIG. 24 Overlay of XRPD (Method 2) of D-BHB erbumine salt Form A and Form B from scale-up.
  • FIG. 25 D-BHB erbumine salt Form A (FR03684-SU4-erbumine-EA) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ' H-NMR spectrum.
  • Figure 26 (A) Overlay of XRPD (Method 3) of D-BHB erbumine salt Form A and Form B from scale-up of process to obtain form B; D-BHB erbumine salt Form B (FR03684-SU8- erbumine-ACN-water-95-5) (B) XRPD (Method 1) (C) DSC (D) TGA and (E) ' H-NMR spectrum [033] Figure 27 Mini-polymorph screening - suspensions equilibrated at 25°C for 1 week : (A) to (C) D-BHB sodium salt Form C (FR03684-SUl-NaOH-1.5-THF) as starting material - XRPD overlay; (D) and (E) D-BHB L-arginine salt Form C (FR03684-SU2-L-arginine-acetone-water- 95-5) as starting material - XRPD overlay; (F) and (G) D-BHB L-
  • FIG. 28 Bulk Stability XRPD (Method 2)
  • A L-arginine salt Form B (FR03684-SU5- L-arginine-acetone-water-95-5);
  • B D-BHB Erbumine salt Form B (FR03684-SU8-erbumine- ACN-water-95-5);
  • C D-BHB sodium salt Form C (FR03684-SUl-NaOH-1.5-THF);
  • D-BHB L-lysine salt form A (FR03684-SU6-L-lysine-EtOH)
  • crystalline forms of D-BHB Disclosed are crystalline forms of D-BHB.
  • Embodiments of crystalline free base forms, salt forms and solvates of D-BHB can be characterized by one or more parameters as described below.
  • D-BHB refers to the compound D-beta-hydrxybutyrate (D-BHB), which may also be referred to as R-beta-hydrxybutyrate or (R)-3-hydroxybutrate and is the conjugate base of D-beta-hydroxybutyric acid (shown below).
  • D-BHB used as starting material and/or the D-BHB crystalline free form or salts are greater than about 95% enantiomerically pure, greater than about 98% enantiomerically pure, or greater than about 99% enantiomerically pure.
  • “Enantiomerically pure” as used herein refers to the percentage of one enantiomer versus the other enantiomer, e.g. the percentage of D-BHD versus L-BHB in a preparation of D-BHB. It is understood that enantiomeric pairs (i.e.
  • the R and S structural configuration of a compound with a chiral atom using absolute configuration convention or D or L structural configuration of a compound with a chiral atom using the convention in relation to L- and D-glyceraldehyde as represented in Fischer projections) may be produced as a mixture having different percentages of the respective enantiomers.
  • D-BHB as starting material to generate the crystalline free form and/or D-BHB crystalline salts is greater than about 95% D-BHB and has less than about 5% L-DHB, greater than about 98% D-BHB and less than about 2% L-BHB, greater than about 99% D-BHB and less than about 1% L-BHB, or greater than about 99.5% D-BHB and less than about 0.5% L-BHB.
  • a crystalline D-BHB having greater than 95% enantiomeric purity is understood as being comprised of greater than 95% D-BHB and less than 5% L-BHB.
  • a crystalline D-BHB having greater than 98% enantiomeric purity is understood as being comprised of greater than 98% D-BHB and less than 2% L-BHB.
  • a crystalline D-BHB having greater than 99% enantiomeric purity is understood as being comprised of greater than 99% D-BHB and less than 1% L-BHB.
  • a crystalline D-BHB having greater than 99.5% enantiomeric purity is understood as being comprised of greater than 99.5% D-BHB and less than 0.5% L-BHB.
  • a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth. All ranges are inclusive of the endpoints of the range. For example, an amount between 1 and 10 includes both 1 and 10.
  • compositions and methods disclosed herein may lack any element that is not specifically disclosed herein.
  • a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of’ and “consisting of’ the components or steps identified.
  • Crystalline free base Form can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 10.93, 12.11, 15.16, 17.53, and 18.88 ⁇ 0.2° 20 using Cu Ka radiation.
  • Form I can be further characterized by having an XRPD pattern having additional peaks at 22.75, 24.30, 26.14, 29.10, and 29.95 ⁇ 0.2° 20 using Cu Ka radiation.
  • Form I can be further characterized by having an XRPD pattern having additional peaks at 30.48, 31.60, 32.96, 33.98, 36.80, and 38.37 ⁇ 0.2° 20 using Cu Ka radiation.
  • Form I has an XRPD pattern substantially as shown in Figure 1(A), wherein by “substantially” is meant that the reported peaks can vary by about ⁇ 0.2°. It is well known in the field of XRPD that while relative peak heights in spectra are depending on a number of factors, such as sample preparation and instrument geometry, peak positions are relatively insensitive to experimental details.
  • Form I may also be characterized by DSC substantially as set forth in Figure 2(A) and/or by the TGA set forth in Figure 3(A).
  • crystalline free base Form I may be characterized by one or more of an XRPD substantially as depicted in Figure 1(A), DSC substantially as set forth in Figure 2(A), and TGA as set forth in Figure 3(A).
  • D-BHB crystalline sodium salt Form A can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 11.82, 17.02, 17.58, 20.34, and 20.83 ⁇ 0.220 using Cu Ka radiation.
  • Sodium salt Form A can be further characterized by having an XRPD pattern having additional peaks at 7.23, 14.39, 24.50, 29.54, and 30.17 ⁇ 0.2° 20 using Cu Ka radiation.
  • Sodium salt Form A can be further characterized by having an XRPD pattern having additional peaks at 12.22, 22.23, 22.60, 30.63, 33.35, and 37.28 ⁇ 0.2° 20 using Cu Ka radiation.
  • sodium salt Form A has an XRPD pattern substantially as shown in Figure 9(A).
  • D-BHB crystalline sodium salt Form A may also be characterized by DSC substantially as set forth in Figure 9(B) and/or by the TGA set forth in Figure 9(C).
  • D- BHB crystalline sodium salt Form A may be characterized by one or more of an XRPD substantially as depicted in Figure 9(A), DSC substantially as set forth in Figure 9(B), and TGA as set forth in Figure 9(C).
  • D-BHB crystalline sodium salt Form B can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 8.98, 11.49, 16.95, 17.53, and 19.38 ⁇ O.2°20 using Cu Ka radiation.
  • Sodium salt Form B can be further characterized by having an XRPD pattern having additional peaks at 7.17, 9.67, 12.16, 20.10, and 26.83 ⁇ 0.2°20 using Cu Ka radiation.
  • Sodium salt Form B can be further characterized by having an XRPD pattern having additional peaks at 6.47, 14.02, 14.35, 18.35, 21.00, and 23.08 ⁇ 0.2°20 using CuKa radiation.
  • sodium salt Form B has an XRPD pattern substantially as shown in Figure 10(A).
  • D-BHB crystalline sodium salt Form B may also be characterized by DSC substantially as set forth in Figure 10(B) and/or by the TGA set forth in Figure 10(C).
  • D- BHB crystalline sodium salt Form B may be characterized by one or more of an XRPD substantially as depicted in Figure 10(A), DSC substantially as set forth in Figure 10(B), and TGA as set forth in Figure 10(C).
  • D-BHB crystalline sodium salt Form C can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 6.40, 7.07, 12.11, 14.08, and 17.05 ⁇ 0.2°20 using Cu Ka radiation.
  • Sodium salt Form C can be further characterized by having an XRPD pattern having additional peaks at 19.16, 22.57, 27.79, 28.75, and 30.11 ⁇ 0.2°20 using Cu Ka radiation.
  • Sodium salt Form C can be further characterized by having an XRPD pattern having additional peaks at 11.98, 30.76, 33.12, 33.63, and 37.08 ⁇ 0.2°20 using Cu Ka radiation.
  • sodium salt Form C has an XRPD pattern substantially as shown in Figure 29(A).
  • D-BHB crystalline sodium salt Form C may also be characterized by DSC substantially as set forth in Figure 29(B) and/or by the TGA set forth in Figure 29(C).
  • D- BHB crystalline sodium salt Form C may be characterized by one or more of an XRPD substantially as depicted in Figure 29(A), DSC substantially as set forth in Figure 29(B), and TGA as set forth in Figure 29(C).
  • D-BHB crystalline magnesium salt Form A can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 5.53, 7.80, 9.61, 11.95, and 13.15 ⁇ 0.2°20 using Cu Ka radiation.
  • Magnesium salt Form A can be further characterized by having an XRPD pattern having additional peaks at 6.11, 11.02, 11.27, 15.61, and 18.98 ⁇ 0.2°20 using Cu Ka radiation.
  • Magnesium salt Form A can be further characterized by having an XRPD pattern having additional peaks at 19.45, 21.13, 21.88, 23.70, and 25.99 ⁇ 0.2°20 using Cu Ka radiation.
  • magnesium salt Form A has an XRPD pattern substantially as shown in Figure 7(A).
  • D-BHB crystalline magnesium salt Form A may also be characterized by DSC substantially as set forth in Figure 7(B) and/or by the TGA set forth in Figure 7(C).
  • D- BHB crystalline magnesium salt Form A may be characterized by one or more of an XRPD substantially as depicted in Figure 7(A), DSC substantially as set forth in Figure 7(B), and TGA as set forth in Figure 7(C).
  • D-BHB crystalline L-lysine salt Form A can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 6.92, 9.16, 12.62, 23.29, and 25.91 ⁇ 0.2°20 using Cu Ka radiation.
  • L-lysine salt Form A can be further characterized by having an XRPD pattern having additional peaks at 10.21, 26.93, 27.64, 28.45, and 29.87 ⁇ 0.2°20 using Cu Ka radiation.
  • L-lysine salt Form A can be further characterized by having an XRPD pattern having additional peaks at 17.87, 18.27, 19.73, 24.58, and 25.02 ⁇ 0.2°20 using Cu Ka radiation.
  • L-lysine salt Form A has an XRPD pattern substantially as shown in Figure 31(A).
  • D-BHB crystalline L-lysine salt Form A may also be characterized by DSC substantially as set forth in Figure 31(B) and/or by the TGA set forth in Figure 31(C).
  • D-BHB crystalline L-lysine salt Form A may be characterized by one or more of an XRPD substantially as depicted in Figure 31(A), DSC substantially as set forth in Figure 31(B), and TGA as set forth in Figure 31(C).
  • D-BHB crystalline L-arginine salt Form A can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 5.28, 7.28, 9.11, 14.99, and 15.82 ⁇ 0.2°20 using Cu Ka radiation.
  • L-arginine salt Form A can be further characterized by having an XRPD pattern having additional peaks at 17.00, 17.76, 18.32, 19.32, and 24.10 ⁇ 0.2°20 using Cu Ka radiation.
  • L-arginine salt Form A can be further characterized by having an XRPD pattern having additional peaks at 10.81, 11.08, 11.27, 11.75, and 26.96 ⁇ 0.2°20 using Cu Ka radiation.
  • L-arginine salt Form A has an XRPD pattern substantially as shown in Figure 14(A).
  • D-BHB crystalline L-arginine salt Form A may also be characterized by DSC substantially as set forth in Figure 14(B) and/or by the TGA set forth in Figure 14(C).
  • D-BHB crystalline L-arginine salt Form A may be characterized by one or more of an XRPD substantially as depicted in Figure 14(A), DSC substantially as set forth in Figure 14(B), and TGA as set forth in Figure 14(C).
  • D-BHB crystalline L-arginine salt Form B can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 7.30, 9.94, 11.28, 14.56, and 15.83 ⁇ 0.2°20 using Cu Ka radiation.
  • L-arginine salt Form B can be further characterized by having an XRPD pattern having additional peaks at 17.27, 18.33, 19.91, 21.90, and 23.49 ⁇ 0.2°20 using Cu Ka radiation.
  • L-arginine salt Form B can be further characterized by having an XRPD pattern having additional peaks at 24.12, 26.45, 26.99, 28.56, 33.56, and 34.16 ⁇ 0.2°20 using Cu Ka radiation.
  • L-arginine salt Form B has an XRPD pattern substantially as shown in Figure 30(A).
  • D-BHB crystalline L-arginine salt Form B may also be characterized by DSC substantially as set forth in Figure 30(B) and/or by the TGA set forth in Figure 30(C)).
  • D-BHB crystalline L-arginine salt Form B may be characterized by one or more of an XRPD substantially as depicted in Figure 30(A), DSC substantially as set forth in Figure 30(B), and TGA as set forth in Figure 30(C).
  • D-BHB crystalline erbumine salt Form A can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 10.04, 10.77, 13.69, 14.71, and 18.24 ⁇ 0.2°20 using Cu Ka radiation.
  • Erbumine salt Form A can be further characterized by having an XRPD pattern having additional peaks at 16.08, 16.95, 17.42, 19.37, and 20.07 ⁇ 0.2°20 using Cu Ka radiation.
  • Erbumine salt Form A can be further characterized by having an XRPD pattern having additional peaks at 22.47, 24.35, 25.60, 26.05 and 28.36 ⁇ 0.2°20 using Cu Ka radiation.
  • erbumine salt Form A has an XRPD pattern substantially as shown in Figure 25(A).
  • D-BHB crystalline erbumine salt Form A may also be characterized by DSC substantially as set forth in Figure 25(B) and/or by the TGA set forth in Figure 25(C).
  • D-BHB crystalline erbumine salt Form A may be characterized by one or more of an XRPD substantially as depicted in Figure 25(A), DSC substantially as set forth in Figure 25(B), and TGA as set forth in Figure 25(C).
  • D-BHB crystalline erbumine salt Form B can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 12.42, 16.07, 16.69, 17.62, and 18.35 ⁇ 0.2°20 using Cu Ka radiation.
  • Erbumine salt Form B can be further characterized by having an XRPD pattern having additional peaks at 8.82, 20.71, 22.22, 23.95, and 27.27 ⁇ 0.2°20 using Cu Ka radiation.
  • Erbumine salt Form B can be further characterized by having an XRPD pattern having additional peaks at 19.76, 21.22, 23.49, 28.46, 31.36, and 38.21 ⁇ 0.2°20 using Cu Ka radiation.
  • D-BHB erbumine salt Form B has an XRPD pattern substantially as shown in Figure 26(B).
  • D-BHB crystalline erbumine salt Form B may also be characterized by DSC substantially as set forth in Figure 26(C) and/or by the TGA set forth in Figure 26(D).
  • D-BHB crystalline erbumine salt Form B may be characterized by one or more of an XRPD substantially as depicted in Figure 26(B), DSC substantially as set forth in Figure 26(C), and TGA as set forth in Figure 26(D).
  • compositions comprising one or more crystalline forms of D-BHB and at least one pharmaceutically acceptable carrier.
  • the carrier can comprise an excipient.
  • the pharmaceutical composition comprises one crystalline form of D-BHB.
  • the pharmaceutical composition comprises two crystalline forms of D-BHB.
  • the pharmaceutical composition comprises two crystalline forms of D-BHB, where one of the two crystalline forms of D-BHB is D-BHB L-arginine salt Form B.
  • the pharmaceutical composition comprises two crystalline forms of D-BHB, where one of the two crystalline forms of D-BHB is D-BHB sodium salt Form C.
  • a carrier as used herein include includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed.
  • the carrier comprises water.
  • the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; and/or nonionic surfactants such as polysorbates (e.g. polysorbate 80 or polysorbate 20), polyethylene glycol (PEG), and poloxamers.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • hydrophilic polymers such as polyvinylpyrrolidone
  • amino acids such as glycine, glutamine, asparagine, arginine or lysine
  • Excipients in the formulation of pharmaceuticals are known in the art and may include diluents, disintegrants, binders, and other classes of excipients known in the art.
  • compositions may be administered orally.
  • Oral administration includes a formulation suitable for swallowing, or buccal or sublingual administration.
  • Formulation suitable for oral administration include solid formulations such as tablets, capsules containing particulates, and powders. Formulations for oral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled targeted- and programmed-release.
  • the pharmaceutical composition may be administered by injection.
  • Administration by injection includes a formulation suitable for subcutaneous injection, intramuscular injection, intradermal injection, or intravenous injection.
  • the pharmaceutical formulation as described herein comprises one or more D-BHB crystalline forms, wherein the one or more crystalline forms as described herein may make up from up to 80 wt (weight) %, up to 70 wt%, up to 50 wt %, up to 40 wt%, up to 30 wt%, or up to 20% of a dosage form.
  • the crystalline form as described herein may make up from 0.5 wt% to 80 wt% of the dosage form, from 0.5 wt% to 70 wt% of the dosage form, from 0.5 wt% to 50 wt% of the dosage form, from 0.5 wt% to 40 wt% of the dosage form, from 0.5 wt% to 30 wt% of the dosage form, or from 0.5 wt% to 20 wt% of the dosage form.
  • the pharmaceutical formulation comprises two or more crystalline forms of D-BHB as described herein make up from from 0.5 wt% to 50 wt% of the dosage form.
  • the pharmaceutical formulation as described herein comprises one or more crystalline forms and may be formulated for administration to a subject as a single dose per day, as two or more doses per day, as three or more doses per day, or up to four doses per day.
  • the crystalline form described herein may be formulated to provide the equivalent of 15 g D-BHB in a single dose or up to 15 g D-BHB in a single dose, 12 g D-BHB in a single dose or up to 12 g D-BHB in a single dose, or 10 g D-BHB in a single dose or up to 10 g D-BHB in a single dose.
  • the crystalline form described herein may be formulated to provide the equivalent of 60 g D-BHB per day or up to 60 g D-BHB per day, 45 g D-BHB per day or up to 45 g D-BHB per day, 48 g D-BHB per day or up to 48 g D-BHB per day, 40 g D-BHB per day or up to 40 g D-BHB per day, 36 g D-BHB per day or up to 36 g D-BHB per day, 30 g D-BHB per day or up to 30 g D-BHB per day, 20 g D- BHB per day or up to 20 g D-BHB per day, 10 g D-BHB per day or up to 10 g D-BHB per day.
  • the amount of a particular crystalline form to be included in a formulation will depend on the molecular weight of the particular crystalline form and that this molecular weight will be considered to deliver the equivalent of the free D-BHB to a subject.
  • the molecule weights of each crystalline form in pharmaceutical formulations are to be taken into account in pharmaceutical formulations that include more than one crystalline form of D-BHB.
  • the invention comprises the following embodiments.
  • Embodiment 1 A crystalline D-BHB (“free Form I”) having an XRPD pattern comprising peaks at 10.93, 12.11, 15.16, 17.53, and 18.88 ⁇ 0.2° 20 using Cu Ka radiation.
  • Embodiment 2 The crystalline D-BHB (“free Form I”) of embodiment 1 wherein the XRPD pattern further comprising peaks at 22.75, 24.30, 26.14, 29.10, and 29.95 ⁇
  • Embodiment 3 The crystalline D-BHB (“free Form I”) of embodiment 1 or embodiment 2 wherein the XRPD pattern further comprising peaks at 30.48, 31.60, 32.96, 33.98, 36.80, and 38.37 ⁇ 0.2° 20 using Cu Ka radiation.
  • Embodiment 4 The crystalline D-BHB (“free Form I”) of any one of embodiments 1 to 3 having an XRPD pattern substantially as shown in Figure 1(A).
