Polymorphs of sepiapterin and salts thereof
11773097 · 2023-10-03
Assignee
Inventors
Cpc classification
C07D475/04
CHEMISTRY; METALLURGY
International classification
A61K31/495
HUMAN NECESSITIES
Abstract
Disclosed are crystalline forms of sepiapterin free base selected from polymorphs A, B, C, D, E, F, and G, and combinations thereof, as well as crystalline polymorphs of salts of sepiapterin. Also disclosed are pharmaceutical compositions containing one or more such polymorphs and methods for preparing such polymorphs. Sepiapterin is useful in the treatment of a number diseases associated with low cellular levels of BH4, for example, phenylketonuria.
Claims
1. A pharmaceutical composition comprising crystalline Form D of sepiapterin free base having peaks at diffraction angle 2θ (°) of about 8.9°, about 10.3°, and about 26.0° as measured by X-ray diffractometry by irradiation with Cu Kα X-rays or calculated from X-ray diffractometry and a pharmaceutically acceptable carrier.
2. The pharmaceutical composition of claim 1, wherein the crystalline Form D is present in an amount of at least 90 percent by weight of the composition.
3. The pharmaceutical composition of claim 1, wherein the crystalline Form D is formulated as particles less than 100 μm in size.
4. The pharmaceutical composition of claim 1, wherein the crystalline Form D has peaks at diffraction angle 2θ (°) of about 8.9°, about 10.3°, about 10.9°, about 17.8°, about 24.9°, about 26.0°, about 26.7°, about 26.8°, and about 28.3° as measured by X-ray diffractometry by irradiation with Cu Kα X-rays or calculated from X-ray diffractometry.
5. A method of treating a tetrahydrobiopterin (BH4)-related disorder in a subject in need thereof, the method comprising administering an effective amount of the pharmaceutical composition of claim 1 to the subject, wherein the BH4-related disorder is selected from primary BH4 deficiency and phenylketonuria.
6. The method of claim 5, wherein the pharmaceutical composition is a liquid suspension.
7. A method of treating a tetrahydrobiopterin (BH4)-related disorder in a subject in need thereof, the method comprising administering an effective amount of crystalline Form D of sepiapterin free base having peaks at diffraction angle 2θ (°) of about 8.9°, about 10.3°, and about 26.0° as measured by X-ray diffractometry by irradiation with Cu Kα X-rays or calculated from X-ray diffractometry to the subject, wherein the BH4-related disorder is selected from primary BH4 deficiency and phenylketonuria.
8. The method of claim 7, wherein the crystalline Form D of sepiapterin free base is administered as a liquid suspension.
9. The method of claim 7, wherein the crystalline Form D has peaks at diffraction angle 2θ (°) of about 8.9°, about 10.3°, about 10.9°, about 17.8°, about 24.9°, about 26.0°, about 26.7°, about 26.8°, and about 28.3° as measured by X-ray diffractometry by irradiation with Cu Kα X-rays or calculated from X-ray diffractometry.
10. A method for decreasing phenylalanine levels in a subject in need thereof, the method comprising administering to the patient an effective amount of the pharmaceutical composition of claim 1 to the subject.
11. The method of claim 10, wherein the pharmaceutical composition is a liquid suspension.
12. A method for decreasing phenylalanine levels in a subject in need thereof, the method comprising administering an effective amount of crystalline Form D of sepiapterin free base having peaks at diffraction angle 2θ (°) of about 8.9°, about 10.3°, and about 26.0° as measured by X-ray diffractometry by irradiation with Cu Kα X-rays or calculated from X-ray diffractometry to the subject.
13. The method of claim 12, wherein the crystalline Form D of sepiapterin free base is administered as a liquid suspension.
14. The method of claim 12, wherein the crystalline Form D has peaks at diffraction angle 2θ (°) of about 8.9°, about 10.3°, about 10.9°, about 17.8°, about 24.9°, about 26.0°, about 26.7°, about 26.8°, and about 28.3° as measured by X-ray diffractometry by irradiation with Cu Kα X-rays or calculated from X-ray diffractometry.
15. A method of preparing a liquid formulation comprising dispersing crystalline Form D of sepiapterin free base having peaks at diffraction angle 2θ (°) of about 8.9°, about 10.3°, and about 26.0° as measured by X-ray diffractometry by irradiation with Cu Kα X-rays or calculated from X-ray diffractometry in a liquid.
16. The method of claim 15, wherein the liquid formulation is a suspension.
17. The method of claim 15, wherein the crystalline Form D has peaks at diffraction angle 2θ (°) of about 8.9°, about 10.3°, about 10.9°, about 17.8°, about 24.9°, about 26.0°, about 26.7°, about 26.8°, and about 28.3° as measured by X-ray diffractometry by irradiation with Cu Kα X-rays or calculated from X-ray diffractometry.
Description
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
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DETAILED DESCRIPTION OF THE INVENTION
(23) The present invention provides a solid form of sepiapterin, wherein the solid form comprises an amorphous form, a crystalline polymorph form, a mixture of amorphous and/or crystalline polymorph forms, a salt of sepiapterin, or a combination thereof, with the proviso that the solid form of sepiapterin is not a pure polymorph Form A and/or pure polymorph Form E.
(24) In an embodiment, in the solid form of sepiapterin the mixture comprises at least one of crystalline polymorph Form B, C, D, F, and G of sepiapterin.
(25) In an embodiment, the solid form comprises at least one crystalline sepiapterin free base selected from polymorph Forms B, C, D, F, and G and crystalline polymorph A or E or both crystalline polymorphs A and E.
(26) The polymorphic forms of sepiapterin free base as well as the polymorphic forms of the salts of sepiapterin may be characterized by any suitable method for studying solid state materials. In an embodiment, the polymorphic forms are characterized by X-ray powder Diffractometry (XRD). The XRD peak positions are expressed as the 2θ°. In the X-ray diagram, the angle of refraction 2θ is plotted on the horizontal axis (x-axis) and the relative peak intensity (background-corrected peak intensity) on the vertical (y-axis). X-ray powder diffraction patterns are measured on, or using instruments comparable to, a PANalytical Empyrean X-ray powder diffractometer with Cu Kα radiation source (Kα1 radiation, wavelength λ=1.54060 Angstrom, Kα2 radiation, wavelength 1.544426 Angstrom; Kα2/Kα1 intensity ratio: 0.50). The optical density of the peaks on the film is proportional to the light intensity. The film is scanned using a peak scanner.
(27) As it relates to any of the peaks of X-ray powder diffraction set forth throughout this application, “about” refers to ±0.1, particularly ±0.05, and more particularly ±0.02 of the 2θ values in degrees.
(28) In an embodiment, the crystalline polymorph Form B of sepiapterin is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 8.4, about 16.9, and about 25.4°.
(29) In a particular embodiment, the crystalline polymorph Form B of sepiapterin is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 8.4, about 14.9, about 16.9, about 25.4, and about 34.1°.