  • Embodiment 5 The crystalline D-BHB (“free Form I”) of any one of embodiments 1 to 4 having substantially the XRPD peaks as shown in Table 2.
  • Embodiment 6. The crystalline D-BHB (“free Form I”) of any one of embodiments 1 to 5, having substantially the DSC of Figure 2(A) and/or substantially the TGA of Figure 3(A).
  • Embodiment 7 A crystalline D-BHB sodium salt designated as “D-BHB sodium salt Form A”, having an XRPD pattern comprising peaks at 11.82, 17.02, 17.58, 20.34, and 20.83 ⁇ 0.2 20 using Cu Ka radiation.
  • Embodiment 8 The crystalline D-BHB sodium salt Form A of embodiment 7, wherein the XRPD pattern further comprises peaks at 7.23, 14.39, 24.50, 29.54, and 30.17 ⁇ 0.2° 20 using Cu Ka radiation.
  • Embodiment 9 The crystalline D-BHB sodium salt Form A of embodiment 7 or embodiment 8, wherein the XRPD pattern further comprises peaks at 12.22, 22.23, 22.60, 30.63, 33.35, and 37.28 ⁇ 0.2° 20 using Cu Ka radiation.
  • Embodiment 10 The crystalline D-BHB sodium salt Form A of any one of embodiments 7 to 9 having an XRPD pattern substantially as shown in Figure 9(A).
  • Embodiment 11 The crystalline D-BHB sodium salt Form A of any one of embodiments 7 to
  • Embodiment 12 The crystalline D-BHB sodium salt Form A of any one of embodiments 7 to
  • Embodiment 13 A crystalline D-BHB sodium salt designated “D-BHB sodium salt Form B” having an XRPD pattern comprising peaks at 8.98, 11.49, 16.95, 17.53, and 19.38 ⁇ O.2°20 using Cu Ka radiation.
  • Embodiment 14 The crystalline D-BHB sodium salt Form B of embodiment 14, wherein the XRPD pattern further comprises peaks at 7.17, 9.67, 12.16, 20.10, and 26.83 ⁇ 0.2° 20 using Cu Ka radiation.
  • Embodiment 15 The crystalline D-BHB sodium salt Form B of embodiment 13 or embodiment 14, wherein the XRPD pattern further comprises peaks at 6.47, 14.02, 14.35, 18.35, 21.00, and 23.08 ⁇ 0.2° 20 using Cu Ka radiation.
  • Embodiment 16 The crystalline D-BHB sodium salt Form B of any one of embodiments 12 to 15 having an XRPD pattern substantially as shown in Figure 10(A).
  • Embodiment 17 The crystalline D-BHB sodium salt Form B of any one of embodiments 12 to
  • Embodiment 18 The crystalline D-BHB sodium salt Form B of any one of embodiments 12 to
  • Embodiment 19 A crystalline D-BHB sodium salt designated as “D-BHB sodium salt Form C” having an XRPD pattern comprising peaks at 6.40, 7.07, 12.11, 14.08, and 17.05 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 20 The crystalline D-BHB sodium salt Form C of embodiment 19, wherein the XRPD pattern further comprises peaks at 19.16, 22.57, 27.79, 28.75, and 30.11 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 21 The crystalline D-BHB sodium salt Form C of embodiment 19 or embodiment 20, wherein the XRPD pattern further comprises peaks at 11.98, 30.76, 33.12, 33.63, and 37.08 ⁇ 0.2° 20 using Cu Ka radiation.
  • Embodiment 22 The crystalline D-BHB sodium salt Form C of any one of embodiments 19 to
  • Embodiment 23 The crystalline D-BHB sodium salt Form C of any one of embodiments 19 to
  • Embodiment 24 The crystalline D-BHB sodium salt Form C of any one of embodiments 19 to
  • Embodiment 25 A crystalline D-BHB magnesium salt designated as “D-BHB , magnesium salt Form A” having an XRPD pattern comprising peaks at 5.53, 7.80, 9.61, 11.95, and 13.15 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 26 The crystalline D-BHB magnesium salt Form A of embodiment 25, wherein the XRPD pattern further comprises peaks at 6.11, 11.02, 11.27, 15.61, and 18.98 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 27 The crystalline D-BHB magnesium salt Form A of embodiment 25 or embodiment 26, wherein the XRPD pattern further comprises peaks at 19.45, 21.13, 21.88, 23.70, and 25.99 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 28. The crystalline D-BHB magnesium salt form A of any one of embodiments 25 to 27 having an XRRD pattern substantially as shown in Figure 7(A).
  • Embodiment 29 The crystalline D-BHB magnesium salt form A of any one of embodiments 25 to 28 having substantially the XRPD peaks shown in Table 13.
  • Embodiment 30 The crystalline D-BHB magnesium salt form A of any one of embodiments 25 to 29 having substantially the DSC of Figure 7(B) and/or substantially the TGA of Figure 7(C).
  • Embodiment 31 A crystalline D-BHB magnesium salt designated as “D-BHB magnesium salt Form B” having an XRPD pattern comprising peaks at 6.12, 8.31, 9.60, 11.87, and 13.45 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 32 The crystalline D-BHB magnesium salt Form B of embodiment 31 , wherein the XRPD pattern further comprises peaks at 11.49, 12.23, 14.09, 14.31, and 18.46 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 33 The crystalline D-BHB magnesium salt Form B of embodiment 31 or embodiment 32, wherein the XRPD pattern further comprises peaks at 17.01, 19.61, 23.70, 24.62, 24.94, and 29.02 and 18.46 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 34 The crystalline D-BHB magnesium salt form B of any one of embodiments 31 to 33 having an XPRD pattern substantially as shown in Figure 8(A).
  • Embodiment 35 The crystalline D-BHB magnesium salt form B of any one of embodiments 31 to 34 having substantially the XRPD peaks shown in Table 14.
  • Embodiment 36 The crystalline D-BHB magnesium salt form B of any one of embodiments 31 to 35 having substantially the DSC of Figure 8(B) and/or substantially the TGA of Figure 8(C).
  • Embodiment 38 The crystalline D-BHB L-lysine salt Form A of embodiment 37, wherein the XRPD pattern further comprises peaks at 10.21, 26.93, 27.64, 28.45, and 29.87 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 39 The crystalline D-BHB L- lysine salt Form A of embodiment 37 or embodiment 38, wherein the XRPD pattern further comprises peaks at 17.87, 18.27, 19.73, 24.58, and 25.02 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 40 The crystalline D-BHB L-Lysine salt Form A of any one of embodiment 37 to
  • Embodiment 41 The crystalline D-BHB L-Lysine salt Form A of any one of embodiment 37 to
  • Embodiment 42 The crystalline D-BHB L-Lysine salt Form A of any one of embodiment 37 to
  • Embodiment 43 A crystalline D-BHB L-arginine salt designated as “D-BHB L-arginine Form A” having an XRPD pattern comprising peaks at 5.28, 7.28, 9.11, 14.99, and
  • Embodiment 44 The crystalline D-BHB L- arginine salt Form A of embodiment 43, wherein the XRPD pattern further comprises peaks at 17.00, 17.76, 18.32, 19.32, and 24.10 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 45 The crystalline D-BHB L- arginine salt Form A of embodiment 43 or embodiment 44, wherein the XRPD pattern further comprises peaks at 10.81, 11.08, 11.27, 11.75, and 26.96 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 46 The crystalline D-BHB L- arginine salt Form A of any one of embodiments 43 to 45 having an XRPD pattern substantially as shown in Figure 14(A).
  • Embodiment 47 The crystalline D-BHB L- arginine salt Form A of any one of embodiments 43 to 46 having substantially the XRPD peaks shown in Table 10.
  • Embodiment 48 The crystalline D-BHB L- arginine salt Form A of any one of embodiments 43 to 47 having substantially the DSC of Figure 14(B) and/or substantially the TGA of Figure 14(C).
  • Embodiment 49 A crystalline D-BHB L-arginine salt designated as “D-BHB L-arginine Form B” having an XRPD pattern comprising peaks at 7.30, 9.94, 11.28, 14.56, and
  • Embodiment 50 The crystalline D-BHB L-arginine salt Form B of embodiment 49, wherein the XRPD pattern further comprises peaks at 17.27, 18.33, 19.91, 21.90, and 23.49 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 51 The crystalline D-BHB L-arginine salt Form B of embodiment 49 or embodiment 50, wherein the XRPD pattern further comprises peaks at 24.12, 26.45, 26.99, 28.56, 33.56, and 34.16 ⁇ 0.2° 20 using Cu Ka radiation.
  • Embodiment 52 The crystalline D-BHB L- arginine salt Form B of any one of embodiments 49 to 51 having an XRPD pattern substantially as shown in Figure 30(A).
  • Embodiment 53 The crystalline D-BHB L- arginine salt Form B of any one of embodiments 49 to 52 having substantially the XRPD peaks shown in Table 25.
  • Embodiment 54 The crystalline D-BHB L- arginine salt Form B of any one of embodiments 49 to 53 having substantially the DSC of Figure 30(B) and/or substantially the TGA of Figure 30(C).
  • Embodiment 55 A crystalline D-BHB erbumine salt designated as “D-BHB erbumine salt Form A” having an XRPD pattern comprising peaks at 10.04, 10.77, 13.69, 14.71, and 18.24 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 56 The crystalline D-BHB erbumine salt Form A of embodiment 55, wherein the XRPD pattern further comprises peaks at 16.08, 16.95, 17.42, 19.37, and 20.07 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 57 The crystalline D-BHB erbumine salt Form A of embodiment 55 or embodiment 56, wherein the XRPD pattern further comprises peaks at 22.47, 24.35, 25.60, 26.05 and 28.36 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 58 The crystalline D-BHB erbumine salt Form A of any one of embodiments 55 to 57 having an XRPD pattern substantially as shown in Figure 25(A).
  • Embodiment 59 The crystalline D-BHB erbumine salt Form A of any one of embodiments 55 to 58 having substantially the XRPD peaks shown in Table 30.
  • Embodiment 60 The crystalline D-BHB erbumine salt Form A of any one of embodiments 55 to 59 having substantially the DSC of Figure 25(B) and/or substantially the TGA of Figure 25(C).
  • Embodiment 61 A crystalline D-BHB erbumine salt designated as “D-BHB erbumine salt Form B” having an XRPD pattern comprising peaks at 12.42, 16.07, 16.69, 17.62, and 18.35 ⁇ 0.2°20 using Cu Ka radiation.
  • Embodiment 62 The crystalline D-BHB erbumine salt Form B of embodiment 61, wherein the XRPD pattern further comprises peaks at 8.82, 20.71, 1. 1, 23.95, and 27.27 ⁇ 0.2° 20 using Cu Ka radiation.
  • Embodiment 64 The crystalline D-BHB erbumine salt Form B of any one of embodiments 61 to 63 having an XRPD pattern substantially as shown in Figure 26(B).
  • Embodiment 65 The crystalline D-BHB erbumine salt Form B of any one of embodiments 61 to 64 having substantially the XRPD peaks shown in Table 31.
  • Embodiment 66 The crystalline D-BHB erbumine salt Form B of any one of embodiments 61 to 65 having substantially the DSC of Figure 26(C) and/or substantially the TGA of Figure 26(D).
  • Embodiment 67 The crystalline D-BHB of any one of embodiments 1 to 66, wherein the D-BHB is greater than 95% enantiomerically pure, greater than 98% enantiomerically pure, or greater than 99% enantiomerically pure.
  • Embodiment 68 The crystalline D-BHB of any one of embodiments 1 to 67, wherein the D-BHB is greater than 99% enantiomerically pure.
  • Embodiment 69 A composition comprising one or more crystalline D-BHB forms as described in any one of embodiments 1 to 68.
  • Embodiment 70 The composition of embodiment 69, wherein the composition comprises D- BHB L-arginine salt Form B.
  • Embodiment 71 The composition of embodiment 70, wherein the composition consists essentially of or consists of D-BHB L-arginine salt Form B.
  • Embodiment 75 The composition of embodiment 74, wherein the composition comprises D- BHB L-arginine salt Form B.
  • Embodiment 79 A pharmaceutical composition comprising one or more crystalline D-BHB forms as described in any one of claims embodiments 1 to 68 and at least one pharmaceutically acceptable carrier.
  • Embodiment 80 The pharmaceutical composition of embodiment 79, wherein the composition comprises D-BHB L-arginine salt Form B.
  • Embodiment 81 The pharmaceutical composition of embodiment 80, wherein the composition consists essentially of or consists of D-BHB L-arginine salt Form B.
  • Embodiment 82 The pharmaceutical composition of embodiment 79, wherein the composition comprises D-BHB sodium salt Form C.
  • Embodiment 83 The pharmaceutical composition of embodiment 80, wherein the composition consists essentially of or consists of D-BHB sodium salt Form C.
  • Embodiment 84 A pharmaceutical composition comprising two or more crystalline D-BHB forms as described in any one of claims embodiments 1 to 68 and at least one pharmaceutically acceptable carrier.
  • Embodiment 85 The pharmaceutical composition of embodiment 84, wherein the composition comprises D-BHB L-arginine salt Form B.
  • Embodiment 86 The pharmaceutical composition of embodiment 84 or embodiment 85, wherein the composition comprises D-BHB sodium salt Form C.
  • Embodiment 87 The pharmaceutical composition of embodiment 85 or embodiment 86, wherein the composition comprises D-BHB L-arginine salt Form B and D- BHB sodium salt Form C.
  • Embodiment 88 The pharmaceutical composition of embodiment 87, wherein the composition consists essentially of or consists of D-BHB L-arginine salt Form B and D- BHB sodium salt Form C.
  • Embodiment 89 The pharmaceutical composition of any one of embodiments 79 to 88 formulated for oral administration.
  • Embodiment 90 The pharmaceutical composition of any one of embodiment 89, wherein the at least one carrier is an excipient.
  • Embodiment 91 The pharmaceutical composition of any one of embodiments 79 to 88 formulated for administration by injection.
  • Embodiment 92 The pharmaceutical composition of any one of embodiment 91, wherein the at least one carrier is water and/or an aqueous pH buffered solution.
  • Embodiment 93 The pharmaceutical composition of any one of embodiments 79 to 92, wherein the composition is formulated to administer up to
  • Embodiment 94 The pharmaceutical composition of any one of embodiments 79 to 92, wherein the composition is formulated to administer about 10 g D-BHB per dose.
  • Embodiment 95 The pharmaceutical composition of any one of embodiments 79 to 94, wherein the composition is formulated to administer up to 60 g D-BHB per day, up to 48 g D-BHB per day, up to 45 g D-BHB per day, up to 40 g D-BHB per day, 36 g D-BHB per day, up to 30 g D-BHB per day, up to 20 g D-BHB per day, or up to 10 g per day.
  • Embodiment 96 The pharmaceutical composition of embodiment 95, wherein the composition is formulated for administration as two doses per day, three doses per day, or four doses per day.
  • Trial 1 50mL of the 40wt% of D-BHB aqueous solution was added into a 500mL glass bottle. Release specifications for D-BHB is greater than 95% D enantiomer (i.e. greater than 95% enantiomeric purity of the D enantiomer). This aqueous solution was treated by lyophilization. Sugar like crystals were obtained after 3 days. Obtained sugar like crystals were very hygroscopic and deliquesced after exposure to ambient condition (20-25°C/40-70%RH) within 30 minutes. Therefore, obtained sugar like crystals were dried under vacuum at 25°C for 2 hours to remove surface moisture. 22.7 g of D-BHB free Form I was obtained. Obtained sugar like crystals were kept in a closed container. Referred to herein as sample: FR03684-1-LP1 (with XRPD shown in Figure 1(A) with the following peaks in Table 2).
  • Trial 2 50mL of the 40wt% of D-BHB aqueous solution was added into a 500mL glass bottle. This aqueous solution was treated by lyophilization. Sugar like crystals were obtained after 6 days. Obtained sugar like crystals were very hygroscopic and deliquesced after exposure to ambient condition (20-25°C/40-70%RH) within 30 minutes. Obtained sugar like crystals were dried under vacuum at 25°C for 1 day to remove surface moisture. 21.0 g of D-BHB free Form I was obtained. Obtained sugar like crystals were kept in a closed container. Referred to herein as sample: FR03684-1-LP2 (with XRPD shown in Figure 1(B) with the following peaks in Table 3).
  • Crystalline salt forms including sodium salt Form A, sodium salt Form B, physical mixtures of magnesium salt Form A and Mg(0H)2, physical mixtures of magnesium salt Form B and Mg(0H)2, L-arginine salt Form A, L- lysine salt Form A and erbumine salt Form A were obtained after equilibration in water, EtOH, acetone, EA, ACN and THF. Screening with Ca(OH)2 only led to gel. XRPD by Method 2 shown in Figure 5.
  • Figure 12(A) shows the XRPD of D-BHB L-lysine salt Form A (FR03684-3-RC7B-EtOH) having peaks shown in TABLE 8.
  • Figure 14(A) shows the XRPD of D-BHB L-arginine salt Form A (FR03684-3-RC6D-EA) having peaks as shown in Table 10.
  • Figure 15(A) shows the XRPD of D-BHB L-arginine salt Form B (FR03684-3-RC6H- acetone- water-95 -5) having peaks as shown in Table 11.
  • Condition 2 To get pure magnesium salt polymorphs for characterization, amorphous magnesium salt was also prepared for re-slurry.
  • Obtained amorphous hemi-magnesium salt was further treated by re-slurry at 25°C for at least 48 hours in 0.1-0.3mL of EtOH, EA, ACN and THF.
  • About 3mg of magnesium salt Form A and Mg(0H) 2 mixture seeds (FR03684-3-RC5B-EtOH and FR03684-3-RC5D-EA) was added into suspension RS9B and RS9D, respectively.
  • About 3mg of the magnesium salt Form B and Mg(0H) 2 mixture seeds (FR03684-3-RC5E-ACN and FR03684-3-RC5F-THF) was added into suspension RS9E and RS9F, respectively.
  • Example 4 Sodium Salt Crystalline Forms A, B, and C
  • the sodium salt Form A obtained from slurry equilibration in THF contained 1.5 equiv. of Na + , which is different from theoretical stoichiometry of the sodium salt. Therefore, screening with different ratios of NaOH were tried and crystalline sodium salt seeds were added during screening to optimize stoichiometry of sodium salt.
  • Sodium salt Form C (FR03684-SUl-NaOH-1.5-THF) was prepared as follows. 2.0g of the D-BHB free Form I (FR03684-1-LP1) and 1.2g of NaOH ( ⁇ 1.5 equivalent by molar ratio) were weighed into a 40mL glass vial. lOmL of THF was added into the vial under stirring at 25°C for about lOmin. A suspension was obtained. About Img of the sodium salt Form A seeds (FR03684-3-RC2D) was added into above suspension. The suspension was kept stirring at 25°C for about 2 days. About 5mL of suspension was taken out and centrifuged.
  • Trail 2 Sodium salt Form C (FR03684-SU9-NaOH-1.5-THF) was prepared as follows. 2.0g of the D-BHB free Form I (FR03684-1-LP2) and 1.2g of NaOH ( ⁇ 1.5 equivalent by molar ratio) were weighed into a 40mL glass vial. lOmL of THF was added into the vial under stirring at 25°C for about lOmin. A suspension was obtained. About Img of the sodium salt Form C seeds (FR03684-SUl-NaOH-1.5-THF as obtained from Trial 1) were added into above suspension. The suspension was kept stirring at 25°C for about 4 days. Solids were collected by filtration and then dried at 50°C under vacuum for about 2 hours. 2.6g of the sodium salt Form C (Figure 16(B) - XRPD Method 2) was obtained as an off-white solid in 81% yield. FR03684-SU9-NaOH-1.5-THF was used for solubility study in Example 12.
  • FIG. 17(A) shows the XRPD for D-BHB sodium salt Form C (FR03684-SU9-NaOH- 1.5-THF) having peaks as shown in Table 22.
  • Figure 19(A) shows the XRPD for D-BHB L-arginine salt Form B (FR03684-SU2-L- arginine-acetone-water-95-5-re) having peaks as shown in Table 24.
  • FR03684-SU5- L-arginine-acetone-water-95-5 was used for bulk stability study, solubility study and hygroscopicity in Examples 11 to 13.
  • the XRPD of D-BHB L-arginine salt Form B (FR03684- SU5-L-arginine-acetone-water-95-5) is shown in Figure 30(A) and has peaks as shown in Table 25. TABLE 25
  • L-lysine salt Form A (FR03684-SU3-L-lysine-EtOH-re) was prepared as follows. 2.0g of the D-BHB free Form I (FR03684-1-LP1) and 2.9g of L-lysine (-1.05 equivalent by molar ratio) were weighed into a lOOmL glass vial. 30mL of EtOH was added into the vial under stirring at 25°C for about lOmin. A suspension was obtained. About 3mg of the L-lysine salt Form A seeds (FR03684-3-RC7B) was added into above suspension. The suspension was kept stirring at 25°C for about 2 days.
  • Figure 31(A) shows the XPRD for D-BHB L-lysine salt Form A (FR03684-SU6-L-lysine- EtOH) having peaks as shown in Table 28. TABLE 28
  • Example 8 Scale up of D-BHB erbumine salt Form A
  • Erbumine salt Form A (FR03684-SU4-erbumine-EA) was prepared as follows. 2.0g of the D-BHB free Form I (FR03684-1-LP1) and 1.5g of erbumine (-1.05 equivalent by molar ratio) were weighed into a 20mL glass vial. 4.0 mL of EA was added into the vial under stirring at 25 °C for about lOmin. A suspension was obtained. About 3 mg of the erbumine salt Form A seeds (FR03684-3-RC9C) were added into above suspension. The suspension was kept stirring at 25°C for about 2 days. About 2mL of suspension was taken out and solid part (wet cake) was characterized by XRPD.
  • erbumine salt Form B ( Figure 24) was obtained. After it was dried at 50°C under vacuum for about 2 hours, the erbumine salt Form B converted to the erbumine salt Form A ( Figure 24). Rest of solids were collected by filtration and then dried at 50°C under vacuum for about 2 hours. 2.5g of the erbumine salt Form A was obtained as an off-white solid in 71% yield. FR03684-SU4-erbumine-EA was used for minipolymorph screening reported in Example 10.
  • Erbumine salt Form B (FR03684-SU8-erbumine-ACN-water-95-5) was prepared as follows. 2.0g of the D-BHB free Form I (FR03684-1-LP2) and 1.5g of erbumine (-1.05 equivalent by molar ratio) were weighed into a 20mL glass vial. 10 m of ACN was added into the vial under stirring at 25°C for about lOmin. A suspension was obtained. About Img of the erbumine salt Form B seeds (FR03684-3-PS8D) were added into above suspension. The suspension was kept stirring at 25°C for about 1 day. About 0. ImL of suspension was taken out and solid part was characterized by XRPD without Kapton film.
  • Salts were placed at 25°C/92.5%RH in an open container, at 25°C/60%RH in an open container, at 40°C/75% RH in an open container and at 60°C in a closed container for 2 weeks. Samples after the stress were characterized by XRPD and IC and inspected for color change.
  • the sesqui-sodium salt Form C and the L-lysine salt Form A were physically stable with no form change after stressed at 60°C in a tight container over 2 weeks. However, each of these were partially deliquesced or deliquesced after stressed at 25°C/92.5%RH in an open container, at 25°C/60%RH in an open container, or at 40°C/75%RH in an open container over 2 weeks.
  • the L-arginine salt Form B and the erbumine salt Form B showed obvious advantage in physical stability at 25°C/60%RH. Each form remained as a solid and showed no physical change after placed at 25°C/60%RH in an open container over 2 weeks.
  • the L-arginine salt Form B was also physically stable with no form change after stressed at 60°C in a tight container over 2 weeks, while the erbumine salt Form B dehydrated and converted to the erbumine salt Form A after stressed at 60°C in a tight container over 2 weeks.
  • Both the L-arginine salt Form B and the erbumine salt Form B are not tolerant to high humidity. They deliquesced after stressed at 25°C/92.5%RH in an open container, or at 40°C/75%RH in an open container over 2 weeks.
  • Solubility of the salts were measured in comparison of the D-BHB free Form I in seven aqueous pH buffers and bio-relevant fluids, including pH 1.2 HC1 buffer, pH 4.5 acetate buffer (50mM), pH 6.8 phosphate buffer (50mM), water, pH 1.6 FaSSGF (Fasted State Simulated Gastric Fluid - Biorelevant), pH 6.5 FaSSIF-vl (Fasted State Simulated Intestinal Fluid - Biorelevant), and pH 5.0 FeSSIF-vl (Fed State Simulated Intestinal Fluid - Biorelevant), at 37°C for 2 hours and 24 hours.
  • pH 1.2 HC1 buffer pH 4.5 acetate buffer (50mM), pH 6.8 phosphate buffer (50mM)
  • water pH 1.6 FaSSGF (Fasted State Simulated Gastric Fluid - Biorelevant)
  • pH 6.5 FaSSIF-vl Feasted State Simulated Intestinal Fluid - Biorelevant
  • pH 5.0 FeSSIF-vl Fed
  • Trial 1 Accurate l.Olmg of the D-BHB free Form I (FR03684-3 -LP2), 1.43mg of the sodium salt Form C (FR03684-SUl-NaOH-1.5-THF), 1.49mg of the sodium salt Form C (FR03684-SU9-NaOH-1.5-THF), 3.04mg of the L-arginine salt Form B FR03684-SU5-L- arginine-acetone- water-95-5), 2.58mg of theL-lysine saltForm A (FR03684-SU6-L-lysine-EtOH) or 1.89mg of the erbumine salt Form B (FR03684-SU8-erbumine-ACN-water-95-5) was weighed into a 2mL glass vial, respectively.
  • solubility medium including pH 1.2 HC1 buffer, pH 4.5 acetate buffer, pH 6.8 phosphate buffer water, FaSSGF, FaSSIF and FeSSIF.
  • the salt amount used are equivalent to 1 mg anhydrous free form.
  • Trial 2 Accurate 5.06mg of the free Form I (FR03684-3-LP2), 7.14mg of the sodium salt Form C (FR03684-SUl-NaOH-1.5-THF), 7.47mg of the sodium salt Form C (FR03684-SU9- NaOH-1.5-THF), 15.18mg of the L-arginine salt Form B (FR03684-SU5-L-arginine-acetone- water-95-5), 12.89mg of the L-lysine salt Form A (FR03684-SU6-L-lysine-EtOH), or 9.43mg of the erbumine salt Form B (FR03684-SU8-erbumine-ACN-water-95-5) was weighed into a 2mL glass vial, respectively.
  • solubility medium including pH 1.2 HC1 buffer, pH 4.5 acetate buffer, pH 6.8 phosphate buffer, water, FaSSGF, FaSSIF andFeSSIF.
  • the salt amount used are equivalent to 5mg anhydrous free form.
  • Trial 3 Accurate 253.04mg D-BHB free Form I (FR03684-3-LP2), 753.54mg L-arginine salt Form B (sample ID FR03684-SU2-L-arginine-acetone-water-95-5-re), 603.66mg L-lysine salt Form A (sample ID FR03684-SU3-L-lysine-EtOH-re), 321.16mg sodium salt Form C (sample ID FR03684-SUl-NaOH-1.5-THF), or 377.25mg of the erbumine salt Form B (sample ID FR03684- SU8-erbumine-ACN-water-95-5) was weighed into a 2mL glass vial, respectively.
  • ImL of pH 1.2 HC1 buffer, pH 4.5 acetate buffer or pH 6.8 phosphate buffer was added in the D-BHB free Form I, L-arginine salt Form B and L-lysine salt Form A.
  • the D-BHB free Form I, L-arginine salt Form B and L-lysine salt Form A amount used are equivalent to 250mg anhydrous free Form I.
  • 0.9mL of pH 1.2 HC1 buffer, pH 4.5 acetate buffer or pH 6.8 phosphate buffer was added in sodium salt Form C.
  • the sodium salt amount used is equivalent to 225mg anhydrous free form.
  • 0.8mL of pH 1.2 HC1 buffer, pH 4.5 acetate buffer or pH 6.8 phosphate buffer was added in erbumine salt Form B.
  • the erbumine salt Form B used is equivalent to 200mg anhydrous free form.
  • D-BHB free Form I the sodium salt Form C, the L-arginine salt Form B, and the L-lysine salt Form A.
  • the D-BHB free Form I, the sodium salt Form C, the L-arginine salt Form B, the L-lysine salt Form A amount used are equivalent to 75mg anhydrous D-BHB free Form I.
  • 0.5mL of water was added to the erbumine salt Form B.
  • the erbumine salt Form B amount used is equivalent to 125mg anhydrous free form.
  • Both the D-BHB crystalline salts and the D-BHB free Form I demonstrated good solubility in selected aqueous media. They showed >10mg/mL solubility in pH 1.6FaSSGF, pH 6.5 FaSSIF- vl, and pH 5.0 FeSSIF-vl. When target concentration for solubility test was increased to 250mg/mL, the D-BHB free Form I showed >250mg/mL solubility in pH 1.2 HC1 buffer, pH 4.5 acetate buffer, pH 6.8 phosphate buffer and water.
  • the D-BHB salts showed 170-240mg/mL solubility in pH 1.2 HC1 buffer, 120-160mg/mL solubility in pH 4.5 acetate buffer and 100- 160mg/mL in pH 6.8 phosphate buffer after 24h.
  • Water uptake water sorption in a specific RH (80% to 95%) - water sorption in 40%RH
  • the criteria are modified from the European Pharmacopeia criteria about hygroscopicity
  • the criteria are modified from the European Pharmacopeia criteria about hygroscopicity
  • the L-arginine salt Form B is moderately hygroscopic below 70%RH. After the DVS test, the L-arginine salt Form B converted to an amorphous form.
  • the erbumine salt Form B underwent dehydration below 20%RH and lost about 9.4% water. The water was absorbed back after the relative humidity (RH) was increased to 50%RH. Then the erbumine salt Form B became very hygroscopic in above 60%RH and deliquesced in high humidity. After the DVS test, and the erbumine salt Form B converted to a liquid state.
  • Sodium salt Form C is a hydrate.
  • Sample FR03684-SUl-NaOH-1.5-THF is of high crystallinity.
  • DSC shows multiple thermal events.
  • TGA shows about 7.8% weight loss at about 130°C.
  • IC shows D-BHB: Na+ is 1: 1.5.
  • 1H-NMR shows no detectable residual solvent.
  • KF shows it contains about 6.7% water by weight, equivalent to 0.6 water molecule.
  • L-arginine salt Form B is a hydrate.
  • Sample FR03684-SU5-L-arginine-acetone-water-95- 5 is of high crystallinity.
  • DSC shows a dehydration T on set of 89.2°C. Melting occurs upon dehydration. TGA shows about 6.3% weight loss at about 130°C.
  • 1H-NMR shows D-BHB: L-arginine is 1: 1.0 and no detectable residual solvent.
  • KF shows it contains about 6.2% water by weight, equivalent to 1.1 water molecule.
  • L-lysine salt Form A is an anhydrate.
  • Sample FR03684-SU6-L-lysine-EtOH is of high crystallinity.
  • DSC shows a melting T on set of 108.4°C with an enthalpy of about 79J/g.
  • TGA shows about 4.1% weight loss at about 100°C.
  • 1H-NMR shows D-BHB: L-lysine is 1: 1.0 and no detectable residual solvent.
  • Erbumine salt Form B is a hydrate.
  • Sample FR03684-SU8-erbumine-ACN-water-95-5 is of high crystallinity.
  • DSC shows a dehydration Tonset of 51.9°C and then a melting T on set of 72.8°C.
  • TGA shows about 10.3% weight loss at about 90°C.
  • 1H-NMR shows D-BHB: erbumine is 1: 1.0 and no detectable residual solvent.
  • KF shows it contains about 9.7% water by weight, equivalent to 1.1 water molecule.
  • D-BHB sodium salt Form C hydrate (C210608011-B/PJ06432-24-B-DRY) and D-BHB L-arginine salt Form B, hydrate (C210608011-FP/PJ06432-23-FP-DRY) (Table 45) were studied at 25°C ⁇ 2°C/60%RH ⁇ 5%RH, 40°C ⁇ 2°C/75%RH ⁇ 5%RH, 50°C ⁇ 2°C/75%RH ⁇ 5%RH. The samples were tested at weeks 1, 2, 3, and 4.
  • the bulk substance was studied in a container closure system that simulates packaging of bulk materials: approximately 1.0 g/package for each of the tests and time points, with the sample packed into double antistatic LDPE bag that was secured with a cable tie. 4 bags of desiccant were then put between two layers of antistatic LDPE bag. Each package (sample in bag with 4 bags of desiccant) into a foil bag that was heat sealed. The foil bag was then stored within a Fiber drum. The drums were then stored in a stability chamber at the conditions noted above with the temperature and humidity monitored and recorded. The tests were Appearance, Related substance by Ion Chromatography (IC), Potency, Coulometric water content determination according to Karl Fischer, XPRD, and Chiral purity.
  • IC Ion Chromatography
  • D-BHB sodium salt Form C, hydrate (C210608011-B/PJ06432-24-B-DRY): Throughout the course of the four-week study, at each of the three temperature/relative humidity conditions, the compound: Maintained a constant appearance, without change: white solid; Maintained chiral purity, without change: 100.0 %; and Maintained XPRD of Form C, without change.
  • D-BHB L-arginine salt Form B hydrate (C210608011-FP/PJ06432-23-FP-DRY) Throughout the course of the four-week study, at each of the three temperature/relative humidity conditions, the compound: Maintained a constant appearance, without change: white solid; Maintained chiral purity, without change: 100.0 %; and Maintained XPRD of Form B, without change.

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Abstract

Provided herein are crystalline forms of D-beta-hydroxybutyrate (D-BHB) and composition comprising one or more crystalline forms of D-BHB.

Description

TITLE
POLYMORPHS AND COMPOSITIONS COMPRISING SAME
TECHNIAL FIELD
[001] The invention relates to polymorphs of D-beta-hydroxybutyrate (D-BHB).
BACKGROUND
[002] The compound D-beta-hydrxybutyrate (D-BHB), which may also be referred to as R-beta- hydrxy butyrate or (R)-3 -hydroxy butrate and is the conjugate base of D-beta-hy dr oxybutyric acid (shown below).
OH O HgC^^OH
[003] D-BHB is of interest as a ketone body that has been associated with a variety of health benefits. However, due to high doses of D-BHB that may be associated with achieving a therapeutic benefit, there is a concern about high mineral intake from safety/compliance perspective. There is a need for various new salt and crystalline forms of D-BHB with different chemical and physical stabilities, and formulations of the same.
SUMMARY
[004] Provided herein are crystalline forms of D-BHB or a salt thereof, including free base crystalline forms, crystalline salts and crystalline solvates. Provided herein are crystalline sodium salt forms A, B, and C; crystalline magnesium salt forms A and C; crystalline L-lysine form A, crystalline erbumine salt forms A and B; and crystalline L-arginine salt forms A and B.
[005] Also provided are pharmaceutical compositions comprising any one or more of the crystalline salt forms disclosed herein and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[006] Examples of the invention will now be described in detail with reference to the accompanying drawings, in which:
[007] Figure 1 depicts an X-ray powder diffraction (“XRPD”) pattern of free base crystalline forms of D-BHB obtained from condition 1 (A) (XRPD Method 2) and condition 2 (B) (XRPD Method 2). [008] Figure 2 depicts a differential scanning calorimetry (“DSC”) thermograph of free base crystalline forms of D-BHB obtained from condition 1 (A) and condition 2 (B).
[009] Figure 3 depicts a thermogravimetric analysis (“TGA”) trace of free base crystalline forms of D-BHB obtained from condition 1 (A) and condition 2 (B).
[010] Figure 4 depicts 'H-NMR spectrum of free base crystalline forms of D-BHB obtained from condition 1 (A) and condition 2 (B).
[Oil] Figure 5 XRPD (Method 2) of (A) sodium salt screening; (B) magnesium salt screening; (C) L-arginine salt screening; (D) L-lysine salt screening; (E) erbumine salt screening; (F) betaine salt screening; and (G) re-slurry screening of L-arginine salt screening.
[012] Figure 6 overlay of XRPD (Method 2) of crystalline magnesium salt forms A and B.
[013] Figure 7 D-BHB magnesium salt Form A (FR03684-3-RC5D-re-EA) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ‘H-NMR spectrum.
[014] Figure 8 D-BHB magnesium salt Form B (FR03684-3 -RC5E-re-ACN) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) 1 H-NMR spectrum.
[015] Figure 9 D-BHB sodium salt Form A (FR03684-3-RC2F-THF) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ‘H-NMR spectrum.
[016] Figure 10 D-BHB sodium salt Form B (FR03684-3-RC2C-Acetone) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ‘H-NMR spectrum.
[017] Figure 11 D-BHB sodium salt Form C (FR03684-3-RC14F-THF) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ‘H-NMR spectrum.
[018] Figure 12 D-BHB L-lysine salt Form A (FR03684-3-RC7B-EtOH) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ‘H-NMR spectrum.
[019] Figure 13 D-BHB erbumine salt Form A (FR03684-3-RC9D-EA) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ‘H-NMR spectrum.
[020] Figure 14 D-BHB L-argimne salt Form A (FR03684-3-RC6D-EA) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ‘H-NMR spectrum.
[021] Figure 15 D-BHB L-arginine salt Form B (FR03684-3 -RC6H- acetone-water-95-5) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ‘H-NMR spectrum.
[022] Figure 16 D-BHB sodium salt Form C XRPD (Method 2) (A) FR03684-SUl-NaOH-1.5- THF and (B) FR03684-SU9-NaOH-1.5-THF. [023] Figure 17 D-BHB sodium salt Form C (FR03684-SU9-NaOH-1.5-THF) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ' H-NMR spectrum.
[024] Figure 18 D-BHB L-arginine salt Form B (FR03684-SU2-L-arginine-acetone-water-95-5- re ) (A) overlay of XRPD (Method 2) of scale up Trial land (B) 'H-NMR spectrum.