(30) In an embodiment, the crystalline polymorph Form C of sepiapterin is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 5.7, about 7.8, and about 25.4°.
(31) In a particular embodiment, the crystalline sepiapterin polymorph Form C is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 5.7, about 7.8, about 9.1, about 11.5, about 15.3, about 16.0, about 20.1, about 25.4, and about 26.6°.
(32) In an embodiment, the crystalline sepiapterin polymorph Form D is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 8.9, about 10.3, and about 26.0°.
(33) In a particular embodiment, the crystalline sepiapterin polymorph Form D is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 8.9, about 10.3, about 10.9, about 17.8, about 24.9, about 26.0, about 26.7, about 26.8, and about 28.3°.
(34) In an embodiment, the crystalline sepiapterin polymorph Form F is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 9.7, about 10.2, and about 11.3°.
(35) In a particular embodiment, the crystalline sepiapterin polymorph Form F is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 9.7, about 10.2, about 11.3, about 14.0, about 14.6, about 19.9, about 22.2, about 25.3, and about 32.4°.
(36) In an embodiment, the crystalline sepiapterin polymorph Form G is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 10.0, about 10.6, and about 25.7°.
(37) In a particular embodiment, the crystalline sepiapterin polymorph Form G is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 10.0, about 10.6, about 11.2, about 15.3, about 15.9, about 22.8, about 24.4, about 25.0, about 25.7, and about 26.6°.
(38) In an embodiment, the solid form comprises at least one crystal sepiapterin free base selected from polymorph forms B, C, D, F, and G; selected from polymorph forms B, C, and D; selected from polymorph forms B, C, and F; selected from polymorph forms D, F, and G; as well as any binary, ternary, or quaternary combinations of the polymorph forms. The solid forms indicated above could further include polymorph A and/or E.
(39) In an embodiment, polymorph Form B, C, D, or G, or a combination thereof, is present in the solid form in an amount of at least 90 percent by weight of the solid form.
(40) In certain embodiments, the crystalline sepiapterin free base is present in at least 70 percent or more by weight, at least 80 percent or more by weight, and preferably at least 90 percent or more by weight, based on the weight of the sepiapterin free base.
(41) The crystalline Form A of sepiapterin free base is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 4.7°, about 7.4°, about 9.5°, about 11.3°, about 15.6°, about 26.2°, and about 27.2°.
(42)
(43) TABLE-US-00001 TABLE 1 Position [2θ°] Relative Intensity 4.7 47.76 7.4 100.00 9.5 33.54 11.3 19.31 12.4 8.49 13.4 3.60 14.2 8.24 15.6 15.08 16.4 11.97 17.6 8.35 18.4 5.03 19.8 9.18 21.5 5.44 24.4 5.56 26.2 35.37 27.2 19.11 28.9 5.93
(44) The crystalline Form B of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ of at least about 8.4, about 16.9, and about 25.4.
(45)
(46) TABLE-US-00002 TABLE 2 Position [2θ°] Relative Intensity 8.4 100.00 14.9 2.34 16.9 10.70 25.4 84.90 34.1 3.00
(47) The crystalline Form C of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ of at least at about 5.7°, about 7.8°, and about 25.4°.
(48)
(49) TABLE-US-00003 TABLE 3 Position [2θ°] Relative Intensity 5.7 48.91 7.8 100.00 9.1 59.49 10.4 8.72 11.5 24.53 12.9 8.50 14.8 9.24 15.3 12.53 16.0 14.09 17.2 7.22 18.2 4.25 19.2 5.78 20.1 14.54 21.5 6.47 22.9 6.85 23.7 4.80 25.4 65.68 26.6 14.53 27.4 8.39 31.5 3.74 34.2 4.36
(50) The crystal Form D of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at least at an angle of refraction 2θ of about 8.9°, about 10.3°, and about 26.0°.
(51)
(52) TABLE-US-00004 TABLE 4 Position [2θ°] Relative Intensity 8.9 100.00 10.3 49.92 10.9 19.96 11.6 2.15 13.6 2.99 14.2 3.45 14.8 2.35 15.4 2.59 16.4 1.55 17.2 2.33 17.8 6.24 19.6 2.62 20.1 2.28 20.5 3.09 20.8 2.27 21.3 3.60 22.3 4.79 23.7 4.31 24.9 5.19 26.0 41.94 26.7 8.58 26.8 9.17 27.4 3.98 28.3 4.75 28.7 6.60 29.8 3.03 31.8 2.72 33.0 2.03 35.5 1.57 37.1 1.09
(53) The crystalline Form E of sepiapterin free base is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 6.0°, about 10.6°, about 12.1°, about 15.9°, about 20.8°, and about 24.6°.
(54)
(55) TABLE-US-00005 TABLE 5 Position [2θ°] Relative Intensity 6.0 100.00 10.6 20.78 12.1 31.95 15.9 12.83 18.1 3.39 20.9 11.63 22.1 2.79 24.6 8.28 26.1 0.88 28.1 7.33 28.9 3.77 32.1 3.57 37.0 1.03
(56) The crystalline Form F of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ of at least about 9.7°, about 10.2°, and about 11.3°.
(57)
(58) TABLE-US-00006 TABLE 6 Position [2θ°] Relative Intensity 9.7 98.27 10.2 100.00 11.3 22.47 14.0 5.01 14.6 12.36 19.9 5.63 21.1 3.72 22.2 5.37 22.7 4.04 24.5 2.99 25.3 17.65 27.2 3.10 32.4 5.29 36.7 2.72
(59) The crystalline form G of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ of at least about 10.0°, about 10.6°, and about 25.7°.
(60) TABLE-US-00007 TABLE 7 Position [2θ°] Relative Intensity 5.3 8.30 6.9 4.54 10.0 100.00 10.6 69.64 11.2 6.59 13.5 7.52 15.3 26.59 15.9 26.43 16.0 23.41 16.9 4.28 18.6 13.02 19.3 11.90 20.1 7.22 20.8 11.01 22.8 16.77 23.5 19.60 24.4 41.45 25.0 23.99 25.7 65.40 26.6 39.64 27.6 13.04 28.7 6.55 30.8 14.76 32.2 9.63 33.7 5.16 37.5 5.80
(61) In the context of stating that crystalline Form A of sepiapterin free base exhibits an X-ray diffraction diagram essentially as in
(62) Alternatively, or in addition, crystalline Form A of sepiapterin free base is characterized by a DSC curve showing endothermal peaks at 82.8° C. and 179.8° C.
(63) In the context of stating that crystalline Form B of sepiapterin free base exhibits an X-ray diffraction diagram essentially as in
(64) Alternatively, or in addition, the crystalline Form B of sepiapterin free base is characterized by a DSC curve showing a melting event at 195.2° C.