[025] Figure 19 D-BHB L-arginine salt Form B (FR03684-SU2-L-arginine-acetone-water-95-5- re) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ' H-NMR spectrum.
[026] Figure 20 D-BHB L -arginine salt Form B (FR03684-SU5-L-arginine-acetone-water-95-5) (A) overlay of XRPD (Method 2) of scale up Trial 2 and (B) ' H-NMR spectrum.
[027] Figure 21 D-BHB L-lysine salt Form A FR03684-SU3-L-lysine-EtOH-re (A) overlay XRPD (Method 2) from scale up and (B) 'H-NMR spectrum.
[028] Figure 22 D-BHB L-lysine salt Form A (FR03684-SU3-L-lysine-EtOH-re) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ' H-NMR spectrum.
[029] Figure 23 D-BHB L-lysine salt Form A (FR03684-SU6-L-lysine-EtOH) (A) XRPD (Method 2) overlay of Trial 2 and (B) 'H-NMR spectrum.
[030] Figure 24 Overlay of XRPD (Method 2) of D-BHB erbumine salt Form A and Form B from scale-up.
[031] Figure 25 D-BHB erbumine salt Form A (FR03684-SU4-erbumine-EA) (A) XRPD (Method 2) (B) DSC (C) TGA and (D) ' H-NMR spectrum.
[032] Figure 26 (A) Overlay of XRPD (Method 3) of D-BHB erbumine salt Form A and Form B from scale-up of process to obtain form B; D-BHB erbumine salt Form B (FR03684-SU8- erbumine-ACN-water-95-5) (B) XRPD (Method 1) (C) DSC (D) TGA and (E) ' H-NMR spectrum [033] Figure 27 Mini-polymorph screening - suspensions equilibrated at 25°C for 1 week : (A) to (C) D-BHB sodium salt Form C (FR03684-SUl-NaOH-1.5-THF) as starting material - XRPD overlay; (D) and (E) D-BHB L-arginine salt Form C (FR03684-SU2-L-arginine-acetone-water- 95-5) as starting material - XRPD overlay; (F) and (G) D-BHB L-lysine salt Form A (FR03684- SU3-L-lysine-EtOH-re) as starting material - XRPD overly; (H) and (I) D-BHB erbumine salt Form A (FR03684-SU4-erbumine-EA) as starting material - XRPD overly. XRPD Method 4 for all except (C) which was Method 2.
[034] Figure 28 Bulk Stability XRPD (Method 2) (A) L-arginine salt Form B (FR03684-SU5- L-arginine-acetone-water-95-5); (B) D-BHB Erbumine salt Form B (FR03684-SU8-erbumine- ACN-water-95-5); (C) D-BHB sodium salt Form C (FR03684-SUl-NaOH-1.5-THF); (D) D-BHB L-lysine salt form A (FR03684-SU6-L-lysine-EtOH)
[035] Figure 29 D-BHB sodium salt Form C (FR03684-SUl-NaOH-1.5-THF) (A) XRPD (Method 1) (B) DSC (C) TGA and (D) 1 H-NMR spectrum.
[036] Figure 30 D-BHB L -arginine salt Form B (FR03684-SU5-L-arginine-acetone-water-95-5) (A) XRPD (Method 1) (B) DSC (C) TGA and (D) 'H-NMR spectrum.
[037] Figure 31 D-BHB L-lysine salt Form A (FR03684-SU6-L-lysine-EtOH) (A) XRPD (Method 1) (B) DSC (C) TGA and (D) 'H-NMR spectrum.
DETAILED DESCRIPTION
[038] Disclosed are crystalline forms of D-BHB. Embodiments of crystalline free base forms, salt forms and solvates of D-BHB can be characterized by one or more parameters as described below.
[039] As used herein “D-BHB” refers to the compound D-beta-hydrxybutyrate (D-BHB), which may also be referred to as R-beta-hydrxybutyrate or (R)-3-hydroxybutrate and is the conjugate base of D-beta-hydroxybutyric acid (shown below).
OH O HgC^^OH
[040] In the context of the invention, D-BHB used as starting material and/or the D-BHB crystalline free form or salts are greater than about 95% enantiomerically pure, greater than about 98% enantiomerically pure, or greater than about 99% enantiomerically pure. “Enantiomerically pure” as used herein refers to the percentage of one enantiomer versus the other enantiomer, e.g. the percentage of D-BHD versus L-BHB in a preparation of D-BHB. It is understood that enantiomeric pairs (i.e. the R and S structural configuration of a compound with a chiral atom using absolute configuration convention , or D or L structural configuration of a compound with a chiral atom using the convention in relation to L- and D-glyceraldehyde as represented in Fischer projections) may be produced as a mixture having different percentages of the respective enantiomers. In the context of the invention, D-BHB as starting material to generate the crystalline free form and/or D-BHB crystalline salts is greater than about 95% D-BHB and has less than about 5% L-DHB, greater than about 98% D-BHB and less than about 2% L-BHB, greater than about 99% D-BHB and less than about 1% L-BHB, or greater than about 99.5% D-BHB and less than about 0.5% L-BHB. A crystalline D-BHB having greater than 95% enantiomeric purity is understood as being comprised of greater than 95% D-BHB and less than 5% L-BHB. A crystalline D-BHB having greater than 98% enantiomeric purity is understood as being comprised of greater than 98% D-BHB and less than 2% L-BHB. A crystalline D-BHB having greater than 99% enantiomeric purity is understood as being comprised of greater than 99% D-BHB and less than 1% L-BHB. A crystalline D-BHB having greater than 99.5% enantiomeric purity is understood as being comprised of greater than 99.5% D-BHB and less than 0.5% L-BHB.
[041] As used herein, “about,” “approximately” and “substantially” are understood to refer to numbers in a range of numerals, for example the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1% to +1% of the referenced number, most preferably -0.1% to +0.1% of the referenced number. All numerical ranges herein should be understood to include all integers, whole or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth. All ranges are inclusive of the endpoints of the range. For example, an amount between 1 and 10 includes both 1 and 10.
[042] As used in this disclosure and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
[043] The words “comprise,” “comprises” and “comprising” are to be interpreted inclusively rather than exclusively. Likewise, the terms “include,” “including” and “or” should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. Nevertheless, the compositions and methods disclosed herein may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of’ and “consisting of’ the components or steps identified.
[044] The terms “at least one of’ and “and/or” used respectively in the context of “at least one of X and Y” and “X and/or Y” should be interpreted as “X without Y,” or “Y without X,” or “both X and Y.” Where used herein, the terms “example” and “such as,” particularly when followed by a listing of terms, are merely exemplary and illustrative and should not be deemed to be exclusive or comprehensive. [045] Crystalline free base form of D-BHB
[046] Proved are crystalline free base forms of D-BHB (D-BHB free Form I).
[047] Crystalline free base Form can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 10.93, 12.11, 15.16, 17.53, and 18.88 ± 0.2° 20 using Cu Ka radiation. Form I can be further characterized by having an XRPD pattern having additional peaks at 22.75, 24.30, 26.14, 29.10, and 29.95 ± 0.2° 20 using Cu Ka radiation. Form I can be further characterized by having an XRPD pattern having additional peaks at 30.48, 31.60, 32.96, 33.98, 36.80, and 38.37 ± 0.2° 20 using Cu Ka radiation. In some embodiments, Form I has an XRPD pattern substantially as shown in Figure 1(A), wherein by “substantially” is meant that the reported peaks can vary by about ± 0.2°. It is well known in the field of XRPD that while relative peak heights in spectra are depending on a number of factors, such as sample preparation and instrument geometry, peak positions are relatively insensitive to experimental details.
[048] Form I may also be characterized by DSC substantially as set forth in Figure 2(A) and/or by the TGA set forth in Figure 3(A). In some embodiments, crystalline free base Form I may be characterized by one or more of an XRPD substantially as depicted in Figure 1(A), DSC substantially as set forth in Figure 2(A), and TGA as set forth in Figure 3(A).
[049] Crystalline sodium salts
[050] D-BHB crystalline sodium salt Form A can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 11.82, 17.02, 17.58, 20.34, and 20.83 ± 0.220 using Cu Ka radiation. Sodium salt Form A can be further characterized by having an XRPD pattern having additional peaks at 7.23, 14.39, 24.50, 29.54, and 30.17 ± 0.2° 20 using Cu Ka radiation. Sodium salt Form A can be further characterized by having an XRPD pattern having additional peaks at 12.22, 22.23, 22.60, 30.63, 33.35, and 37.28 ± 0.2° 20 using Cu Ka radiation. In some embodiments, sodium salt Form A has an XRPD pattern substantially as shown in Figure 9(A).
[051] D-BHB crystalline sodium salt Form A may also be characterized by DSC substantially as set forth in Figure 9(B) and/or by the TGA set forth in Figure 9(C). In some embodiments, D- BHB crystalline sodium salt Form A may be characterized by one or more of an XRPD substantially as depicted in Figure 9(A), DSC substantially as set forth in Figure 9(B), and TGA as set forth in Figure 9(C).
[052] D-BHB crystalline sodium salt Form B can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 8.98, 11.49, 16.95, 17.53, and 19.38 ± O.2°20 using Cu Ka radiation. Sodium salt Form B can be further characterized by having an XRPD pattern having additional peaks at 7.17, 9.67, 12.16, 20.10, and 26.83 ± 0.2°20 using Cu Ka radiation. Sodium salt Form B can be further characterized by having an XRPD pattern having additional peaks at 6.47, 14.02, 14.35, 18.35, 21.00, and 23.08 ± 0.2°20 using CuKa radiation. In some embodiments, sodium salt Form B has an XRPD pattern substantially as shown in Figure 10(A).
[053] D-BHB crystalline sodium salt Form B may also be characterized by DSC substantially as set forth in Figure 10(B) and/or by the TGA set forth in Figure 10(C). In some embodiments, D- BHB crystalline sodium salt Form B may be characterized by one or more of an XRPD substantially as depicted in Figure 10(A), DSC substantially as set forth in Figure 10(B), and TGA as set forth in Figure 10(C).
[054] D-BHB crystalline sodium salt Form C can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 6.40, 7.07, 12.11, 14.08, and 17.05 ± 0.2°20 using Cu Ka radiation. Sodium salt Form C can be further characterized by having an XRPD pattern having additional peaks at 19.16, 22.57, 27.79, 28.75, and 30.11 ± 0.2°20 using Cu Ka radiation. Sodium salt Form C can be further characterized by having an XRPD pattern having additional peaks at 11.98, 30.76, 33.12, 33.63, and 37.08 ± 0.2°20 using Cu Ka radiation. In some embodiments, sodium salt Form C has an XRPD pattern substantially as shown in Figure 29(A).
[055] D-BHB crystalline sodium salt Form C may also be characterized by DSC substantially as set forth in Figure 29(B) and/or by the TGA set forth in Figure 29(C). In some embodiments, D- BHB crystalline sodium salt Form C may be characterized by one or more of an XRPD substantially as depicted in Figure 29(A), DSC substantially as set forth in Figure 29(B), and TGA as set forth in Figure 29(C).
[056] Crystalline magnesium salts
[057] D-BHB crystalline magnesium salt Form A can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 5.53, 7.80, 9.61, 11.95, and 13.15 ± 0.2°20 using Cu Ka radiation. Magnesium salt Form A can be further characterized by having an XRPD pattern having additional peaks at 6.11, 11.02, 11.27, 15.61, and 18.98 ± 0.2°20 using Cu Ka radiation. Magnesium salt Form A can be further characterized by having an XRPD pattern having additional peaks at 19.45, 21.13, 21.88, 23.70, and 25.99 ± 0.2°20 using Cu Ka radiation. In some embodiments, magnesium salt Form A has an XRPD pattern substantially as shown in Figure 7(A). [058] D-BHB crystalline magnesium salt Form A may also be characterized by DSC substantially as set forth in Figure 7(B) and/or by the TGA set forth in Figure 7(C). In some embodiments, D- BHB crystalline magnesium salt Form A may be characterized by one or more of an XRPD substantially as depicted in Figure 7(A), DSC substantially as set forth in Figure 7(B), and TGA as set forth in Figure 7(C).
[059] D-BHB crystalline magnesium salt Form B can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 6.12, 8.31, 9.60, 11.87, and 13.45 ± 0.2°20 using Cu Ka radiation. Magnesium salt Form B can be further characterized by having an XRPD pattern having additional peaks at 11.49, 12.23, 14.09, 14.31, and 18.46 ± 0.2°20 using Cu Ka radiation. Magnesium salt Form B can be further characterized by having an XRPD pattern having additional peaks at 17.01, 19.61, 23.70, 24.62, 24.94, and 29.02 and 18.46 ± 0.2°20 using Cu Ka radiation. In some embodiments, magnesium salt Form B has an XRPD pattern substantially as shown in Figure 8(A).
[060] D-BHB crystalline magnesium salt Form B may also be characterized by DSC substantially as set forth in Figure 8(B) and/or by the TGA set forth in Figure 8(C). In some embodiments, D- BHB crystalline magnesium salt Form B may be characterized by one or more of an XRPD substantially as depicted in Figure 8(A), DSC substantially as set forth in Figure 8(B), and TGA as set forth in Figure 8(C).
[061] Crystalline L-Lysine salt
[062] D-BHB crystalline L-lysine salt Form A can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 6.92, 9.16, 12.62, 23.29, and 25.91 ± 0.2°20 using Cu Ka radiation. L-lysine salt Form A can be further characterized by having an XRPD pattern having additional peaks at 10.21, 26.93, 27.64, 28.45, and 29.87 ± 0.2°20 using Cu Ka radiation. L-lysine salt Form A can be further characterized by having an XRPD pattern having additional peaks at 17.87, 18.27, 19.73, 24.58, and 25.02 ± 0.2°20 using Cu Ka radiation. In some embodiments, L-lysine salt Form A has an XRPD pattern substantially as shown in Figure 31(A). [063] D-BHB crystalline L-lysine salt Form A may also be characterized by DSC substantially as set forth in Figure 31(B) and/or by the TGA set forth in Figure 31(C). In some embodiments, D-BHB crystalline L-lysine salt Form A may be characterized by one or more of an XRPD substantially as depicted in Figure 31(A), DSC substantially as set forth in Figure 31(B), and TGA as set forth in Figure 31(C). [064] Crystalline L-arginine salts
[065] D-BHB crystalline L-arginine salt Form A can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 5.28, 7.28, 9.11, 14.99, and 15.82 ± 0.2°20 using Cu Ka radiation. L-arginine salt Form A can be further characterized by having an XRPD pattern having additional peaks at 17.00, 17.76, 18.32, 19.32, and 24.10 ± 0.2°20 using Cu Ka radiation. L-arginine salt Form A can be further characterized by having an XRPD pattern having additional peaks at 10.81, 11.08, 11.27, 11.75, and 26.96 ± 0.2°20 using Cu Ka radiation.In some embodiments, L-arginine salt Form A has an XRPD pattern substantially as shown in Figure 14(A).
[066] D-BHB crystalline L-arginine salt Form A may also be characterized by DSC substantially as set forth in Figure 14(B) and/or by the TGA set forth in Figure 14(C). In some embodiments, D-BHB crystalline L-arginine salt Form A may be characterized by one or more of an XRPD substantially as depicted in Figure 14(A), DSC substantially as set forth in Figure 14(B), and TGA as set forth in Figure 14(C).
[067] D-BHB crystalline L-arginine salt Form B can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 7.30, 9.94, 11.28, 14.56, and 15.83 ± 0.2°20 using Cu Ka radiation. L-arginine salt Form B can be further characterized by having an XRPD pattern having additional peaks at 17.27, 18.33, 19.91, 21.90, and 23.49 ± 0.2°20 using Cu Ka radiation. L-arginine salt Form B can be further characterized by having an XRPD pattern having additional peaks at 24.12, 26.45, 26.99, 28.56, 33.56, and 34.16 ± 0.2°20 using Cu Ka radiation. In some embodiments, L-arginine salt Form B has an XRPD pattern substantially as shown in Figure 30(A). [068] D-BHB crystalline L-arginine salt Form B may also be characterized by DSC substantially as set forth in Figure 30(B) and/or by the TGA set forth in Figure 30(C)). In some embodiments, D-BHB crystalline L-arginine salt Form B may be characterized by one or more of an XRPD substantially as depicted in Figure 30(A), DSC substantially as set forth in Figure 30(B), and TGA as set forth in Figure 30(C).
[069] Crystalline erbumine salts
[070] D-BHB crystalline erbumine salt Form A can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 10.04, 10.77, 13.69, 14.71, and 18.24 ± 0.2°20 using Cu Ka radiation. Erbumine salt Form A can be further characterized by having an XRPD pattern having additional peaks at 16.08, 16.95, 17.42, 19.37, and 20.07 ± 0.2°20 using Cu Ka radiation. Erbumine salt Form A can be further characterized by having an XRPD pattern having additional peaks at 22.47, 24.35, 25.60, 26.05 and 28.36 ± 0.2°20 using Cu Ka radiation. In some embodiments, erbumine salt Form A has an XRPD pattern substantially as shown in Figure 25(A). [071] D-BHB crystalline erbumine salt Form A may also be characterized by DSC substantially as set forth in Figure 25(B) and/or by the TGA set forth in Figure 25(C). In some embodiments, D-BHB crystalline erbumine salt Form A may be characterized by one or more of an XRPD substantially as depicted in Figure 25(A), DSC substantially as set forth in Figure 25(B), and TGA as set forth in Figure 25(C).
[072] D-BHB crystalline erbumine salt Form B can be characterized by XRPD pattern, obtained as set forth in the Examples, having peaks at 12.42, 16.07, 16.69, 17.62, and 18.35 ± 0.2°20 using Cu Ka radiation. Erbumine salt Form B can be further characterized by having an XRPD pattern having additional peaks at 8.82, 20.71, 22.22, 23.95, and 27.27 ± 0.2°20 using Cu Ka radiation. Erbumine salt Form B can be further characterized by having an XRPD pattern having additional peaks at 19.76, 21.22, 23.49, 28.46, 31.36, and 38.21 ± 0.2°20 using Cu Ka radiation. In some embodiments, D-BHB erbumine salt Form B has an XRPD pattern substantially as shown in Figure 26(B).
[073] D-BHB crystalline erbumine salt Form B may also be characterized by DSC substantially as set forth in Figure 26(C) and/or by the TGA set forth in Figure 26(D). In some embodiments, D-BHB crystalline erbumine salt Form B may be characterized by one or more of an XRPD substantially as depicted in Figure 26(B), DSC substantially as set forth in Figure 26(C), and TGA as set forth in Figure 26(D).
[074] Pharmaceutical Compositions
[075] Also provided herein are pharmaceutical compositions comprising one or more crystalline forms of D-BHB and at least one pharmaceutically acceptable carrier. In some embodiments, the carrier can comprise an excipient. In some embodiments, the pharmaceutical composition comprises one crystalline form of D-BHB. In some embodiments, the pharmaceutical composition comprises two crystalline forms of D-BHB. In some embodiments, the pharmaceutical composition comprises two crystalline forms of D-BHB, where one of the two crystalline forms of D-BHB is D-BHB L-arginine salt Form B. In some embodiments, the pharmaceutical composition comprises two crystalline forms of D-BHB, where one of the two crystalline forms of D-BHB is D-BHB sodium salt Form C. [076] A carrier as used herein include includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or subject being exposed thereto at the dosages and concentrations employed. In some embodiments, the carrier comprises water. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; and/or nonionic surfactants such as polysorbates (e.g. polysorbate 80 or polysorbate 20), polyethylene glycol (PEG), and poloxamers.
[077] Excipients in the formulation of pharmaceuticals, particularly for pharmaceutical formulations formulated for oral administration, are known in the art and may include diluents, disintegrants, binders, and other classes of excipients known in the art.
[078] The pharmaceutical compositions may be administered orally. Oral administration includes a formulation suitable for swallowing, or buccal or sublingual administration.
[079] Formulation suitable for oral administration include solid formulations such as tablets, capsules containing particulates, and powders. Formulations for oral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled targeted- and programmed-release.
[080] The pharmaceutical composition may be administered by injection. Administration by injection includes a formulation suitable for subcutaneous injection, intramuscular injection, intradermal injection, or intravenous injection.
[081] The pharmaceutical formulation as described herein comprises one or more D-BHB crystalline forms, wherein the one or more crystalline forms as described herein may make up from up to 80 wt (weight) %, up to 70 wt%, up to 50 wt %, up to 40 wt%, up to 30 wt%, or up to 20% of a dosage form. The crystalline form as described herein may make up from 0.5 wt% to 80 wt% of the dosage form, from 0.5 wt% to 70 wt% of the dosage form, from 0.5 wt% to 50 wt% of the dosage form, from 0.5 wt% to 40 wt% of the dosage form, from 0.5 wt% to 30 wt% of the dosage form, or from 0.5 wt% to 20 wt% of the dosage form. In one embodiment, the pharmaceutical formulation comprises two or more crystalline forms of D-BHB as described herein make up from from 0.5 wt% to 50 wt% of the dosage form. [082] The pharmaceutical formulation as described herein comprises one or more crystalline forms and may be formulated for administration to a subject as a single dose per day, as two or more doses per day, as three or more doses per day, or up to four doses per day. The crystalline form described herein may be formulated to provide the equivalent of 15 g D-BHB in a single dose or up to 15 g D-BHB in a single dose, 12 g D-BHB in a single dose or up to 12 g D-BHB in a single dose, or 10 g D-BHB in a single dose or up to 10 g D-BHB in a single dose. The crystalline form described herein may be formulated to provide the equivalent of 60 g D-BHB per day or up to 60 g D-BHB per day, 45 g D-BHB per day or up to 45 g D-BHB per day, 48 g D-BHB per day or up to 48 g D-BHB per day, 40 g D-BHB per day or up to 40 g D-BHB per day, 36 g D-BHB per day or up to 36 g D-BHB per day, 30 g D-BHB per day or up to 30 g D-BHB per day, 20 g D- BHB per day or up to 20 g D-BHB per day, 10 g D-BHB per day or up to 10 g D-BHB per day. It is understood that the amount of a particular crystalline form to be included in a formulation will depend on the molecular weight of the particular crystalline form and that this molecular weight will be considered to deliver the equivalent of the free D-BHB to a subject. Likewise, it will be understood that the molecule weights of each crystalline form in pharmaceutical formulations are to be taken into account in pharmaceutical formulations that include more than one crystalline form of D-BHB.
EMBODIMENTS
The invention comprises the following embodiments.
Embodiment 1. A crystalline D-BHB (“free Form I”) having an XRPD pattern comprising peaks at 10.93, 12.11, 15.16, 17.53, and 18.88 ± 0.2° 20 using Cu Ka radiation.
Embodiment 2. The crystalline D-BHB (“free Form I”) of embodiment 1 wherein the XRPD pattern further comprising peaks at 22.75, 24.30, 26.14, 29.10, and 29.95 ±
0.2°20 using Cu Ka radiation.
Embodiment 3. The crystalline D-BHB (“free Form I”) of embodiment 1 or embodiment 2 wherein the XRPD pattern further comprising peaks at 30.48, 31.60, 32.96, 33.98, 36.80, and 38.37 ± 0.2° 20 using Cu Ka radiation.
Embodiment 4. The crystalline D-BHB (“free Form I”) of any one of embodiments 1 to 3 having an XRPD pattern substantially as shown in Figure 1(A).
Embodiment 5. The crystalline D-BHB (“free Form I”) of any one of embodiments 1 to 4 having substantially the XRPD peaks as shown in Table 2. Embodiment 6. The crystalline D-BHB (“free Form I”) of any one of embodiments 1 to 5, having substantially the DSC of Figure 2(A) and/or substantially the TGA of Figure 3(A).
Embodiment 7. A crystalline D-BHB sodium salt designated as “D-BHB sodium salt Form A”, having an XRPD pattern comprising peaks at 11.82, 17.02, 17.58, 20.34, and 20.83 ± 0.2 20 using Cu Ka radiation.
Embodiment 8. The crystalline D-BHB sodium salt Form A of embodiment 7, wherein the XRPD pattern further comprises peaks at 7.23, 14.39, 24.50, 29.54, and 30.17 ± 0.2° 20 using Cu Ka radiation.
Embodiment 9. The crystalline D-BHB sodium salt Form A of embodiment 7 or embodiment 8, wherein the XRPD pattern further comprises peaks at 12.22, 22.23, 22.60, 30.63, 33.35, and 37.28 ± 0.2° 20 using Cu Ka radiation.
Embodiment 10. The crystalline D-BHB sodium salt Form A of any one of embodiments 7 to 9 having an XRPD pattern substantially as shown in Figure 9(A).
Embodiment 11. The crystalline D-BHB sodium salt Form A of any one of embodiments 7 to
10 having substantially the XRPD peaks as shown in Table 16.
Embodiment 12. The crystalline D-BHB sodium salt Form A of any one of embodiments 7 to
11 having substantially the DSC of Figure 9(B) and/or substantially the TGA of Figure 9(C).
Embodiment 13. A crystalline D-BHB sodium salt designated “D-BHB sodium salt Form B” having an XRPD pattern comprising peaks at 8.98, 11.49, 16.95, 17.53, and 19.38 ± O.2°20 using Cu Ka radiation.
Embodiment 14. The crystalline D-BHB sodium salt Form B of embodiment 14, wherein the XRPD pattern further comprises peaks at 7.17, 9.67, 12.16, 20.10, and 26.83 ± 0.2° 20 using Cu Ka radiation.
Embodiment 15. The crystalline D-BHB sodium salt Form B of embodiment 13 or embodiment 14, wherein the XRPD pattern further comprises peaks at 6.47, 14.02, 14.35, 18.35, 21.00, and 23.08 ± 0.2° 20 using Cu Ka radiation.
Embodiment 16. The crystalline D-BHB sodium salt Form B of any one of embodiments 12 to 15 having an XRPD pattern substantially as shown in Figure 10(A). Embodiment 17. The crystalline D-BHB sodium salt Form B of any one of embodiments 12 to
16 having substantially the XRPD peaks as shown in Table 17.
Embodiment 18. The crystalline D-BHB sodium salt Form B of any one of embodiments 12 to
17 having substantially the DSC of Figure 10(B) and/or substantially the TGA of Figure 10(C).
Embodiment 19. A crystalline D-BHB sodium salt designated as “D-BHB sodium salt Form C” having an XRPD pattern comprising peaks at 6.40, 7.07, 12.11, 14.08, and 17.05 ± 0.2°20 using Cu Ka radiation.
Embodiment 20. The crystalline D-BHB sodium salt Form C of embodiment 19, wherein the XRPD pattern further comprises peaks at 19.16, 22.57, 27.79, 28.75, and 30.11 ± 0.2°20 using Cu Ka radiation.
Embodiment 21. The crystalline D-BHB sodium salt Form C of embodiment 19 or embodiment 20, wherein the XRPD pattern further comprises peaks at 11.98, 30.76, 33.12, 33.63, and 37.08 ± 0.2° 20 using Cu Ka radiation.
Embodiment 22. The crystalline D-BHB sodium salt Form C of any one of embodiments 19 to
21 having an XRPD pattern substantially as shown in Figure 29(A).
Embodiment 23. The crystalline D-BHB sodium salt Form C of any one of embodiments 19 to
22 having substantially the XRPD peaks shown in Table 20.
Embodiment 24. The crystalline D-BHB sodium salt Form C of any one of embodiments 19 to
23 having substantially the DSC of Figure 29(B) and/or substantially the TGA of Figure 29(C).
Embodiment 25. A crystalline D-BHB magnesium salt designated as “D-BHB , magnesium salt Form A” having an XRPD pattern comprising peaks at 5.53, 7.80, 9.61, 11.95, and 13.15 ± 0.2°20 using Cu Ka radiation.
Embodiment 26. The crystalline D-BHB magnesium salt Form A of embodiment 25, wherein the XRPD pattern further comprises peaks at 6.11, 11.02, 11.27, 15.61, and 18.98 ± 0.2°20 using Cu Ka radiation.
Embodiment 27. The crystalline D-BHB magnesium salt Form A of embodiment 25 or embodiment 26, wherein the XRPD pattern further comprises peaks at 19.45, 21.13, 21.88, 23.70, and 25.99 ± 0.2°20 using Cu Ka radiation. Embodiment 28. The crystalline D-BHB magnesium salt form A of any one of embodiments 25 to 27 having an XRRD pattern substantially as shown in Figure 7(A).
Embodiment 29. The crystalline D-BHB magnesium salt form A of any one of embodiments 25 to 28 having substantially the XRPD peaks shown in Table 13.
Embodiment 30. The crystalline D-BHB magnesium salt form A of any one of embodiments 25 to 29 having substantially the DSC of Figure 7(B) and/or substantially the TGA of Figure 7(C).
Embodiment 31. A crystalline D-BHB magnesium salt designated as “D-BHB magnesium salt Form B” having an XRPD pattern comprising peaks at 6.12, 8.31, 9.60, 11.87, and 13.45 ± 0.2°20 using Cu Ka radiation.
Embodiment 32. The crystalline D-BHB magnesium salt Form B of embodiment 31 , wherein the XRPD pattern further comprises peaks at 11.49, 12.23, 14.09, 14.31, and 18.46 ± 0.2°20 using Cu Ka radiation.
Embodiment 33. The crystalline D-BHB magnesium salt Form B of embodiment 31 or embodiment 32, wherein the XRPD pattern further comprises peaks at 17.01, 19.61, 23.70, 24.62, 24.94, and 29.02 and 18.46 ± 0.2°20 using Cu Ka radiation.
Embodiment 34. The crystalline D-BHB magnesium salt form B of any one of embodiments 31 to 33 having an XPRD pattern substantially as shown in Figure 8(A).
Embodiment 35. The crystalline D-BHB magnesium salt form B of any one of embodiments 31 to 34 having substantially the XRPD peaks shown in Table 14.
Embodiment 36. The crystalline D-BHB magnesium salt form B of any one of embodiments 31 to 35 having substantially the DSC of Figure 8(B) and/or substantially the TGA of Figure 8(C).
Embodiment 37. A crystalline D-BHB L-lysine salt designated “D-BHB L-lysine salt Form A” having an XRPD pattern comprising peaks at 6.92, 9.16, 12.62, 23.29, and 25.91 ± 0.2°20 using Cu Ka radiation.
Embodiment 38. The crystalline D-BHB L-lysine salt Form A of embodiment 37, wherein the XRPD pattern further comprises peaks at 10.21, 26.93, 27.64, 28.45, and 29.87 ± 0.2°20 using Cu Ka radiation. Embodiment 39. The crystalline D-BHB L- lysine salt Form A of embodiment 37 or embodiment 38, wherein the XRPD pattern further comprises peaks at 17.87, 18.27, 19.73, 24.58, and 25.02 ± 0.2°20 using Cu Ka radiation.
Embodiment 40. The crystalline D-BHB L-Lysine salt Form A of any one of embodiment 37 to
39 having an XRPD pattern substantially as shown in Figure 31(A).
Embodiment 41. The crystalline D-BHB L-Lysine salt Form A of any one of embodiment 37 to
40 having substantially the XRPD peaks shown in Table 28.
Embodiment 42. The crystalline D-BHB L-Lysine salt Form A of any one of embodiment 37 to
41 having substantially the DSC of Figure 31(B) and/or substantially the TGA of Figure 31(C).
Embodiment 43. A crystalline D-BHB L-arginine salt designated as “D-BHB L-arginine Form A” having an XRPD pattern comprising peaks at 5.28, 7.28, 9.11, 14.99, and
15.82 ± 0.2°20 using Cu Ka radiation.
Embodiment 44. The crystalline D-BHB L- arginine salt Form A of embodiment 43, wherein the XRPD pattern further comprises peaks at 17.00, 17.76, 18.32, 19.32, and 24.10 ± 0.2°20 using Cu Ka radiation.
Embodiment 45. The crystalline D-BHB L- arginine salt Form A of embodiment 43 or embodiment 44, wherein the XRPD pattern further comprises peaks at 10.81, 11.08, 11.27, 11.75, and 26.96 ± 0.2°20 using Cu Ka radiation.
Embodiment 46. The crystalline D-BHB L- arginine salt Form A of any one of embodiments 43 to 45 having an XRPD pattern substantially as shown in Figure 14(A).
Embodiment 47. The crystalline D-BHB L- arginine salt Form A of any one of embodiments 43 to 46 having substantially the XRPD peaks shown in Table 10.
Embodiment 48. The crystalline D-BHB L- arginine salt Form A of any one of embodiments 43 to 47 having substantially the DSC of Figure 14(B) and/or substantially the TGA of Figure 14(C).
Embodiment 49. A crystalline D-BHB L-arginine salt designated as “D-BHB L-arginine Form B” having an XRPD pattern comprising peaks at 7.30, 9.94, 11.28, 14.56, and
15.83 ± 0.2°20 using Cu Ka radiation. Embodiment 50. The crystalline D-BHB L-arginine salt Form B of embodiment 49, wherein the XRPD pattern further comprises peaks at 17.27, 18.33, 19.91, 21.90, and 23.49 ± 0.2°20 using Cu Ka radiation.
Embodiment 51. The crystalline D-BHB L-arginine salt Form B of embodiment 49 or embodiment 50, wherein the XRPD pattern further comprises peaks at 24.12, 26.45, 26.99, 28.56, 33.56, and 34.16 ± 0.2° 20 using Cu Ka radiation.
Embodiment 52. The crystalline D-BHB L- arginine salt Form B of any one of embodiments 49 to 51 having an XRPD pattern substantially as shown in Figure 30(A).
Embodiment 53. The crystalline D-BHB L- arginine salt Form B of any one of embodiments 49 to 52 having substantially the XRPD peaks shown in Table 25.
Embodiment 54. The crystalline D-BHB L- arginine salt Form B of any one of embodiments 49 to 53 having substantially the DSC of Figure 30(B) and/or substantially the TGA of Figure 30(C).
Embodiment 55. A crystalline D-BHB erbumine salt designated as “D-BHB erbumine salt Form A” having an XRPD pattern comprising peaks at 10.04, 10.77, 13.69, 14.71, and 18.24 ± 0.2°20 using Cu Ka radiation.
Embodiment 56. The crystalline D-BHB erbumine salt Form A of embodiment 55, wherein the XRPD pattern further comprises peaks at 16.08, 16.95, 17.42, 19.37, and 20.07 ± 0.2°20 using Cu Ka radiation.
Embodiment 57. The crystalline D-BHB erbumine salt Form A of embodiment 55 or embodiment 56, wherein the XRPD pattern further comprises peaks at 22.47, 24.35, 25.60, 26.05 and 28.36 ± 0.2°20 using Cu Ka radiation.
Embodiment 58. The crystalline D-BHB erbumine salt Form A of any one of embodiments 55 to 57 having an XRPD pattern substantially as shown in Figure 25(A).
Embodiment 59. The crystalline D-BHB erbumine salt Form A of any one of embodiments 55 to 58 having substantially the XRPD peaks shown in Table 30.
Embodiment 60. The crystalline D-BHB erbumine salt Form A of any one of embodiments 55 to 59 having substantially the DSC of Figure 25(B) and/or substantially the TGA of Figure 25(C). Embodiment 61. A crystalline D-BHB erbumine salt designated as “D-BHB erbumine salt Form B” having an XRPD pattern comprising peaks at 12.42, 16.07, 16.69, 17.62, and 18.35 ± 0.2°20 using Cu Ka radiation.
Embodiment 62. The crystalline D-BHB erbumine salt Form B of embodiment 61, wherein the XRPD pattern further comprises peaks at 8.82, 20.71, 1. 1, 23.95, and 27.27 ± 0.2° 20 using Cu Ka radiation.
Embodiment 63. The crystalline D-BHB erbumine salt Form B of embodiment 61 or embodiment 62, wherein the XRPD pattern further comprises peaks at 19.76, 21.22, 23.49, 28.46, 31.36, and 38.21 ± 0.2° 20 using Cu Ka radiation.
Embodiment 64. The crystalline D-BHB erbumine salt Form B of any one of embodiments 61 to 63 having an XRPD pattern substantially as shown in Figure 26(B).
Embodiment 65. The crystalline D-BHB erbumine salt Form B of any one of embodiments 61 to 64 having substantially the XRPD peaks shown in Table 31.
Embodiment 66. The crystalline D-BHB erbumine salt Form B of any one of embodiments 61 to 65 having substantially the DSC of Figure 26(C) and/or substantially the TGA of Figure 26(D).
Embodiment 67. The crystalline D-BHB of any one of embodiments 1 to 66, wherein the D-BHB is greater than 95% enantiomerically pure, greater than 98% enantiomerically pure, or greater than 99% enantiomerically pure.
Embodiment 68. The crystalline D-BHB of any one of embodiments 1 to 67, wherein the D-BHB is greater than 99% enantiomerically pure.
Embodiment 69. A composition comprising one or more crystalline D-BHB forms as described in any one of embodiments 1 to 68.
Embodiment 70. The composition of embodiment 69, wherein the composition comprises D- BHB L-arginine salt Form B.
Embodiment 71. The composition of embodiment 70, wherein the composition consists essentially of or consists of D-BHB L-arginine salt Form B.
Embodiment 72. The composition of embodiment 69, wherein the composition comprises D-BHB sodium salt Form C.
Embodiment 73. The composition of embodiment 72, wherein the composition consists essentially of or consists of D-BHB sodium salt Form C. Embodiment 74. A composition comprising two or more crystalline D-BHB forms as described in any one of embodiments 1 to 68.
Embodiment 75. The composition of embodiment 74, wherein the composition comprises D- BHB L-arginine salt Form B.
Embodiment 76. The composition of embodiment 74, wherein the composition comprises D- BHB sodium salt Form C.
Embodiment 77. The composition of embodiment 75 or embodiment 76, wherein the composition comprises D-BHB L-arginine salt Form B and D-BHB sodium salt Form C.
Embodiment 78. The composition of embodiment 77, wherein the composition consists essentially of or consists of D-BHB L-arginine salt Form B and D-BHB sodium salt Form C.
Embodiment 79. A pharmaceutical composition comprising one or more crystalline D-BHB forms as described in any one of claims embodiments 1 to 68 and at least one pharmaceutically acceptable carrier.