(65) In a preferred embodiment, an essentially pure crystalline Form B of sepiapterin free base shows the X-ray diffraction diagram indicated in
(66) In another preferred embodiment, crystalline Form B of sepiapterin free base shows an X-ray diffraction diagram of the type shown in
(67) In the context of stating that crystalline Form C of sepiapterin free base exhibits an X-ray diffraction diagram essentially as in
(68) Alternatively, the crystalline Form C of sepiapterin free base is characterized by a DSC curve showing five endothermal peaks at 58.3° C., 101.8° C., 129.8° C., 156.5° C., and 168.3° C.
(69) In one preferred embodiment, the essentially pure crystalline Form C of sepiapterin free base shows the X-ray diffraction diagram indicated in
(70) In another preferred embodiment, the crystalline Form C of sepiapterin free base shows an X-ray diffraction diagram of the type shown in
(71) In the context of stating that the crystalline Form D of sepiapterin free base exhibits an X-ray diffraction diagram essentially as in
(72) Alternatively, the crystalline Form D of sepiapterin free base is characterized by a DSC curve showing three endotherms at 42.7° C., 66.3° C., and 232.9° C.
(73) In one preferred embodiment, the essentially pure crystalline Form D of sepiapterin free base shows the X-ray diffraction diagram indicated in
(74) In another preferred embodiment, the crystalline Form D of sepiapterin free base shows an X-ray diffraction diagram of the type shown in
(75) In the context of stating that the crystalline Form E of sepiapterin free base exhibits an X-ray diffraction diagram essentially as in
(76) Alternatively, the crystalline Form E of sepiapterin free base is characterized by a DSC curve showing two endothermal peaks at 112.9° C. and 195.8° C.
(77) In the context of stating that the crystalline Form F of sepiapterin free base exhibits an X-ray diffraction diagram essentially as in
(78) Alternatively, the crystalline Form F of sepiapterin free base is characterized by a DSC curve showing two endotherms at 71.6° C. and 233.4° C.
(79) In one preferred embodiment, the essentially pure crystalline Form F of sepiapterin free base shows the X-ray diffraction diagram indicated in
(80) In another preferred embodiment, the crystalline Form F of sepiapterin free base shows an X-ray diffraction diagram of the type shown in
(81) In the context of stating that the crystalline Form G of sepiapterin free base exhibits an X-ray diffraction diagram essentially as in
(82) In one preferred embodiment, the essentially pure crystalline Form G of sepiapterin free base shows the X-ray diffraction diagram indicated in
(83) In another preferred embodiment, the crystalline Form G of sepiapterin free base shows an X-ray diffraction diagram of the type shown in
(84) The invention also provides a crystalline form of sepiapterin hydrochloride salt.
(85) In an embodiment, the crystalline hydrochloride salt is characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 7.8°, about 12.9°, and about 26.2°.
(86) The invention further provides a crystalline polymorph form of a salt of sepiapterin. In certain embodiments, the invention provides a crystalline polymorph form of a salt of sepiapterin, wherein the salt is a salt of sepiapterin with sulfuric acid, p-toluene sulfonic acid, methane sulfonic acid, benzene sulfonic acid, malonic acid, tartaric acid (e.g., L-tartaric acid), phosphoric acid, gentisic acid, fumaric acid, glycolic acid, acetic acid, or nicotinic acid.
(87) In particular embodiments, the crystalline polymorph salt is selected from the group consisting of:
(88) crystalline Form 1 methanesulfonate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 7.8°, about 23.5°, and about 29.0°;
(89) crystalline Form 2 methanesulfonate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 7.9°, about 23.4°, and about 28.9°;
(90) crystalline Form 3 methanesulfonate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 21.7°, about 26.0°, and about 28.9°;
(91) crystalline nicotinate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 9.5°, about 9.9°, and about 24.5°;
(92) crystalline p-toluenesulfonate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 6.5°, about 15.1°, and about 23.4°;
(93) crystalline benzenesulfonate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 6.5°, about 14.8°, and about 19.6°;
(94) crystalline phosphate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 16.6°, about 22.2°, and about 25.6°;
(95) crystalline malonate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 6.9°, about 22.7°, and about 23.8°;
(96) crystalline tartrate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 7.3°, about 14.2°, and about 21.8°;
(97) crystalline gentisate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 7.1°, about 8.7°, and about 26.7°;
(98) crystalline fumarate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at about 11.3°, about 24.0°, and about 28.2°;
(99) crystalline glycolate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 7.6°, about 10.7°, and about 24.0°;
(100) crystalline acetate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 6.2°, about 12.0°, and about 18.1′;
(101) crystalline Form 1 sulfate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 5.1°, about 7.8°, and about 23.0°; and
(102) crystalline Form 2 sulfate salt characterized by an X-ray powder diffraction pattern obtained by irradiation with Cu Kα X-rays having peaks expressed as 2θ at least at about 7.8°, about 8.8°, and about 24.1°.
(103) The crystalline hydrochloride salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ of at least at about 7.8°, about 12.9°, and about 26.2°.
(104)
(105) TABLE-US-00008 TABLE 8 Position [2θ°] Relative Intensity 7.8 100.00 8.9 6.89 12.9 58.56 15.6 8.52 17.9 25.23 19.2 5.48 21.1 10.97 23.6 25.15 25.2 22.66 26.2 45.91 27.6 32.94 30.3 10.50 31.7 7.83 34.2 8.87 36.7 3.67
(106) The crystalline Form 1 methanesulfonate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 7.8°, about 23.5°, and about 29.0°.
(107)
(108) TABLE-US-00009 TABLE 9 Position [2θ°] Relative Intensity 7.9 21.77 11.7 8.20 13.7 8.52 15.7 4.79 16.6 5.34 18.0 5.66 19.8 2.10 20.3 5.36 20.9 2.43 22.3 4.25 22.7 2.15 23.5 100.00 24.7 3.69 25.6 2.70 26.8 1.79 27.2 1.68 28.3 2.75 29.0 57.60 29.8 5.18 30.5 1.37 32.2 4.66 33.0 1.64 36.5 1.29
(109) The crystalline Form 2 methanesulfonate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 7.9°, about 23.4°, and about 28.9°.
(110)
(111) TABLE-US-00010 TABLE 10 Position [2θ°] Relative Intensity 7.9 100.00 11.0 21.32 12.1 22.02 13.5 79.87 15.7 11.87 17.8 9.81 19.7 10.93 21.3 26.79 23.4 96.13 24.1 24.88 24.3 22.10 25.5 9.45 26.0 11.27 27.6 7.63 28.9 95.64 31.2 4.39 36.1 6.65
(112) The crystalline Form 3 methanesulfonate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 21.7°, about 26.0°, and about 28.9°.
(113)
(114) TABLE-US-00011 TABLE 11 Position [2θ°] Relative Intensity 8.2 47.29 10.8 56.14 12.6 16.34 13.2 15.90 14.0 24.39 15.0 12.03 15.9 16.20 18.2 22.97 20.1 25.53 20.5 14.97 21.3 22.70 21.7 71.48 22.2 11.40 23.6 46.37 24.8 44.00 25.5 9.08 26.1 100.00 27.3 3.52 28.9 68.42 31.2 4.49 32.1 6.48 34.8 5.95 35.6 1.67 39.1 2.91
(115) The crystalline nicotinate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 9.5°, about 9.9°, and about 24.5°.
(116)
(117) TABLE-US-00012 TABLE 12 Position [2θ°] Relative Intensity 9.5 10.29 9.9 53.95 11.5 9.31 12.0 11.76 14.7 14.20 15.9 17.61 17.5 7.53 19.0 5.37 20.8 5.88 21.3 6.12 21.7 7.20 23.2 34.05 24.5 100.00 25.2 12.90 28.0 8.51 31.1 5.39 32.3 4.52 33.4 8.02 35.1 5.05
(118) The crystalline p-toluenesulfonate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 6.5°, about 15.1°, and about 23.4°.
(119)
(120) TABLE-US-00013 TABLE 13 Position [2θ°] Relative Intensity 6.5 100.00 12.9 1.79 14.3 1.39 15.1 15.36 16.2 5.33 18.4 8.96 19.6 3.06 20.2 4.86 21.8 2.23 22.5 2.95 23.1 7.99 23.4 9.14 24.5 1.81 26.0 2.48 27.0 4.49 27.3 3.93 28.1 5.31 28.4 5.59 28.8 2.05 30.6 2.24 31.0 1.98 32.6 1.82
(121) The crystalline benzenesulfonate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 6.5°, about 14.8°, and about 19.6°.
(122)
(123) TABLE-US-00014 TABLE 14 Position [2θ°] Relative Intensity 4.9 5.90 6.5 100.00 14.8 16.73 17.8 4.23 19.6 7.98 21.5 2.49 23.7 3.46 24.5 3.84 26.1 3.29
(124) The crystalline phosphate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 16.6°, about 22.2°, and about 25.6°.
(125)
(126) TABLE-US-00015 TABLE 15 Position [2θ°] Relative Intensity 5.5 4.41 8.1 1.21 8.9 2.21 10.3 1.79 10.8 5.80 15.3 1.84 16.6 8.35 17.7 1.95 20.3 1.40 21.2 1.61 22.2 9.77 23.1 1.74 25.6 100.00 30.8 6.31 31.1 4.85 33.5 0.73 36.0 1.70
(127) The crystalline malonate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 6.9°, about 22.7°, and about 23.8°.
(128)
(129) TABLE-US-00016 TABLE 16 Position [2θ°] Relative Intensity 6.9 100.00 8.4 13.11 10.6 7.62 16.4 5.63 17.8 9.73 19.3 8.96 20.1 9.99 22.2 10.50 22.7 20.52 23.8 34.02 24.5 5.82 25.5 24.50 26.6 4.00 27.3 6.96 29.8 5.38 33.1 12.08
(130) The crystalline L-tartrate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 7.3°, about 14.2°, and about 21.8°.
(131)
(132) TABLE-US-00017 TABLE 17 Position [2θ°] Relative Intensity 7.4 100.00 10.1 47.99 14.2 82.76 14.7 27.06 19.1 21.16 20.2 29.91 21.8 85.30 22.1 53.68 23.9 85.30 24.9 19.26 25.5 28.45 26.8 18.58 29.7 21.59 31.6 10.10 32.9 22.18
(133) The crystalline gentisate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 7.1°, about 8.7°, and about 26.7°.
(134)
(135) TABLE-US-00018 TABLE 18 Position [2θ°] Relative Intensity 5.7 17.29 7.1 100.00 8.7 42.69 10.4 3.94 11.3 11.69 12.1 4.13 14.3 21.10 16.0 6.46 16.4 5.94 17.0 5.85 17.6 7.93 19.1 8.27 20.20 3.47 20.7 2.90 21.5 3.37 23.6 2.69 24.4 4.50 26.7 52.20 27.1 35.49 28.2 8.74 28.9 4.31 29.9 2.62 31.4 2.99 34.4 1.28 35.8 3.54 37.6 0.57
(136) The crystalline fumarate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 11.3°, about 24.0°, and about 28.2°.
(137)
(138) TABLE-US-00019 TABLE 19 Position [2θ°] Relative Intensity 6.1 6.43 7.7 5.40 11.4 53.62 11.9 33.37 14.2 8.03 16.5 6.70 18.3 13.86 19.0 6.68 20.7 10.02 21.3 7.02 22.8 24.68 24.0 100.00 28.3 33.26 32.7 6.35 36.0 3.28 38.5 6.02
(139) The crystalline glycolate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 7.6°, about 10.7°, and about 24.0°.
(140)
(141) TABLE-US-00020 TABLE 20 Position [2θ°] Relative Intensity 4.8 6.23 7.6 100.00 10.3 68.06 10.7 70.69 15.3 36.51 18.2 24.25 18.7 27.26 19.9 2.66 21.2 17.11 24.0 96.62 24.4 18.44 28.8 47.57 30.3 7.43 32.5 4.42 33.3 7.49 34.3 5.21 36.3 7.37
(142) The crystalline acetate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 6.2°, about 12.0°, and about 18.1°.
(143)
(144) TABLE-US-00021 TABLE 21 Position [2θ°] Relative Intensity 6.2 100.00 10.2 23.29 12.0 71.59 18.1 31.27 21.1 20.29 24.2 14.92 25.2 23.03 27.3 13.30 29.1 12.95
(145) The crystalline Form 1 sulfate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 5.1°, about 7.8°, and about 23.0°.
(146)
(147) TABLE-US-00022 TABLE 22 Position [2θ°] Relative Intensity 5.1 100.00 6.8 3.33 7.8 43.48 10.2 15.92 15.7 18.13 17.2 8.33 18.7 6.49 19.8 5.19 21.3 5.52 23.0 19.05 23.5 8.29 24.2 5.59 24.8 17.44 25.7 4.97 26.7 10.38 28.7 11.49 30.4 2.88 31.0 3.67
(148) The crystalline Form 2 sulfate salt of sepiapterin free base is characterized by peaks in the X-ray diffraction diagram observed at an angle of refraction 2θ at least at about 7.8°, about 8.8°, and about 24.1°.