Embodiment 80. The pharmaceutical composition of embodiment 79, wherein the composition comprises D-BHB L-arginine salt Form B.
Embodiment 81. The pharmaceutical composition of embodiment 80, wherein the composition consists essentially of or consists of D-BHB L-arginine salt Form B.
Embodiment 82. The pharmaceutical composition of embodiment 79, wherein the composition comprises D-BHB sodium salt Form C.
Embodiment 83. The pharmaceutical composition of embodiment 80, wherein the composition consists essentially of or consists of D-BHB sodium salt Form C.
Embodiment 84. A pharmaceutical composition comprising two or more crystalline D-BHB forms as described in any one of claims embodiments 1 to 68 and at least one pharmaceutically acceptable carrier.
Embodiment 85. The pharmaceutical composition of embodiment 84, wherein the composition comprises D-BHB L-arginine salt Form B.
Embodiment 86. The pharmaceutical composition of embodiment 84 or embodiment 85, wherein the composition comprises D-BHB sodium salt Form C. Embodiment 87. The pharmaceutical composition of embodiment 85 or embodiment 86, wherein the composition comprises D-BHB L-arginine salt Form B and D- BHB sodium salt Form C.
Embodiment 88. The pharmaceutical composition of embodiment 87, wherein the composition consists essentially of or consists of D-BHB L-arginine salt Form B and D- BHB sodium salt Form C.
Embodiment 89. The pharmaceutical composition of any one of embodiments 79 to 88 formulated for oral administration.
Embodiment 90. The pharmaceutical composition of any one of embodiment 89, wherein the at least one carrier is an excipient.
Embodiment 91. The pharmaceutical composition of any one of embodiments 79 to 88 formulated for administration by injection.
Embodiment 92. The pharmaceutical composition of any one of embodiment 91, wherein the at least one carrier is water and/or an aqueous pH buffered solution.
Embodiment 93. The pharmaceutical composition of any one of embodiments 79 to 92, wherein the composition is formulated to administer up to
(a) about 15 g D-BHB per dose;
(b) about 12 g D-BHB per dose; or
(c) about 10 g D-BHB per dose.
Embodiment 94. The pharmaceutical composition of any one of embodiments 79 to 92, wherein the composition is formulated to administer about 10 g D-BHB per dose.
Embodiment 95. The pharmaceutical composition of any one of embodiments 79 to 94, wherein the composition is formulated to administer up to 60 g D-BHB per day, up to 48 g D-BHB per day, up to 45 g D-BHB per day, up to 40 g D-BHB per day, 36 g D-BHB per day, up to 30 g D-BHB per day, up to 20 g D-BHB per day, or up to 10 g per day.
Embodiment 96. The pharmaceutical composition of embodiment 95, wherein the composition is formulated for administration as two doses per day, three doses per day, or four doses per day. EXAMPLES
[083] Characterization of the crystalline forms was carried out by the following methods.
TABLE 1
X-Ray Powder Diffractometer (XRPD)
Figure imgf000022_0001
XRPD method 2 - Used for XRPD shown in Figures 1, 5 to 25, 27(C), and 28
Figure imgf000022_0002
Figure imgf000023_0001
XRPD method 3 - Used for XRPD shown in Figure 26(A)
Figure imgf000023_0002
XRPD method 4 - Used for XRPD shown in Figure 27 (A), (B), and (D) to (I)
Figure imgf000023_0003
Figure imgf000024_0001
Thermogravimetric Analyzer (TGA)
Figure imgf000024_0002
Dynamic Vapor Sorption (DVS)
Method 1 - Results shown in Tables 40 and 41
Figure imgf000024_0003
Method 2 - Results shown in Tables 42 and 43
Figure imgf000024_0004
Figure imgf000025_0001
Method 2 for chemical purity, stoichiometry and solubility
Figure imgf000025_0002
[084] Example 1: D-BHB crystalline free base form (Form I)
[085] Trial 1: 50mL of the 40wt% of D-BHB aqueous solution was added into a 500mL glass bottle. Release specifications for D-BHB is greater than 95% D enantiomer (i.e. greater than 95% enantiomeric purity of the D enantiomer). This aqueous solution was treated by lyophilization. Sugar like crystals were obtained after 3 days. Obtained sugar like crystals were very hygroscopic and deliquesced after exposure to ambient condition (20-25°C/40-70%RH) within 30 minutes. Therefore, obtained sugar like crystals were dried under vacuum at 25°C for 2 hours to remove surface moisture. 22.7 g of D-BHB free Form I was obtained. Obtained sugar like crystals were kept in a closed container. Referred to herein as sample: FR03684-1-LP1 (with XRPD shown in Figure 1(A) with the following peaks in Table 2).
TABLE 2
Figure imgf000026_0001
Figure imgf000027_0001
[086] Trial 2: 50mL of the 40wt% of D-BHB aqueous solution was added into a 500mL glass bottle. This aqueous solution was treated by lyophilization. Sugar like crystals were obtained after 6 days. Obtained sugar like crystals were very hygroscopic and deliquesced after exposure to ambient condition (20-25°C/40-70%RH) within 30 minutes. Obtained sugar like crystals were dried under vacuum at 25°C for 1 day to remove surface moisture. 21.0 g of D-BHB free Form I was obtained. Obtained sugar like crystals were kept in a closed container. Referred to herein as sample: FR03684-1-LP2 (with XRPD shown in Figure 1(B) with the following peaks in Table 3).
TABLE 3
Figure imgf000027_0002
Figure imgf000028_0001
TABLE 4
D-BHB free Form
Figure imgf000029_0001
[087] Example 2: Salt Screening
[088] The following counter-ions were selected for screening.
TABLE 5
Figure imgf000029_0002
Figure imgf000030_0001
[089] About 30mg of the D-BHB free Form I (FR03684-1-LP1) and 1 or 0.5 equivalents of counter ions were added into 0.1-1.2mL of screening solvents (water, ethanol, acetone, ethyl acetate (EA), acetonitrile (ACN) or tetrahydrofuran (THF)) in a 2mL glass vial. Obtained mixtures were stirred at 25°C for at least 48 hours. Obtained suspensions were filtered through a 0.45pm nylon membrane filter by centrifugation at 14,000 rpm. After being dried at 50°C under vacuum for 2h, solids were analyzed by XRPD.
[090] Crystalline salt forms including sodium salt Form A, sodium salt Form B, physical mixtures of magnesium salt Form A and Mg(0H)2, physical mixtures of magnesium salt Form B and Mg(0H)2, L-arginine salt Form A, L- lysine salt Form A and erbumine salt Form A were obtained after equilibration in water, EtOH, acetone, EA, ACN and THF. Screening with Ca(OH)2 only led to gel. XRPD by Method 2 shown in Figure 5.
TABLE 6
Figure imgf000030_0002
Figure imgf000031_0001
11 = Note Carried Out.
[091] Clear solutions obtain in slurry equilibration experiments were cooled to 5°C to precipitate solids. Only clear solutions were obtained.
[092] Clear solutions obtained from cooling experiments were further treated by addition of antisolvent (one or more of heptane, methyl tert-butyl ether (MTBE), or toluene). Only gel, oil or clear solutions were obtained.
[093] Clear solutions obtained from anti-solvent experiments were further treated by slow evaporation under ambient condition (20-30°C/20-70%RH). Only gel or oil was obtained.
[094] Clear solutions obtained from cooling experiments were further treated by slow evaporation under ambient condition (20-30°C/20-70%RH). Only gel was obtained.
[095] Gel samples obtained from slurry equilibration experiments were further treated by reslurry at 25°C for at least 48 hours by addition 0.1-0.5mL of methanol (MeOH), isopropyl alcohol (IP A), MTBE, dichloromethane (DCM), methyl ethyl ketone (MEK), isopropyl acetate (IP Ac), 2- Methyltetrahydrofuran (2-MeTHF), 1,4-di oxane, acetone/water (v:v=95:5), ACN/water (v:v=95:5), THF/water (v:v=95:5) and MeOH/water (v:v=95:5).
[096] Obtained suspensions were filtered through a 0.45 pm nylon membrane filter by centrifugation at 14,000 rpm. After dried at 50°C under vacuum for 2h, solids were analyzed by XRPD (Method 2). A new polymorph of arginine salt, assigned as L-arginine salt Form B, was obtained after re-slurry in acetone/water (v:v=95:5) and in ACN/water (v:v=95:5).
TABLE 7
Figure imgf000032_0001
Figure imgf000033_0001
[097] Figure 12(A) shows the XRPD of D-BHB L-lysine salt Form A (FR03684-3-RC7B-EtOH) having peaks shown in TABLE 8.
TABLE 8
Figure imgf000033_0002
Figure imgf000034_0002
Figure imgf000034_0001
[098] Figure 13(A) shows the XRPD pattern of D-BHB erbumine salt Form A (FR03684-3- RC9D-EA) having peaks as shown in Table 9.
TABLE 9
Figure imgf000035_0001
Figure imgf000036_0001
[099] Figure 14(A) shows the XRPD of D-BHB L-arginine salt Form A (FR03684-3-RC6D-EA) having peaks as shown in Table 10.
TABLE 10
Figure imgf000036_0002
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
[100] Figure 15(A) shows the XRPD of D-BHB L-arginine salt Form B (FR03684-3-RC6H- acetone- water-95 -5) having peaks as shown in Table 11.
TABLE 11
Figure imgf000039_0002
Figure imgf000040_0001
Figure imgf000041_0001
[101] Example 3: Magnesium Salt Forms
[102] Condition 1: To get pure magnesium salt polymorphs for characterization, obtained physical mixtures of crystalline magnesium salt and Mg(0H)2 were used as seeds during screening.
[103] About 30mg of the D-BHB free Form I (FR03684-1-LP1) and 0.5 equiv. of Mg(OH)2 were added into 0.1-0.3mL of EtOH, EA, ACN and THF in a 2mL glass vial. After stirring at 50°C for 10 min, about 3mg of physical mixtures of crystalline magnesium salt and Mg(0H)2 were added into above suspension as seeds.
[104] Obtained mixtures were stirred at 50°C for 2 hours and then at 25°C for at least 48 hours. Hemi-magnesium salt Form A was obtained in EA. Hemi-magnesium salt Form B was obtained in ACN and in THF (Figure 6). TABLE 12
Figure imgf000042_0001
[105] Figure 7 shows D-BHB magnesium salt Form A (FR03684-3 -RC5D-re-EA) with Figure 7(A) showing the XRPD having peaks as shown in Table 13.
TABLE 13
Figure imgf000042_0002
Figure imgf000043_0001
[106] Figure 8 shows D-BHB magnesium salt Form B (FR03684-3-RC5E-re-ACN) with Figure 8(A) showing the XRPD having peaks as shown in Table 14.
TABLE 14
Figure imgf000043_0002
Figure imgf000044_0001
Figure imgf000045_0001
[107] Condition 2: To get pure magnesium salt polymorphs for characterization, amorphous magnesium salt was also prepared for re-slurry.
[108] About 500mg of the D-BHB free Form I (ID FR03684-1-LP1) and 0.5 equiv. of Mg(OH)2 were added into water in a 2mL glass vial. Obtained mixtures were stirred at 25°C for at least 48 hours. About 346mg of amorphous magnesium salt was obtained.
[109] Obtained amorphous hemi-magnesium salt was further treated by re-slurry at 25°C for at least 48 hours in 0.1-0.3mL of EtOH, EA, ACN and THF. About 3mg of magnesium salt Form A and Mg(0H)2 mixture seeds (FR03684-3-RC5B-EtOH and FR03684-3-RC5D-EA) was added into suspension RS9B and RS9D, respectively. About 3mg of the magnesium salt Form B and Mg(0H)2 mixture seeds (FR03684-3-RC5E-ACN and FR03684-3-RC5F-THF) was added into suspension RS9E and RS9F, respectively.
[HO] Obtained suspensions were filtered through a 0.45 pm nylon membrane filter by centrifugation at 14,000 rpm. After dried at 50°C under vacuum for 2h, solids were analyzed by XRPD (Method 2). Based on results, only magnesium salt Form B was obtained after re-slurry in ACN.
[Ill] Condition 3: To get pure magnesium salt polymorphs for characterization, EtOH/water (v:v=95:5), ACN/water (v:v=95:5) and THF/water (v:v=95:5) were also selected as screening solvents. About 150mg of the D-BHB free Form I (sample ID FR03684-1-LP1) and 0.5 equiv. of Mg(0H)2 were added into 0.1-0.5mL of EtOH/water (v:v=95:5), ACN/water (v:v=95:5) and THF/water (v:v=95:5) solvent mixtures in a 2mL glass vial. Obtained mixtures were stirred at 25°C for at least 48 hours. Only emulsion was obtained.
TABLE 15
Figure imgf000046_0001
[112] Example 4: Sodium Salt Crystalline Forms A, B, and C
[113] The sodium salt Form A obtained from slurry equilibration in THF contained 1.5 equiv. of Na+, which is different from theoretical stoichiometry of the sodium salt. Therefore, screening with different ratios of NaOH were tried and crystalline sodium salt seeds were added during screening to optimize stoichiometry of sodium salt.
[114] About 150mg of theD-BHB free FormI (sample ID FR03684-1-LP1) and 1, 1.5 or 2 equiv. of NaOH were added into THF or acetone in a 2mL glass vial. After stirring at 25°C for 10 min, about 3mg of the sodium salt Form A seeds (FR03684-3 -RC2F-THF) were added into suspension RC13F, RC14F and RC2F-re, respectively. About 3mg of the sodium saltForm B seeds (FR03684- 3 -RC2C- Acetone) were added into suspension RC2C-re. Obtained mixtures were stirred at 25°C for at least 48 hours.
[115] Sodium salt Form B was non-reproducible. When screening with 1 equiv. of NaOH in 0.1- 0.5 mL of acetone, sodium salt Form A was obtained. IC showed D-BHB: Na+ is 1 :1.6. When screening with 1.5 or 2 equiv. of NaOH in 0.2-0.5 mL of THF, a new polymorph of sodium salt, assigned as sodium salt Form C, was obtained. IC showed D-BHB: Na+ is 1: 1.5.
[116] The XRPD of sodium salt Form A (FR03684-3-RC2F-THF) is shown in Figure 9(A) having peaks as shown in Table 16.
TABLE 16
Figure imgf000047_0001
Figure imgf000048_0001
[117] The XRPD of sodium salt Form B (FR03684-3-RC2C- Acetone) is shown in Figure 10(A) having peaks as shown in Table 17.
TABLE 17
Figure imgf000049_0001
Figure imgf000050_0002
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000051_0002
[118] The XRPD of sodium salt form C (FR03684-3-RC14F-THF) is shown in Figure 11(A) having peaks as shown in Table 18.
TABLE 18
Figure imgf000051_0003
Figure imgf000052_0001
TABLE 19
Figure imgf000052_0002
Figure imgf000053_0001
// = Not carried out.
[119] Example 5: Scale up of D-BHB Sesqui-sodium salt Form C
[120] Trial 1: Sodium salt Form C (FR03684-SUl-NaOH-1.5-THF) was prepared as follows. 2.0g of the D-BHB free Form I (FR03684-1-LP1) and 1.2g of NaOH (~1.5 equivalent by molar ratio) were weighed into a 40mL glass vial. lOmL of THF was added into the vial under stirring at 25°C for about lOmin. A suspension was obtained. About Img of the sodium salt Form A seeds (FR03684-3-RC2D) was added into above suspension. The suspension was kept stirring at 25°C for about 2 days. About 5mL of suspension was taken out and centrifuged. Obtained THF saturated solution (~5mL) was added back into the 40mL glass vial. Obtained wet cake was dried at 50°C under vacuum for about 2 hours. 585mg of the sodium salt Form C (Figure 16(A) - XRPD Method 2) was obtained. But crystallinity of the sodium salt Form C sample was a little low. To improve crystallinity of the sodium salt Form C, obtained dry cake was added into above-mentioned saturated solution. All of the suspension (lOmL) was kept stirring at 25°C for another 8 days. About ImL of the suspension was taken out. The solid part was collected by filtered through a 0.45pm nylon membrane and characterized by XRPD. Crystallinity of the sodium salt Form C improved (Figure 16(A) - XRPD Method 2). Rest of suspension was collected by filtration and then dried at 50°C under vacuum for about 2 hours. 1 ,9g of the sodium salt Form C was obtained as an off-white solid in 59% yield. Characterization results are reported below in Example 14. FR03684-SUl-NaOH-1.5-THF was used for bulk stability study, solubility study and hygroscopicity reported below in Examples 11 to 13. The XRPD of D-BHB sodium salt Form C (FR03684-SUl-NaOH-1.5-THF) is shown in Figure 29(A) and has peaks as shown in Table 20.
TABLE 20
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
[121] Trail 2: Sodium salt Form C (FR03684-SU9-NaOH-1.5-THF) was prepared as follows. 2.0g of the D-BHB free Form I (FR03684-1-LP2) and 1.2g of NaOH (~1.5 equivalent by molar ratio) were weighed into a 40mL glass vial. lOmL of THF was added into the vial under stirring at 25°C for about lOmin. A suspension was obtained. About Img of the sodium salt Form C seeds (FR03684-SUl-NaOH-1.5-THF as obtained from Trial 1) were added into above suspension. The suspension was kept stirring at 25°C for about 4 days. Solids were collected by filtration and then dried at 50°C under vacuum for about 2 hours. 2.6g of the sodium salt Form C (Figure 16(B) - XRPD Method 2) was obtained as an off-white solid in 81% yield. FR03684-SU9-NaOH-1.5-THF was used for solubility study in Example 12.
TABLE 21
D-BHB sodium salt Form C
Figure imgf000056_0002
[122] Figure 17(A) shows the XRPD for D-BHB sodium salt Form C (FR03684-SU9-NaOH- 1.5-THF) having peaks as shown in Table 22.
TABLE 22
Figure imgf000057_0001
Figure imgf000058_0001
[123] Example 6: Scale up of D-BHB L-arginine salt Form B
[124] Trial 1 : L-arginine salt Form B (FR03684-SU2-L-arginine-acetone-water-95-5-re) was prepared as follows. 2.0g of the D-BHB free Form I (FR03684-1-LP1) and 3.5g of L-arginine (-1.05 equivalent by molar ratio) were weighed into a lOOmL glass vial. 34mL of acetone/water (v:v=95:5) was added into the vial under stirring at 50°C for about lOmin. A gel-like material was obtained. About 3mg of the L-arginine salt Form B seeds (ID FR03684-3 -RC6H) was added. This gel-like material was stirred at 50°C for about 2 hours. This gel-like material was cooled to 25°C and kept stirring at 25°C for about 1 day. The gel-like material gradually converted into a suspension. The suspension was kept stirring at 25°C for another 1 day. Solids were collected by filtration and then dried at 50°C under vacuum for about 2 hours. 4.8g of the L-arginine salt Form B (Figure 19(A)) was obtained (FR03684-3-SU2-L-arginine-acetone-water-95-5). The solid part showed a ratio of free form: L-arginine = 1: 1.2 (Figure 19(B)).
[125] In order to optimize the stoichiometry of L-arginine salt Form B, another lOOmg (-0.05 equivalent by molar ratio) of the D-BHB free Form I (FR03684-1-LP1) and 4.6g of the L-arginine salt Form B (FR03684-3-SU2-L-arginine-acetone-water-95-5) were weighed into a lOOmL glass vial. 5mL of acetone/water (v:v=95:5) saturated solution was added into the vial and kept stirring at 25°C for about 2 days. Solids were collected by filtration and then dried at 50°C under vacuum for about 2 hours. 3.2g of the L-arginine salt Form B (Figure 19(A)) was obtained as an off-white solid in 61% yield. FR03684-SU2-L-arginine-acetone-water-95-5-re was used for solubility study in Example 12. TABLE 23
D-BHB L-arginine salt Form B
Figure imgf000059_0001
[126] Figure 19(A) shows the XRPD for D-BHB L-arginine salt Form B (FR03684-SU2-L- arginine-acetone-water-95-5-re) having peaks as shown in Table 24.
TABLE 24
Figure imgf000059_0002
Figure imgf000060_0001
Figure imgf000061_0001
[127] Trial 2: L-arginine salt Form B (FR03684-SU5-L-arginine-acetone-water-95-5) was prepared using the procedure below. 1.0g of the D-BHB free Form I (FR03684-1-LP1) and 1.7g of L-arginine (~1.0 equivalent by molar ratio) were weighed into a 40mL glass vial. 15mL of acetone/water (v:v=95:5) was added into the vial under stirring at 50°C for about lOmin. A gellike material was obtained. About 3mg of the L-arginine salt Form B seeds (FR03684-3-RC6H) was added. This gel-like material was stirred at 50°C for about 2 hours. This gel-like material was cooled to 25°C and kept stirring at 25°C for about 1 day. The gel-like material gradually converted into a suspension. The suspension was kept stirring at 25°C for another 7 days. About ImL of the suspension was taken out. The solid part was collected by filtered through a 0.45 pm nylon membrane. The L-arginine salt Form B (Figure 20(A)) was obtained. 1 H-NMR showed that a ratio of free form: L-arginine was 1:1.0 (Figure 20(B)). Solids were collected by filtration and then dried at 50°C under vacuum for about 2 hours. 2.4g of the L-arginine salt Form B was obtained as an off-white solid in 89% yield. Characterization results are reported in Example 14. FR03684-SU5- L-arginine-acetone-water-95-5 was used for bulk stability study, solubility study and hygroscopicity in Examples 11 to 13. The XRPD of D-BHB L-arginine salt Form B (FR03684- SU5-L-arginine-acetone-water-95-5) is shown in Figure 30(A) and has peaks as shown in Table 25. TABLE 25
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
[128] Example ?: Scale up of D-BHB of L-lysine salt Form A
[129] Trial 1: L-lysine salt Form A (FR03684-SU3-L-lysine-EtOH-re) was prepared as follows. 2.0g of the D-BHB free Form I (FR03684-1-LP1) and 2.9g of L-lysine (-1.05 equivalent by molar ratio) were weighed into a lOOmL glass vial. 30mL of EtOH was added into the vial under stirring at 25°C for about lOmin. A suspension was obtained. About 3mg of the L-lysine salt Form A seeds (FR03684-3-RC7B) was added into above suspension. The suspension was kept stirring at 25°C for about 2 days. Solids were collected by filtration and then dried at 50°C under vacuum for about 2 hours. 3.7g of the L-lysine salt Form A (Figure 21(A)) was obtained (FR03684-SU3-L-lysine- EtOH). The solid part showed a ratio of free form:L-lysine was 1: 1.2 (Figure 21(B)).
[130] To optimize the stoichiometry of L-lysine salt Form A, another lOOmg (-0.05 equivalent by molar ratio) of the D-BHB free Form I (FR03684-1-LP1) and remaining 3.4g of the L-lysine salt Form A (FR03684-3-SU3-L-lysine-EtOH) were weighed into a lOOmL glass vial. Another 5mL of EtOH saturated solution was added into the vial and kept stirring at 25°C for about 2 days. Solids were collected by filtration and then dried at 50°C under vacuum for about 2 hours. 2.4g of the L-lysine salt Form A (Figure 21(A)) was obtained as an off-white solid in 54% yield. FR03684- SU3-L-lysine-EtOH-re was used for solubility study reported in Example 12.
TABLE 26
D-BHB L-lysine salt Form A
Figure imgf000064_0002
D-BHB L-lysine salt Form A
Figure imgf000065_0001
[131] Figure 22(A) shows the XRPD for D-BHB L-lysine salt Form A (FR03684-SU3-L-lysine- EtOH-re) having peaks as shown in Table 27.
TABLE 27
Figure imgf000065_0002
Figure imgf000066_0001
[132] Trial 2: D-BHB L-lysine salt Form A (FR03684-SU6-L-lysine-EtOH) was prepared as follows. 1.0g of the D-BHB free Form I (FR03684-1-LP1) and 1.4g of L-lysine (~1.0 equivalent by molar ratio) were weighed into a lOOmL glass vial. 30mL of EtOH was added into the vial under stirring at25°C for about lOmin. A suspension was obtained. About 3mg of the L-lysine salt Form A seeds (FR03684-3-RC7B) were added into above suspension. The suspension was kept stirring at 25°C for about 7 days. About ImL of the suspension was taken out. The solid part was collected by filtered through a 0.45 pm nylon membrane. The L-lysine salt Form A (Figure 23(A)) was obtained. ' H-NMR showed a ratio of free form:L-lysine was 1: 1.0 (Figure 23(B)). Solids were collected by filtration and then dried at 50°C under vacuum for about 2 hours. 1.6g of the L- lysine salt Form A was obtained as an off-white solid in 67% yield. Characterization results are reported in Example 14. FR03684-SU6-L-lysine-EtOH was used for bulk stability study, solubility study and hygroscopicity reported in Examples 11 to 13.
[133] Figure 31(A) shows the XPRD for D-BHB L-lysine salt Form A (FR03684-SU6-L-lysine- EtOH) having peaks as shown in Table 28. TABLE 28
Figure imgf000067_0001
Figure imgf000068_0001
[134] Example 8: Scale up of D-BHB erbumine salt Form A
[135] Erbumine salt Form A (FR03684-SU4-erbumine-EA) was prepared as follows. 2.0g of the D-BHB free Form I (FR03684-1-LP1) and 1.5g of erbumine (-1.05 equivalent by molar ratio) were weighed into a 20mL glass vial. 4.0 mL of EA was added into the vial under stirring at 25 °C for about lOmin. A suspension was obtained. About 3 mg of the erbumine salt Form A seeds (FR03684-3-RC9C) were added into above suspension. The suspension was kept stirring at 25°C for about 2 days. About 2mL of suspension was taken out and solid part (wet cake) was characterized by XRPD. A new crystalline form, assigned as erbumine salt Form B (Figure 24) was obtained. After it was dried at 50°C under vacuum for about 2 hours, the erbumine salt Form B converted to the erbumine salt Form A (Figure 24). Rest of solids were collected by filtration and then dried at 50°C under vacuum for about 2 hours. 2.5g of the erbumine salt Form A was obtained as an off-white solid in 71% yield. FR03684-SU4-erbumine-EA was used for minipolymorph screening reported in Example 10.
TABLE 29
D-BHB erbumine salt Form A
Figure imgf000068_0002
D-BHB erbumine salt Form A
Figure imgf000069_0001
[136] The XRPD for erbumine salt Form A (FR03684-SU4-erbumine-EA) is shown in Figure 25(A) having peaks as shown in Table 30.
TABLE 30
Figure imgf000069_0002
Figure imgf000070_0001
Figure imgf000071_0001
[137] Example 9: Scale up of D-BHB erbumine salt Form B
[138] Erbumine salt Form B (FR03684-SU8-erbumine-ACN-water-95-5) was prepared as follows. 2.0g of the D-BHB free Form I (FR03684-1-LP2) and 1.5g of erbumine (-1.05 equivalent by molar ratio) were weighed into a 20mL glass vial. 10 m of ACN was added into the vial under stirring at 25°C for about lOmin. A suspension was obtained. About Img of the erbumine salt Form B seeds (FR03684-3-PS8D) were added into above suspension. The suspension was kept stirring at 25°C for about 1 day. About 0. ImL of suspension was taken out and solid part was characterized by XRPD without Kapton film. A physical mixture of erbumine salt Form A and erbumine salt Form B was obtained after placed under ambient condition (20-25°C/30-60%RH) for about 30min (Figure 26(A)). Another 0. ImL of suspension was taken out and solid part was characterized by XRPD with Kapton film. Erbumine salt Form A was obtained (Figure 26(A)).
[139] Considering that the erbumine salt Form B could be a hydrate, about 0.5mL of water (5% water by volume) was added into above suspension and kept stirring at 25°C for about 4 hours to obtained the erbumine salt Form B. Solids were collected by filtration, and placed under ambient condition (20-25°C/30-60%RH) for 1 day. 1.8g of the erbumine salt Form B (Figure 26(A)). was obtained as an off-white solid in 51% yield. Characterization results are reported in Example 14. FR03684-SU8-erbumine-ACN-water-95-5 was used for bulk stability study, solubility study and hygroscopcity reported in Examples 11 to 13. The XRPD for D-BHB erbumine salt Form B (FR03684-SU8-erbumine-ACN-water-95-5) is shown in Figure 26(B) having peaks as shown in Table 31
TABLE 31
Figure imgf000071_0002
Figure imgf000072_0001
Figure imgf000073_0001
[140] Example 10: Mini-polymorph screening
[141] 30mg of each physical form was added to 50-200pL solvent. Obtained suspensions were equilibrated at 25°C for 1 week. Solids were isolated by centrifugation filtration, and wet cakes were analyzed by XRPD to determine crystal form change.
[142] Clear solutions were evaporated under ambient condition (20-25°C/40-70%RH), and obtained wet cakes were analyzed by XRPD to determine crystal form change.
TABLE 32
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
[143] Example 11: Bulk Stability
[144] Salts were placed at 25°C/92.5%RH in an open container, at 25°C/60%RH in an open container, at 40°C/75% RH in an open container and at 60°C in a closed container for 2 weeks. Samples after the stress were characterized by XRPD and IC and inspected for color change.
TABLE 33
Figure imgf000077_0002
Figure imgf000078_0001
[145] All salts tested were chemically stable under these conditions.
[146] No obvious impurity peaks were observed for the sodium salt Form C, the L-arginine salt Form B, the L-lysine salt Form A, or the erbumine salt Form B by IC.
[147] The sesqui-sodium salt Form C and the L-lysine salt Form A were physically stable with no form change after stressed at 60°C in a tight container over 2 weeks. However, each of these were partially deliquesced or deliquesced after stressed at 25°C/92.5%RH in an open container, at 25°C/60%RH in an open container, or at 40°C/75%RH in an open container over 2 weeks.
[148] The L-arginine salt Form B and the erbumine salt Form B showed obvious advantage in physical stability at 25°C/60%RH. Each form remained as a solid and showed no physical change after placed at 25°C/60%RH in an open container over 2 weeks. The L-arginine salt Form B was also physically stable with no form change after stressed at 60°C in a tight container over 2 weeks, while the erbumine salt Form B dehydrated and converted to the erbumine salt Form A after stressed at 60°C in a tight container over 2 weeks. Both the L-arginine salt Form B and the erbumine salt Form B are not tolerant to high humidity. They deliquesced after stressed at 25°C/92.5%RH in an open container, or at 40°C/75%RH in an open container over 2 weeks.
[149] Example 12: Solubility
[150] Solubility of the salts were measured in comparison of the D-BHB free Form I in seven aqueous pH buffers and bio-relevant fluids, including pH 1.2 HC1 buffer, pH 4.5 acetate buffer (50mM), pH 6.8 phosphate buffer (50mM), water, pH 1.6 FaSSGF (Fasted State Simulated Gastric Fluid - Biorelevant), pH 6.5 FaSSIF-vl (Fasted State Simulated Intestinal Fluid - Biorelevant), and pH 5.0 FeSSIF-vl (Fed State Simulated Intestinal Fluid - Biorelevant), at 37°C for 2 hours and 24 hours.
[151] Trial 1: Accurate l.Olmg of the D-BHB free Form I (FR03684-3 -LP2), 1.43mg of the sodium salt Form C (FR03684-SUl-NaOH-1.5-THF), 1.49mg of the sodium salt Form C (FR03684-SU9-NaOH-1.5-THF), 3.04mg of the L-arginine salt Form B FR03684-SU5-L- arginine-acetone- water-95-5), 2.58mg of theL-lysine saltForm A (FR03684-SU6-L-lysine-EtOH) or 1.89mg of the erbumine salt Form B (FR03684-SU8-erbumine-ACN-water-95-5) was weighed into a 2mL glass vial, respectively. 0.5mL of solubility medium (including pH 1.2 HC1 buffer, pH 4.5 acetate buffer, pH 6.8 phosphate buffer water, FaSSGF, FaSSIF and FeSSIF) was added. The salt amount used are equivalent to 1 mg anhydrous free form.
[152] Obtained clear solutions were stirred at 37°C at 400 rpm and sampled at 2 hours and at 24 hours. Obtained clear solutions were analyzed by pH meter for pH value.
TABLE 34
Figure imgf000080_0001
[153] Trial 2: Accurate 5.06mg of the free Form I (FR03684-3-LP2), 7.14mg of the sodium salt Form C (FR03684-SUl-NaOH-1.5-THF), 7.47mg of the sodium salt Form C (FR03684-SU9- NaOH-1.5-THF), 15.18mg of the L-arginine salt Form B (FR03684-SU5-L-arginine-acetone- water-95-5), 12.89mg of the L-lysine salt Form A (FR03684-SU6-L-lysine-EtOH), or 9.43mg of the erbumine salt Form B (FR03684-SU8-erbumine-ACN-water-95-5) was weighed into a 2mL glass vial, respectively. 0.5mL of solubility medium (including pH 1.2 HC1 buffer, pH 4.5 acetate buffer, pH 6.8 phosphate buffer, water, FaSSGF, FaSSIF andFeSSIF) was added. The salt amount used are equivalent to 5mg anhydrous free form.
[154] Obtained clear solutions were stirred at 37°C at 400 rpm and sampled at 2 hours and at 24 hours. Obtained clear solutions were analyzed by pH meter for pH value.
TABLE 35
Figure imgf000081_0001
Figure imgf000082_0001
[155] Trial 3: Accurate 253.04mg D-BHB free Form I (FR03684-3-LP2), 753.54mg L-arginine salt Form B (sample ID FR03684-SU2-L-arginine-acetone-water-95-5-re), 603.66mg L-lysine salt Form A (sample ID FR03684-SU3-L-lysine-EtOH-re), 321.16mg sodium salt Form C (sample ID FR03684-SUl-NaOH-1.5-THF), or 377.25mg of the erbumine salt Form B (sample ID FR03684- SU8-erbumine-ACN-water-95-5) was weighed into a 2mL glass vial, respectively. ImL of pH 1.2 HC1 buffer, pH 4.5 acetate buffer or pH 6.8 phosphate buffer was added in the D-BHB free Form I, L-arginine salt Form B and L-lysine salt Form A. The D-BHB free Form I, L-arginine salt Form B and L-lysine salt Form A amount used are equivalent to 250mg anhydrous free Form I. 0.9mL of pH 1.2 HC1 buffer, pH 4.5 acetate buffer or pH 6.8 phosphate buffer was added in sodium salt Form C. The sodium salt amount used is equivalent to 225mg anhydrous free form. 0.8mL of pH 1.2 HC1 buffer, pH 4.5 acetate buffer or pH 6.8 phosphate buffer was added in erbumine salt Form B. The erbumine salt Form B used is equivalent to 200mg anhydrous free form. [156] Accurate 75.91mg of the D-BHB free Form I (batch FR03684-3-LP2), 110.85mg of the sodium salt Form C (sample ID FR03684-SU9-NaOH-1.5-THF), 227.76mg of the L-arginine salt Form B (sample ID FR03684-SU5-L-arginine-acetone-water-95-5), 190.97mg of the L-lysine salt Form A (sample ID FR03684-SU6-L-lysine-EtOH) or 235.77mg of the erbumine salt Form B (sample ID FR03684-SU8-erbumine-ACN-water-95-5) was weighed into a 2mL glass vial, respectively. 0.3mL of water was added to the D-BHB free Form I, the sodium salt Form C, the L-arginine salt Form B, and the L-lysine salt Form A. The D-BHB free Form I, the sodium salt Form C, the L-arginine salt Form B, the L-lysine salt Form A amount used are equivalent to 75mg anhydrous D-BHB free Form I. 0.5mL of water was added to the erbumine salt Form B. The erbumine salt Form B amount used is equivalent to 125mg anhydrous free form.
[157] Obtained clear solutions/almost clear solutions were stirred at 37°C at 400 rpm and sampled at 2 hours and at 24 hours. The samples were centrifuged at 37°C at 14,000 rpm for 5min. Supernatants were analyzed by IC and pH meter for solubility and pH value, respectively.
TABLE 36
Figure imgf000084_0001
[158] Both the D-BHB crystalline salts and the D-BHB free Form I demonstrated good solubility in selected aqueous media. They showed >10mg/mL solubility in pH 1.6FaSSGF, pH 6.5 FaSSIF- vl, and pH 5.0 FeSSIF-vl. When target concentration for solubility test was increased to 250mg/mL, the D-BHB free Form I showed >250mg/mL solubility in pH 1.2 HC1 buffer, pH 4.5 acetate buffer, pH 6.8 phosphate buffer and water. The D-BHB salts showed 170-240mg/mL solubility in pH 1.2 HC1 buffer, 120-160mg/mL solubility in pH 4.5 acetate buffer and 100- 160mg/mL in pH 6.8 phosphate buffer after 24h.
[159] Example 13: Hygroscopicity
TABLE 37
Hygroscopicity by DVS at 25°C - METHOD 1
Physical Form
Figure imgf000085_0001
Hygroscopicity by DVS at 25°C - METHOD 1
“//” = Not carried out
Non-hygroscopic water uptake <0.2% Slightly hygroscopic water uptake >0.2% but <2% Moderately hygroscopic water uptake >2% but <15% Very hygroscopic water uptake >15%
Water uptake=water sorption in a specific RH (80% to 95%) - water sorption in 40%RH The criteria are modified from the European Pharmacopeia criteria about hygroscopicity
TABLE 38
Hygroscopicity by DVS at 25°C - METHOD 2
Physical Form
Figure imgf000086_0001
Hygroscopicity by DVS at 25°C - METHOD 2
“//” = Not carried out
Non-hygroscopic water uptake <0.2% Slightly hygroscopic water uptake >0.2% but <2% Moderately hygroscopic water uptake >2% but <15% Very hygroscopic water uptake >15%
Water uptake=water sorption in a specific RH (80% to 95%) - water sorption in 40%RH
The criteria are modified from the European Pharmacopeia criteria about hygroscopicity
[160] Hygroscopicity of the D-BHB salts was evaluated by dynamic vapor sorption (DVS) test at 25°C. The L-arginine salt Form B showed advantage over the other D-BHB salts in hygroscopicity.
[161] The L-arginine salt Form B is moderately hygroscopic below 70%RH. After the DVS test, the L-arginine salt Form B converted to an amorphous form.