(149)
(150) TABLE-US-00023 TABLE 23 Position [2θ°] Relative Intensity 5.0 4.71 7.9 72.24 8.8 100.00 14.5 19.26 15.7 59.40 16.1 8.69 17.2 14.82 17.7 10.89 19.3 9.92 20.2 9.60 23.7 15.38 24.2 43.88 25.0 11.44 26.8 16.81 28.7 16.07 29.4 13.84 31.3 17.14 31.7 7.26 35.7 5.75
(151) In the context of stating that the crystalline hydrochloride salt of sepiapterin free base exhibits an X-ray diffraction diagram essentially as in
(152) In one preferred embodiment, the essentially pure crystalline hydrochloride salt of sepiapterin free base shows the X-ray diffraction diagram indicated in
(153) In another preferred embodiment, the crystalline hydrochloride salt of sepiapterin free base shows an X-ray diffraction diagram of the type shown in
(154) In the context of stating that the crystalline salt forms of sepiapterin free base, such as the crystalline form 1 methanesulfonate salt, crystalline form 2 methanesulfonate salt, crystalline form 3 methanesulfonate salt, crystalline nicotinate salt, crystalline p-toluenesulfonate salt, crystalline benzenesulfonate salt, crystalline phosphate salt, crystalline malonate salt, crystalline L-tartrate salt, crystalline gentisate salt, crystalline fumarate salt, crystalline glycolate salt, crystalline acetate salt, crystalline form 1 sulfate salt, and crystalline form 2 sulfate salt, exhibits an X-ray diffraction diagram such as essentially as in
(155) In preferred embodiments, the essentially pure crystalline hydrochloride salt of sepiapterin free base shows the X-ray diffraction diagram indicated in
(156) In another preferred embodiment, the crystalline form 1 methanesulfonate salt, crystalline form 2 methanesulfonate salt, crystalline form 3 methanesulfonate salt, crystalline nicotinate salt, crystalline p-toluenesulfonate salt, crystalline benzenesulfonate salt, crystalline phosphate salt, crystalline malonate salt, crystalline L-tartrate salt, crystalline gentisate salt, crystalline fumarate salt, crystalline glycolate salt, crystalline acetate salt, crystalline form 1 sulfate salt, and crystalline form 2 sulfate salt of sepiapterin free base shows X-ray diffraction diagrams of the type shown in
(157) Alternatively, the crystalline form 1 methanesulfonate salt of sepiapterin free base is characterized by a DSC curve showing two endotherms at 186.0° C. and 229.1° C.;
(158) the crystalline form 2 methanesulfonate salt of sepiapterin free base is characterized by a DSC curve showing three endotherms at 75.5° C., 182.6° C., and 234.9° C.;
(159) the crystalline form 3 methanesulfonate salt of sepiapterin free base is characterized by a DSC curve showing two endotherms at 195.1° C. and 240.1° C.;
(160) the crystalline nicotinate salt of sepiapterin free base is characterized by a DSC curve showing an endotherm at 221.9° C.;
(161) the crystalline p-toluenesulfonate salt of sepiapterin free base is characterized by a DSC curve showing three endotherms at 77.2° C., 202.4° C. and 260.2° C.;
(162) the crystalline benzenesulfonate salt of sepiapterin free base is characterized by a DSC curve showing two endotherms at 202.3° C. and 265.5° C.;
(163) the crystalline phosphate salt of sepiapterin free base is characterized by a DSC curve showing three endotherms at 125.9° C., 152.1° C., and 157.6° C.;
(164) the crystalline malonate salt of sepiapterin free base is characterized by a DSC curve showing a melting event at 115.8° C.;
(165) the crystalline L-tartrate salt of sepiapterin free base is characterized by a DSC curve showing two endotherms at 97.2° C. and 160.6° C.;
(166) the crystalline gentisate salt of sepiapterin free base is characterized by a DSC curve showing three endotherms at 70.5° C., 128.2° C., and 184.7° C.;
(167) the crystalline fumarate salt of sepiapterin free base is characterized by a DSC curve showing two endotherms at 114.3° C. and 229.7° C.;
(168) the crystalline glycolate salt of sepiapterin free base is characterized by a DSC curve showing two endotherms at 133.9° C. and 147.7° C.;
(169) the crystalline acetate salt of sepiapterin free base is characterized by a DSC curve showing two endotherms at 146.1° C. and 175.4° C.; and
(170) the crystalline form 1 sulfate salt of sepiapterin free base is characterized by a DSC curve showing three endotherms at 94.5° C., 158.3° C., and 209.9° C.
(171) In any of the above embodiments, the crystalline sepiapterin free base or a crystalline polymorph form of a salt of sepiapterin can occur as an anhydrate (e.g., without having any bound water or solvent or hydration or solvation) or as a hydrate, a partial hydrate (e.g., hemihydrate, sesquihydrate, and the like), as a dihydrate, a trihydrate, or the like, wherein the crystalline form binds a water of hydration or a solvent molecule associated with the crystalline form of sepiapterin or salt thereof. In an embodiment, crystalline sepiapterin Form B occurs as an anhydrate. In an embodiment, crystalline sepiapterin Form C occurs as a monohydrate or as a sesquihydrate. In an embodiment, crystalline sepiapterin Form D occurs as a monohydrate or as a sesquihydrate. In an embodiment, crystalline sepiapterin Form F occurs as a monohydrate or as a hemihydrate. In an embodiment, crystalline sepiapterin Form G occurs as an anhydrate.
(172) In an embodiment, the invention provides a method for preparing crystalline Form D of sepiapterin. The method comprises preparing a slurry of sepiapterin in a liquid, wherein the liquid is water, acetone/water, isopropanol/isopropyl acetate, or tetrahydrofuran/n-hexane, and isolating sepiapterin Form D from the slurry. Preferably, the liquid is water. The sepiapterin Form D can be isolated using any suitable isolation method, for example, by centrifugation or by filtration. Preferably, the sepiapterin Form D is isolated by filtration. Typically, the sepiapterin Form D is further freed from solvent (e.g., water) by drying at room temperature.
(173) In an embodiment, the invention provides a method for preparing crystalline Form F of sepiapterin. The method comprises preparing a slurry of sepiapterin in a solvent, wherein the solvent is water, acetone/water, isopropanol/isopropyl acetate, or tetrahydrofuran/n-hexane, and isolating sepiapterin Form D from the slurry. Preferably, the solvent is water. The sepiapterin Form D can be isolated using any suitable isolation method, for example, by centrifugation or by filtration. Preferably, the sepiapterin Form D is isolated by filtration. Sepiapterin Form D is then converted to Form F typically, by heating to 40-60° C. for 0.5-10 hours. Heating may be either at atmospheric pressure or under vacuum. Preferably heating is under vacuum.
(174) Sepiapterin may serve as a useful therapeutic for diseases associated with low intracellular BH4 levels or with dysfunction of various BH4 dependent metabolic pathways including, but not limited to, primary tetrahydrobiopterin deficiency, GTPCH deficiency, 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, DHPR deficiency, sepiapterin reductase deficiency, dopamine responsive dystonia, Segawa Syndrome, tyrosine hydroxylase deficiency, phenylketonuria, DNAJC12 deficiency, Parkinson's Disease, depression due to Parkinson's Disease, impulsivity in Parkinson's patients, major depression, Autism spectrum, ADHD, schizophrenia, Bipolar disorder, cerebral ischemia, restless leg syndrome, Obsessive Compulsive Disorder, anxiety, aggression in Alzheimer's disease, cerebrovascular disorders, spasm after subarachnoidal hemorrhage, myocarditis, coronary vasospasm, cardiac hypertrophy, arteriosclerosis, hypertension, thrombosis, infections, endotoxin shock, hepatic cirrhosis, hypertrophic pyloric stenosis, gastric mucosal injury, pulmonary hypertension, renal dysfunction, impotence, and hypoglycemia. Thus, the various forms of sepiapterin in accordance with the present invention can be administered to a patient in an effective amount to obtain a treatment or amelioration of the disease, disorder or condition.
(175) The present invention further provides a pharmaceutical composition comprising a crystalline sepiapterin free base or a crystalline polymorph form of a salt of sepiapterin as described above and a pharmaceutically acceptable carrier. The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount, e.g., a therapeutically effective amount, including a prophylactically effective amount, of one or more of the aforesaid compounds, or salts thereof, of the present invention.
(176) The pharmaceutically acceptable carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the compound, and by the route of administration. It will be appreciated by one of skill in the art that, in addition to the following described pharmaceutical compositions; the compounds of the present invention can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.
(177) The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, or diluents, are well known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active compounds and one which has no detrimental side effects or toxicity under the conditions of use.
(178) The choice of carrier will be determined in part by the particular active agent, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intra-arterial, intramuscular, intraperitoneal, intrathecal, rectal, and vaginal administration are merely exemplary and are in no way limiting.
(179) The crystalline sepiapterin free base or a crystalline polymorph form of a salt of sepiapterin can be used in the preparation of liquid formulations, such as in the form of a solution, suspension, or emulsion. Formulations suitable for oral administration can consist of (a) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (b) powders; (c) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Preferred are solid oral dosage forms such as capsule forms, tablet forms, and powder forms. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.
(180) Formulations suitable for oral and/or parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, benzyl alcohol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol and other polyethylene alcohols, glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.
(181) Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene-polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazopeak quaternary ammonium salts, and (3) mixtures thereof.
(182) The parenteral formulations will typically contain from about 0.5 to about 25% by weight of the crystalline sepiapterin free base or a crystalline polymorph form of a salt of sepiapterin in solution. Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophilic-lipophilic balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
(183) The crystalline sepiapterin free base or a crystalline polymorph form of a salt of sepiapterin of the present invention may be made into injectable formulations. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986).
(184) Topical formulations, including those that are useful for transdermal drug release, are well-known to those of skill in the art and are suitable in the context of the invention for application to skin. Topically applied compositions are generally in the form of liquids, creams, pastes, lotions and gels. Topical administration includes application to the oral mucosa, which includes the oral cavity, oral epithelium, palate, gingival, and the nasal mucosa. In some embodiments, the composition contains at least one crystalline sepiapterin free base or a crystalline polymorph form of a salt of sepiapterin and a suitable vehicle or carrier. It may also contain other components, such as an anti-irritant. The carrier can be a liquid, solid or semi-solid. In embodiments, the composition is an aqueous solution. Alternatively, the composition can be a dispersion, emulsion, gel, lotion or cream vehicle for the various components. In one embodiment, the primary vehicle is water or a biocompatible solvent that is substantially neutral or that has been rendered substantially neutral. The liquid vehicle can include other materials, such as buffers, alcohols, glycerin, and mineral oils with various emulsifiers or dispersing agents as known in the art to obtain the desired pH, consistency and viscosity. It is possible that the compositions can be produced as solids, such as powders or granules. The solids can be applied directly or dissolved in water or a biocompatible solvent prior to use to form a solution that is substantially neutral or that has been rendered substantially neutral and that can then be applied to the target site. In embodiments of the invention, the vehicle for topical application to the skin can include water, buffered solutions, various alcohols, glycols such as glycerin, lipid materials such as fatty acids, mineral oils, phosphoglycerides, collagen, gelatin and silicone based materials.
(185) The compounds of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.
(186) Additionally, the crystalline sepiapterin free base or a crystalline polymorph form of a salt of sepiapterin of the present invention may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
(187) The crystalline sepiapterin free base or a crystalline polymorph form of a salt of sepiapterin can be used in any suitable dose. Suitable doses and dosage regimens can be determined by conventional range finding techniques. Generally treatment is initiated with smaller dosages, which are less than the optimum dose. Thereafter, the dosage is increased by small increments until optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. In proper doses and with suitable administration of certain compounds, the present invention provides for a wide range of responses. Typically, the dosages range from about 0.001 to about 1000 mg/kg body weight of the patient being treated/day. For example, in embodiments, the crystalline sepiapterin free base or a crystalline polymorph form of a salt of sepiapterin may be administered from about 100 mg/kg to about 300 mg/kg, from about 120 mg/kg to about 280 mg/kg, from about 140 mg/kg to about 260 mg/kg, from about 150 mg/kg to about 250 mg/kg, from about 160 mg/kg to about 240 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
(188) In some embodiments, the crystalline sepiapterin free base or a crystalline polymorph form of a salt of sepiapterin can be formulated into unit solid oral dosage forms such as capsules or tablets. In these embodiments, each unit solid oral dosage form can comprise any suitable amount of the crystalline sepiapterin free base or a crystalline polymorph form of a salt of sepiapterin. For example, each solid oral dosage form can comprise about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 50 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 2000 mg, about 3000 mg, about 4000 mg, about 5000 mg, and the like.
(189) The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
(190) Abbreviations: MeOH—methanol; DMSO—dimethylsulfoxide; EtOAc—ethyl acetate; DMAc—dimethylacetamide; THF—tetrahydrofuran; NMP—N-methylpyrrolidone.
(191) For X-ray powder diffraction analysis, a PANalytical Empyrean X-ray powder diffractometer was used. The parameters are as follows:
(192) TABLE-US-00024 XRD Parameters Parameter Value X-Ray wavelength Cu, kα, Kα1 (Å): 1.540598, Kα2 (Å): 1.544426 Kα2/Kα1 intensity ratio: 0.50 X-Ray tube setting 45 kV, 40 mA Divergence slit Automatic Scan mode Continuous Scan range (°2θ) 3°~40° Scan step time (s) 17.8 Test time (s) 5 min 30 s
(193) DSC was performed using a TA Q200/Q2000 DSC from TA Instruments. Parameters used are as follows:
(194) TABLE-US-00025 Parameters DSC Method Ramp Sample pan Aluminum, crimped Temperature 25° C.-desired temperature Heating rate 10° C./min Purge gas N.sub.2
EXAMPLES
(195) While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention. As such, the following examples are provided to teach various aspects of the present invention. These examples represent individual embodiments of the aspects of this invention and one skilled in the art will recognize that additional examples can be generated in order to equally teach the aspects of the present invention.
Example 1
(196) This example demonstrates a preparation of the crystalline Form B of sepiapterin free base in accordance with an embodiment of the invention.
(197) 73.2 mg of starting material sepiapterin was weighed into a 20-mL glass vial. 2.5 mL of N-methyl pyrrolidone (NMP) was added to dissolve the starting material. The solution was filtered into a new vial. 17 mL of acetonitrile (ACN) was added step-wise, with the sample stirring at RT with a rate of ˜1000 rpm. The suspension was stirred at RT for 2 hrs. The resulting precipitate was isolated by centrifugation and dried in vacuum at RT for 3 hrs to obtain crystalline Form B of sepiapterin free base.