[162] The sesqui-sodium salt Form C and the L-lysine salt Form A became very hygroscopic at above 40%RH and 50%RH, respectively. Then they deliquesced in high humidity. After the DVS test, the sesqui-sodium salt Form C converted to the sesqui-sodium salt Form B and the L-lysine salt Form A converted to a low crystalline form
[163] The erbumine salt Form B underwent dehydration below 20%RH and lost about 9.4% water. The water was absorbed back after the relative humidity (RH) was increased to 50%RH. Then the erbumine salt Form B became very hygroscopic in above 60%RH and deliquesced in high humidity. After the DVS test, and the erbumine salt Form B converted to a liquid state.
[164] Example 14 - Chemical and Physiochemical Properties
[165] Sodium salt Form C is a hydrate. Sample FR03684-SUl-NaOH-1.5-THF is of high crystallinity. DSC shows multiple thermal events. TGA shows about 7.8% weight loss at about 130°C. IC shows D-BHB: Na+ is 1: 1.5. 1H-NMR shows no detectable residual solvent. KF shows it contains about 6.7% water by weight, equivalent to 0.6 water molecule.
[166] L-arginine salt Form B is a hydrate. Sample FR03684-SU5-L-arginine-acetone-water-95- 5 is of high crystallinity. DSC shows a dehydration Tonset of 89.2°C. Melting occurs upon dehydration. TGA shows about 6.3% weight loss at about 130°C. 1H-NMR shows D-BHB: L-arginine is 1: 1.0 and no detectable residual solvent. KF shows it contains about 6.2% water by weight, equivalent to 1.1 water molecule. [167] L-lysine salt Form A is an anhydrate. Sample FR03684-SU6-L-lysine-EtOH is of high crystallinity. DSC shows a melting Tonset of 108.4°C with an enthalpy of about 79J/g. TGA shows about 4.1% weight loss at about 100°C. 1H-NMR shows D-BHB: L-lysine is 1: 1.0 and no detectable residual solvent.
[168] Erbumine salt Form B is a hydrate. Sample FR03684-SU8-erbumine-ACN-water-95-5 is of high crystallinity. DSC shows a dehydration Tonset of 51.9°C and then a melting Tonset of 72.8°C. TGA shows about 10.3% weight loss at about 90°C. 1H-NMR shows D-BHB: erbumine is 1: 1.0 and no detectable residual solvent. KF shows it contains about 9.7% water by weight, equivalent to 1.1 water molecule.
TABLE 39
Figure imgf000089_0001
Stoichiometry by 1H-NMR or IC
Figure imgf000089_0002
Figure imgf000090_0001
[169] Example 15 - DVS Isotherm Plots
[170] (1) DVS Method 1 was used to generate a DVS isotherm plot of D-BHB sodium salt Form C (FR03684-SUl-NaOH-1.5-THF), at 25°C.
TABLE 40
Sample FR03684-3-SU1-2-DVS name:
Batch no.:
Sample mass: 14.986 mg Lowest net
Reference weight: weight
Temperature: 25 °C
Figure imgf000091_0001
[171] (2) DVS Method 1 was used to generate a DVS isotherm plot of D-BHB L-arginine salt Form B (FR03684-SU5-L-arginine-acetone-water-95-5), at 25°C.
TABLE 41
Sample FR03684-3 -SU5-D VS name:
Batch no.:
Sample mass: 8.678 mg
Reference weight: Lowest net weight
Temperature: 25 °C
Figure imgf000091_0002
Figure imgf000092_0001
[172] (3) DVS Method 2 was used to generate a DVS isotherm plot of D-BHB L-lysine salt Form A (FR03684-SU6-L-lysine-EtOH), at 25°C.
TABLE 42
Figure imgf000092_0002
90.0 180.4
95.0 190.7
Figure imgf000093_0002
[173] (4) DVS Method 2 was used to generate a DVS isotherm plot of D-BHB erbumine salt Form B (FR03684-SU8-erbumine-ACN-water-95-5), at 25°C.
TABLE 43
Meth:
STA-SSD Lab DVS-06_40-0-95-0-40%RH_Step 10%_time240min
Sample: FR03684-3-SU8-2-DVS
Temp: 24.7 °C
MRef: 15.0475 from Custom Mass
Figure imgf000093_0001
Cycle 2 0.0 4.0 4.0
10.0 3.6 4.4 0.7
20.0 3.4 4.8 1.5
30.0 3.1 5.4 2.4
40.0 2.7 9.2 6.5
50.0 18.8
60.0 34.7
70.0 76.1
80.0 147.5 90.0 201.2
95.0 194.7
Figure imgf000094_0001
[174] Example 16: Extended Stability Study
[175] D-BHB sodium salt Form C, hydrate (C210608011-B/PJ06432-24-B-DRY) and D-BHB L-arginine salt Form B, hydrate (C210608011-FP/PJ06432-23-FP-DRY) (Table 45) were studied at 25°C±2°C/60%RH±5%RH, 40°C±2°C/75%RH±5%RH, 50°C±2°C/75%RH±5%RH. The samples were tested at weeks 1, 2, 3, and 4. The bulk substance was studied in a container closure system that simulates packaging of bulk materials: approximately 1.0 g/package for each of the tests and time points, with the sample packed into double antistatic LDPE bag that was secured with a cable tie. 4 bags of desiccant were then put between two layers of antistatic LDPE bag. Each package (sample in bag with 4 bags of desiccant) into a foil bag that was heat sealed. The foil bag was then stored within a Fiber drum. The drums were then stored in a stability chamber at the conditions noted above with the temperature and humidity monitored and recorded. The tests were Appearance, Related substance by Ion Chromatography (IC), Potency, Coulometric water content determination according to Karl Fischer, XPRD, and Chiral purity.
[176] D-BHB sodium salt Form C, hydrate (C210608011-B/PJ06432-24-B-DRY): Throughout the course of the four-week study, at each of the three temperature/relative humidity conditions, the compound: Maintained a constant appearance, without change: white solid; Maintained chiral purity, without change: 100.0 %; and Maintained XPRD of Form C, without change.
Other parameters are shown in Table 44, in which the Conditions (abbreviated “Con”) are (A) 25°C±2°C/60%RH±5%RH, (B) 40°C±2°C/75%RH±5%RH, and
(C) 50°C±2°C/75%RH±5%RH.
[177] D-BHB L-arginine salt Form B, hydrate (C210608011-FP/PJ06432-23-FP-DRY) Throughout the course of the four-week study, at each of the three temperature/relative humidity conditions, the compound: Maintained a constant appearance, without change: white solid; Maintained chiral purity, without change: 100.0 %; and Maintained XPRD of Form B, without change.
Other parameters are shown in Table 45, in which the Conditions (abbreviated “Con”) are (A) 25°C±2°C/60%RH±5%RH, (B) 40°C±2°C/75%RH±5%RH, and
(C) 50°C±2°C/75%RH±5%RH. Table 44 - D-BHB sodium salt Form C, hydrate (C210608011-B/PJ06432-24-B-DRY)
Figure imgf000095_0001
Table 45 - D-BHB L-arginine salt Form B, hydrate (C210608011-FP/PJ06432-23-FP-DRY)
Figure imgf000096_0001

Claims

1. A crystalline D-BHB L-arginine salt designated as “D-BHB L-arginine Form B” having an XRPD pattern comprising peaks at 7.30, 9.94, 11.28, 14.56, and 15.83 ± 0.2°29 using Cu Ka radiation.
2. The crystalline D-BHB L-arginine salt Form B of claim 1, wherein one or more of the following
(a) wherein the XRPD pattern further comprises peaks at 17.27, 18.33, 19.91, 21.90, and 23.49 ± 0.2°29 using Cu Ka radiation;
(b) wherein the XRPD pattern further comprises peaks at 24.12, 26.45, 26.99, 28.56, 33.56, and 34.16 ± 0.2° 29 using Cu Ka radiation;
(c) having an XRPD pattern substantially as shown in Figure 30(A);
(d) having substantially the XRPD peaks shown in Table 25; and/or
(e) having substantially the DSC of Figure 30(B) and/or substantially the TGA of Figure 30(C).
3. A crystalline D-BHB sodium salt designated as “D-BHB sodium salt Form C” having an XRPD pattern comprising peaks at 6.40, 7.07, 12.11, 14.08, and 17.05 ± 0.2°29 using Cu Ka radiation.
4. The crystalline D-BHB sodium salt Form C of claim 3, wherein one or more of the following
(a) the XRPD pattern further comprises peaks at 19.16, 22.57, 27.79, 28.75, and 30.11 ± 0.2°29 using Cu Ka radiation;
(b) the XRPD pattern further comprises peaks at 11.98, 30.76, 33.12, 33.63, and 37.08 ± 0.2° 29 using Cu Ka radiation;
(c) having an XRPD pattern substantially as shown in Figure 29(A);
(d) having substantially the XRPD peaks shown in Table 20;
(e) having substantially the DSC of Figure 29(B); and/or
(f) having substantially the TGA of Figure 29(C).
5. A crystalline D-BHB (“free Form I”) having an XRPD pattern comprising peaks at 10.93, 12.11, 15.16, 17.53, and 18.88 ± 0.2° 29 using Cu Ka radiation.
6. The crystalline D-BHB (“free Form I”) of claim 5, wherein one or more of the following
(a) the XRPD pattern further comprising peaks at 22.75, 24.30, 26.14, 29.10, and 29.95
± 0.2°29 using Cu Ka radiation;
(b) the XRPD pattern further comprising peaks at 30.48, 31.60, 32.96, 33.98, 36.80, and 38.37 ± 0.2° 29 using Cu Ka radiation; (c) having an XRPD pattern substantially as shown in Figure 1(A);
(d) having substantially the XRPD peaks as shown in Table 2;
(e) having substantially the DSC of Figure 2(A); and/or
(f) having substantially the TGA of Figure 3(A).
7. A crystalline D-BHB sodium salt designated as “D-BHB sodium salt Form A”, having an XRPD pattern comprising peaks at 11.82, 17.02, 17.58, 20.34, and 20.83 ± 0.2 29 using Cu Ka radiation.
8. The crystalline D-BHB sodium salt Form A of claim 7, wherein one or more of the following
(a) the XRPD pattern further comprises peaks at 7.23, 14.39, 24.50, 29.54, and 30.17 ± 0.2° 29 using Cu Ka radiation;
(b) the XRPD pattern further comprises peaks at 12.22, 22.23, 22.60, 30.63, 33.35, and 37.28 ± 0.2° 29 using Cu Ka radiation;
(c) having an XRPD pattern substantially as shown in Figure 9(A);
(d) having substantially the XRPD peaks as shown in Table 16;
(e) having substantially the DSC of Figure 9(B); and/or
(f) having substantially the TGA of Figure 9(C).
9. A crystalline D-BHB sodium salt designated “D-BHB sodium salt Form B” having an XRPD pattern comprising peaks at 8.98, 11.49, 16.95, 17.53, and 19.38 ± 0.2°29 using Cu Ka radiation.
10. The crystalline D-BHB sodium salt Form B of claim 9, wherein one or more of the following
(a) the XRPD pattern further comprises peaks at 7.17, 9.67, 12.16, 20.10, and 26.83 ± 0.2° 29 using Cu Ka radiation;
(b) the XRPD pattern further comprises peaks at 6.47, 14.02, 14.35, 18.35, 21.00, and 23.08 ± 0.2° 29 using Cu Ka radiation;
(c) having an XRPD pattern substantially as shown in Figure 10(A);
(d) having substantially the XRPD peaks as shown in Table 17;
(e) having substantially the DSC of Figure 10(B); and/or
(f) having substantially the TGA of Figure 10(C).
11. A crystalline D-BHB magnesium salt designated as “D-BHB magnesium salt Form A” having an XRPD pattern comprising peaks at 5.53, 7.80, 9.61, 11.95, and 13.15 ± 0.2°29 using Cu Ka radiation.
12. The crystalline D-BHB magnesium salt Form A of claim 11, wherein one or more of the following
(a) the XRPD pattern further comprises peaks at 6.11, 11.02, 11.27, 15.61, and
18.98 ± 0.2°29 using Cu Ka radiation;
(b) the XRPD pattern further comprises peaks at 19.45, 21.13, 21.88, 23.70, and
25.99 ± 0.2°29 using Cu Ka radiation;
(c) having an XRRD pattern substantially as shown in Figure 7(A);
(d) having substantially the XRPD peaks shown in Table 13;
(e) having substantially the DSC of Figure 7(B); and/or
(f) having substantially the TGA of Figure 7(C).
13. A crystalline D-BHB magnesium salt designated as “D-BHB magnesium salt Form B” having an XRPD pattern comprising peaks at 6.12, 8.31, 9.60, 11.87, and 13.45 ± 0.2°29 using Cu Ka radiation.
14. The crystalline D-BHB magnesium salt Form B of claim 13, wherein one or more of the following
(a) the XRPD pattern further comprises peaks at 11.49, 12.23, 14.09, 14.31, and 18.46 ± 0.2°29 using Cu Ka radiation;
(b) the XRPD pattern further comprises peaks at 17.01, 19.61, 23.70, 24.62, 24.94, and 29.02 and 18.46 ± 0.2°29 using Cu Ka radiation;
(c) having an XPRD pattern substantially as shown in Figure 8(A);
(d) having substantially the XRPD peaks shown in Table 14;
(e) having substantially the DSC of Figure 8(B); and/or
(f) having substantially the TGA of Figure 8(C).
15. A crystalline D-BHB L-lysine salt designated “D-BHB L-lysine salt Form A” having an XRPD pattern comprising peaks at 6.92, 9.16, 12.62, 23.29, and 25.91 ± 0.2°29 using Cu Ka radiation.
16. The crystalline D-BHB L-lysine salt Form A of claim 15, wherein one or more of the following
(a) the XRPD pattern further comprises peaks at 10.21, 26.93, 27.64, 28.45, and 29.87 ± 0.2°29 using Cu Ka radiation;
(b) the XRPD pattern further comprises peaks at 17.87, 18.27, 19.73, 24.58, and 25.02 ± 0.2°29 using Cu Ka radiation;
(c) having an XRPD pattern substantially as shown in Figure 31(A);
(d) having substantially the XRPD peaks shown in Table 28; (e) having substantially the DSC of Figure 31(B); and/or
(f) having substantially the TGA of Figure 31(C).
17. A crystalline D-BHB L-arginine salt designated as “D-BHB L-arginine Form A” having an XRPD pattern comprising peaks at 5.28, 7.28, 9.11, 14.99, and 15.82 ± 0.2°29 using Cu Ka radiation.
18. The crystalline D-BHB L- arginine salt Form A of claim 17, wherein one or more of the following
(a) the XRPD pattern further comprises peaks at 17.00, 17.76, 18.32, 19.32, and 24.10 ± 0.2°29 using Cu Ka radiation;
(b) the XRPD pattern further comprises peaks at 10.81, 11.08, 11.27, 11.75, and 26.96 ± 0.2°29 using Cu Ka radiation;
(c) having an XRPD pattern substantially as shown in Figure 14(A);
(d) having substantially the XRPD peaks shown in Table 10;
(e) having substantially the DSC of Figure 14(B); and/or
(f) having substantially the TGA of Figure 14(C).
19. A crystalline D-BHB erbumine salt designated as “D-BHB erbumine salt Form A” having an XRPD pattern comprising peaks at 10.04, 10.77, 13.69, 14.71, and 18.24 ± 0.2°29 using Cu Ka radiation.
20. The crystalline D-BHB erbumine salt Form A of claim 19, wherein one or more of the following
(a) the XRPD pattern further comprises peaks at 16.08, 16.95, 17.42, 19.37, and 20.07 ± 0.2°29 using Cu Ka radiation;
(b) the XRPD pattern further comprises peaks at 22.47, 24.35, 25.60, 26.05 and 28.36 ± 0.2°29 using Cu Ka radiation;
(c) having an XRPD pattern substantially as shown in Figure 25(A);
(d) having substantially the XRPD peaks shown in Table 30;
(e) having substantially the DSC of Figure 25(B); and/or
(f) having substantially the TGA of Figure 25(C).
21. A crystalline D-BHB erbumine salt designated as “D-BHB erbumine salt Form B” having an XRPD pattern comprising peaks at 12.42, 16.07, 16.69, 17.62, and 18.35 ± 0.2°29 using Cu Ka radiation.
22. The crystalline D-BHB erbumine salt Form B of claim 21, wherein one or more of the following (a) the XRPD pattern further comprises peaks at 8.82, 20.71, 22.22, 23.95, and 27.27 ± 0.2° 29 using Cu Ka radiation;
(b) the XRPD pattern further comprises peaks at 19.76, 21.22, 23.49, 28.46, 31.36, and 38.21 ± 0.2° 29 using Cu Ka radiation;
(c) having an XRPD pattern substantially as shown in Figure 26(B);
(d) having substantially the XRPD peaks shown in Table 31;
(e) having substantially the DSC of Figure 26(C); and/or
(f) having substantially the TGA of Figure 26(D).
23. The crystalline D-BHB of any one of claims 1 to 22, wherein
(a) the D-BHB is greater than 95% enantiomerically pure, greater than 98% enantiomerically pure, or greater than 99% enantiomerically pure; or
(b) the D-BHB is greater than 99% enantiomerically pure.
24. A composition comprising one or more crystalline D-BHB forms as described in any one of claims 1 to 23.
25. A composition comprising two or more crystalline D-BHB forms as described in any one of claims 1 to 23.
26. The composition of claim 24 or claim 25, wherein the composition comprises, consists essentially of, or consists of
(a) D-BHB L-arginine salt Form B;
(b) D-BHB sodium salt Form C; and/or
(c) D-BHB L-arginine salt Form B and D-BHB sodium salt Form C.
27. A pharmaceutical composition comprising one or more crystalline D-BHB as described in any one of claims 1 to 23 and at least one pharmaceutically acceptable carrier.
28. A pharmaceutical composition comprising two or more crystalline D-BHB as described in any one of claims 1 to 23 and at least one pharmaceutically acceptable carrier.
29. The pharmaceutical composition of claim 27 or claim 28, wherein the composition comprises, consists essentially of, or consists of
(a) D-BHB L-arginine salt Form B;
(b) D-BHB sodium salt Form C; and/or
(c) D-BHB L-arginine salt Form B and D-BHB sodium salt Form C.
39. The pharmaceutical composition of any one of claims 27 to 29, wherein the composition is formulated for oral administration or for administration by injection.
31. The pharmaceutical composition of any one of claims 27 to 39, wherein the composition is formulated to administer up to (a) about 15 g D-BHB per dose;
(b) about 12 g D-BHB per dose; or
(c) about 10 g D-BHB per dose.
32. The pharmaceutical composition of any one of claims 27 to 31, wherein the composition is formulated to administer up to about 60 g D-BHB per day, up to about 48 g D-BHB per day, up to about 45 g D-BHB per day, up to about 40 g D-BHB per day, about 36 g D- BHB per day, up to about 30 g D-BHB per day, up to about 20 g D-BHB per day, or up to about 10 g per day.
33. The pharmaceutical composition of claim 32, wherein the composition is formulated for administration as two doses per day, three doses per day, or four doses per day.
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