Example 2
(198) This example demonstrates a preparation of crystalline Form C of sepiapterin free base in accordance with an embodiment of the invention.
(199) 100.4 mg of starting material sepiapterin was weighed into a 20-mL glass vial. 2 mL of ACN was added to form a suspension, which was stirred at 50° C. with a rate of ˜1000 rpm. The resulting solids were isolated by centrifugation for 2 minutes through a 0.25 □m pore size centrifugation filter and drying at RT for approximately 12 hours to obtain crystalline Form C of sepiapterin free base.
Example 3
(200) This example demonstrates a preparation of the crystalline Form D of sepiapterin free base in accordance with an embodiment of the invention.
(201) 200.1 mg of starting material sepiapterin was weighed into a 20-mL glass vial. 5 mL of H.sub.2O was added to form a suspension, which was stirred at 50° C. with a rate of ˜1000 rpm. The resulting solids were isolated by centrifugation for 2 minutes through a 0.25 □m pore size centrifugation filter. One-half of the collected solids were dried at RT for approximately 12 hours at atmospheric pressure to obtain crystalline Form D of sepiapterin free base.
Example 4
(202) This example demonstrates a preparation of the crystalline Form F of sepiapterin free base in accordance with an embodiment of the invention. The other half of the collected solids from Example 3 were dried under vacuum at 50° C. for 0.5 hr to obtain crystalline Form F of sepiapterin free base.
Example 5
(203) This example demonstrates a preparation of the crystalline Form G of sepiapterin free base in accordance with an embodiment of the invention. The crystalline Form G of sepiapterin free base was prepared by heating a sample of crystalline Form F prepared as in Example 4 to 120° C. under N.sub.2 flow.
Example 6
(204) This example demonstrates a preparation of the crystalline hydrochloride salt of sepiapterin free base in accordance with an embodiment of the invention.
(205) 120.4 mg of sepiapterin freebase was weighed into a 20-mL glass vial. 0.8 mL of acetone/H.sub.2O (9:1, v/v) and 42 μL of conc. HCl (37.5%) were added, and the resulting suspension was stirred at RT at a rate of ˜1000 rpm for 5 days. The resulting solids were isolated by vacuum filtration and dried in vacuum at RT for 3 hrs.
(206) The solids obtained above were dispersed in 3 mL of acetone/H.sub.2O (9:1, v/v). 5.5 μL of conc. HCl (37.5%) was added and the suspension was stirred at RT at a rate of ˜1000 rpm for 6 days, following which, the solids were isolated by vacuum filtration and dried in vacuum at RT overnight to obtain the crystalline hydrochloride salt of sepiapterin free base.
Example 7
(207) This example demonstrates a preparation of the crystalline Form 3 methanesulfonate salt of sepiapterin free base in accordance with an embodiment of the invention.
(208) 51.7 mg of methanesulfonic acid was weighed into a 20-mL glass vial. 5 mL of MeOH was added to the vial. 120.7 mg of sepiapterin freebase was weighed into the vial. The resulting suspension was stirred at RT at a rate of ˜1000 rpm for 5 days, following which 20 μL of methanesulfonic acid was added to the vial. The resulting mixture was stirred at RT at a rate of ˜1000 rpm for 1 day. The solids were isolated by vacuum filtration and dried in vacuum at RT overnight. The dried solids were dispersed in 3 mL of MeOH and stirred at RT at a rate of ˜1000 rpm for 1 day. The solids were isolated by vacuum filtration and dried in vacuum at RT overnight to obtain crystalline Form 3 methanesulfonate salt of sepiapterin free base.
Example 8
(209) This example demonstrates a preparation of the crystalline nicotinate salt of sepiapterin free base in accordance with an embodiment of the invention.
(210) 119.5 mg of freebase was weighed into a 20-mL glass vial. 10 mL of MeOH was added to the vial. 100.1 mg of nicotinic acid was weighed into the vial. The resulting suspension was stirred at RT at a rate of ˜1000 rpm for 7 hrs, following which the obtained solids were isolated by vacuum filtration and dried in vacuum at RT for 3 hrs to obtain the crystalline nicotinate salt of sepiapterin free base.
Example 9
(211) This example demonstrates a preparation of the crystalline salt forms of sepiapterin free base in accordance with an embodiment of the invention.
(212) Crystalline form 1 sulfate salt was obtained by slurrying equimolar amounts of starting material and H.sub.2SO.sub.4 in acetone/H.sub.2O (9:1, v/v).
(213) Crystalline form 2 sulfate salt was obtained by slurrying equimolar amounts of starting material and H.sub.2SO.sub.4 in THF/DMAc (9:1, v/v).
(214) Crystalline p-toluenesulfonate salt was obtained by slurrying equimolar amounts of starting material and p-toluene sulfonic acid in methanol.
(215) Crystalline Form 1 methanesulfonate salt was obtained by slurrying equimolar amounts of starting material and methane sulfonic acid in methanol.
(216) Crystalline Form 2 methanesulfonate salt was obtained by slurrying equimolar amounts of starting material and methane sulfonic acid in acetone/H.sub.2O (9:1, v/v).
(217) Crystalline benzenesulfonate salt was obtained by slurrying equimolar amounts of starting material and benzene sulfonic acid in methanol.
(218) Crystalline phosphate salt was obtained by slurrying equimolar amounts of starting material and H.sub.3PO.sub.4 in acetone/H.sub.2O (9:1, v/v).
(219) Crystalline malonate salt was obtained by slurrying starting material and malonic acid (molar ratio of acid/freebase about 5:1) in acetone/H.sub.2O (9:1, v/v).
(220) Crystalline L-tartrate salt was obtained by slurrying starting material and gentisic acid (molar ratio of acid/freebase about 4:1) in acetone/H.sub.2O (9:1, v/v).
(221) Crystalline gentisate salt was obtained by slurrying starting material and L-tartaric acid (molar ratio of acid/freebase about 5:1) in acetone/H.sub.2O (9:1, v/v).
(222) Crystalline fumarate salt was obtained by slurrying starting material and fumaric acid (molar ratio of acid/freebase about 5:1) in acetone/H.sub.2O (9:1, v/v).
(223) Crystalline glycolate salt was obtained by slurrying starting material and glycolic acid (molar ratio of acid/freebase about 4:1) in acetone/H.sub.2O (9:1, v/v).
(224) Crystalline acetate salt was obtained by slurrying starting material and acetic acid (molar ratio of acid/freebase about 5:1) in acetone/H.sub.2O (9:1, v/v).
Example 10
(225) This example demonstrates characterization of the starting sepiapterin used in the preparation of the crystalline polymorphs A, B, C, D, E, F, and G of sepiapterin free base and of the crystalline polymorph forms of salts of sepiapterin described herein.
(226) A sample of sepiapterin free base was obtained commercially. DSC showed two endotherms at 82.8° C. and 179.8° C. The sepiapterin sample contained particles with an average particle size over 100 μm. The XRD pattern was determined before and after grinding to reduce the particle size such that it passes through a 140 mesh screen. The XRD patterns both before and after grinding are shown in
Example 11
(227) This example demonstrates the results of stability studies carried out on the sepiapterin starting material (Form A), crystalline polymorph Form D, and crystalline polymorph Form F at temperatures of room temperature (RT), 35° C., and 50° C.
(228) The purity of initial samples was determined by HPLC and was found to be as follows: Form A=99.3 area %, Form F=99.7 area %, Form D=99.1 area %, wherein area % refers to the area under the curve of the sepiapterin peak as compared with the total area under all of the peaks.
(229) Form A and F samples were placed in chambers with silica gel to remove water (the relative humidity was measured to be ˜10% RH) at different temperatures. Form D samples were placed in chambers with water (relative humidity was estimated to be ˜100% RH) at different temperatures.
(230) The HPLC purities and XRD patterns were obtained for each of Form A/F/D samples stored at various temperatures. The results after 1 week and 4 weeks of storage are set forth in Tables 22 and 23, respectively.
(231) TABLE-US-00026 TABLE 24 Storage after 1 week RT 35° C. 50° C. Purity % of Form Purity % of Form Purity % of Form Sample Humidity (area %) Initial change (area %) Initial change (area %) Initial change Form A 10% RH 98.8 99.5 No 98.0 98.6 No 95.2 95.8 No Form F 10% RH 99.4 99.7 No 99.4 99.7 No 99.2 99.4 No Form D 100% RH 99.0 99.9 No 98.8 99.7 No 98.5 99.3 No
(232) TABLE-US-00027 TABLE 25 Storage after 4 weeks RT 35° C. 50° C. Purity % of Form Purity % of Form Purity % of Form Sample Humidity (area%) Initial change (area%) Initial change (area%) Initial change Form A 10% RH 98.2 98.8 No 96.1 96.7 No 88.5 89.1 No Form F 10% RH 99.5 99.8 No 99.4 99.7 No 98.9 99.1 No Form D 100% RH 98.9 99.8 No 98.7 99.6 No 97.7 98.5 No
(233) As is apparent from the results set forth in Tables 24 and 25, none of the samples exhibited a significant change in crystal structure as observed by XPD. Form A exhibited significantly less stability as determined by HPLC. After storage for 4 weeks at 50° C., the purity of Form A as measured by HPLC peak area % was 89.1% compared to the initial purity. The purities of Forms F and D were 99.1% and 98.5%, respectively, compared to the initial purity.
Example 12
(234) This example demonstrates the stability of polymorphs D and F of sepiapterin free base on storage.
(235) Samples of sepiapterin free base polymorph Forms D, F, and A were stored at room temperature (RT), 35° C., and 50° C. The samples were analyzed by HPLC at 1 week and 4 week intervals. The HPLC parameters were as follows:
(236) TABLE-US-00028 Parameters Solubility Stability (purity) Column Inertsil ODS-3, 4.6 × 250 mm, 5 μm Mobile phase A: 20 mM K.sub.2HPO.sub.4-KH.sub.2PO.sub.4 buffer (pH 7.0) : ACN (98:2) B: 20 mM K.sub.2HPO.sub.4-KH.sub.2PO.sub.4 buffer (pH 7.0) : ACN (50:50) Gradient table Time (min) % B Time (min) % B 0.0 0 0.0 0 3.0 0 5.0 0 10.0 100 25.0 100 10.1 0 25.1 0 12.0 0 35.0 0 Run time 12.0 min 35.0 min Post time 0.0 min Flow rate 1.0 mL/min Injection 5 μL volume Detector UV at 280 nm wavelength Column 40° C. temperature Sampler RT temperature Diluent H.sub.2O
(237) The results for polymorphs A, F, and D of sepiapterin free base are set forth in Tables 26-28.
(238) TABLE-US-00029 TABLE 26 Polymorph A RT/10% RH 35° C./10% RH 50° C./10% RH # RRT Initial 1w 4w 1w 4w 1w 4w Impurity 0.62 0.08 0.16 0.37 0.24 1.03 0.68 2.59 Impurity 0.89 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 0.06 Impurity 0.95 0.42 0.77 1.29 1.36 2.70 3.48 7.67 Sepiapterin 1.00 99.33 98.80 98.18 97.97 96.06 95.15 88.49 Impurity 1.06 <0.05 <0.05 <0.05 <0.05 <0.05 0.45 <0.05 Impurity 1.08 <0.05 0.11 <0.05 0.28 <0.05 0.08 0.85 Impurity 1.17 0.17 0.16 0.16 0.16 0.16 0.16 0.17 Impurity 1.21 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 0.06 Impurity 1.25 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 0.11
(239) TABLE-US-00030 TABLE 27 Polymorph F 35° C./ 50° C./ RT/10% RH 10% RH 10% RH # RRT Initial 1w 4w 1w 4w 1w 4w Impurity 0.60 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 0.12 Impurity 0.95 0.09 0.29 0.30 0.35 0.42 0.58 0.86 Sepiapterin 1.00 99.74 99.42 99.54 99.43 99.43 99.15 98.85 Impurity 1.08 <0.05 0.14 <0.05 0.06 <0.05 0.11 <0.05 Impurity 1.17 0.17 0.16 0.16 0.15 0.15 0.15 0.17
(240) TABLE-US-00031 TABLE 28 Polymorph D RT/ 35° C./ 50° C./ 100% RH 100% RH 100% RH # RRT* Initial 1w 4w 1w 4w 1w 4w Impurity 0.62 <0.05 <0.05 0.09 <0.05 0.09 0.08 0.14 Impurity 0.95 0.69 0.83 0.81 0.86 1.04 1.05 2.03 Sepiapterin 1.00 99.14 99.01 98.92 98.83 98.70 98.45 97.65 Impurity 1.08 <0.05 <0.05 <0.05 0.14 <0.05 0.26 <0.05 Impurity 1.17 0.17 0.16 0.17 0.17 0.17 0.16 0.18 *Relative retention time
(241) As is apparent from the results set forth in Tables 26-28, polymorphs D and F of sepiapterin free base exhibited significantly greater stability than polymorph A. The amount of sepiapterin in polymorph A decreased from 99.33% for 88.49% after storage for 4 weeks at 50° C./10% RH (relative humidity). The amount of sepiapterin in polymorph D decreased from 99.14% to 97.65% after storage for 4 weeks at 50° C./100% RH. The amount of sepiapterin in polymorph F decreased from 99.74% to 98.85% after storage for 4 weeks at 50° C./10% RH.
Example 13
(242) This example demonstrates a preparation of crystalline Form E of sepiapterin free base.
(243) 100.6 mg of starting material was weighed into a 3-mL glass vial. 1 mL of MeOH was added to form a suspension. The sample was stirred at RT with a rate of ˜1000 rpm. The resulting solids were isolated by centrifugation after 3 days and dried at RT overnight.
Other Embodiments
(244) The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
(245) Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.